Currículum - Universidad de Salamanca

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Ministerio de Economía y Competitividad Secretaría de Estado Desarrollo e Innovación

de

Investigación,

Currículum

Nombre: Luis Miguel García Segura Fecha: 10/09/2014

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Plan Nacional de I+D+I ATENCION: Deben firmarse al margen todas las hojas del curiculum Apellidos: García Segura DNI: 647599B

Nombre: Luis Miguel Fecha de nacimiento : 30.10.52

Sexo: V

Situación profesional actual Organismo: CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS Facultad, Escuela o Instituto: INSTITUTO CAJAL Depto./Secc./Unidad estr.: NEUROBIOLOGÍA FUNCIONAL Y DE SISTEMAS Dirección postal: AVENIDA DOCTOR ARCE 37; 28002 MADRID Teléfono (indicar prefijo, número y extensión): 915854729 (directo) Fax: 915854754 Correo electrónico: [email protected] Especialización (Códigos UNESCO): 240113 Categoria profesional: Profesor de Investigación Situación administrativa X Plantilla Contratado Otras situaciones especificar: Dedicación

Fecha de inicio: 1990 Interino

Becario

A tiempo completo X A tiempo parcial

Líneas de investigación Breve descripción, por medio de palabras claves, de la especialización y líneas de investigación actuales. Neuroprotección, neuroendocrinología, hormonas sexuales, receptores nucleares de hormonas esteroides

Formación Académica Titulación Superior Licenciado en Ciencias

Doctorado Ciencias Biológicas

Centro Universidad Complutense de Madrid

Centro Universidad Complutense de Madrid

Fecha 1974

Fecha 03/12/1976

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Actividades anteriores de carácter científico profesional Puesto

Institución

Fechas

BECARIO P.F.P.I.

INSTITUTO CAJAL, C.S.I.C.

1975-1977

PROF. ADJUNTO INTERINO

UNIV.ALCALA, FAC.CIENCIAS

1977-1978

MAITRE-ASSISTANT

UNIV.GINEBRA (SUIZA) FAC. MEDICINA

1978-1980

CHARGÉ D’ENSEIGNEMENT

UNIV. GINEBRA (SUIZA) FAC. MEDICINA

1981-1985

COLABORADOR CIENTÍFICO INSTITUTO CAJAL, C.S.I.C.

1985-1987

INVESTIGADOR CIENTÍFICO

1987-1990

INSTITUTO CAJAL, C.S.I.C.

Idiomas (R = regular, B = bien, C = correctamente) Idioma

Habla

Lee

Escribe

Inglés

B

C

B

Frances

B

C

B

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Participación en Proyectos de I+D financiados en Convocatorias públicas. (nacionales y/o internacionales) TITULO DE PROYECTO: Ultrastructure des membranes et secretion cellulaire. ENTIDAD FINANCIADORA: Swiss National Science Foundation DURACION: 1.10.1980- 30.9.1986 INVESTIGADOR PRINCIPAL: Lelio Orci TITULO DE PROYECTO: Release of neuromodulators from nerve fibers ENTIDAD FINANCIADORA: Swiss National Science Foundation DURACION: 1.10.1980-30.9.1986 INVESTIGADOR PRINCIPAL: R. Straub TITULO DE PROYECTO: Structure de la membrane plasmique neuronale et synaptogenese. ENTIDAD FINANCIADORA: Swiss National Science Foundation DURACION: 1.4.1983-31.3.1985 INVESTIGADOR PRINCIPAL: Luis Miguel GARCIA SEGURA TITULO DE PROYECTO: Mecanismos de acción celular de las hormonas esteroideas en el sistema nervioso central ENTIDAD FINANCIADORA: CAICYT DURACION: 1986-1988 INVESTIGADOR PRINCIPAL: Luis Miguel GARCIA SEGURA TITULO DE PROYECTO: Mecanismos de acción celular de las hormonas esteroideas en el sistema nervioso central ENTIDAD FINANCIADORA: FISS DURACION: 1986 INVESTIGADOR PRINCIPAL: Luis Miguel GARCIA SEGURA TITULO DE PROYECTO: Mecanismos de acción celular de las hormonas esteroideas en el sistema nervioso central ENTIDAD FINANCIADORA: FISS DURACION: 1987-1988 INVESTIGADOR PRINCIPAL: Luis Miguel GARCIA SEGURA TITULO DE PROYECTO: Mecanismos celulares de sinaptogénesis y plasticidad sináptica mediados por las hormonas gonadales ENTIDAD FINANCIADORA: DGICYT DURACION: 1990-1992 INVESTIGADOR PRINCIPAL: Luis Miguel GARCIA SEGURA TITULO DE PROYECTO: Estudio del efecto que las hormonas tiroideas y gonadales tienen sobre el desarrollo de la corteza cerebral de la rata ENTIDAD FINANCIADORA: DGICYT DURACION: 1990-1992 INVESTIGADOR PRINCIPAL: Antonio Ruiz Marcos TITULO DE PROYECTO: Neurotrofismo y plasticidad celular en el sistema nervioso central. Papel del factor de crecimiento tipo insulina I ENTIDAD FINANCIADORA: FISS DURACION: 1992-1993 INVESTIGADOR PRINCIPAL: Ignacio Torres Alemán TITULO DE PROYECTO: Mecanismos de sinaptogénesis y plasticidad sináptica. Interacción entre factores tróficos y hormonales. ENTIDAD FINANCIADORA: DGICYT

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DURACION: 1993-1995 INVESTIGADOR PRINCIPAL: Luis Miguel GARCIA SEGURA TITULO DE PROYECTO: Papel del factor de crecimiento tipo insulina I en los mecanismos de aprendizaje mediados por el cerebelo. ENTIDAD FINANCIADORA: DGICYT DURACION: 1993-1995 INVESTIGADOR PRINCIPAL: Ignacio Torres Alemán TITULO DE PROYECTO: Mecanismos celulares de acción del factor de crecimiento parecido a insulina I (IGFI) en la regulación neuroendocrina ENTIDAD FINANCIADORA: Fundación Ramón Areces DURACION: 1995-1997 INVESTIGADOR PRINCIPAL: Luis Miguel GARCIA SEGURA TITULO DE PROYECTO: Acciones endocrinas y paracrinas del factor de crecimiento tipo insulina I y sus moléculas transportadoras en el sistema nervioso central y posibles implicaciones clínicas ENTIDAD FINANCIADORA: DGICYT DURACION: 1995-1997 INVESTIGADOR PRINCIPAL: Ignacio Torres Alemán TITULO DE PROYECTO: Bases celulares y moleculares de las alteraciones neuroendocrinas asociadas a la anorexia nerviosa: Estudio clínico y básico. ENTIDAD FINANCIADORA: Comunidad de Madrid DURACION: 1998-2000 INVESTIGADOR PRINCIPAL: Luis Miguel GARCIA SEGURA TITULO DE PROYECTO: Factores neurotróficos y enfermedades neurodegenerativas: Mecanismos moleculares de neuroprotección mediada por el IGF-I ENTIDAD FINANCIADORA: Comunidad de Madrid DURACION: 1999-2001 INVESTIGADOR PRINCIPAL: Ignacio Torres Alemán TITULO DE PROYECTO: Mecanismos celulares de protección por esteroides ENTIDAD FINANCIADORA: DGESIC DURACION: 1999-2002 INVESTIGADOR PRINCIPAL: Luis Miguel GARCIA SEGURA TITULO DE PROYECTO: Neuroprotección por dehidroepiandrosterona ENTIDAD FINANCIADORA: Comunidad de Madrid DURACION: 2001-2002 INVESTIGADOR PRINCIPAL: Luis Miguel GARCIA SEGURA TITULO DE PROYECTO: The role of neurosteroids in healthy ageing: Therapeutical perspectives ENTIDAD FINANCIADORA: Comunidad Europea DURACION: 2001-2003 INVESTIGADOR PRINCIPAL: Michael Schumacher TITULO DE PROYECTO: Neuroprotección por neuroesteroides ENTIDAD FINANCIADORA: Instituto de Salud Carlos III DURACION: 2001-2003 INVESTIGADOR PRINCIPAL: Luis M. García Segura TITULO DE PROYECTO: Papel neuroprotector de la esteroidogénesis cerebral y los receptores nucleares de hormonas esteroides ENTIDAD FINANCIADORA: Ministerio de Ciencia y Tecnología DURACION: 2003-2005

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INVESTIGADOR PRINCIPAL: Luis M. García Segura TITULO DE PROYECTO: Bases moleculares para la neuroprotección por estrógenos e IGF-I en el envejecimiento ENTIDAD FINANCIADORA: Comunidad de Madrid DURACION: 2004 INVESTIGADOR PRINCIPAL: Luis M. García Segura TITULO DE PROYECTO: Influencia de la pubertad y los cambios hormonales en la adicción a cannabinoides ENTIDAD FINANCIADORA: Plan Nacional sobre Drogas DURACION: 2004-2006 INVESTIGADOR PRINCIPAL: Luis M. García Segura TITULO DE PROYECTO: Mitochondrial cholesterol transporters and neurosteroid synthesis: regeneration and aging of peripheral nerves ENTIDAD FINANCIADORA: Association francaise contre les myopathies DURACION: 2004-2006 INVESTIGADOR PRINCIPAL: Ghislaine Groyer TITULO DE PROYECTO: Exploratory studies of androgen receptors in axons ENTIDAD FINANCIADORA: National Institutes of Health, USA DURACION: 2004-2007 INVESTIGADOR PRINCIPAL: Lydia L. DonCarlos TITULO DE PROYECTO: Comparative analysis of cerebellar neuropathology in human autistic patients and in cerebellar mouse mutants ENTIDAD FINANCIADORA: National Alliance for Autism Research (USA) DURACION: 2006-2007 INVESTIGADOR PRINCIPAL: Flavio Keller TITULO DE PROYECTO: Acciones neuroprotectoras de los moduladores selectivos de los receptores de estrógeno ENTIDAD FINANCIADORA: Ministerio de Educación y Ciencia DURACION: 2006-2008 INVESTIGADOR PRINCIPAL: Luis M. García Segura TITULO DE PROYECTO: Estrogens and Women Ageing ENTIDAD FINANCIADORA: Comunidad Europea DURACION: 2006-2008 INVESTIGADOR PRINCIPAL: Adriana Maggi TITULO DE PROYECTO: Estrés prenatal: acciones neuroprotectoras de moduladores selectivos de los receptores de estrógeno ENTIDAD FINANCIADORA: Ministerio de Ciencia e Innovación DURACION: 2009-2011 INVESTIGADOR PRINCIPAL: Luis Miguel García Segura TITULO DE PROYECTO: Señalizacion de Notch/Ngn3 en las acciones neuroprotectoras de compuestos estrogenicos. ENTIDAD FINANCIADORA: Ministerio de Ciencia e Innovación DURACION: 2012-2016 INVESTIGADOR PRINCIPAL: Luis Miguel García Segura 1

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Publicaciones o Documentos Científico-Técnicos

PUBLICACIONES 19811981 Garcia-Segura, L.M. and Perrelet, A., Freeze-fracture of developing neuronal plasma membrane in postnatal cerebellum Brain Research, 208: 19-33 (1981). Roth, J., Baetens, D., Norman, A. and Garcia-Segura, L.M. Specific neurons in chick central nervous system stain with an antibody against chick intestinal vitamin D - dependent calcium -binding protein. Brain Research, 222: 452-457 (1981). 1982 Roth, J., Thorens, B., Brown, D., Baetens, D., Garcia-Segura, L.M., Norman, A.W. and Orci, L. Immunocytochemical localization of vitamin D-dependent calcium binding protein (CaBP) in duodenum, kidney, brain and pancreas. In: A.W. Norman, K. Schaefer, D.v. Herrath and H.G. Grigoleit (Eds.), Vitamin D, Chemical, Biochemical and Clinical Endocrinology of Calcium Metabolism, Walter de Gruyer, Berlin, pp. 209214, 1982. Garcia-Segura, L.M., Baetens, D. and Orci., L. Freeze-fracture cytochemistry of neuronal membranes: inhomogeneous distribution of filipin-sterol complexes in perikarya, dendrites and axons. Brain Research, 234: 494-499 (1982). Garcia-Segura, L.M. and Perrelet, A. Climbing fiber destruction affects dendrites and spine membrane organization in Purkinje cells. Brain Research, 236: 253-260 (1982). Baetens, D., Garcia-Segura, L.M. and Perrelet, A. Effects of climbing fiber destruction on large dendrite spines of Purkinje cells. Experimental Brain Research, 48: 256-262 (1982). Perrelet, A., Garcia-Segura, L.M., Singh, A. and Orci, L. Distribution of cytochemically detectable cholesterol in the electric organ of Torpedo marmorata. Proc. Natl. Acad. Sci. USA, 79: 2598-2602 (1982). 1983 Anderson, R.G.W., Orci, L., Brown, M.S., Garcia-Segura, L.M. and Goldstein, J.L. Ultrastructural analysis of crystalloid endoplasmic reticulum in UT-1 cells and its disappearance in response to cholesterol. Journal of Cell Science, 63: 1-20 (1983). Baetens, D., Tribollet, E. and Garcia-Segura, L.M. Colchicine injection in the inferior olivary nucleus increases the number of Purkinje cell dendritic spines. Neuroscience Letters, 38: 239-244 (1983). 1984 Dunant, Y., Muller, D., Jones, G., Loctin, F., Garcia-Segura, L.M. and Parducz, A. Transmission of a nerve impulse at cholinergic synapses: the role of the membrane-bound releasing operator and the role(s) of synaptic vesicles. In: E.S. Vizi and K. Magyar (Eds.), Regulation of Transmitter Function, Akademiai Kiado, Budapest, pp. 107-115, 1984. Dunant, Y., Muller, D., Parducz, A., Jones, G.J. and Garcia-Segura, L.M. Augmentation tres breve du nombre de particules dans la membrane presynaptique pendant la transmission d'un influx nerveux. C. R. Acad. Sci, Paris, 299: 547-552 (1984). Brown, D., Garcia-Segura, L.M. and Orci, L. Carbonic anhydrase is associated with taste buds in rat tongue. Brain Research, 324: 346-348 (1984). Bruhlmann-Papazyan,

M.,

Garcia-Segura,

L.M.,

Pizzolato,

G.P.,

Norman,

A.W.

and

Orci,

L.

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Immunohistochemically detectable vitamin D-dependent calcium-binding protein is reduced in cerebellum of diabetic subjects. Diabetes, 33: 917-922 (1984). Garcia-Segura, L.M. and Perrelet, A. Postsynaptic membrane domains in the molecular layer of the cerebellum: a correlation between presynaptic inputs and postsynaptic plasma membrane organization. Brain Research, 321: 255-266 (1984). Garcia-Segura, L.M. and Perrelet, A. Lateral distribution of intramembrane particles in Purkinje and granule cells of the rat cerebellar cortex. Neuroscience Letters, 48: 37-42 (1984). Garcia-Segura, L.M., Baetens, D., Roth, J., Norman, A. and Orci, L. Immunohistochemical mapping of calciumbinding protein immunoreactivity in the rat central nervous system. Brain Research, 296: 75-86 (1984). Brown, D., Garcia-Segura, L.M. and Orci, L. Carbonic anhydrase is present in olfactory receptor cells. Histochemistry, 80: 307-309 (1984). Orci, L., Brown, M.S., Goldstein, J.L., Garcia-Segura, L.M. and Anderson, R.G.W. Increase in membrane cholesterol: a possible trigger for degradation of HGM CoA reductase and crystalloid endoplasmic reticulum in UT-1 cells. Cell, 36: 835-845 (1984). 1985 Reece, E.A., Pinter, E., Leranth, C., Garcia-Segura, L.M., Sanyal, M.K., Hobbins, J.C., Mahoney, M.J. and Naftolin, F. Ultrastructural analysis of malformations of the embryonic neural axis induced by in vitro hyperglycemic conditions. Teratology, 32: 363-373 (1985). Leranth, C., Garcia-Segura, L.M., Palkovits, M., MacLusky, N.J., Shanabrough, M. and Naftolin, F. The LHRH-containing neuronal network in the preoptic area of the rat: demonstration of LHRH-containing nerve terminals in synaptic contact with LHRH neurons. Brain Research, 345: 332-336 (1985). Garcia-Segura, L.M. Trans-synaptic modulation of Purkinje cell plasma membrane organization by climbing fiber axonal flow. Experimental Brain Research, 61: 186-193 (1985). Garcia-Segura, L.M. and Rakic, P. Differential distribution of intramembrane particles in the plasmalemma of the migrating cerebellar granule cells. Developmental Brain Research, 23: 145-149 (1985). Naftolin, F., Bruhlmann-Papazyan, M., Baetens, D. and Garcia-Segura, L.M. Neurons with whorl bodies have increased number of synapses. Brain Research, 329: 289-293 (1985). Garcia-Segura, L.M., Baetens, D. and Naftolin, F. Sex differences and maturational changes in arcuate nucleus neuronal plasma membrane organization. Developmental Brain Research, 19: 146-149 (1985). 1986 Garcia-Segura, L.M., Baetens, D. and Naftolin, F. Synaptic remodelling in arcuate nucleus after injection of estradiol valerate in adult female rats. Brain Research, 366 131-136 (1986). Garcia-Segura, L.M., De Cozar, M., Moreno, M.C. and Monreal, J. Freeze-fracture characterization of proteolipid and basic protein of myelin incorporated to liposomes. Brain Research, 380: 261-266 (1986). Fournet, N., Garcia-Segura,L.M., Norman, A.W. and Orci, L. Selective localization of calcium-binding protein in human brainstem, cerebellum and spinal cord. Brain Research, 399: 310-316 (1986). Garcia-Segura, L.M., Muller, D. and Dunant, Y. Increase in the number of presynaptic large intramembrane particles during synaptic transmission at the Torpedo nerve-electroplaque junction. Neuroscience, 19: 63-79 (1986). Garcia-Segura, L.M. Harmaline-induced changes in plasma membrane of Purkinje cells: a trans-synaptic effect mediated by climbing fibers. Brain Research, 372: 390-393 (1986).

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Pinter, E., Reece, E.A., Leranth, C.Z., Garcia-Segura, L.M., Hobbins, J.C., Mahoney, M.J. and Naftolin, F. Arachidonic acid prevents hyperglycemia-associated yolk sac damage and embryopathy. American Journal of Obstetrics and Gynecology, 155: 691-702 (1986). Garcia-Segura, L.M., Barnea, E.R., Biggers, W., Naftolin, F. and Sanyal, M.K. Insulin modulates neuronal plasma membrane development in human fetal spinal cord neurons in culture. Neuroscience Letters 65: 283-286 (1986). 1987 Garcia-Segura, L.M., Olmos, G., Tranque, P. and Naftolin, F. Rapid effects of gonadal steroids upon hypothalamic neuronal membrane ultrastructure. Journal of Steroid Biochemistry, 27: 615-623 (1987). Olmos, G., Aguilera, P., Tranque, P., Naftolin, F. and Garcia-Segura, L.M. Estrogen induces synaptic remodelling in adult rat brain is accompanied by the reorganization of neuronal membranes. Brain Research, 425: 57-64 (1987). Parducz, A., Dunant, Y., Loctin, F., Muller, D. and Garcia-Segura, L.M. Presynaptic effects of 4-aminopyridine and changes following a single giant impulse at the Torpedo nerve-electroplaque junction. Neuroscience, 22: 709-718 (1987). Garcia-Segura, L.M., Olmos, G., Tranque, P., Aguilera, P. and Naftolin, F. Nuclear pores in rat hypothalamic arcuate neurons: sex differences and changes during the estrous cycle. Journal of Neurocytology, 16: 469-475 (1987). Suarez, I., Fernandez, B., Bodega, G., Tranque, P., Olmos, G. and Garcia-Segura, L.M. Postnatal development of glial fibrillary acidic protein immunoreactivity in the hamster arcuate nucleus. Developmental Brain Research, 37: 89-95 (1987). Tranque, P., Suarez, I., Olmos, G., Fernandez, B. and Garcia-Segura, L.M. Estradiol-induced redestribution of glial fibrillary acidic protein immunoreactivity in the rat brain. Brain Research, 406: 348-351 (1987). Muller, D., Garcia-Segura, L.M., Parducz, A. and Dunant, Y. Brief occurrence of a population of presynaptic intramembrane particles coincides with transmission of a nerve impulse. Proc. Natl. Acad. Sci., USA, 84: 590-594 (1987). 1988 Garcia-Segura, L.M., Hernandez, P., Olmos, G., Tranque, P.A. and Naftolin, F. Neuronal membrane remodelling during the oestrus cycle: a freeze-fracture study in the arcuate nucleus of the rat hypothalamus. Journal of Neurocytology, 17: 377-383 (1988). Naftolin, F., MacLusky, N.J., Leranth, C.Z., Sakamoto, H.S. and Garcia-Segura, L.M. The cellular effects of estrogens on neuroendocrine tissues. Journal of Steroid Biochemistry, 30: 195-207 (1988). Garcia-Segura, L.M., Perez, J., Tranque, P.A., Olmos, G. and Naftolin, F. Sexual differentiation of the neuronal plasma membrane: neonatal levels of sex steroids modulate the number of exo-endocytotic images in the developing rat arcuate neurons. Neuroscience Letters, 91: 19-23 (1988). Goni, F.M., Cozar, M., Alonso, A., Durrani, A.A., Garcia-Segura, L.M., Lee, D.C., Monreal, J. and Chapman, D. Lipid-protein interaction. The incorporation of myelin proteolipid apoprotein into phosphatidylcholine bilayers. European Journal of Biochemistry, 174: 641-646 (1988). Garcia-Segura, L.M., Suarez, I., Segovia, S., Tranque, P.A., Cales, J.M., Aguilera, P., Olmos, G. and Guillamon, A. The distribution of glial fibrillary acidic protein in the adult rat brain is influenced by the neonatal levels of sex steroids. Brain Research, 456: 357-363 (1988). Carmona, P., de Cozar, M., Garcia-Segura, L.M. and Monreal, J. Conformation of brain proteolipid apoprotein. Effects of sonication and n-octyl-á-D-glucopyranoside detergent. European Biophysics Journal, 16: 169-176

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(1988). 1989 Dunant, Y., Garcia-Segura, L.M., Muller, D. and Parducz, A. Momentary alteration of the postsynaptic membrane during transmission of a single nerve impulse. Proc. Natl. Acad. Sci., USA, 86: 1717-1720 (1989) Garcia-Segura, L.M., Perez, J., Tranque, P.A., Olmos, G. and Naftolin, F. Sex differences in plasma membrane concanavalin A binding in the rat arcuate neurons. Brain Research Bulletin, 22: 651-655 (1989) Garcia-Segura, L.M., Torres-Aleman, I. and Naftolin, F. Astrocytic shape and glial fibrillary acidic protein immunoreactivity are modified by estradiol in primary rat hypothalamic cultures. Developmental Brain Research, 47: 298-302 (1989) Garcia-Segura, L.M., Olmos, G., Robbins, R.J., Hernandez, P., Meyer, J.H. and Naftolin, F. Estradiol induces rapid remodelling of plasma membranes in developing rat cerebrocortical neurons in culture. Brain Research, 498: 339-343 (1989). Olmos, G., Naftolin, F., Perez, J., Tranque, P.A. and Garcia-Segura, L.M. Synaptic remodeling in the rat arcuate nucleus during the estrous cycle. Neuroscience, 32: 663-667 (1989). Garcia-Segura, L.M., Lafarga, M., Berciano, M.T., Hernandez, P. and Andres, M.A. Distribution of nuclear pores and chromatin organization in neurons and glial cells of the rat cerebellar cortex. Journal of Comparative Neurology, 290:440-450 (1989). 1990 Perez, J., Naftolin, F., Garcia-Segura LM. Sexual differentiation of synaptic connectivity and neuronal plasma membrane in the arcuate nucleus of the rat hypothalamus. Brain Research 527:116-122 (1990). Naftolin, F., Garcia-Segura, L.M., Keefe, D., Leranth, C., MacLusky, N.J. and Brawer, J.R. Estrogen effects on the synaptology and neural membranes of the rat hypothalamic arcuate nucleus. Biology of Reproduction, 42: 21-28 (1990) Parducz, A., Garcia-Segura, L.M., Muller, D. and Dunant, Y. Endo-exocytotic images and changes in synaptic transmission induced by diamide at a cholinergic junction. Neuroscience, 37: 227-236 (1990) Perez, J., Tranque, P.A., Naftolin, F. and Garcia-Segura, L.M. Gap junction in the hypothalamic arcuate neurons of ovariectomized and estradiol-treated rats. Neuroscience Letters, 108: 17-21 (1990) Garcia-Segura, L.M., Perez, J., Tranque, P.A. and Naftolin, F. Cellular effects of ovarian secretions on the central nervous system. In: F. Naftolin, J.N. Gutmann, A.H. DeCherney and P.M. Sarrel (Eds.): Ovarian Secretions and Cardiovascular and Neurological Function, Raven Press, New York, pp. 273-286 (1990). Garcia-Segura, L.M., Ferragut, J.A., Ferrer-Montiel, A.V., Escriba, P.V. and Gonzalez-Ros, J.M. Ultrastructural alterations in plasma membranes from drug-resistant P388 murine leukemia cells. Biochimica et Biophysica Acta, 1029: 191-195 (1990). Riquelme, G., Lopez, E. Garcia-Segura, L.M., Ferragut, J.A. and Gonzalez-Ros, J.M. Giant liposomes: a model system in which to obtain patch-clamp recordings of ionic channels. Biochemistry, 29: 11215-11222 (1990) Naftolin, F., Perez, J., Leranth, C.S., Redmond, D.E. and Garcia-Segura, L.M. African green monkeys have sexually dimorphic and estrogen-sensitive hypothalamic neuronal membranes. Brain Research Bulletin, 25: 575-579 (1990) 1991 Naftolin, F., Keefe, D., Apa, R., Palumbo, A. and Garcia-Segura, L.M. The apparent paradox of sexual differentiation of the brain. Contributions to Gynecology and Obstetrics, 18: 24-32 (1991) Muñoz-Cueto, J.A., Garcia-Segura, L.M. and Ruiz-Marcos, A. Regional sex differences in spine density along the

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apical shaft of visual cortex pyramids during postnatal development. Brain Research, 540: 41-47 (1991) Garcia-Segura, L.M., Perez, J., Jones, E. and Naftolin, F. Loss of sexual dimorphism in rat arcuate nucleus neuronal membranes with reproductive aging. Experimental Neurology, 112: 125-128 (1991) Perez, J., Hernandez, P. and Garcia-Segura, L.M. Estradiol increases the number of nuclear pores in the arcuate neurons of the rat hypothalamus. Journal of Comparative Neurology, 303: 225-232 (1991) Garcia-Segura, L.M., Perez, J., Pons, S., Rejas, M.T. and Torres-Aleman, I. Localization of insulin-like growth factor I (IGF-I) like immunoreactivity in the developing and adult rat brain. Brain Research, 560: 167-174 (1991) Morisette, M., Garcia-Segura, L.M., Belanger, A. and DiPaolo, T. Membrane effects of steroid hormones in the rat striatum: changes during the estrous cycle. Posters in Neuroscience, 1: 95-98 (1991) Lafarga, M., Berciano, M.T. and Garcia-Segura, L.M. Freeze-fracture organization of chromatin and cytoplasm in neurons and astroglia of rat cerebellar cortex. Journal of Neurocytology, 20: 533-551 (1991) Torres-Aleman, I., Pons, S. and Garcia-Segura, L.M. Climbing fiber deafferentiation reduces insulin-like growth factor I (IGF-I) content in cerebellum. Brain Research, 564: 348-351 (1991) 1992 Garcia-Segura, L.M., Diolez-Bojda, F., Lenoir, V., Naftolin, F. and Kerdelhue, B., Estrogen-like effects of the mammary carcinogen 7,12-dimethylbenz(a)anthracene on hypothalamic neuronal membranes. Brain Research Bulletin, 28: 625-628 (1992) Castro-Alamancos, M.A., Borrell, J. and Garcia-Segura, L.M., Performance in an escape task induces Fos-like immunoreactivity in a specific area of the motor cortex of the rat. Neuroscience, 49: 157-162 (1992) Morissette, M., Garcia-Segura, L.M., Belanger, A. and Di Paolo, T., Changes of rat striatal neuronal membrane morphology and steroid content during the estrous cycle. Neuroscience, 49: 893-902 (1992) Castro-Alamancos, M.A., Garcia-Segura, L.M. and Borrell, J., Transfer of function to a specific area of the cortex after induced recovery from brain damage. European Journal of Neuroscience, 4: 853-863 (1992) Garcia-Estrada, J., Garcia-Segura, L.M. and Torres-Aleman, I., Expression of insulin-like growth factor I by astrocytes in response to injury. Brain Research, 592: 343-347 (1992) Torres-Aleman, I., Rejas, M.T., Pons, S. and Garcia-Segura, L.M., Estradiol promotes cell shape changes and glial fibrillary acidic protein redistribution in hypothalamic astrocytes in vitro: a neuronal mediated effect. Glia, 6: 180-187 (1992) Garcia-Segura, L.M., Soto, F., Planells-Cases, R., Gonzalez-Ros, J.M. and Ferragut, J.A., Verapamil reverses the ultrastructural alterations in the plasma membrane induced by drug resistance. FEBS Letters, 314: 404-408 (1992) Chowen, J.A., Torres-Aleman, I. and Garcia-Segura, L.M., Trophic effects of estradiol on fetal hypothalamic neurons. Neuroendocrinology, 56: 895-901 (1992) Naftolin, F., Leranth, C. and Garcia-Segura, L.M., Ultrastructural changes in hypothalamic cells during estrogen-induced gonadotrophin feedback. Neuroprotocols, 1:16-26 (1992) 1993 Parducz, A., Perez, J. and Garcia-Segura, L.M., Estradiol induces plasticity of GABAergic synapses in hypothalamus. Neuroscience, 53: 395-401 (1993) Perez, J., Naftolin, F. and Garcia-Segura, L.M., Cycloheximide mimics effects of oestradiol that are linked to synaptic plasticity of hypothalamic neurons. Journal of Neurocytology, 22: 233-243 (1993)

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Perez, J., Luquin, S., Naftolin, F. and Garcia-Segura, L.M., The role of estradiol and progesterone in phased synaptic remodelling of the rat arcuate nucleus. Brain Research, 608: 38-44 (1993) Garcia-Segura, L.M., Luquin, S., Martinez, P., Casas, M.T. and Suau, P., Differential expression and gonadal hormone regulation of histone H1o in the developing and adult rat brain. Developmental Brain Research, 73: 63-70 (1993) Parducz, A. and Garcia-Segura, L.M., Sexual differences in the synaptic connectivity in the rat dentate gyrus. Neuroscience Letters, 161: 53-56 (1993) Naftolin, F., Leranth, C., Perez, J. and Garcia-Segura, L.M., Estrogen induces synaptic plasticity in adult primate neurons. Neuroendocrinology, 57: 935-939 (1993) Naftolin, F., Leranth, C., Keefe, D. and Garcia-Segura, L.M., Estrogen-induced synaptic plasticity: implications for reproduction. In: A. Netter (Ed.), Actualites Gynecologiques, Eska, Paris, pp.45-51 (1993) Luquin, S., Naftolin, F. and Garcia-Segura, L.M., Natural fluctuation and gonadal hormone regulation of astrocyte immunoreactivity in dentate gyrus. Journal of Neurobiology, 24: 913-924 (1993) Garcia-Segura, L.M., Berciano, M.T. and Lafarga, M., Nuclear compartmentalization in transcriptionally activated hypothalamic neurons. Biology of the Cell, 77: 143-154 (1993) Garcia-Estrada, J., Del Rio, J. A., Luquin, S., Soriano, E. and Garcia-Segura, L.M., Gonadal hormones down-regulate reactive gliosis and astrocyte proliferation after a penetrating brain injury. Brain Research, 628: 271-278 (1993) Chowen, J.A., Argente, J., Gonzalez-Parra, S. and Garcia-Segura, L.M., Differential effects of the neonatal and adult sex steroid environments on the organization and activation of hypothalamic growth hormone-releasing hormone and somatostatin release. Endocrinology, 133: 2792-2802 (1993). Chowen, J.A., Argente, J., Torres-Aleman, I., Gonzalez-Parra, S. and Garcia-Segura, L.M., Effects of the neonatal sex steroid environment on growth hormone-releasing hormone and somatostatin gene expression. Journal of Pediatric Endocrinology, 6: 211-218 (1993). Nieto-Bona, P., Garcia-Segura, L.M. and Torres-Aleman, I., Orthograde transport and release of insulin-like growth factor I from the inferior olive to the cerebellum. Journal of Neuroscience Research, 36:520-527 (1993) 1994 Garcia-Segura, L.M., Luquin, S., Parducz, A. and Naftolin, F., Gonadal hormone regulation of glial fibrillary acidic protein immunoreactivity and glial ultrastructure in the rat neuroendocrine hypothalamus. Glia, 10:59-69 (1994) Dueñas, M., Luqu n, S., Chowen J.A., Torres-Aleman, I., Naftolin, F. and Garcia-Segura, L.M., Gonadal hormone regulation of insulin-like growth factor-I-like immunoreactivity in hypothalamic astroglia of developing and adult rats. Neuroendocrinology, 59:528-538 (1994) Garcia-Segura, L.M., Chowen J.A., Dueñas, M., Torres-Aleman, I. and Naftolin, F., Gonadal steroids as promoters of neuro-glial plasticity. Psychoneuroendocrinology, 19:445-453 (1994) Keefe, D., Garcia-Segura, L.M. and Naftolin, F. New insights into estrogen action on the brain. Neurobiology of Aging, 15:495-497 (1994) Garcia-Segura, L.M., Chowen, J.A., Parducz, A. and Naftolin, F., Gonadal hormones as promoters of structural synaptic plasticity: Cellular mechanisms. Progress in Neurobiology, 44: 279-307 (1994) Suarez, I., Bodega, G., Rubio, M., Garcia-Segura, L.M. and Fernandez, B., Astroglial angiogenesis in vivo. Journal of Neural Transplantation and Plasticity, 5:1-10 (1994) Leedom, L., Lewis, C., Garcia-Segura, L.M. and Naftolin F., Regulation of arcuate nucleus synaptology by estrogen.

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Annals New York Academy of Sciences, 743:61-71 (1994) 1995 Lafarga, M., Rodriguez, J.R., Garcia-Segura, L.M. and Suau, P., Transcriptional activation of supraoptic neurons induces changes in the expression of histone H1o. Molecular Brain Research, 29:317-324 (1995). Garcia-Segura, L.M., Cañas, B., Parducz, A., Rougon, G., Theodosis, D., Naftolin, F. and Torres-Aleman, I., Estradiol promotion of astroglial cell shape changes depends on the expression of polysisalic acid on neuronal membranes. Glia, 13:209-216 (1995). Garcia-Segura, L.M., Dueñas, M., Busiguina, S., Naftolin, F. and Chowen, J.A., Gonadal hormone regulation of neuronal-glial interactions in the developing neuroendocrine hypothalamus. Journal of Steroid Biochemistry and Molecular Biology, 53:293-298 (1995). Horvath, T.L., Leedom, L., Lewis, C., Garcia-Segura, L.M., Naftolin, F., Estrogen-induced hypothalamic synaptic plasticity and the regulation of gonadotrophins. Current Opinion in Endocrinology and Diabetes, 2:186-190 (1995). Del Cerro, S., Garcia-Estrada, J. and Garcia-Segura, L.M., Neuroactive steroids regulate astroglia morphology in hippocampal cultures from adult rats. Glia, 14:65-71 (1995). Chowen, J., Busiguina, S. and Garcia-Segura, L.M., Sexual dimorphism and sex steroid modulation of glial fibrillary acidic protein (GFAP) mRNA and immunoreactivity levels in the rat hypothalamus. Neuroscience, 69:519-532 (1995). 1996 Gonzalez-Parra, S., Chowen, J., Garcia-Segura, L.M. and Argente, J., In vivo and in vitro regulation of pituitary transcription factor-1 (Pit-1) by changes in the hormone environment. Neuroendocrinology, 63:3-15 (1996). Leedom, L., Garcia-Segura, L.M. and Naftolin, F., Estrogen effects on neuronal ultrastructure and synaptic plasticity. In: T.W. Stone (Ed.) CNS Neurotransmitters and Neuromodulators: Neuroactive Steroids. CRC Press, New York, 1996, pp. 103-111 (1996). Garcia-Segura, L.M., Chowen, J.A. and Naftolin, F., Endocrine glia: Roles of glial cells in the brain actions of steroid and thyroid hormones and in the regulation of hormone secretion. Frontiers in Neuroendocrinology, 17:180-211 (1996). Chowen, J.A., Evain-Brion, D., Pozo, J., Alsat, E., Garcia-Segura, L.M. and Argente, J., Decreased expression of placental growth hromone in intrauterine growth retardation. Pediatric Research, 39:736-739 (1996). Gahr, M. and Garcia-Segura, L.M., Testosterone-dependent increase of gap-junctions in HVC neurons of adult female canaries. Brain Research 712:69-73 (1996). Garcia-Segura, L.M., Chowen, J.A., Párducz, A. and Naftolin, F., Glial cells and estrogen-induced synaptogenesis and synaptic plasticity. In: A.R. Genazzani, F. Petraglia and R.H. Purdy (Eds.): The Brain: Source and Target for Sex Steroid Hormones, Parthenon, New York, pp. 191-200 (1996) Levy, A., Garcia-Segura, L.M., Nevo, Z., David, Y., Naftolin, F. and Shahar, A., A new organotypic culture method to study the actions of steroid hormones on the nervous system. Journal of Neuroscience Research, 43:719-725 (1996) Gonzalez-Parra, S., Chowen, J.A., Garcia-Segura, L.M. and Argente, J., Ontogeny of pituitary transcription factor-1 (Pit-1), growth hormone (GH) and prolactin (PRL) mRNA levels in male and female rats and the differential expression of Pit-1 in lactotrophs and somatotrophs. Journal of Neuroendocrinology, 8:211-225 (1996) Naftolin, F., Leranth, C., Horvath, T.L., and Garcia-Segura, L.M., Potential neuronal mechanisms of estrogen actions in synaptogenesis and synaptic plasticity. Cellular and Molecular Neurobiology, 16:213-223 (1996)

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Garcia-Segura, L.M., Chowen, J.A., Dueñas, M., Parducz, A., and Naftolin, F., Gonadal steroids and astroglial plasticity. Cellular and Molecular Neurobiology, 16:225-237 (1996) Párducz, A., Szilágyi, T., Hoyk, S., Naftolin, F., and Garcia-Segura, L.M., Neuroplastic changes in the hypothalamic arcuate nucleus: The estradiol effect is accompanied by increased exoendocytotic activity of neuronal membranes. Cellular and Molecular Neurobiology, 16:259-269 (1996) Leucona, E., Luquín, S., Avila, J., Garcia-Segura, L.M., and Martín-Vasallo, P., Expression of the beta1 and beta2 (AMOG) subunits of the Na,K-ATPase in neural tissues: Cellular and developmental distribution patterns. Brain Research Bulletin, 40:167-174 (1996) Chowen, J.A., Argente, J., Busiguina, S., and Garcia-Segura, L.M., The role of glia in the neuroendocrine hypothalamus: Possible implications in hormone secretion. Hormone Research 45 (Suppl 1):15-18 (1996) Chowen J.A., Garcia-Segura, L.M., González-Parra, S., and Argente, J.A., Sex steroid effects on the development and functioning of the growth hormone axis. Cellular and Molecular Neurobiology, 16:297-310 (1996) Dueñas, M., Torres-Aleman, I., Naftolin, F., and Garcia-Segura, L.M., Interaction of insulin-like growth factor-I and estradiol signaling pathways on hypothalamic neuronal differentiation. Neuroscience 76: 531-539 (1996) Argente, J., Garcia-Segura, L.M., Pozo, J. and Chowen, J., Growth hormone-releasing peptides: Clinical and basic aspects. Hormone Research 46:155-159 (1996) Garcia-Segura, L.M., Dueñas, M., Fernandez-Galaz, M.C., Chowen, J., Argente, J., Naftolin, F., and Torres-Aleman, I., Interaction of the signalling pathways of insulin-like growth factor-I and sex steroids in the neuroendocrine hypothalamus. Hormone Research 46:160-164 (1996) Naftolin, F., Mor, G., Horvath, T.L., Luquin, S., Fajer, A.B., Kohen, F. and Garcia-Segura, L.M. Synaptic remodeling in the arcuate nucleus during the estrous cycle is induced by estrogen and precedes the preovulatory gonadotropin surge. Endocrinology 137:5576-5580 (1996) Del Cerro, S., Garcia-Estrada, L.M., and Garcia-Segura, L.M. Neurosteroids modulate the reaction of astroglia to high extracellular potassium levels. Glia 18:293-305 (1996) Fernandez-Galaz, M.C., Torres-Aleman, I. and Garcia-Segura, L.M. Endocrine-dependent accumulation of IGF-I by hypothalamic glia. NeuroReport 8: 373-377 (1996) Levy, A., Garcia-Segura, L.M., Nevo, Z., David, Y., Shahar, A. And Naftolin, F. Action of steroid hormones on growth and differentiation of CNS and spinal cord organotypic cultures. Cellular and Molecular Neurobiology 16: 445-450 (1996) 1997 Fernandez, A.M., Garcia-Estrada, J., Garcia-Segura, L.M. and Torres-Aleman, I., Insulin-like growth factor I modulates c-Fos induction and astrocytosis in response to neurotoxic insult. Neuroscience 76:117-122 (1997) Horvath, T.L., Garcia-Segura, L.M. and Naftolin, F., Lack of gonadotrophin-positive feedback in the male rat is associated with lack of estrogen-induced synaptic plasticity in the arcuate nucleus. Neuroendocrinology 65: 136-140 (1997) Fernandez-Galaz, M.C., Morschl, E., Chowen, J.A., Torres-Aleman, I., Naftolin, F. and Garcia-Segura, L.M., Role of astroglia and insulin-like growth factor-I in gonadal hormone-dependent synaptic plasticity, Brain Research Bulletin 44: 525-531 (1997) Garcia-Segura, L.M., Rodriguez, J.R. and Torres-Aleman, I., Localization of the insulin-like growth factor I receptor in the cerebellum and hypothalamus of adult rats: an electron microscopic study, Journal of Neurocytology 26: 479490 (1997)

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Horvath, T.L., Garcia-Segura, L.M., Naftolin, F., Control of gonadotrophin feedback: the possible role of estrogeninduced synaptic plasticity, Gynecological Endocrinology 11: 139-143 (1997) Nieto-Bona, M.P., Garcia-Segura, L.M., Torres-Aleman, I., Transynaptic modulation by insulin-like growth factor I of dendritic spines in Purkinje cells. International Journal of Developmental Neuroscience 15: 749-754 (1997) Fernandez-Galaz, M.C., Parducz, A., Naftolin, F., Torres-Aleman, I., and Garcia-Segura, L.M., Interaction of gonadal steroids and growth factors in brain sex differentiation. Biomedical Reviews, 7:67-74 (1997) Martín-Vasallo, P., Leucona, E., Luquin, S., Alvarez de la Rosa, D., Avila, J., Alonso, T., and Garcia-Segura, L.M. Cellular and developmental distribution of the Na,K-ATPase  subunit isoforms of neural tissues. Annals of the New York Academy of Sciences 834: 110-114 (1997) 1998 Fernandez Galaz, M.C., Chowen, J.A., and Garcia-Segura, L.M., Role of astroglia in the neural effects of sex hormones and neuroactive steroids. In: Castellano, B., González, B. and Nieto-Sampedro, M. (Eds.). Understanding glial cells. Kluwer, Boston, pp. 185-194 (1998) Garcia-Segura, L.M., Cardona-Gomez, P., Naftolin, F., and Chowen, J.A., Estradiol upregulates Bcl-2 expression in adult brain neurons. NeuroReport 9: 593-597 (1998) Chowen, J.A., González-Parra, S., Garcia-Segura, L.M., and Argente, J., Sexually dimorphic interactions of insulinlike growth factor (IGF)-I and sex steroids in lactotrophs. Journal of Neuroendocrinology 10: 493-502 (1998) González-Parra, S., Argente, J., Garcia-Segura, L.M., and Chowen, J.A. Cellular composition of the adult rat anterior pituitary is influenced by the neonatal sex steroid environment. Neuroendocrinology 68: 152-162 (1998) Lafarga, M., Berciano, M.T., Garcia-Segura, L.M., Andres, M.A., and Carmo-Fonseca, M. Acute osmotic/stress stimuli induce a transient decrease of transcriptional activity in the neurosecretory neurons of supraoptic nuclei. Journal of Neurocytology 27: 205-217 (1998) Azcoitia, I., Sierra, A., and Garcia-Segura, L.M.. Estradiol prevents kainic acid-induced neuronal loss in the rat dentate gyrus. NeuroReport 9: 3075-3079 (1998) 1999 Garcia-Segura, L.M., Wozniak, A., Azcoitia, I., Rodriguez, J.R., Hutchison, R.E. and Hutchison, J.B. Aromatase expression by astrocytes after brain injury: implications for local estrogen formation in brain repair. Neuroscience 89: 567-578 (1999) Fernandez-Galaz, M.C., Naftolin, F., and Garcia-Segura, L.M., Phasic synaptic remodeling of the rat arcuate nucleus during the estrous cycle depends on insulin-like growth factor-I receptor activation. Journal of Neuroscience Research 55:286-292 (1999) Soloaga, A., Veiga, M.P., Garcia-Segura, L.M., Ostolaza, H., Brasseur, R., and Goñi, F.M., Insertion of Escherichia coli -haemolysin in lipid bilayers as a non-transmembrane integral protein: prediction and experiment. Molecular Microbiology 31: 1013-1024 (1999) García-Estrada, J., Luquín, S., Fernández, A.M., and Garcia-Segura, L.M., Dehydroepiandrosterone, pregenenolone and sex steroids down-regulate reactive astroglia in the male rat brain after a penetrating brain injury. International Journal of Developmental Neuroscience 17: 145-151 (1999) Fernandez-Galaz, M.C, Torres-Aleman, I., and Garcia-Segura, L.M., Receptor-mediated internalization of insulin-like growth factor-I in neurons and glia of the central nervous system of the adult rat. European Journal of Anatomy 2: 147-158 (1999) Azcoitia, I., Sierra, A., and Garcia-Segura, L.M.., Localization of estrogen receptor -immunoreactivity in astrocytes of the adult rat brain. Glia 26:260-267 (1999)

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Garcia-Segura, L.M., Chowen, J.A., Naftolin, F., and Torres-Aleman, I., Steroid effects on brain plasticity. Role of glial cells and trophic factors. In: Baulieu, E.E., Robel, P., and Schumacher, M., Contemporary Endocrinology: Neurosteroids: A new Regulatory Function in the Nervous System. Humana Press, Totowa, NJ, pp. 255-268 (1999) Mor, G., Nilsen, J., Horvath, T., Bechmann, I., Brown, S., Garcia-Segura, L.M., and Naftolin, F., Estrogen and microglia: A regulatory system that affects the brain. Journal of Neurobiology 40: 484-496 (1999) Garcia-Segura, L.M., Naftolin, F., Hutchison, J.B., Azcoitia, I. and Chowen, J.A., Role of astroglia in estrogen regulation of synaptic plasticity and brain repair. Journal of Neurobiology 40: 574-584 (1999) Chowen, J.A., Rodriguez de Fonseca, F., Alvarez, E., Navarro, M., Garcia-Segura, L.M., and Blazquez, E., Increased glucagon-like peptide-1 receptor expression in glia after mechanical lesion of the rat brain. Neuropeptides 33: 212-215 (1999) Fernandez-Galaz, M.C., Muñoz, R.M., Villanua, M.A., and Garcia-Segura, L.M., Diurnal oscillation in glial fibrillary acidic protein in a perisuprachiasmatic area and its relationship to the luteinizing hormone surge in the female rat. Neuroendocrinology 70: 368-376 (1999) Azcoitia, I., Sierra, A., and Garcia-Segura, L.M. Neuroprotective effects of estradiol in the adult rat hippocampus: Interaction with insulin-like growth factor-I signalling. Journal of Neuroscience Research 58: 815-822 (1999) Azcoitia, I., Fernandez-Galaz, C., Sierra, A., and Garcia-Segura, L.M. Gonadal hormones affect neuronal vulnerability to excitotoxin-induced degeneration. Journal of Neurocytology 28: 699-710 (1999) 2000 Rodriguez de Fonseca, F., Gorriti, M.A., Bilbao, A., Escuredo, L., Garcia-Segura, L.M., Piomelli, D., and Navarro, M. Role of the endogenous cannabinoid system as a modulator of dopamine transmission: Implications for Parkinson’s disease and schizophrenia. In: Palomo, T., Beninger, R.J., and Archer, T. (Eds.). Neurodegenerative brain disorders. Fundación Cerebro y Mente, Madrid, pp. 387-400 (2000). Garcia-Segura, L.M., Cardona-Gomez, P., Trejo, J.L., Fernandez-Galaz, M.C., and Chowen, J.A. Glial cells are involved in organizational and activational effects of sex hormones in the brain. In: Matsumoto, A. (Ed.). Sexual Differentiation of the Brain. CRC Press, Boca Raton, Fl., pp.83-93 (2000) Busiguina, S., Argente, J., Garcia-Segura, L. M., and Chowen, J. A. Anatomically Specific Changes in the Expression of Somatostatin, Growth Hormone-Releasing Hormone and Growth Hormone Receptor mRNA in Diabetic Rats. Journal of Neuroendocrinology 12: 29-39 (2000) Gonzalez-Parra, S., Argente, J., Garcia-Segura, L. M., and Chowen, J. A. Effect of neonatal and adult testosterone treatment on the cellular composition of the adult female rat anterior pituitary. Journal of Endocrinology 164: 265-276 (2000) Nilsen, J., Horvath, T. L., Levy, A., Chowen, J., Garcia-Segura, L.M., and Naftolin, F. Fetal hypothalamic developmental events presage adult function. In: Bourguignon, J.-P. and Plant, T. M. (Eds.) The Onset of Puberty in Perspective. Elsevier, New York, pp. 233-246 (2000) Cardona-Gómez, G.P., Trejo, J.L., Fernandez, A.M., and Garcia-Segura, L.M. Estrogen receptors and insulinlike growth factor-I receptors mediate estrogen-dependent synaptic plasticity. NeuroReport 11: 1735-1738 (2000) Cardona-Gómez, G.P., Chowen, J.A. and Garcia-Segura, L.M. Estradiol and progesterone regulate the expression of insulin-like growth factor-I and insulin-like growth factor binding protein-2 in the hypothalamus of adult female rats. Journal of Neurobiology 43: 269-281 (2000)

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Cardona-Gomez, G. P., DonCarlos, L., and Garcia-Segura, L. M. Insulin-like growth factor I receptors and estrogen receptors colocalize in female rat brain. Neuroscience 99: 751-760 (2000). Martin-Vasallo, P., Wetzel, R. K., Garcia-Segura, L. M., Molina-Holgado, E., Arystarkhova, E., and Sweadner, K. J. Oligodendrocytes in brain and optic nerve express the beta3 subunit isoform of Na,K-ATPase. Glia 31: 206-218 (2000). Garcia-Segura, L.M., Cardona-Gómez, G.P., Chowen, J.A., and Azcoitia, I. Insulin-like growth factor-I receptors and estrogen receptors interact in the promotion of neuronal survival and neuroprotection. Journal of Neurocytology 29: 425-437 (2000). Chowen, J. A., Azcoitia, I., Cardona-Gomez, G. P. and Garcia-Segura, L. M. Sex steroids and the brain: lessons from animal studies. Journal of Pediatric Endocrinology and Metabolism 13:1045-1066 (2000). Naftolin, F., Zreik,, T.G., Garcia-Segura, L.M., and Horvath, T.L. Neuroendocrine control of reproduction. In: The Physiologic Basis of Gynecology and Obstetrics, Seifer, Samuels, Kniss (eds.), Williams & Wilkins, Baltimore. Pp. 63-74 (2000) 2001 Garcia-Segura, L.M., Azcoitia, I., and DonCarlos, L.L. Neuroprotection by estradiol. Progress in Neurobiology 63: 29-60 (2001). Zsarnovszky, A., Horvath, T. L., Garcia-Segura, L. M., Horvath, B. and Naftolin, F. Oestrogen-induced changes in the synaptology of the monkey (Cercopithecus aethiops) arcuate nucleus during gonadotropin feedback. Journal of Neuroendocrinology, 13: 20-27 (2001). Azcoitia, I., Sierra, A., Veiga, S., Honda, S., Harada, N. and Garcia-Segura, L.M. Brain aromatase is neuroprotective. Journal of Neurobiology 47: 318-329 (2001). Azcoitia, I., Garcia-Ovejero, D., Chowen, J.A. and Garcia-Segura, L.M. Astroglia play a key role in the neuroprotective actions of estrogen. Progress in Brain Research 132: 469-478 (2001) Leitner, S., Voigt, C., Garcia-Segura, L.M., Van't Hof, T. and Gahr, M. Seasonal activation and inactivation of song motor memories in wild canaries is not reflected in neuroanatomical changes of forebrain song areas. Hormones and Behavior 40: 160-168 (2001). Garcia-Ovejero, D., Trejo, J.L., Ciriza, I., Walton, K.D. and Garcia-Segura, L.M. Space flight affects magnocellular supraoptic neurons of young prepuberal rats: transient and permanent effects. Developmental Brain Research 130: 191-205 (2001). Cardona-Gomez, G.P., Mendez, P., DonCarlos, L.L., Azcoitia, I. and Garcia-Segura, L.M. Interactions of estrogens and insulin-like growth factor-I in the brain: implications for neuroprotection. Brain Research Reviews 37:320-334 (2001). Rodriguez De Fonseca, F., Gorriti, M.A., Bilbao, A., Escuredo, L., Garcia-Segura, L.M., Piomelli, D., Navarro, M. Role of the endogenous cannabinoid system as a modulator of dopamine transmission: implications for Parkinson's disease and schizophrenia. Neurotoxicity Research 3: 23-35 (2001). 2002 Ivanova, T., Mendez, P., Garcia-Segura, L.M. and Beyer, C. Rapid stimulation of the PI3-kinase /Akt signalling pathway in developing midbrain neurones by oestrogen. Journal of Neuroendocrinology 14: 73-79 (2002) Chowen, J.A., Goya, L., Ramos, S., Busiguina, S., Garcia-Segura, L.M., Argente, J., and Pascual-Leone, A.M. Effects of early undernutrition on the brain insulin-like growth factor-I system. Journal of Neuroendocrinology 14:163-169 (2002) Garcia-Segura, L.M. Cajal and glial cells. Progress in Brain Research 136: 255-260 (2002)

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Bonansco, C., González de la Vega, A., González Alegre, P., Borde, M., Garcia-Segura, L.M., and Buño, W., Tetanic stimulation of Schäffer collaterals induces rhythmic bursts via NMDA receptor activation in rat CA1 pyramidal neurons. Hippocampus 12: 434-446 (2002) Ribeiro, M.F., and Garcia-Segura, L.M., Dehydroepiandrosterone regulates insulin-like growth factor-1 system in adult rat hypothalamus. Endocrine 17: 129-134 (2002) Azcoitia, I., DonCarlos, L.L., and Garcia-Segura, L.M. Estrogen and brain vulnerability. Neurotoxicity Research 4: 235-245 (2002) Fernandez-Galaz, M.C., Diano, S., Horvath, T.L., and Garcia-Segura, L.M. Leptin uptake by serotonergic neurones of the dorsal raphe. Journal of Neuroendocrinology 14:429-434 (2002) Hansen, H.H., Azcoitia, I., Pons, S., Romero, J., Garcia-Segura, L.M., Ramos, J.A., Hansen, H.S., and Fernandez-Ruiz, J. Blockade of cannabinoid CB(1) receptor function protects against in vivo disseminating brain damage following NMDA-induced excitotoxicity. Journal of Neurochemistry 82:154-158 (2002). Garcia-Ovejero, D., Veiga, S., Garcia-Segura, L.M., and DonCarlos, L.L. Glial expression of estrogen and androgen receptors after rat brain injury. Journal of Comparative Neurology 450: 256-271 (2002). Goya, L., Garcia-Segura, L.M., Ramos, S., Pascual-Leone, A.M., Argente, J., Martin, M.A., and Chowen, J.A. Interaction between malnutrition and ovarian hormones on the systemic IGF-I axis. European Journal of Endocrinology 147: 417-424 (2002). Parducz, A., Hoyk, Z., Kis, Z., and Garcia-Segura, L.M. Hormonal enhancement of neuronal firing is linked to structural remodelling of excitatory and inhibitory synapses. European Journal of Neuroscience 16: 665-670 (2002). Cardona-Gomez, G.P., Mendez, P., and Garcia-Segura, L.M. Synergistic interaction of estradiol and insulin-like growth factor-I in the activation of PI3K/Akt signaling in the adult rat hypothalamus. Molecular Brain Research 107: 80-88 (2002). 2003 Cardona-Gomez, G.P., Mendez, P., DonCarlos, L.L., Azcoitia, I., Garcia-Segura, L.M. Interactions of estrogen and insulin-like growth factor-I in the brain: molecular mechanisms and functional implications. Journal of Steroid Biochemistry and Molecular Biology 83:211-217 (2003). Perez-Martin, M., Cifuentes, M., Grondona, J.M., Bermudez-Silva, F., Arrabal, P.M., Perez-Figares, J.M., Jimenez, A.J., Garcia-Segura, L.M., Fernandez-Llebrez, P. Neurogenesis in explants from the walls of the lateral ventricle of adult bovine brain: role of endogenous IGF-1 as a survival factor. European Journal of Neuroscience 17: 205-211 (2003). Hung, A.J., Stanbury, M.G., Shanabrough, M., Horvath, T.L., Garcia-Segura, L.M., Naftolin, F. Estrogen, synaptic plasticity and hypothalamic reproductive aging. Experimental Gerontology 38: 53-59 (2003). Azcoitia, I., DonCarlos, L.L., Garcia-Segura, L.M. Are gonadal steroid hormones involved in disorders of brain aging? Aging Cell 2: 31-37 (2003). Cowley, M.A., Smith, R.G., Diano, S., Tschop, M., Pronchuk, N., Grove, K.L., Strasburger, C.J., Bidlingmaier, M., Esterman, M., Heiman, M.L., Garcia-Segura, L.M., Nillni, E.A., Mendez, P., Low, M.J., Sotonyi, P., Friedman, J.M., Liu, H., Pinto, S., Colmers, W.F., Cone, R.D., Horvath, T.L., The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis. Neuron 37: 649-661 (2003). Mendez, P., Azcoitia, I., Garcia-Segura, LM., Estrogen receptor alpha forms estrogen-dependent multimolecular complexes with insulin-like growth factor receptor and phosphatidylinositol 3-kinase in the adult rat brain.

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Molecular Brain Research 112:170-176 (2003). Julian, M.D., Martin, A.B., Cuellar, B., Rodriguez De Fonseca, F., Navarro, M., Moratalla, R., Garcia-Segura, L.M., Neuroanatomical relationship between type 1 cannabinoid receptors and dopaminergic systems in the rat basal ganglia. Neuroscience 119: 309-318 (2003). Sierra, A., Azcoitia, I., Garcia-Segura, L.M., Endogenous estrogen formation is neuroprotective in model of cerebellar ataxia. Endocrine 21: 43-52 (2003). Horvath, T.L., Diano, S., Leranth, C., Garcia-Segura, L.M., Cowley, M.A., Shanabrough, M., Elsworth, J.D., Sotonyi, P., Roth, R.H., Dietrich, E.H., Matthews, R.T., Barnstable, C.J., Redmond, D.E., Coenzyme Q induces nigral mitochondrial uncoupling and prevents dopamine cell loss in a primate model of Parkinson's disease. Endocrinology 144: 2757-2760 (2003). DonCarlos, L.L., Garcia-Ovejero, D., Sarkey, S., Garcia-Segura, L.M., Azcoitia, I., Androgen receptor immunoreactivity in forebrain axons and dendrites in the rat. Endocrinology 144: 3632-3638 (2003). Veiga, S., Garcia-Segura, L.M., Azcoitia, I., Neuroprotection by the steroids pregnenolone and dehydroepiandrosterone is mediated by the enzyme aromatase. Journal of Neurobiology 56: 398-406 (2003). Perez-Martin, M., Azcoitia, I., Trejo, J.L., Sierra, A., Garcia-Segura, L.M., An antagonist of estrogen receptors blocks the induction of adult neurogenesis by insulin-like growth factor-I in the dentate gyrus of adult female rat. European Journal of Neuroscience 18: 923-930 (2003). Azcoitia, I., Leonelli, E., Magnaghi, V., Veiga, S., Garcia-Segura, L.M., Melcangi, R.C., Progesterone and its derivatives dihydroprogesterone and tetrahydroprogesterone reduce myelin fiber morphological abnormalities and myelin fiber loss in the sciatic nerve of aged rats. Neurobiology of Aging 24: 853-860 (2003). Sierra, A., Lavaque, E., Perez-Martin, M., Azcoitia, I., Hales, D.B., Garcia-Segura, L.M., Steroidogenic acute regulatory protein in the rat brain: cellular distribution, developmental regulation and overexpression after injury. European Journal of Neuroscience 18: 1458-1467 (2003). Picazo, O., Azcoitia, I., Garcia-Segura, L.M., Neuroprotective and neurotoxic effects of estrogens. Brain Research 990: 20-27 (2003). Chavez-Delgado, M.E., Mora-Galindo, J., Gomez-Pinedo, U., Feria-Velasco, A., Castro-Castaneda, S., LopezDellamary Toral, F.A., Luquin-De Anda, S., Garcia-Segura, L.M., Garcia-Estrada, J. Facial nerve regeneration through progesterone-loaded chitosan prosthesis. A preliminary report. Journal of Biomedical Materials Research 67B: 702-711 (2003). Schumacher, M., Weill-Engerer, S., Liere, P., Robert, F., Franklin, R.J.M., Garcia-Segura, L.M., Lambert, J.J., Mayo, W., Melcangi, R.C., Parducz, A., Suter, U., Carelli, C., Baulieu, E.E., Akwa, Y., Steroid hormones and neurosteroids in normal and pathological aging of the nervous system. Progress in Neurobiology 71: 3-29 (2003). Garcia-Segura, L.M., Veiga, S., Sierra, A., Melcangi, R.C., Azcoitia, I., Aromatase: a neuroprotective enzyme. Progress in Neurobiology 71: 31-41 (2003). Melcangi, R.C., Azcoitia, I., Ballabio, M., Cavarretta, I., Gonzalez, L.C., Leonelli, E., Magnaghi, V., Veiga, S., Garcia-Segura, L.M., Neuroactive steroids influence peripheral myelination: a promising opportunity for preventing or treating age-dependent dysfunctions of peripheral nerves. Progress in Neurobiology 71: 57-66 (2003). Azcoitia I., Sierra, A., Veiga, S., Garcia-Segura, L.M., Aromatase expression by reactive astroglia is neuroprotective. Annals of the New York Academy of Sciences 1007: 298-305 (2003). Azcoitia, I., Ciriza, I., Garcia-Ovejero, D., Mendez, P., Sierra, A., Veiga, S., Naftolin, F., Garcia-Segura, L.M.,

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Estrogen and neurological function: Unsolved and controversial issues. In: Genazzani, A.R. (ed.) Hormone Replacement Therapy and the Brain. The Current Status of Research and Practice. Parthenon, New York, pp. 177-181 (2003). 2004 Schumacher, M., Ibanez, C., Robert, F., Garcia-Segura, L.M., Franklin, R.J.M., Melcangi, R.C., Aging myelin and cognitive decline: a role for steroids. In: Chanson, P., Epelbaum, J., Lamberts, S., Christen, Y. (eds.), Endocrine Aspects of successful aging: genes, hormones and lifestyles. Springer, Berlin, pp. 101-127 (2004). Melcangi, R.C., Azcoitia, I., Galbiati, M., Magnaghi, V., Garcia-Ovejero, D., Garcia-Segura, L.M., Nonneuronal cells in the nervous system: sources and targets of neuroactive steroids. Advances in Molecular and Cell Biology 31: 535-559 (2004). Ciriza, I., Azcoitia, I., Garcia-Segura, L.M., Reduced progesterone metabolites protect rat hippocampal neurones from kainic acid excitotoxicity in vivo. Journal of Neuroendocrinology 16: 58-63 (2004). Garcia-Segura, L.M., McCarthy, M.M., Minireview: Role of glia in neuroendocrine function. Endocrinology 145: 1082-1086 (2004). Cardona-Gomez, P., Perez, M., Avila, J., Garcia-Segura, L.M., Wandosell, F. Estradiol inhibits GSK3 and regulates interaction of estrogen receptors, GSK3, and beta-catenin in the hippocampus. Molecular and Cellular Neuroscience 25: 363-373 (2004). Arango-Davila, C.A., Cardona-Gomez, G.P., Gallego-Gomez, J.C., Garcia-Segura, L.M., Pimienta, H.J. Downregulation of Bcl-2 in rat substantia nigra after focal cerebral ischemia. Neuroreport 15:1437-1441 (2004). Azcoitia, I., Garcia-Segura, L.M., DonCarlos, L.L., Oestradiol signalling in the hippocampus. Current Neuropharmacology 2: 245-259 (2004). Ciriza, I., Carrero, P., Azcoitia, I., Lundeen, S.G., Garcia-Segura, L.M., Selective estrogen receptor modulators protect hippocampal neurons from kainic acid excitotoxicity: differences with the effect of estradiol. Journal of Neurobiology, 61: 209-221 (2004). Hoyk, Z., Parducz, A., Garcia-Segura, L.M., Dehydroepiandrosterone regulates astroglia reaction to denervation of olfactory glomeruli. Glia, 48: 207–216 (2004). Yague, J.G., Lavaque, E., Carretero, J., Azcoitia, I., Garcia-Segura L.M., Aromatase, the enzyme responsible for estrogen biosynthesis, is expressed by human and rat glioblastomas. Neuroscience Letters, 368: 279-284 (2004). Veiga, S., Melcangi, R.C., Doncarlos, L.L., Garcia-Segura, L.M., Azcoitia, I., Sex hormones and brain agin. Experimental Gerontology, 39:1623-1631 (2004). 2005 Azcoitia, I., Sierra, A., Veiga, S., Garcia-Segura, L.M., Brain steroidogenesis: emerging therapeutic strategies to prevent neurodegeneration. Journal of Neural Transmission, 112:171-176 (2005). Azcoitia, I., Perez-Martin, M., Salazar, V., Castillo, C., Ariznavarreta, C., Garcia-Segura, L.M., Tresguerres, J.A., Growth hormone prevents neuronal loss in the aged rat hippocampus. Neurobiology of Aging 26:697-703 (2005). Veiga, S., Azcoitia, I., Garcia-Segura, L.M., Ro5-4864, a peripheral benzodiazepine receptor ligand, reduces reactive gliosis and protects hippocampal hilar neurons from kainic acid excitotoxicity. Journal of Neuroscience Research 80:129-137 (2005). Mendez, P., Azcoitia, I., Garcia-Segura, L.M., Interdependence of oestrogen and insulin-like growth factor-I in the brain: potential for analysing neuroprotective mechanisms. Journal of Endocrinology 185:11-17 (2005).

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Garcia-Ovejero, D., Azcoitia, I., DonCarlos, L.L., Melcangi, R.C., Garcia-Segura, L.M., Glia-neuron crosstalk in the neuroprotective mechanisms of sex steroid hormones. Brain Research Reviews 48:273-286 (2005). Melcangi, R.C., Cavarretta, I.T., Ballabio, M., Leonelli, E., Schenone, A., Azcoitia, I., Garcia-Segura, L.M., Magnaghi, V., Peripheral nerves: a target for the action of neuroactive steroids. Brain Research Reviews 48:328338 (2005). Perez-Martin, M., Salazar, V., Castillo, C., Ariznavarreta, C., Azcoitia, I., Garcia-Segura, L.M., Tresguerres, J.A., Estradiol and soy extract increase the production of new cells in the dentate gyrus of old rats. Experimental Gerontology 40: 450-453 (2005). Alavarez-de-la-Rosa, M., Silva, I., Nilsen, J., Perez, M.M., Garcia-Segura, L.M., Avila, J., Naftolin, F., Estradiol prevents neural tau hyperphosphorylation characteristic of Alzheimer's disease. Annal of the New York Academy of Sciences 1052: 210-224 (2005). Carswell, H.V., Dominiczak, A.F., Garcia-Segura, L.M., Harada, N., Hutchison, J.B., Macrae, I.M., Brain aromatase expression after experimental stroke: Topography and time course. Journal of Steroid Biochemistry and Molecular Biology 96:89-91 (2005). Veiga, S., Azcoitia, I., Garcia-Segura, L.M., Extragonadal synthesis of estradiol is protective against kainic acid excitotoxic damage to the hippocampus. Neuroreport 16:1599-603 (2005). Leonelli, E., Yague, J.G., Ballabio, M., Azcoitia, I., Magnaghi, V., Schumacher, M., Garcia-Segura, L.M., Melcangi, R., Ro5-4864, a synthetic ligand of peripheral benzodiazepine receptor, reduces aging-associated myelin degeneration in the sciatic nerve of male rats. Mechanisms of Ageing and Development 126:1159-1163 (2005). Lorenz, B. Garcia-Segura, L.M., DonCarlos, L.L., Cellular phenotype of androgen receptor-immunoreactive nuclei in the developing and adult rat brain. Journal of Comparative Neurology 492:456-468 (2005). Mendez, P., Cardona-Gomez, G.P., Garcia-Segura, L.M., Interaction of insulin-like growth factor-I and estrogen in the brain. Advances in Experimental Medicine and Biology 567: 285-303 (2005). 2006 Lavaque, E., Mayen, A., Azcoitia, I., Tena-Sempere, M., Garcia-Segura, L.M., Sex differences, developmental changes, response to injury and cAMP regulation of the mRNA levels of steroidogenic acute regulatory protein, cytochrome p450scc, and aromatase in the olivocerebellar system. Journal of Neurobiology 66:308-318 (2006). Darnaudery, M., Perez-Martin, M., Belizaire, G., Maccari, S., Garcia-Segura, L.M., Insulin-like growth factor 1 reduces age-related disorders induced by prenatal stress in female rats. Neurobiology of Aging 27:119-127 (2006). DonCarlos, L.L., Sarkey, S., Lorenz, B., Azcoitia, I., Garcia-Ovejero, D., Huppenbauer, C., Garcia-Segura, L.M., Novel cellular phenotypes and subcellular sites for androgen action in the forebrain. Neuroscience 138: 801-807 (2006). Lavaque, E., Sierra, A., Azcoitia, I., Garcia-Segura, L.M., Steroidogenic acute regulatory protein in the brain. Neuroscience 138: 741-747 (2006). Azcoitia, I., Moreno, A.M., Carrero, P., Palacios, S., Garcia-Segura, L.M., Neuroprotective effects of soy phytoestrogens in the rat brain. Gynecological Endocrinology 22:63-69 (2006). Yague, J.G., Muñoz, A., de Monasterio-Schrader, P., DeFelipe, J. Garcia-Segura, L.M., Azcoitia, I., Aromatase expression in the human temporal cortex. Neuroscience 138:389-401 (2006). D'Astous, M., Mendez, P., Morissette, M., Garcia-Segura, L.M., Di Paolo, T., Implication of the PI3K/Akt

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signaling pathway in the neuroprotective effect of estradiol in the striatum of MPTP mice. Molecular Pharmacology 69:1492-1498 (2006). Mendez, P., Garcia-Segura, L.M., Phosphatidyl Inositol 3 Kinase (PI3K) and Glycogen Synthase Kinase 3 (GSK3) regulate estrogen receptor mediated transcription in neuronal cells. Endocrinology 147:3027-3039 (2006). Azcoitia, I., Garcia-Segura, L.M., The action of oestrogens in the brain. In: Erkkola, R. (ed.), The Menopause. Elsevier, New York, pp. 109-121 (2006). Veiga, S., Leonelli, E., Beelke, M., Garcia-Segura, L.M., Melcangi, R.C., Neuroactive steroids prevent peripheral myelin alterations induced by diabetes. Neuroscience Letters 402:150-153 (2006). Magnaghi, V., Veiga, S., Ballabio, M., Gonzalez, L.C., Garcia-Segura, L.M., Melcangi, R.C. Sex-dimorphic effects of progesterone and its reduced metabolites on gene expression of myelin proteins by rat Schwann cells. Journal of the Peripheral Nervous System 11:111-118 (2006). Melcangi, R.C., Garcia-Segura, L.M., Therapeutic approaches to peripheral neuropathy based on neuroactive steroids. Expert Review of Neurotherapeutics 6:1121-1125 (2006). Ciriza, I., Carrero, P., Frye, C.A., Garcia-Segura, L.M., Reduced metabolites mediate neuroprotective effects of progesterone in the adult rat hippocampus. The synthetic progestin medroxyprogesterone acetate (Provera) is not neuroprotective. Journal of Neurobiology 66:916-928 (2006). Cardona-Gomez GP, Arango-Davila C, Gallego-Gomez JC, Barrera-Ocampo A, Pimienta H, Garcia-Segura LM., Estrogen dissociates Tau and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit in postischemic hippocampus. Neuroreport 17:1337-1341 (2006). Garcia-Segura, L.M., Melcangi, R.C., Steroids and glial cell function. Glia 54:485-498 (2006). Mendez, P., Wandosell, F., Garcia-Segura, L.M., Cross-talk between estrogen receptors and insulin-like growth factor-I receptor in the brain: Cellular and molecular mechanisms. Frontiers in Neuroendocrinology 27:391-403 (2006). Garcia-Segura, L.M., Sanz, A., Mendez, P., Cross-talk between IGF-I and estradiol in the brain: Focus on neuroprotection. Neuroendocrinology 84:275-279 (2006). 2007 Csakvari, E., Hoyk, Z., Gyenes, A., Garcia-Ovejero, D., Garcia-Segura, L.M., Parducz, A., Fluctuation of synapse density in the arcuate nucleus during the estrous cycle. Neuroscience 144:1288-1292 (2007). Leonelli, E., Bianchi, R., Cavaletti, G., Caruso, D., Crippa, D., Garcia-Segura, L.M., Lauria, G., Magnaghi, V., Roglio, I., Melcangi, R.C., Progesterone and its derivatives are neuroprotective agents in experimental diabetic neuropathy: A multimodal analysis. Neuroscience 144:1293-1304 (2007). Roglio I, Bianchi R, Giatti S, Cavaletti G, Caruso D, Scurati S, Crippa D, Garcia-Segura LM, Camozzi F, Lauria G, Melcangi RC. Testosterone derivatives are neuroprotective agents in experimental diabetic neuropathy. Cellular and Molecular Life Sciences 64:1158-1168 (2007). Guerra-Araiza C, Amorim MA, Camacho-Arroyo I, Garcia-Segura LM. Effects of progesterone and its reduced metabolites, dihydroprogesterone and tetrahydroprogesterone, on the expression and phosphorylation of glycogen synthase kinase-3 and the microtubule-associated protein tau in the rat cerebellum. Developmental Neurobiology 67:510-520 (2007). Alejandre-Gomez M, Garcia-Segura LM, Gonzalez-Burgos I. Administration of an inhibitor of estrogen biosynthesis facilitates working memory acquisition in male rats. Neuroscience Research 58: 272-277 (2007).

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DonCarlos LL, Azcoitia I, Garcia-Segura LM. In search of neuroprotective therapies based on the mechanisms of estrogens. Expert Review of Endocrinology and Metabolism 2: 387-397 (2007). Barreto G, Veiga S, Azcoitia I, Garcia-Segura LM, Garcia-Ovejero D. Testosterone decreases reactive astroglia and reactive microglia after brain injury in male rats: role of its metabolites oestradiol and dihydrotestosterone. European Journal of Neuroscience 25:3039-3046 (2007). Naftolin F, Garcia-Segura LM, Horvath TL, Zsarnovszky A, Demir N, Fadiel A, Leranth C, Vondracek-Klepper S, Lewis C, Chang A, Parducz A. Estrogen-induced hypothalamic synaptic plasticity and pituitary sensitization in the control of the estrogen-induced gonadotrophin surge. Reproductive Sciences 14:101-116 (2007). Darnaudery M, Perez-Martin M, Del Favero F, Gomez-Roldan C, Garcia-Segura LM, Maccari S. Early motherhood in rats is associated with a modification of hippocampal function. Psychoneuroendocrinology 32:803-812 (2007). Veiga S, Carrero P, Pernia O, Azcoitia I, Garcia-Segura LM. Translocator protein (18KDa) is involved in the regulation of reactive gliosis. Glia 55:1426-1436 (2007). Garcia-Segura LM, Diz-Chaves Y, Perez-Martin M, Darnaudéry M. Estradiol, insulin-like growth factor-I and brain aging. Psychoneuroendocrinology 32, Suppl 1:557-561 (2007). 2008 Walf AA, Ciriza I, Garcia-Segura LM, Frye CA. Antisense oligodeoxynucleotides for estrogen receptor-beta and alpha attenuate estradiol's modulation of affective and sexual behavior, respectively. Neuropsychopharmacology 33:431-440 (2008). Caruso D, Scurati S, Maschi O, De Angelis L, Roglio I, Giatti S, Garcia-Segura LM, Melcangi RC. Evaluation of neuroactive steroid levels by liquid chromatography-tandem mass spectrometry in central and peripheral nervous system: Effect of diabetes. Neurochemistry International 52:560-568 (2008) Morissette M, Le Saux M, D'Astous M, Jourdain S, Al Sweidi S, Morin N, Estrada-Camarena E, Mendez P, Garcia-Segura LM, Di Paolo T. Contribution of estrogen receptors alpha and beta to the effects of estradiol in the brain. Journal of Steroid Biochemistry and Molecular Biology 108:327-338 (2008) Melcangi RC, Garcia-Segura LM, Mensah-Nyagan AG. Neuroactive steroids: State of the art and new perspectives. Cellular and Molecular Life Sciences 65:777-797 (2008). Roglio I, Giatti S, Pesaresi M, Bianchi R, Cavaletti G, Lauria G, Garcia-Segura LM, Melcangi RC. Neuroactive steroids and peripheral neuropathy. Brain Research Reviews 57:460-469 (2008). Garcia-Segura LM, Lorenz B, DonCarlos LL. The role of glia in the hypothalamus: implications for gonadal steroid feedback and reproductive neuroendocrine output. Reproduction 135:419-429 (2008). Yague JG, Wang AC, Janssen WG, Hof PR, Garcia-Segura LM, Azcoitia I, Morrison JH. Aromatase distribution in the monkey temporal neocortex and hippocampus. Brain Research 1209:115-127 (2008). Sarkey S, Azcoitia I, Garcia-Segura LM, Garcia-Ovejero D, DonCarlos LL. Classical androgen receptors in nonclassical sites in the brain. Hormones and Behavior 53:753-764 (2008). Sanz A, Carrero P, Pernia O, Garcia-Segura LM. Pubertal maturation modifies the regulation of insulin-like growth factor-I receptor signaling by estradiol in the rat prefrontal cortex. Developmental Neurobiology, 68:1018-1028 (2008). Tapia-Gonzalez S, Carrero P, Pernia O, Garcia-Segura LM, Diz-Chaves Y. Selective oestrogen receptor (ER) modulators reduce microglia reactivity in vivo after peripheral inflammation: potential role of microglial ERs. Journal of Endocrinology, 198:219-230 (2008).

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Garcia-Segura LM. Aromatase in the brain: not just for reproduction anymore. Journal of Neuroendocrinology 20:705-712 (2008). Melcangi RC, Garcia-Segura LM. Steroid metabolism in glial cells. In M.S.Ritsner, A. Weizman (eds.) Neuroactive steroids in brain function, behavior and neuropsychiatric disoreders. Chapter 2. Pp. 43-59, Springer, New York, (2008). 2009 Garcia-Segura LM. Hormones and Brain Plasticity. Oxford University Press, New York, 496pp. (2009). Guerra-Araiza C, Amorim MA, Pinto-Almazán R, González-Arenas A, Campos MG, Garcia-Segura LM. Regulation of the phosphoinositide-3 kinase and mitogen-activated protein kinase signaling pathways by progesterone and its reduced metabolites in the rat brain. Journal of Neuroscience Research, 87: 470-481 (2009). Yague JG, Garcia-Segura LM, Azcoitia I. Selective transcriptional regulation of aromatase gene by vitamin D, dexamethasone, and mifepristone in human glioma cells. Endocrine, 35:252-261 (2009). Melcangi RC, Garcia-Segura LM. Glial Steroid Metabolism. In: Squire LR (ed.) Encyclopedia of Neuroscience, volume 4, pp. 871-876. Oxford: Academic Press (2009). Marin R, Díaz M, Alonso R, Sanz A, Arévalo MA, Garcia-Segura LM. Role of estrogen receptor alpha in membrane-initiated signaling in neural cells: Interaction with IGF-1 receptor. Journal of Steroid Biochemistry and Molecular Biology, 114:2-7 (2009) Llorente R, López Gallardo M, Llorente Berzal A, Prada C, Garcia-Segura LM, Viveros MP, Early maternal deprivation in rats induces gender-dependent effects on developing hippocampal and cerebellar cells. International Journal of Developmental Neuroscience 27: 233-241 (2009) Tan O, Fadiel A, Chang A, Demir N, Jeffrey R, Horvath T, Garcia-Segura LM, Naftolin F. Estrogens regulate post-translational modification of neural cell adhesion molecule (NCAM) during the estrogen-induced gonadotrophin surge (EIGS). Endocrinology 150:2783-2790 (2009). Varea O, Garrido JJ, Dopazo A, Mendez P, Garcia-Segura LM, Wandosell F. Estradiol activates beta-catenin dependent transcription in neurons. PLoS ONE 4:e5153 (2009). Garcia-Segura LM, Balthazart J. Steroids and neuroprotection: new advances. Frontiers in Neuroendocrinology 30:V-IX (2009). Doncarlos LL, Azcoitia I, Garcia-Segura LM. Neuroprotective actions of selective estrogen receptor modulators. Psychoneuroendocrinology 34S:S113-S122 (2009) Etgen, AM, Garcia-Segura, L.M. Estrogen Regulation of Neurotransmitter and Growth Factor Signaling in the Brain. In: D.W. Pfaff, A.P. Arnold, A.M. Etgen, S.E. Fahrbach, R.T. Rubin Eds., Hormones, Brain and Behavior, Second Edition, Chapter 3.33, Pp. 1121-1162, Elsevier, San Diego, (2009). Viveros MP, Llorente R, López-Gallardo M, Suarez J, Bermúdez-Silva F, De la Fuente M, Rodriguez de Fonseca F, Garcia-Segura LM. Sex-dependent alterations in response to maternal deprivation in rats. Psychoneuroendocrinology 34S:S217-S226 (2009) Biamonte F, Assenza G, Marino R, D'Amelio M, Panteri R, Caruso D, Scurati S, Yague JG, Garcia-Segura LM, Cesa R, Strata P, Melcangi RC, Keller F. Interactions between neuroactive steroids and reelin haploinsufficiency in Purkinje cell survival. Neurobiology of Disease 36:103-115 (2009) Giatti S, Pesaresi M, Cavaletti G, Bianchi R, Carozzi V, Lombardi R, Maschi O, Lauria G, Garcia-Segura LM, Caruso D, Melcangi RC. Neuroprotective effects of a ligand of translocator protein-18kDa (Ro5-4864) in experimental diabetic neuropathy. Neuroscience 164:520-529 (2009)

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Barreto G, Santos-Galindo M, Diz-Chaves Y, Pernía O, Carrero P, Azcoitia I, Garcia-Segura LM. Selective estrogen receptor modulators decrease reactive astrogliosis in the injured brain: effects of aging and prolonged depletion of ovarian hormones. Endocrinology 150:5010-5015 (2009) 2010 Melcangi RC, Garcia-Segura LM. Sex-specific therapeutic strategies based on neuroactive steroids: In search for innovative tools for neuroprotection. Hormones and Behavior 57:2-11 (2010) Pesaresi M, Maschi O, Giatti S, Garcia-Segura LM, Caruso D, Melcangi RC. Sex differences in neuroactive steroid levels in the nervous system of diabetic and non-diabetic rats. Hormones and Behavior 57:46-55 (2010) Cerciat M, Unkila M, Garcia-Segura LM, Arevalo MA. Selective estrogen receptor modulators decrease the production of interleukin-6 and interferon-gamma-inducible protein-10 by astrocytes exposed to inflammatory challenge in vitro. Glia 58:93-102 (2010) Azcoitia I, DonCarlos LL, Arévalo MA, Garcia-Segura LM. Therapeutic implications of brain steroidogenesis. Hormone Molecular Biology and Clinical Investigation 1:21-26 (2010) Yague JG, Azcoitia I, Defelipe J, Garcia-Segura LM, Muñoz A. Aromatase expression in the normal and epileptic human hippocampus. Brain Research 22:41-52 (2010) García-Cáceres C, Diz-Chaves Y, Lagunas N, Calmarza-Font I, Azcoitia I, Garcia-Segura LM, Frago LM, Argente J, Chowen JA. The weight gain response to stress during adulthood is conditioned by both sex and prenatal stress exposure. Psychoneuroendocrinology 35:403-413(2010) van Gool SA, Wit JM, De Schutter T, De Clerck N, Postnov AA, Kremer Hovinga S, van Doorn J, Veiga SJ, Garcia-Segura LM, Karperien M. Marginal growth increase, altered bone quality and polycystic ovaries in female prepubertal rats after treatment with the aromatase inhibitor exemestane. Hormone Research in Paediatrics 73:49-60 (2010). Giatti S, D'Intino G, Maschi O, Pesaresi M, Garcia-Segura LM, Calza L, Caruso D, Melcangi RC. Acute experimental autoimmune encephalomyelitis induces sex-dimorphic changes in neuroactive steroid levels. Neurochemistry International 56:118-127 (2010). Varea O, Arevalo MA, Garrido JJ, Garcia-Segura LM, Wandosell F, Mendez P. Interaction of estrogen receptors with insulin-like growth factor-I and Wnt signaling in the nervous system. Steroids 75:565-569 (2010). Picazo O, Becerril-Montes A, Huidobro-Perez D, Garcia-Segura LM. Neuroprotective actions of the synthetic estrogen 17alpha-ethynylestradiol in the hippocampus. Cellular and Molecular Neurobiology 30:675-682 (2010). Saraceno GE, Aón Bertolino ML, Galeano P, Romero JI, Garcia-Segura LM, Capani F. Estradiol therapy in adulthood reverses glial and neuronal alterations caused by perinatal asphyxia. Experimental Neurology 223:615-622 (2010). Fernández-Galaz MC, Fernández-Agulló T, Carrascosa JM, Ros M, Garcia-Segura LM. Leptin accumulation in hypothalamic and dorsal raphe neurons is inversely correlated with brain serotonin content. Brain Research 1329:194-202 (2010). Simon-Areces J, Membrive G, Garcia-Fernandez C, Garcia-Segura LM, Arevalo MA. Neurogenin 3 cellular and subcellular localization in the developing and adult hippocampus. Journal of Comparative Neurology 518:18141824 (2010). Grassi D, Amorim MA, Garcia-Segura LM, Panzica G. Estrogen receptor alpha is involved in the estrogenic regulation of arginine vasopressin immunoreactivity in the supraoptic and paraventricular nuclei of ovariectomized rats. Neuroscience Letters 474:135-139 (2010).

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Rodrigues de Amorim MA, Garcia-Segura LM, Goya RG, Portiansky EL. Decrease in PTEN and increase in Akt expression and neuron size in aged rat spinal cord. Experimental Gerontology 45:457-463 (2010). van Gool SA, Wit JM, De Schutter T, De Clerck N, Postnov AA, Kremer Hovinga S, van Doorn J, Veiga SJ, Garcia-Segura LM, Karperien M. Impaired body weight and tail length gain and altered bone quality after treatment with the aromatase inhibitor exemestane in male rats. Hormone Research in Paediatrics 73:376-385 (2010). Garcia-Segura LM, Arévalo MA, Azcoitia I. Interactions of estradiol and insulin-like growth factor-I signalling in the nervous system: new advances. Progress in Brain Research 181:251-272 (2010). Arevalo MA, Santos-Galindo M, Bellini MJ, Azcoitia I, Garcia-Segura LM. Actions of estrogens on glial cells: Implications for neuroprotection. Biochimica Biophysica Acta 1800:1106-1112 (2010) Caruso D, D'Intino G, Giatti S, Maschi O, Pesaresi M, Calabrese D, Garcia-Segura LM, Calza L, Melcangi RC. Sex-dimorphic changes in neuroactive steroid levels after chronic experimental autoimmune encephalomyelitis. Journal of Neurochemistry 114:921-932 (2010) García-Cáceres C, Lagunas N, Calmarza-Font I, Azcoitia I, Diz-Chaves Y, Garcia-Segura LM, Baquedano E, Frago LM, Argente J, Chowen JA. Gender differences in the long-term effects of chronic prenatal stress on the HPA axis and hypothalamic structure in rats. Psychoneuroendocrinology 35:1525-1535 (2010). Amorim MA, Guerra-Araiza C, Pernía O, da Cruz e Silva EF, Garcia-Segura LM. Progesterone regulates the phosphorylation of protein phosphatases in the brain. Journal of Neuroscience Research 88:2826-3832 (2010) Lagunas N, Calmarza-Font I, Diz-Chaves Y, Garcia-Segura LM. Long-term ovariectomy enhances anxiety and depressive-like behaviors in mice submitted to chronic unpredictable stress. Hormones and Behavior 58:786-791 (2010) Caruso D, Pesaresi M, Maschi O, Giatti S, Garcia-Segura LM, Melcangi RC. Effects of short- and long-term gonadectomy on neuroactive steroid levels in the central and peripheral nervous system of male and female rats. Journal of Neuroendocrinology 22:1137-1147 (2010) Azcoitia I, Santos-Galindo M, Arevalo MA, Garcia-Segura LM. Role of astroglia in the neuroplastic and neuroprotective actions of estradiol. European Journal of Neuroscience 32:1995-2002 (2010). De Nicola AF, Pietranera L, Bellini MJ, Goya R, Brocca ME, Garcia-Segura LM, Protective effect of estrogens on the brain of rats with essential and endocrine hypertension. Hormone Molecular Biology and Clinical Investigation 4:549-557 (2010). Amorim MAR, Guerra-Araiza C, Garcia-Segura LM. Progesterone as a regulator of phosphorylation in the central nervous system. Hormone Molecular Biology and Clinical Investigation 4:601-607 (2010). De Marinis E, Ascenzi P, Pellegrini M, Galluzzo P, Bulzomi P, Arevalo MA, Garcia-Segura LM, Marino M. 17β-Estradiol - A new modulator of neuroglobin levels in neurons: Role in neuroprotection against H(2)O(2)induced toxicity. Neurosignals 18:223-235 (2010). 2011 Viveros MP, Marco-López EM, López-Gallardo M, Garcia-Segura LM, Wagner EJ. Framework for sex differences in adolescent neurobiology: A focus on cannabinoids. Neuroscience & Biobehavioral Reviews 35:1740-1751 (2011). Mendez P, Garcia-Segura LM, Muller D. Estradiol promotes spine growth and synapse formation without affecting pre-established networks. Hippocampus 21:1263-1267 (2011)

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Arevalo MA, Santos-Galindo M, Lagunas N, Azcoitia I, Garcia-Segura LM. Selective estrogen receptor modulators as brain therapeutic agents. Journal of Molecular Endocrinology 46:R1-R9 (2011). Pietranera L, Bellini MJ, Arévalo MA, Goya R, Brocca ME, Garcia-Segura LM, De Nicola AF. Increased aromatase expression in the hippocampus of spontaneously hypertensive rats: effects of estradiol administration. Neuroscience 174:151-159 (2011). Pesaresi M, Giatti S, Cavaletti G, Abbiati F, Calabrese D, Bianchi R, Caruso D, Garcia-Segura LM, Melcangi RC. Sex differences in the manifestation of peripheral diabetic neuropathy in gonadectomized rats: A correlation with the levels of neuroactive steroids in the sciatic nerve. Experimental Neurology 228:215-221 (2011). Ruiz-Palmero I, Simon-Areces J, Garcia-Segura LM, Arevalo MA. Notch/Neurogenin 3 signalling is involved in the neuritogenic actions of oestradiol in developing hippocampal neurones. Journal of Neuroendocrinology 23:355-364 (2011). López Rodríguez AB, Mateos Vicente B, Romero-Zerbo SY, Rodriguez-Rodriguez N, Bellini MJ, Rodriguez de Fonseca F, Bermudez-Silva FJ, Azcoitia I, Garcia-Segura LM, Viveros MP Estradiol decreases cortical reactive astrogliosis after brain injury by a mechanism involving cannabinoid receptors. Cerebral Cortex, 21:2046-2055 (2011). Arevalo MA, Garcia-Segura LM, Azcoitia I. Interactions of estradiol and insulin-like growth factor-I in neuroprotection: Implications for brain aging and neurodegeneration. In: Gravanis AG and Mellon SH (Eds.), Hormones in Neurodegeneration, Neuroprotection and Neurogenesis, Chapter 1, pp. 3-11, Wiley-VCH, Weinheim, (2011). Tapia-González S, García-Segura LM, Tena-Sempere M, Frago LM, Castellano JM, Fuente-Martín E, GarcíaCáceres C, Argente J, Chowen JA. Activation of microglia in specific hypothalamic nuclei and the cerebellum of adult rats exposed to neonatal overnutrition. Journal of Neuroendocrinology 23:365-370 (2011). Azcoitia I, Yague JG, Garcia-Segura LM. Estradiol synthesis within the human brain. Neuroscience 191:139147 (2011). Bellini MJ, Hereñú CB, Goya RG, Garcia-Segura LM. Insulin-like growth factor-I gene delivery to astrocytes reduces their inflammatory response to lipopolysaccharide. Jurnal of Neuroinflammation 8:21(2011). Lagunas N, Calmarza-Font I, Grassi D, Garcia-Segura LM. Estrogen receptor ligands counteract cognitive deficits caused by androgen deprivation in male rats. Hormones and Behavior 59:581-584 (2011). Rubio N, Cerciat M, Unkila M, Garcia-Segura LM, Arevalo MA. An in vitro experimental model of neuroinflammation: the induction of interleukin-6 in murine astrocytes infected with Theiler's murine encephalomyelitis virus, and its inhibition by oestrogenic receptor modulators. Immunology 133:360-369 (2011). Garcia-Segura LM, De Nicola F. Hormones and the brain. Hormone Molecular Biology and Clinical Investigation 7:315 (2011) Melcangi RC, Garcia-Segura LM. Sex differences in the injured brain. Hormone Molecular Biology and Clinical Investigation 7:385-391 (2011) Melcangi RC, Panzica G, Garcia-Segura LM. Neuroactive steroids: focus on human brain. Neuroscience 191:15 (2011). Santos-Galindo M, Acaz-Fonseca E, Bellini MJ, Garcia-Segura LM. Sex differences in the inflammatory response of primary astrocytes to lipopolysaccharide. Biology of Sex Differences 2:7 (2011).

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Simon-Areces J, Dopazo A, Dettenhofer M, Rodriguez-Tebar A, Garcia-Segura LM, Arevalo MA. Formin1 mediates the induction of dendritogenesis and synaptogenesis by neurogenin3 in mouse hippocampal neurons. PLoS One 6 (7):e21825 (2011). Azcoitia I, Arevalo MA, De Nicola AF, Garcia-Segura LM. Neuroprotective actions of estradiol revisited. Trends in Endocrinology and Metabolism 22:467-473 (2011) Baquedano E, García-Cáceres C, Diz-Chaves Y, Lagunas N, Calmarza-Font I, Azcoitia I, Garcia-Segura LM, Argente J, Chowen JA, Frago LM. Prenatal stress induces long-term effects in cell turnover in the hippocampushypothalamus-pituitary axis in adult male rats. PLoS ONE 6 (11): e27549 (2011). Arevalo MA, Ruiz-Palmero I, Simon-Areces J, Acaz-Fonseca E, Azcoitia I and Garcia-Segura LM Estradiol meets Notch signaling in developing neurons. Frontiers in Endocrinology 2:21. doi: 10.3389/fendo.2011.00021 (2011) Melcangi RC, Giatti S, Pesaresi M, Calabrese D, Mitro N, Caruso D, Garcia-Segura LM. Role of neuroactive steroids in the pheripheral nervous system. Frontiers in Endocrinology 2:104. doi: 10.3389/fendo.2011.00104 (2011) Pesaresi M, Giatti S, Cavaletti G, Abbiati F, Calabrese D, Lombardi R, Bianchi R, Lauria G, Caruso D, GarciaSegura LM, Melcangi RC. Sex-dimorphic effects of dehydroepiandrosterone in diabetic neuropathy. Neuroscience 199:401-409 (2011).

2012 Arevalo MA, Diz-Chaves Y, Santos-Galindo M, Bellini MJ, Garcia-Segura LM. Selective oestrogen receptor modulators decrease the inflammatory response of glial cells. Journal of Neuroendocrinology 24:183-190 (2012). Wandosell F, Varea O, Arevalo MA, Garcia-Segura LM. Oestradiol regulates β-catenin-mediated transcription in neurones. Journal of Neuroendocrinology 24:191-194 (2012) González-Burgos I, Rivera-Cervantes MC, Velázquez-Zamora DA, Feria-Velasco A, Garcia-Segura LM. Selective estrogen receptor modulators regulate dendritic spine plasticity in the hippocampus of male rats. Neural Plasticity 2012:309494 (2012) Azcoitia I, Garcia-Segura LM. Hormones and the Aging Brain. In: Fink G, Pfaff DW, Levine JE, eds. Handbook of Neuroendocrinology. London, Waltham, San Diego: Academic press, Elsevier; pp. 573-594 (2012). Diz-Chaves Y, Kwiatkowska-Naqvi A, Von Hülst H, Pernía O, Carrero P, Garcia-Segura LM. Behavioral effects of estradiol therapy in ovariectomized rats depend on the age when the treatment is initiated. Experimental Gerontology 47:93-99 (2012). Arevalo MA, Ruiz-Palmero I, Scerbo MJ, Acaz-Fonseca E, Cambiasso MJ, Garcia-Segura LM. Molecular mechanisms involved in the regulation of neuritogenesis by estradiol: Recent advances. Journal of Steroid Biochemistry and Molecular Biology 131: 52-56 (2012). Calmarza-Font I, Lagunas N, Garcia-Segura LM. Antidepressive and anxiolytic activity of selective estrogen receptor modulators in ovariectomized mice submitted to chronic unpredictable stress. Behavioral Brain Research 227:287-290 (2012). Cermenati G, Abbiati F, Cermenati S, Brioschi E, Volonterio A, Cavaletti G, Saez E, De Fabiani E, Crestani M, Garcia-Segura LM, Melcangi RC, Caruso D, Mitro N. Diabetes induced myelin abnormalities are associated with an altered lipid pattern: protective effects of LXR activation. Journal of Lipid Research 53:300-310 (2012).

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Panzica GC, Balthazart J, Frye CA, Garcia-Segura LM, Herbison AE, Mensah-Nyagan AG, McCarthy MM, Melcangi RC. Milestones on steroids and the nervous system: 10 years of basic and translational research. Journal of Neuroendocrinology 24:1-15 (2012). Giatti S, Caruso D, Boraso M, Abbiati F, Ballarini E, Calabrese D, Pesaresi M, Rigolio R, Santos-Galindo M, Viviani B, Cavaletti G, Garcia-Segura LM, Melcangi RC. Neuroprotective effects of progesterone in chronic experimental autoimmune encephalomyelitis. Journal of Neuroendocrinology 24:851-861 (2012). Velázquez-Zamora DA, Garcia-Segura LM, González-Burgos I. Effects of selective estrogen receptor modulators on allocentric working memory performance and on dendritic spines in medial prefrontal cortex pyramidal neurons of ovariectomized rats. Hormones and Behavior 61:512-517 (2012) Barrera-Ocampo A, Gutierrez-Vargas J, Garcia-Segura LM, Cardona-Gómez GP. Glycogen synthase kinase3β/β-catenin signaling in the rat hypothalamus during the estrous cycle. Journal of Neuroscience Research 90:1078-1084 (2012). Mitro N, Cermenati G, Giatti S, Abbiati F, Pesaresi M, Calabrese D, Garcia-Segura LM, Caruso D, Melcangi RC. LXR and TSPO as new therapeutic targets to increase the levels of neuroactive steroids in the central nervous system of diabetic animals. Neurochemistry International 60:616-621 (2012). Diz-Chaves Y, Pernia O, Carrero P, Garcia-Segura LM. Prenatal stress causes alterations in the morphology of microglia and the inflammatory response of the hippocampus of adult female mice. Journal of Neuroinflammation 9:71 (2012). Rubio N, Garcia-Segura LM, Arevalo MA. Survivin prevents apoptosis by binding to caspase-3 in astrocytes infected with the BeAn strain of Theiler's murine encephalomyelitis virus. Journal of Neurovirology 18:354-363 (2012). De Nicola AF, Brocca ME, Pietranera L, Garcia-Segura LM. Neuroprotection and sex steroid hormones: evidence of estradiol-mediated protection in hypertensive encephalopathy. Mini-Reviews in Medicinal Chemistry 12:1081-1089 (2012). Simon-Areces J, Dietrich MO, Hermes G, Garcia-Segura LM, Arevalo MA, Horvath TL. UCP2 induced by natural birth regulates neuronal differentiation of the hippocampus and related adult behavior. PLoS One 7(8):e42911 (2012). 2013 Arevalo MA, Santos-Galindo M, Acaz-Fonseca E, Azcoitia I, Garcia-Segura LM. Gonadal hormones and the control of reactive gliosis. Hormones and Behavior 63:216-221 (2013). Diz-Chaves Y, Baquedano E, Frago LM, Chowen JA, Garcia-Segura LM, Arevalo MA. Maternal stress alters the developmental program of embryonic hippocampal neurons growing in vitro. Psychoneuroendocrinology 38:455-459 (2013). Giatti S, Boraso M, Abbiati F, Ballarini E, Calabrese D, Santos-Galindo M, Rigolio R, Pesaresi M, Caruso D, Viviani B, Cavaletti G, Garcia-Segura LM, Melcangi RC. Multimodal analysis in acute and chronic experimental autoimmune encephalomyelitis. Journal of Neuroimmune Pharmacology 8:238-250 (2013). Grassi D, Lagunas N, Amorim M, Pinos H, Panzica G, Garcia-Segura LM, Collado P. Role of oestrogen receptors on the modulation of NADPH-diaphorase-positive cell number in supraoptic and paraventricular nuclei of ovariectomised female rats. Journal of Neuroendocrinology 25:244-250 (2013). Caruso D, Barron AM, Brown MA, Abbiati F, Carrero P, Pike CJ, Garcia-Segura LM, Melcangi RC. Agerelated changes in neuroactive steroid levels in 3xTg-AD mice. Neurobiology of Aging 34:1080-1089 (2013).

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De Marinis E, Acaz-Fonseca E, Arevalo MA, Ascenzi P, Fiocchetti M, Marino M, Garcia-Segura LM. 17βoestradiol anti-inflammatory effects in primary astrocytes require oestrogen receptor β-mediated neuroglobin upregulation. Journal of Neuroendocrinology 25:260-270 (2013). Diz-Chaves Y, Astiz M, Bellini MJ, Garcia-Segura LM. Prenatal stress increases the expression of proinflammatory cytokines and exacerbates the inflammatory response to LPS in the hippocampal formation of adult male mice. Brain Behavior and Immunity 28:196-206 (2013). Azcoitia I, Arevalo MA, Garcia-Segura LM. Brain aromatase and neuroprotection in mammals. In: Balthazart J and Ball G (eds). Brain Aromatase, Estrogens, and Behavior. Oxford University Press, New York, pp. 371-382 (2013). Simon-Areces J, Acaz-Fonseca E, Ruiz-Palmero I, Garcia-Segura LM, Arevalo MA. A CRM1-mediated nuclear export signal is essential for cytoplasmic localization of neurogenin 3 in neurons. PLoS One 8:e55237 (2013). Ruiz-Palmero I, Hernando M, Garcia-Segura LM, Arevalo MA. G protein-coupled estrogen receptor is required for the neuritogenic mechanism of 17β-estradiol in developing hippocampal neurons. Molecular and Cellular Endocrinology 372:105-115 (2013). Grassi D, Bellini MJ, Acaz-Fonseca E, Panzica G, Garcia-Segura LM. Estradiol and testosterone regulate arginine-vasopressin expression in SH-SY5Y human female neuroblastoma cells through estrogen receptors-α and -β. Endocrinology 154:2092-2100 (2013). Barron AM, Garcia-Segura LM, Caruso D, Jayaraman A, Lee JW, Melcangi RC, Pike CJ. Ligand for translocator protein reverses pathology in a mouse model of Alzheimer's disease. Journal of Neuroscience 33:8891-8897 (2013). Caruso D, Pesaresi M, Abbiati F, Calabrese D, Giatti S, Garcia-Segura LM, Melcangi RC. Comparison of plasma and cerebrospinal fluid levels of neuroactive steroids with their brain, spinal cord and peripheral nerve levels in male and female rats. Psychoneuroendocrinology 38:2278-2290 (2013). Grassi D, Lagunas N, Amorin M, Pinos H, Panzica G, Garcia-Segura LM, Collado P. Estrogenic regulation of NADPH-diaphorase in the supraoptic and paraventricular nuclei under acute osmotic stress. Neuroscience 248:127-135 (2013). Rubio N, Almanza A, Mercado F, Arévalo MA, Garcia-Segura LM, Vega R, Soto E. Upregulation of voltagegated Ca2+ channels in mouse astrocytes infected with Theiler's murine encephalomyelitis virus (TMEV). Neuroscience 247:309-318 (2013). Astiz M, Diz-Chaves Y, Garcia-Segura LM. Sub-chronic exposure to the insecticide dimethoate induces a proinflammatory status and enhances the neuroinflammatory response to bacterial lypopolysaccharide in the hippocampus and striatum of male mice.Toxicology and Applied Pharmacology 272:263-271 (2013). 2014 Astiz M, Acaz-Fonseca E, Garcia-Segura LM. Sex Differences and Effects of Estrogenic Compounds on the Expression of Inflammatory Molecules by Astrocytes Exposed to the Insecticide Dimethoate. Neurotoxicity Research 25:271-285 (2014). Melcangi RC, Giatti S, Calabrese D, Pesaresi M, Cermenati G, Mitro N, Viviani B, Garcia-Segura LM, Caruso D. Levels and actions of progesterone and its metabolites in the nervous system during physiological and pathological conditions. Progress in Neurobiology 113:56-69 (2014). Lopez-Rodriguez AB, Llorente-Berzal A, Garcia-Segura LM, Viveros MP. Sex dependent long-term effects of adolescent exposure to THC and/or MDMA on neuroinflammation and serotoninergic and cannabinoid systems in rats. British Journal of Pharmacology 171:1435-1447 (2014).

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Avila MF, Torrente D, Cabezas R, Morales L, García-Segura LM, Gonzalez J, Barreto GE. Structural insights from GRP78-NF-κB binding interactions: A computational approach to understand a possible neuroprotective pathway in brain injuries. Journal of Theoretical Biology 345:43-51 (2014). Torrente D, Cabezas R, Avila MF, García-Segura LM, Barreto GE, Guedes RC. Cortical Spreading Depression in Traumatic Brain Injuries: is there a role for astrocytes? Neuroscience Letters 565:2-6 (2014). Calabrese D, Giatti S, Romano S, Porretta C, Bianchi R, Milanese M, Bonanno G, Caruso D, Viviani B, Gardoni F, Garcia-Segura LM, Melcangi RC. Diabetic neuropathic pain: a role for testosterone metabolites. Journal of Endocrinology 221: 1-13 (2014). Acaz-Fonseca E, Sanchez-Gonzalez R, Azcoitia I, Arevalo MA, Garcia-Segura LM. Role of astrocytes in the neuroprotective actions of 17β-estradiol and selective estrogen receptor modulators. Molecular and Cellular Endocrinology 389: 48-57 (2014). Garcia-Segura LM, Pérez-Márquez J. A new mathematical function to evaluate neuronal morphology using the Sholl analysis. Journal of Neuroscience Methods 226: 103-109 (2014). Mitro N, Cermenati G, Brioschi E, Abbiati F, Audano M, Giatti S, Crestani M, De Fabiani E, Azcoitia I, GarciaSegura LM, Caruso D, Melcangi RC. Neuroactive steroid treatment modulates myelin lipid profile in diabetic peripheral neuropathy. Journal of Steroid Biochemistry and Molecular Biology 143: 115-121 (2014). Olza-Fernández I, Marín Gabriel MA, Gil-Sanchez A, Garcia-Segura LM, Arevalo MA. Neuroendocrinology of childbirth and mother-child attachment: The basis of an etiopathogenic model of perinatal neurobiological disorders. Frontiers in Neuroendocrinology 35: 459-472 (2014). Pietranera L, Brocca ME, Roig P, Lima A, Garcia-Segura LM, De Nicola AF. 17α-oestradiol-induced neuroprotection in the brain of spontaneously hypertensive rats. Journal of Neuroendocrinology 26:310-320 (2014). Gutierrez-Vargas JA, Muñoz-Manco JI, Garcia-Segura LM, Cardona-Gómez GP. Glun2b N-methyl-D-aspartic acid receptor subunit mediates atorvastatin-Induced neuroprotection after focal cerebral ischemia. Journal of Neuroscience Research 92:1529-1548 (2014). Barreto GE, Santos-Galindo M, Garcia-Segura LM. Selective estrogen receptor modulators regulate reactive microglia after penetrating brain injury. Frontiers in Aging Neuroscience 6:132 (2014). Rubio N, Arevalo MA, Cerciat M, Sanz-Rodriguez F, Unkila M, Garcia-Segura LM. Theiler's virus infection provokes the overexpression of genes coding for the chemokine Ip10 (CXCL10) in SJL/J murine astrocytes, which can be inhibited by modulators of estrogen receptors. Journal of Neurovirology 20:485-495 (2014). Scerbo MJ, Freire-Regatillo A, Cisternas CD, Brunotto M, Arevalo MA, Garcia-Segura LM, Cambiasso MJ. Neurogenin 3 mediates sex chromosome effects on the generation of sex differences in hypothalamic neuronal development. Frontiers in Cellular Neuroscience 8:188 (2014). Avila Rodriguez M, Garcia-Segura LM, Cabezas R, Torrente D, Capani F, Gonzalez J, Barreto GE. Tibolone protects T98G cells from glucose deprivation. Journal of Steroid Biochemistry and Molecular Biology 144:294303 (2014). Ghorbanpoor S, Garcia-Segura LM, Haeri-Rohani A, Khodagholi F, Jorjani M. Aromatase inhibition exacerbates pain and reactive gliosis in the dorsal horn of the spinal cord of female rats caused by spinothalamic tract injury. Endocrinology 155:4341-4355 (2014). Cabezas R, Avila M, Gonzalez J, El-Bachá RS, Báez E, García-Segura LM, Jurado Coronel JC, Capani F, Cardona-Gomez GP, Barreto GE. Astrocytic modulation of blood brain barrier: perspectives on Parkinson's disease. Frontiers in Cellular Neuroscience 8:211 (2014).

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Diz-Caves Y, Garcia-Segura LM. Sex differences in affective disorders: Interaction between stress and ovarian hormones. In: Estrogens and Cognition. Psychobiological and Clinical Aspects (I. Gonzaález-Burgos, Ed), Research Signpost, Kerala, pp. 119-152 (2014). Grassi D, Lagunas N, Calmarza-Font I, Diz-Chaves Y, Garcia-Segura LM, Panzica GC. Chronic unpredictable stress and long-term ovariectomy affect arginine-vasopressin expression in the paraventricular nucleus of adult female mice. Brain Research 1588:55-62 (2014). 2015 Pietranera L, Brocca ME, Roig P, Lima A, Garcia-Segura LM, De Nicola AF. Estrogens are neuroprotective factors for hypertensive encephalopathy. Journal of Steroid Biochemistry and Molecular Biology 146C: 15-25 (2015). Lopez-Rodriguez AB, Siopi E, Finn DP, Marchand-Leroux C, Garcia-Segura LM, Jafarian-Tehrani M, Viveros MP. CB1 and CB2 Cannabinoid receptor antagonists prevent minocycline-induced neuroprotection following traumatic brain injury in mice. Cerebral Cortex 25:35-45 (2015). Arevalo MA, Azcoitia I, Garcia-Segura LM. The neuroprotective actions of oestradiol and oestrogen receptors. Nature Reviews Neuroscience 16:17-29 (2015). Chacón PJ, Del Marco Á, Arévalo Á, Domínguez-Giménez P, García-Segura LM, Rodríguez-Tébar A. Cerebellin 4, a synaptic protein, enhances inhibitory activity and resistance of neurons to amyloid-β toxicity. Neurobiology of Aging 36:1057-1071 (2015). Lopez-Rodriguez AB, Avila-Rodriguez M, Vega-Velez NE, Capani F, Gonzalez J, Garcia-Segura LM, Barreto E. Neuroprotection by exogenous estrogenic compounds following traumatic brain injury. In K.A. Duncan (Ed.), Estrogen effects on traumatic brain injury. Mechanisms of neuroprotection and repair, Academic Press, Amsterdam, pp. 73-90 (2015). Giatti S, Rigolio R, Romano S, Mitro N, Viviani B, Cavaletti G, Caruso D, Garcia-Segura LM, Melcangi RC. Dihydrotestosterone as Protective Agent in Chronic Experimental Autoimmune Encephalomyelitis. Neuroendocrinology, in press Lopez-Rodriguez AB, Acaz-Fonseca E, Giatti S, Caruso D, Viveros MP, Melcangi RC, Garcia-Segura LM. Correlation of brain levels of progesterone and dehydroepiandrosterone with neurological recovery after traumatic brain injury in female mice. Psychoneuroendocrinology 56:1-11 (2015). Giatti S, Garcia-Segura LM, Melcangi RC. New steps forward in the neuroactive steroid field. Journal of Steroid Biochemistry and Molecular Biology, in press Cermenati G, Audano M, Giatti S, Carozzi V, Porretta-Serapiglia C, Pettinato E, Ferri C, D'Antonio M, De Fabiani E, Crestani M, Scurati S, Saez E, Azcoitia I, Cavaletti G, Garcia-Segura LM, Melcangi RC, Caruso D, Mitro N. Lack of Sterol Regulatory Element Binding Factor-1c Imposes Glial Fatty Acid Utilization Leading to Peripheral Neuropathy. Cell Metabolism, 21: 571-583 (2015). Zancan M, Dall'Oglio A, Sarzenski TM, Maher MI, Garcia-Segura LM, Rasia-Filho AA. Glial and axonal perikaryal coverage and somatic spines in the posterodorsal medial amygdala of male and cycling female rats. Journal of Comparative Neurology, in press Giatti S, Romano S, Pesaresi M, Cermenati G, Mitro N, Caruso D, Tetel MJ, Garcia-Segura LM, Melcangi RC. Neuroactive steroids and the peripheral nervous system: An update. Steroids, in press Arevalo MA, Azcoitia I, Gonzalez-Burgos I, Garcia-Segura LM. Signaling mechanisms mediating the regulation of synaptic plasticity and memory by estradiol. Hormones and Behavior, in press

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Participación en contratos de I+D de especial relevancia con Empresas y/o Administraciones (nacionales y/o internacionales) Título del contrato/proyecto: SERMs and neuroprotection Tipo de contrato:Research agreement Empresa/Administración financiadora: Johnson & Johnson Pharmaceutical Research & Development Entidades participantes:Instituto Cajal Duración, desde: 01.01.03 hasta: 31.12.04 Investigador responsable: Luis M. García Segura Número de investigadores participantes: 3 PRECIO TOTAL DEL PROYECTO: 72.000 $ Título del contrato/proyecto:Efectos neuroprotectores de la genisteina y la vitamina E Tipo de contrato:Investigación Empresa/Administración financiadora:Laboratorios Rovi/Fundación Endocrinología y Nutrición Entidades participantes:Instituto Cajal, CSIC; Instituto Palacios. Duración, desde: 01.01.04 hasta: 31.12.04 Investigador responsable: Luis M. García Segura Número de investigadores participantes: 3 PRECIO TOTAL DEL PROYECTO: 36.000 Euros

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Nota: Si necesita más casos, añádalos utilizando las funciones de copiar y pegar con el 2º caso.

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Patentes y Modelos de utilidad

Inventores (p.o. de firma): Título: N. de solicitud: País de prioridad: Entidad titular: Países a los que se ha extendido: Empresa/s que la están explotando:

Fecha de prioridad:

Inventores (p.o. de firma): Título: N. de solicitud: País de prioridad: Entidad titular: Países a los que se ha extendido: Empresa/s que la están explotando:

Fecha de prioridad:

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Nota: Si necesita más casos, añádalos utilizando las funciones de copiar y pegar con el 2º caso.

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Estancias en Centros extranjeros

(estancias continuadas superiores a un mes) CLAVE: D = doctorado, P = postdoctoral, I = invitado, C = contratado, O = otras (especificar).

CENTRO: Laboratoire de Cytologie. Université de Paris VI LOCALIDAD: París PAIS: Francia AÑO: 1976 DURACION: 3 meses TEMA: Aprendizaje de técnicas de inmunocitoquímica. CLAVE: Postdoctoral CENTRO: Departamento de Morfología. Escuela de Medicina. Universidad de Ginebra. LOCALIDAD: Ginebra PAIS: Suiza AÑO: 1978 - 1985 DURACION: 6 años y 4 meses TEMA: Reconocimiento neuronal y sinaptogénesis CLAVE: Contratado/Plantilla CENTRO: Yale University Medical School. LOCALIDAD: New Haven PAIS: Estados Unidos AÑO: 1984 DURACION: 4 meses TEMA: Mecanismos de acción celular de las hormonas gonadales en el sistema nervioso. CLAVE: Invitado CENTRO: Yale University Medical School. LOCALIDAD: New Haven PAIS: Estados Unidos AÑO: 1988 DURACION: 3 meses TEMA: Mecanismos de acción celular de las hormonas gonadales en el sistema nervioso. CLAVE: Invitado CENTRO: Yale University Medical School. LOCALIDAD: New Haven PAIS: Estados Unidos AÑO: 1991 DURACION: 2 meses TEMA: Mecanismos de acción celular de las hormonas gonadales en el sistema nervioso. CLAVE: Invitado CENTRO: The Babraham Institute. MRC Neuroendocrine, Development and Behaviour Group. LOCALIDAD: Babraham, Cambridge PAIS: Reino Unido AÑO: 1997 DURACION: Cuatro meses TEMA: Regulación de la expresión de la enzima aromatasa CLAVE: Sabático CENTRO: Kennedy Space Center, NASA LOCALIDAD: Cape Canaveral, Florida

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PAIS: USA AÑO: 1998 DURACION: Un mes TEMA: Participación en el proyecto Neurolab CLAVE: Co-investigador

4

Nota: Si necesita más casos, añádalos utilizando las funciones de copiar y pegar con el 2º caso.

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Contribuciones a Congresos (Presentaciones por invitación) TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 11th Annual Meeting Union of Swiss Societies of Experimental Biology LUGAR DE CELEBRACION: Ginebra (Suiza) AÑO: 1979 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 6th Lectures in Neurobiology LUGAR DE CELEBRACION: Gif-sur-Yvette (Francia) AÑO: 1981 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 13th Annual Meeting Union of Swiss Societies of Experimental Biology LUGAR DE CELEBRACION: Lausana (Suiza) AÑO: 1981 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Tables Rondes Roussel Uclaf: ‘’Steroids and the Brain’’ LUGAR DE CELEBRACION: París (Francia) AÑO: 1985 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 3es Jornades de Neurobiologia. Societat Catalana de Biologia. LUGAR DE CELEBRACION: Barcelona AÑO: 1986 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: VII International Congress on Hormonal Steroids LUGAR DE CELEBRACION: Madrid AÑO: 1986 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Conference on Neurosciences GDR/Spain LUGAR DE CELEBRACION: Berlín (República Democática Alemana) AÑO: 1987 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 8th International Symposium of the Journal of Steroid Biochemistry LUGAR DE CELEBRACION: París (Francia) AÑO: 1987 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: XXIII Congreso Nacional de la Sociedad Española de Ciencias Fisiológicas LUGAR DE CELEBRACION: Tenerife AÑO: 1988 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: International Serono Symposium on "Cardiovascular and Neurological Functions and Ovarian Secretions" LUGAR DE CELEBRACION: Dubrovnik (Yugoslavia) AÑO: 1989 TIPO DE PARTICIPACION: Conferencia invitada

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CONGRESO: Geoffrey Harris Memorial Symposium on Neuroendocrinology LUGAR DE CELEBRACION: Phoenix (Arizona, USA) AÑO: 1989 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 4es Jornades de Neurobiologia. Societat Catalana de Biologia. LUGAR DE CELEBRACION: Barcelona AÑO: 1989 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Symposium Satelite sobre Neurobiología. XXIV Congreso Nacional de la Sociedad Española de Ciencias Fisiológicas. LUGAR DE CELEBRACION: Madrid AÑO: 1990 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: First Swedish-Spanish Symposium "Frontiers in Neurobiology" LUGAR DE CELEBRACION: Estocolmo (Suecia) AÑO: 1991 TIPO DE PARTICIPACION: Presentación invitada CONGRESO: Primera reunión CSIC-INSERM LUGAR DE CELEBRACION: Madrid AÑO: 1992 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: International Conference on "Hormones, Brain and Behaviour". LUGAR DE CELEBRACION: Tours (Francia) AÑO: 1993 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 10th Biennial Meeting of the International Society for Developmental Neuroscience LUGAR DE CELEBRACION: San Diego (California, USA) AÑO: 1994 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: IX International Congress on Hormonal Steroids LUGAR DE CELEBRACION: Dallas (Texas, USA) AÑO: 1994 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: The role of glia in synapse development, synapse loss, and synaptic plasticity. LUGAR DE CELEBRACION: Tagernsee (Alemania) AÑO: 1994 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: International Workshop on What Steroid Hormones Tell the Brain LUGAR DE CELEBRACION: Puerto de la Cruz, Tenerife. AÑO: 1994 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 1995 Workshop on Steroid Hormones and Brain Function LUGAR DE CELEBRACION: Breckenridge (Colorado, USA) AÑO: 1995 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Sociedad Española de Neurociencias

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LUGAR DE CELEBRACION: Valladolid AÑO: 1995 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: XIVth International Symposium on Endocrinology and Development LUGAR DE CELEBRACION: Palma de Mallorca AÑO: 1995 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Simposio Avances Recientes en Criometodos. LUGAR DE CELEBRACION: Lerida AÑO: 1995 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Semana Marañon 95. La Evolución de la Sexualidad y los Estados Intersexuales. LUGAR DE CELEBRACION: Madrid AÑO: 1995 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: The 7th International hGH Symposium LUGAR DE CELEBRACION: Madrid AÑO: 1996 TIPO DE PARTICIPACION: Conferencia plenaria CONGRESO: Second European Meeting on Glial Cell Function in Health and Disease LUGAR DE CELEBRACION: Arcachon, Bordeaux (Francia) AÑO: 1996 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: First Tuscania Conference on Reproductive Medicine. The Brain: Source and Target for Sex Steroid Hormones LUGAR DE CELEBRACION: Pisa (Italia) AÑO: 1996 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 11th European Society for Neurochemistry Meeting LUGAR DE CELEBRACION: Grooningen (Holanda) AÑO: 1996 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Hormonal and Genetic Disorders in Gender-Specific Brain Differentiation 2nd EU Biomed Meeting LUGAR DE CELEBRACION: Salamanca AÑO: 1996 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Similarities and interactions between reproductive endocrinology and neuroscience roundtable workshop. Satellite symposium to the Xth World Congress of Psychiatry LUGAR DE CELEBRACION: Madrid AÑO: 1996 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Vth International Conference on Hormones, Brain, and Behavior LUGAR DE CELEBRACION: Torino (Italia) AÑO: 1996 TIPO DE PARTICIPACION: Conferencia invitada

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CONGRESO: Primera reunión nacional sobre IGFs LUGAR DE CELEBRACION: Madrid AÑO: 1996 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 8th International Congress on the Menopause LUGAR DE CELEBRACION: Sydney (Australia) AÑO: 1996 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO:Oestrogens and the brain and breast LUGAR DE CELEBRACION: Rotorua, (Nueva Zelanda) AÑO: 1996 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Symposium on Long Term Modulation of Synaptic Interactions in Neuronal Networks LUGAR DE CELEBRACION: Göttingen (Alemania) AÑO: 1996 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: International Joint Meeting of Physiology (Sociedad Española de Ciencias Fisiológicas y The American Physiological Society) LUGAR DE CELEBRACION: Malaga AÑO: 1997 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Graduiertenkolleg ‘Organisation und Dynamik neuronaler Netzwerke’, Georg-August-Universität Göttingen. Spring Meeting on Regulatory Mechanisms in Neural Plasticity LUGAR DE CELEBRACION: Bogensee, Berlin (Alemania) AÑO: 1997 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: EC Biomed Workshop. Genetic and Epigenetic Factors in Sexual Differentiation of thr Brain. LUGAR DE CELEBRACION: Gargellen (Austria) AÑO: 1997 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: International symposium on ‘Psychoneuroendocrine and Immunological Correlates of Aging’. LUGAR DE CELEBRACION: Pavia (Italia) AÑO: 1997 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: INSERM Philippe Laudat Conference ‘Neuroactive Steroids’ LUGAR DE CELEBRACION: Aix-les-Bains (Francia) AÑO: 1997 TIPO DE PARTICIPACION: Conferencia plenaria CONGRESO: XVIIIèmes Journées de l’Association Française pour l’étude de la Ménopause. LUGAR DE CELEBRACION: París (Francia) AÑO: 1997 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: II Annual ESHRE Winter Training Course in Reproductive Endocrinology. LUGAR DE CELEBRACION: Barcelona AÑO: 1998 TIPO DE PARTICIPACION: Conferencia invitada

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CONGRESO: Third Spain-Israel Scientific and Cultural Meeting LUGAR DE CELEBRACION: Salamanca AÑO: 1998 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 1998 Forum of European Neuroscience LUGAR DE CELEBRACION: Berlin AÑO: 1998 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 4th International Congress of Neuroendocrinology LUGAR DE CELEBRACION:Kitakyushu (Japón) AÑO: 1998 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 4th International Congress of Neuroendocrinology Satellite Symposium: Steroids in the Brain From Transcription Factor to Behavior. LUGAR DE CELEBRACION: Kyoto (Japón) AÑO: 1998 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: The 14th TMIN International Symposium. Development and Differentiation of Neuroendocrine System: Novel answers to Classic Questions. LUGAR DE CELEBRACION: Tokyo (Japón) AÑO: 1998 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Experimental Biology 1999 LUGAR DE CELEBRACION: Washington (USA) AÑO: 1999 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Steroids meet growth factors. LUGAR DE CELEBRACION: Günzburg (Alemania) AÑO: 1999 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 2000 Workshop on Steroid Hormones and Brain Function LUGAR DE CELEBRACION: Breckenridge, Colorado (USA) AÑO: 2000 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 2000 Neuroendocrine Workshop: Estrogen, growth factors and neuroplasticity. American Neuroendocrine Society. LUGAR DE CELEBRACION: Toronto (Canada) AÑO: 2000 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Pannon Symposium: Tissue-selective estrogen action: from basic science to clinical practice. LUGAR DE CELEBRACION: Pecs (Hungría) AÑO: 2000 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Graduiertenkolleg ‘Organisation und Dynamik neuronaler Netzwerke’, Georg-August-Universität Göttingen. Symposium on: ‘Cell Biological Aspects of Neurobiology’ LUGAR DE CELEBRACION: Tihany (Hungría) AÑO: 2000

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TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: International meeting: Steroids and nervous system. LUGAR DE CELEBRACION: Turín (Italia) AÑO: 2001 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Segundo symposio de IGFs. IGFs/Insulina: Del desarrollo al envejecimiento. LUGAR DE CELEBRACION: Madrid AÑO: 2001 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Cajal Club/Cajal Institute International Conference ‘’Changing views of Cajal’s Neuron’’ LUGAR DE CELEBRACION: Madrid AÑO: 2001 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Perspectivas para la salud femenina en el siglo XXI: Papel de las hormonas sexuales. Universidad de Verano de La Gomera. LUGAR DE CELEBRACION: La Gomera AÑO: 2001 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Gordon Research Conference: Biology of Aging LUGAR DE CELEBRACION: Oxford (Reino Unido) AÑO: 2001 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: International Society of PsychoNeuroendocrinology XXXII Annual Meeting. LUGAR DE CELEBRACION: Quebec (Canada) AÑO: 2001 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: International Meeting on Neurodevelopmental Labilities in Brain Disease States LUGAR DE CELEBRACION: Mojacar AÑO: 2001 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Endogenous neuroprotective factors- novel approaches to treatment of CNS diseases. MaxPlanck-Institute for Exoperimental Medicine. LUGAR DE CELEBRACION: Göttingen (Alemania) AÑO: 2001 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Workshop on Genomic vs non-genomic steroid actions: encountered or unified views. Instituto Juan March de Estudios e Investigaciones. Centre for International Meetings in Biology LUGAR DE CELEBRACION: Madrid AÑO: 2001 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Recent Advances in Steroid Biochemistry and Molecular Biology LUGAR DE CELEBRACION: Munich (Alemania) AÑO: 2002 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 10th World Congress on the Menopause LUGAR DE CELEBRACION: Berlín (Alemania)

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AÑO: 2002 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Society for Behavioral Neuroendocrinology Annual Meeting LUGAR DE CELEBRACION: Amherst, Massachusetts (USA) AÑO: 2002 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: European Society for Pediatric Endocrinology (ESPE), 41st Annual Meeting. LUGAR DE CELEBRACION: Madrid AÑO: 2002 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: II Congreso de la Sociedad de Neurociencias de Castilla y León. LUGAR DE CELEBRACION: Salamanca AÑO: 2002 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Satellite Symposia. The role of Neuroactive steroids in healthy ageing: therapeutical perspectives. 2nd International Meeting Steroids and Nervous System LUGAR DE CELEBRACION: Turín (Italia) AÑO: 2003 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: International Symposium. Neurobiology of Aging: Molecular approaches. LUGAR DE CELEBRACION: Szeged (Hungría) AÑO: 2003 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 3rd Pisa Workshop ‘’HRT in Climateric and Aging Brain’’. The International Menopause Society. LUGAR DE CELEBRACION: Pisa (Italia) AÑO: 2003 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: New Frontiers in Neuroendocrinology LUGAR DE CELEBRACION: Milán (Italia) AÑO: 2003 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: XXXIII Annual Congress of the International Society of Psychoneuroendocrinology LUGAR DE CELEBRACION: Pisa (Italia) AÑO: 2003 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 45 Congreso de la Sociedad Española de Endocrinología y Nutrición LUGAR DE CELEBRACION: Cáceres AÑO: 2003 TIPO DE PARTICIPACION: Conferencia inaugural CONGRESO: 8th Meeting of the Portuguese Society for Neurosciences. LUGAR DE CELEBRACION: Curia (Portugal) AÑO: 2003 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Sixth IBRO World Congress of Neuroscience LUGAR DE CELEBRACION: Prague (Czech Republic)

44

AÑO: 2003 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Simposium Internacional sobre Neuroplasticidad LUGAR DE CELEBRACION: Guadalajra, Jalisco (México) AÑO: 2003 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: X Congreso de la Sociedad Española de Biología Celular LUGAR DE CELEBRACION: Santander AÑO: 2003 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: International Symposium: Neural communication, neurotransmitters and aging. LUGAR DE CELEBRACION: Madrid AÑO: 2003 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 23rd Joint Meeting of the British Endocrine Societies with the European Federation of Endocrine Societies LUGAR DE CELEBRACION: Brighton (UK) AÑO: 2004 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 6th Neurochemistry Winter Conference LUGAR DE CELEBRACION: Sölden, Ötztal (Austria) AÑO: 2004 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: VIII Congreso de la Asociación Española para el Estudio de la Menopausia LUGAR DE CELEBRACION: Granada AÑO: 2004 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 5th International Symposium on Women’s Health and Menopause. New Findings, New Strategies, Improved Quality of Life LUGAR DE CELEBRACION: Florencia (Italia) AÑO: 2004 TIPO DE PARTICIPACION: Conferencia plenaria CONGRESO: 11th Meeting of the European Neuroendocrinology Association LUGAR DE CELEBRACION: Nápoles (Italia) AÑO: 2004 TIPO DE PARTICIPACION: Conferencia plenaria CONGRESO: 1st International OASI workshop: neurodegeneration/ neuroprotection LUGAR DE CELEBRACION: Troina (Italia) AÑO: 2004

Glia-neuron

crosstalk

in

neuroinflammation,

TIPO DE PARTICIPACION: Conferencia plenaria CONGRESO: Seventh International Symposium on Neurobiology and Neuroendocrinology of Aging LUGAR DE CELEBRACION: Bregenz (Austria) AÑO: 2004 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 12th International Congress of Endocrinology

45

LUGAR DE CELEBRACION: Lisboa (Portugal) AÑO: 2004 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: IV Simposio Nacional de Insulina/IGFs: Avances recientes y nuevos conceptos LUGAR DE CELEBRACION: Valdeganga, Albacete AÑO: 2004 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 15º Congreso de la Sociedad Andaluza de Ginecología y Obstetricia LUGAR DE CELEBRACION: Málaga AÑO: 2004 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Current knowledge on neurosteroids: correlation with the treatment of neurodegeneration, pain, anxiety, depression and memory deficiency. Eltem-Neurex Workshop. LUGAR DE CELEBRACION: Engelberg (Switzerland) AÑO: 2004 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 3rd International Meeting on: "Steroids and Nervous System" LUGAR DE CELEBRACION: Turín (Italia) AÑO: 2005 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 5º Encuentro Nacional de Salud y Medicina de la Mujer LUGAR DE CELEBRACION: Madrid AÑO: 2005 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Workshop on Steroid Homones and Brain Function LUGAR DE CELEBRACION: Breckenridge, Colorado (USA) AÑO: 2005 TIPO DE PARTICIPACION: Conferencia invitada (en simposio) CONGRESO: Envejecimiento: El desafío del siglo XXI. Una mirada endocrinológica. LUGAR DE CELEBRACION: Buenos Aires (Argentina) AÑO: 2005 TIPO DE PARTICIPACION: Conferencia plenaria CONGRESO: Envejecimiento: El desafío del siglo XXI. Una mirada endocrinológica. LUGAR DE CELEBRACION: Buenos Aires (Argentina) AÑO: 2005 TIPO DE PARTICIPACION: Mesa Redonda CONGRESO: Envejecimiento: El desafío del siglo XXI. Una mirada endocrinológica. LUGAR DE CELEBRACION: Buenos Aires (Argentina) AÑO: 2005 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Society for Behavioral Neuroendocrinology Annual Meeting LUGAR DE CELEBRACION: Austin, Texas (USA) AÑO: 2005 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: XLVIII Congreso Nacional de Ciencias Fisiológicas LUGAR DE CELEBRACION: Guadalajara, Jalisco (México)

46

AÑO: 2005 TIPO DE PARTICIPACION: Conferencia plenaria CONGRESO: XI Congreso de la Sociedad Española de Neurociencia LUGAR DE CELEBRACION: Torremolinos AÑO: 2005 TIPO DE PARTICIPACION: Conferencia inaugural CONGRESO: 56º Congreso Nazionale de la Societá Italiana di Fisiologia e Joint Symposium SIF-Physiological Society. LUGAR DE CELEBRACION: Palermo (Italia) AÑO: 2005 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Aging women – comprehension before treatment. Satellite meeting of the EMBO conference Nuclear receptors: from chromatin to disease. LUGAR DE CELEBRACION: Gardone Riviera, Lake Garda (Italia) AÑO: 2005 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Symposium Neurology, Neuroprotection and HRT. 11th World Congress on the Menopause LUGAR DE CELEBRACION: Buenos Aires (Argentina) AÑO: 2005 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Symposium Aging Brain. 11th World Congress on the Menopause LUGAR DE CELEBRACION: Buenos Aires (Argentina) AÑO: 2005 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Symposium Brain Protection and Estrogen. 11th World Congress on the Menopause LUGAR DE CELEBRACION: Buenos Aires (Argentina) AÑO: 2005 TIPO DE PARTICIPACION: Conferencia plenaria CONGRESO: IV Encuentro Nacional de Neurociencias – V Seminario Internacional de Neurociencias LUGAR DE CELEBRACION: Medellín (Colombia) AÑO: 2005 TIPO DE PARTICIPACION: Conferencia plenaria CONGRESO: Eventi neurodegenerativi durante l'invecchiamento e possibili strategie terapeutiche. Center of Excellence on Neurodegenerative Diseases. University of Milan. LUGAR DE CELEBRACION: Milán (Italia) AÑO: 2005 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Symposium Neurosteroids and Neuroendocrinology LUGAR DE CELEBRACION: Pittsburgh (USA) AÑO: 2006

Neuroprotection.

Sixth

International

Congress

on

TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Symposium One hundred years after the first Nobel Prize to the neurosciences in 1906. 5th Forum of European Neuroscience LUGAR DE CELEBRACION: Viena (Austria) AÑO: 2006

47

TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Lille Summer School in Neurosciences: "Brain Plasticity in Life Span", LUGAR DE CELEBRACION: Lille (Francia) AÑO: 2006 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 1st ESE Postgraduate Course in Molecular and Cellular Endocrinology. European Society of Endocrinology. LUGAR DE CELEBRACION: Córdoba AÑO: 2006 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Simposio Transtornos Psicóticos en la Mujer: de los Datos a las Hipótesis. X Congreso Nacional de Psiquiatría. LUGAR DE CELEBRACION: Sevilla AÑO: 2006 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: International Workshop. Role of locally-synthesized steroids in synaptic plasticity and neuroprotection. LUGAR DE CELEBRACION: Estrasburgo (Francia) AÑO: 2006 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Satellite symposium: Selective estrogen receptor modulators and the brain. 4th International Meeting on: "Steroids and Nervous System" LUGAR DE CELEBRACION: Turín (Italia) AÑO: 2007 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Symposium: Basic to Clinical: Hormones and the brain. 9th European Congress of Endocrinology LUGAR DE CELEBRACION: Budapest (Hungría) AÑO: 2007 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Tne Endocrine Society 89th Annual Meeting LUGAR DE CELEBRACION: Toronto (Canadá) AÑO: 2007 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Programme European Neuroscience Schools (PENS). Summer School: Advanced Courses in Neuroplasticity LUGAR DE CELEBRACION: Roma (Italia) AÑO: 2007 TIPO DE PARTICIPACION: Conferencia inaugural CONGRESO: 6º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC) LUGAR DE CELEBRACION: Cádiz AÑO: 2007 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Simposio IBRO. “Steroids and Central Nervous System”. Sociedad Argentina de Investigación en Neurociencias. XXII Reunión Anual. LUGAR DE CELEBRACION: Los Cocos, Córdoba (Argentina) AÑO: 2007 TIPO DE PARTICIPACION: Conferencia invitada

48

CONGRESO: Simposio “Mecanismos Innovadores en Neurociencias”. LII Reunión Científica de la Sociedad Argentina de Investigación Clínica. LUGAR DE CELEBRACION: Mar del Plata (Argentina) AÑO: 2007 TIPO DE PARTICIPACION: Conferencia Lanari CONGRESO: Conferencia de Clausura. LII Reunión Científica de la Sociedad Argentina de Investigación Clínica. LUGAR DE CELEBRACION: Mar del Plata (Argentina) AÑO: 2007 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 12 World Congress of Gynecological Endocrinology. LUGAR DE CELEBRACION: Florencia (Italia) AÑO: 2008 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 12 World Congress on the Menopause LUGAR DE CELEBRACION: Madrid AÑO: 2008 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Nobel Conference on “Recent Advances in Understanding Estrogen Signaling: From Molecular Insights to Clinical Implications” LUGAR DE CELEBRACION: Sånga Säby (Suecia) AÑO: 2008 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 18th International Symposium of the Journal of Steroid Biochemistry and Molecular Biology. “Recent Advances in Steroid Biochemistry and Molecular Biology” LUGAR DE CELEBRACION: Seefeld (Austria) AÑO: 2008 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 13th International Congress of Endocrinology LUGAR DE CELEBRACION: Rio de Janeiro (Brasil) AÑO: 2008 TIPO DE PARTICIPACION: Conferencia pleanria CONGRESO: 5th International Meeting Steroids and Nervous System LUGAR DE CELEBRACION: Turín (Italia) AÑO: 2009 TIPO DE PARTICIPACION: Dos conferencias pleanrias CONGRESO: Simposio: “Sistema Neuroendocrino: Fisiología Reproductiva, Patología y Envejecimiento” Centro Científico Tecnológico Mendoza. CONICET. LUGAR DE CELEBRACION: Mendoza (Argentina) AÑO: 2009 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Rapid Responses to Steroid Hormones 2009 LUGAR DE CELEBRACION: Elche AÑO: 2009 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Reunión conjunta de la Red Glial Española (RGE) y de la Red Glial Iberoamericana (RGIA) LUGAR DE CELEBRACION: Tarragona

49

AÑO: 2009 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: XIII National Congress of the Italian Society for Neuroscience. LUGAR DE CELEBRACION: Milán (Italia) AÑO: 2009 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: XIII Congreso Nacional Sociedad Española de Toxicomanías-Foro Internacional LUGAR DE CELEBRACION: Las Palmas de Gran Canaria AÑO: 2009 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Brain/Body Communication: The Endocrine Connection LUGAR DE CELEBRACION: Instituto Cervantes, Chicago (USA) AÑO: 2009 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 14th World Congress of Gynecological Endocrinology LUGAR DE CELEBRACION: Florencia (Italia) AÑO: 2010 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: World Summit on Interdisciplinary Aging Medicine LUGAR DE CELEBRACION: Monte-Carlo (Monaco) AÑO: 2010 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 12th European Congress of Endocrinology LUGAR DE CELEBRACION: Praga (República Checa) AÑO: 2010 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: International Behavioral Neuroscience Society (IBNS) 19th Annual Meeting LUGAR DE CELEBRACION: Villasimius, Cerdeña (Italia) AÑO: 2010 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: INRA Symposium Plasticity of Neuroendocrine Systems LUGAR DE CELEBRACION: Tours (Francia) AÑO: 2010 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: First international symposium of the journal: Hormone Molecular Biology and Clinical Investigation LUGAR DE CELEBRACION: Seefeld (Austria) AÑO: 2010 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 14th International Congress on Hormonal Steroids and Hormones & Cancer LUGAR DE CELEBRACION: Edinburgh (Reino Unido) AÑO: 2010 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 6th International Meeting Steroids and Nervous System LUGAR DE CELEBRACION: Turín (Italia) AÑO: 2011

50

TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 1st Yale-Cajal Joint symposium on Neurobiology LUGAR DE CELEBRACION: Madrid AÑO: 2011 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Spring Hippocampal Research Conference LUGAR DE CELEBRACION: Verona (Italia) AÑO: 2011 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: VI International Symposium on the Extra-Cellular Matrix LUGAR DE CELEBRACION: Búzios (Brasil) AÑO: 2011 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 10th European Meeting on Glial Cells in Health and Disease. LUGAR DE CELEBRACION: Praga (República Checa) AÑO: 2011 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Simposio Enfermedades Neurodegenerativas y Neuroplasticidad. XXXV Aniversario del Centro de Investigación Biomédica de Occidente del Instituto Mexicano del Seguro Social. LUGAR DE CELEBRACION: Guadalajara (México) AÑO: 2011 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Opening doors: Workshop on “The Biology of Ageing”. British Council/CSIC LUGAR DE CELEBRACION: Carmona, Sevilla AÑO: 2012 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 2012 Winter Meeting, Cajal Institute, CSIC. LUGAR DE CELEBRACION: Madrid AÑO: 2012 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Tne Endocrine Society 95th Annual Meeting LUGAR DE CELEBRACION: San Francisco (USA) AÑO: 2013 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: XLVIII Congresso Anual da Sociedade Brasileira de Fisiologia LUGAR DE CELEBRACION: Ribeirão Preto (Brasil) AÑO: 2013 TIPO DE PARTICIPACION: Conferencia plenaria CONGRESO: XLVIII Congresso Anual da Sociedade Brasileira de Fisiologia LUGAR DE CELEBRACION: Ribeirão Preto (Brasil) AÑO: 2013 TIPO DE PARTICIPACION: Mesa redonda CONGRESO: Spanish young neuroscientists symposium. 15 Congreso Nacional de la Sociedad Española de NeuroCiencia. LUGAR DE CELEBRACION: Oviedo AÑO: 2013

51

TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: Cajal at Yale, II Cajal-Yale Meeting. LUGAR DE CELEBRACION: New Haven (USA) AÑO: 2013 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 8th International Conference on Hormones, Brain and Behavior. LUGAR DE CELEBRACION: Lieja (Belgica) AÑO: 2014 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 27th Conference of European Comparative Endocrinologists LUGAR DE CELEBRACION: Rennes (Francia) AÑO: 2014 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 2nd International Workshop on Biomedical Research & Innovation LUGAR DE CELEBRACION: Vigo AÑO: 2014 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: III International Symposium Frontiers in Neuroscience. LUGAR DE CELEBRACION: Buzios (Brasil) AÑO: 2014 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: 8th International Meeting Steroids and Nervous System. LUGAR DE CELEBRACION: Turín (Italia) AÑO: 2015 TIPO DE PARTICIPACION: Conferencia invitada CONGRESO: I Workshop en Neurociencias. Universidad Europea de Madrid. LUGAR DE CELEBRACION: Villaviciosa de Odón, Madrid AÑO: 2015 SEMINARIOS Y OTRAS CONFERENCIAS INVITADAS (sin incluir presentaciones en congresos) I. NACIONALES 1984 MADRID. Seminario. Instituto Cajal. Invitado por el Dr. J. del Río. 1985 PALMA DE MALLORCA. Seminario. Departamento de Biofísica. Facultad de Ciencias. Universidad de las Islas Baleares. Invitado por el Dr. J.M. Gonzalez-Rós. 1985 MADRID. Seminario. Departamento de Psicobiología. Universidad Nacional de Educación a Distancia. Invitado por el Dr. A. Guillamón. 1986 ALICANTE. Seminario. Facultad de Medicina. Universidad de Alicante. Invitado por el Dr. C. Belmonte. 1986 VALENCIA. Seminario. Facultad de Ciencias. Universidad de Valencia. Invitado por el Dr. C. López-García. 1987 MADRID. Conferencia en el Curso Interuniversitario para Postrgraduados sobre Neurobiología. Fundación Ramón Areces. Invitado por el Dr. F. Valdivieso. 1987 SEVILLA. Seminario. Facultad de Biología. Universidad de Sevilla. Invitado por el Dr. J.M. Delgado García.

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1988 MADRID. Seminario. Departamento de Morfología. Facultad de Medicina. Universidad Autónoma de Madrid. Invitado por el Dr. C. Avendaño. 1988 MADRID. Conferencia en el II Curso Interuniversitario para Postgraduados sobre Neurobiología. Fundación Ramón Areces. Invitado por el Dr. F. Valdivieso. 1988 SANTANDER. Seminario. Departamento de Anatomía y Biología Celular. Facultad de Medicina. Universidad de Cantabria. Invitado por el Dr. M. Lafarga. 1989 MADRID. Seminario. Departamento de Biología Molecular. Facultad de Ciencias. Universidad Autónoma de Madrid. Invitado por la Dra. E. Bogonez. 1990 LA RABIDA. Conferencia-coloquio en el I Curso Nacional de Neurociencia. Universidad Hispanoamericana Santa María de la Rábida. Invitado por el Dr. J.M. Delgado García. 1990 BARCELONA. Conferencia en el Seminario de Técnicas de Inmunocitoquímica en Microscopía Electrónica. Facultad de Biología Univerrsidad de Barcelona. Invitado por el Dr. S. Vilaró. 1990 MALAGA. Conferencia en Cursos Abiertos de la Universidad de Málaga. Neurociencia. Invitado por el Dr. J.C. Dávila Cansino. 1990 ALICANTE. Conferencia en el Curso de Doctorado: Biomembranas. Departamento de Neuroquímica, Facultad de Medicina, Universidad de Alicante. Invitado por el Dr. J. Ferragut. 1990 ALICANTE. Seminario. Instituto de Neurociencias. Invitado por el Dr. J.M. González-Rós. 1990 MADRID. Seminario. Departamento de Morfología. Facultad de Medicina. Universidad Autónoma. Invitado por el Dr. J. Regadera. 1990 BILBAO. Seminario. Departamento de Neurociencias. Universidad del País Vasco. Invitado por el Dr. C. Matute. 1991 MADRID. Conferencia en el Curso de Doctorado sobre "Actualización en Endocrinología Pediátrica". Departamento de Pediatría. Facultad de Medicina. Universidad Autónoma. Invitado por el Dr. J. Argente. 1991 MADRID. Conferencia en el III Curso Interuniversitario para Postgraduados sobre Fundamentos Moleculares del Sistema Endocrino. Implicaciones Fisiopatológicas. Fundación Ramón Areces. Invitado por el Dr. E. Blázquez Fernández. 1991 SANTIAGO DE COMPOSTELA. Seminario. Departamento de Fisiología. Facultad de Medicina. Invitado por el Dr. C. Acuña. 1992 MADRID. Seminario. Museo Nacional de Ciencias Naturales. Invitado por el Dr. J. Rodrigo y la Asociación de Amigos del Museo Nacional de Ciencias Naturales. 1992 LA LAGUNA. Seminario. Departamento de Fisiología. Facultad de Medicina. Invitado por el Dr. M. Más. 1992 MADRID. Conferencia en el curso: Avances en Inmunocitoquímica y Técnicas Relacionadas. Organizado por el Departamento de Morfología de la Facultad de Medicina de la Universidad Autónoma de Madrid. Invitado por el Dr. L. Santamaría Solís. 1993 EL ESCORIAL. Cursos de Verano de la Universidad Complutense de Madrid. Participación en Mesa Redonda en el curso: Mecanismos Funcionales del Cerebro. Invitado por los Dres. C. Belmonte y F. Rubia. 1994 MURCIA. Seminario. Departamento de Oftalmología. Facultad de Medicina. Invitado por el Dr. M. Vidal-Sanz

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1994 MADRID. Seminario. Instituto de Investigaciones Biomédicas. C.S.I.C. Invitado por la Dra. M.A. Rodríguez Peña. 1994 EL ESCORIAL. Cursos de Verano de la Universidad Complutense de Madrid. Ponencia en el curso: Hormonas, Instintos y Emociones. Invitado por los Dres. J. Botella LLusiá y J.A.F. Tresguerres. 1995 MADRID. Seminario en el curso: Bases Moleculares de la Plasticidad Sináptica. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Invitado por el Dr. Javier Diez Guerra. 1995 LA LAGUNA. Seminario. Departamento de Anatomía, Facultad de Medicina. Universidad de La Laguna. Invitado por la Dra. M. del Mar Pérez-Delgado. 1996 OVIEDO. Seminario. Departamento de Psicología. Universidad de Oviedo. Invitado por el Dr. Jorge L. Arias. 1996 SALAMANCA. Seminario. Departamento de Anatomía. Facultad de Medicina. Universidad de Salamanca. Invitado por el Dr. J. Amat. 1996-2012 MADRID. Participación (2 horas/año) en la asignatura Desarrollo y Plasticidad del Sistema Nervioso (alumnos de 4º y 5º cursos, especialidad Neurobiología, licenciatura de Biología), Departamento de Biología Celular, Facultad de Biología, Universidad Complutense de Madrid (Profesora Responsable: Dra. Rosa Paz). 1996 JAEN. Ponencia en el Curso Sobre Inmunocitoquímica y Técnicas Relacionadas. Universidad de Jaén. Area de Biología Celular. Departamento de Biología Experimental y Ciencias de la Salud. Invitado por los Dres. M.A. Peinado, J. A. Pedrosa y J. Rodrigo. 1996 SANTANDER. Seminario. Departamento de Anatomía y Biología Celular. Facultad de Medicina. Universidad de Cantabria. Invitado por el Dr. M. Lafarga y la Dra. M. Berciano. 1996 MADRID. Seminario. Departamento de Fisiología. Facultad de Medicina, Universidad Complutense de Madrid. Invitado por la Dra. A. Colino. 1996 SEVILLA Conferencia en el XIII Curso sobre Perspectiva Actual de la Neurociencia. Departamento de Fisiología y Biología Animal, Facultad de Biología, Universidad de Sevilla. Invitado por el Dr. Blas Torres. 1997 MALAGA Conferencia en el Ciclo de Conferencias de Biología Celular y Molecular, Curso 1996-1997 del Programa de Doctorado ‘’Métodos experimentales en Biología Celular y Molecular’’ Facultad de Ciencias, Universidad de Málaga. Invitado por el Dr. Salvador Guirado. 1997 LA RABIDA Conferencia en la II Maestria de Neurociencia. Universidad Internacional de Andalucia. Sede Iberoamericana de La Rábida. Invitado por el Dr. J. M. Delgado García. 1999 MADRID Conferencia en el curso monográfico de doctorado ‘’Aproximación Clínica y Molecular al Crecimiento Humano Normal y Patológico’’. Departamento de Pediatría de la Universidad Autónoma de Madrid y Hospital Infantil ‘’Niño Jesús’’. Invitado por el Dr. Jesús Argente. 1999 SANTA CRUZ DE TENERIFE Conferencia en el curso: ‘’Presente y futuro de la investigación en biología: Beneficios económicos y científicos’’. Universidad Internacional Menéndez Pelayo. Invitado por el Dr. Pablo Martín-Vasallo. 2000 PAMPLONA. Seminario. Facultad de Medicina, Universidad de Navarra. Invitado por el Dr. José Manuel Giménez-Amaya. 2000 SEVILLA Conferencia en el programa de doctorado ‘’Neurociencia y Biología del Comportamiento’’ de la Universidad Pablo de Olavide. Laboratorio Andaluz de Biología. Invitado por la Dr. Agnès Gruart.

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2000 ALICANTE. Conferencia en el minisimposio: ‘’Avances en los mecanismos de señalización celular por esteroides sexuales’’. Universidad Miguel Hernandez. Instituto de Neurociencias-CSIC. Invitado por el Dr. Félix Viana. 2000 MADRID. Seminario. Departamento de Biología Celular y Molecular. Facultad de Medicina. Universidad Complutense. Invitado por el Dr. J. Javier Fernández Ruiz. 2000 SANTA CRUZ DE TENERIFE. Conferencia en el curso: ‘’Salud femenina en el próximo milenio’’. Universidad Internacional Menéndez Pelayo. Directores: Dr. G. Hernández y Dr. R. Alonso Solis. 2000 SEVILLA. Seminario en el Ciclo de Seminarios sobre ‘’Perspectiva de la fisiología en el nuevo milenio’’. Universidad de Sevilla. Invitado por la Dr. Rosario Pásaro. 2001 MADRID. Conferencia en el curso: ‘’Psicoanálisis y Biología’’Universidad Complutense de Madrid, Facultad de Biología. Invitador por la Dra. Rosario García Cordovilla. 2001 MADRID. Conferencia en el curso: ‘’Inteligencia y Emoción’’ CosmoCaixa, Fundación La Caixa, Invitado por el Dr. Alberto Ferrús. 2001 LLEIDA. Seminario. Departament de Cienccies Mediques Basiques, Universitat de Lleida. Invitado por el Dr. Joan X. Comella. 2001, 2002 MADRID Charla en el ciclo “Charlando con nuestros sabios” para escolares, organizado por el Museo Nacional de Ciencia y Tecnología. 2002 BARCELONA. Jornada de Seminarios Instituto de Neurociencias-Sociedad Española de Neurociencias. Facultad de Medicina, Universidad Autónoma de Barcelona. Invitado por el Dr. Lluis Ferrer. 2002 MALAGA. Seminario. Servicio de Neuroendocrinología y Nutrición. Hospital Civil de Málaga. Invitado por el Dr. Federico Soriguer. 2002 MADRID. Conferencia en el ciclo: Envejecimiento y Neurociencias. Circulo de Bellas Artes, Madrid. Invitado por la Dra. Jorgina Satrústegui Gil-Delgado. 2003 MADRID. Seminario. Instituto de Investigaciones Biomédicas Alberto Sols, CSIC. Invitado por la Dra. Isabel Varela. 2003 MADRID. Departamento de Farmacología, Facultad de Medicina, Univeridad Autónoma de Madrid. Invitado por el Dr. Dr. Antonio G. García. 2003 MADRID. Conferencia en el curso: ‘’Psicoanálisis y Biología’’Universidad Complutense de Madrid, Facultad de Biología. Invitador por la Dra. Rosario García Cordovilla. 2003 MADRID. Conferencia en el curso: ‘’Comportamiento Humano’’Universidad Complutense de Madrid, Facultad de Biología. Invitador por la Dra. Rosario García Cordovilla. 2003 MADRID. Seminario. Departamento de Morfología. Facultad de Medicina. Universidad Autónoma de Madrid. Invitado por los Dres. Carmen Cavada y Miguel Garzón. 2003 MADRID. Seminario. Departamento de Bioquímica, Facultad de Farmacia, Universidad Complutense de Madrid. Invitado por la Dra. María Jesús Oset. 2003 ALICANTE. Conferencia en el curso: “Temas Actuales en Neurocienxias”. Clínica Mediterránea de Neurociencias. Invitado por el Dr. Pedro Antón y el Dr. Juan Vicente Sánchez Andrés. 2003 ALBACETE. Seminario. Facultad de Medicina, Universidad de Castilla La Mancha. Invitado por el Dr. Valentín Ceña y el Dr. Pedro Tranque.

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2004 MADRID. Conferencia en el curso: ‘’Comportamiento Humano’’Universidad Complutense de Madrid, Facultad de Biología. Invitador por la Dra. Rosario García Cordovilla. 2004 MADRID. Conferencia en el curso de doctorado: ‘’Enfermedades neurodegenerativas’’. Departamento de Bioquímica, Facultad de Farmacia, Universidad Complutense de Madrid. Invitado por la Dra. María Jesús Oset. 2004 SANTANDER. Conferenciante y co-organizador del curso: “El cerebro mutable. Cambios del entorno y modificación del cerebro adulto”. Curso Magistral, Universidad Internacional Menéndez Pelayo. 2004 VÉLEZ-MÁLAGA. Conferenciante en el “Seminario de la Red de Investigación en Ciencias Neurológicas”. Invitado por el Dr. Fernando Rodríguez de Fonseca. 2005 MADRID. Conferencia en el curso de doctorado: ‘’Enfermedades neurodegenerativas’’. Departamento de Bioquímica, Facultad de Farmacia, Universidad Complutense de Madrid. Invitado por la Dra. María Jesús Oset. 2005 MADRID. Conferencia en el curso: “Prevención del envejecimiento y hormonas”. Facultad de Medicina, Universidad Complutense de Madrid. Coordinadores: Dr. Jesús A.F. Tresguerres y Dr. Julián Bayon Plaza. 2005 CÁDIZ. Seminario. Facultad de Medicina, Universidad de Cádiz. Invitado por la Dra. Cármen Estrada. 2006 MADRID. Conferencia en el curso de doctorado: ‘’Enfermedades neurodegenerativas’’. Departamento de Bioquímica, Facultad de Farmacia, Universidad Complutense de Madrid. Invitado por la Dra. María Jesús Oset. 2006 TOLEDO. Seminario. Hospital Nacional de Parapléjicos. Invitado por el Dr. Eduardo Molina-Holgado. 2006 OVIEDO. Seminario. Departamento de Psicología. Universidad de Oviedo. Invitado por el Dr. Jorge L. Arias. 2006 LEGANES (MADRID). Conferencia a alumnos del IES La Fortuna en el Acto de Conmemoración de la Concesión del Premio Nobel a Santiago Ramón y Cajal. Invitado por el equipo directivo del centro. 2006 SANTIAGO DE COMPOSTELA. Conferencia en el ciclo: “Homenaje a Santiago Ramón y Cajal”. Facultad de Biología, Universidad de Santiago de Compostela. Invitado por el Dr. Héctor Caruncho. 2006 EL ESCORIAL (MADRID). Conferencia en el Curso: La Corteza Cerebral: Aspectos Evolutivos, Cognición y Enfermedades Cerebrales. Cursos de Verano de la Universidad Complutense. Invitado por los Dres. Javier De Felipe y Antonio Gil-Nagel. 2006 MADRID Charla en el ciclo “Charlando con nuestros sabios” para escolares, organizado por el Museo Nacional de Ciencia y Tecnología. 2006 MADRID Conferencia en el Maratón Científico: Santiago Ramón y Cajal y la Neurociencia del Siglo XXI, Museo Nacional de Ciencia y Tecnología. 2006 MALAGA. Conferencia. La obra de Santiago Ramón y Cajal en el centenario del premio Nobel. Facultad de Ciencias, Universidad de Málaga. Invitado por el Dr. Salvador Guirado. 2006 A CORUÑA. Conferencia. V Encuentros Atlánticos de Neurociencia. Los Estados del Cerebro. Oleiros, A Coruña. Invitado por el Dr. F. Javier Cudeiro. 2007 MADRID. Conferencia en el curso de doctorado: ‘’Enfermedades neurodegenerativas’’. Departamento de Bioquímica, Facultad de Farmacia, Universidad Complutense de Madrid. Invitado por la Dra. María Jesús Oset. 2007 MADRID. Conferencia en el curso: ‘’III Jornadas de Encuentro Psicoanálsis y Biología’’Universidad Complutense de Madrid, Facultad de Biología. Invitador por la Dras. Silvia Pérez Galdós y Rosario García Cordovilla.

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2007 ZAMORA. Conferencias en la II Semana de la Ciencia. Museo Etnográfico de Castilla y León. 2008 MADRID. Conferencia en el curso de doctorado: ‘’Enfermedades neurodegenerativas’’. Departamento de Bioquímica, Facultad de Farmacia, Universidad Complutense de Madrid. Invitado por la Dra. María Jesús Oset. 2008 LA CORUÑA. Conferencia. Semana Internacional del Cerebro. Domus. 2008 LA LAGUNA. Seminario. Departamento de Fisiología, Facultad de Medicina, Universidad de La Laguna, Tenerife. Invitado por la Dra. Raquel Marín. 2008 SEVILLA. Seminario. Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER). Invitado por el Dr. Alfredo Rodríguez Tebar. 2008 VELEZ-MALAGA. Conferencia en el Curso: “Los Nuevos Desafíos de la Biología”. Cursos de Verano de la Universidad de Málaga. Invitado por el Dr. Pedro Fernández-Llebrez. 2008 MADRID. Charla en la Reunión CiberNed en el Instituto Cajal: Aplicaciones Terapéuticas de los factores tróficos. Instituto Cajal, CSIC. Invitado por el Dr. Ignacio Torres-Alemán 2008 BARCELONA. Conferencia en el curso: Dones vs Homes: Cervell i Intel·ligència. CosmoCaixa. 2008 LLEIDA. Conferencia en el ciclo “Els Vespres de la Ciència”. CaixaForum. 2008 BARCELONA. Seminario. Institut de Recerca, Hospital Universitari Vall d’Hebron. Invitado por el Dr. Juan Emilio Feliú. 2009 MADRID. Conferencia en el curso de doctorado: ‘’Enfermedades neurodegenerativas’’. Departamento de Bioquímica, Facultad de Farmacia, Universidad Complutense de Madrid. Invitado por la Dra. María Jesús Oset. 2009 MADRID. Seminario. Facultad de Biología. Universidad Complutense de Madrid. Invitado por la Dra. Paz Viveros. 2009. TOLEDO. Seminario. Hospital Nacional de Parapléjicos. Invitado por los Dres. Fernando de Castro Soubriet y Juan de los Reyes Aguilar. 2010. MADRID. Presentación al público en general. Semana Mundial del Cerebro. Instituto Cajal. 2010. ALICANTE. Conferencia en el curso: “Neurociencias y Sexología”. Instituto de Neurociencias. Invitado por el Dr. Salvador Martínez. 2011. MADRID. Seminario. Instituto de Investigaciones Sanitarias Fundación Jiménez Díaz. Invitado por la Dra. Marina Sanchez García. 2011. CIUDAD REAL. Conferencia Semana Mundial del Cerebro. Facultad de Medicina, Universidad de Castilla la Mancha. Invitado por la Dra. Inmaculada Ballesteros. 2011. LA LAGUNA. Seminario. Facultad de Biología. Universidad de La Laguna, Tenerife. Invitado por la Dra. Raquel Marín. 2011. MADRID. Conferencia en el curso: “Actualización en Neurorregulación y bases moleculares de la Pubertad”. Instituto de Investigaciones Sanitarias Fundación Jiménez Díaz. Invitado por las Dras. Carmen Ayuso y Mercedes Ruíz Moreno. 2011. SALAMANCA. Seminario. Instituto de Neurociencias de Castilla y León. Invitado por el Prof. Miguel Merchán.

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2011, 2012, 2013, 2015 MADRID. Conferencia en el Master de Neurociencia UAM-CSIC. Curso: Neurobiología del envejecimiento cerebral y las enfermedades del sistema nervioso. Instituto Cajal, CSIC. Invitado por la Dra. María Ceballos. 2012. MADRID. Conferencia en el Master de Bioquímica, Biología Molecular y Biomedicina. Universidad Complutense de Madrid. Invitado por el Prof. Javier Fernández-Ruiz. 2013, 2014. MADRID. Conferencia en el Master de Neurociencia UAM-CSIC, Curso de Neuroendocrinología, Facultad de Medicina de la UAM. Invitado por el Prof. Jesús Argente y la Dra. Julie A. Chowen. 2013. BILBAO. Seminario. Achucarro Basque Center for Neuroscience. Invitado por la Dra. Amanda Sierra. 2014. CÓRDOBA. Seminario. Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC). Invitado por el Prof. Manuel Tena-Sampere. 2014. LA LAGUNA. Seminario. Centro de Investigaciones Biomédicas de Canarias. Universidad de La Laguna. Invitado por el Prof. Rafael Alonso. 2015. VALENCIA. Seminario. Instituto de Biomedicina de Valencia, C.S.I.C. Invitado por la Dra. Nuria Flames.

II. EXTRANJERO 1978 GINEBRA (Suiza). Seminario. Institut d'Histologie. Ecole de Médecine. Universidad de Ginebra. Invitado por el Dr. L. Orci. 1981 GINEBRA (Suiza). Seminario. Departement de Physiologie. Ecole de Médecine. Universidad de Ginebra. Invitado por el Dr. C. Bader. 1981 LAUSANA (Suiza). Seminario. Institut d'Anatomie. Faculté de Médecine. Universidad de Lausana. Invitado por el Dr. H. Van der Loos. 1983 NEW HAVEN (USA). Seminario. Department of Obstetrics and Gynecology. Yale-New Haven Hospital. Invitado por el Dr. A.H. De Cherney. 1983 NEW HAVEN (USA). Seminario. School of Medicine. Yale University. Invitado por el Dr. F. Naftolin. 1983 GINEBRA (Suiza). Seminario. Département de Biochimie. Faculté de Sciences II. Universidad de Ginebra. Invitado por la Dra. A. Zurn. 1984 GINEBRA (Suiza). Seminario. Département de Pharmacologie. Centre Medical Universitaire. Universidad de Ginebra. Invitado por el Dr. D. Muller. 1986 GINEBRA (Suiza). Seminario. Département de Physiologie. Centre Médical Universitaire. Invitado por el Dr. J.J. Dreifuss. 1988 QUEBEC (Canada). Seminario. Centre de Recherches en Endocrinologie Moléculaire. Le Centre Hospitalier de l'Université Laval. Invitado por la Dra. T. Di Paolo. 1988 SANTA MARGHERITA LIGURE (Italia). Conferencia en la Autum School del European Training Programme in Brain and Behaviour Research. European Science Foundation. Invitado por el Dr. B. Droz. 1988 PARIS (Francia). Seminario. Faculte de Medecine Paris-Sud. Laboratoire de Hormones. Bicêtre. Invitado por el Dr. E.E. Baulieu.

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1988 ESTRASBURGO (Francia). Seminario. Centre de Neurochimie du C.N.R.S. Invitado por el Dr. J.P. Zanetta. 1991 LAUSANA (Suiza). Seminario. Institut de Physiologie. Faculté de Médecine. Universidad de Lausana. Invitado por el Dr. P. Magistretti. 1994 BURDEOS (Francia). Seminario. INSERM/Université de Bordeaux II. Invitado por la Dra. D. Theodosis. 1994 JOUY EN JOSAS (Francia). Seminario. Laboratoire de Physiologie Sensorielle. Institut National de la Recherche Agronomique. Invitado por el Dr. Jacques Servière. 1995 BOSTON (USA). Seminario. Department of Biology. Boston University. Invitado por el Dr. Michael Baum. 1995 PROVIDENCE (USA). Seminario. Department of Neuroscience, Brown University. Invitado por el Dr. M. A. Castro-Alamancos. 1995 SEEWIESEN (Alemania). Conferencia. International Summer School at the Max-Planck-Institut für Verhaltensphysiologie: Steroid hormone-dependent control of sexual behaviour: From behaviour to mechanisms. Invitado por el Dr. Manfred Gahr. 1996 MILAN (Italia) Seminario. Instituto de Endocrinologia, Universidad de Milan. Invitado por el Dr. Luciano Martini. 1997 ULM (Alemania) Seminario. Abteilung Anatomie und Zellbiologie, Universidad de Ulm. Invitado por el Dr. Christof Pilgrim. 1997 CAMBRIDGE (Reino Unido). Seminario. The Babraham Institute. Invitado por los Dres. R.J. Bicknell y J.B. Hutchison. 1998 NUEVA YORK (USA). Seminario. The Mount Sinai Medical Center, Mount Sinai School of Medicine of the City University of New York. Invitado por el Dr. J. L. Roberts. 1999 GUADALAJARA (México). Conferencia. ‘’Curso Internacional en Neurociencias Santiago Ramón y Cajal’’. Invitado por la Dra. M. Dueñas. 2000 AMERSTH (Massachusetts, USA). Seminario. Center for Neuroendocrine Studies. University of Massachusetts. Invitado por la Dra. Nancy G. Forger. 2000 CHICAGO (Illinois, USA). Seminario. Department of Cell Biology, Neurobiology and Anatomy. Loyola University Chicago, Stritch School of Medicine. Invitado por la Dra. Lydia DonCarlos. 2000 QUEBEC (Canada). Seminario. Centre de Recherche. Centre Hospitalier Universitaire de Québec. Invitado por la Dra. Thérèse Di Paolo. 2003 MÉXICO DF (México). Conferencia invitada. Curso Taller de Investigación en Medicina: Bases Experimentales y Bioética. Escuela Superior de Medicina, Instituto Politécnico Nacional. Invitado por el Dr. Ofir Picazo. 2004 SZEGED (Hungría). Seminario. Institute of Biophysiscs, Biological Research Center, Hungary Academy of Sciences. Invitado por el Dr. Arpad Parducz. 2004 BRUSELAS (Bélgica). Curso para estudiantes de doctorado (3 h): Role of hormones and growth factors in development, plasticity and repair of the CNS. Medical School, Free University of Brussels (VUB). Invitado por la Dra. Elisabeth Hooghe-Peters. 2005 BUENOS AIRES (Argentina). Seminario. Instituto de Biología y Medicina Experimental. Invitado por el Dr. Alejandro DeNicola.

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2005 SAN ANTONIO (Texas, USA). Seminario. Center for Biomedical Neuroscience and the Barshop Center for Aging. The University of Texas Health Science Center at San Antonio. Invitado por el Dr. James Roberts. 2005 BRUSELAS (Bélgica). Curso para estudiantes de doctorado (3 h): Role of hormones and growth factors in development, plasticity and repair of the CNS. Medical School, Free University of Brussels (VUB). Invitado por la Dra. Elisabeth Hooghe-Peters. 2006 ESTOCOLMO (Suecia). Seminario. Department of Biosciences and Nutrition and Clinical Research Centre, Karolinska Institute, Novum. Invitado por el Dr. J.A. Gustafsson. 2006 PARIS (Francia). Seminario. Institut National de la Sante et de la Recherche Medicale, Unite Mixte de Recherche 788. Invitado por el Dr. Michael Schumacher. 2006 ROMA (Italia). Seminario. Laboratory of Developmental Neuroscience and Neural Plasticity. Università Campus Bio-Medico di Roma. Invitado por el Dr. Flavio Keller. 2006 LA PLATA (Argentina). Seminario. Facultad de Medicina, Universidad Nacional de La Plata. Invitado por el Dr. Rodolfo Goya. 2006 BRUSELAS (Bélgica). Curso para estudiantes de doctorado (3 h): Role of hormones and growth factors in development, plasticity and repair of the CNS. Medical School, Free University of Brussels (VUB). Invitado por la Dra. Elisabeth Hooghe-Peters. 2007 LOS ANGELES (USA). Charles H. Sawyer Distinguished Lecture. Brain Research Institute, UCLA. Invitado por el Prof. Arthur P. Arnold. 2007 LOS ANGELES (USA). Seminario. Laboratory of Neuroendocrinology. UCLA. Invitado por el Prof. Arthur P. Arnold. 2008 SASSARI (Italia). Seminario. Dipartamento di Biologia Animale. Università degli Studi di Sassari. Invitado por el Prof. Gianluca Lepore. 2010 TORINO (Italia). Seminario. Dipartamento de Anatomia, Farmacologia e Medicina Legale. Invitado por el Prof. GianCarlo Panzica. 2010 MAGDEBURGO (Alemania). Seminario. Institute of Biology, Otto von Guericke University Magdeburg. Invitado por el Dr. Volker Korz. 2011 LAUSANA (Suiza). Seminario. École polytechnique Fédérale de Lausanne (EPFL). Invitado por la Dra. Carmen Sandi. 2011 SASSARI (Italia). Seminario. Dipartamento di Biologia Animale. Università degli Studi di Sassari. Invitado por el Prof. Gianluca Lepore. 2011 SAN JUAN (Puerto Rico). Seminario. Reciento de Ciencias Médicas, Universidad de Puerto Rico. Invitado por el Dr. Jorge Miranda González. 2011 RIO DE JANEIRO (Brasil). Seminario. Biomedical Sciences Institute. Federal University of Rio de Janeiro. Invitado por el Prof. Vivaldo Moura Neto. 2011 FORTALEZA (Brasil). Seminario. School of Medicine. Federal University of Ceara. Invitado por el Prof. Vivaldo Moura Neto. 2011 CRACOVIA (Polonia). Seminario. Institute of Pharmacology. Polish Academy of Sciences. Invitado por los profesores Władysław Lasoń y Krzysztof Wędzony.

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2011 AQUISGRÁN (Alemania). Seminario. Institute of Neuroanatomy. UKA-RWTH Aachen. Invitado por el Prof. Cordian Beyer. 2011 GUADALAJARA (México). Conferencia Magistral. XXII Semana de la Investigación Científica y Tecnológica, Centro Universitario de Ciencias Biológicas y Agropecuarias (CUCBA). Invitado por el Dr. S. Mena Munguía (Rector), Dr. E. Pimienta Barrios (Secretario Académico) y Dr. A. Feria y Velasco (Coordinador de Investigación). 2011 ROMA (Italia). Seminario. Dipartimento di Biologia, Uniniversitá Roma Tre. Invitado por la Prof. Maria Marino. 2012 HAMBURGO (Alemania). Seminario. Institute of Neuroanatomy, University Medical Center HamburgEppendorf. Invitado por la Prof. Gabriele M. Rune. 2012 LYON (Francia). Seminario. Centre de Recherche en Neurosciences de Lyon. Invitado por el Prof. Jérôme Honnorat. 2012 ROTTERDAM (Holanda). Erasmus Lecture on Endocrinology. Erasmus Medical Center. Invitado por el Prof. Joseph Janssen. 2013 NUEVA YORK (EEUU). Seminario. Departments of Environmental Medicine and Obstetrics and Gynecology, New York University Langone Medical Center. Invitado por el Prof. Frederick Naftolin. 2014 BOGOTÁ (Colombia). Seminario. Facultad de Ciencias. Universidad Pontificia Javeriana. Invitado por el Prof. George Barreto. 2014 BOGOTÁ (Colombia). Seminario. Instituto de Genética. Universidad Nacional de Colombia. Invitado por el Prof. Gonzalo Humberto Arboleda. 2014 MEDELLÍN (Colombia). Seminario. Facultad de Medicina. Universidad de Antioquia. Invitado por la Prof. Patricia Cardona Gómez. 2014 RIO DE JANEIRO (Brasil). Seminario. Institute of Biomedical Sciences. Universidad Federal de Rio de Janeiro. Invitado por el Dr. Mychael Vinicius Lourenco. 5

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Tesis Doctorales dirigidas

TITULO: Acciones de las hormonas gonadales sobre la membrana neuronal y las sinapsis del núcleo arcuato de la rata. DOCTORANDO: Gabriel OLMOS BONAFE UNIVERSIDAD: Complutense de Madrid FACULTAD/ESCUELA: Ciencias Biológicas AÑO: 1988 CALIFICACION: Apto cum laude TITULO: Acciones de las hormonas sexuales sobre el citoesqueleto de los astrocitos. DOCTORANDO: Pedro TRANQUE GOMEZ UNIVERSIDAD: Complutense de Madrid FACULTAD/ESCUELA: Ciencias Biológicas AÑO: 1989 CALIFICACION: Apto cum laude TITULO: Estudio de la distribución de los poros nucleares en las neuronas y células gliales del sistema nervioso central de la rata DOCTORANDO: Pedro HERNANDEZ ACOSTA UNIVERSIDAD: Complutense de Madrid FACULTAD/ESCUELA: Ciencias Biológicas AÑO: 1991 CALIFICACION: Apto cum laude TITULO: Mecanismos celulares de sinaptogénesis y plasticidad sináptica en el núcleo arcuato del hipotálamo. DOCTORANDO: Julio PEREZ MARQUEZ UNIVERSIDAD: Complutense de Madrid FACULTAD/ESCUELA: Ciencias Biológicas AÑO: 1991 CALIFICACION: Apto cum laude TITULO: Diferencias sexuales en la estructura de la corteza cerebral de la rata. Acción de las hormonas esteroides gonadales. DOCTORANDO: José Antonio MUÑOZ CUETO UNIVERSIDAD: Complutense de Madrid FACULTAD/ESCUELA: Ciencias Biológicas AÑO: 1992 CALIFICACION: Apto cum laude (Codirigida con A. Ruiz Marcos) TITULO: Sistemas transportadores de calcio en organulos intracelulares de cerebro de rata: efectos de la edad e implicaciones en la pérdida de memoria asociada al envejecimiento. DOCTORANDO: Pablo BLANCO MORALES UNIVERSIDAD: Autónoma de Madrid FACULTAD/ESCUELA: Ciencias AÑO: 1992 CALIFICACION: Apto cum laude (Codirigida con J. Satrústegui Gil-Delgado) TITULO: Influencia de hormonas gonadales y neuroesteroides sobre la reorganización del tejido cerebral tras lesión. DOCTORANDO: Joaquín GARCIA ESTRADA UNIVERSIDAD: Complutense de Madrid

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FACULTAD/ESCUELA: Ciencias Biológicas AÑO: 1995 CALIFICACION: Apto cum laude (Codirigida con I. Torres Alemán) TITULO: Hormonas gonadales y plasticidad neuroglial en la rata adulta. DOCTORANDO: Sonia LUQUIN de ANDA UNIVERSIDAD: Complutense de Madrid FACULTAD/ESCUELA: Ciencias Biológicas AÑO: 1995 CALIFICACION: Apto cum laude TITULO: La glia y su papel en la regulación neuroendocrina. Interacción entre el factor de crecimiento semejante a insulina tipo-1 y las hormonas gonadales. DOCTORANDO: Marcela DUEÑAS JIMENEZ UNIVERSIDAD: Complutense de Madrid FACULTAD/ESCUELA: Ciencias Biológicas AÑO: 1995 CALIFICACION: Apto cum laude (Codirigida con I. Torres Alemán) TITULO: Influencia del factor de crecimiento similar a insulina tipo I (IGF-I) en el cerebelo de rata. DOCTORANDO: María Paz NIETO BONA UNIVERSIDAD: Complutense de Madrid FACULTAD/ESCUELA: Ciencias Biológicas AÑO: 1997 CALIFICACION: Apto cum laude (Codirigida con I. Torres Alemán) TITULO: Mecanismos celulares y moleculares de interacción in vivo entre el estrógeno y el factor de crecimiento parecido a insulina tipo I (IGF-I) en el cerebro de rata DOCTORANDO: Patricia CARDONA GÓMEZ UNIVERSIDAD: Autónoma de Madrid FACULTAD/ESCUELA: Ciencias Biológicas AÑO: 2001 CALIFICACION: Sobresaliente cum laude TITULO: Neuroesteroidogénesis y supervivencia neuronal DOCTORANDO: Amanda SIERRA SAAVEDRA UNIVERSIDAD: Complutense de Madrid FACULTAD/ESCUELA: Ciencias Biológicas AÑO: 2003 CALIFICACION: Sobresaliente cum laude TITULO: La glía reactiva como diana de andrógenos y estrógenos DOCTORANDO: Daniel GARCÍA OVEJERO UNIVERSIDAD: Complutense de Madrid FACULTAD/ESCUELA: Ciencias Biológicas AÑO: 2004 CALIFICACION: Sobresaliente cum laude TITULO: Mecanismos moleculares de interacción entre estrógenos y el factor de crecimiento similar a la insulina de tipo I (IGF-I) DOCTORANDO: Pablo MÉNDEZ GARCÍA UNIVERSIDAD: Autónoma de Madrid FACULTAD/ESCUELA: Ciencias AÑO: 2005

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CALIFICACION: Sobresaliente cum laude TITULO: Acciones neuroprotectoras de la progesterona, el estradiol y los moduladores selectivos de los receptores de estrógenos DOCTORANDO: Iratxe CIRIZA EREÑO UNIVERSIDAD: Complutense de Madrid FACULTAD/ESCUELA: Ciencias Biológicas AÑO: 2006 CALIFICACION: Sobresaliente cum laude TITULO: Expresión y regulación transcripcional de la aromatasa en el sistema nervioso en condiciones normales y patológicas DOCTORANDO: Josué GARCÍA YAGÜE UNIVERSIDAD: Autónoma de Madrid FACULTAD/ESCUELA: Medicina AÑO: 2008 CALIFICACION: Sobresaliente cum laude (Coodirigida con I. Azcoitia) TITULO: Regulación por andrógenos y estrógenos de la gliosis tras lesiones cerebrales en la rata DOCTORANDO: George Emilio Sampaio BARRETO UNIVERSIDAD: Complutense de Madrid FACULTAD/ESCUELA: Ciencias Biológicas AÑO: 2008 CALIFICACION: Sobresaliente cum laude (Coodirigida con I. Azcoitia) TITULO: Regulación por la ovariectomía, la síntesis local de estradiol y la terapia hormonal, del estado de activación de quinasas implicadas en señalización intracelular, en el cerebro de rata DOCTORANDO: Amaya SANZ RODRÍGUEZ UNIVERSIDAD: Autónoma de Madrid FACULTAD/ESCUELA: Medicina AÑO: 2009 CALIFICACION: Sobresaliente cum laude TITULO: Efecto de los mediadores metabólicos leptina, IL-6 y estradiol en la activación microglial e implicación en la homeostasis energética en diferentes modelos de inflamación sistémica crónica DOCTORANDO: Silvia TAPIA GONZÁLEZ UNIVERSIDAD: Autónoma de Madrid FACULTAD/ESCUELA: Medicina AÑO: 2010 CALIFICACION: Sobresaliente cum laude (Codirigida con J. Chowen) TITULO: Efectos emocionales y cognitivos de la privación de hormonas gonadales en roedores DOCTORANDO: Luisa Natalia LAGUNAS GARCÍA UNIVERSIDAD: Complutense de Madrid FACULTAD/ESCUELA: Psicología AÑO: 2011 CALIFICACION: Sobresaliente cum laude TITULO: Dimorfismo sexual y efecto de las hormonas ováricas y moduladores selectivos de los receptores de estrógeno en modelos de trastornos afectivos y psicóticos en roedores. DOCTORANDO: Isabel María CALMARZA FONT UNIVERSIDAD: Complutense de Madrid FACULTAD/ESCUELA: Biología AÑO: 2011

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CALIFICACION: Sobresaliente cum laude TITULO: La respuesta glial a estímulos inflamatorios: diferencias sexuales y regulación por moduladores de la esteroidogénesis y compuestos estrogénicos. DOCTORANDO: María SANTOS GALINDO UNIVERSIDAD: Autónoma de Madrid FACULTAD/ESCUELA: Medicina AÑO: 2012 CALIFICACION: Sobresaliente cum laude TITULO: Diferencias sexuales y regulación de la ruta de Notch durante la gliosis reactiva. DOCTORANDO: Estefanía Acaz Fonseca. UNIVERSIDAD: Autónoma de Madrid FACULTAD/ESCUELA: Ciencias AÑO: 2015 CALIFICACION: Sobresaliente cum laude

6

Participación en comités y representaciones internacionales

Título del Comité: International Scientific Committee of the European Training Program in Brain and Behavior Research Entidad de la que depende: European Science Foundation Tema: Neurociencia Fecha: 1987-1991 Título del Comité: Council of the Federation of European Neuroscience Societies Entidad de la que depende: Federation of European Neuroscience Societies Tema: Neurociencia Fecha: 2001-2003 Título del Comité: Governing Council of the International Brain Research Organization (IBRO) Entidad de la que depende: International Brain Research Organization Tema: Neurociencia Fecha: 2002-2003 Título del Comité: International Scientific Advisory Board of the Biological Research Center, Szeged, Hungary Entidad de la que depende: Hungarian Academy of Sciences Tema: Neurociencia Fecha: 2004 Título del Comité: Comité de evaluación externo del Instituto de Investigación Médica Mercedes y Martín Ferreyra (INIMEC), Córdoba, Argentina.

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Entidad de la que depende: Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina Tema: Neurociencia Fecha: 2010 Título del Comité: Scientific Advisory Board of GIGA-Neurosciences Entidad de la que depende: Liege University, Belgium Tema: Neurociencia Fecha: 2011-ACTUAL Título del Comité: CIMUS External Advisory Board Entidad de la que depende: Centro Singular de Investigación en Medicina Molecular y Enferemedades Crónicas, Universidad de Santiago de Compostela Tema: Biomedicina Fecha: 2014-ACTUAL

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Experiencia en organización de actividades de I+D Organización de congresos, seminarios, jornadas, etc., científicos-tecnológicos Título: Tipo de actividad:

Ambito:

Fecha: Título: Tipo de actividad:

Ambito:

Fecha:

Organizador. Mesa redonda. Plasticidad y Desarrollo Neuronales. II Congreso de la Sociedad Española de Neurociencia. Barcelona, 1987 Miembro del Comité Organizador. International Conference Ramón y Cajal. Madrid, 1990 Miembro del Comité Organizador. First Swedish-Spanish Symposium "Frontiers in Neurobiology". Estocolmo (Suecia), 1991 Miembro del Comité Organizador. Primera reunión CSIC-INSERM en Neurociencias. Madrid, 1992 Miembro del Comité Científico Internacional. International Workshop on What Steroid Hormones Tell the Brain. Puerto de la Cruz, Tenerife, 1994 Miembro del Comité Organizador. Similarities and interactions between reproductive endocrinology and neuroscience roundtable workshop. Satellite symposium to the Xth World Congress of Psychiatry. Madrid, 1996 Miembro del Comité Científico Internacional. Third European Meeting on Glial Cell Function in Health and Disease. Atenas, Grecia 1998 Miembro del Comité Organizador. Joint meeting of the Sixth International Conference on Hormones, Brain and Behavior and the Society for Behavioral Neuroendocrinology. Madrid, 2000. Miembro del Comité Científico Internacional. International meeting: Steroids and nervous system.Turín, Italia, 2001 Organizador. Symposium on: Sex steroids and the brain: new signaling, new functions. Instituto Cajal, Madrid, 2001 Organizador. Workshop. Steroid hormones in regeneration, plasticity and protection. V European Meeting on Glial Cell Function in Health and Disease, Roma, Italia, 2002 Miembro del Comité Científico Internacional (International Program Organizing Committee) International Congress on Hormonal Steroids and Hormones and Cancer. Fukuoka, Japón, 2002. Miembro del Comité Científico Internacional. 2nd International Meeting on: "Steroids and Nervous System" Turín, Italia, 2003. Organizador. Symposium. Novel mechanisms of estrogen signalling. XXXII Congress of the Spanish Society of Physiological Sciences. International Joint Meeting with the Physiological Society. Puerto de la Cruz, Tenerife, 2003. Co-organizador. Mesa Redonda. Fronteras entre la Neurología y la Neurociencia básica en España. XXXII Congreso de la Sociedad Española de Fisiología. Puerto de la Cruz, Tenerife, 2003. 67

Organizador. Symposium. New cellular localizations and functions of steroid hormone receptors in the brain. XXXIII Annual Congress of the International Society of Psychoneuroendocrinology. Pisa, Italia, 2003. Co-organizador. Symposium. Neuroactive steroids: A clue for the identification of new pharmacological treatments for neurodegeneration. Sixth IBRO World Congress of Neuroscience. Prague, Czech Republic, 2003. Organizador. Symposium. Control of brain development and plasticity by sex hormones. 4th Forum of European Neuroscience. Lisboa, Portugal, 2004. Co-organizador del curso: “El cerebro mutable. Cambios del entorno y modificación del cerebro adulto”. Curso Magistral, Universidad Internacional Menéndez Pelayo, Santander, 2004. Organizador. Short Course in Unbiased Stereology: "Applications Neural Systems". Instituto Cajal, Madrid, 2004

of

Unbiased

Stereology To

Miembro del Comité Científico Internacional. 3rd International Meeting on: "Steroids and Nervous System" Turín, Italia, 2005. Co-organizador. Symposium. “Body-brain communication: the endocrine loop”. Instituto Cajal, Madrid, 2005. Co-organizador. Sympoium. “Comunicación cuerpo-cerebro: nuevos avances en neuroendocrinología”. XI Congreso de la Sociedad Española de Neurociencia. Torremolinos, Málaga, 2005. Co-organizador. International Symposium: “Glial cells as key elements for brain function, protection and regeneration”. Fundación Ramón Areces, Madrid, 2005. Miembro del Comité Científico Internacional. 4th International Meeting on: "Steroids and Nervous System" Turín, Italia, 2007. Co-organizador. Symposium. “Regulation of glial function by steroids”. IBRO World Congress of Neuroscience. Melbourne, Australia, 2007. Presidente del Comité Organizador. IV Reunión de la Red Glial Española. Madrid, 2008. Miembro del Comité Científico Internacional. 5th International Meeting on: "Steroids and Nervous System" Turín, Italia, 2009. Co-organizador. Stellite Symposium. Gender-Specific Therapeutic Strategies Based on Neuroactive Steroids: In Search for Innovative Tools for Neuroprotection. 5th International Meeting on: "Steroids and Nervous System" Turín, Italia, 2009. Presidente del Comité Científico. XIII Congreso de la Sociedad Española de Neurociencia, Tarragona, 2009. Co-organizador: Symposium. Estrogen signaling in brain and neuroinflammatory cells. XIII National Congress of the Italian Society for Neuroscience. Milán, Italia, 2009. Miembro del Programme Organising Committee. International Congress of Neuroendocrinology. Rouen, Francia, 2010. Miembro del comité de programas de la Sociedad Española de Neurociencia, 2010-2013. Presidente del Comité Científico. V Reunión de la Red Glial Española, Málaga, 2010. Miembro del Comité Científico Internacional. 6th International Meeting Steroids and Nervous System. Turín, Italia, 2011. 68

Co-organizador. Neurocomplutum. 1ª Jornada de Neurociencia de la UCM en el Instituto Cajal. Madrid, 2012. Co-organizador. Symposium “Frontiers in Glial Research” Satellite symposium of the 8th FENS Forum of Neuroscience, Barcelona, 2012. Miembro del Comité Científico Internacional. 7th International Meeting Steroids and Nervous System. Turín, Italia, 2013. Miembro del Comité Científico Internacional. 8th International Meeting Steroids and Nervous System. Turín, Italia, 2015.

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Experiencia de gestión de I+D Gestión de programas, planes y acciones de I+D Título: Tipo de actividad: Fecha: Título: Tipo de actividad: Fecha:

#1986-1990. Coordinador del Programa de Neurociencias del CSIC #1988-1990. Secretario, Instituto Cajal, CSIC, Madrid. #1991. Director, Instituto Cajal, CSIC, Madrid. #1994-1996. Coordinador Científico (area de Fisiología), Agencia Nacional de Evaluación y Prospectiva (ANEP), Madrid. #1997-2004 Miembro de la Comisión de Area de Biomedicina, CSIC. #2000. Coordinador Científico, Consejería de Educación, Comunidad de Madrid. #2003. Miembro del panel de expertos en el área de Farmacología/Fisiologia y Medicina de la Agencia Valenciana de Ciencia y Tecnologia. #2003. Vocal de la Comisión de Selección de Proyectos de Investigación del Programa Nacional de Promoción General del Conocimiento. Area de Fisiología. #2006-2007 Jefe del Departamento de Plasticidad Neural, Instituto Cajal, CSIC, Madrid. #2007-2013 Jefe del Departamento de Neurobiología Funcional y de Sistemas, Instituto Cajal, CSIC, Madrid. #2012- Colaborador de la Subdirección General de Proyectos de Investigación, Departamento Técnico de Ciencias de la Vida y Agroalimentación, Area de Biología Fundamental: Biología Integrativa y Fisiología, Ministerio de Economía y Competitividad.

7

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Otros méritos o aclaraciones que se desee hacer constar (utilice únicamente el espacio equivalente a una página).

ACTIVIDAD EDITORIAL Miembro del comité editorial de: - European Journal of Anatomy (1996-) - Gynecological Endocrinology (1997-2014) - Endocrinology (2005-2008) (2015-) - Brain Research (Reviewing Editor) (2007-) - Brain Research Reviews (2007-) - Investigación y Ciencia de la Universidad Autónoma de Aguas Calientes, México (2007-) - Glia (2009-) - Hormone Molecular Biology and Clinical Investigation (2009-) - Frontiers in Neuroendocrine Science (2010-) - Journal of Neuroendocrinology (2010-) - Journal of Chemical Neuroanatomy (2011-2015) - Frontiers in Neuroendocrinology (2011-) - Frontiers in Systems and Translational Endocrinology (2014-) Guest Editor: Jordan C.L. and Garcia-Segura, L.M. Special Issue. Glia and Steroids. Journal of Neurobiology 40 (4), 1999 Garcia-Segura, L.M. and Wise, P.M. Special Issue. Neuroprotection by steroids: New perspectives. Journal of Neurocytology 29 (5/6), 2000 McCarthy, M.M., Guillamón, A., Garcia-Segura, L.M., Segovia, S. Special Issue. ICHB&B/SBN Proceedings. Hormones and Behavior 40 (2), 2001 Garcia-Segura LM. Special issue: Steroids and Neuroprotection. Frontiers in Neuroendocrinology 30 (2), 2009 Melcangi RC, Garcia-Segura LM, Panzica G. Special issue: Neuroactive steroids: Focus in the human brain. Neuroscience 191, 2011 Garcia-Segura L.M., De Nicola A.F. Special Issue. Hormones and the brain. Hormone Molecular Biology and Clinical Investigation 7, 2011 Beyer, C. and Garcia-Segura L. Special Isuue: Sex steroids and brain disorders. Journal of Steroid Biochemistry and Molecular Biology 146:1-80, 2015 PORTADAS DE REVISTAS Cell 36 (1984); Journal Comparative Neurology 303 (1991); Journal of Neurocytology 20 (1991); Neuroendocrinology 57 (1993); Neuroendocrinology 59 (1994); Journal of Neurocytology 27 (1998); European Journal of Anatomy 2 (1999); Neuroscience 138 (2006) OTRAS ACTIVIDADES CIENTÍFICAS/ADMINISTRATIVAS #1984, Research Associate, Department OB/GYN, Yale University School of Medicine, New Haven, CT, USA. #1988, 1991, Visiting Professor, Yale University School of Medicine, Department of Obstetrics and Gynecology, New Haven, CT, USA. #1996- Miembro del Patronato de la Fundación Endocrinología y Nutrición. 71

#1997, Visiting Professor, Babraham Institute and St. John’s College, Cambridge, United Kingdom. #1999-2001 Presidente Electo de la Sociedad Española de Neurociencia #2001- 2003 Presidente Ejecutivo de la Sociedad Española de Neurociencia #2008- Coordinador de la Red Glial Española #2010- Professor Adjunct in the Section of Comparative Medicine,Yale University School of Medicine, New Haven, CT USA #2011, Visiting Professor. Dipartamento di Biologia Animale. Università degli Studi di Sassari, Italy.

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CURRÍCULUM ABREVIADO (CVA) Lea detenidamente las instrucciones que figuran al final de este documento para rellenar correctamente el CVA

06/07/2014 Fecha del CVA Parte A. DATOS PERSONALES Nombre y apellidos RAFAEL LUJAN MIRAS DNI/NIE/pasaporte 27519894-A Edad 48 Researcher ID G-2555-2015 Núm. identificación del investigador 0000-0003-2001-9545 Código Orcid A.1. Situación profesional actual Organismo UNIVERSIDAD DE CASTILLA-LA MANCHA Dpto./Centro Facultad de Medicina, Departamento de Ciencias Médicas Dirección Campus Biosanitario, Avenida de Almansa, 14, 02006 Albacete Teléfono 689 012255 correo electrónico [email protected] Categoría profesional CATEDRATICO DE UNIVERSIDAD Fecha inicio 04/12/2009 Espec. cód. UNESCO NEUROCIENCIAS Receptores, canales iónicos, microscopía electrónica, Palabras clave inmunohistoquímica, hipocampo, cerebelo, neuroanatomía A.2. Formación académica (título, institución, fecha) Licenciatura/Grado/Doctorado Universidad LICENCIADO EN BIOLOGIA UNIVERSIDAD DE GRANADA DOCTOR EN BIOLOGIA UNIVERSIDAD DE GRANADA

Año 1990 30/06/1993

A.3. Indicadores generales de calidad de la producción científica (véanse instrucciones) - Tres sexenios de investigación de tres posibles (último 2005-2010). - Tesis dirigidas en los últimos 10 años: cinco. - Nº artículos indexados publicados en los últimos 5 años (2010-2014): 47. - Nº de artículos en los últimos 5 años en primer cuartil (Q1): 35. - Nº de artículos en los últimos 5 años en primer decil (D1): 23. - Nº de artículos en los últimos 5 años en segundo cuartil (Q2): 8. - Índice de impacto acumulado en los últimos 5 años: 387,450 (Índice de impacto medio: 8,244). - Nº artículos totales indexados publicados en revistas internacionales: 119. - Citas totales: 6.008 (Cita media por artículo: 49,65) - Citas/año en los últimos 5 años: 575 (2010), 572 (2011), 667 (2012), 617 (2013), 599 (2014). - Promedio citas/año en los últimos 5 años (2010-2014): 606 citas/año. - Índice h: 37 Parte B. RESUMEN LIBRE DEL CURRÍCULUM (máximo 3500 caracteres) Rafael Luján was born on 9th April 1967 in Almería, Spain. In 1985, he was admitted to the Biology School, University of Granada, Spain, to study biological sciences. Following his graduation in 1990, he enrolled to the Department of Cell Biology as a graduate student. His aim was to understand how administration of ethanol affects the number and structure of thalamic nerve cells. He completed his thesis in 1993 and received Ph.D. From 1993 to 1997 he was a Postdoctoral Research Fellow in Peter Somogyi’s lab at University of Oxford, where he learned high-resolution immunoelectron microscopy. His aim was to establish the molecular architecture of central synapses. During these years, he developed novel quantitative, immunolabeling EM techniques to reveal the precise subcellular location of neurotransmitter receptors and to tell how many of them are segregated in certain synapses. In 1998, Dr Luján returned to Spain and he spent two years at the Instituto de Neurociencias, Alicante, Spain. In 2000, he moved to Albacete to establish his independent research group in the Medicine School, Universidad de Castilla-La Mancha, where he setup the Laboratory of Synaptic Structure, and at present he is full Professor of Histology at this University. In 2008 and 2010, he spent several months in Shigemoto’s lab at NIPS, Japan, where he learned SDS-FRL techniques. Dr. Luján´s research interest focuses on unravelling several aspects of the neuronal functional structure with particular emphasis in dissecting the molecular cytology of neurons through the analysis of cellular and subcellular localization of receptors and ion channels. This knowledge is crucially important to understand the basic mechanisms by which the brain functions and, therefore, the consequences deriving from its dysfunction under pathological conditions. In particular, the main 1 de 4 / Currículum abreviado

 

CURRÍCULUM ABREVIADO (CVA) Lea detenidamente las instrucciones que figuran al final de este documento para rellenar correctamente el CVA

emphasis of the research in the laboratory is on the fundamental principles that regulate the cellular and subcellular localization taking place in neuronal signalling processes in the brain, specially focused in understanding how neurotransmission mediated by neurotransmitter receptors and ion channels occurs in normal and pathological conditions. The methodology of the laboratory is based in the use of immunohistochemical techniques at the light microscopic level, and specifically highresolution immunohistochemical techniques at the electron microscopic level, combined with quantitative analysis and 3D reconstruction of immunolabelling. More recently, we have introduced two newly developed cutting-edge methodologies (SDS-FRL and FIB/SEM immunogold techniques), which are considered to be the two best immunoelectron microscopic techniques to study the subcellular localization of receptors and ion channels at a level of detail never previously attained. Actually, the Synaptic Structure Laboratory is the only worldwide so far applying FIB/SEM to immunogold electron microscopy. As a result of the use of this new technology, we have been granted with a research project of the European Union in the context of the ramp-up phase (01/04/2014 30/03/2016) of the Human Brain Project. Our high-resolution ultrastructural efforts have revealed that receptors and ion channels are not uniformly distributed along the neuronal surface and each of them has a specific spatial distribution fingerprint. Recently, we have revealed a cell-to-cell heterogeneity in content, and clustering of GIRK and SK channels along the axo-somato-dendritic plasma membranes of hippocampal neurons. Ongoing efforts are seeking to: 1) reveal the structural, molecular and functional heterogeneity of central synapses, 2) determine the subcellular localization of neurotransmitter receptors and ion channels along the neuronal surface, 3) provide the morphological evidence for the interaction and functional coupling between neurotransmitter receptors, effector ion channels and associated signalling molecules in specific subcellular compartments, and 4) define the molecular and cellular mechanisms underlying pathological conditions in neuronal signalling. Parte C. MÉRITOS MÁS RELEVANTES (ordenados por tipología) C.1. Publicaciones 1- Ballesteros-Merino C, Martínez-Hernández J, Aguado C, Watanabe M, Adelman JP, Luján R. (2014). Localization of SK2 channels relative to excitatory synaptic sites in the mouse developing Purkinje cells. Frontiers in Neuroanatomy 8:154. 2- García-Negredo G, Soto D, Llorente J, Morató X, Galenkamp KM, Gómez-Soler M, FernándezDueñas V, Watanabe M, Adelman JP, Shigemoto R, Fukazawa Y, Luján R*, Ciruela F*. (2014). Coassembly and coupling of SK2 channels and mGlu5 receptors. Journal of Neuroscience 34(44):14793-802. 3- Ballesteros-Merino C, Watanabe M, Shigemoto R, Fukazawa Y, Adelman JP, Luján R. (2014). Differential subcellular localization of SK3-containing channels in the hippocampus. European Journal of Neuroscience 39(6):883-92. 4- Luján R, Marron Fernández de Velasco E, Aguado C, Wickman K. (2014). New Insights into the therapeutic potential of GIRK channels. Trends in Neuroscience 37(1):20-9, 2014. 5- Fajardo-Serrano A, Wydeven N, Young D, Watanabe M, Shigemoto R, Martemyanov KA, Wickman K, Luján R. (2013). Association of Rgs7/Gβ5 complexes with girk channels and GABAB receptors in hippocampal CA1 pyramidal neurons. Hippocampus 23(12):1231-45. 6- Ballesteros-Merino C, Lin M, Wu WW, Ferrandiz-Huertas C, Cabañero MJ, Watanabe M, Fukazawa Y, Shigemoto R, Maylie J, Adelman JP, Luján R. (2012). Developmental profile of SK2 channel expression and function in CA1 neurons. Hippocampus 22(6):1467-80. 7- Allen D*, Bond CT*, Luján R*, Ballesteros-Merino C, Lin MT, Wang K, Klett N, Watanabe M, Shigemoto R, Stackman RW Jr, Maylie J, Adelman JP. (2011). The SK2-long isoform directs synaptic localization and function of SK2-containing channels. Nature Neuroscience 14(6):744-749. 8- Lin MT*, Luján R*, Watanabe M, Frerking M, Maylie J, Adelman JP. (2010). Coupled activitydependent trafficking of synaptic SK2 channels and AMPA receptors. Journal of Neuroscience 30(35):11726-34. 9- Lin MT*, Lujan R*, Watanabe M, Adelman JP and Maylie J. (2008). SK channel plasticity contributes to LTP at Shaffer Collateral–CA1 synapses. Nature Neuroscience 11(2):170-7.

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10- Luján R, Maylie J, Adelman JP. (2009). New sites of action for GIRK and SK channels. Nature Reviews Neuroscience 10(7):475-80. C.2. Proyectos 1- MICINN, BFU2006-01896/BFI. “Bases celulares y subcelulares que median la interacción entre los canales de potasio GIRK y los receptores de GABAB en el cerebelo”. Investigadores: Rafael Luján (IP). Financiación: 117.000 €. Duración: 01/10/2006 – 30/09/2009. Concedido. 2- MICINN, CSD2008-00005. Programa CONSOLIDER-INGENIO 2010. The Spanish Ion Channel Initiative (SICI). Grupos de investigación: 26. Coordinador General: Antonio Vicente Ferrer Montiel. IP del grupo Cellular Neurotransmision: Rafael Luján (IP). Financiación total: 6.000.000 €. Financiación Grupo: 175.000 €. Duración: 6 años, 15/12/08-15/12/14. Concedido. 3- MICINN, BFU2009-08404/BFI. “Mecanismos celulares y subcelulares implicados en la interacción entre los receptores de GABAB y los canales de potasio GIRK y en el hipocampo”. Investigadores: Rafael Luján (IP). Financiación: 178.000 €. Duración: 01/10/2010 – 30/09/2012. Concedido. 4- MICINN, BFU2012-38348/BFI. “Receptores acoplados a proteínas G y sus canales iónicos efectores: bases celulares y subcelulares de su asociación morfológica y funcional en el hipocampo”. Investigadores: Rafael Luján (IP). Financiación: 163.800 €. Duración: 01/01/2013 – 31/12/2015. Concedido. 5- JUNTA COMUNIDADES CASTILLA-LA MANCHA, PPII11-0284-9301. “Mecanismos subcelulares que median la asociación entre los canales de potasio SK y los receptores NMDA en el hipocampo”. Investigadores: Rafael Luján (IP). Financiación: 70.000 €. Duración: 27/09/2014 30/09/2017. Concedido. 6- EUROPEAN COMMISSION, Human Brain Project (HBP), Project Ref. 604102. “Localization of ion channels and receptors by two- and three-dimensional immunoelectron microscopic approaches”. Investigadores: Rafael Luján (IP, Coordinator) and Ryuichi Shigemoto (Co-IP, IST Austria). Financiación: 467.508 €. Duración: 01/04/2014 - 30/03/2016. Concedido. 7- NATIONAL INSTITUTES OF HEALTH (NIH), 1R01MH093599-01A1. “Coupled LTPdependent trafficking of synaptic SK channels and NMDARs”. Investigadores: John Adelman (IP), James Maylie (Co-IP) and Rafael Luján (Subcontrato). Financiación: 29.560 €. Duración: 01/06/2012 – 31/05/2016. Concedido. C5. Otros méritos de relevancia - Evaluador externo de más de 150 trabajos/artículos de investigación para 15 revistas neurocientíficas especializadas, entre las que destacan Neuron, Journal of Neurochemistry, European Journal of Neuroscience, Trends in Neuroscience, Nature Neuroscience, Journal of Neuroscience. - Evaluador externo de proyectos para la ANEP (Agencia Nacional de Evaluación y Prospectiva). Proyectos del Plan Nacional de I+D y Regionales/Autonómicos. - Evaluador de proyectos científicos de al menos 15 instituciones internacionales, entre las que destacan Medical Research Council, Wellcome Trust), Austrian Science Fundation, Sexto y Séptimo Programa Marco de la Unión Europea, European Research Council, Czech Science Foundation, BBSRC (UK), Israel Science Foundations, Swiss National Science Foundation, Philip Morris External Program y Agencia Francesa Agence Nationale de la Recherche. - Miembro de la Comisión de Profesorado de la Universidad de Castilla-La Mancha, desde 2012. - Participación en 26 proyectos de investigación, tanto de agencias nacionales como extranjeras, además de lo cual he dirigido/dirijo 12 proyectos de investigación internacional, nacional o de comunidades autónomas. - Dirección como Investigador Principal de un Subcontrato del Proyecto QLG3-CT-1999-00192 financiado por la Unión Europea (1999-2002). - En la actualidad tengo 119 publicaciones indexadas en la base de datos ISI Web of Knowledge, 16 publicaciones no indexadas, 8 capítulos de libro, y 2 libros editados, 147 ponencias/posters en congresos internacionales y 42 en congresos nacionales, 47 seminarios/conferencias invitadas en Universidades y Centros de Investigación y 13 estancias en centros de investigación internacionales. - Diez portadas en revistas de investigación. 3 de 4 / Currículum abreviado

 

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- Cuatro quinquenios docentes (último 2006-2011). - Distinción como Reconocido 2013 del Personal Docente e Investigador de la UCLM – Consejo Social de la Universidad de Castilla-La Mancha. - Tesorero de la Sociedad Española de Neurociencia desde septiembre de 2011 hasta septiembre 2013. - Vocal de la Sociedad Española de Neurociencia desde septiembre de 2013 hasta septiembre 2015. - Cinco primeros premios a la mejor investigación científica. Edición de los Premios de Investigación CHUA (Complejo Hospitalario Universitario de Albacete) y Bienestar Social, Junta de Comunidades de Castilla-La Mancha. 2006, 2007, 2008, 2010 y 2010. - Premio a la mejor publicación del año (2008), otorgado por la Sociedad de Neurociencias Suiza: Mameli y cols. Science 317:530-533 (2007). - Miembro fundador (2000) del Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete. - Miembro fundador (2009) y actual miembro del Instituto de Investigación en Discapacidades Neurológicas (IDINE), Universidad de Castilla-La Mancha, Albacete. - Miembro del comité organizador del “XV Congreso Nacional y III Internacional de la Sociedad Española de Histología e Ingeniería Tisular”, celebrado del 8 al 11 de Julio de 2009 en la Facultad de Medicina de la Universidad de Castilla-La Mancha, Albacete. - Organizador de la IV Reunión Internacional RECI. “New horizons in ion channel research”. Cuenca, Spain, 12-13 Febrero 2013. - Miembro de la comisión de Valoración de plazas de Personal de Administración y Servicios (PAS) para el Departamento de Ciencias Médicas, en la Facultad de Medicina de la UCLM. - Participación como miembro de tribunal en comisiones de contratación y plazas docentes de la Universidad de Castilla-La Mancha y de otras Universidades Españolas. - Participación como miembro de tribunal de Tesis doctorales en Universidades y Centros nacionales y extranjeros. - Supervisión académica y científica de estudiantes de doctorado y postdoctorales en el Division of Cerebral Structure, National Institute for physiological Sciences, Okazaki, Japón. Curso académico: marzo a mayo de 2000. - Tutor externo de médicos cirujanos (D. Arturo Ignacio Ortíz Torres y Dña. Leyla Guadarrama López), que realizaban estudios de Doctorado en Ciencias Biomédicas en el Departamento de Fisiología (Facultad de Medicina, Universidad Nacional Autónoma de México, México). - Distinción como mejor profesor de la Licenciatura de Medicina por parte de la tercera promoción de la Facultad de Medicina (2 de junio de 2006) y de la décimo-primera promoción de la Facultad de Medicina (6 de junio de 2014). - Asesoramiento de alumnos: tutorías personalizadas a estudiantes del Grado de Medicina desde el curso académico 2000/2001 hasta la actualidad. - Director de cinco DEAS y un Proyecto Fin de Master de Técnicas Experimentales en Biomedicina. C6. Otras publicaciones de relevancia de mi laboratorio 1- Reyes et al. (1998) Nature Neuroscience 4: 275-285. 2- Nusser et al. (1998) Neuron 21: 545-559. 3- Vigot et al. (2006). Neuron 50:589-601. 4. Pérez-Otaño et al. (2006) Nature Neuroscience 9:611-21. 5- Mameli et al. (2007) Science 317(5837):530-3. 6. Labouèbe et al. (2007) Nature Neuroscience 10:1559-68. 7. Rebola et al. (2008) Neuron 57:121-34. 8. Cueni et al., (2008) Nature Neuroscience 11:683-92. 9. Englomd et al. (2008) Neuron 59:497-508. 10. Roberts et al. (2009) Neuron 63:342-56. 11- Fazzari et al. (2010) Nature 464(7293):1376-80, 2010. 12- Huang et al. (2011) Nature Neuroscience 14:478-86. 13. Padgett et al. (2012) Neuron 73:978-89. 14- Rozas et al. (2012) Neuron 74:151-65. 15- Marcos et al. (2013) Nature Medicine 19(8):1030-8. 16- Hearing et al. (2013) Neuron 80:159-70, 2013. 17- Soden et al. (2013) Neuron 80:997-1009. 18- Ito et al. (2014) Nature Communications 5:4450. 19- Devedsa et al. (2014) PNAS 111(51):18345-50 20- Martinello et al. (2015) Neuron 85:346-63. 4 de 4 / Currículum abreviado

Indice Impacto IF: 8.863 IF 16.505 IF: 13.894 IF: 14.805 IF: 26.372 IF: 15.664 IF: 14.170 IF: 14.164 IF: 14.170 IF: 13.260 IF: 36.104 IF: 15.531 IF: 15.766 IF: 15.766 IF: 28.054 IF: 15.982 IF: 15.982 IF: 11.470 IF: 9.674 IF: 15.054

 

CURRÍCULUM ABREVIADO (CVA) Lea detenidamente las instrucciones que figuran al final de este documento para rellenar correctamente el CVA

Parte A. DATOS PERSONALES Nombre y apellidos Isabel Varela Nieto DNI/NIE/pasaporte 05233327-E Núm. identificación del investigador

Fecha del CVA

Researcher ID Código Orcid

22-06-2015

Edad 56 G-5289-2015 0000-0003-3077-0500

A.1. Situación profesional actual Organismo Consejo Superior de Investigaciones Científicas Dpto./Centro Instituto de Investigaciones Biomédicas “Alberto Sols” (IIBm) Dirección C/Arturo Duperier 4 Teléfono 91-5854421 correo electrónico [email protected]  Categoría profesional Profesor de Investigación Fecha inicio 2008 Espec. cód. UNESCO 240105, 240701¸240108, 240111, 240113 Fisiopatología audición, envejecimiento, modelos animales, hipoacusia, ototoxicidad, ruido, Hcy, p38, neurobiología audición, Palabras clave neuroinflamación, apoptosis, autofagia, senescencia, señalización, IGF-1, IGF1R, RAF, AKT, p38, MEF2. A.2. Formación académica (título, institución, fecha) Licenciatura/Grado/Doctorado Universidad Ciencias Químicas Universidad Complutense de Madrid Doctora en Químicas. Sección Universidad Complutense de Madrid Bioquímica

Año 1986 1988

A.3. Indicadores generales de calidad de la producción científica (véanse instrucciones) - Autora de 102 artículos (24 D1, 51 Q1, 20 otros Q & 6 pendientes de adscribir) - Autora de 12 revisiones de divulgación en revistas nacionales y de 25 capítulos en libros. - Editora de 3 libros. - Autora de 4 patentes - Editora de 6 números monográficos en revistas científicas internacionales. - 5 Sexenios concedidos (último 2006-2011). - Producción científica completa e índices disponibles en las direcciones: PubMed: Varela-Nieto I:http://www.ncbi.nlm.nih.gov/pubmed/?term=varela-nieto+i, y Varela I antes del 1991. Research Gate:http://www.researchgate.net/profile/Isabel_Varela-Nieto/reputation. Google Scholar:http://scholar.google.es/citations?user=saHG7jEAAAAJ&hl=en Frontiers:http://community.frontiersin.org/people/IsabelVarela-Nieto/13829 Citation indices All Since 2010 Citations

2495 1071

h-index

31

20

i10-index

64

39

- Actividad científica reflejada en Internet:  URL: http://www2.iib.uam.es/ivarela_lab/  URL: http://www.ciberer.es/fichagrupos/grupo.aspx?unidad=u761  URL: www.targear.eu

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Parte B. RESUMEN LIBRE DEL CURRÍCULUM (máximo 3500 caracteres, incluyendo espacios en blanco) La Dra. Isabel Varela Nieto se licenció (1981) y doctoró (1985) en CC Químicas, sección Bioquímica, en la Universidad Complutense de Madrid. Es EMBO fellow ya que disfrutó de dos ayudas EMBO postdoctorales en las Universidades de Glasgow (RU) y Lovaina (Bélgica) (1986-1989). Ha sido científico invitado visitante en las Facultades de Medicina de las Universidades de Upsala (FEBS fellow; 1993, Suecia) y San Diego (19992000, EEUU). Ha sido Profesor contratado en la Facultad de Medicina de la UAM (19911992), científico titular CSIC (1992-2002), investigador científico (2002-2004) y en la actualidad es Profesor de Investigación desde el año 2005 en el Instituto de Investigaciones Biomédicas “Alberto Sols” del CSIC en Madrid. Desde principios de los 90 se interesa en el estudio de la neurobiología de la audición, inicialmente en la regulación del desarrollo embrionario del oído interno y en la neurogénesis del ganglio auditivo. Estudios que continúan en el grupo, en paralelo con los de avanzar en el conocimiento de la fisiopatología de modelos genéticos y ambientales de sorderas humanas. Aspecto en el que se ha especializado desde hace algo más de diez años. El grupo ha caracterizado la participación del sistema y dianas del IGF-1 en la pérdida auditiva, y ha demostrado que el estado oxido-inflamatorio es clave para el desarrollo del daño. Ha descrito nuevos aspectos de la biología del oído como son la importancia de la autofagia y la senescencia, de su metabolismo con el ciclo de la metionina e identificado nuevos factores de transcripción importantes para la función auditiva como los MEF2. Recientemente, en el marco de colaboraciones con empresas y con el apoyo de proyectos europeos del FP7, su trabajo ha tomado un sesgo traslacional y se ha enfocado al estudio de moléculas con potencial actividad otoprotectora, desde el nivel funcional hasta la caracterización de su efecto en el inflamasoma y transcriptoma. Estos estudios son abordados mediante el desarrollo y evaluación de modelos celulares y animales, aplicación de micrométodos para obtener datos masivos, y la puesta a punto de técnicas de microcirugía y biología no invasiva para el estudio de la función auditiva. Nuestros objetivos son aumentar el conocimiento básico en el campo de la neurobiología de la audición, contribuir al desarrollo de nuevas terapias de reparación y regeneración auditiva, y finalmente realizar aportaciones que a medio o largo plazo puedan mejorar la salud humana. Parte C. MÉRITOS MÁS RELEVANTES (ordenados por tipología) C.1. Publicaciones Se presenta una selección de un total de 125 artículos, revisiones y capítulos de libro: Editor. Autores (p.o. de firma): Magariños, M., Contreras, J. and Varela-Nieto, I. Título: “Early Development of the Vertebrate Inner Ear” Capítulo de Libro: Development of Auditory and Vestibular Systems: 1–30. Editora del Libro: Romand R & Varela-Nieto I. Portada. Fecha: 2014 Editorial: Academic Press ISBN: 9780124080881 Artículos. 1.- Autores (p.o. de firma): de Iriarte Rodríguez R, Magariños M, Pfeiffer V, Rapp UR, Varela-Nieto I. Título: C-Raf deficiency leads to hearing loss and increased noise susceptibility through mechanisms involving increased JNK activity and cochlear apoptosis. Revista: Cell Mol Life Sci. 2015 May 15. [Epub ahead of print] (Grupo A: primer cuartil de su campo) Fecha: 2015 2.- Autores (p.o. de firma): Fuentes-Santamaría V, Alvarado JC, Rodríguez-de la Rosa L, Murillo-Cuesta S, Contreras J, Juiz JM and Varela-Nieto I. Título: Insulin-like growth factor 1 deficiency causes atrophy and upregulation of vesicular glutamate transporter 1 in the mouse cochlear nuclei. Revista: Brain Struct Funct. 2014 Nov 7. [Epub ahead of print] (Grupo A: primer decil) Fecha: 2014 3.- Autores (p.o. de firma): Martínez-Vega R, Garrido F, Partearroyo T, Cediel R, Zeisel SH, Martínez-Álvarez C, Varela-Moreiras G, Varela-Nieto I* and Pajares MA (*corresponding. Equal senior contribution). 2 de 4 / Currículum abreviado

 

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Título: Folic Acid Deficiency Induces Premature Hearing Loss through Mechanisms Involving Cochlear Oxidative Stress and Impairment of Homocysteine Metabolism. Revista FASEB J. 2015 Feb;29(2):418-32. doi: 10.1096/fj.14-259283. (Grupo A: primer decil de su campo) Fecha: 2015 4.- Autores (p.o. de firma): Muñoz-Espín D, Cañamero M, Maraver A, Gómez G, Contreras J, Murillo-Cuesta S, Rodríguez A, Varela-Nieto I, Ruberte J, Collado M and Serrano M. Título: Programmed cell senescence during mammalian embryonic development. Revista: Cell.;155(5):1104-18. Fecha: 2013 (Grupo A: primer decil de su campo). 5.- Autores (p.o. de firma): Varela-Nieto I, Murillo-Cuesta S, Rodríguez-de la Rosa L, Lassatetta L, Contreras J. Título: IGF-I deficiency and hearing loss: molecular clues and clinical implications. Revista: Pediatr Endocrinol Rev.;10(4):460-72. Revisión invitada Fecha: 2012 (Todavía sin indexar) 6.- Autores (p.o. de firma): Rodriguez Aburto M, Sanchez-Calderón H, Hurlé J.M.,VarelaNieto I* and Magariños M* (*co-senior) Título: “Early otic development depends on autophagy for apoptotic cell clearance and neural differentiation” Revista: Cell Death Disease 3, e; doi:10.1038/cddis.2012.132 Fecha: 2012 (Grupo A: primer cuartil de su campo) 7.-  Autores (p.o. de firma): Murillo-Cuesta S., Camarero G., González-Rodríguez A., Rodríguez-de la Rosa L., Burks DJ, Avendaño C., Valverde A and Varela-Nieto I. Título: IRS2-deficient mice show sensorineural hearing loss before the onset of diabetes that is delayed by concomitant PTP1B loss of function. Revista: Mol. Med. Dec 1. doi: 10.2119/molmed.2011.00328. Fecha: 2011 (Grupo A: primer cuartil de su campo) 8.- Autores (p.o. de firma): Magariños M, Rodriguez Aburto M, Sánchez-Calderón H, Muñoz Agudo C, Rapp U and Varela–Nieto I. Título: “RAF kinase activity regulates neuroepithelial cell proliferation and neuronal progenitor cell differentiation during early inner ear development” Revista: PLoS ONE 5(12): e14435. doi:10.1371/journal.pone.0014435 Fecha: 2010 (Grupo A: primer cuartil de su campo) 9.- Autores (p.o. de firma): Riquelme R, Cediel R, Contreras J, Rodriguez de la Rosa L, Murillo-Cuesta S, Hernandez-Sanchez C, Zubeldia JM, Cerdan S and Varela-Nieto I. Título: A comparative study of age-related hearing loss in wild type and insulin-like growth factor I deficient mice. Revista: Front. Neuroanat. vol.4, 27:1-13. Fecha: 2010 (Grupo A: primer cuartil de su campo). C.2. Proyectos Se presentan 6 de un total de 31 proyectos públicos y 3 de fundaciones como IP. 1.-Título del proyecto: “TARGEAR-targeting challenges of active ageing: innovative integrated strategies for the healing of age-related hearing loss (“Marie Curie” FP7People-2013-IAPP: ID: 612261)”. Entidad financiadora: Proyecto financiado por la Comisión Europea en el marco de Marie Curie Industry-Academia Partnerships and Pathways (IAPP) Duración, desde: 2014 hasta: 2017 Investigador responsable: Dra. Isabel Varela Nieto. IIBm 2.-Título del proyecto: “AFHELO-preclinical proof of concept of AF243 potency to prevent and/or treat sensorineural hearing loss. (FP7-Health-2012-Innovation-2: AFHELO 304900)”. Entidad financiadora: European Union. Health Duración, desde: 2013 hasta: 2015 Investigador responsable: Dra. Isabel Varela Nieto. IIBm 3.-Título del proyecto: “Interacciones ambiente-genotipo en la perdida auditiva de diferente etiología. Bases moleculares, factores de protección ótica e implicaciones en terapia: SAF2011-24391)”. Entidad financiadora: Ministerio de Ciencia e Innovación. 3 de 4 / Currículum abreviado

 

CURRÍCULUM ABREVIADO (CVA) Lea detenidamente las instrucciones que figuran al final de este documento para rellenar correctamente el CVA

Duración, desde: 2012 hasta: 2014 Investigador responsable: Dra. Isabel Varela Nieto. IIBm 4.- Título del proyecto: “Células madre mesenquimales de placenta: posibles aplicaciones en medicina regenerativa y reparadora (FMM2012-201222005)” Entidad Financiadora: Fundación Mutua Madrileña. Fecha de inicio y finalización: 2012-2014. Investigador responsable: Dra. Isabel Varela Nieto. IIBm 5.- Título del proyecto: “Modelos animales y celulares para el estudio de la hipoacusia neurosensorial, metabólica y por exposición a ruido excesivo (SAF2008-00470)”. Entidad financiadora: Programa Nacional de Salud. Ministerio de Ciencia e Innovación. Duración, desde: 2009 hasta: 2011 Investigador responsable: Dra. Isabel Varela Nieto. IIBm 6.- Título del proyecto: “Development of a European network for preclinical testing of interventions in mouse models of age and age-related diseases (MouseAGE; BM1402)”. Entidad financiadora: COST Duración, desde: 2015 hasta: 2017 Comité ejecutivo. Dra. I Varela Nieto. CIBERER. ISCii. Madrid. 7. y 8. - Proyectos SAF2014 (CSIC) y CDTI (CIBERER) concedidos. C.3. Contratos de I+D+i Se presenta uno de un total de 6 contratos con empresas: Título del proyecto: Potencial otoprotector de moléculas con actividad inhibidora TGFbeta. Empresa: Digna Biotech. Duración, desde: 2006 hasta: 2010 Investigador responsable: Dra. Isabel Varela Nieto. IIBm C.4. Patentes Se presenta una de un total de 4 patentes: Inventores: Cobo Parra P., Varela Nieto I., Murillo Cuesta S., Cediel Algovia R. Título: Cámara acústica reverberante para ensayos auditivos con animales Nº referencia: PCT/ES2009/070433. Países de prioridad: All except USA. Fecha: 2008. Entidad titular: CSIC-UCM. C.5.Otros - Docencia. Dirección de 12 Tesis Doctorales (+3 en marcha), 7 tesinas y 6 proyectos fin de master. Es Profesor Honorario de la UAM desde el año 1997 y su participación en Docencia ha sido reconocida con 5 quinquenios. - Premios. Joven Investigador SEBBM (1994) y Juan Gassó Bosch de la SEORL (2010). - Comités Internacionales y Advisory Boards.  Spanish delegate @ European Medical Research Council (EMRC) of the European Science Foundation (ESF) (2007-2009).  Member @ EMRC Core group (2009-2012)  ESF delegate @ DH-BIO (Council of Europe) (since 2009)  Member @ ESF Scientific Review Group for the Biomedical Sciences (SRG-MED) (since 2013).  Spanish delegate @ European Cooperation in Science and Technology (COST) domain of Biomedicine and Molecular Biosciences (BMBS) (since 2013).  Scientific advisor Wellcome Trust-EUROPE-PMC. - Invited Speaker Inner Ear Biology (Montpelier, 2006; Prague 2010; Lisbon, 2011; Kyoto 2014; Rome, 2015); UIMP (Santander, 2011); FENS (Barcelona 2012); IBRO (Florence, 2011); IGF research society (Munich, 2012); ARO (Baltimore, 2013; San Diego, 2014); Gordon Research Conference IGFs (Ventura 2013); Ageing Summit (London, 2015). - Ad hoc reviewer de revistas internacionales (J. Neurosci, BMC Medicine, etc.) - Ad hoc reviewer para agencias internacionales y nacionales de evaluación. 4 de 4 / Currículum abreviado

 

CURRÍCULUM ABREVIADO (CVA)

INSTRUCCIONES PARA RELLENAR EL CVA AVISO IMPORTANTE En virtud del artículo 11 de la convocatoria NO SE ACEPTARÁ NI SERÁ SUBSANABLE EL CURRÍCULUM ABREVIADO que no se presente en este formato.

Este documento está preparado para que pueda rellenarse en el formato establecido como obligatorio en las convocatorias (artículo 11.7.a): letra Times New Roman o Arial de un tamaño mínimo de 11 puntos; márgenes laterales de 2,5 cm; márgenes superior e inferior de 1,5 cm; y espaciado mínimo sencillo. La extensión máxima del documento (apartados A, B y C) no puede sobrepasar las 4 páginas. Parte A. DATOS PERSONALES Researcher ID (RID) es una comunidad basada en la web que hace visibles las publicaciones de autores que participan en ella. Los usuarios reciben un número de identificación personal estable (RID) que sirve para las búsquedas en la Web of Science. Los usuarios disponen de un perfil donde integrar sus temas de investigación, sus publicaciones y sus citas. Acceso: Web of Science > Mis herramientas > Researcher ID Código ORCID es un identificador compuesto por 16 dígitos que permite a los investigadores disponer de un código de autor inequívoco que les permite distinguir claramente su producción científico-técnica. De esta manera se evitan confusiones relacionadas con la autoría de actividades de investigación llevadas a cabo por investigadores diferentes con nombres personales coincidentes o semejantes. Acceso: www.orcid.org Si no tiene Researcher ID o código ORCID, no rellene estos apartados. A.3. Indicadores generales de calidad de la producción científica Se incluirá información sobre el número de sexenios de investigación y la fecha del último concedido, número de tesis doctorales dirigidas en los últimos 10 años, citas totales, promedio de citas/año durante los últimos 5 años (sin incluir el año actual), publicaciones totales en primer cuartil (Q1), índice h. Adicionalmente, se podrán incluir otros indicadores que el investigador considere pertinentes. Para calcular estos valores, se utilizarán por defecto los datos recogidos en la Web of Science de Thomson Reuters. Cuando esto no sea posible, se podrán utilizar otros indicadores, especificando la base de datos de referencia. Parte B. RESUMEN LIBRE DEL CURRÍCULUM (máximo 3500 caracteres, incluyendo espacios en blanco) Describa brevemente su trayectoria científica, los principales logros científico-técnicos obtenidos, los intereses y objetivos científico-técnicos a medio/largo plazo de su línea de investigación. Indique también otros aspectos o peculiaridades que considere de importancia para comprender su trayectoria. Si lo considera conveniente, en este apartado se puede incluir el mismo resumen del CV que se incluya en la solicitud, teniendo en cuenta que este resumen solo se utilizará para el proceso de evaluación de este proyecto, mientras que el que se incluye en la solicitud podrá ser difundido.

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CURRÍCULUM ABREVIADO (CVA)

Parte C. MÉRITOS MÁS RELEVANTES (ordenados por tipología) Teniendo en cuenta las limitaciones de espacio, detalle los méritos más relevantes ordenados por la tipología que mejor se adapte a su perfil científico. Los méritos aportados deben describirse de una forma concreta y detallada, evitando ambigüedades. Los méritos aportados se pondrán en orden cronológico inverso dentro de cada apartado. Salvo en casos de especial importancia para valorar su CV, se incluirán únicamente los méritos de los últimos 10 años. C.1. Publicaciones Incluya una reseña completa de las 5-10 publicaciones más relevantes. Si es un artículo, incluya autores por orden de firma, año de publicación, título del artículo, nombre de la revista, volumen: pág. inicial-pág. final. Si se trata de un libro o de capítulo de un libro, incluya, además, la editorial y el ISBN. Si hay muchos autores, indique el número total de firmantes y la posición del investigador que presenta esta solicitud (p. ej., 95/18). C.2. Participación en proyectos de I+D+i Indique los proyectos más destacados en los que ha participado (máximo 5-7), incluyendo: referencia, título, entidad financiadora y convocatoria, nombre del investigador principal y entidad de afiliación, fecha de inicio y de finalización, cuantía de la subvención, tipo de participación (investigador principal, investigador, coordinador de proyecto europeo, etc.) y si el proyecto está en evaluación o pendiente de resolución. C.3. Participación en contratos de I+D+i Indique los contratos más relevantes en los que ha participado (máximo 5-7), incluyendo título, empresa o entidad, nombre del investigador principal y entidad de afiliación, fecha de inicio y de finalización, cuantía. C.4. Patentes Relacione las patentes más destacadas, indicando los autores por orden de firma, referencia, título, países de prioridad, fecha, entidad titular y empresas que las estén explotando. C.5, C.6, C.7… Otros Mediante una numeración secuencial (C.5, C.6, C.7...), incluya los apartados que considere necesarios para recoger sus principales méritos científicos-técnicos: dirección de trabajos, participación en tareas de evaluación, miembro de comités internacionales, gestión de la actividad científica, comités editoriales, premios, etc. Recuerde que todos los méritos presentados deberán presentarse de forma concreta, incluyendo las fechas o período de fechas de cada actuación. El currículum abreviado pretende facilitar, ordenar y agilizar el proceso de evaluación. Mediante el número de identificación individual del investigador es posible acceder a los trabajos científicos publicados y a información sobre el impacto de cada uno de ellos. Si considera que este currículum abreviado no recoge una parte importante de su trayectoria, puede incluir voluntariamente el currículum en extenso en la documentación aportada, que será facilitado también a los evaluadores de su solicitud.

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CURRÍCULUM ABREVIADO (CVA)

20/09/2014 Fecha del CVA Parte A. DATOS PERSONALES Nombre y apellidos José Antonio Esteban García DNI/NIE/pasaporte 07528695J Edad 48 Researcher ID L-4944-2014 Núm. identificación del investigador Código Orcid 0000-0002-3759-3300 A.1. Situación profesional actual Organismo Consejo Superior de Investigaciones Científicas Dpto./Centro Centro de Biología Molecular “Severo Ochoa” Dirección Nicolás Cabrera 1, Madrid Teléfono 91-1964637 correo electrónico [email protected] Categoría profesional Profesor de Investigación Fecha inicio 15/03/2013 Espec. cód. UNESCO synaptic plasticity, hippocampus, membrane trafficking, Palabras clave neurotransmitter receptors A.2. Formación académica (título, institución, fecha) Licenciatura/Grado/Doctorado Universidad Licenciado en Biología Univ. Complutense de Madrid Doctorado en Biología Univ. Autónoma de Madrid

Año 1988 1993

A.3. Indicadores generales de calidad de la producción científica (véanse instrucciones) - Sexenios (4): 1988-1993, 1994-1999, 2000-2005, 2006-2011 - Quinquenios (5): 1988-1992, 1993-1997, 1998-2002, 2003-2007, 2008-2012 - Dirección de tesis doctorales (4): 2007 Tyler Brown. University of Michigan Medical School, USA Endosomal trafficking of AMPA receptors at hippocampal synapses Apto cum laude 2008 Kristin Arendt. University of Michigan Medical School, USA Role of PI(3,4,5)P3 in hippocampal synaptic plasticity Apto cum laude 2014 María Royo Cantabrana. Universidad Autónoma de Madrid, Spain Papel de la proteína efectora Rab11-FIP2 en el transporte de receptores AMPA durante plasticidad sináptica Sobresaliente cum laude 2014 Rocío Palenzuela Muñoz. Universidad Autónoma de Madrid, Spain The light chain of MAP1B: a novel modulator of AMPA receptor trafficking in hippocampal neurons Sobresaliente cum laude - Número de publicaciones por cuartil de la revista (número total de publicaciones: 53) Q1: 43 Q2: 7 Q3: 3 - Número total de citas: 4446 - Número de citas por año en los últimos 5 años: 2009: 333 2010: 330 2011: 316 2012: 332 2013: 309 -

Índice H: 29

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CURRÍCULUM ABREVIADO (CVA)

Parte B. RESUMEN LIBRE DEL CURRÍCULUM (máximo 3500 caracteres, incluyendo espacios en blanco) My research group has a longstanding interest in the molecular and cellular mechanisms of synaptic plasticity, and their contribution to cognitive processes such as learning and memory. Using electrophysiological, imaging and molecular techniques, we have made important contributions to understand how the membrane trafficking machinery of the neuron controls synaptic function by shuttling neurotransmitter receptors in and out of the synaptic membrane. We are also particularly interested in how these processes are altered in human pathologies associated to cognitive disorders. Indeed, we have found that some signaling cascades controlling this trafficking machinery are defective in Alzheimer’s disease. We are now exploiting this molecular information, together with behavioral assays on mouse models, to develop potential therapeutic avenues for cognitive enhancement. As a whole, we are trying to understand how molecular mechanisms operating at the synapse contribute to brain function in health and disease. These studies have been funded by several international agencies, such as the National Institutes of Health (NIMH) and the Dana Foundation, and since my return to Spain, the Spanish Ministry for Science, the Carlos III Health Institute, Ramón Areces Foundation and the French Science Institute. Parte C. MÉRITOS MÁS RELEVANTES (ordenados por tipología) C.1. Selección de 10 publicaciones recientes más relevantes (sobre un total de 53) 1. Benoist M, Palenzuela R, Rozas C, Rojas P, Tortosa E, Morales B, González-Billault C, Ávila J, Esteban JA. MAP1B-dependent Rac activation is required for AMPA receptor endocytosis during long-term depression. EMBOJ 32, 2287-2299, 2013. 2. Fernández-Monreal M, Brown TC, Royo M, Esteban JA. The balance between receptor recycling and trafficking towards lysosomes determines synaptic strength during longterm depression. J Neurosci 32, 13200-13205, 2012. 3. Knafo S, Esteban JA. Common Pathways for Growth and for Plasticity. Curr Opin Neurobiol 22, 405-412, 2012. 4. Knafo S, Venero C, Sánchez-Puelles C, Pereda-Peréz I, Franco A, Sandi C, Suárez LM, Solís JM, Alonso-Nanclares L, Martín ED, Merino-Serrais P, Borcel E, Li S, Chen Y, Gonzalez-Soriano J, Berezin V, Bock E, DeFelipe J, Esteban JA. Facilitation of AMPA receptor synaptic delivery as a molecular mechanism for cognitive enhancement. PLoS Biology 10, doi:10.1371/journal.pbio.1001262, 2012. 5. Jurado S, Benoist M, Lario A, Knafo S, Petrok CN and Esteban JA. PTEN is recruited to the postsynaptic terminal for NMDA receptor-dependent long-term depression. EMBOJ 29:2827-2840, 2010. 6. Arendt KL, Royo M, Férnandez-Monreal M, Knafo S, Petrok CN, Martens JR and Esteban JA. PIP3 controls synaptic function by maintaining AMPA receptor clustering at the postsynaptic membrane. Nat Neurosci 13:36-44, 2010. 7. Correia SS, Bassani S, Brown TC, Lisé M-F, Backos DS, El-Husseini A, Passafaro M, Esteban JA. Motor protein-dependent transport of AMPA receptors into spines during long-term potentiation. Nat Neurosci 11:457-466, 2008. 8. Brown TC, Correia SS, Petrok CN, Esteban JA. Functional compartmentalization of endosomal trafficking for the synaptic delivery of AMPA receptors during LTP. J Neurosci 27:13311-13315, 2007. 9. Gerges NZ, Backos DS, Rupasinghe CN, Spaller MR, Esteban JA. Dual role of the exocyst in AMPA receptor targeting and insertion into the postsynaptic membrane. EMBOJ 25:1623-1634, 2006. 10. Brown TC, Tran, IC, Backos DS, Esteban JA. NMDA receptor-dependent activation of the small GTPase Rab5 drives the removal of synaptic AMPA receptors during hippocampal LTD. Neuron 45:81-94, 2005. C.2. Proyectos (selección de los últimos 5 años) 2009-2011 Ministerio de Ciencia e Innovación, SAF2008-04616. “Regulación de la plasticidad sináptica por fosfoinosítidos”. Cuantía: 210.000 €. Investigador principal. 2010-2011 Ministerio de Ciencia e Innovación, SAF2009-05558-E. “¿Se pueden revertir enfermedades mentales del desarrollo?”. Cuantía: 75.000 €. Investigador principal. 2 de 2 / Instrucciones para rellenar el currículum abreviado

 

CURRÍCULUM ABREVIADO (CVA)

2010-2012 Fundación Ramón Areces. “Bases moleculares del retraso mental asociado al síndrome “X Frágil”. Cuantía: 125.832 €. Investigador principal. 2010-2011 Institut de France-NRJ Fondation. ”Regulation of the PIP3 pathway as a strategy to correct synaptic dysfunction in Alzheimer’s disease”. Cuantía: 50.000 €. Investigador principal. 2011-2015 Ministerio de Ciencia e Innovación, CSD2010-00045. “BrainAge: Brain dysfunction during aging. Relevance for Alzheimer’s disease”. Cuantía para el laboratorio del Dr. Esteban: 561.550 €. Investigador principal. 2012-2014 Ministerio de Ciencia e Innovación, SAF2011-24730. “Molecular mechanisms of synaptic plasticity, cognitive function and age-dependent cognitive decline, controlled by the PI3K-PTEN pathway”. Cuantía: 310.000 €. Investigador principal. C.3. Contratos C.4. Patentes INVENTORES (p.o. de firma): Mark R. Spaller, José A. Esteban, César Venero, Shira Knafo TITULO: Method for mitigating or alleviating synaptic and cognitive deficits Nº DE SOLICITUD: DC520USL PAÍS DE PRIORIDAD: EEUU FECHA DE PRIORIDAD: 26/11/2013 ENTIDADES TITULARES: Geisel School of Medicine at Darmouth Alla Kan Consejo Superior de Investigaciones Científicas Universidad Nacional de Educación a Distancia C.5. Organización de Congresos Científicos Nov. 2005 Symposium on “Cellular Biology of Postsynaptic Receptor Trafficking and Synaptic Plasticity”, 35th Annual Meeting of the Society for Neuroscience, Washington, DC, USA. Dic. 2008 Symposium on “Cell Biology of the Synapse”, 48th Annual Meeting of the American Society for Cell Biology (ASCB), San Francisco, California, USA. Oct. 2010 Cantoblanco Workshop on “Memory Formation and Memory Loss”. Centro de Biología Molecular “Severo Ochoa”, Madrid, Spain. Oct. 2013 Current Trends in Biomedicine Workshop on “Membrane Traffic at the Synapse. The Cell Biology of Synaptic Plasticity”, UNIA, Baeza, Spain. C.6. Evaluador para agencias científicas National Institutes of Health (NIH, EEUU) Wellcome Trust (UK) Alzheimer’s Association (EEUU) Human Frontier Science Program (HFSP) Netherlands Organisation for Scientific Research (Holanda) Agence d’Évaluation de la Recherche et de l’Enseignement Supérieur (AERES, Francia) Fédération pour la Recherche sur le Cerveau (FRC, Francia) Agence Nationale de la Recherche (ANR, Francia) FONDECYT Program (Chile) Agencia Nacional de Evaluación y Prospectiva (ANEP, España) C.7. Miembro de paneles evaluadores para institutos científicos Interdisciplinary Institute for Neuroscience, Bordeaux, Francia Leloir Institute, Buenos Aires, Argentina.

3 de 2 / Instrucciones para rellenar el currículum abreviado

 

Short CV Before proceeding, please read carefully the instructions at the end of the document.

07/17/2015

DATE

Part A. Personal Information Surname(s) Bovolenta Forename Paola Sex Female Age 57 Researcher ID L-6256-2014 Open Researcher and 0000-0002-1870-751X Contributor ID (ORCID) A.1. Professional Situation Now Post/ Professional Category UNESCO Code Key Words Name of Institution

Start date

Full Professor and Chair of the Development and Differentiation Dept. 2490 Pattern formation, neural differentiation; axon guidance; cell-cell communication Consejo Superior de Investigaciones Cientificas Centro de Biologia Molecular Department/Center Severo Ochoa c/ Nicolas Cabrera 2, Cantoblanco Full Address Madrid Email Address [email protected] Phone Number 911964718 01/28/2005

A.2. Academic Preparation (title, institution, date) 1981 1984 1986

Universitá di Firenze, First Degree Firenze, Italia. New York University, Master School of Medicine New York University, PhD School of Medicine

Biology Biology Biology

A.3. Indicators of Quality in Scientific Production (See the instructions) - Total number of citations: 5040 (WOK); 5930 (Google Scholar Citation) - Mean number citation /year: 306 (2010-2014) (WOK) 420 (GSC) -Total number publications: 103/97 en PubMed - Q1 Publications: 86 - H index: 42 (WOK); 45 (Google Scholar Citation) - Sexenios: 5 (last 2008-2013) - Quinquenios CSIC: 6 (last 2007-2011) - Number of supervised PhD thesis: 11 Part B. Free Summary of CV (Max. of 3.500 characters, including spaces) Paola Bovolenta is Research Professor and Head of the Development and Differentiation Dept at the Center for Molecular Biology Severo Ochoa, (CSIC-UAM). She obtained her PhD degree at the New York University, School of Medicine, NY under the supervision of Prof. CA Mason. She worked as a postdoctoral fellow at Columbia University, Center for Neurobiology and Behaviour, NY under the supervision of Profs. T. Jessell and J, Dodd. She then moved to the Instituto Cajal, CSIC, Madrid, first under the supervision of ProF. M. Nieto-Sampedro and after directing her own research group. She also spent long training periods at the DIBIT in Milan and in the ENS in Paris. Since 1996 she directs her research group aimed at the understanding of the molecular mechanisms underlying early 1 of 4 / Short CV

 

Short CV Before proceeding, please read carefully the instructions at the end of the document.

development of the nervous system with particular focus on those processes that may lead to congenital defects and neurodegenerative diseases. She is author of about 100 publications, most of them in high impact journals of her field and she is often invited to international symposia and conferences. She has been the PI of about 30 research grants obtained in national and international competitive calls. She acts as reviewer for national and international granting agencies and for major journals, including, Nature, Science, Nature Neuroscience, Neuron, Cell and Dev. Cell. She acts as European Editor of the journal Developmental Neurobiology and is member of the editorial board of different journals and is a Member of the Faculty of 1000. She is currently Treasurer of the SEBD, member of the Scientific Advisory Committee of the Institut de la Vision in Paris, of the Neuroscience Committee of the ANR, of the International Affairs Committee of the SfN and of the selection committee of the “The Remedio Caro Amela Prize” and “Premios Fundación Biogen Idec Jóvenes Investigadores”. She acted as a member of the Comunication Committee of FENS, of the Telethon Advisory Board and of the AERES in France. Between 2008-2012 she acted as Head of the Biology and Biomedicine area of the Spanish Research Council (CSIC). She received the Fundaluce Price in 2009 and 2012. In 2012 she has been elected member of EMBO. Her early work set the ground to identify the mechanisms of visual axon sorting at the chiasm and the role of the floor plate in commissural axon guidance and ventral neural tube specification. She also established the inhibitory role of specific proteoglycans on axon elongation and regeneration and defined the contribution of neurotrophins in the determination of retinal ganglion cell number. Her lab has thereafter focused on the role of transcription factors and cell signaling pathways, defining essential mechanisms for the specification and differentiation of ocular structures, including the retina pigmented epithelium, the neural retina and the optic disc. Her lab also provided the first demonstration that Sonic hedgehog acts as an axon guidance cue, thus contributing to open a new area of research related to the role of morphogenetic signaling pathways in brain wiring. She further identified novel roles for the family of the Secreted Frizzled Related Proteins -initially characterized as Wnt inhibitors- as Frizzled ligands, modulators of Wnt diffusion and regulators of ADAM10 metalloprotease activity, thus defining and important link between Wnt and Notch signaling. Her lab is currently expanding the latter line of research to neurodegeneration. Part C. Accomplishments (Order by typology) C.1. Publications Marcos S., Nieto-Lopez F., Sandonis A., Di Marco, F., Cardozo M., Esteve, P. and Bovolenta P. (2015) Secreted Frizzled Related Proteins modulate pathfinding and fasciculation of mouse retina ganglion cell axons by direct and indirect mechanisms. J. Neurosci. 35, 4729-4740 (cover caption article) Cardozo M., Sánchez-Arrones L., Sandonis A., Sánchez-Camacho C., Gestri G., Wilson SW, Guerrero, I. and Bovolenta P. (2014) Cdon acts as a Hedgehog decoy receptor during proximal-distal patterning of the optic vesicle. Nature Comm. 5:4272. doi: 10.1038/ncomms5272. (Selected in The Faculty of 1000) Cdon and Boc are cell surface glycoproteins implicated in Hegehog (Hh) signalling. Heteromeric interaction between either one of these proteins and the Hh receptor Patched (Ptc) is responsible for high-affinity Hh binding and presentation of Ptc at the cell surface. This manuscript together with the J. Neurosci. listed below demonstrates that Cdon and Boc, when expressed in the absence of the Ptc recptor, act as a Hh decoy receptors, thereby limiting pathway activation. Sanchez-Arrones, L.*, Nieto-Lopez, F.*, Sanchez-Camacho, C., Carreres M.I., Herrera, E., Okada, A. and Bovolenta, P. (2013) Shh/Boc signaling is required for sustained generation of ipsilateralprojecting ganglion cells in the mouse retina. J. Neurosci. 33, 8596-8607. (Featured article). *Equally contributing. Esteve P., Sandonìs A., Cardozo M., Malapeira J., Ibañez C., Crespo I., Marcos S., Gonzalez-Garcia S., Toribio M.L., Arribas J., Shimono A., Guerrero I. and Bovolenta P. (2011). Sfrps act as negative modulators of ADAM10 to regulate retinal neurogénesis. Nat. Neurosci. 14, 562-569. (Selected in The Faculty of 1000) Secreted Frizzled Related Proteins have been characterized as Wnt modulators. In this manuscript we provide evidence that Sfrps are negative modulators of ADAM10, metalloprotease involved in the activation of Notch signalling. The manuscript defines important links between Wnt and Notch signalling during retinal development. It further raises the possibility that Sfrp1 might be involved in

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Short CV Before proceeding, please read carefully the instructions at the end of the document.

the regulation of additional ADAM10 substrates, including APP. This opened a new line of research in Alzheimer Disease that we are currently and successfully pursuing. Esteve, P., Sandonìs A., Ibañez C., Shimono A., Guerrero I. and Bovolenta P. (2011) Secreted Frizzled-Related Proteins are required for Wnt/βcatenin signalling activation in the vertebrate optic cup. Development, 138, 4179-4184. (Highlighs in “In this issue Development”) Bovolenta P, Esteve P., Ruiz J.M., Cisneros E. and Lopez-Rios, J. (2008) Beyond Wnt inhibition: new functions of secreted Frizzled-related proteins in development and disease. J. Cell Sci. 121: 737-746 This is a highly cited review (287, GSC) on the Wnt non-related functions of SFRPs, one of the most recent and successful lines of research of our lab. Sánchez-Camacho C. and Bovolenta P. (2008) Autonomous and non-autonomous Shh signalling mediate the in vivo growth and guidance of mouse retina ganglion cell axons. Development, 135, 3531-3540 (Cover caption article and Highlighs “In this issue”). In 2001 our laboratory provided the first demonstration that Sonic hedgehog acts as an axon guidance cue, contributing to open a new area of research related to the role of morphogenetic signaling pathways in brain wiring. This manuscript provides additional insight of this Shh function demonstrating a novel cell autonomous mechanism by which Shh regulates the growth of retinal ganglion cell axons. Morcillo J., Martinez-Morales, J.R, Trousse, F., Fermin Y., Sowden J. and Bovolenta P. (2006). Proper patterning of the optic fissure requires the sequential activity of Bmp7 and Shh. Development 133, 3179-3190. (Highlighs in “In this issue Development” and in Nat. Rev. Neurosc. 2006, 7, 684; Selected in The Faculty of 1000) In this manuscript we demonstrate that progenitors of the optic disc have unique properties that distinguish them from cell of the optic stalk and that the development of OD progenitors depends on BMP7. Microphthalmia seen in the Bmp7-null mice resembles a rare congenital condition known as true aplasia of the optic nerve, indicating that absence of BMP7 might be the cause of such malformations in humans. Rodríguez, J., Esteve, P., Weinl, C., Ruiz, J.M., Fermin Y., Trousse, F., Dwivedy A, Holt C.E. and Bovolenta P. (2005) SFRP1 regulates the growth of retinal ganglion cell axons through the Fz2 receptor. Nat Neurosci. 8, 1301-1309. (News and Views 8, 1281-1282) Sfrp1 was considered an inhibitor of Wnt signalling. This manuscript provides the first demonstration that, independently of its interaction with Wnt, Sfrp1 can act as a repulsive guidance molecule for retinal axons on their way to the tectum, signalling through the Fz2 receptor. Thus, it demonstrates that Sfrp1 can act as a positive modulator of non-canonical Wnt signalling. The article was resigned with a N&V in the journal in which it was published. Martinez-Morales, J.R, Del Bene, F., Nica G., Hammerschmidt M., Bovolenta P.* and Wittbrodt, J.* (2005) Differentiation of the vertebrate retina is coordinated by an FGF signaling center. Dev Cell 8, 565-574. *co-senior authors (Selected in The Faculty of 1000) This manuscript demonstrates that the onset of retinal neurogenesis in vertebrates depends for a species-specific signalling center activated by Fgf signalling. C.2. Projects Estudio de Sfrp1 como un nuevo componente que contribuye a la patogénesis de la enfermedad de Alzheimer. Fundación Tatiana Pérez de Guzmán el Bueno. 2015-2017. Paola Bovolenta (108.900€) Generando diversidad neuronal. MINECO 2015-2016. (BFU2014-55738-REDT). IP. Paola Bovolenta. (32.000 €) Control de la comunicación celular: desde el neurodesarrollo a la neurodegeneracion. MINECO 20142016. IP. Paola Bovolenta and Pilar Esteve (508.200 €) Linajes y competición celular en el desarrollo y la enfermedad (CEL-DD). Comunidad Autonoma de Madrid (CAM, S2010/BMD-2315). 2011-2014. IP. Gines Morata. Paola Bovolenta (PI partner). 540.000 € (total, 5 grupos) Regulación de la morfogénesis del sistema visual: papel de vías de señalización celular y redes transcripcionales. MICINN (BFU2010-16031). 2011- 2013. IP. Paola Bovolenta (544.500 €) Estudio de la función del dominio NTR de Sfrp1 en la degeneración de los fotorreceptores en modelos de distrofias retinianas. Fundación ONCE. 2012-2013. Paola Bovolenta and Pilar Esteve (10.000 €)

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C.3. Contracts CIBER en Enfermedades Raras (CIBERER). Instituto de Salud Carlos III. U-709. IP. Paola Bovolenta. 2007-actualmente COST Action. ECMNET “Brain Extracellular Matrix in Health and Diseases”. UE. BM1001. (20112014) IP. Dityatev Alexander. Paola Bovolenta (Representante español) C.4. Patents “Efecto mitógeno de Sonic hedgehog (Shh) sobre precursores de oligodendrocitos y su uso en enfermedades desmielinizantes”. nº 200600697. Inventores. Fernando de Castro (US), Paola Bovolenta (CSIC), Paloma Merchán (US), Cristina Sánchez-Camacho (CSIC), Ana Bribián (US). “Método de diagnóstico de la enfermedad de Alzheimer que emplea Sfrp1 como biomarcador". PCT/ES2012/070226. Inventores: Paola Bovolenta, Pilar Esteve, África Sandonis, Inmaculada Crespo, Isabel Guerrero, Carmen Ibáñez, Jordi Malapeira, Joaquín Arribas y Akiko Shimono. C.5 Recent Honor and Distinctions 2000 2009 2012 2012

Invited Professorship, Ecole Normale Superioure. Paris, Francia Premio a la Investigación “Fundaluce” Premio a la Investigación “Fundaluce” Miembro Electo de la European Molecular Biology Organization (EMBO)

C.6 Editorial Activity European Editor for “Developmental Neurobiology” (July 2008-present) Contributing Editor for the European Journal of Neuroscience (July 2008- present) Miembro del Editorial Board de “Development” (desde Septiembre 2005-2013) Contributing Faculty Member en la Sección de Neurodesarrollo, de la Sección de Neurociencia de la “Faculty of 1000”. Responsables de la sección: Andrew Lumsden and Joshua Sanes. Noviembre de 2010-pres. Miembro del Editorial Board de “Frontiers in Neuroscience” (desde Octubre 2008-presente) Miembro del Editorial Board del “The Tohoku J Exp. Medicine” (desde Enero 2002) Miembro del Editorial Board de “Developmental Neurobiology” (anteriormente Journal of Neurobiology) (Noviembre 2001-June 2008) Miembro Editorial Board de The Open Cell Development & Biology Journal. (Octubre 2008-pres) Guest Editor: Journal of Neurobiology Special Issue “Unexpected roles for morphogens in the development and regeneration of the CNS” (issue de 15 September, 2005) C.7 Experience in I+D administration: Scientific Advisory Committee of the Institut de la Vision in Paris (2011-present) Scientific Advisory Committee "Institut de Biologie de Dev de Marseille (IDBM)" (2014-present) Society for Neuroscience International Affairs Committee (IAC). (2011-present) Comité de Selección Premio Nacional de Investigación “Santiago Ramón y Cajal” (2014) Tesorera de la Sociedad Española de Biologia del Desarrollo (SEBD) (2011-presente) Comité de Selección “The Remedio Caro Amela Prize for Res in Dev Neurobiology” (2011-present) Comité de Selección de los “Premios Fundación Biogen Idec Jóvenes Investigadores” (2011-presente) Coordinadora del Área de Biología y Biomedicina (Área 2), CSIC- (June 2008-May2012) Coordinadora Adjunta del Área de Biología y Biomedicina (Área 2), CSIC (May 2012-2014) Miembro del FENS Comunication/Publication Commitee. 2009-2013 Adjunto ANEP. Área de BMC. (2004-2008) Miembro del panel de Selección “LS4–Neurosciences” European Research Council (ERC) Starting Grants. (2007-2010) Miembro del Scientific Commitee Telethon Combatti la Distrofia Muscolare e altre Malattie Genetiche, Italy (2006-2009). Miembro del panel de Selección ANR -SVSE4-Neurosciences. Paris, 2010, 2012, 2013 Scientific Commitee Site Visit: Centre de Biochimie, Université Nice, Nice, January 201 4 of 4 / Short CV

IMPRESO NORMALIZADO DE CURRICULUM VITAE

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FECHA DE CUMPLIMENTACION: 15 de julio de 2015 APELLIDOS: MADRID CUEVAS NOMBRE: JUAN FRANCISCO SEXO: Varón Nº D.N.I.: 27.446.029-Z FECHA DE NACIMIENTO: 24-6-1964 DIRECCION PARTICULAR: Plaza Fuensanta 1, 5ºB TELEFONO: 968935904 CODIGO POSTAL: 30005 LOCALIDAD: Murcia PROVINCIA: Murcia ESPECIALIZACION CODIGO UNESCO: 2 4 0 7

F O R M A C I O N A C A D E M I C A Licenciatura/Ingenería Centro Fecha Ldo. Ciencias Biológicas Universidad de Murcia 1987 Doctorado Doctor en Biología Universidad de Murcia 1989 DIRECTOR(ES) DE TESIS: Francisco Hernández Calvo y José Ballesta Germán SITUACION PROFESIONAL ACTUAL (cargo, categoría, etc.): Catedrático de Universidad. ORGANISMO: Universidad de Murcia CENTRO: FACULTAD DE MEDICINA DEPT/SECC/UNIDAD ESTR: Biología Celular e Histología DIRECCION POSTAL: 30100 Espinardo - MURCIA TELEFONO ( Indicar prefijo, ciudad y extensión): 868884691 CONTRATO INTERINO OTRAS SITUACIONES (especificar) PLANTILLA X BECARIO

EXCEDENCIA JUBILADO

ACTIVIDADES ANTERIORES DE CARACTER CIENTIFICO O PROFESIONAL Fechas Sep 87/sep-91 Oct- 91/ene-01 Ene-01/Ene-07 Ene-07/Actualidad

Puesto Profesor Colaborador Profesor Titular Universidad Profesor Titular Universidad Catedrático Universidad

Institución Universidad de Murcia Universidad del País Vasco Universidad de Murcia Universidad de Murcia

IDIOMAS (R = regular; B = bien; C = correctamente) HABLA LEE R B C Inglés Francés

x

ESCRIBE

R B C x

R B C x

PUBLICACIONES (no incluir proceeding ni abstracts de congresos) CLAVES: ( CLAVE: L = libro completo, CL = capítulo de libro, A = artículo, R = “review”, E = editor, S = Documento Científico-Técnico restringido. ) AUTORES : (Consignese por orden de firma) CLAVE: A L.M. Pastor, J. Ballesta, R. Pérez-Tomás, J.A. Marín, F. Hernández, and J.F. Madrid TITULO: Immunocytochemical localization of serotonin in the reptilian lung. REVISTA/LIBRO : Cell and Tissue Research Vol.: 248 Pág.: 713 a 715 Año: 1987 EDITOR: País y ciudad: Alemania CODIGO UNESCO:

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AUTORES : (Consignese por orden de firma) CLAVE: A L.M. Pastor, J. Ballesta, J.F. Madrid, R. Pérez-Tomás, and F. Hernández. TITULO: A histochemical study of the mucins in the digestive tract of the chicken REVISTA/LIBRO : Acta Histochemica . Vol.: 83 Pág.: 91 a 97 Año: 1988 EDITOR: Hoachim Scharff País y ciudad: Alemania CODIGO UNESCO:

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AUTORES : (Consignese por orden de firma) CLAVE: A L.M. Pastor, J. Ballesta, M.T. Castells, R. Pérez-Tomás, J.F. Madrid and J.A. Marín. TITULO: A light and electron microscopic study of the epithelium of the extrapulmonary airways of Mauremys caspica and Lacerta lepida (Reptilia). REVISTA/LIBRO : Journal of Submicroscopy Cytology and Pathology Vol.: 20 Pág.: 25 a 36 Año: 1988 EDITOR: País y ciudad: Italia CODIGO UNESCO:

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AUTORES : (Consignese por orden de firma) CLAVE: A J.F. Madrid, J. Ballesta, L.M. Pastor, R. Pérez-Tomás and F. Hernández TITULO: Distribution of mucins in the mucosa of the digestive tract of reptiles. A histochemical study REVISTA/LIBRO : Acta Histochemica Vol.: 85 Pág.: 117 a 129 Año: 1989 EDITOR: Hoachim Scharff País y ciudad: Alemania CODIGO UNESCO: 2 4 0 7

AUTORES : (Consignese por orden de firma) CLAVE: A L.M. Pastor, J. Ballesta, M.T. Castells, R. Pérez-Tomás, J.A. Marín and J.F. Madrid. TITULO: A light and electron microscopic study of the lung of Testudo graeca (Chelonia). REVISTA/LIBRO : Journal of Anatomy Vol.: 164 Pág.: 19 a 39 EDITOR: País y ciudad: Reino Unido CODIGO UNESCO: 2 4 0 7

Año: 1989

AUTORES : (Consignese por orden de firma) CLAVE: A J.F. Madrid, J. Ballesta, M.T. Castells, J.A. Marín and L.M. Pastor. TITULO: Characterization of glycoconjugates in the intestinal mucosa of vertebrates by lectin histochemistry. REVISTA/LIBRO : Acta Histochemica et Cytochemica Vol.: 22 Pág.: 1 a 14 Año: 1989 EDITOR: País y ciudad: Japón CODIGO UNESCO:

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AUTORES : (Consignese por orden de firma) CLAVE: A J.F. Madrid, J. Ballesta, T. Galera, M.T. Castells, and R. Pérez-Tomás. TITULO: Histochemistry of glycoconjugates in the gallbladder epithelium of ten animal species. REVISTA/LIBRO : Histochemistry Ahora, Histochemistry and Cell Biology Vol.: 91 Pág.: 437 a 449 Año: 1989 EDITOR: T.H. Schiebler País y ciudad: Alemania CODIGO UNESCO:

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AUTORES : (Consignese por orden de firma) CLAVE: A R. Pérez-Tomás, J. Ballesta, L.M. Pastor, J.F. Madrid and J.M. Polak. TITULO: A comparative study of the gastroenteropancreatic (GEP) endocrine system of three reptiles. REVISTA/LIBRO : General and Comparative Endocrinology Vol.: 76 Pág.: 171 a 179 Año: 1989 EDITOR: País y ciudad: USA CODIGO UNESCO: 2 4 0 7 AUTORES : (Consignese por orden de firma) CLAVE: A M.T. Castells, J. Ballesta, L.M. Pastor, J.F. Madrid and J.A. Marín. TITULO: Histochemical Characterization of Glycoconjugates in the Epithelium of the Extrapulmonary Airways or Several Vertebrates. REVISTA/LIBRO : Histochemical Journal Vol.: 22 Pág.: 24 a 35 Año: 1990 EDITOR: País y ciudad: Reino Unido CODIGO UNESCO:

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AUTORES : (Consignese por orden de firma) CLAVE: A R. Pérez-Tomás, J. Ballesta, J.F. Madrid, L.M. Pastor and F. Hernández. TITULO: Histochemical and ultrastructural study of the digestive tract of the tortoise Testudo graeca (Testudines). REVISTA/LIBRO : Journal of Morphology Vol.: 204 Pág.: 235 a 245 Año: 1990 EDITOR: País y ciudad: USA CODIGO UNESCO: 2 4 0 7 AUTORES : (Consignese por orden de firma) CLAVE: A J.F. Madrid, J. Ballesta, M.T. Castells, M. Avilés, J.A. Martínez-Menárguez and F. Hernández. TITULO: Characterization of carbohydrate residues in the digestive tract of the rabbit by means of lectins and conventional histochemistry. REVISTA/LIBRO : Histologia médica Vol.: 6 Pág.: 63 a 72 Año: 1990 EDITOR: Pérez de Vargas País y ciudad: Málaga, España CODIGO UNESCO:

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AUTORES : (Consignese por orden de firma) CLAVE: A J.F. Madrid, J. Ballesta, M.T. Castells and F. Hernández. TITULO: Glycoconjugate distribution in the human fundic mucosa revealed by lectin- and glycoproteingold cytochemistry. REVISTA/LIBRO : Histochemistry Ahora, Histochemistry and Cell Biology Vol.: 95 Pág.: 179 a 187 Año: 1990 EDITOR: T.H. Schiebler País y ciudad: Alemania CODIGO UNESCO:

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AUTORES : (Consignese por orden de firma) CLAVE: A M.T. Castells, J. Ballesta, J.F. Madrid, F. Hernández, J.A. Martínez-Menárguez and M. Avilés. TITULO: Histochemistry of glycoconjugates in the lung of several vertebrates. REVISTA/LIBRO : Zoologische Jahrbucher Anatomie Vol.: 120 Pág.: 331 a 346 Año: 1990 EDITOR: País y ciudad: Alemania CODIGO UNESCO:

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AUTORES : (Consignese por orden de firma) J. Figols, J.F. Madrid and J. Cervós-Navarro. TITULO: Lectins as differentiation marker of human gliomas. REVISTA/LIBRO : Histology and Histopathology Vol.: 6 Pág.: 79 a 85 Año: 1991 EDITOR: F. Hernández País y ciudad: Murcia, España CODIGO UNESCO: 2 4 0 7

CLAVE: A

AUTORES : (Consignese por orden de firma) CLAVE: A M.T. Castells, J. Ballesta, J.F. Madrid, M. Avilés and J.A. Martínez-Menárguez. TITULO: Characterization of glycoconjugates in developing rat respiratory system by means of lectin histochemistry. REVISTA/LIBRO : Histochemistry Ahora, Histochemistry and Cell Biology Vol.: 95 Pág.: 419 a 426 Año: 1991 EDITOR: T.H. Schiebler País y ciudad: Alemania CODIGO UNESCO: 2 4 0 7 AUTORES : (Consignese por orden de firma) CLAVE: A J. Ballesta, J.A. Martínez-Menárguez, L.M. Pastor, M. Avilés, J.F. Madrid and M.T. Castells. TITULO: Lectin binding pattern in the testes of several tetrapode vertebrates. REVISTA/LIBRO : European Journal of Basic and Applied Histochemistry . Ahora, European Journal of Histochemistry Vol.: 35 Pág.: 107 a 117 Año: 1991 EDITOR: Manfredi Romanini País y ciudad: Italia CODIGO UNESCO: 2 4 0 7 AUTORES : (Consignese por orden de firma) CLAVE: A M.T. Castells, J. Ballesta, J.F. Madrid, J.A. Martínez-Menárguez and M. Avilés. TITULO: Ultrastructural localization of glycoconjugates in human bronchial glands: The subcellular organization of N- and O-linked oligosaccharide chains REVISTA/LIBRO : Journal of Histochemistry and Cytochemistry Vol.: 40 Pág.: 265 a 274 Año: 1992 EDITOR: País y ciudad: USA CODIGO UNESCO:

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AUTORES : (Consignese por orden de firma) CLAVE: A J.A. Martínez-Menárguez, J. Ballesta, M. Avilés, J.F. Madrid and M.T. Castells. TITULO: Influence of sulphate groups in the binding of Peanut agglutinin (PNA). Histochemical demonstration with light and electron microscopy. REVISTA/LIBRO : Histochemical Journal Vol.: 24 Pág.: 207 a 216 Año: 1992 EDITOR: País y ciudad: Reino Unido CODIGO UNESCO:

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AUTORES : (Consignese por orden de firma) CLAVE: A J.A. Martínez-Menárguez, J. Ballesta, M. Avilés, M.T. Castells and J.F. Madrid. TITULO: Cytochemical characterization of glycoproteins in the developing acrosome of rat. An ultrastructural study using lectin histochemistry, enzymes and chemical deglycosylation REVISTA/LIBRO : Histochemistry. Ahora, Histochemistry and Cell Biology Vol.: 98 Pág.: 265 a 274 Año: 1992 EDITOR: J Roth País y ciudad: Alemania CODIGO UNESCO:

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AUTORES : (Consignese por orden de firma) CLAVE: A J.A. Martínez-Menárguez, M. Avilés, J.F. Madrid, M. T. Castells and J. Ballesta. TITULO: Glycosilation in Golgi apparatus of early spermatids of rat. A high resolution lectin cytochemical study. REVISTA/LIBRO : European Journal of Cell Biology Vol.: 61 Pág.: 21 a 33 Año: 1993 EDITOR: País y ciudad: Reino Unido CODIGO UNESCO: 2 4 0 7 AUTORES : (Consignese por orden de firma) CLAVE: A M.T. Castells, J.F. Madrid, M. Avilés, J.A. Martínez-Menárguez and J. Ballesta. TITULO: Cytochemical characterization of sulpho- and sialo-glycoconjugates of human laryngeal glandular cells. REVISTA/LIBRO : Journal of Histochemistry and Cytochemistry Vol.: 42 Pág.: 485 a 496 Año: 1994 EDITOR: País y ciudad: USA CODIGO UNESCO: 2 4 0 7 AUTORES : (Consignese por orden de firma) CLAVE: A M. Avilés, J.A. Martínez-Menárguez, M.T. Castells, J.F. Madrid and J. Ballesta. TITULO: Cytochemical characterization of oligosaccharide side chains of the glycoproteins of rat zona pellucida. An ultrastructural study. REVISTA/LIBRO : Anatomical Record Vol.: 239 Pág.: 137 a 149 Año: 1994 EDITOR: País y ciudad: USA CODIGO UNESCO:

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AUTORES : (Consignese por orden de firma) CLAVE: A J.F. Madrid, M.T. Castells, J.A. Martínez-Menárguez, M. Avilés, F. Hernández and J. Ballesta. TITULO: Subcellular characterization of glycoproteins in the principal cells of human gallbladder. A lectin cytochemical study. REVISTA/LIBRO : Histochemistry. Ahora, Histochemistry and Cell Biology Vol.: 101 Pág.: 195 a 204 Año: 1994 EDITOR: J. Roth País y ciudad: Alemania CODIGO UNESCO:

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AUTORES : (Consignese por orden de firma) CLAVE: R J.F. Madrid, F. Hernández y J. Ballesta. TITULO: Characterization of glycoproteins in the epithelial cells of human and other mammals gallbladder. REVISTA/LIBRO : Microscopy Research and Technique Vol.: 38 Pág.: 616 a 630 Año: 1997 EDITOR: País y ciudad: USA CODIGO UNESCO:

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AUTORES : (Consignese por orden de firma) CLAVE: A O. Leis, J.F. Madrid, J. Ballesta and F. Hernández TITULO: N- and O-linked oligosaccharides in the secretory granules of rat Paneth cells. An ultrastructural cytochemical study REVISTA/LIBRO : Journal of Histochemisry and Cytochemistry Vol.: 45 Pág.: 285 a 293 Año: 1997 EDITOR: País y ciudad: USA CODIGO UNESCO: 2 4 0 7 AUTORES : (Consignese por orden de firma) CLAVE: A Y. Robledo, J.F. Madrid, O. Leis y M.P. Cajaraville TITULO: Analysis of the distribution of glycoconjugates in the digestive gland of the bivalve mollusc Mytilus galloprovincialis by conventional and lectin histochemistry REVISTA/LIBRO : Cell and Tissue Research Vol.: 288 Pág.: 591 a 602 Año: 1997 EDITOR: País y ciudad: Alemania CODIGO UNESCO: 2 4 0 7 AUTORES : (Consignese por orden de firma) CLAVE: A L. Díaz-Flores, H. Alvarez-Argüelles, J.F. Madrid, H. Varela, M.P. González and R. Gutiérrez TITULO: Satellite cells in a peculiar form of skin granular cell tumor. Perineurial cell tumor (perineuroma) with granular cells. REVISTA/LIBRO : Journal of Cutaneous Pathology Vol.: 24 Pág.: 575 a 579 Año: 1997 EDITOR: País y ciudad: USA CODIGO UNESCO:

2 4 0 7

AUTORES : (Consignese por orden de firma) CLAVE: CL J.F. Madrid, F. Hernández and J. Ballesta. TITULO: Amplification of lectin-gold histochemistry. REVISTA/LIBRO : Methods in Molecula Medicine Series. Lectins in Medical Research Vol.: Pág.: 121 a 131 Año: 1998 EDITOR: Rhodes J.M. and Milton J.D. Humana Press. País y ciudad: Reino Unido CODIGO UNESCO: 2 4 0 7 AUTORES : (Consignese por orden de firma) CLAVE: A J.F. Madrid, L. Díaz-Flores, R. Gutiérrez, H. Varela, F. Valladares, N. Rancel and F. Rodríguez TITULO: Participation of angiogenesis from rat femoral veins in the neovascularization of adjacent occluded arteries. REVISTA/LIBRO : Histology and Histopathology Vol.: 13 Pág.: 1 a 11 Año: 1998 EDITOR: País y ciudad: España CODIGO UNESCO:

2 4 0 7

AUTORES : (Consignese por orden de firma) CLAVE: A J.F. Madrid, O. Leis, L. Díaz-Flores y F. Hernández TITULO: Secretion of fucosylated oligosaccharides related with the H-antigen by the human gastric cells REVISTA/LIBRO : Histochemistry and Cell Biology Vol.: 110 Pág.: 295 a 301 Año: 1998 EDITOR: País y ciudad: USA CODIGO UNESCO: 2 4 0 7 AUTORES : (Consignese por orden de firma) CLAVE: A M.I. Arenas, J.F. Madrid, F.R. Bethencourt, B. Fraile y R. Paniagua TITULO: Identification of N- and O-linked oligosaccharides in the human epididymis. REVISTA/LIBRO : Journal of Histochemistry and Cytochemistry Vol.: 46 Pág.: 1185 a 1188 Año: 1998 EDITOR: Baskin País y ciudad: USA CODIGO UNESCO:

2 4 0 7

AUTORES : (Consignese por orden de firma) CLAVE: A J.F. Madrid, O. Leis, L. Díaz-Flores, F.J. Sáez and F. Hernández TITULO: Lectin-gold localization of fucose residues in human gastric mucosa REVISTA/LIBRO : Journal of Histochemistry and Cytochemistry Vol.: 46 Pág.: 1311 a 1320 Año: 1998 EDITOR: Baskin País y ciudad: USA CODIGO UNESCO:

2 4 0 7

AUTORES : (Consignese por orden de firma) M.I. Arenas, J.F. Madrid, F.R. Bethencourt, B. Fraile y R. Paniagua TITULO: Lectin histochemical study of the human testes REVISTA/LIBRO : International Journal of Andrology Vol.: 21 Pág.: 332 a 342 Año: 1998 EDITOR: País y ciudad: CODIGO UNESCO:

CLAVE: A

AUTORES : (Consignese por orden de firma) M.I. Arenas, J.F. Madrid, F.R. Bethencourt, B. Fraile y R. Paniagua TITULO: Lectin histochistry study in the human vas deferens REVISTA/LIBRO : Glycoconjugate Journal Vol.: 15 Pág.: 1085 a 1091 Año: 1998 EDITOR: País y ciudad:

CLAVE: A

CODIGO UNESCO:

AUTORES : (Consignese por orden de firma) CLAVE: A F.J. Sáez, J.F. Madrid, R. Aparicio, O. Leis and B. Oporto. TITULO: Lectin histochemical localization of N- and O-linked oligosaccharides during the spermiogenesis of the urodele amphibian Pleurodeles waltl. REVISTA/LIBRO : Glycoconjugate Journal Vol.: 16 Pág.: 639 a 648 Año: 1999 EDITOR: País y ciudad: CODIGO UNESCO:

AUTORES : (Consignese por orden de firma) CLAVE: A J.F. Madrid, R. Aparicio, F.J. Sáez and G. Hernández. TITULO: Lectin cytochemical characterization of the N- and O-linked oligosaccharides in the human rectum. REVISTA/LIBRO : Histochemical Journal Vol.: 32 Pág.: 281 a 289 Año: 2000 EDITOR: País y ciudad: CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: A F.J. Sáez, J.F. Madrid, R. Aparicio, O. Leis and F. Hernández TITULO: Glycan residues of N- and O-linked oligosaccharides in the premeiotic spermatogenetic cells of the urodele amphibian pleurodeles waltlcharacterized by means of lectin histochemistry. REVISTA/LIBRO : Tissue and Cell Vol.: 32 Pág.: 302 a 311 Año: 2000 EDITOR: País y ciudad: CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: A F.J. Sáez, J.F. Madrid, E. Alonso y F. Hernández TITULO: Lectin histochemical identification of the carbohydrate moieties on N- and O-linked oligosaccharides in the duct cells of the testis of an amphibian urodele, the Sapnish newt (Pleurodeles waltl). REVISTA/LIBRO : Histochemical Journal Vol.: 32 Pág.: 717 a 724 Año: 2000 EDITOR: País y ciudad: CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: A F.J. Sáez, J.F. Madrid, R. Aparicio, F. Hernández and E. Alonso. TITULO: Carbohydrate moieties of the interstitial and glandular tissues of the amphibian Pleurodeles waltl testis showed by lectin histochemistry. REVISTA/LIBRO : Journal of Anatomy Vol.: 198 Pág.: 47 a 56 Año: 2001 EDITOR: País y ciudad: CODIGO UNESCO:

AUTORES : (Consignese por orden de firma) CLAVE: A L. Díaz-Flores, J.F. Madrid, R. Gutiérrez, F. Valladares, M. Díaz, H. Varela y L. Díaz-Flores Jr. TITULO: Arterial wall neovascularization induced by glycerol REVISTA/LIBRO : Histology and Histopathology Vol.: 16 Pág.: 1175 a 1181 EDITOR: País y ciudad:

Año: 2001

CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: A F.J. Sáez, J.F. Madrid, E. Alonso and F. Hernández TITULO: Glycan composition of follicle (Sertoli) cells of the amphibian Pleurodeles waltl. A lectin cytochemical study REVISTA/LIBRO : Journal of Anatomy Vol.: 198 Pág.: 673 a 681 Año: 2001 EDITOR: País y ciudad: CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: A E. Alonso, F.J. Sáez, J.F. Madrid y F. Hernández TITULO: Galactosides and sialylgalactosides in the O-linked oligosaccharides of the primordial germ cells in Xenopus embryos. REVISTA/LIBRO : Glycoconjugate Journal Vol.: 18 Pág.: 225 a 230 Año: 2001 EDITOR: País y ciudad: CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: A E. Alonso, F.J. Sáez, J.F. Madrid y F. Hernández TITULO: GalNAc moieties in O-linked oligosaccharides of the primordial cells of Xenopus embryos REVISTA/LIBRO : Histochemistry and Cell Biology Vol.: 117 Pág.: 345 a 349 Año: 2002 EDITOR: País y ciudad: CODIGO UNESCO:

AUTORES : (Consignese por orden de firma) CLAVE: A E. Alonso, F.J. Saez, J.F. Madrid and F. Hernández TITULO: Lectin histochemistry show fucosylated glycoconjugates in the primordial germ cells of Xenopus embryos REVISTA/LIBRO : Journal of Histochemistry and Cytochemistry Vol.: 51 Pág.: 239 a 243 Año: 2003 EDITOR: País y ciudad: USA CODIGO UNESCO:

AUTORES : (Consignese por orden de firma) CLAVE: A B. García Pérez, I. Ayala, M.T. Castells, J.F. Madrid, M.R. Ortega, J.V. Ortega, J. Ballesta y M. Valdés TITULO: Planimetric and histological study of he aortae in atherosclerotic chickens treated with nifedipine, verapamil and diltiazem REVISTA/LIBRO : Histology and Histopathology Vol.: 18 Pág.: 1027 a 1033 Año: 2003 EDITOR: País y ciudad: CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: R F.J. Sáez, J.F. Madrid, S. Cardoso, L. Gómez and F. Hernández TITULO: Glycoconjugates in the urodele amphibian testis showed by lectin cytochemical methods - A Review REVISTA/LIBRO : Microscopic Research and Technique Vol.: 64 EDITOR: País y ciudad:

Pág.: 63

a 76

Año: 2004

CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: R L. Diaz-Flores Jr., J.F. Madrid, R. Gutiérrez, H. Varela, F. Valladares, H. Alvarez-Argüelles and L. DíazFlores TITULO: Adult stem and transit-ampifying cell locaton REVISTA/LIBRO : Histology and Histopathology Vol.: 21 Pág.: 995 a 1027 Año: 2006 EDITOR: País y ciudad: CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: A E. Alonso. L. Gómez, J.F. Madrid and F.J. Sáez TITULO: Identification of mannose moieties in N- and O-linked oligosaccharides of the primordial germ cells of Xenopus embryos REVISTA/LIBRO : Microscopic Research and Technique Vol.: 69 Pág.: 595 a 599 Año: 2006 EDITOR: País y ciudad: CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: A Gomez-Santos L, Alonso E, Ferrer C, Zuasti A, Saez FJ, Madrid JF. TITULO: Histochemical demonstration of two subtypes of O-linked oligosaccharides in the rat gastric glands REVISTA/LIBRO : Microscopic Research and Technique Vol.: 70 Pág.: 809 a 815 Año: 2007 EDITOR: País y ciudad: CODIGO UNESCO:

AUTORES : (Consignese por orden de firma) CLAVE: A Diaz-Flores L Jr, Madrid JF, Gutierrez R, Varela H, Valladares F, Diaz-Flores L. TITULO: Cell contribution of vasa-vasorum to early arterial intimal thickening formation REVISTA/LIBRO : Histology and Histopathology Vol.: 22 Pág.: 1379 a 1386 EDITOR: País y ciudad:

Año: 2007

CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: A Valbuena G, Hernández F, Madrid JF, Sáez FJ. TITULO: biosynthesis in spermatocytes and spermatids revealed by HPA lectin cytochemistry REVISTA/LIBRO : Anatomical Record Vol.: 291 Pág.: 1097 a 1015 EDITOR: País y ciudad: CODIGO UNESCO:

Año: 2008

AUTORES : (Consignese por orden de firma) CLAVE: R Diaz-Flores L, Gutiérrez R, Madrid JF, Varela H, Valladares F,Acosta E, Martín-Vasallo f and DíazFlores L Jr TITULO: Pericytes. Morphofunction, interactions and pathology in a quiescent and activated mesenchymal cell niche REVISTA/LIBRO : Histology and Histopathology Vol.: 24 Pág.: 909 a 969 EDITOR: País y ciudad:

Año: 2009

CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: R Díaz-Flores L Jr, Gutiérrez R, Madrid JF, Varela H, Valladares F and Díaz-Flores L TITULO: Adult stem cells and repair through granulation tissue REVISTA/LIBRO : Frontiers in Bioscience Vol.: 14 Pág.: 1433 a 1470 EDITOR: País y ciudad:

Año: 2009

CODIGO UNESCO: AUTORES : (Consignese por orden de firma) Alonso A, Gómez-Santos L, Madrid JF and Sáez FJ TITULO: The expression of a novel cxcr4 gene in Xenopus embryo REVISTA/LIBRO : Histology and Histopathology Vol.: 24 Pág.: 1097 a 1103 EDITOR: País y ciudad: CODIGO UNESCO:

Año: 2009

CLAVE: A

AUTORES : (Consignese por orden de firma) CLAVE: Díaz-Flores L, Gutiérrez R, Madrid JF, Alvarez-Argüelles H, Valladares F, Spreafico M, Díaz-Flores L. TITULO: Choroid plexus papilloma with stromal deposition of amyloid and elastic material REVISTA/LIBRO : Amyloid Vol.: 17 Pág.: 69 a 74 Año: 2010 EDITOR: País y ciudad: CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: Valbuena G, Madrid JF, Hernández F, Sáez FJ. TITULO: Identification of fucosylated glycoconjugates in Xenopus laevis testis by lectin histochemistry. REVISTA/LIBRO : Histochemistry and Cell Biology Vol.: 134 Pág.: 215 a 225 Año: 2010 EDITOR: País y ciudad: CODIGO UNESCO: AUTORES : (Consignese por orden de firma) Valbuena G, Alonso E, Díaz-Flores L Jr, Madrid JF, Sáez FJ.

CLAVE:

TITULO: Identification of N-acetylgalactosamine in carbohydrates of Xenopus laevis testis. REVISTA/LIBRO : Anatomical Record Vol.: 294 Pág.: 363 a 371 EDITOR: País y ciudad:

Año: 2011

CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: Díaz-Flores L Jr, Gutiérrez R, Madrid JF, Sáez FJ, Valladares F, Villar J, Díaz-Flores L. TITULO: Peg-and-socket junctions between smooth muscle cells and endothelial cells in femoral veins are stimulated to angiogenesis by prostaglandin E₂ and glycerols. REVISTA/LIBRO : Histology and Histopathology Vol.: 26 Pág.: 623 a 630 EDITOR: País y ciudad:

Año: 2011

CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: Valbuena G, Alonso E, Madrid JF, Díaz-Flores L, Sáez FJ. TITULO: Galactosides in glycoconjugates of Xenopus laevis testis shown by lectin histochemistry REVISTA/LIBRO : Microscopy Research and Techniques Vol.: 74 EDITOR: País y ciudad:

Pág.: 778

CODIGO UNESCO:

a 787

Año: 2011

AUTORES : (Consignese por orden de firma) CLAVE: Díaz-Flores L, Gutiérrez R, García MP, Alvarez-Argüelles H, Díaz-Flores L Jr, Madrid JF. TITULO: Myopericytoma and arterial intimal thickening: the relationship between myopericytes and myointimal cells. REVISTA/LIBRO : Journal of Cutaneous Pathology Vol.: 38 Pág.: 857 a 864 Año: 2011 EDITOR: País y ciudad: CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: Díaz-Flores L, Gutiérrez R, García MP, Díaz-Flores L Jr, Valladares F, Madrid JF TITULO: Ultrastructure of myopericytoma: a continuum of transitional phenotypes of myopericytes. REVISTA/LIBRO : Ultrastructural Pathology Vol.: 36 Pág.: 189 a 194 Año: 2012 EDITOR: País y ciudad: CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: Diaz-Flores L Jr, Gutierrez R, Madrid JF, Acosta E, Avila J, Diaz-Flores L, Martin-Vasallo P TITULO: Cell sources for cartilage repair; contribution of the mesenchymal perivascular niche. REVISTA/LIBRO : Frontiers in Bioscience (Scholar edition) Vol.: 4 Pág.: 1275 a 1294 Año: 2012 EDITOR: País y ciudad: CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: Valbuena G, Alonso E, de Ubago MM, Madrid JF, Díaz-Flores L, Sáez FJ. TITULO: Histochemical identification of sialylated glycans in Xenopus laevis testis REVISTA/LIBRO : Journal of Anatomy Vol.: 221 Pág.: 318 a 330 Año: 2012 EDITOR: País y ciudad: CODIGO UNESCO: AUTORES : (Consignese por orden de firma) Díaz-Flores L, Gutiérrez R, Sáez FJ, Díaz-Flores L Jr, Madrid JF. TITULO: Telocytes in neuromuscular spindles. REVISTA/LIBRO : Journal of Cellular and Molecular Medicine Vol.: 17 Pág.: 457 a 465 Año: 2013 EDITOR: País y ciudad: CODIGO UNESCO:

CLAVE:

AUTORES : (Consignese por orden de firma) CLAVE: Seco-Rovira V, Beltrán-Frutos E, Ferrer C, Sánchez-Huertas M, Madrid J, Saez F, Pastor L. TITULO: Lectin Histochemistry as a Tool to Identify Apoptotic Cells in the Seminiferous Epithelium of Syrian Hamster (Mesocricetus auratus) Subjected to Short Photoperiod. REVISTA/LIBRO : Reproduction Domestic Animal Vol.: 48 Pág.: 974 a 983 Año: 2013 EDITOR: País y ciudad: CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: Díaz-Flores L, Gutiérrez R, García MP, Sáez FJ, Díaz-Flores L Jr, Valladares F, Madrid JF TITULO: CD34+ stromal cells/fibroblasts/fibrocytes/telocytes as a tissue reserve and a principal source of mesenchymal cells. Location, morphology, function and role in pathology. REVISTA/LIBRO : Histology and Histopathology Vol.: 29 Pág.: 831 a 870 EDITOR: País y ciudad:

Año: 2014

CODIGO UNESCO: AUTORES : (Consignese por orden de firma) Seco-Rovira V, Beltrán-Frutos E, Ferrer C, Sáez FJ, Madrid JF, Pastor LM.

CLAVE:

TITULO: The death of sertoli cells and the capacity to phagocytize elongated spermatids during testicular regression due to short photoperiod in Syrian hamster (Mesocricetus auratus). REVISTA/LIBRO : Biology of Reproduction Vol.: 90 Pág.: 107 a EDITOR: País y ciudad:

Año: 2014

CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: Díaz-Flores L, Gutiérrez R, García MP, Sáez FJ, Aparicio F, Díaz-Flores L Jr, Madrid JF. TITULO: Uptake and intracytoplasmic storage of pigmented particles by human CD34+ stromal cells/telocytes: endocytic property of telocytes. REVISTA/LIBRO : Journal of Cellular and Molecular Medicine Vol.: 18 EDITOR: País y ciudad:

Pág.: 2478

CODIGO UNESCO:

a 2487

Año: 2014

AUTORES : (Consignese por orden de firma) CLAVE: Díaz-Flores L, Gutiérrez R, Lizartza K, Goméz MG, García Mdel P, Sáez FJ, Díaz-Flores L Jr, Madrid JF. TITULO: Behavior of in situ human native adipose tissue CD34+ stromal/progenitor cells during different stages of repair. Tissue-resident CD34+ stromal cells as a source of myofibroblasts. REVISTA/LIBRO : Vol.: 298 Pág.: 917 EDITOR: País y ciudad:

a 930

Año: 2014

CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: Díaz-Flores L, Gutiérrez R, García MP, González M, Sáez FJ, Aparicio F, Díaz-Flores L Jr, Madrid JF. TITULO: Human resident CD34+ stromal cells/telocytes have progenitor capacity and are a source of αSMA+ cells during repair. REVISTA/LIBRO : Histology and Histopathology Vol.: 30 Pág.: 615 a 627 EDITOR: País y ciudad:

Año: 2015

CODIGO UNESCO: AUTORES : (Consignese por orden de firma) CLAVE: Seco-Rovira V, Beltrán-Frutos E, Ferrer C, Saez FJ, Madrid JF, Canteras M, Pastor LM. TITULO: Testicular histomorphometry and the proliferative and apoptotic activities of the seminiferous epithelium in Syrian hamster (Mesocricetus auratus) during regression owing to short photoperiod. REVISTA/LIBRO : Andrology Vol.: 3 Pág.: 598 EDITOR: País y ciudad:

a 610

Año: 2015

CODIGO UNESCO: AUTORES : (Consignese por orden de firma)

CLAVE:

TITULO: REVISTA/LIBRO : Vol.: Pág.: EDITOR: País y ciudad:

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Año:

CODIGO UNESCO: AUTORES : (Consignese por orden de firma)

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TITULO: REVISTA/LIBRO : Vol.: Pág.: EDITOR:

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Participación en proyectos de investigación financiados 1.- "Filogenia, ontogenia y control nervioso de las secreciones glucoproteicas en los sistemas respiratorio y digestivo". Facultad de Medicina. Universidad de Murcia. IP: José Ballesta Financiado por la Dirección General de Investigación Científica y Técnica (DGICY T). Nº del proyecto: PB87-0701 Duración: 1988-1991 Cuantia: 2.100.00pts Número de integrantes del grupo: 5 2.- “Patrón de unión de lectinas en el sistema neuroendocrino difuso y sus tumores”. Facultad de Medicina. Universidad de Murcia. IP: Francisco Hernández Ayuda de Investigación del Fondo de Investigaciones Sanitarias de la Seguridad Social (FISS). Nº del proyecto: 88/0993 Duración: 1988. Cuantia: 500.000pts Número de integrantes del grupo: 4 3.- "Localización ultraestructural de glucoproteínas en el tracto digestivo y respiratorio de la rata wistar empleando lectinas marcadas con oro coloidal. Facultad de Medicina. Universidad de Murcia. IP: José Ballesta Ayuda de Investigación de la Comunidad Autónoma de la Región de Murcia. Nº del proyecto: 90/5 Duración: 1991. Cuantia: 1.250.000pts Número de integrantes del grupo: 5 4.- "Estudio citoquímico de los procesos de glucosilación proteica en los sistemas digestivo y respiratorio humano". Facultad de Medicina. Universidad de Murcia. IP: José Ballesta Proyecto financiado por la Dirección General de Investigación Científica y Técnica (DGICYT). Nº del proyecto: PB90-0310 Duración: 1991-1994. Cuantia: 5.050.000pts Número de integrantes del grupo: 4

5.- "Preservación de injertos en un modelo de autotransplante experimental en perro". Facultad de Medicina. Universidad del País Vasco. IP: Jaime Méndez Proyecto financiado por el FIS. Nº del proyecto:93/0703 Duración: 1994-1997. Cuantia: 10.000.000 Número de integrantes del grupo: 10 6.- "Biología celular y molecular de la interfase nucleocitoplásmica". Facultad de Medicina. Universidad del País Vasco. IP: Juan Aréchaga Proyecto financiado por la Universidad del País Vasco. Nº del proyecto: UPV075.310-EB14/92 Duración: 1992-1995 Cuantia: 4.900.000pts Número de integrantes del grupo: 6 7.- "Papel del los carbohidratos en el bloqueo de la poliespermia durante el proceso de la fertilización". Facultad de Medicina. Universidad del País Vasco. IP: Juan F. Madrid Proyecto co-financiado por la Dirección General de Investigación Científica y Técnica (DGICYT) y por la Universidad del País Vasco. Nº del proyecto: PB93-1123-C02-02 y UPV075.327-EC236/95 Duración: 1994-1997. Cuantia: 6.200.000pts Número de integrantes del grupo: 4 8.- "Estudio citoquímico con lectinas de las secuencias oligosacarídicas de las células germinales primordiales (CGPs)" Facultad de Medicina. Universidad del País Vasco. IP: Francisco J. Sáez Financiado por la Universidad del País Vasco. Nº del proyecto: UPV075.310-EA137/97 Duración: 1997-1998. Cuantia: 1.500.000pts Número de integrantes del grupo: 3 9.- "Estudio mediante citoquímicaa con lectinas de los glicoconjugados del testículo del anfibio urodelo Pleurodeles Waltl: Formación del acrosoma. IP: Francisco J. Sáez Financiado por el Gobierno Vasco Nº del proyecto: PI-1997-48 Facultad de Medicina. Universidad del País Vasco. Duración: 1998-2000. Cuantia: 5.700.000pts Número de integrantes del grupo: 4

10.- “Estudio microscópico del proceso de diferenciación de las células principales del estómago humano y de rata” IP: Juan F. Madrid Financiado por la Dirección General de Enseñanza Universitaria (MECD) Nº del proyecto: PM98-0067 Duración: 1999-2002 Cuantia: 7.000.000pts Número de integrantes del grupo: 5 11.- “Ayuda a Grupos Competitivos” Entidad financiadora: Universidad del País Vasco Entidades participantes: Universidad del País Vasco, Duración, desde: 1999-01-01 hasta: 2000-12-31 Cuantía de la subvención: 9.000.000pts Investigador responsable: Juan Francisco Madrid Cuevas Número de investigadores participantes: 5 12.- “Caracterización citoquímica de las secreciones glicoproteicas gástricas en el estado adulto y durante el desarrollo e identificación de los factores de crecimiento implicado” IP: Juan F. Madrid Financiado por la Fundación Séneca, dependiente de la comunicad Autónoma de la Región de Murcia Nº del proyecto: PI-88/00836/FS/01 Duración: 2002-2004 Cuantia: 32.395,55 euros Número de integrantes del grupo: 5 13.- “Evaluación histológica, histoquímica e inmunocitoquímica de un modelo animal de inducción de úlcera gástrica en estado adulto y lactante IP: Juan F. Madrid Financiado por el FIS (Fondo de Investigación Sanitaria) Nº del proyecto: PI-021653 Duración: 2002-2005 Cuantia: 105.340 euros Número de integrantes del grupo: 7 14.- Miembro del grupo de Investigación de Calidad subvencionado por la Comunidad Autónoma de la Región de Murcia Duración: 1-1-08 – 31-12-12 Cuantía: 300.000 euros Número de integrantes: 12

Participación en contratos de I+D de especial relevancia con Empresas y/o Administraciones (nacionales y/o internacionales) Título del contrato/proyecto: Publicación HISTOLOGY AND HISTOPATHOLOGY

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revista

internacional

revista

internacional

revista

internacional

revista

internacional

Tipo de contrato: Empresa/Administración financiadora: Fundación Séneca Entidades participantes: Universidad de Murcia Duración, desde: abril 2004 hasta: abril 2005 Investigador responsable: Juan F Madrid Número de investigadores participantes: 2 PRECIO TOTAL DEL PROYECTO: 9.000 euros Título del contrato/proyecto: Publicación HISTOLOGY AND HISTOPATHOLOGY

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Tipo de contrato: Empresa/Administración financiadora: Fundación Séneca Entidades participantes: Universidad de Murcia Duración, desde: abril 2005 hasta: abril 2006 Investigador responsable: Juan F Madrid Número de investigadores participantes: 2 PRECIO TOTAL DEL PROYECTO: 12.000 euros Título del contrato/proyecto: Publicación HISTOLOGY AND HISTOPATHOLOGY

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Tipo de contrato: Empresa/Administración financiadora: Fundación Séneca Entidades participantes: Universidad de Murcia Duración, desde: abril 2006 hasta: abril 2007 Investigador responsable: Juan F Madrid Número de investigadores participantes: 2 PRECIO TOTAL DEL PROYECTO: 12.000 euros Título del contrato/proyecto: Publicación HISTOLOGY AND HISTOPATHOLOGY

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Tipo de contrato: Empresa/Administración financiadora: Fundación Séneca Entidades participantes: Universidad de Murcia Duración, desde: septiembre de 2007 hasta: septiembre de 2008 Investigador responsable: Juan F Madrid Número de investigadores participantes: 1 PRECIO TOTAL DEL PROYECTO: 12.000 euros

Título del contrato/proyecto: Publicación HISTOLOGY AND HISTOPATHOLOGY

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internacional

Tipo de contrato: Empresa/Administración financiadora: Fundación Séneca Entidades participantes: Universidad de Murcia Duración, desde: enero de 2009 hasta: diciembre de 2009 Investigador responsable: Juan F Madrid Número de investigadores participantes: 1 PRECIO TOTAL DEL PROYECTO: 12.000 euros Título del contrato/proyecto: Publicación HISTOLOGY AND HISTOPATHOLOGY

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Tipo de contrato: Empresa/Administración financiadora: Fundación Séneca Entidades participantes: Universidad de Murcia Duración, desde: enero de 2010 hasta: diciembre de 2010 Investigador responsable: Juan F Madrid Número de investigadores participantes: 1 PRECIO TOTAL DEL PROYECTO: 12.000 euros

internacional

CONGRESOS (Comunicaciones orales o posters) Congresos nacionales 1.-

Estudio comparativo del epitelio faveolar de Testudo graeca en estado activo y de hibernación. L.M. Pastor, J. Ballesta, R. Pérez-Tomás, F. Hernández y J.F. Madrid. IV Congreso Nacional de Histología. Granada. Junio, 1985.

2.-

Development of the peptidergic innvervation in the blood vessels of the wistar rats. J. Ballesta, L.M. Pastor, F. Hernández y J.F. Madrid. First Congress of the Spanish Society for Cell Biology. Madrid. 12-14 December, 1985.

3.-

Localización y distribución de mucinas en el tracto digestivo de diversos reptiles. R. Pérez-Tomás, L.M. Pastor, J.F. Madrid y J. Ballesta. IV Reunión Nacional de la Sociedad Española de Histoquímica. Valencia. 24-26 Abril, 1986.

4.-

Determinación histoquímica de las mucinas epiteliales en la mucosa digestiva de Gallus gallus. L.M. Pastor, R. Pérez-Tomás, J.F. Madrid y J. Ballesta. IV Reunión de la Sociedad Española de Histoquímica. Valencia. 24-26 Abril, 1986.

5.-

Estudio histoquímico de las mucinas epiteliales de las vías aéreas de Testudo graeca, Pseudemys scripta elegans y Gallus gallus. L.M. Pastor, J.F. Madrid, R. Pérez-Tomás y J. Ballesta. IV Reunión Nacional de la Sociedad Española de Histoquímica. Valencia. 24-26 Abril, 1986.

6.-

Estudio microscópico del epitelio laringotraqueal de Lacerta lepida. L.M. Pastor, R. Pérez-Tomás, J.F. Madrid, F. Hernández y J. Ballesta. Congreso Nacional de Microscopía Electrónica. Avila. 29-31 Octubre, 1986.

7.-

Estudio microscópico del epitelio del intestino grueso de Testudo graeca. R. Pérez-Tomás, L.M. Pastor, J.F. Madrid, F. Hernández y J. Ballesta. Congreso Nacional de Microscopía Electrónica. Avila. 29-31 Octubre, 1986.

8.-

Estudio inmunocitoquímico y ultraestructural del pulmón de Testudo graeca (Chelonia). L.M. Pastor, J. Ballesta, M.T. Castells y J.F. Madrid. V Congreso Nacional de Histología. Santiago de Compostela. 1-4 Julio, 1987.

9.-

Identificación de células endocrinas en el aparato digestivo de Testudo graeca con inmunocitoquímica, microscopía electrónica e inmunomicroscopía electrónica. J. Ballesta, R. Pérez-Tomás, L.M. Pastor, J.F. Madrid y F. Hernández. V Congreso Nacional de Histología. Santiago de Compostela, 1-4 Julio, 1987.

10.- Estudio histológico e histoquímico de la lengua de diversos vertebrados. R. Pérez-Tomás, J. Ballesta, M. Carvajal, J.F. Madrid y L.M. Pastor. V Congreso Nacional de Histología. Santiago de Compostela. 1-4 Julio, 1987. 11.- Localización de glucoconjugados en la mucosa esofago-gástrica de diversos vertebrados empleando lectinas. J.F. Madrid, J. Ballesta, T. Galera, L.M. Pastor y R. Pérez-Tomás. V Congreso Nacional de Histología. Santiago de Compostela. 1-4 Julio, 1987. 12.- Identificación de los componenetes glucídicos de la mucosa intestinal de diversos vertebrados utilizando lectinas. J.F. Madrid, J. Ballesta, L.M. Pastor, R. Pérez-Tomás y A. Zuasti. V Congreso Nacional de Histología. Santiago de Compostela. 1-4 Julio, 1987. 13.- Estudio de la distribución de glucoconjugados en las vesículas biliares de diversos vertebrados utilizando lectinas. J.F. Madrid, T. Galera, J.A. Martínez-Menárguez, J. Ballesta y F. Hernández. V Congreso Nacional de Histología. Santiago de Compostela. 1-4 Julio, 1987. 14.- Inmunocytochemical localization of Neuromidin U in the rat central nervous system. J. Ballesta, S.J. Gibson, J.H. Steel, J.F. Madrid y J. Polak. Second Congress of the Spanish Society for Cell Biology. Barcelona. 16-19 December, 1987. 15.- Lectin-binding pattern in testis and epididymis of several non-mammalian vertebrates. L.M. Pastor, J.A. Martínez-Menárguez, J.F. Madrid y J. Ballesta. Second Congress of the Spanish Society for Cell Biology. Barcelona. 16-19 December, 1987. 16.- Lectin histochemistry of the esophageal and gastric mucosa of vertebrates. J.F. Madrid, J. Ballesta, L.M. Pastor y T. Galera. Second Congress of the Spanish Society for Cell Biology. Barcelona. 16-19 December, 1987. 17.- Lectin binding pattern in the intestinal mucosa of several vertebrates. J.F. Madrid, J. Ballesta, L.M. Pastor y F. Hernández. Second Congress of the Spanish Society for Cell Biology. Barcelona. 16-19 December, 1987. 18.- Estudio de la evolución de los glucoconjugados en el pancreas de rata durante el desarrollo. J.F. Madrid, M. Carvajal, M.T. Castells y J. Ballesta. VI Congreso Nacional de Histología. Cadiz. 7-10 Junio, 1989. 19.- Estudio ontogenético de los carbohidratos de la mucosa esofago-gástrica de la rata. J.F. Madrid, T. Galera y J. Ballesta. VI Congreso Nacional de Histología. Cadiz. 7-10 Junio, 1989.

20.- Caracterización de glucoconjugados en la vejiga urinaria de rata durante el desarrollo. J.F. Madrid, T. Galera, M.T. Castells y J. Ballesta VI Congreso Nacional de Histología. Cadiz. 7-10 Junio, 1989. 21.- Identificación de carbohidratos en el tracto digestivo de reptiles utilizando lectinas y técnicas convencionales de mucinas. J.F. Madrid, J. Marín y J. Ballesta. VI Congreso Nacional de Histología. Cadiz. 7-10 Junio, 1989. 22.- Estudio histoquímico ultraestructural de las células de Paneth. J. Ballesta, J.F. Madrid y R. Pérez-Tomás. VI Congreso Nacional de Histología. Cadiz. 7-10 Junio, 1989. 23.- Caracterización de carbohidratos en el intestino de rata durante el desarrollo. Aplicación de técnicas convencionales y lectinas. J.F. Madrid y J. Ballesta. VI Congreso Nacional de Histología. Cadiz. 7-10 Junio, 1989. 24.- Identificación de receptores glucídicos mediante la aplicación de neoglucoconjugados en el tracto digestivo de la rata wistar. J.F. Madrid, J. Ballesta y J.A. Martínez-Menárguez. VI Congreso Nacional de Histología. Cadiz. 7-10 Junio, 1989. 25.- Identificación simultanea de glucoconjugados y lisozima en las glándulas pilóricas y de Brunner humanas. J.F. Madrid, F. Hernández y J. Ballesta. VI Congreso Nacional de Histología. Cadiz. 7-10 Junio, 1989. 26.- Caracterización ultraestructural de glucoconjugados en la vesícula biliar humana. R. Pérez-Tomás, J.F. Madrid, F. Hernández y J. Ballesta. VI Congreso Nacional de Histología. Cadiz 7-10 Junio, 1989. 27.- Localización ultraestructural de lisozima y glucoproteinas en la mucosa bronquial human. Relación con los procesos de N- y O-glucosilación. M.T. Castells, J.F. Madrid, J. Garrido, F. Hernández y J. Ballesta. XVI Reunión bienal de la Sociedad Española de Microscopía Electrónica. Cadiz, 10-13 Diciembre, 1990. 28.- Influencia de la presencia de radicales sulfato en la unión de la lectina peanut agglutinin (PNA). J.A. Martínez-Menárguez, A. Avilés, J.F. Madrid y J. Ballesta. XVI Reunión bienal de la Sociedad Española de Microscopía Electrónica. Cadiz, 10-13 Diciembre, 1990. 29.- Caracterización histoquímica ultraestructural de residuos glucídicos en la mucosa laríngea humana. M.T. Castells, J.F. Madrid, J.A. Martínez-Menárguez y J. Ballesta. XVI Reunión bienal de la Sociedad Española de Microscopía Electrónica. Cadiz, 10-13 Diciembre, 1990.

30.- Localización ultraestructural de residuos GlNAc, GalNAc, Man y Glc en las células principales y parietales de la mucosa fúndica humana. J.F. Madrid, M.T. Castells, M. Avilés, F. Hernández y J. Ballesta. XVI Reunión bienal de la Sociedad Española de Microscopía Electrónica. Cadiz, 10-13 Diciembre, 1990. 31.- Estudio ultraestructural de las secreciones glucoproteicas de las células superficiales y mucosas del cuello del estómago humano. J.F. Madrid, M.T. Castells, J.V. Ortega, F. Hernández y J. Ballesta. XVI Reunión bienal de la Sociedad Española de Microscopía Electrónica. Cadiz, 10-13 Diciembre, 1990. 32.- Caracterización citoquímica de las glucoproteínas presentes en la zona pelúcida. M. Avilés, J.A. Martínez-Menárguez, J.F. Madrid, F. Hernández y J. Ballesta. VII Congreso Nacional de Histología. Lugo, 11-13 Septiembre, 1991. 33.- Caracterización de N- y O-glucosil residuos en el epitelio traqueobronquial de la rata wistar. M.T. Castells, J.F. Madrid, F. Hernández y J. Ballesta. VII Congreso Nacional de Histología. Lugo, 11-13 Septiembre, 1991. 34.- Localización citoquímica de sialoglucoconjugados en el epitelio laríngeo humano. M.T. Castells, J.F. Madrid, J.A. Martínez-Menárguez y J. Ballesta. VII Congreso Nacional de Histología. Lugo, 11-13 Septiembre, 1991. 35.- Diferenciación ultraestructural de los tipos celulares de la mucosa gástrica de ratas lactantes mediante el marcaje con lectinas. J.F. Madrid, M.T. Castells, C. Belda y J. Ballesta. VII Congreso Nacional de Histología. Lugo, 11-13 Septiembre, 1991. 36.- Secuenciación citoquímica de los carbohidratos de las glucoproteínas epiteliales de las vesícula biliar humana. J.F. Madrid, M.T. Castells, M. Avilés, F. Hernández y J. Ballesta. VII Congreso Nacional de Histología. Lugo, 11-13 Septiembre, 1991. 37.- Estudio histoquímico ultraestructural de los fotorreceptores de rana (Rana perezi) utilizando lectinas. J.A. Marín, J.F. Madrid, M.T. Castells y J. Ballesta. VII Congreso Nacional de Histología. Lugo, 11-13 Septiembre, 1991. 38.- Estudio citoquímico de los procesos de glucosilación en el aparato de Golgi durante la espermiogénesis. J.A. Martínez-Menárguez, M. Avilés, J.F. Madrid, F. Hernández y J. Ballesta. VII Congreso Nacional de Histología. Lugo, 11-13 Septiembre, 1991. 39.- Lectin binding properties of the lysosomal compartment of bivalve digestive cells. Y. Robledo, J.F. Madrid, J. Ballesta, E. Angulo y M.P. Cajaraville. V Congreso Nacional de la Sociedad Española de Biología Celular. Badajoz, 14-17 diciembre, 1993.

40.- Caracterización citoquímica de N- y O-glucoproteínas en células de Paneth. O. Leis, J.F. Madrid, J. Ballesta y F. Hernández. XVII Reunión Bienal de la Sociedad Española de Microscopía Electrónica. Oviedo, 5-8 abril, 1995. 41.- Origin of the chief cells of the human fundic glands. J.F. Madrid, O. Leis, R. Aparicio y M.J. González-Díaz. I Congreso de la Sociedad Española de Biología del Desarrollo. Bilbao, 7-11 diciembre, 1996. 42.- Secreción del antígeno H en el estómago humano independiente del status secretor. J.F. Madrid, O. Leis, y F. Hernández. IX Congreso Nacional de Histología. Pamplona, 4-6 julio, 1997. 43.- Identificación histoquímica de los carbohidratos del intestino grueso. R. Aparicio, O. Leis, F. Hernández y J.F. Madrid IX Congreso Nacional de Histología. Pamplona, 4-6 julio, 1997. 44.- Estudio histoquímico con lectinas de los glicoconjugados del testículo de Pleurodeles Waltl (Amphibia, caudata). F.J. Sáez, J.F. Madrid, O. Leis y J. Aréchaga. IX Congreso Nacional de Histología. Pamplona, 4-6 julio, 1997. 45.- Localización del receptor del epidermal growth factor (EGFR) en las células de Paneth. O. Leis, R. Aparicio y J.F. Madrid. IX Congreso Nacional de Histología. Pamplona, 4-6 julio, 1997. 46.- Reflexiones sobre la histología. F. Hernández y J.F. Madrid. IX Congreso Nacional de Histología. Pamplona, 4-6 julio, 1997. 47.- Los residuos de fucosa se localizan en el estómago humano en O- y en N-oligosacáridos. J.F. Madrid, O. Leis y F. Hernández. VII Congreso Nacional de la Sociedad Española de Biología Celular. Córdoba, 15-19 septiembre, 1997. 48.- Los gránulos de secreción de las células de Paneth presentan dos compartimentos de composición oligosacarídica distinta. O. Leis, R. Aparicio y J.F. Madrid. VII Congreso Nacional de la Sociedad Española de Biología Celular. Córdoba, 15-19 septiembre, 1997. 49.- Localización de factores de crecimiento (EGF y TGF-α) en las células de Paneth. O. Leis, J.F. Madrid, R. Aparicio, F.J. Sáez y F. Hernández. XIX Reunión Bienal de la Sociedad Española de Microscopía Electrónica. Murcia, 28-30 abril, 1999.

50.- Identificación de manosa en el acrosoma de Pleurodeles waltl durante la espermiogenesis. F.J. Sáez, J.F. Madrid, R. Aparicio y O. Leis. X Congreso Nacional de la Sociedad Española de Histología. Alicante, 18-20 noviembre, 1999. 51.- Localización ultrastructural de residuos de ácido siálico unidos por enlace α(2-6) en las glándulas fúndicas del estómago humano. J.F. Madrid, F.J. Sáez, O. Leis, R. Aparicio and F. Hernández. X Congreso Nacional de la Sociedad Española de Histología. Alicante, 18-20 noviembre, 1999. 52.- (Sialil)galactósidos en O-oligosacáridos de las células germinales primordiales (CGPs) del embrión de Xenopus. E. Alonso, M. Sierra,, F.J. Sáez, J.F. Madrid y F. Hernández XI Congreso Nacional de la Sociedad Española de Histología. La Rábida (Huelva), 1114 septiembre, 2001 53.- Composición oligosacarídica de las secreciones mucosas fúndicas de rata. L. Gómez-Santos, G. Larrauri, J.F. Madrid, F.J. Sáez y A. Delgado. XI Congreso Nacional de la Sociedad Española de Histología. La Rábida (Huelva), 1114 septiembre, 2001 54.- Patrón de unión de lectinas en las células principaes y parietales del estómago de rata. L. Gómez-Santos, G. Larrauri, J.F. Madrid, F.J. Sáez y A. Delgado. XI Congreso Nacional de la Sociedad Española de Histología. La Rábida (Huelva), 1114 septiembre, 2001 55.- Identificación de los carbohidratos de los glicoconjugados de las células caliciformes de pacientes con enfermedad celíaca. G. Larrauri, L.Gómez-Santos, J.F. Madrid, F.J. Sáez, E. Martín-Bejarano y A. Delgado. XI Congreso Nacional de la Sociedad Española de Histología. La Rábida (Huelva), 1114 septiembre, 2001 56.- Patrón de unión de lectinas en los enterocitos de pacientes con enfermedad celíaca. G. Larrauri, L.Gómez-Santos, J.F. Madrid, F.J. Sáez, E. Martín-Bejarano y A. Delgado. XI Congreso Nacional de la Sociedad Española de Histología. La Rábida (Huelva), 1114 septiembre, 2001 57.- Identificación de N-acetilgalactosamina en cadenas O-glicosídicas en las células germinales primordiales (CGPs) del embrión de Xenopus. E. Alonso, M. Sierra, J.F. Madrid, y F. Hernández XI Congreso Nacional de la Sociedad Española de Histología. La Rábida (Huelva), 1114 septiembre, 2001 58.- Sobre el posible origen común de las células foliculares y de los conductos eferentes de urodelos. F.J. Sáez, J.F. Madrid, E. Alonso, M. Sierra y F. Hernández XI Congreso Nacional de la Sociedad Española de Histología. La Rábida (Huelva), 1114 septiembre, 2001 59.- Modificaciones en la composición glicídica de las células testiculares productoras de esteroides de urodelos F.J. Sáez, J.F. Madrid, E. Alonso, M. Sierra y F. Hernández XI Congreso Nacional de la Sociedad Española de Histología. La Rábida (Huelva), 1114 septiembre, 2001

60.- Incorporación de N-acetilgalactosamina a los glicoconjugados acrosómicos duratne la espermiogénesis de Pleurodeles walt. M. Sierra, E. Alonso, F.J. Sáez, J.F. Madrid y F. Hernández. XI Congreso Nacional de la Sociedad Española de Histología. La Rábida (Huelva), 1114 septiembre, 2001 61.- Modificaciones de los glicoconjugados fucosilados en el acrosomoa del urodelo Pleurodeles walt. M. Sierra, E. Alonso, F.J. Sáez, J.F. Madrid y F. Hernández. XI Congreso Nacional de la Sociedad Española de Histología. La Rábida (Huelva), 1114 septiembre, 2001 62.- Caracterización de los carbohidratos sulfatados de las secreciones mucosas del intestino grueso humano. J.F. Madrid, F.J. Sáez, L. Gómez-Santos, G. Larrauri y F. Hernández. XI Congreso Nacional de la Sociedad Española de Histología. La Rábida (Huelva), 1114 septiembre, 2001 63.- Caracterización de oligosacáridos unidos por enlace de tipo N y tipo O en las secreciones mucosas del intestino grueso humano. J.F. Madrid, F.J. Sáez, G. Larrauri, L. Gómez-Santos y F. Hernández. XI Congreso Nacional de la Sociedad Española de Histología. La Rábida (Huelva), 1114 septiembre, 2001 64.- Identificación de glicoconjugados con N-Acetil-Galactosamina en el acrosoma del urodelo Pleurodeles wwaltl F.J. Sáez, S. Cardoso, M. Hervella, E. Fernaández, M. Sierra y J.F. Madrid. XII Congreso Nacional de la Sociedad Española de Histología e Ingeniería Tisular. Valencia 17-20 septiembre, 2003 65.- Identificación de galactosa en oligosacáridos de la mucosa gástrica de rata. L. Gómez, J.F. Madrid, F.J. Sáez y F. Hernández X Congreso de la Sociedad Española de Biología Celular. Santander, 18-21 diciembre, 2003. 66.- Distribución de N-acetilgalactosamina en el acrosoma del espermatozoide de Pleurodeles waltl. M. Hervella, F.J. Sáez, E. Fernández, L. Gómez y J.F. Madrid X Congreso de la Sociedad Española de Biología Celular. Santander, 18-21 diciembre, 2003. 67.- Localización de N-acetilgalactosamina en los glicoconjugados de las vesículas precursoras del acrosoma durante la espermiogénesis de Pleurodeles waltl. E. Fernández, F.J. Sárz, M. Hervella, L. Gómez y J.F. Madrid X Congreso de la Sociedad Española de Biología Celular. Santander, 18-21 diciembre, 2003. 68.- Identificación de lGalNAc en oligosacáridos de las células germinales primordiales embrionarias de Xenopus laevis. E. Alonso, F.J. Sáez, J.F. Madrid y F.J. Sáez X Congreso de la Sociedad Española de Biología Celular. Santander, 18-21 diciembre, 2003.

69. - Parietal cells show a different maturation pattern depending on their location in the gland J.F. Madrid, L. Gómez-Santos, F. Hernández, L. Díaz-Flores and F.J. Sáez XIII Congreso de la Sociedad Española de Biología Celular. Murcia. 18-20 de diciembre de 2009. 70.- Changes in Leydig cell and pericyte population of the Syrian hamster during testicular regression after exposure to short photoperiod E. Beltrán-Frutos, V. Seco-Rovira, M. Canteras, C. Ferre, J.F. Madrid, F.J. Sáez, D Llor y L.M. Pastor 12º Congreso Internacional de la Asociación Española de Reproducción Animal (AERA). Alicante, 16-18 de octubre de 2014. 71.- Determination by proteomic analysis of some overexpressed proteins in testes of regressed hámster subjected to short photoperiod V. Seco-Rovira, E. Beltrán-Frutos, C. Ferrer, J.F. Madrid, F.J. Sáez y LM Pastor 12º Congreso Internacional de la Asociación Española de Reproducción Animal (AERA). Alicante, 16-18 de octubre de 2014. 72.- Presence of GAP junctions in Leydig cells in the Syrian hamster during aging and after exposure to short photoperiod E. Beltrán-Frutos, V. Seco-Rovira, C. Ferrer, J.F. Madrid, F.J. Sáez, D. Llor y L.M. Pastor 12º Congreso Internacional de la Asociación Española de Reproducción Animal (AERA). Alicante, 16-18 de octubre de 2014. 74.- Apoptosis of Sertoli cells and elimination of late spermatids during testicular regression due to short photoperiod V. Seco-Rovira, E. Beltrán-Frutos, C. Ferrer, J.F. Madrid, F.J. Sáez y L.M. Pastor 12º Congreso Internacional de la Asociación Española de Reproducción Animal (AERA). Alicante, 16-18 de octubre de 2014.

Congresos internacionales 1.- Ultrastructural localization of lectin binding sites in human and rat Paneth cells. J. Ballesta, J.F. Madrid, R.Pérez-Tomás y M.T. Castells. Congreso Ibérico de Microscopía Electrónica. Lisboa. 14-16 Diciembre, 1988. 2.- Cytochemical localization of glycoconjugates in human bronchial glands using lectingold complexes. M.T. castells, J.F. Madrid, J.A. Martínez-Menárguez, M. Avilés y J. Ballesta. Interlec 13. Berlin. August 1991. 3.- Lectin cytochemistry of the epithelial glycoproteins of human gallbladder. J.F. Madrid, M.T. Castells, M. Avilés, J.A. Martínez-Menárguez y J. Ballesta. Interlec 13. Berlin. August 1991. 4.- Cytochemical characterization of carbohydrates in zona pellucida of rat ovarian follicles. M. Avilés, J.A. Martínez-Menárguez, J.F. Madrid, M.T. Castells y J. Ballesta. Interlec 13. Berlin. August 1991.

5.- In situ characterization of glycoproteins in Golgi apparatus and acrosomes of rat spermatogenic cells. J.A. Martínez-Menárguez, M. Avilés, M.T. Castells, J.F. Madrid y J. Ballesta. Interlec 13. Berlin. August 1991. 6.- Subcellular localization of sialoglycoconjugates in human laryngeal glands. M.T. Castells, J.F. Madrid, J.A. Martínez-Menárguez y J. Ballesta. 5th International Congress on Cell Biology. Madrid. 26th-31st July 1992. 7.- Localization and characterization of N-linked oligosaccharides in Golgi apparatus of early spermatids. J.A. Martínez-Menárguez, M. Avilés, M.T. Castells, J.F. Madrid y J. Ballesta. 5th International Congress on Cell Biology. Madrid. 26th-31st July 1992. 8-

Cytochemical characterization of the glycoproteins in the goblet cells of human large intestine. J.F. Madrid, M.T. Castells, M. Avilés, F. Hernández y J. Ballesta. 5th International Congress on Cell Biology. Madrid. 26th-31st July 1992.

9.- Cytochemical localization of carbohidrate residues in zona pellucida and ooplasm of rat ovarian follicles. M. Avilés, J.A. Martínez-Menárguez, M.T. Castells, J.F. Madrid y J. Ballesta. 5th International Congress on Cell Biology. Madrid. 26th-31st July 1992. 10.- Ultrastructural localization of UEA-I and AAA binding sites in the human fundic glands. Implications in the fucosylation processes. J.F. Madrid, M.T. Castells, M. Avilés, J.A. Martínez-Menárguez y J. Ballesta. 9th International Congress of Histochemistry anf Cytochemistry. Maastricht August 30September 5, 1992. 11.- Ultrastructural characterization of terminal oligosaccharide sequences in the glycoproteins of human laryngeal glandular cells. M.T. Castells, J.F. Madrid, J.A. Martínez-Menárguez, M. Avilés y J. Ballesta. 9th International Congress of Histochemistry anf Cytochemistry. Maastricht August 30September 5, 1992. 12.- Application of galactose oxidase, neuraminidase and lectin cytochemistry for ultrastructural detection of penultimate residues in zona pellucida and acrosome of rats. J.A. Martínez-Menárguez, M. Avilés, M.T. Castells, J.F. Madrid y J. Ballesta. 9th International Congress of Histochemistry anf Cytochemistry. Maastricht August 30September 5, 1992. 13.- Cytochemical characterization of the oligosaccharide biosynthesis of rat acrosomal glycoproteins M. Avilés, J.A. Martínez-Menárguez, M.T. Castells, J.F. Madrid y J. Ballesta. 9th International Congress of Histochemistry anf Cytochemistry. Maastricht August 30September 5, 1992.

14.- Ultrastructural demonstration of terminal sialyl and fucosyl residues in the glycoproteins of human laryngeal glands. M.T. Castells, J.F. Madrid, M. Avilés, M. García y J. Ballesta. X European Congress on Electron Microscopy. Granada. September 7-11, 1992. 15.- Localization of galactose residues in Golgi apparatus and acrosomes of rat spermatids J. Martínez-Menárguez, M. Avilés, J.F. Madrid, M.T. Castells, D. Sancho y J. Ballesta. X European Congress on Electron Microscopy. Granada. September 7-11, 1992. 16.- Localization of penultimate carbohidrate residues by enzymatic treatment and lectin cytochemistry. Application of neuraminidase and galactose oxidase. M. Avilés, J.A. Martínez-Menárguez, M.T. Castells, J.F. Madrid, F. Hernández y J. Ballesta. X European Congress on Electron Microscopy. Granada. September 7-11, 1992. 17.- Identification of fucosyl residues in human fundic glands by means of lectin-gold cytochemistry. J.F. Madrid, M.T. Castells, J.A. Martínez-Menárguez, F. Hernández y J. Ballesta. X European Congress on Electron Microscopy. Granada. September 7-11, 1992. 18.- Phagocytic activity and lectin binding properties of molluscan haemocytes. M.P. Cajaraville, Y. Robledo, I. Olabarrieta, I. Marigómez, J.F. Madrid y J. Ballesta. IVth European Cell Biology Congress. Prague. June 26th-July 1st, 1994. 19.- Subcellular identification of fucosyl residues in N- and O-linked glycoproteins in human laryngeal glands. M.T. Castells, J.F. Madrid y J. Ballesta. 13th International Congress on Electron Microscopy. Paris. July 17-22, 1994. 20.- Expression of H-antigen in human fundic glands. An ultrastructural study using immunocytochemistry and lectin histochemistry. J.F. Madrid, O. Leis, F. Hernández, M.T. Castells y J. Ballesta. 13th International Congress on Electron Microscopy. Paris. July 17-22, 1994. 21.- Cytochemical localization of fucose residues in Paneth cells. J.F. Madrid, O. Leis, F. Hernández y J. Ballesta. 13th International Congress on Electron Microscopy. Paris. July 17-22, 1994. 22.- Lectin and enzyme cytochemistry of the lysosomal-vacuolar system of bivalve digestive cells. Y. Robledo, J.F. Madrid, O. Leis, E. Angulo y M.P. Cajaraville. ECBO Meeting. Stuttgart. April 5-8, 1995 23.- The Paneth cells contain TGF-α O. Leis, J.F. Madrid, M.J. González-Díaz y R. Aparicio 6th International Congress on Cell Biology. San Francisco. December 7-11, 1996. 24.- Characterization of N- and O-linked oligosaccharides of the Pleurodeles watls testis. R. Aparicio, F.J. Sáez, J.F. Madrid and O. Leis

The Frontiers of the Biology of Amphibia. The International Symposium. Hiroshima. March 22-24, 1999. 25.- N- and O-linked oligosaccharides in the human rectum. A lectin cytochemical study. J.F. Madrid, R. Aparicio, F.J. Sáez y F. Hernández Xith International Congress of Histochemistry and Cytochemistry. York. September 3-8, 2000 26.- (Sialyl)galactosides in O-linked oligosaccharides of the primordial germ cells in Xenopus embryos E. Alonso, F.J. Sáez, J.F. Madrid y F. Hernández Xith International Congress of Histochemistry and Cytochemistry. York. September 3-8, 2000 27.- A new method that reveals the existence of two subtypes of O-linked oligosaccharides in rat gastric mucosa. L. Gómez, J.F. Madrid, F.J. Sáez, M.J. García-García, J.M. Gódinez y F. Hernández 12th International Congress of Histochemistry and Cytochemistry. La Jolla, San Diego. USA. July 24-28, 2004. 28.- N- and O-linked galactosides in the rat gastric mucosa shown by lectins histochemistry. L. Gómez, J.F. Madrid, F.J. Sáez, M.J. García-García, J.M. Godínez y F. Hernández 12th International Congress of Histochemistry and Cytochemistry. La Jolla, San Diego. USA. July 24-28, 2004. 29.- SNA-binding sites in the human gastric glands. A lectin ultrastructural localization of sialic acid. J.F. Madrid, L. Gómez, F.J. Sáez, J.M. Godínez, M.J. García-García and F. Hernández 13th European Microscopy Congress. Antwerp, Bélgica. August 22-27, 2004. 30.- Unequal distribution of GalNAc-glycoconjugates in the acrosome of the spermatozoon of the urodele Pleurodeles waltl. J.F. Madrid, M. Hervella, F.J. Sáez, E. Fernández, L. Gómez, G. Valbuena and F. Hernández 13th European Microscopy Congress. Antwerp, Bélgica. August 22-27, 2004. 31.- Identification of GalNAc-glycoconjugates in pro-acrosomal vesicles during spermiogenesis in the urodele Pleurodeles waltl. J.F. Madrid, E. Fernández, F.J. Sáez, M. Hervella, L. Gómez, G. Valbuena and F. Hernández 13th European Microscopy Congress. Antwerp, Bélgica. August 22-27, 2004. 32.- Histology and Histopathology: a challenge J.F. Madrid and F. Hernández First International Congress of Histology and Tisuee Engineering. Alcalá de Henares, September 14-17, 2005. 33.- Telematic resources of the Spanish Society of Histology and Tissue Engineering (SEHIT) F.J. Sáez and J.F. Madrid First International Congress of Histology and Tisuee Engineering. Alcalá de Henares, September 14-17, 2005.

34.- PCNA and EGFR ommunohistochemical expresión in gastric ulder healing M.F. García-García, J.M. Godínez, A. Zuasti, G. Valbuena, F.J. Sáez and J.F. Madrid First International Congress of Histology and Tisuee Engineering. Alcalá de Henares, September 14-17, 2005. 35.- A pattern of distribution of fucose residues in stomach during development in an animal model, Sprague-Dawley rat J.M. Godínez, M.J. García-García, C. Ferrer, E. Poblet, F.J. Sáez and J.F. Madrid First International Congress of Histology and Tisuee Engineering. Alcalá de Henares, September 14-17, 2005. 36.- Identification of GalNAc by lectin cytochemistry in the acrosome of Pleurodeles waltl at the ultrastructural level G. Valbuena, E. Alonso, L. Gómez, F. Hernández, J.F. Madrid and F.J. Sáez First International Congress of Histology and Tisuee Engineering. Alcalá de Henares, September 14-17, 2005. 37.- The expresion of a novel gene, CXCR4B, In Xenopus embryo development E. Alonso, M.L. King, T. Venkataraman, L. Gómez, J.F. Madrid and F.J. Sáez First International Congress of Histology and Tisuee Engineering. Alcalá de Henares, September 14-17, 2005. 38.- Stem cell markers in the human hair follicle E. Poblet, J.M. Godínez, J.M. Madrid and E. Escario First International Congress of Histology and Tisuee Engineering. Alcalá de Henares, September 14-17, 2005. 39.- GalNAc containing oligosaccharides in the rat gastric glands. A lectin histochemical charaterization L. Gómez, G. Valbuena, M.J. García-García, A. Zuasti, F.J. Sáez and J.F. Madrid First International Congress of Histology and Tisuee Engineering. Alcalá de Henares, September 14-17, 2005. 40.- Demonstration of two sybtypes of O-linked oligosaccharides in the rat gastric glands L. Gómez, E. Alonso, J.M. Godínez, C. Ferrer, F.J. Sáez and J.F. Madrid First International Congress of Histology and Tisuee Engineering. Alcalá de Henares, September 14-17, 2005. 41.- CD10 in hair follicle development E. Poblet, J.M. Godínez, P.J. Benito Castellanos and J.F. Madrid Second International Congress of Histology and Tissue Engineering. Córdoba, 1013 de septiembre de 2007 42.- Acrosome biogenesis in premeiotic cells shown by means of HPA-lectin cytochemistry G. Valbuena, H. Lafuente, J.F. Madrid, F. Hernández and F.J. Sáez Second International Congress of Histology and Tissue Engineering. Córdoba, 1013 de septiembre de 2007 43.- GlcNAc in the oligosaccharidic chains fo the rat gastric mucosa. Histochemical approach L. Gómez-Santos, F.J. Sáez, H. Lafuente, G. Valbuena, E. Poblet and J.F. Madrid Second International Congress of Histology and Tissue Engineering. Córdoba, 1013 de septiembre de 2007

44.- Glycosidic differences in parietal cells: Implication in their migration throughout the gastic mucosa L. Gómez-Santos, F.J. Sáez, G. Valbuena, F. Hernández, L. Díaz-Flores and J.F. Madrid Second International Congress of Histology and Tissue Engineering. Córdoba, 1013 de septiembre de 2007 45.- The expression of a CXCR4 gene in Xenopus embryo E. Alonso, J.F. Madrid, G. Valbuena and F.J. Sáez Joing Meeting of the British and Spanish Developmental Biology Societies. Sevilla, 24-27 de septiembre de 2008. 46.- Modification of glycoconjugate content in male germ cells during spermatogenesis G. Valbuena, J.F. Madrid, F. Hernández and F.J. Sáez 9th International Congress of Andrology. Barcelona, 7-10 de marzo de 2009. 47.- Acrosome biogenesis in primary spermatocytes shown by lectin cytochemistry G. Valbuena, J.F. Madrid and F.J. Sáez 9th International Congress of Andrology. Barcelona, 7-10 de marzo de 2009. 48.- Characterization of sialic acid in the rat gastric mucosa J.F. Madrid, L. Gómez-Santos, F. Hernández and F.J. Sáez Third International Congress of Histology and Tissue Engineering. Albacete, 8-11 de julio de 2009. 49.- Expression fo fucosylated oligosaccharides in the rat gastric mucosa J.F. Madrid, L. Gómez-Santos, F. Hernández and F.J. Sáez Third International Congress of Histology and Tissue Engineering. Albacete, 8-11 de julio de 2009. 50.- Modification of glycoconjugate content during spermatogenesis in Xenopus laevis shown by lectin histochemistry J.F. Madrid, G. Valbuena, F. Hernández and F.J. Sáez Third International Congress of Histology and Tissue Engineering. Albacete, 8-11 de julio de 2009. 51. Interactions between pericytes and transmigrating leukocytes during angiogenesis and granulation tissue formation F. Valladares, R. Gutiérrez, J.F. Madrid, H. Varela, L. Díaz-Flores Jr, M.N. Rancel and L. Díaz-Flores Third International Congress of Histology and Tissue Engineering. Albacete, 8-11 de julio de 2009. 52.- Pericytes as a source of adipose-derived stromal stem cells (ASCs). Confirmation of the perivascular mesenchymal cell niche hypothesis R. Gutiérrez, L. Días-Flores Jr, E. Acosta, J.F. Madrid, P. Martín-Vasallo, F. Valladares, H. Varela and L. Díaz-Flores Third International Congress of Histology and Tissue Engineering. Albacete, 8-11 de julio de 2009. 53.- Verification of treatment time for O-linked oligosaccharide removal by ßelimination G. Valbuena, F. Hernández, J.F. Madrid and F.J. Sáez Primer Encuentro Internacional Virtual de Educación e Investigación en Ciencias Morfológicas. Córdoba, Argentina. 11-30 de septiembre de 2009. 54.- Lectin histochemical characgerization of different populations of mucous and parietal cells in the rat gastric epithelium

L. Gómez-Santos, F.J. Sáez, E. Alonso, M. Martínez de Ubago, E. Sagredo, B. Urcelay, L. Díaz-Flores and J.F. Madrid TERMIS EU 2011 Annual Meeting. Granada, España, 7-10 de junio de 2011 55.- Lectin histochemical identification of fucosylated glycoconjugates during Xenopus laevis spermiogenesis G. Valbuena, E. Alonso, M. Martínez de Ubago, E. Sagredo, B. Urcelay, J.F. Madrid and F.J. Sáez TERMIS EU 2011 Annual Meeting. Granada, España, 7-10 de junio de 2011 56.- HPA is no an universal marker for amphibian acrosome G. Valbuena, E. Alonso, M. Martíenz de Ubago, J.F. Madrid and F.J. Sáez TERMIS EU 2011 Annual Meeting. Granada, España, 7-10 de junio de 2011 57.- Partial loos of glycoconjugate content during acrosome biognesis in a sterile GOPC deficient mouse M. Martínez de Ubago, E. Alonso, L. Gómez-Santos, J.F. Madrid, T. Noda, F.J. Sáez V International Congress of Histology and Tissue Engineering. Logroño, 12-14 de septiembre de 2013. 58.- Proliferation of spermatogonia and Sertoli cells during testicular recrudescence of Syrian hámster after exposure to short photoperiod M.M. Sánchez-Huertas, V. Seco-Rovira, E. Beltrán-Frutos, J.F. Madrid, F.J. Sáez, C. Ferrer, M. Canteras M, V. Quesada-Cubo, L.M. Pastor V International Congress of Histology and Tissue Engineering. Logroño, 12-14 de septiembre de 2013. 59.- Morphological and morphometric changes in testes of hámster Mesocricetus Auratus durint testicular recrudescence after exposure to short pohotoperiod M.M. Sánchez-Huertas, V. Seco-Rovira, E. Beltrán-Frutos, J.F. Madrid, F.J. Sáez, C. Ferrer, M. Canteras M, V. Quesada-Cubo, L.M. Pastor V International Congress of Histology and Tissue Engineering. Logroño, 12-14 de septiembre de 2013. 60.- Morphometric changes in Syrian hámster (Mesocricetus auratus) testes during testicular regression after exposure to shorto photoperiod V. Seco-Rovira, M.M. Sánchez-Huertas, E. Beltrán-Frutos, J.F. Madrid, F.J. Sáez, C. Ferrer, M. Canteras M, V. Quesada-Cubo, L.M. Pastor V International Congress of Histology and Tissue Engineering. Logroño, 12-14 de septiembre de 2013. 61.- Glycoconjugate content in the acrosomte of TNP2-/- knockout mouse M. Martínez de Ubago, E. Alonso, L. Gómez-Santos, M.E. Sagredo, B- Urcelay, J.F. Madrid, F.J. Sáez V International Congress of Histology and Tissue Engineering. Logroño, 12-14 de septiembre de 2013. 62.- Characterization of the differentiation process in the human fundic glands by sialicacid binding lectins J.F. Madrid, L. Gómez-Santos, E. Alonso, M. Martínez de Ubago, L. DíazFlores, F.J. Sáez V International Congress of Histology and Tissue Engineering. Logroño, 12-14 de septiembre de 2013.

63.- The use of the virtual microscope as a tool histology teaching in medicine. A two year experience J.F. Madrid, M. Avilés, J.A. Martínez-Menárguez, L.M. Pastor, M. MartínezMovilla, M.J. Izquierdo-Rico, E. Beltrán, V. Seco, C. Ferrer V International Congress of Histology and Tissue Engineering. Logroño, 12-14 de septiembre de 2013.

Experiencia de gestión de I+D Gestión de programas, planes y acciones de I+D Título: Revista Científica QATAR UNIV. SCI. JOURNAL Tipo de actividad: Referee Fecha: 1992 Título: Revista Científica Kuwait Journal of Science and Engineering Tipo de actividad: Referee Fecha: 1993 Título: Revista Internacional Histochemical Journal Tipo de actividad: Referee Fecha: 1995, 2005 Título: Revista Internacional Microscopy Research and Technique Tipo de actividad: Referee Fecha: 1998, 2006, 2007 Título: Revista Internacional INTERNATIONAL JOURNAL OF CANCER Tipo de actividad: Referee Fecha: 1999 Título: Universidad de Kuwait Tipo de actividad: Evaluador para la calificación a aspirantes a la categoría de “Profesor” Fecha: 1999 Título: Universidad de Otago (Australia) Tipo de actividad: Evaluador para la calificación a aspirantes a la categoría de “Profesor” Fecha: 2004 Título: Agencia Nacional de Evaluación y Prospectiva Tipo de actividad: Evaluador de Proyectos de Investigación Fecha: 2001

Título: FIS Tipo de actividad: Evaluador de Proyectos de Investigación Fecha: 2002, 2003 Título: FONCYT (Argentina) Tipo de actividad: Evaluador de Proyectos de investigación Fecha: 1999, 2000, 2003, 2004, 2005 Título: Comisión Europea Tipo de actividad: Experto evaluador y “Rapporteur” en el Programa Marie Curie Fecha: 2005-actualidad Título: Programa ACADEMIA Tipo de actividad: Experto evaluador para el sistema de acreditación nacional Fecha: 2008-2009 Título: Tipo de actividad: Fecha: Título: Tipo de actividad: Fecha:

Experiencia en organización de actividades de I+D Organización de congresos, seminarios, jornadas, etc., científicos-tecnológicos Título: I Congreso de la Sociedad Española de Biología del Desarrollo Tipo de actividad: Miembro del Comité Ambito: Nacional Organizador Fecha: 7-11 de diciembre de 1996 Título: IX Congreso Nacional de Histología Tipo de actividad: Miembro del Comité Científico

Ambito: Nacional

Fecha: 4-6 julio de 1997 Título: X Congreso Nacional de Histología Tipo de actividad: Miembro del Comité Científico

Ambito: Nacional

Fecha: 18-20 de noviembre de 1999 Título: XI Congreso Nacional de Histología Tipo de actividad: Miembro del Comité Científico

Ambito: Nacional

Fecha: 11-14 de septiembre de 2001 Título: XIII Congreso Nacional de Histología e Ingeniería Tisular y I International Congres of Histolgy and Tissue Engineering

Tipo de actividad: Miembro del Comité Cientifico

Ambito: Internacional

Fecha: 14-17 septiembre 2005 Título: XIV Congreso Nacional de Histología e Ingeniería Tisular y I International Congres of Histolgy and Tissue Engineering

Tipo de actividad: Miembro del Comité Cientifico Fecha: 10-13 de septiembre de 2007

Ambito: Internacional

Participación en comités y representaciones internacionales Título del Comité: Editor de la revista Internacional HISTOLOGY AN DHISTOPATHOLOGY Factor de impacto 2,502 Entidad de la que depende: Tema: Publicación científica Fecha: 1989-actualidad

Título del Comité: Miembro del equipo editorial de la revista internacional BIOMEDICAL RESEARCH Entidad de la que depende: Tema: Publicación científica Fecha: 1995-acutalidad Título del Comité: Miembro del equipo editorial de la revista internacional CURRENT NEUROVASCULAR RESEARCH Entidad de la que depende: Tema: Publicación científica Fecha: 2004-acutalidad Título del Comité: Miembro del equipo editorial de la revista internacional JOURNAL OF CELLULAR AND MOLECULAR MEDICINE Entidad de la que depende: Tema: Publicación científica Fecha: 2014-actualidad Título del Comité: Entidad de la que depende: Tema: Fecha:

Otros méritos o aclaraciones que desee hacer constar Expediente académico: Aprobados Notables Sobresalientes Matrículas de Honor

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69 Comunicaciones a Congresos Nacionales 63 Comunicaciones a Congresos Internacionales Premio Nacional Jóvenes Investigadores otorgado por la Universidad de La Laguna, dotado con 1 millón de pesetas. 1993 Expert Evaluator para la Comisión Europea en el Programa Marie Curie Evaluador de la ANEP del Ministerio de Ciencia y Tecnología Evaluador del FONCYT (Fondo Nacional de Ciencia y Técnica) de Argentina. Evaluador del FIS Evaluador de la ANECA para el programa ACREDITA Concesión de tres sexenios de investigación (todos los posibles).

Indice Pág. Curriculum Vitae Abreviado Datos personales Situación profesional Actual Formación Académica Indicadores Generales de Calidad de la Produccion Científica Resúmen biográfico Méritos más relevantes Publicaciones Proyectos Contratos Patentes Becas Premios y Distinciones Consejos Editoriales Gestión

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Curriculum normalizado completo Informacion personal Formación Académica Situación Profesional Actual Actividades Anteriores de carácter Cientifico o Profesional Idiomas de interés científico Participación de Proyectos de Investigación Competitiva Publicaciones Participación en Contratos de especial relevancia para empresas Patentes y Modelos de Utilidad Estancias en Centros Extranjeros Congresos Tesis Doctorales Dirigicas Grandes equipos que utiliza o ha utilizado Otros méritos

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10 Publicaciones seleccionadas Archivo informático CDROM (con publicaciones en pdf)

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CURRÍCULUM ABREVIADO (CVA)

15/09/2014 Fecha del CVA Parte A. DATOS PERSONALES Nombre y apellidos Sebastián Cerdán García-Esteller DNI/NIE/pasaporte 02186071J Edad 61 L-1124-2014 Researcher ID Núm. identificación del investigador 0000-0001-9965-0270 Código Orcid A.1. Situación profesional actual Organismo Consejo Superior de Investigaciones Científicas Dpto./Centro Modelos Animales de Enfermedades Humanas/IIBm CSIC Dirección c/Arturo Duperier 4, Madrid 28029 Teléfono 915854444 correo electrónico [email protected] Categoría profesional Profesor de Investigación CSIC Fecha inicio 1/10/2010 Espec. cód. UNESCO 240699 (NMR), 240606, 24013,24901,24902, 320602 Resonancia Magnética, Neuroquímica, Imagen Multimodal, Palabras clave Imagen Multiparamétrica, Agentes de contraste A.2. Formación académica (título, institución, fecha) Licenciatura/Grado/Doctorado Universidad Licenciado en Farmacia Complutense de Madrid Doctor en Farmacia Complutense de Madrid A.3. Indicadores generales de calidad de instrucciones) Sexenios de investigación: Tesis Doctorales Dirigidas últimos 10 años: Citas Totales/Promedio citas año últimos 5 años: Publicaciones Totales Primer Cuartil (Q1): Indice h:

Año 1975 1978

la producción científica (véanse 6, último 2007-2013 10 Tesis Defendidas/4 en curso 2736/ 32,4 citas promedio 70 31

Parte B. RESUMEN LIBRE DEL CURRÍCULUM (máximo 3500 caracteres, incluyendo espacios en blanco) Sebastián Cerdán García-Esteller, es Profesor de Investigación del CSIC y responsable del Laboratorio de Imagen y Espectroscopía por Resonancia Magnética de Alto Campo (LIERM) del CSIC desde 1989. Su trayectoria científica contiene estancias postdoctorales en los Estados Unidos (Universidad de Pennsylvania, John E. Fogarty International Fellow 1980-4) y en Suiza (Biozentrum der Universität Basel, EMBO Long Term Fellow 1986-8). Es autor de ciento treinta artículos de investigación, más de doscientas comunicaciones a congresos y cinco patentes. Su objetivo general es el desarrollo y evaluación de nuevas aplicaciones biomédicas de la Resonancia Magnética, siendo uno de los investigadores más consolidados en este campo en nuestro país. Entre sus contribuciones más relevantes destacan la caracterización de la interacción metabólica entre neuronas y células de glía en el cerebro adulto, el desarrollo de espectroscopías multinucleares para estudios metabólicos y más recientemente el desarrollo de nuevos abordajes para la caracterización de la obesidad mediante Espectroscopia e Imagen por Resonancia Magnética. Más concretamente ha desarrollado en los últimos cinco años una serie extensa de nuevas aplicaciones de la Imagen por Resonancia Magnética a la Biomedicina. En su vertiente de investigación básica, ha probado que las Tecnologías de Resonancia Magnética pueden proporcionar información no accesible a otras tecnologías sobre diversos aspectos del metabolismo cerebral in situ y sobre los mecanismos fisiopatológicos involucrados en enfermedades neurodegenerativas como la epilepsia ó la isquemia y más recientemente en el diagnóstico precoz de la obesidad y su tratamiento. En su vertiente de investigación aplicada, ha patentado y transferido a la industria una nueva serie de indicadores del pHi y pHe para 1H RMN y descrito y patentado una nueva 3

CURRÍCULUM ABREVIADO (CVA)

serie de complexonas de Gd(III) con eficacia mejorada con potencial utilidad como agentes de contraste en enfermedades que transcurren con modificaciones de pH. Sus publicaciones incluyen aportaciones a Nature Comm, Ann. Rev. Byophys. Bioeng., PNAS, Neuroimage, J. Cereb Blood Flow and Metab,, J. Neurochem., J. Biol. Chem., NMR in Biomed., Mag. Res. in Med., Progress in NMR Spectr. Actualmente, forma parte de paneles editoriales de revistas de difusión internacional de RM Biomédica (NMR in Biomedicine), de bioenergetica cerebral (Frontiers in Neuroenergetics), y metabolismo cerebral (Journal of Cerebral Blood Flkow and Metabolism), ha sido Director del Instituto de Investigaciones Biomédicas “Alberto Sols” CSIC/UAM, Secretario de la Sociedad de Biofísica de España (1996-2002), Vocal y Presidente de la Sociedad Europea de Resonancia Magnética (1996-2000, 2004-10), miembro del Consejo Directivo de la Sociedad Internacional de Resonancia Magnética en Medicina. y Académico correspondiente de la Real Academia Nacional de Farmacia (2010).Su laboratorio mantiene además una intensa actividad a nivel internacional con numerosos intercambios y visitas reciprocas de investigadores europeos. Mención especial merecen la coordinación de los Proyectos MULTIMAG-CM y I2M2-CM que articulan el funcionamiento de la Resonancia Magnética Biomédica en la Comunidad de Madrid y su participación en Proyectos Europeos como MEDITRANS y BIBA (Euronanomed). Parte C. MÉRITOS MÁS RELEVANTES (ordenados por tipología) C.1. Publicaciones más relevantes (2009-15) 1. Calle, D., Negri,V., Ballestero,P., Cerdan, S. (2015) Magnetoliposomes loaded with Poly-unsaturated Fatty Acids as novel theranostic anti-inflammatory formulations. Theranostics 5: 489-503. I.F. 7,89 (9/124 Medicine Research and Experrimental Q1) 2. Frost, G., Sleeth, M.L., Meliz A., Arisoylu,M.A. Lizarbe, B., Cerdan,S., Brody,L., Anastasovska, J., Ghourab, S., Hankir, M., Zhang, S., Carling, D., Swan, J., Gibson, G., Viardot, A., Morrison,D., Louise Thomas,E., Bell, J.D. (2014) The short chain fatty acetate reduces appetite via a central homeoistatic mechanism. Nature Comm. DOI 10.1038/ncomms4611). I.F. 10,472 (3/55 Multidisciplinary Sciences Q1) 3. Lizarbe,B., Benitez, A., Sanchez-Montañes, M., Lago-Fernández,L., Garcia-Martín, M.L., LopezLarrubia,P., Cerdan, S. (2013) Imaging Hypothalamic Activity using Diffusion Weighted Magnetic Resonance Imaging in the mouse and human brains. Neuroimage 64: 448-457. I.F. : 5.895 (1/14 Neuroimaging Q1) 4. Lizarbe,B., Benitez,A., Peláez-Brioso, G.A., Sánchez-Montañés, M., López-Larrubia,P., Ballesteros,P., Cerdán,S. (2013) Hypothalamic metabolic compartmentation during apetite regulation as revealed by magnetic resonance imaging and spectroscopy methods. Frontiers in Neuroenegetics doi: 10.3389/fnene 2013 ecollection 2013. 5. Delgado,T.C., Violante,I.R., Nieto-Charques, L., Cerdán, S. (2011) Neuroglial metabolic compartmentation underlying leptin deficiency in the obese ob/ob mice as detected by magnetic resonance imaging and spectroscopy methods. J. Cereb Blood Flow and Metabolism. 31 :2257-2266. I.F.: 5.46 (29/231 Q1 Neurosciences) 6. Pacheco-Torres J, Calle D, Lizarbe B, Negri V, Ubide C, Fayos R, Larrubia PL, Ballesteros P, Cerdan S. (2011) Environmentally Sensitive Paramagnetic and Diamagnetic Contrast agents for Nuclear Magnetic Resonance Imaging and Spectroscopy. Curr Top Med Chem 11: 115-30. I.F.: 4.47 (5/46 Q1 Chemistry Medicinal) 7. Negri,V., Cerpa,A., Lopez-Larrubia,P., Cerdan,S., Ballesteros,P. (2011) Nanotubular Paramagnetic Probes as contrast agents for Magnetic Resonance Imaging based in the Diffusion Tensor. Angewandte Chemie Int. Ed. (2010) 49(10):1813-5. I.F.: 10.88 (4/127 Q1 Chemistry Multidisciplinary).

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8. Rodrigues,T.B., Violante,I., Cerdán,S. (2010) Unambiguous assignment of the H3S and H3R deuterations of cerebral (2-13C) glutamate by 13C NMR at 18.8 Tesla. Magnetic Resonance in Medicine (2010) 63(4):1088-91. I.F.: 3.44 (16/92 Q1 Radiology, Nuclear Medicine, Medical Imaging) 9. Cerdán,S., Sierra,A., Fonseca, L.L., Ballesteros, P., Rodrigues.,T.B. (2009) The turnover of the H3 deuterons from (2-13C) glutamate and (2-13C) glutamine reveals subcellular trafficking in the brain of partially deuterated rats. J. Neurochem. (2009) 109 Suppl 1: 63-72. I.F.: 4.500 (40/219 Q1 Neurosciences, 60/276 Q1 Biochemistry and Molecular Biology) 10. Rodrigues, T.B., López-Larrubia,P., Cerdán,S. (2009) Redox Dependence and Compartmentation of (13C) pyruvate in the brain of deuterated rats bearing C6 gliomas. J. Neurochem. (2009) 109 Suppl. 1: 237-45. I.F.: 4.500 (40/219 Q1 Neurosciences, 60/276 Q1 Biochemistry and Molecular Biology). C.2. Proyectos 1. Imagen Multimodal de la inflamación y de las cascadas inflamatorias en la salud y la enfermedad 2012-14. Ministerio de Ciencia e Innovación SAF2011-23622. Entidades participantes: IIBm, CSIC. Cuantìa de la Subvención: 254.100 euros. I.P.: Sebastián Cerdán. Nº de investigadores participantes: 8 2. Imagen Molecular Multimodal de la Inflamación-I2M2 2012-15. Comunidad de Madrid. Entidades participantes: CSIC, UCM, UNED, UAH, UAM. Cuantía de la subvención: 968.610 euros. I.P. y Coordinador: Sebastián Cerdán. Nº de grupos participantes: 10 grupos de investigacion, 2 Laboratorios de referencia, 3 grupos asociados y cinco empresas (60 investigadores). 3. Nuevas tecnologías para la producción de concentrados de alta pureza de acidos grasos omega3, EPA y DHA (2013-15) Entidad Financiadora: MINECO- INNPACTO 2012- IPT-2012-1331060000 Coordinador: Gerard Bannemberg, Grupos CSIC: Sebastián Cerdán y Lisardo Boscá. Precio del Proyecto: 1.244.246 euros (CSIC 330.000 euros). Número de Investigadores participantes (Grupo SC): 5 4. Acoplamiento neuroglial y neurovascular durante la activacion cerebral por apetito detectada mediante métodos de Resonancia Magnética 2009-2011. Ministerio de Ciencia e Innovación. Entidades participantes: CSIC. Cuantía de la subvención: 210.000 euros. Investigador responsable y coordinador: Sebastián Cerdán. Nº de investigadores participantes: 7. 5. Targeted Delivery of Nanomedicine MEDITRANS. Comunidad de Europea (Integrated Project) 2007-.2010. 300.000 euros. IP: Sebastián Cerdán. Coordinador: Gert Storm (Universidad de Utrech) Nº de investigadores participantes: 30 grupos 6. Nanopharmaceuticals delivery acroos Biological Barriers (BIBA) 2011-2014. Union Europea (UE)-Instituto de Salud Carlos III (EURONANOMED). Entidades participantes: CSIC, Hospital Universitario La Paz (IdIPAZ) Cuantía de la subvención: 100.000 euros. I.P. Sebastian Cerdan, Coordinador: Patrick Boisseau (CEA, FR). Nº de grupos europeos participantes: 7 (40 investigadores). 7. Imagen Multiparamétrica de la competencia vascular (MULTIMAG-CM) 2007-.2010. Comunidad de Madrid- Convocatoria de grupos cooperativos en Biocoencias. Entidades participantes: UNED-CSIC-UCM-UAH. 600.000 euros. I.P. y Coordinador: Sebastián Cerdán. Nº de investigadores participantes: 13 grupos y 4 empresas.

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C.3. Contratos 1. Nuevas nanoparticulas superparamagnéticas como agentes de contraste para imagen por Resonancia Magnética (2009-13), Entidad Financiadora: SOLUTEX S.L., I.P. S. Cerdán. Precio del Proyecto: 470.000 euros. Número de Investigadores participantes: 5 C.4. Patentes 1. Cerdán, S., López-Larrubia, P., Nieto-Charques, L.,Negri, V., PerezMayoral,E., Ballesteros,P. Materiales nanoestructurados tubulares con propiedades magnéticas anisotrópicas. Procedimiento de Obtención y sus aplicaciones. N. Solicitud: P200800024. Fecha de prioridad: 8-01-2008. Entidad Titular: UNED-CSIC 2. Cerdán, S., Calle,D., Moreno, F. (2012) Superparamagnetic Nanoparticles as contrast agents for Magnetic Resonance Imaging (MRI) of Magnetic Susceptibility. PCT ES2012/070044. Fecha prioridad: 27/1/2012. Entidad Titular: SOLUTEX S.L. 3. Cerdán, S., Calle,D., Moreno,F. (2014) Composiciones que contienen liposomas, acidos grasos poliinsaturados omega-3 de cadena larga y nanoparticulas superparamagneticas y su uso en el tratamiento de tumores malignos. P201430035. Fecha prioridad: 15/1/2014. C5. Becas 1976-78: Beca Predoctoral MICINN 1979-80: Beca Postdoctoral MICINN 1980-81: Beca Postdoctoral Fullbright 1982-84: Beca postdoctoral NIH, John E. Fogarty International Fellow 1986-88: Beca EMBO Long Term C6. Premios y Distinciones 1975: Premio Carracido de Licenciatura, Facultad de Farmacia, Universidad Complutense, Madrid. 1978: Premio Extraordinario de Doctorado, Facultad de Farmacia, Universidad Complutense, Madrid. 2010: Académico Correspondiente Real Academia Nacional de Farmacia C7. Consejos Editoriales 1. NMR in Biomedicine (desde 1998, Miembro del Editorial Board, http://www3.interscience.wiley.com/journal/13087/home/EditorialBoard.html ). 2. Frontiers in Neuroenergetics (desde 2008, Editor Asociado, http://frontiersin.org/neuroscience/profiles/sebastiancerdan/). 3. Journal of Cerebral Blood Flow and Metabolism (desde 2013, Miembro del Editorial Board http://www.nature.com/jcbfm/about.html#Editors).

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C8. Gestión 2004-8: Director del Instituto de Investigaciones Biomédicas “Alberto Sols”, Madrid. 2006: Board of Trustees International Society of Magnetic Resonance Medicine ISMRM. 2000-8: Presidente Sociedad Europea de Resonancia Magnetica en Medicina y Biología (ESMRMB). 2010-presente: Colaborador Subdirección General Recursos Humanos MINECO (Programas FPI, PTA, PTQ).

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INFORMACION PERSONAL

APELLIDOS: CERDÁN GARCÍA ESTELLER NOMBRE: SEBASTIÁN SEXO: VARÓN DNI: 2186071J FECHA DE NACIMIENTO: 17/04/53 Nº FUNCIONARIO: 0218607168 A5403 DIRECCION PARTICULAR: FRANCISCA MORENO 1 CIUDAD: MADRID CODIGO POSTAL:28001 TELEFONO:914353961 ESPECIALIZACIÓN: BIOFÍSICA FORMACIÓN ACADEMICA ___________________________________________________________________________ TITULACIÓN SUPERIOR CENTRO FECHA Licenciatura en Farmacia Grado de Licenciado Doctorado en Farmacia

Facultad de Farmacia, UCM Facultad de Farmacia, UCM Facultad de Farmacia, UCM

1970-1975 1976 1978

SITUACIÓN PROFESIONAL ACTUAL

ORGANISMO: Consejo Superior de Investigaciones Científicas FACULTAD, ESCUELA o INSTITUTO DEL C.S.I.C.: Instituto de Investigaciones Biomédicas “Alberto Sols” CSIC/UAM DEPT./SECC./ UNIDAD ESTR.: Modelos Animales Enfermedades Humanas CATEGORIA PROFESIONAL Y FECHA DE INICIO: Profesor de Investigación, 30/06/2010 DIRECCION POSTAL: Arturo Duperier 4, Madrid 28029 TELEFONO (indicar prefijo, número y extensión) 91-585-4444 (4445) ACTIVIDADES ANTERIORES DE CARACTER CIENTíFICO O PROFESIONAL

FECHAS

PUESTO

INSTITUCION

20102008-2009 2004-2008 2001-2004 1985-2001 1986-1988 1985 1981-1984 1980-1981 1979-1980 1978-1979 1977-1978

Profesor de Investigación CSIC CSIC Investigador Científico CSIC CSIC Director de Instituto A CSIC CSIC Investigador Científico CSIC Científico Titular CSIC Investigador Postdoctoral /EMBO Fellow Univ. de Basilea Prof. Titular Universidad Univ. de Cádiz Investigador Postdoctoral/Fogarty Fellow Univ. Pennsylvania Prof. Agregado Interino Univ. Autónoma Madrid Prof. Adjunto Contratado Univ. Autónoma Madrid Prof. Encargado de Curso Univ. Autónoma Madrid Becario Predoctoral MEC Univ. Complutense Madrid

IDIOMAS DE INTERES CIENTíFICO (R=regular,B=bien,C=correctamente)

IDIOMA

HABLA

Inglés Francés Alemán

C R R

LEE C C R

ESCRIBE C R R

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PARTICIPACION EN PROYECTOS DE INVESTIGACION FINANCIADOS 30 Proyectos, 23 como Investigador Principal, Recursos Movilizados: 4.683 M euros en convocatorias públicas de investigación competitiva __________________________________________________________________________________

Título del Proyecto: Metabolismo hepático y cerebral por RMN Entidad Financiadota: FISss Duración, desde: 1988 HASTA: 1990 Cuantía de la subvención: 6 MPts Investigador Principal: Dr. Sebastián Cerdán Centro: Instituto de Investigaciones Biomédicas Número de Investigadores participantes: 3 ___________________________________________________________________________ Título del Proyecto: Aplicaciones Biomédicas de la Resonancia Magnética Entidad Financiadora: DGICYT PM 89-010 Duración, desde: 1990 HASTA; 1992 Cuantía de la subvención: 9 MPts Investigador Principal: Dr. Sebastián Cerdán Centro: Instituto de Investigaciones Biomédicas Número de Investigadores participantes: 3 ___________________________________________________________________________ Título del Proyecto: Implementación infraestructura RMN en el area Biología y Biomedicina del CSIC (Actualización instrumento AM-360) Entidad Financiadora: Comunidad de Madrid / CSIC (Infraestructura) Entidades participantes: CSIC Duración, desde : 1991 HASTA: Cuantía de la Subvención: 12 MPts Investigador Principal: Dr. Sebastián Cerdán CENTRO: Instituto de Investigaciones Biomédicas Número de investigadores participantes: 3 ___________________________________________________________________________ Titulo del proyecto: Sondas extrínsecas para medir el pH y pCa por Resonancia Magnética Protónica. Entidad Financiadora: Comunidad de Madrid C015/91 Durabción, desde: 1992 HASTA; 1994 Cuantía de la subvención: 9 MPts Investigador Principal: Prof. Paloma Ballesteros Centro: Dept. Química Orgánica y Biología, UNED/Instituto de Investigaciones Biomédicas CSIC Número de investigadores participantes: 3 ___________________________________________________________________________ Título del proyecto: Nuevas Aplicaciones de la Resonancia Magnética Nuclear en Biología y Medicina Entidad financiadora: DGICYT Entidades participantes: CSIC Duración, desde: 1992 hasta: 1995 Cuantía de la subvención: 15 Mpts Investigador responsable: Sebastián Cerdán Número de investigadores participantes: 4 Título del proyecto: Resonancia Magnética Heteronuclear del Cerebro Entidad financiadora: Fundación Eugenio Rodríguez-Pascual Entidades participantes: CSIC Duración, desde: 1992 hasta: 1993 Cuantía de la subvención: 4 Mpts Investigador responsable: Sebastián Cerdán Número de investigadores participantes: 3

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Título del proyecto: Caracterización de Tejidos por RMN Entidad financiadora: Union Europea, Acción Concertada BIOMED-2 PL92/0432 Entidades participantes: CSIC+15 Institutciones Europeas Duración, desde: 1993 hasta: 1995 Cuantía de la subvención: Investigador responsable: Franca Podo , Isituto Superiore di Sanitá, Roma Número de investigadores participantes: 50 Título del proyecto: Dinámica Molecular de barbitúricos e hidantoínas como base de sus propiedades neuroactivas Entidad financiadora: DGICYT, Acciones Integradas Hispano-Alemanas BHA-93/018 Entidades participantes: CSIC, UNED, RTWA Duración, desde: 1994 hasta: 1995 Cuantía de la subvención: 1,2 Mpts Investigador responsable: Paloma Ballesteros/Manfred Zeitler Número de investigadores participantes: 8 Título del proyecto: Implementacón Accesorio de microimagen por Resonancia Magnética Entidad financiadora: DGICYT, CSIC Entidades participantes: CSIC Duración, desde: 1994 hasta: 1995 Cuantía de la subvención: 25 MPts Investigador responsable: Sebastián Cerdán Número de investigadores participantes: 5 Título del proyecto: Microimagen multinuclear del oocito de Xenopus Entidad financiadora: Comunidad de Madrid Entidades participantes: CSIC Duración, desde: 1995 hasta: 1995 Cuantía de la subvención: 1,5 MPts Investigador responsable: Sebastián Cerdán Número de investigadores participantes: 4 Título del proyecto: Heterogeneidad Metabólica Resuelta en el espacio mediante espectroscopía e imagen por Resonancia Magnética Entidad financiadora: DGICYT PB 94-011 Entidades participantes: CSIC Duración, desde: 1995 hasta: 1997 Cuantía de la subvención: 12 MPts Investigador responsable: Sebastián Cerdán Número de investigadores participantes: 3 Título del proyecto: Dinámica Molecular de Ligandos Heterocíclicos de relevancia en RMN Biomédica Entidad financiadora: DGICYT, Acción Integrada Hispano-Alemana 14B Entidades participantes: CSIC, UNED, RTWH Duración, desde: 1995 hasta: 1997 Cuantía de la subvención: 1,2 MPts Investigador responsable: Paloma Ballesteros Número de investigadores participantes: 3 Título del proyecto: Desarrollo y Evaluación de la Espectroscopía de Resonancia magnética en Oncología. Aplicación al Diagnóstico Precoz del Cáncer Entidad financiadora: Comunidad de Madrid, Acción Concertada 105/96 Entidades participantes: CSIC, UNED, Hospital Universitario La Paz Duración, desde: 1996 hasta: 1997 Cuantía de la subvención: 6 MPts Investigador responsable: Sebastián Cerdán Número de investigadores participantes: 9

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Título del proyecto: Bases Biológicas del contraste intrínseco en imágenes por Resonancia Magnética Entidad financiadora: DGES PB96-0864-C02 Entidades participantes: CSIC,UNED Duración, desde: 1997 hasta: 1999 Cuantía de la subvención: 10 MPts Investigador responsable: Sebastián Cerdán Número de investigadores participantes: 5 Título del proyecto: Resonancia Magnética en Oncología. Aplicación al Diagnóstico Precoz del Cáncer. Entidad financiadora: Comunidad de Madrid 08.1/0023/97 Entidades participantes: CSIC, UNED, Hospital Universitario La Paz Duración, desde: 1998 hasta: 2000 Cuantía de la subvención: 18 MPts Investigador responsable: Sebastián Cerdán Número de investigadores participantes: 10 Título del proyecto: Nuevas Aplicaciones de la Resonancia Magnética en Oncología. Alicación al Diagnóstico Precoz del Cáncer. Entidad financiadora: Comunidad de Madrid 08.1/0046/1998 Entidades participantes: CSIC Duración, desde: 1999 hasta: 2000 Cuantía de la subvención: 12 MPts Investigador responsable: Sebastián Cerdán Número de investigadores participantes: 10 Título del proyecto: Nuevos Agentes de Contraste para el Diagnóstico y Pronóstico del Cáncer mediante métodos de Resonancia Magnética Entidad financiadora: Comunidad de Madrid, Plan Regional de Investigación 2000-3, Contrato Programa Grupo Estratégico de Investigación Entidades participantes: UNED, CSIC Duración, desde: 2000 hasta: 2003 Cuantía de la subvención: 600.000 Euros/3 años Investigador responsable: Paloma Ballesteros (UNED), Sebastián Cerdán (CSIC) Número de investigadores participantes: 10 Título del proyecto: Interacciones Metabólicas entre Neuronas y Células de glía en el cerebro de roedores y de seres humanos detectadas mediante Resonancia Magnética de 13C Entidad financiadora: Ministerio de Ciencia y Teconología, DGI, Plan Nacional de Biomedicina, SAF 2001-2245 Entidades participantes: CSIC Duración, desde: 2002 hasta: 2004 Cuantiía de la subvención: 97.000 Euros Investigador responsable: Sebastián Cerdán Número de investigadores participantes: 5 Título del proyecto: Red de Centros de Investigación en Cáncer Entidad financiadora: Instituto de salud Carlos III, Fondo de Investigaciones Sanitarias, FIS C03/010 Entidades participantes: CSIC, UNED, Hospirtal Universitario La Paz Duración, desde: 2003 hasta: 2005 Cuantiía de la subvención: 42.000 Euros/año Investigador responsable: Amparo Cano (coordinadora del nodo), Sebastián Cerdán Número de investigadores participantes: 6

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Título del proyecto: Red de Centros de Investigación en Enfermedades del Metabolismo Entidad financiadora: Instituto de Salud Carlos III, Fondo de Investigaciones Sanitaría, FIS C03/08 Entidades participantes: CSIC, UNED, Hospital Universitario La Paz Duración, desde: 2003 hasta: 2005 Cuantía de la subvención: 32.000 Euros/año Investigador responsable: Juan José Aragón (coordinador del nodo), Sebastián Cerdán Número de investigadores participantes: 6 Título del proyecto: Red de grupos de Investigación en “Bases celulares, moleculares y fisiopatológicas de la Encefaloptía Hepática. Implicaciones Diagnósticas” Entidad financiadora: Instituto de Salud Carlos III, Fondo de Investigaciones Sanitaria, FIS G03/155 Entidades participantes: CSIC, UNED, Hospital Universitario La Paz Duración, desde: 2003 hasta: 2005 Cuantía de la subvención: 24.000 Euros/año Investigador responsable: Vicente Felipo (coordinador), Sebastián Cerdán Número de investigadores participantes: 6 Título del proyecto: Acoplamiento Funcional y Heterogeneidad Metabólica en Modelos de Cáncer Detectada mediante métodos de Resonancia Magnética Entidad financiadora: Plan Nacional Biomedicina SAF2004-03197 Entidades participantes: CSIC Duración, desde: 2005 hasta: 2007 Cuantía de la subvención: 220.000 Euros Investigador responsable: Sebastián Cerdán Número de investigadores participantes: 6 Título del proyecto: Nuevas gliconanopartículas superparamagnéticas como trazadores no invasivos de la migración celular in vivo mediante imagen por Resonancia Magnética Entidad financiadora: Acción Estratégica en Nanotecnología NAN2004-09125-C07-03 Entidades participantes: CSIC Duración, desde: 31/12/2005 hasta: 31/12/2008 Cuantía de la subvención: 165.600 Euros Investigador responsable: Sebastián Cerdán Número de investigadores participantes: 6 Título del proyecto: Exploración de las aplicaciones biomédicas de gliconanopartículas magnéticas bifuncionales Entidad financiadora: Proyectos Intramurales de Frontera CSIC Entidades participantes: CSIC Duración, desde: 1/9/2005 hasta: 1/9/2007 Cuantía de la subvención: 22.500 Euros Investigador responsable: Sebastián Cerdán (Coordinadora: Soledad Penedés Ullate) Número de investigadores participantes: 5 Título del proyecto: Targeted Delivery of Nanomedicine MEDITRANS Entidad financiadora: Comunidad de Europea (Integrated Proyect) Duración, desde: 2007 hasta: 2010 Cuantía de la subvención: 300.000 euros Investigador responsable: Sebastián Cerdán Coordinador: Gert Storm (Universidad de Utrech) Nº de investigadores participantes: 30 grupos Título del proyecto: Imagen Multiparamétrica de la competencia vascular (MULTIMAG-CM) Entidad financiadora: Comunidad de Madrid- Convocatoria de grupos cooperativos en biocoencias Entidades participantes: UNED-CSIC-UCM-UAH Duración, desde: 2007 hasta: 2010 Cuantía de la subvención: 600.000 euros Investigador responsable y coordinador: Sebastián Cerdán Nº de investigadores participantes: 13 grupos y 4 empresas 12

CURRÍCULUM NORMALIZADO COMPLETO (CVNC)

Título del proyecto: Acoplamiento neuroglial y neurovascular durante la activacion cerebral por apetito detectada mediante métodos de Resonancia Magnética

Entidad financiadora: Ministerio de Ciencia e Innovación

Entidades participantes: CSIC Duración, desde: 2009 HASTA: 2011 Cuantía de la subvención: 210.000 euros Investigador responsable y coordinador: Sebastián Cerdán Nº de investigadores participantes: 7 Título del proyecto: Nanopharmaceuticals delivery acroos Biological Barriers (BIBA)

Entidad financiadora: Union Europea (UE)-Instituto de Salud Carlos III

Entidades participantes: CSIC, Hospital Universitario La Paz (IdIPAZ) Duración, desde: 2010 HASTA: 2013 Cuantía de la subvención: 100.000 euros Investigador responsable y coordinador: Patrick Boisseau (CEA, FR) Nº de grupos europeos participantes: 7 (40 investigadores) Título del proyecto: Imagen Multimodal de la inflamación y de las cascadas inflamatorias en la salud y la enfermedad. Entidad financiadora: Ministerio de Ciencia e Innovación, Plan Nacional Investigación SAF2011-23622

Entidades participantes: IIBm, CSIC Duración, desde: 2011 HASTA: 2014 Cuantìa de la Subvención: 254.100 euros Investigador responsable y coordinador: Sebastián Cerdán Nº de investigadores participantes: 8 Título del proyecto: Imagen Molecular Multimodal de la Inflamación-I2M2

Entidad financiadora: Comunidad de Madrid

Entidades participantes: CSIC, UCM, UNED, UAH, UAM Duración, desde: 2012 HASTA: 2015 Cuantía de la subvención: 968.610 euros Investigador responsable y coordinador: Sebastian Cerdan Nº de grupos participantes: 10 grupos de investigacion, 2 Laboratorios de referencia, 3 grupos asociados y cinco empresas (60 investigadores).

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PUBLICACIONES (CLAVE: L = libro completo, CL = capítulo de libro, A = artículo, R = “review”, E = editor, S = Documento Científico-Técnico restringido. ) Perfil general: Investigación Multidispplinar en aplicaciones biomédicas de la Resonancia Magnética. Areas de actividad/Publicaciones (algunas publicacioones figuran en mas de una area de especialización JCR): Biochemistry and Molecular Biology/ 38, Neurosciences/ 26, Radiology, Nuclear Medicine, Medical Imaging/24, Chemistry (all)/20/, Biophysics/15, Spectroscopy/16, Oncology/3) Una búsqueda reciente (Septiembre, 2014) en la bases de datos Web of Science proporciona en siguiente perfil (solo desde 1995) Published items per year Citations in each year

Numero total de publicaciones ISI WOK: 136 Numero total de citas: 2736 Promedio de citas por articulo: 20,12 Valor bibliométrico h= 31 1 AUTORES: M. Cascales, B. Feijoo, S. Cerdan, E.M. Robles-Chillida, A. Santos-Ruiz. TITULO: Effect des coenzymes UDP-glucose, CDP-choline, S-adenosylmetionine et CoA-SH sur le foie de rat traite avec de l'ethanol at en regime alimentaire lipidique. REF. REVISTA/LIBRO: Annuales Pharmaceutiques Francaises (1977), 35, 447-459. A ISSN 0003-4509 2 AUTORES: S. Cerdan, M. Cascales, P.Chacon, F. Cascales and A. Santos-Ruiz. TITULO: Hepatotoxic effect of thioacetamide on NADP-linked enzymes, aminotransferases and glutamate dehydrogenase. REF. REVISTA/LIBRO: Archives Toxicology (1978), 1, 221- 224. A I.F.: 2.626 (22/75 Q2 Toxicology) ISSN 0171-9750

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3. AUTORES: M. Cascales, S.Cerdán, Santos-Ruiz, M.R., Feijoo, B. and A. Santos-Ruiz. TITULO: Alcohol and aldehyde dehydrogenase on chronic ethanol intoxication. Effect of acetaldehyde and fat diet REF. REVISTA/LIBRO: Archives Toxicology (1978), 2, 497. A I.F.: 2.626 (22/75 Q2 Toxicology) ISSN: 0171-9750 4. AUTORES: M. Cascales, B .Feijoo, S. Cerdan, C. Cascales and A. Santos-Ruiz. TITULO: Effect of thioacetamide on rat liver urea cycle enzymes. REF. REVISTA/LIBRO: Journal of Clinical Chemistry and Clinical Biochemistry (1979) 17, 129-132. A ISSN: 0340-076X 5 AUTORES: S. Cerdan, S.S. Robles, M.S. Ayuso and R. Parrilla. TITULO: Acute effects of ethanol on the rate of hepatic protein synthesis in the rat in vivo REF. REVISTA/LIBRO: I.R.C.S. (1980) 8, 229. A 6 AUTORES: S. Cerdan, M. Cascales and A. Santos-Ruiz. TITULO: Effect of thioacetamide on the Pentose Phosphate Pathway and other NADPlinked enzymes of rat liver cytosol. Chronology of the perturbations and metabolic significance. A REF. REVISTA/LIBRO: Molecular Pharmacology (1981) 19, 451-455. IF: 4.71 (22/216 Q1 Pharmacology) ISSN: 0026-895X 7 AUTORES: C.J. Martinez-Honduvilla, M. Cascales, S. Cerdán, L. Riesco and A.SantosRuiz . TITULO: Effect of thioacetamide on the levels of nucleic acid fraction and soluble proteins of rat liver. REF. REVISTA/LIBRO: Oral directed toxicity: Chemical Indices and Mechanisms" (IUPAC) Ed. S.S.. Braum and D.S. Davies, Pergamon Press, Oxford and New York. (1981), 183-9 CL 8 AUTORES: M. Cascales, E.M. Robles-Chillida, B. Feijoo, S. Cerdan, P.Martin-Sanz y A. Santos-Ruiz. TITULO: Alteraciones producidas por la tioacetamida sobre las aminotransferasas GOT y GPT y la glutamato deshidrogenasa en hígado de rata. Estudio estructural. A REF. REVISTA/LIBRO: Revista Española de Fisiologia (1982), 38, 105-112. ISSN: 0034-9402 9 AUTORES: S. Cerdan, S.S. Robles, A. Martin-Requero, M. S. Ayuso and R. Parrilla. TITULO:Role of the state of reduction of the NAD-system on the regulation of hepatic protein synthesis in the rat in vivo. REF. REVISTA/LIBRO: International Journal of Biochemistry (1982) 14, 615-620. A 10 AUTORES: A. Martin-Requero, B.E. Corkey, S. Cerdan, E. Walajtis-Rode, R. Parrilla and J.R. Williamson. TITULO: Interactions between alpha-ketoisovalerate metabolism and the pathways of gluconeogenesis and urea synthesis in isolated hepatocytes. A REF. REVISTA/LIBRO: Journal of Biological Chemistry (1983) 258, 3673-3681. I:F: 5.520 (41/276, Q1 Biochemistry and Molecular Biology) ISSN: 0021-9258

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11 AUTORES: S. Cerdan, C.J. Lusty, K. Davies, J. Jacobson and J.R. Williamson. TITULO: Role of Calcium as an inhibitor of rat liver carbamyl-phosphate synthetase. A REF. REVISTA/LIBRO:Journal of Biological Chemistry (1984) 259,323-331. I.F.: 5.520 (41/276, Q1 Biochemistry and Molecular Biology) ISSN: 0021-9258 12 AUTORES: S. Cerdan, R. Parrilla, J.Santoro and M. Rico. 1H NMR detection of cerebral myo-inositol. TITULO: A REF. REVISTA/LIBRO: FEBS Letters (1985) 187, 167-172. I.F.: 3.264 (101/276, Q2 Biochemistry and Molecular Biology) ISSN: 0014-5793 13 AUTORES: S. Cerdan, V. Harihara-Subramanian, M. Hilberman, J. Cone, J. Egan, B. Chance and J.R. Williamson 31P NMR detection of dog brain phospholipids. TITULO: REF. REVISTA/LIBRO: Magnetic Resonance in Medicine (1986) 3, 432-439. A I.F.: 3.449 (16/90, Q1 Radiology, Nuclear Medicine and Medical Imaging) ISSN: 0740-3194 14 AUTORES: S. Cerdan, C. Hansen, R. Johanson and J.R. Williamson TITULO: Nuclear Magnetic Resonance Spctrsocopic analysis of myo-inositol phosphates including inositol 1,3,4,5-tetrakisphosphate. REF. REVISTA/LIBRO: Journal of Biological Chemistry (1986) 261, 14676-14680. A I.F.: 5.520 (41/276, Q1 Biochemistry and Molecular Biolology) ISSN:0014-5793 15 AUTORES: D. Perez-Sala, S. Cerdan, P.Ballesteros, M.S. Ayuso and R. Parrilla TITULO: Pyruvate decarboxylating action of L-cycloserine. The significance of this in understanding its metabolic inhibitory action. REF. REVISTA/LIBRO:Journal of Biological Chemistry (1986) 261, 13969-13972. A I.F.: 5.520 (41/276, Q1 Biochemistry and Molecular Biology ISSN: 0014-5793 16 AUTORES: S. Cerdan, B. Kunnecke, A. Dölle and J. Seelig TITULO: In situ metabolism of 1, dicarboxylic acids in the liver of intact rats as detected 13 1 by C and H NMR. REF. REVISTA/LIBRO: Journal of Biological Chemistry (1988) 263, 11664-11674. A I.F.: 5.520 (41/276, Q1 Biochemistry and Molecular Biology) ISSN: 0014-5793 17 AUTORES: S. Cerdan, H.R. Lötcher, B. Kunnecke and J. Seelig TITULO: Monoclonal antibody coated particles as contrast agents in Magnetic Resonance Imaging of tumors. REF. REVISTA/LIBRO: Magnetic Resonance in Medicine (1989) 12, 151-163. A I.F: 3.449 (16/90 Q1 Radiology, Nuclear Medicine and Medical Imaging) ISSN: 0740-3194 18 AUTORES: B. Künnecke and S. Cerdan TITULO: Use of 13C-multilabelled substrates in metabolic studies by 13C NMR. REF. REVISTA/LIBRO: NMR in Biomedicine (1989) 2, 274-277. A I.F.: 4.329 (3/39 Spectroscopy, 9/90 Radiology, Nuclear Medicine and Medical Imaging, Q1) ISSN: 0952-3480 16

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19* AUTORES: S. Cerdan and J. Seelig TITULO: NMR Studies of Metabolism. REF. REVISTA/LIBRO: Annual Reviews of Biophysics and Biomolecular Structure (1990) 19, 43-67. A I.F.: 17.049 (4/276 Q1 Biochemistry and Molecular Biology) ISSN:0083-9182, DOI: 10.1146/annurev.bb.19.060190.000355 20* AUTORES: S. Cerdan, B. Künnecke and J. Seelig 13 TITULO: Cerebral metabolism of (1,2- C2) acetate as detected by in vivo and in vitro 13C

NMR techniques. A REF. REVISTA/LIBRO: Journal of Biological Chemistry (1990) 265, 12916-12926. I.F.: 5.520 (41/276 Q1 Biochemistry and Molecular Biology) ISSN:0021-9258 21 AUTORES: R. Nieto, F. Cruz, J.M. Tejedor, G. Barroso and S. Cerdán. TITULO: Origin of the ammonia used for mitochondrial citrulline synthesis as revealed by 13C-15N spin coupling patterns observed by 13C NMR. Biochimie (1992) 74, 903-11. A REF. REVISTA/LIBRO: I.F.: 3.071 (111/276 Q2 Biochemistry and Molecular Biology) ISSN: 0300-9084 ___________________________________________________________________________ 22 AUTORES: M.S. Gil, F. Cruz, S. Cerdán and P. Ballesteros TITULO: Imidazol-1-yl-alkanoate esters and their corresponding acids. A novel series of extrinsic 1H NMR probes for intracellular pH. REF. REVISTA/LIBRO: Bioorganic and Medicinal Chemistry Letters (1992) 2, 1717-1722. A I.F.: 2.531 (16/40 Q2 Chemistry Medicinal, 22/55 Q2 Chemistry, Organic) ISSN: 0968-0896 23 AUTORES: R.J. Gillies, J.-P. Galons, K.A. Mc Govern, P.G. Scherrer, Y.-H. Lien, C. Job, R. Ratcliff, F. Chapa, S. Cerdán and B.E. Dale TITULO: Dessign and Application of NMR-compatible bioreactor circuits for extended Perfusion of High Density Mammalian Cell Cultures. A REF. REVISTA/LIBRO: NMR in Biomedicine (1993), 6, 95-104. I.F.: 4.329 (3/39 Spectroscopy, 9/90 Radiology, Nuclear Medicine and Medical Imaging, Q1) ISSN: 0952-5480 ___________________________________________________________________________ 24 AUTORES: M. A. Navas, S. Cerdán and J.M. Gancedo TITULO:Futile cycles in Saccharomyces cerevisiae strains expressing the gluconeogenic enzymes during growth on glucose. A REF. REVISTA/LIBRO: Proc. Natl. Acad. Sci. U.S.A. (1993) 90, 1290-1294. I.F.: 9.38 (3/42 Q1 Multidisciplinary Sciences) ISSN: 0027-8424 25 AUTORES:B. Künnecke, S. Cerdán and J. Seelig TITULO: Cerebral metabolism of (1,2-13C2) glucose and (U-13C4) 3-hydroxybutyrate as detected by 13C NMR spectroscopy. REF. REVISTA/LIBRO: NMR in Biomedicine (1993) 6, 264-277. A I.F.: 4.329 (3/39 Spectroscopy, 9/90 Radiology, Nuclear Medicine and Medical Imaging, Q1) ISSN: 0952-3480

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26 AUTORES: N.E. Preece and S. Cerdán TITULO:Determining 14N/15N ratios in body fluids by single pulse 1H NMR. A REF. REVISTA/LIBRO: Anal. Biochem. (1993) 215, 180-3 I.F.: 3.088 (14/70 Q1 Chemistry Analytical) ISSN: 0003-2697 27 AUTORES:M.A Günther-Sillero, A. de Diego, S. Cerdán, G. Criel and A. Sillero TITULO:Occurence of millimolar concentrations of guanosine-(5´)-tetraphospho- (5´)guanosine(GP4G) in encysted embrios of Thamnocephalus platyurus. A REF. REVISTA/LIBRO: Comp. Biochem. Physiol. (1994) 108B, 41-45. I.F.: 1.709 (196/276 Q3 Biochemistry and Molecular Biology) 28 AUTORES: M. Moldes, S. Cerdán, P. Erhard and J. Seelig TITULO:1H-2H exchange in the perfused rat liver metabolizing (3-13C) alanine and 2H2O as detected by multinuclear NMR spectroscopy. A REF. REVISTA/LIBRO: NMR in Biomedicine (1994), 7, 249-262 I.F.: 4.329 (3/39 Spectroscopy, 9/90 Radiology, Nuclear Medicine and Medical Imaging, Q1) ISSN: 0952-5480 29 AUTORES: M.S. Gil, P. Zaderenko, F. Cruz, S. Cerdán and P. Ballesteros TITULO:Imidazol-1-yl-alkanoic acids as extrinsic probes for intracellular pH, extracellular pH and cell volume. REF. REVISTA/LIBRO: Bioorganic and Medicinal Chemistry (1994), 2, 305-14 A I.F.: 3.075 (10/40 Q1 Chemistry, Medicinal, 110/276 Q2 Biohemistry and Molecular Biology) ISSN: 0968-0896 30 AUTORES: P. Zaderenko,M.S. Gil, P. Ballesteros and S. Cerdán TITULO:Synthesis and Regioselective hydrolysis of 2-Imidazol-1-yl-succinic acid esters. REF. REVISTA/LIBRO: J. Org. Chem. (1994) 59,6268-73 A I.F.: 3.952 (7/55 Q1 Chemistry, Organic) 31 AUTORES: F. Chapa, F. Cruz, M. Moldes, P. Grühlke, B. de Mateo, M.J. Maté, S. Cerdán TITULO:13C NMR detected spin-coupling patterns and isotopic shifts as novel metabolic tools REF. REVISTA/LIBRO: Quarterly of Magnetic Resonance in Medicine and Biology (1994) 1, 107-116 A 32 AUTORES: F. Chapa, B. Künnecke, R. Calvo, F. Escobar del Rey, G. Morreale de Escobar, S. Cerdán TITULO:Adult onset hypothyroidism and the cerebral metAbolism of (1,2-13C2) acetate REF. REVISTA/LIBRO: Endocrinology (1995) 136, 296-305 A I.F.: 4.945 (20/93 Q1 Endocrinology and Metabolism) ISSN:0013-7227 ___________________________________________________________________________ 33 AUTORES:M.Moldes, F. Cruz, F. Chapa, S. Cerdán TITULO:The perfused mouse liver as a model system for the NMR study of intracellular pH homeostasis. REF. REVISTA/LIBRO: Quarterly of Magnetic Resonance in Medicine and Biology (1995) 2, 517 A ___________________________________________________________________________

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34 AUTORES: P. Grhülke,C. Seipelt,A.Dölle,M.Zeitler,P.Zaderenko,P.Ballesteros, S.Cerdán TITULO:Molecular dynamics of Barbiturics and hydantoins as a basis for their neuroactive properties. REF. REVISTA/LIBRO: J. Chem.Soc.Perkin Trans. II (Org. Biomol. Chem) (1995) 1215-1219 A I.F.: 3.550(10/51 Q1 Chemistry Organic) 35 AUTORES: E. López-Beltrán, M.J. Maté, S. Cerdán TITULO: Dynamics and environment of mitochondrial water as detected by 1H NMR. A REVISTA/LIBRO: J. Biol. Chem. (1996) 271, 10648-10653. I.F.: 5.520 (41/276 Q1 Biochemistry and Molecular Biology) ISSN: 0021-9258 36 AUTORES: N. Preece, S. Cerdán TITULO:Metabolic Precursors and compartmentation of cerebral GABA in Vigabratin treated rats REF. REVISTA/LIBRO: J. Neurochem. (1996) 67,1718-1725 A I.F.: 4.500 (40/219 Q1 Neurosciences, 60/276 Q1 Biochemistry and Molecular Biology) ISSN: 0022-3042 37 AUTORES: B. de Mateo, P. Zaderenko, P. López, P. Ballesteros, S. Cerdán TITULO:Interactions between intracellular pH and cellular volume as detected by 1H NMR. REF. REVISTA/LIBRO: Eurospin Annual (1996) Editores: F. Podo,W.M.M.J. Bovée, J. de Certaines, O. Henricksen, M.O. Leach, D. Leibfritz. Istituto Superiore di Sanitá Pub,pg 397406 CL 38 AUTORES: M. Moldes, F. Cruz, M.L. García-Martín, M.A. García-Espiniosa, J. Alvarez, S. Cerdán TITULO:Effects of heavy water on hepatic intracellular pH and phosphatidylcholine turnover. A 31P NMR study REF. REVISTA/LIBRO: Cell and Mol. Biol. (1997) 43, 731-740. A I.F.: 1.333 (217/276 Q4 Biochemistry and Molecular Biology) 39 AUTORES: F. Cruz, M.L. García-Martín, M.A. García-Espinosa, S. Cerdán TITULO: 13C NMR: Methods and Applications REF.REVISTA/LIBRO: Syllabus: I Spectroscopy-II Methodology (1997) ESMRMB’97 Educational Programm, Editor: J. Bittoun, European Society of Magnetic Resonance Medicine and Biology. Guerbet Pub. (1997) Pg 1-14. CL 40 AUTORES: Cerdán, S. TITULO: Hepatic metabolism of pyruvate as detected by in vivo and in vitro 13C NMR. A quantitative stud REF.REVISTA/LIBRO:Medical and Biological Papers V, Fundación Eugenio RodriguezPascual (1997), Pg 103-133. CL 41 AUTORES: Cerdán, S. TITULO: Substrate selection in the neuronal and glial compartments of the intact rat brain as detected by 13C NMR spectroscopy. REF.REVISTA/LIBRO:Medical and Biological Papers V, Fundación Eugenio RodriguezPascual (1997), Pg 165-189. CL

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42 AUTORES: JM Pascual, F. Carceller, S. Cerdán, J.M. Roda TITULO: Diagnóstico de tumores in vitro por espectroscopía de Resonancia Magnética de protón. Método de los cocientes espectrales. A REF.REVISTA/LIBRO: Neurocirugía (1998) 9, 4-10 I.F.: 0.277 (142/148 Q4 Surgery) 43 AUTORES: R.S. Maxwell, I. Martínez-Pérez, S. Cerdán, M. Cabañas, C. Arús, C. Moreno, J.M. Roda, F. Carceller, J.M. Pascual, S.L. Howells, R. Mazzuco, J. Griffiths TITULO: Pattern Recognition Analysis of 1H NMR spectra from perchloric acid extracts of human brain tumor biopsies. A REF.REVISTA/LIBRO: Mag. Res. in Med. (1998) 39, 869-877 I.F.: 3.449 (16/90 Q1 Radiology, Nuclear Medicine and Medical Imaging) ISSN: 0740-3194 44 AUTORES: Gray,H., Maxwell,R.J., Martinez-Pérez,I., Arús,C., Cerdán,S. TITULO: Genetic Programming for classification and feature selection: analysis of 1H NMR spectra from human brain tumor biopsies. A REF.REVISTA/LIBRO: NMR in Biomedicine (1998) 11, 217-224 I.F.: 4.329 (3/39 Spectroscopy, 9/90 Radiology, Nuclear Medicine and Medical Imaging, Q1) ISSN: 0952-3480 45 AUTORES:J.M. Pascual, F. Carceller, J.M. Roda, S. Cerdán TITULO:Glutamate, Glutamine and GABA as substrates for the neuronal and glial compartments after focal cerebral ischemia in the rat. REF.REVISTA/LIBRO: Stroke (1998) 29, 1048-1057. A I.F.: 6.449 (6/156 Q1 Clinical Neurology, 7/56 Q1 Perypheral Vascular Disease) ISSN: 0039-2499 46 AUTORES: F. Cruz, R. Scott, I. Barroso, P. Santisteban, S. Cerdán TITULO:Ontogenesis and cellular localization of the pyruvate recycling system in rat brain A REF.REVISTA/LIBRO: J. Neurochem (1998) 70, 2613-2619. I.F.: 4.500 (40/219 Q1 Neurosciences, 60/276 Q1 Biochemistry and Molecular Biology) ISSN: 0022-3042 47 AUTORES: Protection by pyruvate and malate against glutamate mediated neurotoxicity. TITULO: F. Ruiz, G. Alvarez, R. Pereira, M. Hernández, M. Villalva, F. Cruz, S. Cerdán, E. Bogonez, J. Satrústegui REF.REVISTA/LIBRO: Neuroreport (1998) 9, 1277-1282 A I.F.: 1.904 (150/219 Q3 Neurosciences) ISSN: 0059-4965 48 AUTORES: M. García-Martin, M.A. García-Espinosa, S. Cerdan TITULO:Biochemistry detectable by MRS REF.REVISTA/LIBRO: Sillabus: Methodology, Spectroscopy and Clinical MRI. Cerdán,S., Haase,A., Terrier,F. Eds. Springer Verlag 1988, pg 7-13 E, CL 49 AUTORES: F. Cruz, S. Cerdan TITULO:Quantitative 13C NMR studies of metabolic compartmentation in the adult mammalian brain A REF.REVISTA/LIBRO: NMR in Biomedicine (1999) 12, 1-12 I.F.: 4.329 (3/39 Q1 Spectroscopy, 9/90 Q1 Radiology, Nuclear Medicine and Medical Imaging) ISSN: 0952-3480 ___________________________________________________________________________ 20

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50 AUTORES:P. López, C. G. Seipelt, P. Merkling, L. Sturz, A. Dölle, M. D. Zeidler, J. Álvarez, S. Cerdán, P. Ballesteros TITULO:N-2-(azol-1(2)-yl)-ethyliminodiacetic acids; a novel series of Gd(III) chelators as relaxation agents for magnetic resonance imaging. REF.REVISTA/LIBRO: Bioorg. Med. Chem. (1999) 7, 517.527 A I.F.: 3.075 (110/276 Q2 Biochemistry and Molecular Biology, 10/40 Chemistry Medicinal) ISSN: 0968-0896 51 AUTORES: R. Van Sluis, Z.M. Bhujwala, N. Raghunand, P. Ballesteros, J. Alvarez, S. Cerdán, J.P. Gallons, R.J. Gillies. TITULO:In vivo Imaging of extracellular pH using 1H MRSI REF.REVISTA/LIBRO: Mag. Res. Med. (1999) 41, 743-750. A I.F.: 3.449 (16/90 Q1 Radiology, Nuclear Medicine and Medical Imaging) ISSN:0740-3194 52 AUTORES: García-Pérez,A.I., Lopez-Beltran,E., Klünner,P., Luque,J.,Ballestros,P., Cerdán,S. TITULO:Subcellular compartmentation and molecular crowding as determinants of intracellular diffusion of metabolites REF.REVISTA/LIBRO: Arch. Biochem. Biophys. (1999), 362, 329-338 A I.F.: 2.226 (137/276 Q2 Biochemistry and Molecular Biology, 30/70 Q2 Biophysics) ISSN: 0003-9861 53 AUTORES: A. Pestaña, S. Cerdan TITULO:La Resonancia Magnética Nuclear en España REF.REVISTA/LIBRO: Mundo Científico (1999) 206, 70-75. A 54 AUTORES: F. Cruz, S. Cerdan TITULO:13C NMR Methods and Applications: 13C Isotopomer Analysis REF.REVISTA/LIBRO: EMBO Course Sillabus on " In vivo Magnetic Resonance Spectroscopy and Imaging in Biological Research", Seelig, J. Ed., 1999, 1-14 CL 55 AUTORES: F. Morales, P. Ballesteros, S. Cerdán TITULO:Neural Networks in Automatic Diagnosis malignant brain tumors REF.REVISTA/LIBRO: Engineering Applications of Bio-Inspired Artificial Neural Networks. Mira,J. Ed., Springer, 1999, pp 778-787. ISBN: 978-3-540-48772-2 CL 56 AUTORES: F. Cruz, S. Cerdan TITULO: NMR of cells REF.REVISTA/LIBRO: Encyclopedia of Spectroscopy and Spectrometry. Lindon J. Ed., Academic Press, 2000, 180-9. CL ISBN:978-0-12-374413-5_______________________________________________________ 57 AUTORES: Chapa, F., Cruz, F. García-Martín, M.L., García-Espinosa, M.A., S. Cerdán TITULO: Metabolism of (1-13C) glucose and (2-13C, 2-2H3) acetate in the neuronal and glial compartments of adult mammalian brain as detected by 13C-2H NMR spectroscopy. A REF.REVISTA/LIBRO: Neurochem. Int. (2000) 37, 217-228. I.F.: 3.228 (192/276 Q2 Biochemistry Molecular Biology, 76/219 Q2 Neurosciences) ISSN: 0197-0186

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58 AUTORES: García-Martín,M.L., S. Cerdán TITULO:13C NMR Spectroscopy REF.REVISTA/LIBRO: Sillabus: Methodology, Spectroscopy and Clinical MRI. Norris,D. Ed., Schering Diagnostics Pub, 2000, pg.39-48 CL ___________________________________________________________________________ 59 AUTORES:A. Pestaña, S. Cerdán TITULO: Spanish scientific productivity and equipment in magnetic resonance from a regional and European perspective REF.REVISTA/LIBRO: Scientometrics (2000) 49, 215-231 A I.F. 2.328 (13/94 Q1 Computer Science, Interdisciplinary Applications) ISSN : 0138-9130 60* AUTORES: Roda,J.M., Pascual, J.M., Carceller,F., González-Llanos,F., Barrios,L., S. Cerdán. TITULO:Non histological diagnosis of human cerebral tumors by 1H Magnetic Resonance Spectroscopy and amino acid analysis. A REF.REVISTA/LIBRO: Clin. Cancer Res.. (2000) 6, 3983-3993 I.F.: 6.448 (16/141 Q1 Oncology) ISSN: 1078-0432 ___________________________________________________________________________ 61 AUTORES: Pascual,J.M., González-Llanos,F., Cerdán,S., Carceller,F., Roda,J.M. TITULO:Fisiopatología de las células gliales en la isquemia cerebral REF.REVISTA/LIBRO: Neurocirugía (2000) 11, 247-259 A I.F.: 0.277 (210/219 Q4 Neurosciences, 142/148 Q4 Surgery) ISSN :1130-1473 62* AUTORES: García-Martín, M.L., Ballesteros, P., S. Cerdán TITULO: Dynamics and metabolism of water in cells and tissues as detected by Magnetic Resonance Methods REF.REVISTA/LIBRO: Progr. NMR Spectroscopy (2001) 39, 41-77 A I.F.: 6.162 (8/113 Q1 Chemistry, Physical , 1/31 Q1 Physics, Atomic Molecular) ISSN: 0079-6565 63 AUTORES: García-Martín, M.L., Herigault, G., Remy, C., Flarion. R., Ballesteros, P., Coles, J., Cerdán, S., Ziegler, A. TITULO: Mapping extracellular pH in rat brain gliomas in vivo by 1H magnetic resonance spectroscopy: comparison with maps of other metabolites. REF.REVISTA/LIBRO: Cancer Res. (2001) 61, 6524-6531 A I.F.: 7.514 (12/141 Q1 Oncology) ISSN:008-5472 ___________________________________________________________________________ 64 AUTORES: Hortelano, S.,Garcia-Martin, M. L.,Cerdan, S., Castrillo, A., Alvarez, A. M., Bosca, L. (2001) TITULO: Intracellular water motions decrease in apoptotic macrophages after caspase activation. REF.REVISTA/LIBRO: Cell Death Diff 8, 1022-8. A I.F.: 7.548 (24/276 Q1 Biochemistry and Molecular Biology, 22/157 Q1 Cell Biology) ISSN: 1350-9047, doi: 10.1038/sj.cdd.4400913

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65 AUTORES: Bhujwala,Z., Artemov, D., Ballesteros, P., Cerdán,S., Gillies, R.J., Solaiyappan, M. TITULO: Combined vascular and extracellular pH imaging of solid tumors A REF.REVISTA/LIBRO: NMR in Biomedicine (2002) 15, 114-119 I.F.: 4.329 (3/39 Spectroscopy, 9/90 Radiology, Nuclear Medicine and Medical Imaging, Q1) ISSN: 0952-3480 66* AUTORES:García-Martín,M.L., García-Espinosa,M.A., Ballesteros,P. Bruix,M., S. Cerdán. TITULO: Hydrogen turnover and subcellular compartmentation of hepatic (2-13C) glutamate and (3-13C) aspartate as detected by high field 13C NMR. A REF.REVISTA/LIBRO: J. Biol. Chem. (2002) 277, 7799-7807 I.F.: 5.520 (41/276 Biochemistry and Molecular Biology) ISSN:0014-5793_____________________________________________________________ 67 AUTORES: Cruz,F., Villalba,M., García-Espinosa,M.A., Bogónez,E. Satrústegui,J., Cerdán,S. TITULO: Intracellular pyruvate compartmentation in prymary cultures of cortical neurons as detected with 13C NMR with multiple 13C labels A REF.REVISTA/LIBRO: J. Neurosci. Res. (2001) 66, 771-781 I.F.: 3.086 (84/219 Q2 Neuroscience) ISSN: 0360-4012 68 AUTORES: Cerdan,S., Brandao Rodrigues,T., Benito,M., Sierra,A., P. López, Ballesteros,P., Perez-Mayoral,E. TITULO: The subcellular Metabolism of water and its implications for Magnetic Resonance Image Contrast REF.REVISTA/LIBRO: Magnetic Resonance in Food Science, Rutledge, D., Belton, P.S., Gil, A.M., Webb, G.A., Eds., Royal Society of Chemistry, 2003, pg 121-135. ISBN:978-1-84973-233-9 CL. 69 AUTORES: García-Espinosa,M.A., García-Martín,M.L., Cerdán,S. TITULO: Role of glial metabolism in diabetic encephalopathy as detected by high resolution 13C NMR REF.REVISTA/LIBRO: NMR in Biomedicine (2003), 16: 440-9 A I.F.: 4.329 (3/39 Spectroscopy, 9/90 Radiology, Nuclear Medicine and Medical Imaging, Q1) ISSN: 0952-3480 70 AUTORES: Cerdán,S. TITULO: 13C NMR Studies of Cerebral Metabolism. REF.REVISTA/LIBRO: NMR in Biomedicine (2003) 16: 301-2. A I.F.: 4.329 (3/39 Spectroscopy, 9/90 Radiology, Nuclear Medicine and Medical Imaging, Q1) ISSN: 0952-3480 71 AUTORES: Mayoral, E.P., García-Amo, M., P. López, E. Soriano, S. Cerdán, P. Ballesteros TITULO: A novel series of complexones with bis- or biazole structure as mixed ligands of paramagnetic contrast agents for MRI REF.REVISTA/LIBRO: Bioorg. Med. Chem. (2003) 11: 5555-5567. A I.F.: 3.075 (12/55 Q1 Chemistry Organic) ISSN: 0968-0896 23

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72 AUTORES: Cavero, S., Vozza, A., del Arco, A., Palimieri, L., Villa, A., Blanco, E., Runswick, M.J., Walker, J.E., Cerdán, S., Palmieri,F., Satrústegui,J. TITULO: Identification and metabolic role of the mitochondrial aspartate-glutamate transporter in Saccharomyces cerevisiae. REF.REVISTA/LIBRO: Molecular Microbiology (2003), 50: 1257-1269 A I.F.: 5.513 (46/276 Q1 Biochemistry Molecular Biology, 13/91 Q1 Microbiology) ISSN: 0950-382X 73 AUTORES: López-Larrubia, P., García-Amo, M., Perez Mayoral, E., Gillies, R.J., Cerdán, S., Ballesteros, P. TITULO: -Regioselective addition of (-)-N-tert-butoxycarbonyl-L-hystidine methyl ester to diethyl fumarate A REF.REVISTA/LIBRO: ARKIVOC (2004) 3: 26-31 I.F.: 1.377 (33/55 Q3 Chemistry Organic) ISSN: 1424-6376 74 AUTORES: García-Espinosa,M.A., Brandao Rodrigues,T., Sierra,A., Benito,M., C. Fonseca, Gray,H., Bartnik, B., Ballesteros,P., Cerdán,S. TITULO: Cerebral Glucose Metabolism and the Glutamine cycle as revealed by in vivo and in vitro 13C NMR spectroscopy. REF.REVISTA/LIBRO: Neurochemistry International (2004), 45: 297-303 A I.F.: 3.228 (102/276 Q2 Biochemistry Molecular Biology, 76/219 Q2 Neurosciences) ISSN: 0197-0186, doi: 10.1016/j. Neuint.2003.08.014 75 AUTORES: Pascual-Garvi, J.M., Gonzalez-Llanos, F., Prieto-Arribas, R., Cerdán, S., Roda, J.M. TITULO: La barrera hematoencefálica: desarrollo de una estructura que permite la heterogeneidad del sistema nervioso central. A REF.REVISTA/LIBRO: Rev. Neurol. (2004) 38, 565-581. I.F.: 1.083 (126/156 Q4 Clinical Neurology) 76 AUTORES: E. Soriano, S. Cerdan, P. Ballesteros, TITULO: Computational determination of pKa Values. A comparison of theoretical approaches and a novel procedure REF.REVISTA/LIBRO: J. Mol. Struc. (Theochem) (2004) 684: 121-8. A I.F.: 1.594 (66/113 Q3 Chemistry Physical) ISSN: 0022-2860_____________________________________________________________ 77 AUTORES: Rodrigues, T.B., Gray, H.L., Benito, M., Garrido, S., Sierra, A., Geraldes, C.F., Ballesteros, P., Cerdán, S. TITULO: Futyle Cycling of Lactate through the plasma membrane of C6 glioma cells as detected by (13C,2H) NMR spectroscopy. REF.REVISTA/LIBRO: J. Neurosci. Res. (2005) 79, 119-127 A I.F.: 3.086 (84/219 Q2 Neuroscience) ISSN: 0360-4012, doi: 10.1002/jnr.20308 78 AUTORES: Rodrigues, T.B., Cerdán, S. TITULO: A Fast and Sensitive (1H, 2H) NMR Method to Measure the Turnover of the H2 Hydrogen of Lactate A REF.REVISTA/LIBRO: Magn. Res. Med.(2005) 54: 1014-1019 I.F.: 3.449 (16/90 Q1 Radiology, Nuclear Medicine and Medical Imaging) ISSN: 0740-3194 ___________________________________________________________________________ 24

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79 AUTORES: Cerdan, S., Rodrigues, T.B. TITULO: The cerebral tricarboxylic acid cycles REF.REVISTA/LIBRO: Handbook of Neurochemistry and Molecular Neurobiology. Vol. 5: Brain Energetics from Genes to Cells, Integration of Molecular and Cellular Processes. Lajtha A., Gybson G., Dienel G., editors. Springer, New York. pp. 63-91 (2007). ISBN: 978-0-387-30349-9 CL 80 AUTORES: Rodrigues, T.B., Cerdan, S. TITULO: 13C NMR an outstanding tool for metabolic studies. A REF.REVISTA/LIBRO: Concepts in Magnetic Resonance (2005) 27A: 1-16 I.F.: 1.431 (23/39 Q3 Spectroscopy, 73/113 Q3 Chemistry Physical) ISSN: 1552-5023 ___________________________________________________________________________ 81 AUTORES: Ballesteros, P., Soriano, E., Pérez-Mayoral, E., García-Amo, M., Cerdán, S. TITULO: Flexible tetraazamacrocycles as metal ligands. Their relevance in Magnetic Resonance Imaging. REF.REVISTA/LIBRO: Structural analysis of cyclic systems, Iriepa,I. Ed., Research Singpost, 2005, 69-86 CL 82 AUTORES: Alvarez, J., Ballesteros, P., Cerdán, S. TITULO: Microscopic images of intraspheroidal pH by 1H Magnetic Resonance Chemical Shift Imaging of pH Sensitive indicators REF.REVISTA/LIBRO: MAGMA (2005), 18: 293-301. A I.F.: 1.888 (47/90 Q3 Radiology, Nuclear Medicine, Medical Imaging) ISSN: 0968-5243, doi: 10.1007/s10334-005-0013-z ___________________________________________________________________________ 83 AUTORES: Rodríguez, E., Roig, A., Molins, E., Arús, C., Quintero, M.R., Cabañas, M., Cerdan, S., Lopez-Larrubia. P., Sanfeliu, C. TITULO: In vitro Characterization o fan Fe8 cluster as potential MRI contrast agent REF.REVISTA/LIBRO: NMR in Biomedicine (2005), 18: 300-307. A I.F.: 4.329 (3/39 Spectroscopy, 9/90 Radiology, Nuclear Medicine and Medical Imaging, Q1) ISSN: 0952-3480 ___________________________________________________________________________ 84 AUTORES: Pérez-Mayoral, E., García-Amo, M.,Lopez-Larrubia, P., Cerdán, S., Ballesteros, P. TITULO: Synthesis of a new family of ligands with bispyrazole structure. Reactivity of bispyrazolilmethyl esthers. A REF.REVISTA/LIBRO: Heterocycles (2005), 65: 1691-1704. I.F.: 0.980 (39/55 Q3 Chemistry Organic) ISSN: 0385-5414 85 AUTORES: Cerdan, S., Rodrigues, T.B., Sierra, S., Benito, M., Fonseca, L.L, Fonseca, C., García-Martín, M.L. TITULO: The red-ox switch red-ox coupling hypothesis A REF.REVISTA/LIBRO: Neurochem. Int. (2006) 48: 523-530. I.F.: 3.228 (102/276 Q2 Biochemistry Molecular Biology, 76/219 Q2 Neurosciences) ISSN:0187-0186, doi:10.1016/j.neuint.2005.12.036

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86 AUTORES: Torija, P., Vicente, J.J., Rodrigues, T.B., Robles, A., Cerdan, S., Sastre, L., Calvo, R.M., Escalante, R. TITULO: Functional genomics in Dictiostelium: MidA, a new conserved protein, is required for mitochondrial function and development. REF.REVISTA/LIBRO: J. Cell Sci. (2006) 119: 1154-64 A I.F.: 6.247 (27/157 Q1 Cell Biology) ISSN: 0021-9533, doi:10.1242/jcs02819 87 AUTORES: Perez-Mayoral, E., Soriano, E., Cerdán, S. TITULO: Experimental and theoretical study of lanthanide complexes based on linear macrocyclic polyaminopolycarboxylic acids containing pyrazolil ethyl arms. A REF.REVISTA/LIBRO: Molecules (2006) 11: 345-56 I.F.: 1.252 (36/55 Q3 Chemistry Organic) ISSN:1420-3049 88 AUTORES: Pacheco-Torres, J., Pérez-Mayoral, E., Soriano, E., López-Larrubia, P., Ouari, O., González-Cortés, A., Cerdán, S., Ballesteros, P. TITULO: A convenient and efficient síntesis of the First (Nitroimidazolyl)succinic Esters and theirs Diacids REF.REVISTA/LIBRO: Synthesis-Stuttgart (2006) 22: 3859-3864. A I.F.: 2.470 (23/55 Q2 Chemistry Organic) ISSNs: 0039-7881 89 AUTORES: Diekmann, C., Simoes, R., Pohmann,R., Cerdan,S., Arús, C. TITULO: Proton Chemical Shift Imaging of Mouse Brain Tumors at 7T REF.REVISTA/LIBRO: Bruker Spin Report (2006) 6: 18-22. A 90 AUTORES: Rodriguez, E., Simoes, R., Roig, A., Molins, E., Nedelko, N., Slawska-Wanievska, A., Aime, S., Arús, C., Cabañas, M., Sanfeliu, C., Cerdán, S., García-Martin, M.L. TITULO: An iron based T1 contrast agent made of iron-phosphate complexes. In vitro and in vivo studies REF.REVISTA/LIBRO: MAGMA (2007) 20: 27-37. A I.F.: 1.888 (47/90 Q3 Radiology, Nuclear Medicine, Medical Imaging) ISSN: 0968-5243, doi:10.1007/s10334-006.0066-7 91 AUTORES: Prata, M.I.M., Santos, A.C., Torres, S., André, J.P., Martins, J.A., Neves, M., García-Martín, M.L., Rodrigues, T.B., Lopez-Larrubia, P., Cerdan, S., Geraldes, C.F.G.C. TITULO: Targeting of lanthanide (III) chelates of DOTA type glycoconjugates to the hepatic asyaloglycoprotein receptor: cell internatilzation and animal imaging studies REF.REVISTA/LIBRO: Contrast Media and Molecular Imaging (2006) 1: 246-258 A I.F.: 3.255 ( 19/90 Q1 Radiology, Nuclear Medicine, Medical Imaging) ISSN:1555-4317, doi: 10.1002/cmmi.111 _________________________________________ 92. AUTORES: Provent, P., Benito, M., Hiba, B., Farion, R., Lopez-Larrubia, P., Ballesteros, P., Remy, C., Segebarth, C., Cerdan, S., Coles, J., García-Martín, M.L. TITULO: Serial in vivo spectroscopic NMR imaging of lactate and extracellular pH in rat gliomas shows redistribution of protons away from sites of glycolisis REF.REVISTA/LIBRO: Cancer Res. (2007) 67: 7638-45. A I.F.: 7.514 (12/141 Q1 Oncology) ISSN:0008-5472, doi: 10.1158/008-5472.CAN-06-3459

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93 AUTORES: Rodrigues, T.B., Granado, N., Ortiz, O., Cerdan, S., Moratalla, R. TITULO: The metabolic interactions between gflutamatergic and dopaminergic neurotransmitter systems are mediated through D1 dopamine receptors A REF.REVISTA/LIBRO: J. Neurosci. Res. (2007) 85: 3284-93. I.F.: 3.086 (84/219 Q2 Neuroscience) ISSN: 0360-4012, doi:10.1002/jnr.21302 94 AUTORES: Ramírez, B.G., Rodrigues, T.B., Violante, I., Cruz, F., Fonseca, L.L., Ballesteros, P., Castro,l M.M., García-Martín, M.L., Cerdán,S. TITULO: Kinetic propertiews of the redox switch/redox coupling mechanisms as determined in prymary cultures of neurons and glial cells from rat brain. REF.REVISTA/LIBRO: J. Neurosci. Res. (2007) 85: 3244-53. A I.F.: 3.086 (84/219 Q2 Neurosciences) ISSN: 0360-4012, doi:10.1002/jnr.21386 95 AUTORES: Pérez-Mayoral, E., Soler-Padrós, J., Negri, V., Cerdán, S., Ballesteros, P. TITULO: Synthetic Approaches to heterocyclic ligands for Gd(III)-based contrast agents. A REF.REVISTA/LIBRO: Molecules (2007) , 12: 1771-95. I.F.: 1.252 (36/55 Q3 Chemistry Analytical) ISSN: 1420-3049 96 AUTORES: Rodrigues, T.B., Cerdán, S., García-Martín, M.L. TITULO: A method to measure lactate recycling in cultured cells by edited 1H nuclear magnetic resonance spectroscopy A REF.REVISTA/LIBRO: Anal. Biochem. (2007) 370:246-8 I.F.: 3.088 (14/70 Q1 Chemistry Analytical) ISSN: 0003-2697, doi: 10.1016/j.ab.2007.08.015 97 AUTORES: Caulí, O., Lopez-Larrubia, P., Rodrigues, T.B., Cerdán, S., Felipo, V. TITULO: Magnetic Resonance analysis of the effect of acute ammonia intoxication on rat brain. Role of NMDA receptors. REF.REVISTA/LIBRO: J. Neurochem. (2007) 103: 1334-43. A I.F.: 4.500 (40/219 Q1 Neurosciences, 60/276 Q1 Biochemistry and Molecular Biology) ISSN: 1471-4159, doi: 10.1111/j.1471-4159.2007.04878.x 98 AUTORES: Soler-Padrós, J., Pérez-Mayoral, E., Domínguez, L., López-Larrubia, P., Soriano, E., Marco-Contelles, J.L., Cerdan,S., Ballesteros, P. TITULO: Novel generation of pH indicators for 1H magnetic resonance spectroscopic imaging REF.REVISTA/LIBRO: J. Med. Chem. (2007) 50: 4359-62. A I.F.: 4.898 (40/219 Q1 Neurosciences, 60/276 Q1 Biochemistry and Molecular Biology) ISSN:0022-2623, doi:10.1021/jm070044j 99 AUTORES: Pascual, J.M., Solivera, J., Prieto, R., Barrios, L., López-Larrubia, P., Cerdán, S., Roda, J.M. TITULO: Time course of early metabolic changes following diffuse traumatic brain injury in rats as detected by 1H NMR spectroscopy A REF.REVISTA/LIBRO: J. Neurotrauma (2007) 24: 944-59 I.F.: 3.528 (29/156 Q1 Clinical Neurology) ISSN: 0897-7151, doi:10.1089/neu.2006.0190

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100 AUTORES: Ballesteros, P., Perez-Mayoral, E., Benito, M., Cerdán, S. TITULO: Imagen molecular de pH mediante metodos de Resonancia Magnética. Implicaciones Clínicas. A REF.REVISTA/LIBRO: Radiología (2008) 50: 463-70. I.F.: 0.955 (77/90 Q4 Radiology, Nuclear Medicine, Medical Imaging) ISSN:0033-8338 101 AUTORES: Pérez-Mayoral, E., Negri, V., Soler-Padros, J., Cerdán, S., Ballesteros, P. TITULO: Chemistry of paramagnetic and diamagnetic contrast agents for Magnetic resonance Imaging and Spectroscopy. pH responsive contrasta agents. REF.REVISTA/LIBRO: Eur. J. Radiol. (2008) 67: 453-8. I.F.: 2.339 (36/90 Q2 Radilogy, Nuclear Medicine, medical Imaging) A ISSN: 0720-048X, doi:10.1016/j.ejrad.2008.02.048 102 AUTORES: Simoes, R.V., Martínez-Aranda,A., Martín,B., Cerdán,S. Sierra,A., Arús, C. TITULO: Preliminary Charac terization of experimental breast cancer cells brain metastasis mouse model by MRI/MRS REF.REVISTA/LIBRO: MAGMA (2008) 21: 237-49 I.F.: 1.888 (47/90 Q3 Radiology, Nuclear Medicine, Medical Imaging) A ISSN: 0968-5243, doi:10.1007/s10334-008-0114-6 103 AUTORES: Fonseca, C.P., Sierra, A., Geraldes, C.F., Cerdán,S., Castro, M.M. TITULO: Mechanisms underlying Li+ effects in glutanmatergic and gabergic neurotransmissions in the adult rat brain and in prymary cultures of neural cells as revealed by 13C NMR REF.REVISTA/LIBRO: J. Neurosci. Res. (2009) 87: 1046-55 I.F.: 3.086 (84/219 Q2 Neuroscience) A ISSN:1097-4547, doi:10.1002/jnr21900 104 AUTORES: Delgado, T.C., Pinheiro, D., Caldeira, M., Castro, M.M., Geraldes, C.F., LopezLarrubia,P., Cerdán,S., Jones, J.G. TITULO: Sources of Hepatic triglyceride accumulation during high fat feeding in the healthy rat REF.REVISTA/LIBRO: NMR in Biomedicine (2009) 22:310-7. I.F.: 4.329 (3/39 Spectroscopy, 9/90 Radiology, Nuclear Medicine and Medical Imaging, Q1) A ISSN: 0952-3480, doi:10.1002/nbm.1327 105 AUTORES: Righi,V., Roda, J.M., Paz, J., Mucci,A., Tugnoli, V., Rodriguea- Tarduchi, G., Barrios, L., Schenetti, L., Cerdán,S., García.Martín, M.L. TITULO: 1H HR-MAS and Genomic Análisis of human tumor biopsies discriminates between high and low grade astrocytomas REF.REVISTA/LIBRO: NMR in Biomedicine (2009) 22: 629-37. I.F.: 4.329(3/39 Spectroscopy, 9/90 Radiology, Nuclear Medicine and Medical Imaging, Q1) A ISSN: 1099-1492, doi:10.1002/nbm.1377 106 AUTORES: Solivera,J., Cerdán,S., Pascual, J.M., Barrios,L., Roda,J.M. TITULO: Assesment of 31P NMR phospholipid profiles for potential differential,diagnosis of human cerebral tumors REF.REVISTA/LIBRO: NMR in Biomedicine (2009) 22: 663-674. I.F.: 4.329 (3/39 Spectroscopy, 9/90 Radiology, Nuclear Medicine and Medical Imaging, Q1)A ISSN: 1099-1492, doi:10.1002/nbm.1387

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107 AUTORES: Cerdán,S., Sierra,A., Fonseca, L.L., Ballesteros, P., Rodrigues.,T.B. TITULO: The turnover of the H3 deuterons from (2-13C) glutamate and (2-13C) glutamine reveals subcellular trafficking in the brain of partially deuterated rats. REF.REVISTA/LIBRO: J. Neurochem. (2009) 109 Suppl 1: 63-72. I.F.: 4.500 (40/219 Q1 Neurosciences, 60/276 Q1 Biochemistry and Molecular Biology)A ISSN: 1471-4159, doi:10.1111/j.1471-4159.2009.05962x 108 AUTORES: Rodrigues, T.B., López-Larrubia,P., Cerdán,S. TITULO: Redox Dependence and Compartmentation of (13C) pyruvate in the brain of deuterated rats bearing C6 gliomas REF.REVISTA/LIBRO: J. Neurochem. (2009) 109 Suppl. 1: 237-45. I.F.: 4.500 (40/219 Q1 Neurosciences, 60/276 Q1 Biochemistry and Molecular Biology) A ISSN: 1471-4159, doi:10.1111/j.1471-4159.2009.05935x 109 AUTORES: Violante, I., Anastasovska, J., Sanchez-Canon, G.J., Rodrigues, T.B., Righi, V.,, Nieto-Charques, L., Parkinson, J.R., Bloom, S.R., Bell, J.D., Cerdán,S. TITULO: Cerebral activation by fasting induces lactate accumulation in the hypothalamus REF.REVISTA/LIBRO: Magn. Res. Med. (2009), Jun 12 (Epub ahead of print) PMID: 19526502 I.F.: 3.449 (16/90 Q1 Radiology, Nuclear Medicine and Medical Imaging) A ISSN:1522-2594, doi: 10.1002/mrm.22010 110 AUTORES: Prieto, R., Gutiérrez-GFonzález, R., Pascual, J.M., Roda, J.M., Cerdán,S., Matías-Guiu, Barcia, J.A. TITULO: Experimental Models of Traumatic Brain Injury REF.REVISTA/LIBRO: Neurocirujía (2009) 20: 225-244. I.F.: 0.277 (142/148 Q4 Surgery) A ISSN:1130-1473 111 AUTORES: T. B. Rodrigues, A. Sierra, P. Ballesteros, S. Cerdán TITULO: Pyruvate Oxidation and Mitochondrial Substrate Transport in the Central Nervous System (2014) REF.REVISTA/LIBRO: Advances in Neurobiology, vol. 4. Neural Metabolism in vivo, I.Y. Choi, Gruetter, R., Eds., Springer, pg 715-761. CL ISSN: 2190-5215, ISBN: 978-1-4614-1787-0_______________________________________ 112 AUTORES: Marradi, M., Alcántara, D., Martínez de la Fuente, J., García-Martín, M.L., Cerdán, S., Penadés, S.L. TITULO: Paramagnetic Gd-based gold glyconanoparticles as probes for MRI: tuning relaxivities with sugars REF.REVISTA/LIBRO: Chem. Commun. (2009) 3922-3924 A I.F.: 5.34 (13/125 Q1 Chemistry Organic) ISSN: 1359-7345, doi:10.1039/B900957D ___________________________________________________________________________ 113 AUTORES: Rodrigues, T.B., Fonseca, C.P., Castro,M.M., Cerdan,S,, Geraldes, C. TITULO: 13C NMR tracers in neuriochemistry: implications for molecular imaging REF.REVISTA/LIBRO: Quarterly Journal Medical Molecular Imaging (2009) 53: 631-45 A ISSN:1824-4785

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114 AUTORES: Novakowsky, A., Alonso-Martín, S., Gonzalez-Manchón, C., Larrucea, S., Fernández, D., Vilar, M., Cerdán, S., Ayuso, M.S., Parrilla, R. TITULO: Ventricular enlargement associated with the panneural ablation of the podocalyxin gene REF.REVISTA/LIBRO: Mol. Cell. Neurosci (2010) 43: 90-97. A I.F.: 3.57 (13/125 Q2 Neurosciences) ISSN:1095-9327, doi:10.1016/jmcn.2009.09.011 ___________________________________________________________________________ 115 AUTORES: Rodrigues,T.B., Violante,I., Cerdán,S. TITULO: Unambiguous assignment of the H3S and H3R deuterations of cerebral (2-13C) glutamate by 13C NMR at 18.8 Tesla REF.REVISTA/LIBRO: Magnetic Resonance in Medicine (2010) 63(4):1088-91. A I.F.: 3.44 (16/92 Q1 Radiology, Nuclear Medicine, Medical Imaging) ISSN: 1522-2594, doi: 10.1002/mrm.22277 116 AUTORES: Pacheco-Torres,J., Lopez-Larrubia,P., Ballesteros,P., Cerdan,S. TITULO: Imaging tumour hypoxia by Magnetic Resonance Methods REF.REVISTA/LIBRO: NMR in Biomedicine (2011) 24 (1) 1-16 A I.F.: 4.329 (3/39 Q1 Spectroscopy, 9/90 Radiology, Nuclear Medicine and Medical Imaging) ISSN:1099-1492, doi:101002/nbm.1558 117 AUTORES: Negri,V., Cerpa,A., Lopez-Larrubia,P., Cerdan,S., Ballesteros,P. TITULO: Nanotubular Paramagnetic Probes as contrast agents for Magnetic Resonance Imaging based in the Diffusion Tensor REF.REVISTA/LIBRO: Angewandte Chemie Int. Ed. (2010) 49(10):1813-5 A I.F.: 10.88 (4/127 Q1 Chemistry Multidisciplinary) ISSN: 1521-3773, doi:10.1002/anie.200906415 118 AUTHORS: Riquelme R, Cediel R, Contreras J, la Rosa Lourdes RD, Murillo-Cuesta S, Hernandez-Sanchez C, Zubeldia JM, Cerdan S., Varela-Nieto I. TITLE: A comparative study of age-related hearing loss in wild type and insulin-like growth factor I deficient mice. A REF.JOURNAL/BOOK: Frontiers in Neuroanatomy Volume:13 Pages:4-27 Date: 2010 ISSN:1662-5129, doi: 10.3389/fnana.2010.00027 119 AUTHORS: Pardo B, Rodrigues TB, Contreras L, Garzón M, Llorente-Folch I, Kobayashi K, Saheki T, Cerdan S, Satrústegui J. TITLE: Brain glutamine synthesis requires neuronal-born aspartate as amino donor for glial glutamate formation. A REF.JOURNAL/BOOK: J. Cereb. Blood Flow Metab. Volume:31 Pages: 90-101 Date: 2011 I.F.: 5.46 (29/231 Q1 Neurosciences) ISSN: 1559-7016, doi:10.1038/jcbfm.2010.146

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120 AUTHORS: Pacheco-Torres J, Calle D, Lizarbe B, Negri V, Ubide C, Fayos R, Larrubia PL, Ballesteros P, Cerdan S. TITLE: Environmentally Sensitive Paramagnetic and Diamagnetic Contrast agents for Nuclear Magnetic Resonance Imaging and Spectroscopy. REF.JOURNAL/BOOK: Curr Top Med Chem A Date: 2011 Volume: (11) . Pages: 115-30 I.F.: 4.47 (5/46 Q1 Chemistry Medicinal) ISSN: 1873-4294 121 AUTHORS: Cauli O, López-Larrubia P, Rodrigo R, Agusti A, Boix J, Nieto-Charques L, Cerdán S, Felipo V. TITLE: Brain Region-Selective Mechanisms Contribute to the Progression of Cerebral Alterations in Acute Liver Failure in Rats. A REF.JOURNAL/BOOK: Gastroenterology. Volume: e-published ahead of print Pages: Date: 2010 I.F.: 12.89 (1/66 Q1 Gastroenteroly & Hepatology) ISSN: 1528-0012, doi: 10.1053/gastro.2010.10.043 122 AUTHORS: Muñoz S, Franckhauser S, Elias I, Ferré T, Hidalgo A, Monteys AM, Molas M, Cerdán S, Pujol A, Ruberte J, Bosch F. TITLE: Chronically increased glucose uptake by adipose tissue leads to lactate production and improved insulin sensitivity rather than obesity in the mouse. REF.JOURNAL/BOOK: Diabetologia A Volume: 11 Pages: 2417-30 Date: 2010 I.F.: 6.55 (9/105 Q1 Endocrinology and Metabolism) ISSN:1432-0428, doi:10.1007/s00125-010-1840-7 123 AUTHORS: Delgado,T.C., Violante,I.R., Nieto-Charques, L., Cerdán, S. TITLE: Neuroglial metabolic compartmentation underlying leptin deficiency in the obese ob/ob mice as detected by magnetic resonance imaging and spectroscopy methods REF.JOURNAL/BOOK: Journal of Cereb Blood Flow and Metabolism. A Volume: 31 Pages:2257-2266 Date: 2011 I.F.: 5.46 (29/231 Q1 Neurosciences) ISSN: 1559-7016, doi: 10.1038/jcbfm.2011.134 124 AUTHORS: Fernández-Trillo,F., Pacheco-Torres,J., Correa, J., Ballesteros, P., LopezLarrubia, P., Cerdán, S., Riguera,R., Fernández-Mejía, E. TITLE: Dendritic MRI contrast agents: an efficient prelabelling approach based on CuAAC REF.JOURNAL/BOOK: Biomacromolecules A Volume: 12 Pages:2902-2907 Date: 2011 I.F.: 5.479 (7/56 Q1 Chemistry) ISSN:1526-4602, doi: 10.1021/bm2004466 125 AUTHORS: Borges, A.R., Lopez-Larrubia, P., Marques, J.B., Cerdán, S. TITLE: MR Imaging Features of High Grade Gliomas in Murine Models: How they Compare with human disease, Reflect Tumor Biology and Play a Role in Preclinical Trials. REF.JOURNAL/BOOK: American J. Neuroradiology A Volume: Pages: Epub ahead of print Date: 2011 I.F.: 2.928 (27/116 Q1 Radiology, Nuclear Medicine, Medical Imaging) ISSN:1936-959X, doi:10.3174/ajnr.A2959

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126 AUTHORS: López-Larrubia,P., Cañadillas-Cardenas, E., Metelo, A.M., Arias, N., MartínezMaestro,M., Salguero, A., Cerdán, S. TITLE: Magnetic resonance Imaging of Gliomas BOOK: Advances in the Biology, Imaging and Therapies of Glioblastoma CL Editor: Clark. Chen Chapter 12, Date: 2011 Published electrionically : http://www.intechopen.com/articles/show/title/magnetic-resonanceimaging-of-gliomas ISBN : 978-953-307-284-5 127 AUTHORS: Delgado, T,C., Barosa, C., Nunes, P.M., Cerdan, S., Geraldes, C.F., Jones, J.G. TITLE: Resolving the sources of plasma glucose excursions following a glucose tolerance tests in the rat with deuterated water and (U-13C6) glucose JOURNAL: PlosOne A Date: 2012 Volume : 7 (3) Pages : epub a of print PMID 22479514 IF : 4.092 (12/84 Q1 Biology) ISSN :1932-6203, doi :10.1371/journal.pone.00344042 128 AUTHORS: Esteras,N., Alquezar, Bartolomé,F., Antequera, D., Barrios, L., Carro,E., Cerdan,S., Martin-Requero,M.A. TITLE: Systematic Evaluation of Magnetic resonance Imaging and Spectroscopy for imaging a transgenic mouse model of Alzheimer disease (APP/PS1) JOURNAL: Journal of Alzheimer Disease A Volume : 30 (2) Pages : 337-53 Date: 2012 I.F. : 3.745 (73/243 Neurosciences Q2) ISSN : 1875-8908, doi : 10.3233/JAD-2012-111967 129 AUTHORS: Lizarbe,B., Benitez, A., Sanchez-Montañes, M., Lago-Fernández,L., GarciaMartín, M.L., Lopez-Larrubia,P., Cerdan, S. TITLE: Imaging Hypothalamic Activity using Diffusion Weighted Magnetic Resonance Imaging in the mouse and human brains. JOURNAL: Neuroimage A Volume :64 Pages : 448-57 Date: 2013 I.F. : 5.895 (1/14 Neuroimaging Q1) ISSN :1662-6247, doi : 10.1016/j.neuroimage.2012.09.033 130 AUTHORS: Miguel F. Ferreira, André F. Martins, Catarina I. O. Martins, Paula M. Ferreira, Éva Tóthc, Tiago B. Rodriguesd, Daniel Calle, Sebastian Cerdan, Pilar López-Larrubia, José A. Martins, Carlos F. G. C. Geraldes TITLE: Amide conjugates of the DO3A-N-(a-amino) propionate ligand: leads for stable, high relaxivity contrast agents for MRI? JOURNAL: Contrast Media and Molecular Imaging A Volume: 8(1) Pages: 40-9 DOI: 10.1002/cmmi.1492 Date:2013 I.F.: 3.328 (19/116 Radiology, Nucelar Medicine, Medical Imaging Q1) ISSN:1555-4317, doi:10.1002/cmmi.1492

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131 AUTHORS: Lizarbe,B., Benitez, A., Lago, L., Sanchez-Montañes, M., Lopez-Larrubia, P,, Cerdan, S. TITLE: Intelligent Image Analysis of Diffusion Data Sets: A new tool for functional imaging. BOOK: Combinatorial Image Analaysis, Pgs 9-12. Date: 2011 CL Editors: Aggarwal, J.K., Barnera, R.P., Brimkov, R.E., Koroutchev, K,N.,Koroutcheva, E. Publisher : Springer ISBN 978-3-642-21073-0 132 AUTHORS: Delgado, T.C., Nunes, P.M., Scott, D.K., O´Doherty, R.M., Cerdán, S., Geraldes C.F., Jones. J.G. TITLE: Effect of Cyclosporine A on hepatic carbohydrate metabolism and hepatic gene expression in rat. JOURNAL: Expert Opin. Drug Metab. Toxicol. A Volume: 8(10) Pgs 1223-30. Date: 2012 I.F.: 3.119 (73/261 Pharmacology & Pharmacy Q2) ISSN: 1744-7607, doi: 10.1517/17425255.2012.709500 ___________________________________________________________________________ 133 AUTHORS: Rodrigues,T.B., Ceballos,A.,Grijota-Martínez,C., Nuñez,B., Refetoff,S. Cerdán,S., Morte,B., Bernal,J. TITLE: Increased oxidative metabolism and neurotrnamitter cycling in the brain of mice lacking the thyroid hormone transporter SLC16A2 (MCT8) JOURNAL: PlosOne 2013 Volume: 8 (10) Pags: e74621 A I.F. 3.70 (7/56 Multidisciplinary Sciences Q1) ISSN:1932-6203, doi: 10.1371/journal.pone0074621 ___________________________________________________________________________ 134 AUTHORS: García-Palmero,I., Lopez-Larrubia,P., Cerdán,S. and Villalobo, A. TITLE: Nuclear Magnetic Resonance Imaging of tumor growth and neovasculature performance in vivo reveals GrB7 as a novel antiangiogenic target. JOURNAL: NMR in Biomedicine 2013, Volume: 26(9): Pages: 1059-69. A I.F. 3.45 (Radiology, Nuclear Medicine, Medical Imaging 20/120 Q1, Spectroscopy 5/43 Q1) ISSN: 1099-1492, doi:10.1002/nbm.2918 ___________________________________________________________________________ 135 AUTHORS: Lizarbe,B., Benitez,A., Peláez-Brioso, G.A., Sánchez-Montañés, M., LópezLarrubia,P., Ballesteros,P., Cerdán,S. TITLE: Hypothalamic metabolic compartmentation during apetite regulation as revealed by magnetic resonance imaging and spectroscopy methods JOURNAL: Frontiers in Neuroienegetics doi: 10.3389/fnene 2013 ecollection 2013 A ISSN:1662-6427, doi: 10.3389/fnene.2013.0006 ___________________________________________________________________________ 136 AUTHORS: Lizarbe,B. and Cerdán,S. TITLE: Control hipotalámico de las interacciones neuroendocrinas JOURNAL: Anales de la Real Academia de Farmacia Volumen: 2013, 79(1) Pags: 90-110 A ISSN: 1697-4271 ___________________________________________________________________________

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137 AUTHORS: Cauli,O., Liansola,M., Agustí,A., Rodrigo,R., Hernández-Rabaza,V., Rodrigues,T.B., Lopez-Larrubia,P., Cerdán,S., Felipo,V. TITLE: Cerebral oedema is not responsible for motor and cognitive deficits in rats with hepatic encephalopathy. JOURNAL: Liver Int. Vol: Pags: A ISSN: 1478-3231, doi: 10.1111/liv.12258 ___________________________________________________________________________ 138 AUTHORS: Cerpa, A., Köber,M., Calle,D., Negri, V., Gavira, .M., Hernanz, A., Cerdán, S., Ballesteros,P. (2013) TITLE: Single Wall Carbon Nanotubes as anisotropic relaxation probes for magnetic resonance imaging. JOURNAL: Med. Chem. Comm. Volumen: 4 Pags: 90-110 A I.F. 2.72 (25/59 Chemistry Medicinal Q2) ISSN: 2040-2511, doi: 10.139/C3MD2023F ___________________________________________________________________________ 139 AUTHORS: Gutiérrez-Fernández M, Rodríguez-Frutos B, Ramos-Cejudo J, Teresa VallejoCremades M, Fuentes B, Cerdán S, Díez-Tejedor E. (2013) TITLE: Effect of intravenous administration of allogenic bone.-marrow and adipose tissuederived messenquimal stem cells on functional recovery and brain repair markers in experimental ischemic stroke JOURNAL: Stem Cell Res.Ther. VOL: 4 Pags:1-11 A I.F. 4.63 ( 19/124 Medicine, Research and Experimental, Q1) ISSN: 1757-6512, doi: 10.1186/scrt159._________________________________________ 140 AUTHORS: Gary Frost , Michelle L. Sleeth, Meliz A. Arisoylu, Blanca Lizarbe, Sebastian Cerdan, Leigh Brody, Jelena Anastasovska, Samar Ghourab, Mohammed Hankir,Shuai Zhang, David Carling, Jonathan Swan, Glenn Gibson, Alexander Viardot, Douglas Morrison, E. Louise Thomas, Jimmy D. Bell (2014) TITLE: The short- chain fatty acid acetate reduces appetite via a central homeostatic mechanism”. JOURNAL: Nature Comm. Vol: 5 Pags:1-11 A I.F. 10,15 (3/56 Multidisciplinary Sciences Q1) ISSN:2041-1723 , doi: 10.1038/ncomms4611 ___________________________________________________________________________ 141 AUTHORS: Ramos-Cejudo J, Gutiérrez-Fernández M, Otero-Ortega L, Rodríguez-Frutos B, Fuentes B, Vallejo-Cremades MT, Hernanz TN, Cerdán S, Díez-Tejedor E. (2015) TITLE: Brain derived neurotrophic factor administration mediated oligodendrocyte differentiation and myelin formation in experimental ischemic stroke. JOURNAL: Stroke , Vol 46 Pags: 221-8 A I.F. 6,02 (5/65 Perypheral Vascular Disease Q1, Clinical Neurology 11/194 Q1) ISSN:1524-4628, doi:10.1161/strokeaha.114.006692 ___________________________________________________________________________ 142 AUTHORS: Calle, D., Negri,V., Ballesteros,P., Cerdán,S. TITLE: Magnetoliposomes loaded with Poly-unsaturated Fatty Acids as Novel Theranostic Anti-Inflammatory Formulations (2015) JOURNAL: Theranostics , Vol 5, Pags: 489-503 A I.F. 7,827 (9/124 Medicine, Research&Experimental Q1) ISSN:1838-7640 34

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___________________________________________________________________________ 143 AUTHORS: Ferreira,M.F., Gonzalves,J., Mousavi,B., Prata, M.I., Rodrigues,S.P., Calle, D., López-Larrubia,P., Cerdán, S., Rodrigues, T.B., Ferreira, P.M., Helm.L., Martins,J.A., Geraldes, C.F. TITLE: Gols Nanoparticles functionalized with fast water exchange Gd(3+) chelate: linker effects on the relaxivity (2015) JOURNAL: Dalton Transactions , Vol 44, Pags: 4016-31 A I.F. 4,097 (6/46 Chem istry, Inorganic & Nuclear Q1) ISSN: 1477-9234, doi: 10.1039/c4dt03210a ___________________________________________________________________________

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PARTICIPACION EN CONTRATOS DE INVESTIGACION DE ESPECIAL RELEVANCIA CON EMPRESAS Y/O ADMINISTRACIONES 6 Proyectos de investigación translacional, 5 proyectos como investigador principal, Total recursos movilizados: 5.664.246 euros) Título del Contrato: Evaluación de derivados de Imidazol como agentes de contraste para imagen de pH mediante métodos de Resonancia Magnética Entidad financiadora: Laboratorios Farmacéuticos ROVI SA Entidades participantes: CSIC, UNED Duración, desde: 1999 hasta: 2000 Precio del Proyecto: 20 Mpts Investigador responsable: Sebastián Cerdán Número de investigadores participantes: 5 Título del Contrato: Nuevos Agentes de Contraste de Alta eficacia para el diagnóstico por imagen de Resonancia Magnética. Complexotas Heterocíclicas como ligandos de lantánidos. Entidad financiadora: Laboratorios Farmacéuticos ROVI S.A. Entidades participantes: CSIC, UNED Duración, desde: 1999 hasta: 2000 Precio del Proyecto: 20 Mpts Investigador responsable: Sebastián Cerdán Número de investigadores participantes: 5 Título del Contrato: Una nueva generación de Agentes de Contraste para Diagnóstico por Imagen Entidad financiadora: JUSTESA IMAGEN S.A. Entidades participantes: CSIC, UNED Duración, desde: 2003 hasta: 2007 Precio del Proyecto: 3.000.000 Euros Investigador responsable: Sebastián Cerdán Número de investigadores participantes: 6 Titulo del Contrato: Modelización Molecular y Evaluación in vivo mediante imagen por Resonancia Magnética de Promotores de absorción intestinal Entidad Financiadora: Laboratorios Farmacéuticos ROVI, S. A. Proyecto CENIT Consorcio NanoFarma 2005 Duración, desde: 2006 hasta: 2007 Investigador Principal: Sebastián Cerdán Precio del Proyecto: 710.000 euros Numero de Investigadores participantes: 7 ___________________________________________________________________________ Titulo del Contrato: Nuevas nanoparticulas superparamagnéticas como agentes de contraste para imagen por Resonancia Magnética Entidad Financiadora: SOLUTEX S.A. Duración, desde: 2009 hasta: 2013 Investigador Principal: Sebastián Cerdán Precio del Proyecto: 470.000 euros Número de Investigadores participantes: 5 ___________________________________________________________________________

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Titulo del Contrato: Nuevas tecnologías para la producción de concentrados de alta pureza de acidos grasos omega-3, EPA y DHA Entidad Financiadora: CEDETI- INNPACTO 2012- IPT-2012-1331-060000 Duración, desde: 2013 hasta: 2015 Investigador Principal: Gerard Bannemberg Precio del Proyecto: 1.244.246 euros Número de Investigadores participantes: 5 ___________________________________________________________________________

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PATENTES Y MODELOS DE UTILIDAD 6 Patentes (5 Europeas, 1 Americana), 1 transferida ___________________________________________________________________________ Inventores (p.o. de firma): P. Ballesteros, P. López Larrubia, S. Cerdán Titulo: Complexonas de naturaleza de ácidos N-2-(azol-1(2)-il)etiliminodiacéticos. Síntesis, estudio analítico y aplicaciones biológicas N. de solicitud: ES96/129 PAÍS DE PRIORIDAD: España Fecha de prioridad: 11-Junio-1996 Entidad Titular: UNED-CSIC Paises a los que se ha extendido: Europa, EEUU, Canadá y Japón Empresa/s que la están explotando: ROVI S.A. Inventores (p.o. de firma): P. Ballesteros, M. S. Gil, P. Zaderenko, S. Cerdán, J. Álvarez, R. J. Gillies, R. Natarajan, R. Van Sluis, Z. Bhujwala Título: Procedimientos para la obtención de imágenes y espectros del pH extracelular por resonancia magnética con indicadores extrínsecos conteniendo 1H ó 19F N. Solicitud: ES97/496, PCT/ES98/00045 PAÍS DE PRIORIDAD: España Fecha de prioridad: 6-Marzo-1997 Entidad Titular: UNED-CSIC-U. Arizona-U. John Hopkins Paises a los que se ha extendido: EEUU (aceptada 22/7/2003- REEL 013749/FRAME:0761, Patent No 6,596,258 B1), Europa, EEUU, Canadá Empresa/s que la están explotando: Laboratorios Farmacéuticos ROVI S.A. (Transferido 6 de Junio de 2000) Inventores (p.o. de firma): P. Ballesteros, M. S. Gil, P. Zaderenko, S. Cerdán, J. Álvarez, R. J. Gillies, R. Natarajan, R. Van Sluis, Z. Bhujwala Titulo: Productos de naturaleza de imidazol y derivados N. de solicitud: ES98/801 PAÍS DE PRIORIDAD: España Fecha de prioridad: 20-Noviembre-2000 Entidad Titular: UNED-CSIC-U. Arizona-U. John Hopkins Paises a los que se ha extendido: Empresa/s que la están explotando: Laboratorios Farmacéuticos ROVI S.A. (Transferido 6 de Junio de 2000) Inventores (p.o. de firma): P. Ballesteros, S. Cerdán Titulo: Nuevos ligandos de Gd(III) con estructura bi- y bisazólica N. Solicitud: ES2001/00167 PAÍS DE PRIORIDAD: España Fecha de prioridad: 25-Enero-2001 Entidad Titular: UNED-CSIC-Laboratorios Farmacéuticos ROVI Paises alos que se ha extendido: Empresa/s que la están explotando: Laboratorios Farmacéuticos ROVI S.A. ___________________________________________________________________________ Inventores (p.o. de firma): P. Ballesteros, P. López, L. Domínguez, Marco, J.L., Cerdán, S. Titulo: 1,w-di-imidazol-1-ylpolioles como indicadores extrínsecos de pH para Espectroscopía e Imagen por Resonancia Magnética N. Solicitud: P200300936 PAÍS DE PRIORIDAD: España Fecha de prioridad: 23-Abril-2003 Entidad Titular: UNED-CSIC Paises alos que se ha extendido: Empresa/s que la están explotando: ___________________________________________________________________________

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Inventores (p.o. de firma): Cerdán, S., López-Larrubia, P., Nieto-Charques, L.,Negri, V., PerezMayoral,E., Ballesteros,P. Titulo: Materiales nanoestructurados tubulares con propiedades magnéticas anisotrópicas. Procedimiento de Obtención y sus aplicaciones N. Solicitud: P200800024 PAÍS DE PRIORIDAD: España Fecha de prioridad: 8-01-2008 Entidad Titular: UNED-CSIC Paises alos que se ha extendido: Empresa/s que la están explotando: ___________________________________________________________________________ Inventores (p.o. de firma): Cerdán, S., Calle, D., Moreno, F. Titulo: Superparamagnetic nanoparticles as contrast agents for Magnetic Resonance Imaging of Magnetic Susceptibility. N. Solicitud: PCT ES2012/070044 PAÍS DE PRIORIDAD: España Fecha de prioridad: 8-01-2008 Entidad Titular: SOLUTEX SL Paises alos que se ha extendido: Empresa/s que la están explotando: SOLUTEX SL ___________________________________________________________________________

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ESTANCIAS EN CENTROS EXTRANJEROS 2 Estancias larga duración, 1 USA, 1 Suiza, Total: 6 años CLAVE: D = doctorado, P = postdoctoral, I = invitado, C = contratado, O = otras (especificar). Centro: Department of Biochemistry and Biophysiscs, University of Pennsylvania Localidad: Philadelphia País:U.S.A. Fecha:1981-4 Duración: (semanas) 216 Tema: Aplicaciones Biologicas de la Resonancia Magnética Clave: P Centro:

Abteilung Biophysikalische Chemie, Biozentrum der Universität Basel

Localidad: Basel País: Suiza Fecha: 1986-8 Duración: (semanas): 108 Tema: Aplicaciones de la Resonancia Magnética Nuclear de 13C in vivo Clave: P ___________________________________________________________________________

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CONGRESOS 225 Comunicaciones Congresos, 195 Comunicaciones Internacionales, 26 Comunicaciones Nacionales, 4 Conferencias Plenarias Internacionales de Apertura/Clausura (45 min-1h) P: Poster, PO: Comunicación Oral (< 15 min), IS: Speaker Invitado, Ponencia a Simposium (>30 min), CP: Conferencia Plenaria 1. Cascales,M, Cerdán,S, Feijoo,B, Santos-Ruiz,A (1976) Efecto de coenzimas nucleotidicos sobre higado de rata tratado con Tioacetamida. Proceedings 1st Congress Federation of Societies of Experimental Biology FESBE. Madrid, Spain. P 2. Cerdán,S, Cascales,M, Chacon,P, Cascales,C y Santos-Ruiz,A (1977). Hepatotoxic effect of thioacetamide on NADP-linked enzimes, aminotransferases and glutamate dehydrogenase. Proceedings IX International Meeting of the European Society of Toxycology. Copenhague, Denmark. P 3. Cerdán,S, Chacon,P, Cascales,M, Santos-Ruiz, A (1977) Efecto de la tioacetamida sobre niveles de nucleotidos energeticos y metabolismo del glutamato. Proceedings II eme Rencontre Franco-Espagnole de Pharmacologie. Madrid, Spain. PO 4. M. Cascales, B. Feijoo, S. Cerdán, D. Velasco, M.Robles-Chillida, A. Santos-Ruiz (1977) Efecto de coenzimas nucleotidicos sobre hígado de rata tratado con etanol y dieta grasa. Proceedings II eme Rencontre Franco-Espagnole de Pharmacologie. Madrid, Spain. P 5. Cascales,M, Cerdán,S, Santos-Ruiz,MR, Santos-Ruiz,A (1978) Alcohol and aldehyde dehydrogenases in chronic ethanol intoxication. Effect of acetaldehyde and fat diet. Proceedings X International Meeting of the European Society of Toxicology. Berlin, Germany. P 6. Cerdán, S, Cascales,M, Santos-Ruiz,A (1978) Efecto hepatotóxico de la tioacetamida sobre la ruta de los Fosfatos de Pentosa. Proceedings VII Meeting of the Spanish Society of Biochemistry. Madrid, Spain. P 7. Cascales,C, Santos-Ruiz,MR, Cascales,C, Cerdán,S, Santos-Ruiz,R (1979) Efecto hepatotóxico del alcohol y acetaldehido sobre enzimas NADP-dependientes, aminotransferasas y glutamato deshidrogenasa. Proceedings VIII Congress Spanish Society of Biochemistry. Murcia, Spain. P 8. Cerdán, S. Sanchez-Robles,S, Ayuso, MS, Parrilla,R (1980). Efecto del etanol sobre sintesis de proteinas hepaticas de rata "in vivo". Proceedings 1st Protuguese-Spanish Congress of Biochemistry. Coimbra, Portugal. P 9. Robles,SS, Cerdán,S, Parrilla,R, Ayuso,MS (1980) Efecto diferencial de la oxidación de octanoato y oleato sobre el estado de fosforilación del sistema hepático de nucleótidos de adenina en la rata "in vivo". Proceedings 1st Protuguese-Spanish Congress of Biochemistry. Coimbra, Portugal. P *10. Cerdán,S, Lusty, C, Davies,K, Williamson,J (1983) Role of calcium as an inhibitor of rat liver Carbamyl-Phosphate Synthetase I. Proceedings 74th Annual Meeting of the American Society of Biological Chemists. San Francisco, USA. Federation Proceedings (1983): 42, 2076. P

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*11. Williamson,JR, Cerdán,S, Kobayashi,K, Seeholtzer,S (1983) 13C NMR study of pyruvate metabolism in perfused rat liver. Proceedings 74th Annual Meeting of the American Society of Biological Chemists. San Francisco, USA. Federation Proceedings (1983): 42, 2166. P 12. Cerdán,S, Egan,J, Hilberman,M (1984) "In vivo" and "in vitro" measurements of brain phospholipids by 31P NMR. Proceedings of the Symposium on Medical Applications of Magnetic Resonance. Interlaken, Switzerland. PO 13. Hilberman,M, Cerdán,S, Cone,J, Egan,J, Williamson,JR (1984) Measurements of brain phospholipids by 31P NMR. (1984) Proceedings 3rd Annual Meeting of the Society of Magnetic Resonance in Medicine, New York, USA. pg 325. P 14. Cerdán,S (1984) Resonancia Nuclear magnetica de 1H, 13C y 31P en cerebro de perro.Proceedings XI Congress of the Spanish Society of Biochmistry. Tenerife, Spain. P 15. Kobayashi,K, Cerdán,S, Chance,EM, Williamson, JR (1984) Mathematical model of gluconeogenesis using 13C NMR data from perfused rat liver. Proceedings 75th Annual Meeting of the American Society of Biological Chemists. San Louis, USA. Federation P Proceedings (1985):43, 1517. 16. Cerdán,S, Parrilla,R, Santoro,J, Rico,M (1985) 1H NMR detection of cerebral myo-inositol. Proceedings 13th International Congress of Biochemistry (IUB) Amsterdam, The Netherlands. P 17. Cerdán,S (1985) Estudios metabólicos realizados con Resonancia Nuclear Magnética. Proceedings XII Congress Spanish Society of Biochemistry. Valencia, Spain. SI 18. Cerdán,S, Parrilla,R (1985) Determinacion "in situ" del flujo a traves de piruvato deshidrogenasa, piruvato carboxilasa y ciclo tricarboxilico a partir de medidas de Resonancia Nuclear Magnetica. Proceedings XII Congress Spanish Society of Biochemistry. Valencia,Spain. P 19. Cerdán,S (1986) Análisis por Resonancia Nuclear Magnetica de algunos derivados fosforilados del myo-inositol de interes biologico. Proceedings XIII Congress Spanish Society of Biochemistry. Zaragoza, Spain. P 13

20. Kunnecke,B, Cerdán,S, and Seelig,J (1989) Cerebral metabolism of (1,2- C ) acetate as 2 13

detected by in vivo and in vitro C NMR techniques .Proceedings VIII Meeting of the Society of Magnetic Resonance in Medicine. Amsterdam, The Netherlands. P 21. Cerdán,S (1989) Estudios de RMN sobre la estructura en solucion de polifosfo-inositoles y polifosfo-inositidos. Proceedings XVI Congress Spanish Society of Biochemistry. Alicante, Spain. SI 22. Cerdán,S. (1989) Resonancia Magnética en Biología. Congerencia Clausura II Jornadas del Grupo Química Orgánica de la Real Sociedad de Física y Química, Barco de Avila, España. SI 13

23. Kunnecke,B, Cerdán,S and Seelig,J (1990) Cerebral metabolism of (1,2- C ) acetate and 2 13 13 (1,2- C ) glucose in the in vivo rat brain as detected by C NMR spectroscopy. Proceedings 2

2nd Congress of the European Society of Magnetic Resonance in Medicine. Strasbourg 1990. P 42

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24. Moldes,M, Cerdán,S (1990) Comportamiento in vivo del sistema Avidina-Insulina Biotinilada. Proceedings XVII Congress of Spanish Society of Biochemistry. Oviedo, Spain.P 25. Chapa,F, Ballesteros,P, Cerdán,S (1990) A new method for the 13C NMR study of the pyruvate recicling system based the homonuclear spin-coupling analysis. Proceedings XVII Congress Spanish Society of Biochemistry. Oviedo,Spain. P 26. Chapa,F, Ballesteros,P, Villa,A, Aranda,A and Cerdán,S (1991) Metabolism of glucose and amino acids in Neuroblastoma and Glioma cell cultures as detected by 1H NMR spectroscopy. Proceedings IV Portuguese-Spanish Biochemistry Congress. Póvoa de Varzim, Portugal. P 27. Cruz,F, Chapa,F, Moldes,M, Cerdán,S (1991) Cerebral synaptosomes as models for the study of metabolism in the nerve terminals using 1H MRS. Proceedings IV PortugueseSpanish Biochemistry Congress. Povoa de Varzim, Portugal. P 28. Moldes,M, Cruz, F, Chapa,F, Cerdán,S (1991) Short term interactions of 2H20 with hepatic metabolic pathways". Proceedings IV Portuguese-Spanish Biochemistry Congress. Póvoa de Varzim, Portugal. P 29. Moldes,M, Cruz,F, Chapa,F, Cerdán,S (1991) Sustitución de protones específicos de metabolitos hepáticos por deuterones del solvente, detectada por RMN de 2H, 1H y 13C". Proceedings III Congress of the Spanish Society of Biophysics. Madrid, Spain. P 30. Nieto,R, Barroso,G, Tejedor,JM, Cerdán,S (1991) Difusion restringida de amonio en la matriz mitocondrial detectada a través de los acoplamientos 13C-15N observados en 13C RMN. Proceedings III Congress of the Spanish Society of Biophysics. Madrid, Spain. P *31. Chapa,F, Calvo,RM, Escobar del Rey,F, Morreale de Escobar,G, and Cerdán,S (1992) Effect of surgical thyroidectomy on the in situ metabolism of (1,2-13C2) acetate in adult rat brain as detected by 13C NMR spectroscopy. Proceedings 20th Annual Meeting of the European Thyroid Association. Dublin (Ireland). Jounal of Endocrinological Investigation 15, P 93,106. 32. Gil,MS, Cruz,F, Cerdán,S, Ballesteros,P (1992) Imidazol-1-yl-Alcanoate esters and their corresponding acids. A novel series of 1H NMR probes for intracellular pH". Proceedings XVth European Colloquium on Heterocyclic Chemistry. Noordwijkerhout, The Netherlands. P 33. Cabañas,ME, Cruz, F, Arús,C, Cerdán,S (1992) 1-13C and U-13C6 Glucose metabolism in the perfused bovine retina as detected by 13CNMR spectroscopy. Proceedings XI Annual Scientific Meeting and Exhibition Society Magnetic Resonance Medicine SMRM. Berlin, Germany. P 34. Moldes, M, Cerdán, S, Erhard, P, Seelig,J (1992) 1H-2H exchange in the perfused rat liver metabolising (3-13C) alanine and D2O. Proceedings 11th Annual Scientific Meeting and Exhibition Society Magnetic Resonance Medicine SMRM. Berlin, Germany. P 35. Cerdán, S. (1992) 13C-13C, 13C-2H and 13C-15N spin coupling patterns as novel tools in the study of the tricarboxylic acid cycle in liver and brain. VII PAABS-SEB Congress. Itxapa, México. SI 43

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36. Cerdán,S, Künnecke,B, Chapa,F, Cruz,F, Gil,A, Moldes,M (1993) 13C Isotopomer analysis. A novel tool in the study of neuronal-glial interactions in the adult brain in situ. Cancer and brain disease characterization an therapy assessment by quantitive magnetic resonance spectroscopy. First Plenary Meeting. Paris (France). SI 37. Cerdán,S, Kunnecke,B, Chapa,F, Cruz,F, Gil,A (1993) 13C Isotopomer analysis. A novel tool in the study of neuronal-glial interactions in the adult brain in situ". Cancer and Brain Disease Characterization and Therapy Assessment by Quantitative Magnetic Resonance Spectroscopy. Paris, France. SI 38. Ballesteros,P, Gil, MS, Zaderenko,P, Cruz,F, Cerdán,S (1993) Imidazol-1-yl-Alkanoic acids. A novel series of extrinsic 1H NMR probes for intracellular pH. Proceedings 12th Annual Scientific Meeting and Exhibition Society Magnetic Resonance Medicine SMRM. New York, USA. P 39. Cruz,F, Villalba,M, Cerdán,S (1993) Actividad de los enzimas involucrados en el reciclaje cerebral de piruvato. Compartimentación celular y patrón de desarrollo". Proceedings XVIII Congress of the Spanish Society of Biochemistry. San Sebastián, Spain. P 40. Gil, A, Moldes, M, Cerdán, S (1993). Localizacion del receptor de insulina in situ utilizando insulina biotinilada y avidina ferromagnética. Proceedings XVIII Congress of the Spanish Society of Biochemistry. San Sebastián, Spain. P 41. Chapa,F, Cerdán,S (1993) Determinación in situ de la Selección de sustratos en el compartimento neuronal y glial del cerebro de rata adulta por resonancia magnetica heteronuclear de carbono-13 (13C-2H). Proceedings XVIII Congress of the Spanish Society of Biochemistry. San Sebastián, Spain. P 42. Moldes,M, Cruz,F, Chapa,F, Cerdán,S (1994) Intercambios 2H-1H como una nueva herramienta en el estudio de la canalización de sustratos in situ por 13C RMN. Proceedings IV Congress of the Spanish society of Biophysiscs. Cáceres (Spain). SI 43. Cerdán, S. (1994) 13C NMR observed spin coupling patterns and isotopic shifts as novel metabolic tools. Conferencia Invitada. International Course “NMR in Biology and Medicine: SI From models to in vivo”, Altavilla Milicia, Sicilia, Italia. 44. Cerdán,S. (1994) The perfused mouse liver as a model for 31P NMR studies of intracellular pH homeostasis. Conferencia Invitada. International Course “NMR in Biology and Medicine: From models to in vivo”, Altavilla Milicia, Sicilia, Italia. SI 45. Cerdán, S (1995) The Physiological Basis of 13C NMR. III Meeting and Exhibition of the International Society of Magnetic Resonance in Medicine and Biology ISMRMB. Nice. France. SI 46. Cerdán,S, Moldes,M, Lopez-Beltran,E (1995) The metabolism of water in mammalian tissues as detected by multinuclear NMR spectroscopy. Proceedings 23rd FEBS Meeting. Basel, Switzerland. SI 47. Lopez-Beltrán,E, Maté,MJ, Cerdán,S (1995) Water exchange across the inner mitochondrial membrane as detected by 1H NMR. Proceedings 23rd FEBS Meeting. Basel, Switzerland. P 48. Roda,JM, Carceller,F, Pascual,JM, Cerdán,S (1995) Time course of metabolic responses in ischemic and spared hemispheres after focal cerebral ischemia in rats. Proceedings 10th European Congress of Neurosurgery, Berlin. PO 44

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49. Carceller,F, Roda,JM, Pascual,JM, Cerdán,S. (1995) A 13C NMR study of the effects of focal cerebral ischemia in the rat. Proceedings 4th International Workshop on Cerebrovascular surgery. Chicago, USA. PO 50. Gray,HF, Maxwell,RJ, Martinez-Pérez,I, Arús,C., Cerdán,S (1996) Genetic programming for classification of brain tumors from 1H NMR biopsy spectra. Proceedings International Society for Magnetic Resonance in Medicine. Fourth Scientific Meeting and Exhibition. New York. USA. PO 51. López-Beltrán,E, Maté, MJ, Cerdán,S (1996) Dynamics and environment of mitochondrial water detected by 1H NMR relaxation measurements. Proceedings International Society for Magnetic Resonance in Medicine. Fouth Scientific Meeting and Exhibition. New York. USA. P 52. Alvarez,J., Lopez-Beltran,E, Cerdán,S (1996) Microscopic NMR imaging as a novel tool in the diagnosis of hepatocellular necrosis. Proceedings 24th FEBS Meeting. Barcelona, Spain.P 53. Sanz,S, Pinilla,M, Luque,J, Cerdán,S (1996) Effects of eryhthocytes containing encapsulated glutamate dehydrogenase on hepatic ammonia metabolism as detected by 13C15N couplings. Proceedings 24th FEBS Meeting. Barcelona, Spain. P 54. Cerdán,S (1996) 13C MRS. Proceedings 13th Annual Meeting of the European Society for Magnetic Resonance in Medicine and Biology. Prague. Check Republik. MAGMA. 4, 58. SI 55. García-Martin, ML, García-Espinosa, MA, Cerdán,S (1996) Relative rates of 1H-2H exchange in the perfused mouse liver dtected by 13C NMR. Proceedings 13th Annual Meeting of the European Society for Magnetic Resonance in Medicine and Biology. Prague. Check Republic.MAGMA. 4, 105. PO 56. García-Espinosa,MA, García-Martín,ML, Cerdán,S (1996) Neuronal-Glial Interactions in the adult brain during experimental diabetes. Proceedings 13th Annual Meeting of the European Society for Magnetic Resonance in Medicine and Biology. Prague. Check Republic MAGMA 4, 235 P 57. Chapa,F, Cruz,F, Moldes,M, Cerdán,S (1996) 13C-2H spin coupling patterns as tools to study sibstrate selection in the neuronal and glial compartments of rat brain. Proceedings 2nd Meeting European Neuroscience Association. Strasbourg, France. SI 58. Pascual,JM, Roda,JM, Carceller, F., Cerdán S. (1996) Analysis por Espctroscopía de 1H de extractos de tumores cerebrales. Proceedings XLVI Meeting of the Spanih-Portuguese Society of Neurosurgery. Algarve. Portugal. PO 59. Pascual,JM, Roda,JM,Carceller,F,Cerdán,S (1996) Efecto de la Isquemia Focal sobre el metabolismo cerebral detectado por espectroscopia de 13C RMN. Proceedings XLVI Meeting of the Spanih-Portuguese Society of Neurosurgery. Portugal. P 60. Pascual,JM, Roda,JM, Carceller,F, Cerdán,S (1996) Cinetica de la respuesta metabólica en los hemisferios normoxico e isquémico tras la isquemia cerebral focal en ratas. Proceedings XLVI Meeting of the Spanih-Portuguese Society of Neurosurgery. Algarve. Portugal. P 61. Pascual,JM, Carceller,S., Cerdán,S y Roda JM (1996) Cambios metabolicos sequenciales despues de la Isquemia cerebral focal. Proceedings 1st Meeting Spanish Society of Neurosurgery. Salamanca, P 45

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62. Carceller,F, Pascual,JM, Cerdán,S, Roda, JM (1996) Interaccion Neuronal y glial despues de la isquemia cerebral focal. Proceedings IX Congress Spanish Society of Surgical Investigations. León, Spain. P 63. Roda,JM, Pascual,JM, F. Carceller y Cerdán,S. (1996) Tumores cerebrales: Análisis con espectroscopía de de Resonancia Magnética de Proton. Proceedings IX Congress Spanish Society of Surgical Investigations. León, Spain. PO 64. Roda,JM,Pascual,JM,García,S, Cerdán,S, Carceller,F (1997) Glutámico, Glutamina y Prolina como criterio diagnóstico en tumores cerebrales humanos. Proceedings II Congress Spanish Society of Neurosurgery. Barcelona, Spain. PO 65. Pascual,JM, Carceller,F, Cerdán,S, Roda, JM (1997) 1H MRS Analysis of extracts from human brain tumors. Prtoceedings 11th International Congress of Neurological Surgery. Amsterdam. The Netherlands. PO 66. Roda,JM, Pascual,JM, Cerdán,S,Carceller,F (1997) Neuronal-glial interactions in both hemispheres of rat brain after focal cerebral ischemia. Proceedings 11th International Congress of Neurological Surgery. Amsterdam. The Netherlands. P 67. Cerdán,S, Cruz,F (1997) Ontogeny and cellular location of the pyruvate recycling system of rat brain. Proceedings 3rd International Conference of Brain Energy metabolism, Waterville Valley, New Hampshire, USA. CP 68. Cerdán,S, Pascual,M, Carceller,F, Roda,JM (1997) Glutamate, glutamine and GABA as substrates in both hemispheres of rat brain after focal cerebral ischemia. Proceedings 3rd International Conference of Brain Energy metabolism, Waterville Valley, New Hampshire, USA. J. Neurochem. 69, Suppl S-277D., P 69. Scott,R, Cerdán,S (1997) Development of cerebral compartmentation of acetate metabolism in the rat. Proceedings 3rd International Conference of Brain Energy metabolism, Waterville Valley, New Hampshire, USA. J. Neurochem. 69, Suppl S-277C. IS 70. García-Espinosa,MA, García-Martín,ML, Cerdán,S (1997) Effect of experimental diabetes on the neuronal and glial metabolisms of adult rat brain. Proceedings 3rd International Conference of Brain Energy metabolism, Waterville Valley, New Hampshire, USA. J. Neurochem. 69, Suppl S-277A. P 71. Cerdán,S (1997) 13C MRS: Methods and Applications. Proceedings 14th Annual Meeting of the European Society for Magnetic Resonance in Medicine and Biology. Brussels. Belgium. MAGMA 5, 6. SI 72. García-Martin, ML, García-Espinosa,MA,Cerdán,S (1997) Role of the Na+/H+ exchanger in the regulation of intracellular pH in chemical carcinogenesis of the liver. Proceedings 14th Annual Meeting of the European Society for Magnetic Resonance in Medicine and Biology. Brussels. Belgium. MAGMA 5,59. PO 73. García-Espinosa,MA, García-Martín,ML, Cerdán,S (1997) Effects of experimental diabetes on neuronal and glial metabolisms of adult rat brain. Proceedings 14th Annual Meeting of the European Society for Magnetic Resonance in Medicine and Biology. Brussels. Belgium. MAGMA 5,87. PO 74. Scott,R., Planas,A, Cerdán,S (1997) (1,2-13C2) acetete metabolism in different regions of the adult rat brain. Proceedings 14th Annual Meeting of the European Society for Magnetic Resonance in Medicine and Biology. Brussels. Belgium. MAGMA 5,88. PO 46

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75. García-Pérez,A, López-Beltrán,E, Cerdán,S. (1997) Apparent diffusion coefficients of water and other metabolites in different subcellular compartments. Proceedings 14th Annual Meeting of the European Society for Magnetic Resonance in Medicine and Biology. Brussels. Belgium. MAGMA 5,159. P 76. Alvarez-Perez,J, López-Beltrán,E, Cerdán, S (1997) Microscopic images of pH using 1H NMR. Proceedings 14th Annual Meeting of the European Society for Magnetic Resonance in Medicine and Biology. Brussels. Belgium. MAGMA 5,164-5. P 77. C.G. Seipelt, P. López, P. Merkling, S. Cerdán y P. Ballesteros (1997) Magnetic Resonance Contrast Agents. Relaxivities, stability constants and theoretical calculations. Tenth European Symposium on Organic Chemistry, ESOC 10. Basilea. P 78. Pascual,J.M., Carceller,F., Roda,J.M., Cerdán (1998) Glutamate, glutamine and GABA as substrates for the neuronal and glial compartments of rat brain after focal cerebral ischemia. XII Congress of the European Society of Neurochemistry, San Petersburgo, J. Neurochem 71, Suppl 1, S53A. SI 79. M.L. García Martín, M.A. García Espinosa, S. Cerdán (1998) Role of endogenous bicarbonate production in the regulation of intracellular pH in the perfused mouse liver. Proceedings 15th Annual Meeting of the European Society for Magnetic Resonance in Medicine and Biology. Ginebra (Suiza).MAGMA 6, Suppl 1, 21. PO 80. M.A. García-Espinosa, M.L. García-Martín, S. Cerdán (1998) Relative contributions of plasma glucose and glial glutamine as precursors of neuronal glutamate. Proceedings 15th Annual Meeting of the European Society for Magnetic Resonance in Medicine and Biology. Ginebra (Suiza). MAGMA 6, Suppl 1, 223. P 81. F. Cruz, S.Cerdán (1998) A comparison of the metabolism of (13C) glucose in primary cultures of neurons and in the neuronal compartment of the adult brain. Proceedings 15th Annual Meeting of the European Society for Magnetic Resonance in Medicine and Biology. Ginebra (Suiza). MAGMA 6, Suppl 1, 113. PO 82. M.L. García Martín, M.A. García Espinosa, S. Cerdán (1998) Biochemistry detectable by MRS. Proceedings 15th Annual Meeting of the European Society for Magnetic Resonance in Medicine and Biology. Ginebra (Suiza). MAGMA 6, Suppl 1, 5 . SI 83. S. Cerdán, P. Ballesteros (1998) Novel contrast agents for functional magnetic resonance imaging (fMRI). 2nd Potuguese-Spanish Biophysics Congress. VI Congreso de la Sociedad de Biofísica de España. Madrid (Spain). SI 84. M.L. García-Martín, M.A. García-Espinosa, M. Bruix, P. Ballesteros, S. Cerdán (1998) The turnover of hydrogens from hepatic metabolites as detectred by 13C NMR. 2nd Potuguese-Spanish Biophysics Congress. VI Congreso de la Sociedad de Biofísica de España. Madrid (Spain). P 85. M.A. García-Espinosa, M.L. García-Martín,S. Cerdán (1998) Relatice contributions of plasma glucose and glial glutamine as precursors of neuronal glutamate. 2nd PotugueseSpanish Biophysics Congress. VI Congreso de la Sociedad de Biofísica de España. Madrid (Spain). P 86. J. Alvarez-Pérez, E.A. López-Beltrán,E.A., S. Cerdán (1998) Microscopic pH imaging using 1H NMR. 2nd Potuguese-Spanish Biophysics Congress. VI Congreso de la Sociedad de Biofísica de España. Madrid (Spain). P

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87. F. Cruz, S. Cerdán (1998) (1-13C) glucose metabolism in prymary cultures of cortical neurons and in the adult brain. 2nd Potuguese-Spanish Biophysics Congress. VI Congreso de la Sociedad de Biofísica de España. Madrid (Spain). P 88.F. Cruz, García-Espinosa,M.A., Cerdán,S. (1999) Absolute values for TCA cycle flux and exchange of glutamate, glutamine and GABA between the neuronal and glial compartments of adult rat brain during (1,2-13C2) acetate metabolism. 30th Annual Meeting of The American Society for Neurochemistry. New Orleans (USA). J. Neurochemistry 72, Suppl., S25A. P 89. Pascual, J.M., Carceller,F., García-Martín,M.L., Roda,J.M., Cerdán,S. (1999) Neuronalglial interactions in the adult brain during ischemia. A multinuclear nmr approach. 30th Annual Meeting of The American Society for Neurochemistry. New Orleans (USA). J. Neurochemistry 72, Suppl., S3B. SI 90. García-Martín,M.L., García-Espinosa,M.A., Ballesteros,P., Bailey,L.E., Bruix,M., Cerdán,S. (1999) Hydrogen turnover in Hepatic Metabolites as detected by 13C NMR. VII Scientific Meeting International Society of Magnetic Resonance in Medicine and Biology. Philadelphia (USA). PO 91. Alvarez-Pérez,J., van Sluis,R., Raghunand,N., Bhujwala,Z., Cerdán,S., Ballesteros,P. (1999) pH heterogeneity in spheroids by 1H NMR. VII Scientific Meeting International Society of Magnetic Resonance in Medicine and Biology. Philadelphia (USA). P 92. López, P, Seipelt, C.G., Merkling,P.,Sturz,L., Dölle,A.,Zeidler,M.D.,Álvarez,J., Cerdán,S., and P. Ballesteros. (1999) N-2-(Azol-1(2)-yl) ethyliminodiacetic Acids; A Novel Series of Gd(III) Chelators as T2 Relaxation Agents for High Field Magnetic Resonance Imaging. VII Scientific Meeting International Society of Magnetic Resonance in Medicine and Biology. Philadelphia (USA). P 93. Cruz,F.,García-Espinosa,M.A., Barroso,I.,Santisteban,P., Sebastian Cerdán,GozálezLlanos,F., Pascual,J.M., Carceller,F. and Roda,J.M. (1999) Cerebral Pyruvate Recycling after Global Cerebral Ischemia. Gene Expression and in situ Flux. Proceedings International Symposium on “Animal Models of Cerebral Ischemia: A reappraisal”, La Cristalera, Madrid (Spain). P 94. Pascual,J.M., Carceller,F., Cruz,F., Roda,J.M., Cerdán,S. (1999) Cerebral metabolism under ischemic conditions: from cultured cells to the human brain. . Proceedings International Symposium on “Animal Models of Cerebral Ischemia: A reappraisal”, La Cristalera, Madrid (Spain). SI 95. Cerdán,S. (1999) (1,2-13C2) Acetate Metabolism in different Brain Regions. International Symposium on Glutamate, glutamine and GABA homeostasis in the CNS. Wierzba, Polonia.SI 96. Cerdán, S. (1999) Subcellular metabolism of water and heavy water as detected by 13C NMR. Proceedings of the 14th International Meeting of the Royal Society of Chemistry on NMR Spectroscopy, Edinburgh (U.K.). SI 97. Cerdán,S. (1999) The NMR visibility of water. Worshop on Magnetic Resonance Relaxation in vivo. Kuopio, Finlandia. SI 98. Cerdán,S. (1999) Molecular crowding and viscosity as determinants of translational diffusion in subcellular organelles. Workshop on Magnetic Resonance Relaxation in vivo. Kuopio, Finlandia. SI 99. Cerdán, S. (1999) Metabolism of water and heavy water by the perfused mouse liver. Worshop on Magnetic Resonance Relaxation in vivo. Kuopio, Finlandia. SI 48

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100. Cruz, F., Cerdán,S. (1999) 13C Nuclear Magnetic Resonance studies of metabolic compartmentation “ex vivo”. 44 Congreso de la Sociedad Italiana de Bioquímica y Biología Molecular. Symposium conjunto con Sociedad Española de Bioquímica y Biología Molecular. Alghero, Cerdeña, Italia. SI 101. Cruz, F., Carceller,F., García-Espinosa,M.A., García-Martín,M.L., Cerdán,S., Roda,J.M. (1999) Neuronal pyruvate recycling after global certebral ischemia. Gene expression and in situ flux. 16th Annual Meeting European Society of Magnetic Resonance in Medicine and Biology ESMRMB. Sevilla, España, MAGMA 8, Suppl 1, p 75. PO 102. García-Martín,M.L., García-Espinosa,M.A., Cruz,F., Cerdán,S. (1999) The activity of the malate aspartate shuttle as measured by the turnover of specific hydrogens from individual 13C isotopomers. 16th Annual Meeting European Society of Magnetic Resonance in Medicine and Biology ESMRMB. Sevilla, España, MAGMA 8, Suppl 1, p 113. PO 103. Cruz, F., Cerdán,S. (1999) Glucose and lactate as competitive substrates for neuronal metabolism. Procveedings of the Harden conference on Brain Energy Metabolism, Oxford. PO 104. García-Espinosa,M.A., García-Martín,M.L., Cruz,F., Cerdán,S. (1999) Cerebral pyruvate recycling under hypoglycemic conditions. 16th Annual Meeting European Society of Magnetic Resonance in Medicine and Biology ESMRMB. Sevilla, España, MAGMA 8, Suppl 1, p 76 PO 105. F. Cruz, S. Cerdán (1999) 13C NMR methods and Applications: 13C Isotopomer Analysis. EMBO Course on "In vivo Spectroscopy and Imaging in Biomedical Research". SI 106. García-Martín,M.L., García-Espinosa,M.A., Cerdán,S. (2000) Hierchical contribution of hepatic intracellular alkalinization systems during chemical carcinogenesis. Proceddings of the VIII Scientific Congress and Exhibition International Society for Magnetic Resonance in Medicine. Denver (Co). Oral presentation-253. PO 107. García-Martín,M.L., Herigault, G., Rémy,C., Farion,R,., Ballesteros,P., Coles,J.A., Cerdán,S., Ziegler,A. (2000) 1H Magnetic Resonance Spectroscopic imaging of extracellular pH in an intracerebral glioma. VIII Scientific Congress and Exhibition International Society for Magnetic Resonance in Medicine. Denver (Co). Abstract-1029. P 108. Brandao,T., Cruz.F., García-Martín, M.L., García-Espinosa,M.A., Cerdán,S. (2000) A convenient focussed microwave apparatus built from a commercial microwave oven. Congress of Biomedical Technology, Coimbra, Portugal. PO 109. García-Martín,M.L., S. Cerdán (2000) 13C NMR Spectroscopy, ESMRMB ´00 Meeting, Paris, Teaching Course on Biological Spectroscopy, MAGMA 11, pg 3. SI 110. Cruz,F., García-Espinosa,M.A, S. Cerdán (2000) Glucose, lactate, pyruvate and glutamine as competitive substrates for neuronal metabolism in vitro. ESMRMB ´00 Meeting Paris, MAGMA 11, pg 97. PO 111. García-Martín,M.L., Herigault, G., Rémy,C., Farion,R,., Ballesteros,P., Coles,J.A., Cerdán,S., Ziegler,A. (2000) 1H spectroscopic imaging of extracellular lactate, choline and lipids in rat brain glioma in vivo. ESMRMB ´00 Meeting Paris, MAGMA 11, pg 99. PO 112. García-Martín, M.L., García-Espinosa,M.A., Brandao,T., Ballesteros,P., Cerdán,S. (2000) Heterogeneidad de pH en tumores revelada por imagen espectroscopica de RM. IV Congreso Iberoamericano de Biofisica, SOBLA-SBE, Alicante. PO 49

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113. M. L. García-Martín, G. Herigault, C. Remy, R. Farion, P. Ballesteros, J. Coles, S. Cerdán, A. Ziegler (2001) Combined 1H MR spectroscopic imaging of extracellular pH and metabolites in rat glioma: high lactate is not associated with lower extracellular pH. Proceedings IX ISMRM Meeting and exhibition, Glasgow, Abstract 724. PO 114. García-Martín, M. A. García-Espinosa, P. Ballesteros, S. Cerdán (2001) Choline kinease modulates proton extrussion in precarcinogenoic livers, Proceedings IX ISMRM Meeting Glasgow, Abstract 721. PO 115. Z. Bhugwala, D. Artemov, P. Ballesteros, S. Cerdán, R. J. Gillies, M. Solaiyappan (2001) Identifying high metastatic risk vascular and physioilogical regions in solid tumors. Proceedings IX ISMRM Meeting Glasgow, Abstract 2249. PO 116. Intracellular water motions decrease in apoptotic macrophages after caspase activation (2002) S. Hortelano, M.L. García-Martín, S. Cerdán, A. Castaño, A. Alvarez, L. Boscá. Intracellular water motions decrease in apoptotic macrophages after caspase activation. Proceddings X ISMRM Meeting Honolulu, Abstract 128. PO 117. M.L. García-Martín, M.A. García-Espinosa, M. Benito, A. Sierra, P. Ballesteros, S. Cerdán (2002) Slow exchange of a-ketoglutarate/glutamate between mitochondrial and cytosolic compartments of perfused mouse liver as detected by 1H and 2H decoup0led 13C NMR spectroscopy. Proceedings X ISMRM Meeting Honolulu, Abstract 133. PO 118. M.A. García-Espinosa, M.L. García-Martín, M. Benito, P. Ballesteros, S. Cerdán (2002) Neuronal and glial tricarboxylic acid cycles and the glutamine cycle as precursors of cerebral glutamine and GABA in the adult rat brain, Proceedings X ISMRM Meeting Honolulu, Abstract 587. PO 119. P. Hernández-Vargas, S. Garrido, M. Benito, A. Sierra, P. Ballesteros, S. Cerdán (2002) Non stoichiometric productions of protons and lactate in cultures of C6 cells. ESMRMB 19th Annual Meeting Cannes, Mag. Res. Mat. Sci. MAGMA 2002, 15, Suppl. 1, 183, pp 86. PO 120. E. Pérez-Mayoral, P. López-Larrubia, M. García-Amo, E. Soriano, S. Cerdán, P. Ballesteros (2002) Mixed Heterocyclic ligands for Gd(III) as novel contrast agents for MRI. ESMRMB 19th Annual Meeting Cannes, Mag. Res. Mat. Sci. MAGMA 2002, 15, Suppl. 1, 412, pp 197. P 121. A. Sierra, M. Benito, C. Benedicto, S. Garrido, F. Cruz, P. Ballesteros, S. Cerdán (2002) Turnover of the H3 hydrogens from (2-13C) glutamate in the adult rat brain during (2-13C) acetate metabolism. ESMRMB Annual Meeting Cannes, Mag. Res. Mat. Sci. MAGMA 2002, 15, Suppl. 1, 62, p 26. PO 122. M. Benito, M.L. García-Martín, P. Ballesteros, S. Cerdán (2002) Estudio termodinámico de la dependencia del pH en los procesos de transferencia de magnetizacion. VIII Congreso Luso-Español de Biofísica, Tenerife, España, Junio 2002. P 123. P. Lopez-Larrubia, M. Benito, S. Garrido, P. Sánchez, P. Ballesteros, S. Cerdán (2003) ISUCA a novel indicator for extracellular pH measurements in tumors by 1H magnetic resonance spectroscopy. XI ISMRM Meeting Toronto, Canada PO 124. A. Sierra, T.B. Rodrigues, M. Benito, P. Ballesteros, S. Cerdán (2003) Turnover of the H3 hydrogens of (2-13C) glutamate and (2-13C) glutamine during the cerebral metabolism of (113C) glucose. XI ISMRM Meeting Toronto, Canada. PO

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125. A thermodynamic approach to magnetisation transfer processes between water and the exchangeable hydrogens of biomolecules” M. Benito, A. Sierra, P. Ballesteros, S. Cerdan (2003) A thermodynamic approach to magnetisation transfer processes between water and the exchangeable hydrogens of biomolecules. XI ISMRM Meeting Toronto, Canada. PO 126. M. Benito, A. Sierra, S. Garrido, P. Ballesteros, S. Cerdán (2003) Glucose stimulated lactate metabolism in C6 glioma cells in culture. XI ISMRM Meeting Toronto, Canada. PO 127. S. Cerdán (2003) Metabolism Cerebral y RMN. Conferencia de clausura. "Resonancia Magnetica Nuclear, una tecnica de vanguardia en Quimica y Biología (2003) Cursos de verano de la Universidad Comoplutense de Madrid. El Escorial, Septiembre. CP 128. M. Benito, P. López, A. Sierra, T.B. Rodrigues, P. Ballesteros, S. Cerdán (2003) Water difusión influences Magnetisation Transfer Processes in a pH dependent manner. 20th Meeting European Society of Magnetic Resonante in Medicine and Biology, Rotterdam, Proceedings 93, 416. P 129. H. Gray, T.B. Rodrigues, S. Garrido, R.J. Gillies, S. Cerdán (2003) Futyle cycling of lactate through the plasma membrana of C6 glioma cells as detected by 2H-13C NMR. 20th Meeting European Society of Magnetic Resonante in Medicine and Biology, Rotterdam, Proceedings 38, 128. PO 130. T.B. Rodrigues, P. Sánchez, A. Sierra, M. Benito, P. Ballesteros, C. Geraldes, A.B. Martín, R, Moratalla, S. Cerdán. 13C NMR chacterization of transgenic knock-out mice in D1 dopamine receptirs as models of Parkinson Disease. 20th Meeting European Society of Magnetic Resonante in Medicine and Biology, Rótterdam, Proceedings 66, 267. PO 131. A. Sierra, S. Garrido, M. Benito, T.B. Rodrigues, P. Ballesteros, S. Cerdán (2003) Hydrogen turnover in prymary cucltures of cerebral cortical astrocytes as detected by 2H-13C NMR. 20th Meeting European Society of Magnetic Resonante in Medicine and Biology, Rótterdam, Proceedings 70, 285. PO 132. Rodrigues, T.B., Gray, H.L., Garrido, S., Benito, M., Cerdán, S. (2004) Red-ox dependence of lactate C2 deuteration and lactate recycling in C6 glioma cells in culture. 22 Meeting of the European Society of Magnetic Resonance in Medicine and Biology ESMRMB. Copenhaguen, Denmark. Presentation 105. PO 133. Rodrigues, T.B., Sierra, A., Cerdán, S. (2004) 13C MRS. 22 Meeting of the European Society of Magnetic Resonance in Medicine and Biology ESMRMB. Copenhaguen, Denmark. Presentation 15. IS 134. Sierra, A., Lopes da Fonseca, L. Ballesteros, P., Cerdán, S. (2004) Quantitative modelling of H3 hydrogen turnover in (2-13C) glutamate and glutamina during (2-13C) acetate metabolism in the adult rat brain. 22 Meeting of the European Society of Magnetic Resonance in Medicine and Biology ESMRMB. Copenhaguen, Denmark. Presentation 103. PO 135. Benito, M., Domínguez, L., Ballesteros,P., Cerdán, S. Marco, J.L. (2004) A new series of H NMR indicators for extracellular pH measuremnents based on di-imidazolic structures. 22 Meeting of the European Society of Magnetic Resonance in Medicine and Biology ESMRMB. Copenhaguen, Denmark. Presentation 203. PO

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136. Rodrigues, T.B., Martín, A.B., S´ñanchez, P., Sierra, A., Geraldes, C., Ballesteros,P., Cerdán, S., Moratalla, R. (2004) transgenic Knock-out mice in D1 Dopamine receptors have increased glutamine cycle activity as detected by 1H NMR. ISMRM 12th Scientific Meeting and Exhibition, Kyoto, Japón. Presentación 29. PO 51

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137. Rodrigues, T.B., Gray, H.L., Benito, M., Garrido, S., Ballesteros, P., Cerdán, S. (2004) Futile cycling of lactate and Protons through the plasma membrane of C6 glioma cells as detected by 2H-13C NMR. ISMRM 12th Scientific Meeting and Exhibition, Kyoto, Japón. Presentación 2027. PO 138. Benito, M., López, P., Sierra, A., Rodrigues, T.B., Ballesteros,P., García-Martín, M.L., Cerdán, S. (2004) Translational water difusión influences Magnetization Transfer Processes in a pH dependent manner, Kyoto, Japón. 12th Scientific Meeting and Exhibition. Presentación 2336. PO 139. Cerdan, S. (2004) Subcellular transfer of reducing equivalents in the neuronal and glial compartments of the adult rat brain after protocaval anastomosis as detected by ( 13C, 2H) NMR. VI International Symposium on Hiperammoniemia and Hepatic Encephaloptahy. Valencia. IS 140. Cerdan,S. (2004) Metabonomica. II Reunion de la Red de Metabolismo. Sitges. 2004.IS 141. Fonseca, C.P., Montezinho, L.P., Sierra,A., Cerdán, S., Geraldes, C.F.G.C. and Castro M.M.C.A. (2004) Studies of lithium effects in cellular models: towards the understanding of the therapeutic action of this drug. III Congresso Ibérico de Espectroscopia. Las Palmas de Gran Canaria. P 142. C.P. Fonseca, L.P. Montezinho, A. Sierra, S. Cerdán, C.F.G.C. Geraldes and M.M.C.A. Castro. Spectroscopic studies of lithium effects on neuronal models and on brain cells metabolism. 3rd Portuguese-Spanish Biophysics Congress. October, 2004. Lisboa (Portugal). P 143. Cerdan, S., Ballesteros,P., Mayoral, E.P. (2004) The Intracellular water environment as the basis for intrinsic and extrinsic MRI Contrast . From structural genomIcs to molecular medicine. The challenging role of NMR. Workshop SAPIO symposium. June 2004, Torino.P 144. A.Sierra, L. M. Lopes da Fonseca, T.B. Rodrigues, M. Benito, P. Ballesteros and S. Cerdán (2005) Quantitative modeling of H3 turnover in (2-13C) glutamate and (2-13C) glutamine during (1-13C) glucose metabolism in the adult rat brain. 13th Annual Scientific Meeting and Exhibition ISMRM, South Beach, Miami, Florida (USA). PO 145. T.B. Rodrigues, M. Benito, A. Sierra, P. Ballesteros, C.F.G.C. Geraldes and S. Cerdán (2005) A Fast and Sensitive (1H, 2H) NMR Method to Measure the Turnover of the H2 Hydrogen of Lactate, 13th Annual Scientific Meeting and Exhibition ISMRM. South Beach, Miami, Florida (USA). PO 146. M. Benito, P. Sanchez, T.B. Rodrigues, A. Sierra, S. Garrido, P. López-Larrubia and S. Cerdán (2005) Heavy Water (D2O) Inhibits the Growth of C6 Gliomas. 13th Annual Scientific Meeting and Exhibition ISMRM. South Beach, Miami, Florida. P 147. M. Benito, E. Soriano, T.B. Rodrigues, A. Sierra, P. López-Larrubia, P. Ballesteros, S. Cerdán and M.L. García-Martín (2005) The ADC of Water Limits Magnetization Transfer Process under Physiological Conditions. 13th Annual Scientific Meeting and Exhibition ISMRM. South Beach, Miami, Florida (USA). P

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148. Cerdan,S., Sierra, A., Rodrigues, T.B. (2005) Cerebral activation as detected by in vivo ad in Vitro multinuclear NMR spectroscopy. I Iberoamerican Meeting on NMR Spectroscopy. Angra dos Reis, Brasil. IS 149. Cerdan, S. (2005) The energetics of glutamatergic neurotransmissions in the adult mammalian brain. Biomedical Magnetic Resonance: Molecular Imaging and Metabolism. Coimbra, Portugal. IS 150. Cerdán, S. (2005) The tumor metabolome as detected by 1H NMR, Biomedical Magnetic Resonance: Molecular Imaging and Metabolism. Coimbra, Portugal. IS 151. Cerdán, S. (2005) Subcellular compartmentation in the brain as detected by 13C,2H NMR spectroscopy, Biomedical Magnetic Resonance: Molecular Imaging and Metabolism. Coimbra, Portugal. S 152. Cerdán, S. (2005) Modalidades de Imagen Biomédica. Resonancia Magnética Nuclear e Imagen Molecular. Cátedra Severo Ochoa de Medicina Molecular. Bases Moleculares y Celulares de la Medicina. Universidad de Cuyo, Mendoza, Argentina. Junio. IS 153. Cerdán, S. (2005) Excitación y Relajación Magnética. Cátedra Severo Ochoa de Medicina Molecular. Bases Moleculares y Celulares de la Medicina. Universidad de Cuyo, Mendoza, Argentina. Junio. IS 154. Cerdán, S. (2005) La estructura tridimensional de macromoléculas biologicas: determinación mediante espectroscopia de Resonancia Magnética Nuclear. Cátedra Severo Ochoa de Medicina Molecular. Bases Moleculares y Celulares de la Medicina. Universidad de Cuyo, Mendoza, Argentina. Junio. IS 155. Cerdán, S. (2005) Imagen por Resonancia Magnética y Espectroscopia Localizada. Cátedra Severo Ochoa de Medicina Molecular. Bases Moleculares y Celulares de la Medicina. Universidad de Cuyo, Mendoza, Argentina. Junio. IS 156. Pérez-Mayoral, García-Amo, M., Cerdán, S., Ballesteros,P.(2005) Gadolinium complexes of pyrazolylethyl DPA derivatives. Effect of pyrazole substitution on paramagnetic metl complexation. 14th European Symposium on Organic Chemistrty, Helsinki, Finland. Julio.P 157. Pérez-Mayoral,E., Soriano, E., Cerdán, S., Ballesteros, P.(2005). Experimental and theoretical study of lanthanide complexes based on linear macrocyclic polyaminopolycarboxylic acid of pyrazolylethyl arms. 14th European Symposium on Organic Chemistrty, Helsinki, Finland. Julio. P 158. Pacheco, J., Soriano, E., Perez-Mayoral, E., Lopez-Larrubia, P., Cerdán, S., Ballesteros, P. (2005) Síntesis of new oxygen tension (pO2) probes for 1H Magnetic Resonance Spectroscopy and Imaging. IX International Electronic Conference on Synthetic Organic Chemistry (ECSOC-9). Electronic Congress. P 159. Rodrigues, T.B., Felipo, V. and Cerdan, S (2005) Subcellular Transfer of Reducing Equivalents in the Neuronal and Glial Compartments of the Adult Rat Brain Under Hepatic Encephalopathy as Detected by {13C,2H} NMR. ESMRMB, Basle, Switzerland. Septiembre PO 160. Rodrigues, T.B., Sanchez, P. and Cerdan, S (2005) Lactate Metabolism in the Brain of Rats Bearing C6 Gliomas as Detected by {13C,2H} NMR. ESMRMB, Basle, Switzerland, Septiembre PO 53

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161. Sierra, A., Lopes da Fonseca, L., Rodrigues, T.B., Cerdán, S. (2005) Cerebal hydrogen (deuterium) turnover and the redox switch/redox coupling hypothesis. Third Wierzba Conference. Glutaate in CNS Metabolism and Neurotransmission. Interactions at the Intra and intercellular level. Wierzba, Poland. August. IS 162. Cerdan, S. (2005) NMR as a novel tool to study cerebral physiology and pathology. Gruppo Italiano de Discuzzione di Resonancia Magnética GIDRM. Monte Portio Catone, Roma. Septiembre. IS 163. Cerdan, S. (2005) The Physiological Basis of MRI Contrast.. First joined Course Sociedad Mexinacana de Resonancia Magnetica y ISMRM, Ciudad de Mexico, Mexico. October, 2005. IS 164. Cerdan, S. (2005) MR Sectroscopy and cerebral biochemistry. First joined Course Sociedad Mexinacana de Resonancia Magnetica y ISMRM, Ciudad de Mexico, Mexico. October, 2005. IS 165. Cerdan, S., García-Martín, M.L. (2005) 13C NMR detected deuterium turnover: a novel metabolic tool to investigate metabolism. Fifth biannual Minnesotta Workshops on High Field NMR spectroscopy and imaging of brain function. Minneapolis, Octubre IS. 166. Torija, P., Vicente, J.J., Rodrigues, T.B., Robles, A., Cerdan, S, Sastre, L., Calvo, R.M. and Escalante, R. (2005) Human versus Dictyostelium: comparative genomics to study the function of highly conserved genes of unknown function. Annual International Dyctiostelium Conference, Autrans, France. P 167. Cerdan, S. and Rodrigues, T.B. (2006) 13C NMR detected deuterium turnover: a novel metabolic tool to investigate subcellular glutamate and lactate recycling in the brain. European Winter Conference Brain Reasearch EWCBR. Villar sur Ollon, Switzerland, Marzo. IS 168. J. Soler Padrós, E. Pérez-Mayoral, Pilar López-Larrubia, Elena Soriano, José Marco-Contelles, Sebastián Cerdán and Paloma Ballesteros (2006) A novel generation of pH indicators for Proton Magnetic Resonance Spectroscopic Imaging (1H MRSI), 23th European Society for Magnetic Resonance in Medicine and Biology (ESMRMB), Varsovia, Septiembre P 169. Pacheco, J.; Lopez-Larrubia, P.; Soriano, E.; Perez-Mayoral, E.; Cerdán, S. Ballesteros, P. (2006), Characterization of New Oxygen Tension Probes (PO2) for 1H Magnetic Resonance Spectroscopy Imaging, 23th European Society for Magnetic Resonance in Medicine and Biology (ESMRMB), Varsovia, Septiembre P

170. C. F. Geraldes, S. Torres, J. A. Martins, J. P. André, M. I. Prata, A. C. Santos, T. B. Rodrigues, P. López-Larrubia, M. L. Garcia-Martin, S. Cerdán (2006) Evaluation of Gd(III) chelates of DOTA-type glycoconjugates as liver targeted MRI agents: studies on a Hep-G2 cell line, gamma scintigraphy and MRI of mice 23th European Society for Magnetic Resonance in Medicine and Biology (ESMRMB), Varsovia, Septiembre. P 171. Pacheco, J., Lopez-Larrubia, P., Soriano, E., Perez-Mayoral, E., Cerdán, S., Ballesteros, P. (2006) Synthesis of new oxygen tension probes (pO2) for 1H MRSI magnetic resonance spectroscopy imaging. European Society for Molecular Imaging Kick-Off Meeting, París, September. P 172. Rodrigues, T.B. and Cerdan, S. Detection of Pyruvate compartmentation in the adult rat 13 2 PO brain by ( C, H) NMR. ESMRMB, Warsaw, Poland (2006).

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173. Rodrigues, T.B., Martín, A.B., Moratalla, R.. and Cerdan, S. Metabolic interactions between glutamatergic and dopaminergic neurotransmiter systems are mediated through D1, but not D2, dopamine receptors. International Conference in Brain Energy Metabolism, Lausanne, Switzerland (2006). PO

174. Torres, S., Martins, J.A., André, J.P., Prata, M.I., Santos, A.C., Neves, M., Rodrigues, T.B., López-Larrubia, P., Cerdán, S. and Geraldes, C.F.G.C. Glycoconjugated and Micellar Gd Chelates as Potential Liver MRI Contrast Agents. 10th Bi-Annual Conference New Developments in Contrast Agent Research, Vilnius , Lithuania (2006). P 13

175. Rodrigues, T.B. and Cerdan, S. C NMR Detected Deuterium Turnover: a Novel Metabolic Tool to Investigate Subcellular Glutamate and Lactate Recycling in the Brain. European Winter Conference on Brain Research, Villars sur Ollon, Switzerland (2006). IS 176. Cerdán, S. The redox/switch/redox coupling hypothesis. Gordon Research Conference on Glial Biology. Ventura, CA (USA), March (2007). C.P. 177. Cerdan, S. Molecular Imaging of the tumor microenvironment. XVII Meeting of the Japaneese Society of Magnetic Resonance in Medicine, Osaka, September (2007). C.P. 178. Cerdán, S. The subcellular compartmentation of latate and glutamate and the redox switch redox coupling hypothesis. XIII Brain Energy Meeting. Peking. July (2008) IS

179. T. B. Rodrigues, I. R. Violante, R. Fayos, S. Cerdan (2008) Resolution of H3proS and H3proR deuterations in (2-13C) glutamate by high-field 13C NMR . 25th Meeting European Society of Magnetic Resonance in Medicine and Biology. Valencia, October PO 180. J. Pacheco-Torres, D. Zhao, A. Contero, S. Cerdan, R. P. Mason (2008). DOCENT (Dynamic Oxygen Challenge Evaluated by NMR T1 and T2*) of Tumors - New methodology for measuring tumor oxygenation. 25th Meeting European Society of Magnetic Resonance in Medicine and Biology. Valencia, October PO 181. J. Pacheco-Torres, P. Ballesteros, S. Cerdan, P. Lopez-Larrubia (2008) Hypoxia measurements by 1H-Magnetic Resonance Spectroscopy : Development of a new series of nitroimidazoles probes as molecular markers of hypoxia. 25th Meeting European Society of Magnetic Resonance in Medicine and Biology. Valencia, October PO 182. I. R. Violante, L. Nieto-Charques, T. B. Rodrigues, S. Cerdan (2008) Regional hypothalamic neuronal activity and intermediary metabolism in normal and obese mice. 25th Meeting European Society of Magnetic Resonance in Medicine and Biology. Valencia, October. PO 183. M. R. Fayos Carrió, V. Righi, A. Mucci, L. Schenetti, S. Cerdán (2008) Quantitative Magic Angle Spinning detection of deuteration in small biopsies of rat brain. 25th Meeting European Society of Magnetic Resonance in Medicine and Biology. Valencia, October. P 184. V. Negri, J. Soler, E. Pérez-Mayoral, P. López-Larrubia, L. Nieto-Charques, S. Cerdán, P. Ballesteros (2008) Single Wall Carbon Nanotubes and Magnetic Resonance Imaging. 25th Meeting European Society of Magnetic Resonance in Medicine and Biology. Valencia, October P 185. T. B. Rodrigues, S. Cerdán (2008) Accurate values for 2H induced isotopic shifts and 2H13C coupling constants for in vivo and ex vivo 13C NMR applications. 25th Meeting European Society of Magnetic Resonance in Medicine and Biology. Valencia, October. P 55

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186. I. R. Violante, J. Anastasovska, G. J. Sanchez-Cannon, T. B. Rodrigues, L. NietoCharques, S. R. Bloom, J. R. Parkinson, J. D. Bell, S. Cerdán (2008) Fasting induces lactate and GABA accumulation in the hypothalamus. 25th Meeting European Society of Magnetic Resonance in Medicine and Biology. Valencia, October. PO 187. V. Righi, P. Lopez-Larrubia, L. Schenetti, V. Tugnoli, M. L. García-Martín, S. Cerdán (2008) High resolution 13C HR-MAS spectroscopy analysis of different brain regions from rats bearing C6 implanted gliomas. 25th Meeting European Society of Magnetic Resonance in Medicine and Biology. Valencia, October. PO 188. Jesus Pacheco-Torres , Paloma Ballesteros, Sebastian Cerdan, Pilar Lopez-Larrubia (2009) Novel 1 Nitroimdazolyl Derivatives and Its Reduction Products Reveal Hypoxia in Cultures of C6 Cells Using H HR MAS. ISMRM 17th Meeting and Exhibition, Honolulu, April. PO 189. Tiago Brandao Rodrigues, Pilar Lopez-Larrubia, Sebastián Cerdán (2009) Redox Dependence and 13 Compartmentation of [ C]Pyruvate in the Brain of Deuterated Rats Bearing Implanted C6 Gliomas. ISMRM 17th Meeting and Exhibition, Honolulu, April. PO 190. Tiago Brandao Rodrigues, Inês Ribeiro Violante, Sebastián Cerdán (2009) Resolution of H3proS and 13 H3proR Deuterations in [2- C]glutamate. ISMRM 17th Meeting and Exhibition, Honolulu, April. PO 191. Jesús Pacheco-Torres, Francisco Fernández-Trillo, Eduardo Fernández-Megía, Ricardo J. Riguera, Paloma Ballesteros, Pilar Lopez-Larrubia, Sebastian Cerdan (2009) New Family of Dendrimeric Ligands as MRI Contrast Agents. ISMRM 17th Meeting and Exhibition, Honolulu, April. P 192. Pilar Lopez-Larrubia, Viviana Negri, Laura Nieto-Charques, Sebastian Cerdan, Paloma Ballesteros (2009) Characterization of Single Wall Carbon Nanotubes as Anysotropic Contrast Agents for MRI. ISMRM 17th Meeting and Exhibition, Honolulu, April. P 193. Valeria Righi, Pilar Lopez-Larrubia, Luisa Schenetti, Vitaliano Tugnoli, Maria-Luisa Garcia-Martin, 13 Sebastian Cerdan (2009) High Resolution C HR-MAS Spectroscopy Analysis of Different Brain Regions from Rats Bearing C6 Implanted Gliomas. ISMRM 17th Meeting and Exhibition, Honolulu,April. P 194. Inês R. Violante, Jelena Anastasovska, Gina J. Sanchez-Canon, Tiago B. Rodrigues, Valeria 2 Righi, Laura Nieto-Charques, James RC Parkinson, Stephen R. Bloom, Jimmy D. Bell Sebastián Cerdán (2009) Cerebral Activation by Fasting Induces Lactate Accumulation in the Hypothalamus. ISMRM 17th Meeting and Exhibition, Honolulu, April. PO 195. Laura Nieto-Charques, Pilar López-Larrubia, Sebastián Cerdán (2009) Orthogonal Diffusion Anisotropy Measurements in the Dorsal Third Ventricle of Adult Rat Brain Reveal Cerebrospinal Fluid Flow. ISMRM 17th Meeting and Exhibition, Honolulu, April. P 196. Jesus Pacheco-Torres, Dawen Zhao, Debu Saha, Pilar Lopez-Larrubia, Sebastian Cerdan, Ralph P. Mason (2009) Evaluation of Lung Tumor Oxygenation Using FREDOM and TOLD. ISMRM 17th Meeting and Exhibition, Honolulu, April. P 197. Inês R. Violante, Tiago B. Rodrigues, Laura Nieto-Charques, Sebastián Cerdán (2009) Hypothalamic Neuronal Activity and Intermediary Metabolism in ob/ob Mice. ISMRM 17th Meeting and Exhibition, Honolulu, April. P 198. Tiago Brandao Rodrigues, Regina Rodrigo, Omar Cauli, Vicente Felipo, Sebastián Cerdán (2009) Subcellular Transfer of Reducing Equivalents in the Neuronal and Glial Compartments of the Adult Rat 13 2 Brain After Portocaval Anastomosis and Chronic Moderate Hyperammonemia as Detected by ( C, H) NMR. ISMRM 17th Meeting and Exhibition, Honolulu, April. P 199. Pilar Lopez-Larrubia, Omar Cauli, Tiago Brandao Rodrigues, Sebastian Cerdan, Vicente Felipo (2009) Magnetic Resonance Analysis of the Effects of Acute Ammonia Intoxication on a Rat Brain. ISMRM 17th Meeting and Exhibition, Honolulu, April. P 200. Maria Rosa Fayos Carrio, Valeria Righi, Adele Mucci, Luisa Schenetti, Sebastián Cerdán (2009) Quantitative Magic Angle Spinning Detection of Deuteration in Small Biopsies of Rat Brain. ISMRM 17th Meeting and Exhibition, Honolulu, April. P 201. S. Cerdan (2009) Advanced Techniques for the investigation of Cerebral Brain Injury. National Neurotrauma Society (U.S.) Annual Meeting. Santa Barbara, September. IS th 202. S. Cerdan (2009) Perfused cells as models for ex vivo and in vivo NMR. 26 Meeting European Society of Magnetic Resonance in Medicine and Biology. Antalya, Turkey, October. IS

203. Jesus Pacheco-Torres , Rocio Pérez-Carro , Paloma Ballesteros , Pilar Lopez-Larrubia , Sebastian Cerdan (2010) In Vivo Detection of the Metabolism of Novel Hypoxia Probes in Models of Glioma by 1 H NMR. ISMRM 18th Meeting and Exhinition. Stockholm. May PO 204. Viviana Negri, Arisbel Cerpa, Laura Nieto, Pilar Lopez-Larrubia , Sebastian Cerdan , Paloma Ballesteros (2010) Superparamagnetic Probes as Contrast Agents for Diffusion Tensor Magnetic Resonance Imaging. ISMRM 18th Meeting and Exhinition. Stockholm. MaP 56

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205. Jesus Pacheco-Torres, Paloma Ballesteros , Pilar Lopez-Larrubia ,Sebastian Cerdan (2010) Reduced Glutathion Concentration Limits the Reduction Rate of Nitoimidazol Derivatives /in Vitro. ISMRM 18th Meeting and Exhinition. Stockholm. May P 206. Jesus Pacheco-Torres, Paloma Ballesteros , Pilar Lopez-Larrubia , Sebastian Cerdan (2011) Reduced Glutathione Rather than Oxygen Concentration Determines the Reduction Rate of Nitroimizadol Probes Used as Hypoxia Markers. ISMRM 19th Meeting and Exhibition. Montreal, May P 207. Blanca Lizarbe, Ania Benitez , Pilar Lopez-Larrubia , Sebastian Cerdan (2011) Orthogonal Diffusion Measurements in the Mouse Hypothalamus by MRI Reveal Cerebral Activity in the Fed or Fasted States. ISMRM 19th Meeting and Exhibition. Montreal, May P 208. Ania Benítez, Blanca Lizarbe , Luis Lago-Fernández , Pilar López-Larrubia, Sebastian Cerdán, Manuel Sánchez-Montañés (2011) An Automatic Protocol to Detect the Fed & Fasted Brain using Multivariate Analysis of Diffusion Weighted Data Sets. ISMRM 19th Meeting and Exhibition. Montreal, May P 209. B. Lizarbe, P. Lopez-Larrubia, S. Cerdan (2011) 2-Deoxy-D-glucose mimics the effects of hypothalamic activation by fasting as detected by ortoghonal measurements of water diffusion by MRI. ESMRMB 2011 Congress. Leipzig, October. P 210. A. Martin-Recuero, P. López-Larrubia, S. Cerdan (2011) Multi-parametric MRI identifies selective hippocampal vulnerability to cerebral inflammation. ESMRMB 2011 Congress. Leipzig, October. PO 211. B. Lizarbe, M. Garcia-Martin, P. Lopez-Larrubia, S. Cerdan (2011) Activation by fasting increases the slow diffusion parameters of the hypothalamus in the adult human brain as detected by DWI. ESMRMB 2011 Congress. Leipzig, October. PO 212. A. R. Borges, P. L. Lopez, J. B. Marques, J. L. Antunes, S. Cerdan (2011) Longitudinal fMRI characterization of the Gl261 murine model of high grade glioma. ESMRMB 2011 Congress. Leipzig, October. P 213. D. Calle, V. Negri, A. Sierra, P. Ballesteros, S. Cerdán (2011) Anisotropic relaxivity of multiwalled carbon nanotubes magnetically oriented in agarose gels. ESMRMB 2011 Congress. Leipzig, October. P

214. A. Benítez, B. Lizarbe, L. Lago-Fernández, P. López-Larrubia, S. Cerdán (2011) Automatic classification of fed and fasted mouse brain using multivariate analysis of diffusion weighted images. ESMRMB 2011 Congress. Leipzig, October. P 215. D. Calle, A. Cerpa, V. Negri, E. Agostinelli, S. Cerdan, S. Laureti, P. Ballesteros, G. Varvaro (2012) Anisotropic MRI Relaxivity of magnetically aligned Carbon Nanotubes. ERSMRMB Congress 2012, Lisbon, October P 216. A.B. Martin-Recuero, A. Krzyzanowska, P. Lopez-Larrubia, C. Avendano, S. Cerdan (2012) In-vivo PRESS spectroscopy reveals changes in osmolite and phospholipid turnover anticipating those observed by MRI in a model of LPSinduced cerebral inflammation. ESMRMB Congress 2012, Lisbon, October P 217. A.R. Borges, J.M. Bravo Marques, G. Rodriguez-Tarduchy, L. Barrios, P. Lopez-Larrubia, S. Cerdan (2012) The impact of anti-VEGF mAb antiangiogenic therapy on MRI biomarkers in a mouse model of HGG. ESMRMB Congress 2012, Lisbon, October PO 218. A.R. Borges, J.M. Bravo Marques, G. Rodriguez-Tarduchy, L. Barrios, P. Lopez-Larrubia, S.Cerdan (2012) Correlations between MRI biomarkers and gene expression in a mouse model of high grade glioma. ESMRMB Congress 2012, Lisbon, October P 219. B. Lizarbe, P. Lopez-Larrubia, S. Cerdan, V. Caz, M. Tabernero, E. De Miguel, C. Largo (2012) fDWI predicts obesity development in rats. ESMRMB Congress 2012, Lisbon, October P 57

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220. B. Lizarbe, P. Sanchez, P. Lopez-Larrubia, S. Cerdan (2012) fDWI reveals reduced feeding impulse in NPY knockout mice. ESMRMB Congress 2012, Lisbon, October. P 221. A. Benitez, A.R. Borges, P. Lopez-Larrubia, L. Lago-Fernandez, M. Sanchez-Montanes, S. Cerdan (2012) A quantitative analysis of glioma response to antiangiogenic therapy using intelligent image processing. ESMRMB Congress 2012, Lisbon, October. P 222. D. Calle, G. Bannenberg, S. Cerdan, F. Moreno (2012) A novel superparamegnetic particle shows no splenic accummulation, improved pharmacokinetics and optimal properties for perfusion imaging. ESMRMB Congress 2012, Lisbon, October. P 223. A. Benítez, G. Peláez-Brioso, B. Lizarbe, P. Lopez-Larrubia, S. Cerdán, L. LagoFernández, M. Sánchez-Montañés (2012) My appetite: A novel software tool to identify appetite disorders. ESMRMB Congress 2012, Lisbon, October. PO 224. S. Cerdan (2010) pH in Cancer (+`Sillabus),ISMRM Teaching Courses “MRI and IS MRS Preclinical models of Cancer”, ISMRM Stockholm, May 225. S. Cerdan (2011) pH and Cancer (+ Sillabus), ISMRM Teaching Courses, “MR of preclinical Models of Cancer”, ISMRM Montreal, May IS 226. S. Cerdan (2012) Tumor Energy Metabolism (+ Sillabus), ISMRM Teaching Courses, “ IS MR of Preclinical Models of Cancer”, ISMRM Melbourne, May

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TESIS DOCTORALES DIRIGIDAS Resumen 20 Tesis Doctorales dirigidas o en curso

1. TITULO:Metabolismo del agua deuterada en hígado de roedores detectado por Resonancia Magnética. DOCTORANDO:Milagros Moldes Rodriguez UNIVERSIDAD: Alcalá de Henares FACULTAD: Farmacia AÑO:1990-4 CALIFICACION: Sobresaliente “Cum Laude” por Unanimidad 2. TITULO:Compartimentación Metabólica en el cerebro de rata adulta detectada por Resonancia Magnética Nuclear de carbono-13 DOCTORANDO:Francisco Chapa Gabriel (Co-dirección Prof. Paloma Ballesteros) UNIVERSIDAD:Autónoma de Madrid FACULTAD: Ciencias Biológicas AÑO:1990-4 CALIFICACION: Sobresaliente “Cum Laude” por Unanimidad 3. TITULO:Metabolismo intermediario en neuronas, astrocitos y sinaptosomas detectado por Resonancia Magnética de 13C RMN DOCTORANDO: Fátima Cruz Fernández-Castañeda UNIVERSIDAD: Alcalá de Henares FACULTAD: Farmacia AÑO: 1991-5 CALIFICACION: Sobresaliente “Cum Laude” por Unanimidad 4. TITULO:Bases biológicas del contraste intrínseco y extrínseco en imagen por Resonancia Magnética. DOCTORANDO: José Alvarez-Pérez UNIVERSIDAD: Autónoma de Madrid FACULTAD: Ciencias Biológicas AÑO: 1996-9 CALIFICACION: Sobresaliente “Cum Laude” por Unanimidad 5. TITULO:Interacciones metabólicas neurona-glía durante la isquemia cerebral focal detectadas por Resonancia Magnética de carbono-13. DOCTORANDO: José María Pascual Garvi (Co- dirección Dr. J.M. Roda Frade) UNIVERSIDAD:Autónoma de Madrid FACULTAD: Medicina AÑO: 1995-9 CALIFICACION: Sobresaliente “Cum Laude” por Unanimidad 6. TITULO: Regulación del pH en modelos experimentales de tumores detectada por Resonancia Magnética DOCTORANDO: María Luisa García-Martín UNIVERSIDAD: Autónoma de Madrid FACULTAD: Ciencias Biológicas AÑO: 1996-2001 CALIFICACION: Sobresaliente "Cum Laude" por unanimidad 7. TITULO: Metabolismo neuronal y glial en el cerebro de rata adulta durante la diabetes experimental detectado por 13C RMN. DOCTORANDO: María Antonia García-Espinosa UNIVERSIDAD: Autónoma de Madrid FACULTAD: Ciencias Biológicas AÑO: 1996-2001 CALIFICACION: Sobresaliente "Cum Laude" por unanimidad 8. TÍTULO: Los intercambios 2H-1H detectados por 13C RMN como una nueva herramiente para el estudio de la compartimentación cerebral DOCTORANDO: Alejandra Sierra Lopez Coodirección: Paloma Ballesteros García (UNED) UNIVERSIDAD: UAM FACULTAD: MEDICINA AÑO: 27 de abril de 2006 CALIFICACION SOBRESALIENTE "Cum Laude" Unanimidad

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9. TÍTULO: Bases Moleculares y Fisiopatológicas del Contraste por Transferencia de Magnetización en Imágenes por Resonancia Magnética DOCTORANDO: Marina Benito Vicente Co-dirección: Dr. Paloma Ballesteros (UNED) y Dra. Mª Luisa García Martín (CSIC) UNIVERSIDAD: UAM FACULTAD: MEDICINA FECHA: 20 de diciembre de 2006 CALIFICACION Sobrsaliente "Cum Laude" por Unanimidad _________________________________________________________________________ 10. TITULO: Metabolismo de lactato en células normales y tumorales detectado por 13C,2H RMN DOCTORANDO: Tiago Brandao Rodrigues FACULTAD: Ciencias Biológicas UNIVERSIDAD: Universidad de Coimbra FACULTAD: MEDICINA CALIFICACION: Sobresaliente “Cum laude” por Año: 7 Diciembre 2009 Unanimidad ___________________________________________________________________________ 11. TÍTULO: Nuevos aboradjes de 13 RMN para el estudio de la diabetes DOCTORANDO: Teresa de Jesús Delgado Cardoso Co-dirección: Dr. John Jones UNIVERSIDAD: Universidad de Coimbra FACULTAD: MEDICINA AÑO: Diciembre 2010 CALIFICACION: Sobresaliente “Cum Lause” por Unanimidad ___________________________________________________________________________ 12. TÍTULO: Nuevos Agentes de Contraste de Tensión de Oxígeno por Resonancia Magnética DOCTORANDO: Jesús Pacheco Torres Co-dirección: Dra. Paloma Ballesteros y Dra. Pilar López Larrubia (CSIC) UNIVERSIDAD: UAM FACULTAD: MEDICINA AÑO: EN CURSO 2013 CALIFICACION: Sobresaliente cum Laude (Mencion Europea) ___________________________________________________________________________ 13. TÍTULO: Analisis Multiparametrico de Espectros de Resonancia Magnetica de tejidos y extractos. DOCTORANDO: Juan Solivera Co-dirección: J.M. Roda, J.M. Pascual, Hospital La Paz UNIVERSIDAD: Universidad de Barcelona FACULTAD: Facultad de Medicina AÑO: 2011 CALIFICACION: Sobresaliente cum Laude ___________________________________________________________________________ 14. TÍTULO: Alteraciones cerebrales después del traumatismo craneoencefálico DOCTORANDO: Ruth Prieto Co-dirección: J.M. Roda, J.M. Pascual, Hospital La Paz UNIVERSIDAD: Universidad Autonoma de Madrid FACULTAD: Facultad de Medicina AÑO: EN CURSO 2012 CALIFICACION: Sobresaliente cum Laude

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15. TÍTULO: Nanotubos marcados magnéticamente como agentes de contraste para imagen vascular por Resonancia Magnética. DOCTORANDO: Viviana Negri Co-dirección: P. Ballesteros UNIVERSIDAD: Universidad Complutense de Madrid FACULTAD: Facultad de Química AÑO: En curso, prevista lectura 2014 ___________________________________________________________________________ 16. TÍTULO: Activacion hipotalámica detectada mediante Imagen por Resonancia Magnetica pesada en difusion DOCTORANDO: Blanca Lizarbe Co-dirección: Pilar Lopez-Larrubia UNIVERSIDAD: Universidad Autonoma de Madrid FACULTAD: Facultad de Ciencias Fisicas AÑO: 2013 CALIFICACION: Sobresaliente cum Laude ___________________________________________________________________________ 17. TÍTULO: Propiedades Magneticas de Nanotubos de Carbono DOCTORANDO: Daniel Calle Co-dirección: Pilar Lopez-Larrubia UNIVERSIDAD: Universidad Autonoma de Madrid FACULTAD: Facultad de Ciencias Fisicas AÑO: En curso, prevista lectura 2014 18. TÍTULO: Relaciones entre expresión génica e imagen por resonancia magnetica en modelos murinos de glioma DOCTORANDO: Alexandra dos Reis Borges Co-dirección: Pilar Lopez-Larrubia UNIVERSIDAD: Universidade Nova de Lisboa FACULTAD: Facultad de Medicina AÑO: En curso, prevista lectura 2014 19. TÍTULO: Imagen inteligente de la inflamación cerebral DOCTORANDO: Ana Belén Martin-Recuero Co-Direccion: Francisco del Pozo UNIVERSIDAD: Universidad Politécnica de Madrid FACULTAD: Escuela Técnica Superior de Ingenieros de Telecomunicación AÑO: En curso, prevista lectura 2015 20. TÍTULO: Analisis inteligente de imágenes médicas DOCTORANDO: Ania Benitez sanchez del Campo Co-dirección: Manuel Sánchez-Montañés UNIVERSIDAD: Universidad Autónoma de Madrid FACULTAD: Escuela Politecnica Superior AÑO: En curso, prevista lectura 2014

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GRANDES EQUIPOS QUE UTILIZA O HA UTILIZADO

CLAVE : R= responsable, UA = usuario asiduo, UO = usuario ocasional

EQUIPO Espectrómetro RMN BRUKER WH 360 Espectrómetro RMN BRUKER BioSpec 1,9 T Espectrómetro RMN BRUKER XP WB 360 Espectrómetro RMN BRUKER AM360 Espectrómetro RMN BRUKER AVANCE WB500 Espectrómetro RMN BRUKER PharmaScan 7T

FECHA:

CLAVE:

1981-1983 1986-1988 1986-1988 1991-2003 20032003-

UA UA UA R R R

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OTROS MÉRITOS O ACLARACIONES QUE SE DESEE HACER CONSTAR

Becas, Premios y Distinciones 1975: Premio Carracido de Licenciatura 1976-78: Beca Predoctoral del Plan de Formación de Personal Investigador 1977: Beca FEBS Corta duración (Gröningen, Holanda) 1978: Premio Extraordinario de Doctorado, F. Farmacia, U. Complutense 1980: Profesor Adjunto de Biofísica por oposición 1981: Beca Postdoctoral del MEC (Dept. Biochem/Biophys-Philadelphia) 1982: Beca Postdoctoral Comité Conjunto Hispano-Norteamericano para la Cooperación Científica y Tecnológica (Dept. Biochem/Biophys-Philadelphia) 1983-4: Beca Postdoctoral John E. Fogarty (NIH) (Dept. Biochem/Biophys-Philadelphia) 1985: Beca Postdoctoral CSIC (Madrid-CIB) 1985: Profesor Titular Biofísica, Facultad de Medicina, Universidad de Cádiz 1987-8: Beca EMBO Larga duración (Biozentrum Basel) 1989: Académico correspondiente Real Academia de Doctores 1991:Creación del Laboratorio de Resonancia Magnética del IIB “Alberto Sols”. Puesta en Funcionamiento del espectrómetro Bruker AM-360 1991: Miembro del Panel de reviewers de NMR in Biomedicine (a propuesta del panel de editores y del editor en jefe) 1994: Editor del Journal of Magnetic Resonance Analysis 1995-6: Director del Departamento de Biología Molecular y Celular de la Transducción de señales, IIB-CSIC 1996: Secretario Electo de la Sociedad de Biofísica Española (SBE) 1998: Editor de NMR in Biomedicine Editor del Libro Sillabus on Methodology,Spectroscopy and Clinical MRI Springer-Verlag Vocal del Council de la Sociedad Europea de Resonancia Magnética en Biología y Medicina. 1999: Co-Presidente (con Dr. Luis Martí-Bonmatí) del Comité Local del XVI Congreso ESMRMB en Sevilla. 2001: Dotación de dos nuevos instrumentos de Resonancia Magnética, Bruker AVANCE 500WB y Pharmascan 7T para el Laboratorio de Resonancia Magnética 2002: Miembro del Comité Cientifico del Congresos de la ISMRM Toronto 2003 y Kioto 2004. 2003: Editor invitado del número especial de la revista NMR in Biomedicine "13C NMR studies of cerebral metabolism" 2003: Coordinador (en colaboración con Francisca Pons y Emili Martinez Miralles) del Programa de Metodos No Invasivos en el Diagnostico e Investigacion en Cancer, Red Tematica de Centros de Investigacion en Cancer, Instituto de Salud Carlos III. 2004-8: Director del Instituto de Investigaciones Biomédicas "Alberto Sols" CSIC/UAM 2004: Presidente Electo de la Sociedad Europea de Resonancia Magnetica en Medicina y Biologia 2006: Presidente de la Sociedad Europea de Resonancia Magnética en Biología y Medicina 2008: Past-President Sociedad Europea de Resonancia Magnética en Biología y Medicina 2009: Editor Asociado “Frontiers in Neuroenergetics” 2010: Academico correspondiente Real Academia Nacional de Farmacia Actividad docente 1978-1980: Docencia de primer curso de Licenciatura, Facultad de Medicina. Universidad Autónoma de Madrid. Asignatura: Biofísica (Programa completo) 1989-presente: Colaborador en diversos cursos del Tercer Ciclo; El Método Científico en las Ciencias Biomédicas (Coordinadores; Prof. C. Gancedo, Prof. JJ Aragón,UAM) Biología Celular y Molecular I y II (Coordinadores; rotación entre varios Profs) Metodologías aplicadas a la investigación de las membranas neuronales. (Organizador: Prof. Jaime Monreal, I. Cajal, Programa de neurociencias UCM no 94021, UCM) Organizador Curso Internacional “Resonancia Magnética en Biología y Medicina”, 1991 Fundación Ramón Areces (en colaboración con Prof. C. Arús, Univ. Autónoma de Barcelona) 63

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1992-2003 Coordinador del Curso de doctorado “Resonancia Magnética en Biología y (anual) Medicina”, Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de Madrid, Programa de postgrado del CSIC 2003-6 Coordinador de la Asignatura Metodos No Invesivos de Diagnostico e Investigación, Master en Biofisica, Facultad de Ciencias Fisicas, Universidad Autonoma de Madrid.

Otros servicios Servicios editoriales como Editor o referee especializado en revistas de difusión internacional Revistas servidas: BBA, Biochimie , Brain Res., Cell&Mol Biol., Dev. Neurosci, (orden alfabético) Dev. Brain Research, Eur. J. Biochem, J. American Chemical Society , J. Bacteriol, J. Mag. Res., J. Neurochem, Neurosci Lett, Magnetic Resonance in Medicine, NMR in Biomedicine (Editor), Proc. Natl. Acad. Sci. USA. Servicios al CSIC como miembro de Tribunal examinador en oposiciones 1989: Vocal del Tribunal del Concurso Oposición de Colaboradores Científicos “Bioquímica Física” 1991: Vocal del Tribunal del Concurso Oposición de Colaboradores Científicos “Estructura de proteinas por RMN” 1996: Vocal del Tribunal del Concurso Oposición de Colaboradores Científicos “Estructura tridimensional de macromoléculas biológicas” 2002: Presidente del Tribunal del Concurso Oposición para promoción interna a titulados superiores del CSIC en el Area de Biología y Biomedicina. 2003: Presidente del Tribunal del concurso oposicion libre de Titulados Superiores Especializados del CSIC en la especialidad Resonancia Magnética. Biología Servicios a la Agencia Nacional de Evaluación y actividades relacionadas 1992-presente: Evaluador anónimo de proyectos de investigación para PGC, Planes Nacionales, Comunidades Autónomas, Agencias Privadas (La Caixa, etc…), FISss, FEDER, Evaluador anónimo de Proyectos de Transferencia de Tecnología y PETRI Servicios al Instituto de Investigaciones Biomédicas CSIC como miembro de comisiones específicas 1990-3 Comisión de Compras 1990-4 Comisión Científica 1993-6 Comisión de Equipamiento 1997-00 Comisión de Bioseguridad Servicios al MICINN, MINECO y a la ANEP Colaborador del MICINN (MINECO), en la Subdirección General de Formación e Incorporación de Investigadores, en seguimiento de programas Torres-Quevedo, Ramón y Cajal, Juan de la Cierva, Becas FPI y Técnicos de Apoyo (Infraestructuras y Proyectos de Investigacion) Colaborador de la ANEP, en la evaluación de solicitudes de Proyectos de Investigacion. Colaborador MINECO, Seguimiento de Programas de la Subdireccion Gral. De Recursos Humanos, PTQ, PTA, Becas FPI, Programa Juan de la Cierva y Programa Ramón y Cajal. Pertenencia a Sociedades Científicas 1978-1999: Socio adherido Sociedad Española de Bioquímica 1984-presente: Socio Fundador Sociedad Española de Biofísica 1994: Socio de la European Society of Magnetic Resonance in Medicine and Biology (ESMRMB) 2000: Socio ordinario de la Sociedad de Española de Bioquimica y Biologia Molecular. 2001: Socio Ordinario de la Real Sociedad Española de Fisica y Quimica 2002: Socio ordinario de la International Society for Magnetic Resonance in Medicine (ISMRM)

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Pertenencia a Consejos Editoriales NMR in Biomedicine (desde 1998, Miembro del Editorial Board, http://www3.interscience.wiley.com/journal/13087/home/EditorialBoard.html). Frontiers in Neuroenergetics (desde 2008, Editor Asociado, http://frontiersin.org/neuroscience/profiles/sebastiancerdan/). Journal of Cerebral Blood Flow and Metabolism (desde 2013, Miembro del Editorial Board http://www.nature.com/jcbfm/about.html#Editors). Pertenencia a Comités Científicos en Organización de Congresos 1996: Miembro del Comité Científico del Congreso de la ESMRMB´96 en Praga. 1997: Miembro del Comité Científico del Congreso Spectroscopy of Biological Molecules, Sociedad Internacional del Espectroscopía, El Escorial, Madrid. 1997: Miembro del Comité Científico del Congreso de la ESMRMB´97 en Bruselas. 1998: Miembro del Comité Científico del Congreso SBE´98 Madrid. 1998: Miembro del Comité Científico del Congreso de la ESMRMB´98 en Ginebra. 1999 : Co-Organizador (en colaboración con C. Avendaño, J.M. Roda) del Symposium “Animal models of cerebral ischemia: A reappraisal” La Cristalera, Mayo 1999. 1999: Co-Presidente (junto con Luis Martí-Bonmatí) de Comité Científico del XVI Congreso de la Sociedad Europea de Resonancia Magnética (Sevilla). 2000: Miembro del Comité Científico del IV Congreso Iberoamericano de Biofisica, Alicante, Octubre 2000 2003-5: Miembro del Comité Científico de los Congresos ISMRM en Toronto (2003), Kyoto (2004) y Miami (2005) 2004: Miembro del Comité Científico del Congreso "Brain Enery Metabolism: mitochondria, transporters, neurodegeneration", Creta, Mayo 2004. 2005-7 Miembro del Comité Científico del Congreso ESMRMB Basel (Suiza), Warsaw (Polonia), Octubre 2006 y 2007.

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Publicaciones Representativas Selecciondas (1977-2015)

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S. Cerdan, C.J. Lusty, K. Davies, J. Jacobson and J.R. Williamson. Role of Calcium as an inhibitor of rat liver carbamyl-phosphate synthetase. Journal of Biological Chemistry (1984) 259,323-331. I.F.: 5.520 (41/276, Q1 Biochemistry and Molecular Biology) ISSN: 0021-9258

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: Role of calcium as an inhibitor of rat liver carbamylphosphate synthetase I. S Cerdan, C J Lusty, K N Davis, J A Jacobsohn and J R Williamson J. Biol. Chem. 1984, 259:323-331.

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CURRÍCULUM NORMALIZADO COMPLETO (CVNC)

S. Cerdan, C. Hansen, R. Johanson and J.R. Williamson Nuclear Magnetic Resonance Spctrsocopic analysis of myo-inositol phosphates including inositol 1,3,4,5-tetrakisphosphate. Journal of Biological Chemistry (1986) 261, 14676-14680. I.F.: 5.520 (41/276, Q1 Biochemistry and Molecular Biolology) ISSN:0014-5793

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S. Cerdan, B. Künnecke and J. Seelig 13 Cerebral metabolism of (1,2- C ) acetate as detected by in vivo 2

and in vitro

13

C NMR

techniques. Journal of Biological Chemistry (1990) 265, 12916-12926. I.F.: 5.520 (41/276 Q1 Biochemistry and Molecular Biology) ISSN:0021-9258

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S. Cerdan and J. Seelig NMR Studies of Metabolism. Annual Reviews of Biophysics and Biomolecular Structure (1990) 19, 43-67. I.F.: 17.049 (4/276 Q1 Biochemistry and Molecular Biology) ISSN:0083-9182, DOI: 10.1146/annurev.bb.19.060190.000355

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Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

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NMR STUDES OF ETABOLS Sebastian Cerdan  and Joachim Seelig

Bicente  the niveiy  Bae Klineleae  CH Bael itzelan KEY WOD

noninvasive methods, stable sotopes meaolc reguaon n vvo NMR loclzaion echniqe.

COS PPV D VVW         . . . . . . . . . . . . . . . . . . . .  . . . . . . . . . . . . . 

3

SPAAL OCALZAON METHODS FOR I VVO SPCROSCOy

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

7

Surface oils    Imge-Gdd Spetoopy............... Metbote Detetbe b  NMR.. ntaa Cmatmntatn  .  n tu onto of Oxdtve Phophoton .... .... ...  ...

13C NMR..

ial id l ad lad ali ai............................

Goneoee................ Fe Faty Ad Mtabm and Ktgn ...    yogen Snthe and egadaton  ......   ........  . Tgyede nd hphopd Metbom   ........  NM..

  .............

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Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

44

ERDAN & SEELIG

and hesry In he as en year ne NMR aans have eered as serndin ane ih a hrzna re are enh  adae ana r hans have een deveed r he rs e  ha ee se  sd a ehans and mea aha n an and ana n a nnnvave and nnderve anner ya e nde he dy  he enery ea  eea and hear e y  NMR he dy  he rarxy ad ye  en snhesi and deradain and  neenei and e enes n he ver   NMR and s reen he eareen  eae n ran y H NMR Mdern NMR de  n vv eas ray ean n  2 hen Ean e a (8 eyed  NMR  nr he eas  ue in a eary e e n   Mn & Rihards (2 ued 3 NMR  eare nran hhae and 2dhh erae (D  n erhre and  deerne he inraear H rm he earain  he hhr renane Larr (06 denraed he e  H NMR ain  an X-ray-ie iae  ivn ie and as sianey he rs    R er  ania e a ishe ( The s  in viv eai y R i arary araive r severa rean Fr NMR exerens rvde he runy  err reeive, nnnvaive eareen in he ae iia yse end niqe NMR rerie h a he  and  reaan e r he hmnear and heernear sin n aerns an e easred These araeer nain eifi inran  he hys a r aha sae  he ise nder nsderain ( n ea arenain ( r n he x hrh se ai ahay (08,     hrd nidrena rean raes  enzya reans an e ed n s sn anean ranser ehnqes (. Fnay i is n se  an saiay resved ea nr ain yiedin he diriin  eae hin a isue r ran ( 2 42  hs enn a ne avenue  sd ah and heay a he ear eve (0  8 . n h revie e reen aan  NMR  he sd  dfferen ae  eaim We en ih a rie ine  azain ehd ha are ny sed  ain in viv NMR sera. We hen dere in re dea eai inran reeny ained  NMR  ered ran ina ana and han revis reve have aready vered he aans  NMR  he d  ea n rranis ( iaed r ivaed es (4  2 4 and r (64 NMR er  he rain ( 48 and han n vv NMR ery (3  8  have as een reveed reeny

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D O MAOLM

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L LOLZO O O

Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

 VVO EOOY

 mjo pobm ssocid wih in vivo spcoscopy is h pop oc izion of h ionofins (ROI Gidd by  poon MR im of h bc, i shd b pssib  dn h ROI dic n h imin sn nd o ms H_, , o MR spc of h scd vom  psn, no simp mhod iss  sov hi pm Mn din sis hv bn dvod (  0, 3  , 79,  79, dpndin, in p on h chnooc s o h MR nsmn n h oown sions w smmiz fo mhods h hv povn sf in  viy of ppicins  sfc cis,  vm sciv ciin V,  SEM, nd d ISIS hoh  sfc oi sd n is spi sciv, i cn so b cmbind wih on of h  ps sns o fh impov sciviy Surfae l

Sf os  pobby sd n mo hn 0 of h bshd n vivo MR sdis hoh h hv n inhmonos d nd  imid pnion dph hi hih in fco ss in  od sn onois io Fhmo, sf ois  sy o hnd nd h  h mhod of choic fo h msmn of spci iss h snsiviy nd sviy of sf os hs bn invsid hoy nd pimny 2, 9,  25 Rcn impovmns ind h s o dibi pss (  70,  83,  8 h pnn dph   sc o cosponds h o h dim of h sph Spi iss my b sppssd y hoosin   0 ps nh  h posiion o h sc  s vin o h sf oi hni s sposop infm imin (9,  79 Th ps n fo iin is ncmnd in sm sps, nd fo c ps n h copondin findcon dcy (D is od A wodimnsion Foi nsfoion wi hn sov h chi shi on on is nd h posiion on h oh Image-Guded Sperpy

Impovd cono of h si shp nd posiion of h RO  obnd by ombnn in mn d dns wih fnysiv pss h mhods nd ps sncs mpoyd fo ocizion b  cos smbn o MR imin oins n vomsciv ciion spcosopy (S c i    nysiv ps ppid n h psn of  si dn  ss



C & 



Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

Acq. Gx



SIice1



Gz

Gy

 90



 90

   

Sie3

Slie 2  





 90

!





 -   q------       

gna

 

G-2  c 

 







                





- -



- _





Shms fo muidd in vvo sposoy.  Volumsliv xittion

(VSE ( I, 1 5 1 6)  Simultd o quiston mod (STEAM) (75

a omoou c of  am ad oa  mazao o  a A oc u  caad w  "o u I oa  mazao o  wo am   ad a o  lc c  a aoa ° A   of  u adwc  maao of  cd c  oad    ad oed alo  a z ax  dca w  coo  laao m.   o ad  maao oud  cd c  oad o  x  a ad  ad dad   ad ad. T quc  ad   ma wo oooal co a  RI  u fu aowd fi o a od ad  o a mall cu      2  2. T mao dcul  al  V quc o a o od m  a  ° ad u uua cao  mad o ou w u wood co  om  aod   PARS quc wc a a u o   ac o  u coda duc  adw   2. Lk VSE  SPARS quc    aao of  cd l alo   . x 

Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

N MR STUIS O TAOISM

47

Te ue o timuted eo ppe to be te mtod o oi o voume-eted potonMR petoop C H MR) 737)  SM tniqu timulated o aquition mod) onit o t un-lete 90° pue tat ae appd in te peen o oto gona gadient and tu eet a ube-aped egion o wel-dned dimen ion Fig ) STM avoid boad-bandd adio un pul wi a iffiult to genate wit lag bod oil and it allow a eate mpe mmng o e egon o nee. Su mmng  abolutl mandato in 'HMR wit t mall ema  ang S  extm nv to edd uen ndued b gaden w­ ng ne t magnetiation o t egion o nteet depa and i old n  pn o -gadn  good ompnaion o edd uent i teeoe equid.  potonNM ptum o te ban o one o e auto gu 3) a obtand t SM  ISIS uene 137) lo poide  ee-dmenonal loaiton tt  fqt 8 pe n t pne o  gad nt wit noneltive 90 quiition pue in te abene o gadint e woe eqene ompe egt deent xpement and te egon o-intet i gnated b an add-and-ubtat m Sine t magnet­ iaton o intt  tod along t ax SS  mu l nitve to dd uent ultivolume eletiv petoop n be onideed a a viant o SS 8 124 127) T epimnt u lie-eletie ineion pul tat imultaneoul eite vea diffnt li o te ame objt u deent egon witn te ame ogan g te eat 1) an be montoed at t ame point in tme.  R

Te majoit o MR tudi in living tm ave been onned wit  MR invtigtion o eneg mtabolm and it egulaton in a aiet o tu nluding pma keltal mul 4810143144) at 9 18) and bain 148)  olowng apet a onded e: ) metabote tat an b obvd in itu b  MR  ntalula metboi ompatmentation nd  MR vibilt  in itu ontol o oxidti popoltion a viewd b  MR Meboes Deecbe by  NMR

n in vivo  NMR ptum obtained om uman l mu at   n a woe bod magn outinl ud o MR imaging  own in iue 2 2 iv onan wi n be aigned to te -  and  popat o  TP popoeain P) and inogan popate i) peiv ae obved e nuotide tipopat a nomall

48

D & SEEL



Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

 

 NM sem  human

a musl and a 2 n a wh d nsrumen. esnanes are assned 

AP,  and P (2)













present in musce in muc smer concentrtions tn  TP nd re not beid o conibe sinicn o e in io 3 MR spcr Wi few remrbe ecptions 7 117) te  nd -pospt rsonnces of P ovrp in vvo wit t  nd pospt rsonncs of  TP Terfor te contibuton of DP to t  MR spctrum cnnot be determined drct n most cses n indirect procedure to ccute P concentrtons wen te TPTP rto s ver cose to unt is describd er. n ddton to resonncs of nuceotds r nd Pi two futr sins r norm obsrd in   MR spr of issus d r int preted s pospomonoester nd pospodester signs. Te pospo­ monoester resonnce is  composite nd contins contributions from pos poretnomine poporcoin fructos-P MP tc.  min component n brin undr bs conditions s posportnomin 86) nd n iver is posporcoline 66 95) but sinicnt contrbutions fro noito-   n brn ) or ro ugr popts in ivr or musce 44 93) e been detected foowing pocrpine tretment fructose nfusions or rcisnducd cogenoss More rcnt vr ow fid sins 16. ppm) risin from nosto 2-ccc pospt v bn dtectd in tumor cs 8) T popodster rsonnce in vivo is so  coposit sin con­ tning contributions mn fom gcroposporcoine GPC) nd gceroposporetnomine GPE) 36) Intresting in te in situ spctr of do brn  trd spcis i i mob pospoipids so contribut to t rsonnc 41) Tis is vidncd b two fcts ) n ueous etrcts of te brin t intenst o te pospodstr reonnce s distinct smer tn pedced on te bsis of te in vvo spctr

M U OF MBOM

49

Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

which inicates the presence of a iester tha is hyrophobic an cannot

 xtac nto h aquous has;  th hoshos sonanc oans sctvy wh ncasng  sngh 15) whch s yca o axaon o hoshos va chmca sh ansooy 8). cnty, a chaactsc hoshos sgna was oun n cuu tumo acno cacnoma cs an was shown o as om hghy mo hoshos 84) Quanttav nomaon concnng h atv concnatons o th n maos monto aov can also  otan  on assms tha h voum c s h sam o a maos, av vaaons n concntaton can asy  olow  th sca a acqu n nonsatuang contons o a coct or h ncs  laaton tmes 113    1) Th tmnaton o asou concnatons y n vvo  s mo ct Th mos coon ocu comas h sonanc o ns wth h aa o an nognou mtao o nown concenraon, g th AT ak 180) Howv, oh mhos nvov ng comaon wh an xognous sana snt n an xtal callay o accumula n h ss o neres or wh the H MR sgnal o wat tc y a ouy-tun suac co can  us 11) asumnt o th av o aso concntaons o  R osva mtaos aows th cacuaon o h n stu concntaon o  and o the hoshor aton potenta. Ths proceres assum ha th can hoshoknas acon s at qum whch mgh not aways  he case) an qu knowg o h nacla H th catn concntaon o h toa catn  hoshocatn oo) an th vau o h quu consant o h catn hoshoknas ac ton 185). h oowng xssons a s [A] =

[AT] C] C] K· H]

AT] [A] []

[C KH]  P] 

1 .

where A, [C, , an [H+] represent h fee concentraons o maos whch can  mn om th   scta an [C] or  a known om ancay nomaon Fo a gvn conon h ao ]{· H]} s constan ho a quatav nx ctng th lav changs n h concnaton o A o th hoshoyato otnta can  oan y a sm xamnaon o h AT/C o / rao n aton to cng hoshous-conanng mao   aso alows he measremen of  the nraceuar H an ) he 

50

D & 

M concentaon We do no addess ese questons ee. Petnent eeences ae (12, 77885103146).

Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

IntraceuLar Com artmentation

eas one o e os neesn esus obaned b    e deence between te DP and P concentatons easued n vvo b NMR ad ose easued  classcal enac eto n ecoc acd  solube eaboe exacts Table 1 esents an ustave coason We e concentaons o  TP and  ae sa wen easued b  NMR and casscal etods e n stu concentatons o P and DP ae eakabl lowe wt 31p NMR as coaed to conventonal acd exacon eods. Te ncease n otal P and P n e  extacts cannot be accouted o  a equvaen decease n e  P o  total concentaons. Ts ndcaes tat te excess DP and P cannot be oduced b a beadown o  ene osates dun e etac­ ton ocedue and suests e exsence o ools o P ad P tat ae not detecae b n vvo P NMR Tsc nvse oos account o aoxatel 50% (75%) o te total P and 70% 100%) o e DP  ve ea  80). Tee ae seveal easons o beleve a onl e ctosolc P s NMR vsbe s t s known o exeents wt as subcelula ac onaton tnus tat aoxal 50% o  total cellula P s oconda n eused a lve (7 72) and anendo eued eat (100) a ecee tt el atce e obsevao esented b 

Cmprin f th cncntrtin f phphltd mtb­

lit i pfud h d pfud iv  mud i iu b

31 P

NM nd in vit b cnvntin nzmtic sss f prchlic cid xtrct Livr

 O tchnu

NMR

Extrct

NMR

tt

T

323 ±OOSb

3S5024

28±02

27±02

D

OoJ 001

07009

0301

1101

104019

186 01O'

20

3802

Cr Pi

00

O

" A concetratos re give  mromoles per grm wet weght b Results re expressed s me ± stdrd errr lues f hert d le were tke frm reereces (8) d (66), respectely 'Te rom efeene  nd oeted int mo/ wet weht n he wedry weht rto of 637 ±  recmmended  (8)

Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

NMR SUDES O METAOLSM

51

n Tabl 1  cond P vbl by  NMR rc  a racllular pH a doygluco-phopha, an ecluvly cyoolc eabole ( 1 ) hird, ochodria ocupy oly 1 00 of he oal elllar volue and conan l waer han h cyool Hgh vcoy low organll cocenaon, ad ao a pobably hghe conen of paaagec eal on can ak ochondral eabol nvble o NMR n cll or u ( 1 7). kw ADP  nally undcabl undr n vvo condon n he hear (8), dney ( 1 78), ul (4), or bra (4 1 , 4), and oly barely dcabl   lvr (). Th raon ar lar o hoe dcrbed or P I paricular, h cyoolc ATP/ADP rao derved ro ubellular fracoao ud  1 0  h prud ler ( 1 7 and bwen 0 and 1  n h angendorf perfued har ( 1 7 1 ). Aung a cyoolc ATP ocerao of 35 M  bo a, h ea ha he free ADP concenraon  a h dg of  NMR vbily  he lvr ad  far below he level of deeion  he hear urhore ADP boe nvbl o NM whn   bound o maoolul lk anyon n ucl or ha, or o he nurou phophorylanrae  h lver. Renly, he vbly of ATP by  NMR ha ben nvegad n perfued ra lver ( 1 ). Aer 1  i of aoa he lvel o ATP vible by NMR dcrad o 0% of  ial value whl h oal AP, a der­ nd  era, dcayed o only 84% A prerenal dreae o he cyoolic ATP durng anoa wh no alraon of h ochodral ATP wa rpored alo ulaoly, by Aw e al ( 1 4). Ta oghr, he rul ugg ha oly h cyoolc AP  vbl by NMR. hi poi of vew i no a varae wh he fa ha he ATP reoane av been obervd   NMR pera of olaed iac ohodra penon (). The prn ued ochondral concnraon ha wee gcanly hgher han hoe hough o occur  he ac u ad ha wr prepard n h prnc of chlang agn Th iiial approaio ha eeally all AP i NMR vible ill hold ru undr oroc condon bcau he ajor par o cllular ATP  deed cyool ( 1 7) In Su nr  Oxdave Phsphran

 order o prode or, eeal ad arda ule hydrole AP gnrag ADP and P n he eergon raco:  Th MgAT  dialy rynhed fro ADP ad P n h ochondra by odave phophoylaon accordng o h quaon

5

RAN & I

gAD  +ADH+H

Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.



MgT 2  ND + H

4

Th chmr f P r !Oz   a arma (  0) Th w csss (uas  a 4) a gh cu  sa usc a ha wh h gA T  cca mag ssa csa hf masums f h wk fm r f h g c sum v quaav fma  h A hs b ­ av hha If h g su  h mcha s m ATP ca as b ga hugh h ca hshkas qubum a h s f r a MgAD 5 Th r f hs rac  cua rgc a h masurm f s fwa a vs uca flus usg maga asf chqus   ass h uh frma  hs c ca b fu  fcs ( 0 6 1 1 16 16).  h as sa mcha c aas  us gas w us fr su  av hsha hr h av f  M chu ma hs rcs amab   u aass 444 9  20  49     has fcus  h ash bw mchaca k a mabc g u  ska musc (4  1   161 16)  har (76 6)   r   usa h facs crg av hshra  su hac  a (6) fu ha h ra sh bw mchaca wk a h A ccra  h  su ska muc ( a rasfr fuc) fw Mchas kcs wh a  f 5 M f AD. Ths sus w v sma  hs us  f susss f ac sa mcha (49).  hc cs h aa  f AD cas  arma 77 M a rguar vau a g u h bcam h mg fac Chac  a 46) as fu ha h c cras f A a P a rs as rm b M  su wr bw h "  rra whch suggs a b rguar r   f AD bu as f   h c f musc sa. Sma sus bas  h us f asfr fucs w cfm   ch (07) a c ch g har raras (  8 1) a  h  su bra    7). Tak ghr hs sus ca ha av hshra cu b cr   b h AP ccra bu b sva h facs sch as h  ccra h lvr ra f h subsas a f g a h hsha a. Ths ccuss w as sur b sus  mcaa wrk  uc a fr AD ccra  Lagrf rfus hars u

Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

NMR SUDES O MEAOLSM

5

dffrn ur ondon 7 Work roduon w dndn on  ADP onnron ony wn uo w  ur  m ndndn  ADP w gluo lu nun or w yruv  ura, ndan aan a faor or an  ADP onnraon m onro mord rron n ummary p NMR a onrud gnnly o our urrn g  h l   phphyl   h  u onnron of fr ADP nd P wr found o  nnly lowr n  ol vu murd y onvnonl ron nu. T n vvo ooryaon ona  Euaon  wa u aou an ordr  mgnud gr an rvou m , 1 1 , 1 85. T rul  nonn w  yo of rmodynm onro of odv ooryon [ (7)   rvw. In onr,     P NMR rmn on o kn onro nd mul o o ura maon n w AD   dvr r o ur and oyn and NADH ranfr o  moondra oud om  onrolln aor dndn on  yolol rumn

 dvomn of n vvo C NMR rooy a n owr an   ounrr u of  ow nur undan of  3C,  unfvorl gyromn ro of  C nuu nd  nd o u bl h  m h g C_H plg h om  undrould C r. T lmon an  ovr om n r y  u  vy h ur  y l douronan douln dv, u a onnrl or add d H o 4, , douy und l 3 nd H o  1 5, or H mgn ro  1 . T ma  ran of  NMR  ou 00 ppm nd  3C NMR  onlly m nformv n 3p MR,  amo  ao onan aron u ony a fw onan ooru aally,   3C NMR aro  mlr o  lal 4C rdo av oo rmn. Howvr, C NMR offr rn advana ovr  onvnona radooo nu. r, he n of  3 lng n ndvdul ron from  vry of maol n  followd mulnoy nd nonnvvly. Sondly vn f h ny  p formd n r,  C NMR aroa vod, n mo a, romaogra aron and ma dgradaon, w r n al n onvnona radoav oo nu. nally,  rn nd rlav rooron of dffrn 3C ooomr of  m m o n  vud y  ny o IC_IC omonur n pg p  1 08, l ,  ml ppy h    

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C & I

y NM metd. n te te and 3 NM teniqe ae le eniive an nvenina adiipe eniqe and ny metalie peent in te millimla nentatin ane an e nveniently deteted

Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

m VIVO.

3  die ave een epted f a vaiety  metali patway, inldin te itate yle and elated anapleti eatin te lneenei patway and elated ftile yle, te yntei and deadatin f lyen te idatin f t ain and medim ain fee fatty aid and ketenei and te metalim f tilyeide and pplipid Trrox A e n Ree Anpero Reons

3 NM tdie f te tiayli aid yle ave mainly een pe fmed wit Lanendpefed eat ( 1 , 0, 1 1 , 1 1 , 1 , 1 0). e invetiatin ave fed n te tate pefeene y te eat and n te qantitatin f metalic fle t te idative and ana­ pleti e f te itate yle in eveal peially 3enied tate. T           ltamate in eat etat wa meaed f anendff eat pefed wit eite nlaeled gle and [  3Cpyvate  wit [3aetate (50) A pitiated matematial mdel f te tayl aid yl allwed te allatin f  te ttal fl t te yle  te elative ntitin f laeled and nlaeled fm f aetylCA, and c te eane e t te alanine  apatate amintanfeae n a late tdy, dieent itpme pplatin in ltamate  ld e deteted n viv dn pefn f te eat wit 5 M[ 3latate and  mM nlaeled le ( 1 0) Ti itpme analyi iinally pineeed f ateia ( 1 08), pved etemely elpfl in fte eli datin te metalim f te eat. F eample, in pefin in 5 mM _3Cpyvate   mM [ 3Catate t wa fnd tat app mately 0% f te iti aid yle  wa deived m e [_  enied aetylA pl Ntaly, wen nlaeled le pl inlin wee added t te ave mentined 3enied pefate, nly a i deeae in t peentae f 3Caeled aetylCA entein te yle wa mead, nmin te pefeene f te eat f pyvate ve le a tate Te ame appa wa late ed t  te elative fle t te mined anapleti eatin ve te  t  tate yntae  te intat eat ( 1 1 5 5. Te mined  t te anapleti patway wa appmately 1 0% f te tate yntae  wit aetate a te nly tate it ineaed t 0  0% wen pyvate  ppnate wee added, t indiatin iniant 

N MR STIES O METAOLISM

5

couo o pyu coxyl ux  popo coxyl ux  u

Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

uoeogee

lucoog   co wh h coxylc c cycl h  ly  ol  hpocy 558)  pu ou o  lv 5 57   c l   ) y  NR  y  y o 3-ch glucoogc pcuo o u h ocu o h  u uo o ux w pyu coxyl  pyu hyog whch pov lv ou o h c c cycl o o h  u cvy o pyuv k  h phophoolpyuv cycl whch  pol oulo o h  glucoogc ux  u.  xp wh pu ou o  v 5 5 c h wh l w h oly u   h coxylc c cyc  pproxy u popoo hough  pyu hyog  pyuv coxyl ou. Howv wh h lv w pu wh  u o l  hol oly o o whch w  ll) l  h c cycl xcluvly hough pyuv coxyl whl hol w ulz ly o llg h cyl-oA pool Th  NR ul w uh l y  c xp 58). Th u how h  NR coul pov  co l o h oc oop ho whch  o­ lly u o luc h ch o glucoog  8) o  w]. Glucoog w lo g wh 3]l [2 ho o 2]pyu pu o  pu v o pooo c  Th ul w cop wh g o l xp wh l o o  o  l  5 5) A ol ffc o ulh ulo o pyu k ux  uw c  h u I co o ffc o ul o h l chlg o u hough pyu oxyl o pyuv hyog coul  c. Free Fa d Meabolsm and Keogeness

 u o o ho ch  u ch y c  h  o c  h  u y  NR chu o ff uo o hoo o cg g g  ox o pozooz-uc    2 1 4 4 1 ). Eph w plc o h  u ch  h hool gulo o kog  -oxo wo phwy o cocylo oo o uy w co  1 40  copl oxo o wo olcul o  1  cyloA  yh o  cocyloA hough h hyoxy

Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

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CE & EE

myyl-CA phwy    p  yl-CA w y  wh h mhl pl  ylA. h w mhm   h m  h y h li C l   Cl  C  hyxyy hwy  p  C   C l  C  phw  lw ymm ll  C   C    m ff p . Alhh h fi mhm  lly hh   h mj phwy   m h  ymmy    w h li  h C l  C    hyxyy pi  i   j i  h i phwy    h mhi  m mp  h -x phwy  w m h xy i    w ly y  i      i  C NMR hq (39).  h  pily pp h h -x  xyi i i h  w  i x mhm. Hw CMR hw h [C]   1  1 1  1 3] ii  w    mlly. Wh p  h l  i  h ihl  pxl -xii y  h  xii  lw i hi ixy i,  C ll w  i i  h ixy i y  i h   S h l w  phwy  lz  h  h h l  h h x  mm h  y  p pmy  h hp pm  NMR h p h   i   h iy  h pxl -xii ym ye Syte ad Deadat

C   h        98.0  96.0 pp, py  h -l-4 y k  ly Cl y  100. pp (6)   pl  y h hl   y yhi      p   hh m wih, ly w hw   plly ii y MR    l  i ly  3 p (69) Gly y­ h   h    p    l  4 , 66 69)   h (OS 33, 34). M y 3C   ll l ly h l    hm (3). Iiil i ly h i  [-C] ipi i ly   i lyyi i h p   (69) y yh  h       w   h y   60 m   l  [3C]l  0% ly pi w   ppxly 60 m  

Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

NMR STES O METAOLISM

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glucgn dmnn ( 1 4) Smll glcgn nh m  1 3Cgluc w nhbd b  glucgn wh nuln ln  n cm­ bnn wh glucgn hd n mul  nhb ffc n glcgn y (  66) Sv C NM udi hv cn ddd h mchnim f  gyg r ſt  g d  d r r pmn wh dcv c hd hwn h glcgn nh qud h cv  h glucngnc phw nd h h ndc u g  r  gcgn cud mk  ignicn cnbuin  gyg r r   y d dr ph­ w gluc  gluc-l  Dgluc  glcgn (99 1 5  6. h cnbun f h dc nd ndc phw  glcgn pln w cmpd b  dmnn f  1  C]guc   1C]gluc plu unbld nn ( 1 58, 1 6 , 1 64) Th dc nd h indc phw ccund f  nd - pcivl, f h gcgn pducn Hwv h cnibun f h indic phw  dmind n h NM ud w gnficnl ml hn pvu mumn p fmd wh dcv p (\ 5). Th dicpnc cn pbb b xpnd b h dn   h guc ld l    f yrd gyg y   rd w   r viy  glyg y d ryl ud dr d  rrd  rg y  d micd  nclul lv   h crdg r d c   1 0 Th in iu  f gcgn nh c pd h cv f glcgn nh md n v Th l ugg h glcgn nh  h rg p n glcgn nh n vv In r  ug  y  ry  rd  r w   gr    gyg y  g gy­ gy w dd   Udr   d ry b pp  b inhibd b n unknwn c h  n pn n h n v  Th ul d dub n h cnncl vw h p ry  r  d    d dud b  pryli d ( 1 6) rglcerde ad Posold Meabolsm

C NM h bn ud  ud h gld nd phphlpd cm pn  nl nd dd mucl (0  1 , 67) f d iu nd ld dc (  68) nd f bin (9, 22 Th f cd cmpn  d   r w w   dd udr dffr dry d   rur  dgr  ur  h y cid  ud  drd r  r   00/2  , which c h i f mnunud  plunud f cd n 

8

C & 

Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

s ss Ths  ls s h   ms     s s l  sh msls  b, l s s mbl s  b  s   ml sl) (9)   sml  s sb f  smul musl (20). M l, sl mbl NMR sb s s    b  msl (22 R

H NMR s  hl hh ss  _  NMR,   l mb  lul mbls su b bl H,  ms   b s: a T  sl  h ss hs  b m,  b h mx sl s h  u    ss   b s B bs  b m,  , b  lm   suss  qus (89 90  8)   ms f -msl   msl sl   ) Cb bsm s b s b H MR      () l x  x 4 ) hlm 24) l sus  1 7  fis 78  um s   . T m us  b  u b b l­ ss, l hx sls s m qull b m h s f h l mh s   f h mhl  f Nls  N) ssm  ml 'H NMR sbl  bh ms   ()    f hs sus s ffiul; ,  hs b sh h bl  m  b mll sbl b NMR ()  h fs   l   ls  h s f h l mh s (8)  ,  lx ms f h ml u f N  b ms     m 88   s  h   N mhl s m b   h s f h   m  b h l  H NMR s f h um b s b  su b  S hqu    s sh  u   llus ss  s m h h hh su 'H NMR sum f  ­ hl  x b m  b  84 ) Fu  m s   s    s  l h  s umb  ss  m Nls, ,  s  hslhl,  bbl l  b sl  h hum b sum  hh slu su shs  s­     4  b mbs (40)  48) h H NMR sus h s  sl msl mblsm 

9

NR STIES  TALIS



A

Ncsp H

Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

 J Cr

Gy?

 C/GC

B 

 CH 

aa'

N-A -Asp

C r

 (Ha C

Ha Ha

i

,

 Ha

Gn

a



Ha a- glucose  



'

l

 





I

GPC T aa Tu





I

[H

! Th C



HH{ 

i 

BBA



V C



Leu 



0  

0



'H M spectroscoy of the bain.



0





Localized H NMR spc o human

brn n u tned ung the  ehnque t   (qutn me  mn, lum lctd:

4

c3) (coutsy o H P Hn B Hgh oluton H M pca of 

perhlr  exr bne rm rt brn   T  4.

CERDAN & SEELIG

Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

6

pc,  w pol o moo mol popoc ol  lc poco  ppo o p og  o  gocm cl. clll p col lo   mi om  cmcl i o  mizolc poo o co 8 i mo iiill popo i 9  x o clll pH mo  m kll mcl  womiol (2D NMR g mo  lo    c cl  8 i xc 99  o fli 8 9  x   o   o 9  T mo low  igow gm o  oc   imlo g o ll mol   x o  lo cqo m W l  ppoc w  o o  Jo­  ccoxlc c o   o    9    oc w  moo  o,     o  io po  ml w cific   pcloc c xc o  li w oi Fi 4 ow  SY pcm o  xc 

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ontou plot of a  OY 45 spectra obtaind rom a perchloric acid xtract

o th livr o an alloan diabtic rat inusd with [l,2-1zdodcadicrboxylc acid. Rlvant assgns a ndicad  th x Fro (39)

N SD ES O AS



Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

i with tl t  how) l iott d  b dttd tbly th     A B C d D d    th lt I d M. Th lti w hw t b ily did  dii id lio A d D) d th i id -yi ltio  C , d M)  iditd blo D

A

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n +NHCHCHCHCHCHCOO

I

NH h o  Cdddiboyli d ld     th ht tti  lyi d di id A ihibity t  d d th  dt  th -dt  dddii id  th thwy  lyi ddtio t th ll o -iodii d diti) ld b d  th xit Oh lo o H NR h b y  45 9F 

Th 9 l h  lihtly ll ti ot th th to wh  hl h   l by  to  0 No ­ tibtio o ti bd l  obd whih  9  itti ti lbl  tbli tdi U itd idit 9F NMR h b tily ld t  th itlll H  wll  th  C  d  M ottio ( 2 Oth litio o 9 NR to th tdy  i it tboli iol a td thti d t d d b oitd l diti loh tyu d i i wllld F NMR t i bbt bi  0 d th ti   hlth liit o bbit bi  b ollod th tdi hod bixotil dy  i th bl hoth hot hi i ily ot ith o dt hil it ( . Th tboli  5 ooyl  tdid i  li 7 itt il 75, o 

Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

62

CERDAN & SEELIG

as ( 88. Mar ealies i de lier i si  i persed se ie wee -flalaie FL ad fl-reidp­ piic acid FUPA.  cras e l ealie deeced i a was FBL   case cld diidflracl FUH2 e seed ee  i is a c precrsr  FBAL ad FUPA. e relaie criis   aldse edcas pawa ad  e pese pspae s  e erea eais  iaes ised 2flrdelcse 2FDG wee sdied i a rai    e i i  F NMR spec swed sials  e isae ad   fl2delcel FDGL ad  2-fl2-de6psp­ lcae D-6-PG w ke ealies i e aldse edcase pawa ad e pese pspae s especiel Sriil a iii  e aldse edcase pawa esseiall eiiaed 2FDG ealis s dcai a is ealic e is e ai pawa  cereal caais   FDG e sesiii   9F is sciel i  allw  9F MR ai ( 1 3 1).  9 iaes  2flr-2-delcse6pspae 2FDG-P a ke eaie  e lci pawa reealed i cse iiai i ai sia cd ad ear. Liewise  F iaes  2-flr--de sil -FDL a eaie  e adse edcase pawa eealed a spaial eeeei i e disrii  e aldse redcase e acii  is ee was ies i rai ad es.

Deei MR is a ece addii  e eperie  i i NMR ecies. e aeic e  e deeri ces is saller a a  e pr  a ac  6. 5 wic redces e sesiii  e easree csideral Frerre e aral adace  deeri  lw 002%, ad      i i dies apea ealiic a s si  e e ad deeri is a spi  =  cles ad de  is adrple e as a e sr reaai ie.  ilica isses e  reaai ie is picall  e rder   00 s wic allws a er rapid repeii  e NMR easree.  ac waer ad a resaces ca e sered  i i    ee a e aal adace leel  deei e disic adaae  e H cles wee is s peial as a aei lael silar   a  9   1 6  earl ealic appicais  NMR specra  e ade  a se were aied a 47 esla wi acisii ies  les a  i (33). e esaces cd e assied  D ad  e elee­ Desaces  a e ies  ese esaces iceased akedl p addii   0% D   e driki wa e rer  waer

Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

NM STUDS OF TABOSM

6

d  cld e deeed  e dec  e H MR sl es e eplcg e deeed we w ml dkg we [ 1 - 2H]­  [6 H ]cse ve ee epled  sd e lce ewee e  glclc pw d e pll pw  e ee les ( H NM clic sdies ve ls ee eped  e diec d idiec l ce sess pws   lve 82 d  e  s melsm  -(l-  H eiie d -(mel- H  meiie (09 0) D  s ee sed s  ee dsle ce  deeme e  s ld w i  c  live () d i ipled de mse s (    . H N iges cld e ed m s   sevel weeks eceived diki we dped w D   fm s  d ied   peel ec f D  (  8) H ges ve ls ee epcd  c  (70) ese sdies ses  D   lw dses m ls ecme  sel cs ge  medcl esec  ms CKNOWLDGMNS

s wk ws spped  G 4889 8 5   8 f e wiss Nil ciece Fd ( . d  88/ 87  e pis Fd de vesicies is de  eidd cil ( 

Literature Cited

I Aerman    Ey   Bee N. N., Shlt, R. A. 98. Poc Nal A ca Sci USA 8 99- 2 2  Aema 1  1  . Grove . H .  Wong G . C. Gadan D. G . , Radda G K 1 9 Naue 28 1  . Aguayo   .  McLennan I.    Aguiar  Ch en  H. M.  98 Bochem. Bo­ phys Res Commun  2 9- . Aler  R. Beha K L, Rothman . L, Shlan R G. 98.  Magn Reson   4- . g J. R. Shulman, R. G 1 98. Q Rev Bophys    82  Aler J R   Slld L   Bear K L   Glle R. J. Shlman R. G. et al. 1 98  . Scnce 2   2 . Alonso,  As  West, W. M . , Mk      98. Magn Reson Me   :  1   8 . Arus,  arany M.  8. Bochm Bophs Aca 88   -2 9. As   Wesle, W. M   Baany M .  Maey  .  . Bochemsy 2     1 0 . Ae W. P  9. Rev Magn Reson Me   2  2 I   Ae W  Mlle S Cross T A.

2.

1

. IS

1

. 18.

9. 20



eel   98.  Magn Rson    Ason M  J .  Hthengon H  hulman R. G. 1 9. nnu Rev Bo phys Bophys Chem.   2 Ason   Rotma D.  Nadel  Shulan R. G  9. Po Nat! A ca Sc SA 8  4- A T.  Andsson B. S ons, D.  98. m  Phso 22 C C Baley, I A. Gadan D. G. Mathes  M .  Radda G. K Seeley   1 98  . FEBS Le 1 2   -  8 Baey   Wllms S  R   Rada G. K. Gadan  . G. 98  . Bochem  19 1  8 Balaan R. S. 198. m.  hso 2 C IC 9 als  R. Bell J D. Nholson J. K Sadle P  Tmrell, J . A.  a. 1 988. Magn Reson Med   Baany M .  Chan Y  Ars   . Bochemsy    1-   Baany M .  oye, . . aff  .  Weste W. M .  Marley,  L  982.  Bo Chem 2 2 -4 Barany M   Doyle, D D. Gra, G.

 22.

3

Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

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2

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9

CERDAN & SEELIG Wele, W M . , Mkle  . 1984 Magn Resn Med I: 33 Bn M Vnkubmnn, P. N . 1987 Bichim. Biphs. A ca 93 33946 Beckmn, N . , M ull, S., Sel, . 1 989 Man. Resn Md. 9 39-94 Beh, .  . , den ollnde, .  Peo,   c eenon  .  cd,  W  , Sulmn, . G  985  Nercm 44  04555 Be, K ., den ollnde, .  Somk, M . E no, T Slmn, . G et  1 983 Prc Na!. A cad. Sci. USA 80 494548 Be, K.  .  omn . ., Sulmn . G   Peoff  A C ichd  W  984 Prc Nal. Acad Sci. USA 8  : 2 5 1 7 1 9 Belon, P . S, cl, . G 1 985 Prg N! Magn Rsn. Specrs. 1 7  414 Beden  A . , Cull, P R. oul, .    Mcun  . dd G K . , cd, . . 1 974 FES e 46 5558 Bekowtz, B. A . , Wolf, S. ., Blbn, . S.  988  Magn. Rsn. 79 47



30 Bemn, S . P. Cente C. .  985 Annu. Re Bihm 4 83 16 3 \  Botomle, P.  1 989 Radilgy 1 70: 11 3   Botomle P    Chl  . c oeme, P B .  Flamig,  . , Eneseth ., et  1 988 Magn. Rsn. Md. 7  3  936  Beeon  M .  n M. G   eld ., oddell, . M.  986 Bihm Bi phys Res. mmn.  37 7984 34 Bnd K. M  , Cmbll .   987 Q  Bphys 19: 1 5982 3 Buk, S. , Sulmn . . 1 987 Annu. R. Mirbil. 4   9  6 36 But, C T  1 985 rn Bichm. Sci. 1 0 4046 37 Cbnc S   MletMno M . ., Bon, M .  Mno, . Nedelec .  mcol . F 1 988 NMR imed I:   320 38 Canioni P  Ale,  ., Sulmn, . G 1 983 Bichmisr  497480 39 Cdn, S Kunnck B . , oll,  Seel,  1 988  Bi Chem. 63 1  66474 40 Cedn, S., Pll , Snoo,    co, M. 985 BS .  87  677 4 1  Cedn, S. Submnn V.  • l­ bn M Cone  En  et l  986 Magn. Res. Med. 3 4339 4 Chiss  . A., Blcklde, M.  dd, G K .  988 Am.  Phsil 54 C41 7C4

43 Cl, . .  e  ., dd  K 1 987 Bichem  246 1 6372 44 Chnc, B . , lff S., Bnk, W., eh, . S. ., Wnell, . 198 Prc. Nal A cad. Sci. USA 79 7 7  4  8 4 Cnc, B  , l, S . , ,  S.  1 98 Pr Na. A ad. S i USA 77 743034 46 Chnce B. leff S   e . S  . Sokolow . Se A  98 1  Prc. Nal A cad. Sci. USA 78 67 1 4 18 47 Cnc B h,   S Clk, B    M,  Ken,  . , e l. 1 98 Prc Nal. A ca Sci. USA 82: 838488 48 Cnc B  , e . S . Ken  McCll M  K   Nok S .  et l. 1 986 Prc. Nal A cad. Sci. USA 83 9458 62 49 Cnc, B . , Wllm, G  1 956 J Bi. Chem.   7 4097 0 Chnc, E M  , So, S.  . , Kob K .  Wllmon,    1 983  Bi. Chm. 58 1 378594  1  Cn   .  Pee, B. M . , Wen­ sten P.  .  on M A .  Muh Boesc,  . ,  l. 1 987 Magn. Rsn. Med. 4 78 1  oen, S. M. 983  B hm 58 49414308 3 Coen, S M. 1987 B ihm isy 6 56372 54 Coen S M 1 987 Bichmisry 26: 57380 55 Cohen, S. M.  987 Bichmisr 6 58 89 56 Con, S . M, lnn, P, Sulmn, . G 1 98 1  Pr. Nal A ad. Sci. USA 78 4 7 Coen S. M . , w, S Sulmn  . . 1 979 Prc al. A cad Sci. USA 76  6037 58 Coen, S. M . , onsd, ., Sulmn, . . K  .  8    Bi. Ch 56 3483 9 o, K.  . , Sle,  983  Magn. Resn.  1 669 60 Co K.  .  olme . T. Mon­ od   Wt   1984 B i­ chemisry 23: 8997 6 1  Cross T A., M ulle, S., Aue, W. P 1 985 J Magn. Rsn. 6 898 6 Co,  A., Phl,  .  benl,  . , Aue, W . P . , Kelle, U . , See,   984 Bichm isr 3 6398640 63 Cunnnhm, C. c Mllo, C.  .  dd G   .  986 Bichim. Biphys. A ca 885:  22 64  P  Cohen  S  989 Canr Res 49 77079 65 mdn, . 1 97 1  Scinc 1  7    1 53 66 emouln, F., Cozzone, P  

 U OF MBOM

67

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Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

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76.

77.

78.

79.

80.

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86.

o P. 1 987. Eu.  Bhem 1 6 1 5 159 Dol D. D   Calo  M   aa M. 1 98 1 . FEBS Lett. 1  1  1 4750 Eak R T. Moga L.  Ggg C T M awo N . A. 1 97 FEBS  28 259-64 Evloh J L   Crowl M . G .  Ak­ m   H 1 984  an en 6 1 1 -4 Ew C S  Akrm   H   l­ ba R S  1 988 Man Rn Me 8 4 a T WM Higa R M   La  N .  Jrtk . 1 986 Bhm Bphy Ata 882 1 54-67 ihr M. J. Dillo P F 1 988 MR Bme  : 1 2 1-26 Frahm J. ruh H. Gglb M L Mrbol K D .  Hik W Saur R. 1 989. Man R M 9 79-93 Frahm  Mbolt K. D   Hk W. \ 987 . Man Ren 72 028 Frahm J   Mrbol K. D. Hk W Haa A 1 98 5   Man Ren 64 8 1 93 From . H. L. Pi M .  Mihur­ ki S. P Zimmr S. D .  Ugurbil K . 1 986 FEBS Ltt 206 257-61 Gaia D G. 1 982. I MR an t Appatn t Lvn Sytem, pp 1 1 37  xfor Claro Pr Gai D G Proor D L William S. R. Ca E. . Ga M. R. 1 986. Ma Rn M 3 1 0 6 Gallowa G. J. rro  M .  rook W  M .  il  .  Irig M    a l  1 987 Ma. Re. . 4 998 Gar J K. Kihur G  M Akr­ ma J J H   Eibrg . . ia­ lo J. J  al. 1 985 Bph J 48 803 1 3 GozalzMz R   Hah G  M .  WaJarzk  . G Jark  . 1 988 Man R Me 6:  780 Gooma M. N   Mauok L. K    Rop J S Jo A. D 1 989 B­ hem. Bphy R Cmmn 1 9: 22 27 Graham R  Mr R. . Swr­ gol . S row  R. 1 987 J B Chm. 26 3 37 Guioi L   Mariui G Ramplli G. M .  Roi  Vii V. 1 98 Ma Rn  M 5 57885 Gupa R K Gupa P Moo R D . 1 984 Ann Rv Bphy Bn. 1 3: 2216 Gulai L. oligr L. Ligh   S. alow  Cha   1 98 FEBS Lett 1 78 1 3 72

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87. Ha H . P .  Mll S   Slg  1 989. Mn n M    88. Haok C c Rohma D L Prihar J  W   Ju T. Shulma R. G. 1 988. P at. A a. S. USA 85 1 8 15 89. Ho P   1 98.  Mn Re. 54 592 90 Hr P. J. 1 98. J Man Ren 5 5  8-00 9 1  Houl D  .  u S 1 W. Gaa D G Raa G K Ra R E   Sl P  J  1 974 atu  85-87 9 Huo S M   rkik D Willi G  D L Nou K   rigg R W 1 989 Bhemty 8 435 9 l R A Griffi  . R Stv A . Gaia D. G Poru R. 1 980. Bhm. J 1 92 1  1202 94. Il R. . Jago J. R .  William S R .  Calmr R . 1 986 FEBS Lett 20 49-3 95 Il R A. Stv A. N. Gt J . R. Morri P. G. 1 985. Bhm J  229 1 4 1 -5 1 96 gwall J . S  1 982 Am  Phy/ 24 H729-H744 97 Jaobu W J 1 98   An Rv Ph  47: 707- 98 K J 1 98 5  Am J Phy/ 248 R39 1 R399 99 Kaz J Kuwajima M For D. W   MGarr J. D . 1 988 n Bhem. S 1 1  1 60 100 Kaup R. A. Hlu  K   Hai  . E .  980. FBS Ltt 1 1 2: 273 76 1 0 1 . Kim S. G .  krma   H . 1 988. ae Re. 48 4495 1 02 KiglHikma P  .  Sako E. Y   Mohaakriha P. Robiaill P  M  L. From A H  L     al 1 987. B hemty 26: 750 1  1 0 1 0 3  Kuhmrik M . J .  Dillo P F .  Mr R     row  R Kraa J. M   Sw H L. 1 986. J B/ Chm 26 1 : 1 44229 1 04. Laau . R. War J 1 988. FASB  2 236875 1 0  Laughli M. R Pi W A. Dizo J. M. Shulma R. G 1 988.  B/ Chm 263 228-91 1 06. Laurbur P  C. 1 973.  24 1 90 91 1 07. Ligi L .  bakk M . D .  Clark  J   Shall M .  oligr L.  a' 1 987. Man Re. Me 4 1 1 2-1 9 1 08  Loo R .   1 988. P . u. Ma Rn. Spet. 20: 33783 1 09. Loo R  E. Gabl S  1 988. B­ hemty 27 786469 1 1 0 Loo R E .  Gabl S   uk A  1 987 Bhmsty  7  7864-69

Annu. Rev. Biophys. Biophys. Chem. 1990.19:43-67. Downloaded from www.annualreviews.org Access provided by CSIC - Consejo Superior de Investigaciones Cientificas on 02/26/15. For personal use only.

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CD  SG

 l   oo R E Koma V.  .   twyo N  1 975  A m  Ch oc 97 3565-73 1 1 2  uy  R Mae A      ma   e ollae   A  986  Ma so 67 1 48-55 1  . Malloy C R Cnning, C. c R G K. 1 986 Bochm Boph A 885   1 1 4 Malloy C R hey A.  .  Jeffey     1 98 7  BS tt 2 1 2  862 1 1  Malloy C R hey A.  .  eey F  . H  1 988  Bo Ch. 63 6364-7 1 1 1 6 Mash     ato  .  mh I. C . 1 97. Pro. N M o. ptros. 1  : 2 1 17 1  1 7 Mahews P. M   hen   F oxlI D Masour T. E  1 985 Biohm. Bo phs. Act 845: 1 7888 1 1 8  May G. L Wi gh L  olmes K . T   Wllam  P. G. mh I. C P e al. 1 986. J BoI. Chm. 26 1  30483 1 19  Mee R. A .  own, T R. Klow   Ke   986 Am J Pho 246: 264274 1 2 Meye  A   Kusmick M .  ow T R 982  J Pho. 4

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1 2 1  Moo R . . Rhads    . 1 973. J oI Ch 48  8  22  M o G. A eema R. 98 J Ma. so. 29 433-62 1 23  ois P. G 1 988 A. p Nu M o Spro 0 1 -60   .  u . 988 M. o Md. 6 364-7 1 1 2 . M lle  Aue W P eel J 1 98 J M. so. 63: 53043 1 26. M e , Aue W. . eeg  1 85  M so 6 33-3 1 7 . Mlle . aute R Webe  .  lg   1 988. J M o 6 1  51 1 28. Me  eel   1 7  M so  466 1 29  uph y  E   Gabe . A unk A. Loo R E  1 98 8  Bohm istr 7 56-8 1 30 Nakada T Kwee  L. 1 987. M. . Md 4 36-7 1 1 3 1 . Nakaa T. Kwee I. L. C .  Mawyo N . A. Gey  .  Gy R  . 1 988 Ma. so Md 6 7-3 1 3. Neuoh K.  1 984.  M. o 5 5 1 1  1 4 1 33  Neuoh K .  a   hulma R G. 983. Pro N d. S S 80 1 6037 1 34. Neo K . Goln G Neuoh    . , Rohma  . L   ln R G. 1 984 m  55

1 3 5 . Newa C  .   L    o s  . W. MGay   . 1 983. J BoI Ch. 258 804652 1   Newa C  .  Mooe  V   oste . W .  MGay    98 . o. Chm. 259 65863 1 3 i g  R  Conn A on  A.  1 986.  M o. 66 283 94 1 38 Osbake M  1 988.  NM Th­  iqu i th Stud of ardoasur Strutr ad  to    . Osbak­ ke   aselgove pp. 0737 n Kso NY ua 1 39. Os bakke M . Lge L. lk,  .    olge L .  ubama    e  al. 1 986. M. so. M 3 8017 140 PahlWosl  eel  1 986 Bio chmstr 2 6799-6807 1 4 1  Pahl-Wstl  Slg   98  BoI Ch. HoppSr 368: 205 1 4 1 4 2 . Pa   W   am  R .  Rohma  . L. hulma R . G 1 988 Pr N.  S US 8 78339 1 43 . Park    ow R. . Pak  R   Coh   . , Chae   1 988. Pro. at. A S USA 78 67 1 - 1 8 44 Pa      ow R   Pak   R    .   .  et al.   Pro. Nt A d. S. SA 84 897 80  Peo O . A.  988 mp ohm Phsio. 908: 24960 1 46 Perff  A     Pchard  W Bar K  L Ale  . e lla­ de  A. huma R   98 5 . r oo 3 5  78 1 -88 1 47 Peo O. A   Y  R K Oo T 1 986. . Nroh 4  0-6 1 48  a  W   ln, R G. 1 86. A  N 9 6 1 - 1 49. Rad G. K. 1 986. Bohm So Tr 1 4 5  75 1 5. Raa G K 1 98. Si 233 640 4 1 5 1  Remy  Alba  P eabd A . L. eops M .  Jaot M   e a l . 1 8. Ma o. Md. 4 1 4-2  1 5  Rehaw P F. h .  .  Leh  . 1 987 M. o. Md. 4 2 2 1  6 1 53  Reo . V. Ewy C  .  ege   A Akema      1 984 J M so. 8 76-84  54 Reo N. V   efe  A Akema    1 984 J Bo Ch 9 1 36667 1 55  Rhad T L. Ky M  A. e e P R Pea   .  Adews . T e al. 1 987. M so. Md 5 335 1 6 aue A. Rud M. 1 98. Ma  Md 4 1 8

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 TDIS O TBOIS

1 57. Shn M. D Yshk, K   Chn B h 1 S r 1 988. Magn Reson. Me. 6 1 -23 1 58 Shlwiz R A o N V   Br N  N   Hll A   Ewy C S Ackrmn   H. 1 989.  Bio. Chm. 64 393034 1 59 Sh A D Mll, C . oby   ajol A J ffy ,  M    1 988 Bihem.  54 59398 1 60. hrry A D Nunnally .  shok  M 1985.  Bio Chem. 60: 9 9 1 6 1 . Shoubrd E. A. Bland J  adda  K. 1 984. Biochim. Biophys Acta 85: -8 1 6  hou rd    A   dd  K 1 984 Biochim. Biophys. Acta 85 7988 1 63 . Shulman G. !" osstt    othman D. . Bl   .  mth D. 1987  Clin. nvet 80 38-93 1 64. hulman G. I. Rhn D L. mth D ohnson  Blar  B    al. 1985.  Clin. nvest. 76: 1 936 1 65 Shulman . G 1 988. Trnds Biochem. c i 1 3 : 339 1 66 Sgfrid B A   N. V. Ew, C. S   Shlwi, R. A Akn,  J H   McDonad . M . 1 986   Bio Chem 60 1 6 1 374 1 67 S  A Brock D G .  Wlnd O. H. 1 98. In Metaolic Compartmen tation d. H  Ss . 35 57. Nw Yok Acdmc 1 68 . Sllcrud .  Hn C H .  B nky  M. W. Fnnds, A. A. 1986. . Bil hm 6 1  438088 1 69. Sllru .  Shulman  G. 1983. Biochem istry  1 08794 1 0 lv M  S osh    Hoult D  1 984.  Magn. Reson. 59 3475 1 1 7 1 . Soboll S Bunr  1 98 1 . Hoppe Seylers Z Physiol Chem. 36 1 53 1 72. Soboll S Scholt    Hldt H W 1978 r . Biochem 87 3770 1 73. Sotack C H .  rmn D M . , Hurd D  1 988  Magn. eson. 8 355 61

6

1 74 Srnr C . . 1987. Ann ev Bio­ phys. Biophys. Chem 1 6 375-99 1 75 , A N M  rr  P G ,   A Shdon P W   Grffith J    1984. Br.  Cancer 50 1 1 3- 1 7 1 6 Stol  A .  lsn      Aldrman D. W. Schwz M  1 98. Magn. Reson Me. 4 14452 1 77 Sk M  E AiMndz F  , Alr   Shulmn . G . 1986. Magn eson. Med 3: 4-3 1 8. Sbbs M   rman D. Rss B. D. 1984. Biohem. . 224 24146 1 79. Sls P 1 988 n Phsics of MR

1 80. 181. 1 8. 183.

1 84.  85

1 86. 1 87. 188

1 89.

1 9

191

pectroscopy in B iology an Meicine d B  vl  4 1 39 Amt­ dm NothHollnd hysc Ts, P, Wry S. 1985. . Physiol. 359 4 1 79 Tots P. Way  1 988. c!. Magn. son Biomd. 1  1 - 1 0 Turnr C    1 984. Prog c!. Magn eson. Specrosc 1 6 3 1 170 Ugb1 K   Grwood M . , Bndall M.  1 98.  Magn eson.  1 7785  gurb l  K   wd M . , Rh A .  1 988.   Magn Reso 8 44869 Vch  L andolhLawson J W Conll N  W Ks H  A . 1 979.  Bio. Chm 54 6538-47 Vnk  McIntoh T dn A I . 1 988. Magn. son. M 7 9599 Wn M W. 1 988. nst adiol. 3 53-61 Wl W. Albigh M  J.  M . S .  Wr H .  Rhd V   Sr,  . 1 987. Magn Reson mag 5 1 65-69 Wyz A M. onvoy .  Nchols B.  .  bak K. . sl P 198 Biochim. Biophs Acta 929 27 177 Wrwi, A M   snn M H   Sh­ fild J.  Tllman P  Goron    M  t  n P A 1 98 Sciene 3: 48-30 Yshk K  , S, Y., shkw H . 1 98 1 . Biochim. Biophys. Acta 678 83  91

CURRÍCULUM NORMALIZADO COMPLETO (CVNC)

E. López-Beltrán, M.J. Maté, S. Cerdán Dynamics and environment of mitochondrial water as detected by 1H NMR. J. Biol. Chem. (1996) 271, 10648-10653. I.F.: 5.520 (41/276 Q1 Biochemistry and Molecular Biology) ISSN: 0021-9258

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THE JOURNAL OF BIOLOGICAL CHEMISTRY © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 271, No. 18, Issue of May 3, pp. 10648 –10653, 1996 Printed in U.S.A.

Dynamics and Environment of Mitochondrial Water as Detected by 1 H NMR* (Received for publication, December 1, 1995, and in revised form, February 8, 1996)

Emilio A. Lo´pez-Beltra´n, Marı´a J. Mate´‡, and Sebastia´n Cerda´n§ From the Instituto de Investigaciones Biome´dicas, Consejo Superior de Investigaciones Cientı´ficas, Arturo Duperier 4, 28029 Madrid, Spain

Adequate understanding of the dynamics of water at the cellular level involves the determination of the water exchange rate across the different intracellular membranes and the study of the water environment within the different organelles (1– 4). Considerable evidence has been accumulated with erythrocytes on the kinetics of water exchange across the plasma membrane (2, 4 –10), as well as on the physical properties of the cytosolic compartment (11). However, the transport of water across the inner mitochondrial membrane and the environmental properties of the intramitochondrial space remain poorly understood. These two aspects are of particular metabolic relevance since a general belief indicates that viscosity and slow diffusion of metabolites in the matrix could affect the activity of a variety of intramitochondrial enzymes (12–16). However, to our knowledge no direct measurements on the physical prop-

* This work was supported in part by Grants PM 92/0011–PB 94/0011 from the Spanish DGICYT and AE-00219/94 from the Community of Madrid. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Present address: Departamento de Cristalografı´a, Centro de Investigacio´n y Desarrollo, C.S.I.C., Avda. Jordi Girona, 18 –26, 08031 Barcelona, Spain. § To whom correspondence and reprint requests should be addressed. Tel.: 34-1-585-46-33; Fax: 34-1-585-45-87.

erties of the intramitochondrial environment exist to support these hypotheses. In this report we study the dynamics of water in mitochondrial suspensions, providing values for the mean residence time1 (17) of water in the intramitochondrial space and estimates for matrix viscosity. Our methodology is based on comparisons of the 1H NMR longitudinal (T1)2 and transversal (T2) relaxation times of the water protons in the mitochondrial suspensions with those observed in the resuspension medium without mitochondria. This approach is specially suited for the noninvasive study of the water environment in biological systems since the magnetic relaxation properties of the water protons are a direct consequence of their translational and rotational motions. MATERIALS AND METHODS

Preparation and Characterization of Rat Liver Mitochondria—Mitochondria were prepared by differential centrifugation (18) from the liver of adult male Wistar rats (250 –300 g) fed ad libitum using 300 mM mannitol, 0.5 mM EDTA (pH 7.2) as isolation medium and 300 mM mannitol, 20 mM HEPES/KOH (pH 7.2) as resuspension medium. Protein concentration was measured immediately after preparation by the biuret method using bovine serum albumin as a standard. Respiratory control ratios (RCR) were routinely determined polarographically in all mitochondrial preparations prior to NMR experiments using glutamate and malate as substrates (19). Preparations with RCRs smaller than 4 were discarded. Intramitochondrial volume was measured essentially as described by Dawson et al. (20). Mitochondrial suspensions (at 75 mg of protein/ml) were incubated for 3 min at 25 °C, in a medium containing 3H2O (100,000 cpm/ml) and [14C]sucrose (100,000 cpm/ml) to determine the total water volume and the extramitochondrial volume, respectively. The difference between both volumes was taken as the matrix space. Electron microscopy was performed on three mitochondrial preparations following the protocol of Lang (21). Mitochondria showed the expected spherical morphology with internal cristae (17, 22). 1 H NMR Spectroscopy—Longitudinal (T1) and transversal (T2) relaxation times of the water protons were measured on the resuspension medium without mitochondria and on mitochondrial suspensions prepared as described above. An AM-360 Bruker spectrometer (360.13 MHz, 1H frequency) equipped with a 5-mm 1H selective probe was used. T1 was determined by the inversion-recovery sequence (␲–␶–␲/2– acquire). T2 was measured by the Carr-Purcell-Meiboom-Gill spin-echo sequence (␲/2–␶–␲–␶–acquire). Acquisition conditions were: 7 ␮s (␲/2) pulse, 10-ppm spectral width, 8K words data table, and 4 scans per spectrum. The temperature dependence of T1 and T2 was measured using a Bruker variable temperature unit (B-VT 1000, temperature stability ⫾ 0.1 °C). In these experiments, a temperature-calibrated coaxial capillary containing ethylene glycol (100%) was inserted in

1 The water exchange time or water mean residence time is the reciprocal of the first order rate constant for water leaving a given compartment, defined as the time span for the population of water molecules of the compartment to fall to 1/e. 2 The abbreviations used are: T1, spin-lattice relaxation time; T2, spin-spin relaxation time; RCR, respiratory control ratio; cP, centipoise (10⫺2 poise); ␶c, rotational correlation time.

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The dynamics and environment of water in suspensions of isolated rat liver mitochondria have been investigated by 1H NMR. NMR longitudinal and transversal relaxation times (T1 and T2) were measured in the resuspension medium (2.65 s and 44.57 ms) and in mitochondrial suspensions (1.74 s and 23.14 ms), respectively. Results showed monoexponential relaxation in both cases, suggesting a fast water exchange across the inner mitochondrial membrane. Ferromagnetically induced shift of the extramitochondrial water with nonpermeant ferromagnetic particles revealed no detectable water signal from the intramitochondrial compartment, confirming the fast exchange case. Simulations on a twocompartment model indicated that the intramitochondrial water residence time has an upper limit of approximately 100 ␮s. Calculated intramitochondrial relaxation times revealed that the intramitochondrial environment has an apparent viscosity 30 times larger than the resuspension medium and 15 times larger than the cytosol of erythrocytes. The higher apparent viscosity of the mitochondrial matrix could account for reductions of more than one order of magnitude in the diffusion coefficient of water and other substrates, limitations in the rate of enzymatic reactions which are diffusion controlled and a more favorable formation of multienzyme complexes.

Dynamics and Environment of Mitochondrial Water

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RESULTS

Fig. 1 summarizes the results of T1 (A) and T2 (B) measurements, performed on mitochondrial suspensions (closed circles) and on the resuspension medium without mitochondria (open circles). The water relaxation times observed in these suspensions, are determined by the decay of magnetically labeled water in the intramitochondrial space, in the external resuspension medium and by the exchange of magnetically labeled water between these two environments. T1 (T2) measurements of the resuspension medium (n ⫽ 6) and of the mitochondrial suspensions (n ⫽ 5) were always monoexponential with values of 2.65 ⫾ 0.2 s (44.57 ⫾ 2.2 ms) and 1.74 ⫾ 0.1 s (23.14 ⫾ 1.6 ms), respectively. These results indicate that the relaxation times of intramitochondrial water are significantly shorter than those of water in the resuspension medium. Furthermore, the results shown in Fig. 1 provide information on the rate of exchange of water between the two environments. A fast exchange rate between the intramitochondrial space and the resuspension medium would yield a single exponential relax3 A linear relationship exists between the absolute temperature T (K) and the chemical shift difference (⌬ppm) between the OH and CH2 resonances of pure ethylene glycol. In the range of 294 –325 K, our measurements gave a straight line, where the following linear equation was fitted: (⌬ppm)⫽ 4.74 ⫺ 0.0104 ⫻ (K) (r ⫽ ⫺0.9998).

FIG. 1. Longitudinal (T1) and transversal (T2) equilibrium magnetization recoveries in the resuspension medium (open symbols) and in mitochondrial suspensions (closed symbols). Mitochondrial suspensions had a protein content of 100 mg of protein/ml. The results are mean ⫾ S.E. of six measurements in the resuspension medium and five measurements on different mitochondrial preparations.

ation behavior, while a slow or intermediate exchange would result in a more complex behavior. We fitted the data of Fig. 1 to both a single exponential and a double exponential. There was no significant difference in ␹2 from both single and double exponential fits, and when double exponential was used, the values of both fitted rate constants were virtually identical. However, there was a consistent tendency for the ␹2 of the single exponential fit to be slightly lower. The fast exchange situation is confirmed in the experiment of Fig. 2, which shows the water resonance of the resuspension medium (A and C) and of mitochondrial suspensions (B and D), before (A and B) and after (C and D) the addition of 7.5 mg of iron/ml of nonpermeant, dextran-coated ferromagnetic particles. Ferromagnetic shift of extramitochondrial water revealed no detectable water signal from the intramitochondrial space (D). This result confirms that the exchange of water across the mitochondrial membrane must occur faster than the difference in frequency between the shifted and unshifted resonances (26). Since the frequency difference between these two resonances is 936 Hz, the exchange of water across the mitochondrial membrane must be faster than 1.07 ms.

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every sample for accurate temperature determination. Activation energies (Ea) were calculated using the Arrhenius equation (k1, 2 ⫽ Ae ⫺ Ea/RT, where R ⫽ 2 cal K⫺1 mol⫺1, and T is the absolute temperature) from the slope of the linear portions of the plots of log k1, 2 versus 1/T, where k1 ⫽ 1/T1 and k2 ⫽ 1/T2. Nonpermeant dextran-coated ferromagnetic iron particles were prepared by alkalinizing (pH 11.0) a solution of Cl2Fe (10 mg/ml) and Cl3Fe (15 mg/ml) in the presence of dextran (Mr 40,000, Pharmacia, Uppsala) (23). The ferromagnetic particles coated with dextran were separated from free dextran prior to use using a Sephacryl S-300 column (23). Electron microscopy of these preparations revealed spherical particles with diameters in the range of 30 –50 nm. Iron concentration of preparations was determined by the thiocyanate method, measuring the absorbance of the iron-thiocyanate complex at 460 nm (24). Viscosity Measurements—Model solutions of resuspension medium with increasing concentration of glycerol (0, 10, 30, 60, and 75%) were prepared. The dynamic viscosity of the resuspension medium was measured (22 °C) for three of these samples (i.e., partial substitution by glycerol at 0, 30, and 60%, v/v), using a Brookfield viscometer (model RVT, Brookfield Engineering Laboratories, Stoughton, MA). T1 and T2 measurements were performed on all the model solutions and compared with T1 and T2 measurements performed in mitochondrial suspensions. Data Analysis and Model Simulations—T1 and T2 values were obtained from non linear three parameter fits of the longitudinal and transversal equilibrium magnetization recoveries to the equations M(t) ⫽ M(⬁) (1 ⫺ K0 exp (⫺␶/T1)) and M(t) ⫽ K1 ⫹ M(0)exp (⫺␶/T2), respectively. Curve fitting was performed using a nonlinear least squares regression program (IGORTM, WaveMetrics, Lake Oswego, OR). Double exponential magnetization recoveries for T1 and T2 measurements were also fitted to evaluate the data for the existence of double exponential as opposed to single exponential behavior. The following equations were used: M(t) ⫽ Ma(⬁)(1 ⫺ K0exp(⫺␶/T1a)) ⫹ Mb(⬁)(1 ⫺ K1exp(⫺␶/T1b)), and M(t)⫽ K0 ⫹ K1 ⫹ Ma(0)exp(⫺␶/T2a) ⫹ Mb(0)exp(⫺␶/T2b), where the a and b subscripts represent two magnetically different water environments. Chi square values (␹2) obtained from the monoexponential and biexponential fits, were analyzed using the Student’s t test. A kinetic model was developed to estimate the water residence time in the intramitochondrial environment from measurements of the relaxation times of the mitochondrial suspension and the resuspension medium. For this purpose, we used a package for modeling system dynamics in biology, based on the Euler algorithm for the numerical integration of simultaneous differential equations (STELLATM, High Performance Systems, Lyme, NH). T2 relaxation curves were simulated for different values of the water residence time in the intra- and extramitochondrial space and compared to the experimentally observed T2 values of mitochondrial suspensions, using ␹2 criterion for the goodness of fit. These comparisons were further completed with a test for randomness or trends of residuals in every fit (25).

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Water exchange across the mitochondrial membrane was further characterized with a study of the variation of T1 and T2 in mitochondrial suspensions with protein concentration and temperature. The dependence of the relaxation times on the mitochondrial protein content is shown in Fig. 3. In these experiments, the final incubation volume was maintained constant at 0.5 ml and the amount of liver mitochondria increased up to 110 mg/ml. An approximately linear decrease in T1 (Fig. 3A) and T2 (Fig. 3B) was observed for increasing protein concentrations. The decrease is due to the shorter relaxation times of intramitochondrial water and to the progressive increase in the relative contribution of intramitochondrial space to the overall relaxation observed. Measurements of the intramitochondrial water in the mitochondrial suspensions gave values of 1.3 ⫾ 0.1 ␮l/mg of protein (n ⫽ 7). Thus, the titration shown in Fig. 3 covers an intramitochondrial volume range of up to 143 ␮l/ml. Total water content was determined in the same samples used for T1 and T2 measurements, measuring the dry/wet weight ratio. A total H2O content of 8.4 ⫾ 1 ␮l of H2O/mg of protein or 840 ␮l of H2O/ml of sample was determined for mitochondrial suspensions of 100 mg of protein/ml. For this protein content, the intramitochondrial volume represents approximately 12–16% of the total water volume, the

FIG. 3. Effects of the increase in mitochondrial protein concentration on the relaxation times T1 (A) and T2 (B) of water. T1 and T2 values were determined in mitochondrial suspensions containing increasing protein concentrations in a final incubation volume of 0.5 ml. For each mitochondrial sample, several measurements of T1 and T2 were taken (n ⫽ 3 to 6). The S.E. is very small and cannot be appreciated from the plot.

proportion of mitochondrial matrix space normally found in liver cells (27). Fig. 4 depicts measurements of the temperature dependence for T1 (A) and T2 (B) of the resuspension medium alone and of a typical mitochondrial suspension. In the resuspension medium, T1 increased linearly, whereas T2 increased in a nonlinear fashion. This result suggest that T2 relaxation in the resuspension medium is dependent on more than one process. The activation energies (Ea) calculated from the temperature dependence of T1 and T2 in the resuspension medium, for the 22–50 °C interval, were ⫺4.8 kcal/mol and ⫺6.3 kcal/mol, respectively. These activation energies are within the range previously reported for the self-diffusion of water (28, 29). In the mitochondrial suspension, T1 showed a biphasic behavior. It increased up to 32 °C and decreased at higher temperatures. Additional experiments with different mitochondrial suspensions (n ⫽ 3) confirmed this behavior, but the transition temperature varied slightly between the samples (32–35 °C). The Ea values calculated below the transition temperature (from ⫺3.8 to ⫺4.8 kcal/mol) for these mitochondrial suspensions are also consistent with the values for the self diffusion of water, being similar to the Ea of the resuspension medium. In con-

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FIG. 2. Chemical shifts of the water resonance before and after the addition of ferromagnetic particles to the resuspension medium and to mitochondrial suspensions. A, resuspension medium; B, mitochondrial suspension (90 mg of protein/ml); C, same as in A with ferromagnetic particles (7.6 mg of iron/ml); D, same as in B with ferromagnetic particles (7.6 mg of iron/ml). Note the absence of intramitochondrial residual water signal after the shift of the extramitochondrial water; E, stock solution of ferromagnetic particles (15.3 mg of iron/ml).

Dynamics and Environment of Mitochondrial Water

trast, Arrhenius analysis of the linear portion above the transition temperature gave much higher Ea values in the range of 3.8 – 6.4 kcal/mol (35–57 °C). The biphasic trend suggests the presence of two different relaxation mechanisms having dominant effects below or above the transition temperature. The values of intramitochondrial relaxation times are thought to be dependent mainly on the mitochondrial content of paramagnetic ions and on the viscosity of the matrix microenvironment. We investigated the potential contribution of free paramagnetic ions to the relaxation times observed by measuring the T1 and T2 relaxation times in mitochondrial suspensions prepared in the absence or presence of 0.5 mM EDTA in the isolation and resuspension medium. Values of 1.46 s (29.91 ms) or 1.63 s (33.34 ms) were obtained for T1 (T2) in the absence or presence of 0.5 mM EDTA, respectively. Increasing the concentration of EDTA to 1 mM did not further increase the values of T1 or T2. These results indicate that free paramagnetic ions contribute at most 10% of the observed relaxation in suspensions of rat liver mitochondria. In addition, EPR measurements on typical mitochondrial suspensions (n ⫽ 2) showed no free Mn2⫹ signal detectable at maximum EPR sensitivity (results not shown). Our results confirm previous findings indicating very small contributions of paramagnetic ions to mitochondrial relaxation times (30, 31). Thus, intramitochondrial viscosity may be considered as the main determinant of intramitochondrial relaxation under our experimental conditions. To inves-

FIG. 5. T1 (A) and T2 (B) dependence of viscosity. Measurements were performed on model solutions consisting of the resuspension medium with partial substitutions of glycerol (0, 10, 30, 60, 75%, v/v). Interpolation of calculated intramitochondrial T1 and T2 values (arrows) indicate that the mitochondrial matrix has an apparent viscosity similar to that of a 55% glycerol solution.

tigate the influence of viscosity on intramitochondrial relaxation times, measurements of T1 and T2 were performed on model solutions of the resuspension medium containing increasing concentrations of added glycerol (Fig. 5). These measurements were compared with intramitochondrial T1 and T2 values calculated for a fast water exchange situation (see “Discussion”). Calculated intramitochondrial T1 (T2) values were 0.6 ⫾ 0.06 s (5.8 ⫾ 0.6 ms) (n ⫽ 6). Interpolation of these values (arrows) on the calibration curve obtained with model solutions, indicated that the matrix space behaves similarly to a solution of the resuspension medium containing approximately 52–59% glycerol. We performed measurements of the dynamic viscosity of the resuspension medium without glycerol, and with two concentrations of added glycerol, namely 30 and 60% (v/v). The viscosities were (mean ⫾ S.E., n ⫽ 4) 1.3 ⫾ 0.1, 20.0 ⫾ 1.4, and 44.5 ⫾ 2.5 cP, respectively. Thus, intramitochondrial viscosity would be similar to that of a 55% glycerol solution, approximately 40 cP. DISCUSSION

Our results indicate that the water exchange time between the intramitochondrial matrix and the external resuspension medium is faster than the NMR relaxation time scales. To provide a more quantitative estimate of the water residence time in the intramitochondrial matrix, we implemented a model consisting of two compartments, A and B, representing the extra- and intramitochondrial environments respectively, separated by a semipermeable membrane (Fig. 6). The model

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FIG. 4. Temperature dependence of T1 (A) and T2 (B) values of the resuspension medium (open symbols) and of a representative mitochondrial suspension (closed symbols). The mitochondrial suspension had a protein content of 100 mg/ml. The data points of the resuspension medium are expressed as mean ⫾ S.E. with at least six measurements for every temperature. One representative mitochondrial sample is shown.

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Dynamics and Environment of Mitochondrial Water

considered the T2 relaxation of magnetically labeled protons of free water in the extramitochondrial (Ma) and intramitochondrial (Mb) compartments, the experimentally observed relaxation of all water molecules in the sample (MT) and the exchange of water across the inner mitochondrial membrane. This exchange is expressed in terms of the water residence times in the external medium and in the mitochondrial matrix, ␶a and ␶b, respectively. The decay of the NMR signal observed experimentally (MT), contains the addition of the decays of the macroscopic magnetization from both compartments, Ma and Mb, dMT/dt ⫽ dMa/dt ⫹ dMb/dt,

(Eq. 1)

where dMa/dt and dMb/dt are given by Ref. 32. dMa/dt ⫽ ⫺ Ma/T2a ⫺ Ma/␶a ⫹ Mb/␶b

(Eq. 2)

dMb/dt ⫽ ⫺ Mb/T2b ⫺ Mb/␶b ⫹ Ma/␶a

(Eq. 3)

T2a and T2b refer to the transversal relaxation times of water in the external medium and in the mitochondrial matrix, respectively. The relaxation times of the water in the external medium (T2a) and in mitochondrial suspensions (T2) can be determined experimentally (Fig. 1). The relaxation time of water (T2b) in the mitochondrial matrix can be calculated, because of the fast water exchange, using the expression: 1/T2 ⫽ Pa/T2a ⫹ Pb/T2b

(Eq. 4)

where Pa and Pb represent the fractional contributions to the total volume of compartments A and B, determined by the 3 H2O and [14C]sucrose distributions, respectively. Using Equation 4 we obtained values of 0.6 ⫾ 0.06 s (5.8 ⫾ 0.6 ms) for intramitochondrial T1 (T2). With these values it became possible to simulate the T2 relaxation behavior of the water resonance in mitochondrial suspensions (dMT/dt) as a function of ␶a and ␶b (Fig. 7). Values of ␶b were varied iteratively to mimic the

⫺1 T2b ⫽



⫺1 pi T2i

(Eq. 5)

i

Accordingly, the effective intramitochondrial correlation time for water (␶c) calculated above from T2b, contains the weighted average of the contributions from the different correlation times experienced by the water molecule during its intramitochondrial relaxation. As indicated in the results section, viscosity is thought to be the main determinant of reduced water mobility in the matrix. Intramitochondrial relaxation times were found to be similar to those of glycerol suspensions of 40 cP. This apparent matrix viscosity is approximately 15 times larger than the apparent viscosity of the cytoplasm in human erythrocytes (2.10 cP) (11). Finally, an important aspect of the present study relates to the influence that elevated matrix viscosity can have on the

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FIG. 6. Two compartment model for the analysis of transversal relaxation data from intra- and extramitochondrial compartments in the presence of water exchange. Compartment A refers to the extramitochondrial medium and compartment B to the intramitochondrial space. The magnetically labeled water protons in these compartments are Ma and Mb, with relative populations Pa and Pb. The mean residence time of water in both compartments, ␶a and ␶b, and the relaxation times in each compartment, T2a and T2b, determine the observed relaxation behavior (MT) of the sample. Simulations of the experimental behavior of MT can be obtained by adjusting the values of ␶a and ␶b (cf. Fig. 7).

experimental magnetization recoveries. The corresponding values of ␶a were calculated for every simulation to satisfy the equilibrium condition Pa/␶a ⫽ Pb/␶b. Fig. 7A shows model simulations of the observed relaxation behavior for the limiting cases of very slow or absent water exchange (␶b, ␶a 3 ⬁) and of fast water exchange (␶b ⬍⬍ T2b, ␶a ⬍⬍ T2a). In addition, the figure depicts, superimposed to the simulations, the experimental points for T1 and T2 relaxation of mitochondrial suspensions and their corresponding single exponential fits. The figure shows that the fast exchange simulation resembles closely the experimental data, while the no water exchange situation is clearly different from the observed results. We performed additional simulations in the fast exchange regime, for the interval 0.5 ms ⬎ ␶b ⬎ 0.02 ms. In these cases, the plot of residuals of every fit (Fig. 7B) revealed more clearly the goodness of the fit than the direct superposition of simulated data over experimental values. Fig. 7B shows that values of ␶b smaller than 0.1 ms give very similar residual trends to those of the experimental values, indicating that ␶b must be smaller than 0.1 ms. To our knowledge, this estimate represents the first approximation to the water residence time in the mitochondrial matrix. Insight about the mobility of the water molecule in the intramitochondrial space may be obtained from the relationship of the relaxation times with the rotational correlation time (␶c) (26). T2 values of the extra (intra) mitochondrial medium were 44.57 ⫾ 2.15 ms (5.79 ⫾ 0.59 ms), which led to calculated values of ␶c of 4.5 ⫻ 10⫺10 s (6.2 ⫻ 10⫺9 s), respectively. Thus, water rotational mobility in the matrix is significantly restricted as compared to the extramitochondrial medium. Moreover, water protons in distilled water at 20 °C have a ␶c of about 3 ⫻ 10⫺12 s (33, 35), approximately three orders of magnitude shorter than the matrix correlation times. The dynamic interpretation of intramitochondrial relaxation times deserves further attention. Several reports have shown that water in tissues, cells (34 –37), or even in intact mitochondria (38), is heterogeneously distributed in different phases. Phase heterogeneity is thought to be the result of the different physical properties of water molecules in the “bulk solvent” and those “bound or adsorbed” to macromolecules or cellular surfaces (39). While bulk solvent water is able to rotate freely, water bound or adsorbed on macromolecular surfaces is though to adopt the correlation time of the host macromolecule (33). The exchange of water molecules between these different phases is thought to be fast in the NMR time scales (33, 40). Thus, during the T2b relaxation period, water molecules have a defined probability (0 ⬍ pi ⬍1) to relax in a variety of intramitochondrial microenvironments, including bulk rotational freedom and an array of restricted macromolecular rotations (T2i). Thus, T2b can be expressed as:

Dynamics and Environment of Mitochondrial Water

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kinetics of intramitochondrial reactions. The diffusion coefficients (D) of water and substrates are inversely related to the viscosity (␩) by the Einstein-Stokes relationship D ⫽ kT/6␲␩r0 where k is the Boltzman constant, T the absolute temperature, and r0 the Stokes radius of the molecule under study (11). Thus, a 15 times increase in the average viscosity of the intramitochondrial environment as compared to the cytoplasm can account for an identical reduction in the diffusion coefficient of water and even for a larger reduction in the diffusion coefficient for larger substrates. These reductions can introduce kinetic limitations in those mitochondrial reactions which are diffusion controlled, mainly hydration-dehydration and proton transfer reactions (41). Notably, recent evidence indicates that cytosolic and mitochondrial aminotransferases of alanine and aspartate experience different solvent exchange environments in the perfused liver (42). Further effects of intramitochondrial viscosity would be to favor the formation of enzyme aggregates or multienzyme complexes. These complexes have been also proposed to occur in the mitochondrial matrix (13–15). However, to our knowledge no direct evidence on the physical properties of the intramitochondrial environment was previously available. Acknowledgments—We thank Dr. Julio San Roma´n and Dr. Jose´ Manuel Peren˜a for their collaboration and facilities in the viscosity measurements, Dr. Paloma Calle for measurements of free Mn2⫹ by EPR, and Dr. Juana M. Gancedo and Dr. Juan J. Arago´n for helpful discussion and critical reading of the manuscript. REFERENCES 1. Haines, T. H. (1994) FEBS Lett. 346, 115–122 2. Latour, L. L., Svoboda, K. Mitra, P. P., and Sotak, C. H. (1994) Proc. Natl Acad. Sci. U. S. A.. 91, 1229 –1233 3. Poxleitner, M., Seitz-Beywl, J. and Heinzinger, H. (1993) Z. Naturforsch. 48c, 654 – 665 4. Solomon, A. K. (1989) Methods Enzymol. 173, 192–222 5. Benga, G., Pop, V. I., Popescu, O. and Borza, V. (1990) J. Biochem. Bioph. Methods 21, 87–102 6. Benga, G. (1989) Int. Rev. Cytol. 114, 273–316 7. Chien, D. Y., and Macey, R. I. (1977) Biochim. Biophys. Acta 464, 45–52 8. Andrasko, J. (1976) Biochim. Biophys. Acta 428, 304 –311 9. Conlon, T., and Outhred, R. (1972) Biochim. Biophys. Acta 288, 354 –361 10. Shporer, M., and Civan, M. M. (1975) Biochim. Biophys. Acta 385, 81– 87 11. Price, W. S., Kuchel, P. W., and Cornell, B. A. (1989) Biophys. Chem. 33, 205–215

12. Ova´di, J., and Srere, P. A. (1992) Trends Biochem. Sci. 17, 445– 447 13. Srere, P. A. (1987) Annu. Rev. Biochem. 56, 89 –124 14. Srere, P. A., Sumegi, B., and Sherry, A. D. (1987) in Krebs’ Citric Acid Cycle. Half a Century and Still Turning (Kay, J., and Weitzman, P. D. J., eds) Vol. 54, pp. 173–182, Cambridge University Press, New York 15. Welch, G. R., and Easterby, J. S. (1994) Trends Biochem. Sci. 19, 193–197 16. Welch, G. R. (1986) Symp. Biol. Hung. 30, 217–226 17. Herbst, M. D., and Goldstein, J. H. (1989) Am. J. Physiol. 256, C1097– C1104 18. Rickwood, D., Wilson, M. T., and Darley-Usmar, V. M. (1987) in Mitochondria: A Practical Approach (Darley-Usmar, V. M., Rickwood, D., and Wilson, M. T., eds) pp. 1–16, IRL Press, Washington, D. C. 19. Nieto, R., Cruz, F., Tejedor, J. M., Barroso, G., and Cerda´n, S. (1992) Biochimie (Paris) 74, 903–911 20. Dawson, A., Klingenburg, M, and Kra¨mer, R. (1987) in Mitochondria: A Practical Approach (Darley-Usmar, V. M., Rickwood, D., and Wilson, M. T., eds) pp. 35–78, IRL Press, Washington D. C. 21. Lang, R. D. A. (1987) in Mitochondria: A Practical Approach (Darley-Usmar, V. M., Rickwood, D., and Wilson, M. T., eds) pp. 17–34, IRL Press, Washington, D. C. 22. Scarpa, A. (1979) in Membrane Transport in Biology II: Transport Across Single Biological Membranes (Giebisch, G., Tosteson, D. C., and Ussing, H. H., eds) pp. 263–355, Springer-Verlag, Berlin 23. Molday, R. S., and Mackenzie, D. (1982) J. Immunol. Methods 52, 353–367 24. Renshaw, P. F., Owen, C. S., Evans, A. E., and Leigh, J. S., Jr. (1986) Magn. Reson. Imaging 4, 351–357 25. Straume, M., and Johnson, M. (1992) Methods Enzymol. 210, 87–105 26. Gadian, D. G. (1982) Nuclear Magnetic Resonance and Its Application to Living Systems, pp. 125–129, Oxford University Press, Oxford, UK 27. LaNoue, K. F., Strzelecki, T., and Finch, F. (1984) J. Biol. Chem. 259, 4116 – 4121 28. Wang, J. H. (1951) J. Am. Chem. Soc. 73, 510 –513 29. Benga, G., Popescu, O., Pop, V. I., Hodor, P., and Borza, T. (1992) Eur. J. Cell Biol. 59, 219 –223 30. Hutson, S. M., Williams, G. D., Berkich, D. A., LaNoue, K. F., and Briggs, R. W. (1992) Biochemistry 31, 1322–1330 31. Gunter, T. E., and Puskin, J. S. (1972) Biophys. J. 12, 625– 635 32. McConnell, H. M. (1958) J. Chem. Phys. 28, 430 – 431 33. Koenig, S. H., and Schillinger, W. (1969) J. Biol. Chem. 244, 3283–3289 34. Foster, M. A. (1984) Magnetic Resonance in Medicine and Biology, pp. 137–147, Pergamon Press Ltd., Oxford, UK 35. Cooke, R., and Kuntz, I. D. (1974) Annu. Rev. Biophys. Bioeng. 3, 95–126 36. Cleveland, G. G., Chang, D. C, Hazlewood, C. F., and Rorschach, H. E. (1976) Biophys. J. 16, 1043–1053 37. Neville, M. C., Paterson, C. A., Rae, J. L., and Woessner, D. E. (1974) Science 184, 1072–1074 38. Garlid, K. D. (1979) in Cell Associated Water (Drost-Hansen, W., and Cleg, J., eds), pp. 293–361, Academic Press, New York 39. Israelachvili, J., and Wennerstro¨m, H. (1996) Nature 379, 219 –225 40. Koenig, S. H., Hallenga, K., and Shporer, M. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 2667–2671 41. Fehrst, A. (1977) Enzyme Structure and Mechanism, pp. 126 –132, W. H. Freeman & Co., New York 42. Moldes, M., Cerda´n, S., Erhard, P., and Seelig, J. (1994) NMR Biomed. 7, 249 –262

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FIG. 7. Representative model simulations of the kinetics of water exchange across the inner mitochondrial membrane. Simulations of experimental T2 (A) relaxation curves for different mitochondrial preparations (100 mg/ml, n ⫽ 4) were obtained using the model of Fig. 6. The continuous line with open symbols (䡺) is a simulation for the case of no water exchange (␶a, ␶b 3 ⬁). The dashed line is a simulation for the case of fast water exchange with ␶b ⬍ 100 ␮s. Closed symbols (f) indicate experimental measurements. B, residuals plot obtained for the experimental results fitted to a single exponential (fOOf) and to simulations performed in the interval 0.5 ms ⬎ ␶b ⬎ 0.02 ms. Note that the residuals obtained from the simulations mimic better those of the experimental data for the interval 100 ⬎ ␶b ⬎ 20 ␮s.

Cell Biology and Metabolism: Dynamics and Environment of Mitochondrial Water as Detected by H NMR

J. Biol. Chem. 1996, 271:10648-10653. doi: 10.1074/jbc.271.18.10648

Access the most updated version of this article at http://www.jbc.org/content/271/18/10648 Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites. Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 34 references, 5 of which can be accessed free at http://www.jbc.org/content/271/18/10648.full.html#ref-list-1

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Emilio A. López-Beltrán, María J. Maté and Sebastián Cerdán

CURRÍCULUM NORMALIZADO COMPLETO (CVNC)

J.M. Pascual, F. Carceller, J.M. Roda, S. Cerdán Glutamate, Glutamine and GABA as substrates for the neuronal and glial compartments after focal cerebral ischemia in the rat. Stroke (1998) 29, 1048-1057. I.F.: 6.449 (6/156 Q1 Clinical Neurology, 7/56 Q1 Perypheral Vascular Disease) ISSN: 0039-2499

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Glutamate, Glutamine, and GABA as Substrates for the Neuronal and Glial Compartments After Focal Cerebral Ischemia in Rats Jose´ M. Pascual, MD; Fernando Carceller, MD, PhD; Jose´ M. Roda, MD, PhD; Sebastia´n Cerda´n, PhD Background and Purpose—Even though the utilization of substrates alternative to glucose may play an important role in the survival of brain cells under ischemic conditions, evidence on changes in substrate selection by the adult brain in vivo during ischemic episodes remains very limited. This study investigates the utilization of glutamate, glutamine, and GABA as fuel by the neuronal and glial tricarboxylic acid cycles of both cerebral hemispheres after partially reversible focal cerebral ischemia (FCI). Methods—Right hemisphere infarct was induced in adult Long-Evans rats by permanent occlusion of the right middle cerebral artery and transitory occlusion of both common carotid arteries. (1,2-13C2) acetate was infused for 60 minutes in the right carotid artery immediately after carotid recirculation had been re-established (1-hour group) or 23 hours later (24-hour group). Extracts from both cerebral hemispheres were prepared and analyzed separately by 13C nuclear magnetic resonance and computer-assisted metabolic modeling. Results—FCI decreased the oxidative metabolism of glucose in the brain in a time-dependent manner. Reduced glucose oxidation was compensated for by increased oxidations of (13C) glutamate and (13C) GABA in the astrocytes of the ipsilateral hemispheres of both groups. Increased oxidative metabolism of (13C) glutamine in the neurons was favored by increased activity of the neuronal pyruvate recycling system in the 24 – hour group. Conclusions—Data were obtained consistent with time-dependent changes in the utilization of glutamate and GABA or glutamine as metabolic substrates for the glial or neuronal compartments of rat brain after FCI. (Stroke. 1998;29:1048-1057.) Key Words: cerebral ischemia, focal 䡲 cerebral metabolism 䡲 middle cerebral artery occlusion 䡲 neuronal damage 䡲 stroke, experimental

S

ome of the metabolic mechanisms underlying the development of ischemic brain damage have been investigated using highly enriched cultures of neural cells1– 4 or cerebral cortex slices.5,6 However, the metabolic events occurring in vivo in the neuronal and glial compartments of the adult brain during the development of ischemic damage remain less understood. The mammalian brain contains essentially two different metabolic compartments: the glial and the neuronal. Classic radiolabeling experiments and more recent 13C NMR evidence have shown that these two compartments differ in their utilization of particular substrates, their metabolic products, and the presence of characteristic enzymes.7–10 The glial compartment uses glucose or acetate as its main substrates, is characterized by the presence of glutamine synthase, and produces glutamine, which is transferred to the neuronal compartment and metabolized. The neuronal compartment uses glucose and glial glutamine as its main substrates, is characterized by the presence of high glutaminase and GABA

See Editorial Comment, page 1056 decarboxylase activities, and releases glutamate and GABA, which are transferred to the glial compartment and metabolized.11 In addition, glial lactate has been proposed recently as an important substrate for the neuronal compartment, at least in neural cell cultures of fetal or neonatal origin during neuronal activation.12 The metabolisms of neurons and glial cells interact closely, competing for glucose as a primary substrate, and using the glutamate, glutamine, and GABA produced in the other cell type as an alternative substrate for oxidation in the tricarboxylic acid cycle. Thus, several substrates are available to neurons and glial cells in situ, allowing to compensate deficits in one substrate with increased utilization of others. The aim of this study was to investigate the use of glutamate, glutamine, and GABA as alternative substrates to glucose by the neuronal and glial compartments in both rat brain hemispheres after partially reversible FCI. To this end we used a combination of previously validated 13C NMR and

Received October 12, 1997; final revision received February 5, 1998; accepted February 19, 1998. From the Servicio de Neurocirugı´a, Hospital La Paz (J.M.P., F.C., J.M.R.), and Instituto de Investigaciones Biome´dicas del C.S.I.C. (S.C.), Madrid, Spain. Correspondence to Dr Sebastian Cerda´n, Instituto de Investigaciones Biome´dicas del C.S.I.C., c/Arturo Duperier 4, E-28029 Madrid, Spain. E-mail [email protected] © 1998 American Heart Association, Inc.

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Pascual et al

Ac AcCoA Cr PCr FCI GABA Gln Glc Glu HPLC Ino Lac MABP NAA NMR OAA Succ TSP

Selected Abbreviations and Acronyms ⫽ acetate ⫽ acetyl-CoA ⫽ creatine ⫽ phosphocreatine ⫽ partially reversible focal cerebral ischemia ⫽ ␥-aminobutyryc acid ⫽ glutamine ⫽ glucose ⫽ glutamate ⫽ high-performance liquid chromatography ⫽ myo-inositol ⫽ lactate ⫽ mean arterial blood pressure ⫽ N-acetyl-aspartic acid ⫽ nuclear magnetic resonance ⫽ oxalacetate ⫽ succinate ⫽ 2,2⬘-3,3⬘ tetradeutero trimethyl-silyl-propionate

mathematical modelling techniques.13–15 The approach used (1,2-13C2) acetate as a substrate and computer-assisted analysis of extracts prepared from the ipsilateral and contralateral hemispheres, 1 hour or 24 hours after the ischemic insult. It has been shown that after FCI, glutamate and GABA behave, in addition to their role as excitatory or inhibitory transmitters, as true alternative substrates to glucose in the glial compartment of the in situ brain, while glutamine assumes a similar role in the neuronal compartment. These alterations in substrate selection in both compartments are time dependent and affect both the ipsilateral and contralateral hemispheres.

Materials and Methods Animals, Surgical Procedures, and Experimental Design The experimental protocols used in this study were approved by appropriate institutional review committees, and meet the guidelines of the responsible governmental agency. Long-Evans female rats (250⫾25 g, n⫽10), receiving rat chow and water ad libitum, were subjected to focal cerebral ischemia after an overnight fasting period. Anesthesia was induced with 2.5 mL/kg body weight of a mixture of ketamine hydrochloride (25 mg/mL), diazepam (2 mg/mL), and atropine (0.1 mg/mL) injected intraperitoneally, and prolonged when necessary with one third of the initial dose. Animals were allowed to breathe unassisted. The surgical procedure was essentially that described by Chen et al in 198616 and Liu et al in 1989,17 with the modifications previously reported.18,19 Briefly, under a surgical microscope, a small craniectomy was made over the main trunk of the right middle cerebral artery and above the rhinal fissure. The right middle cerebral artery was permanently ligated just before its bifurcation between the frontal and parietal branches. During this occlusion period, the right external carotid artery was cannulated retrogradely, and the catheter was placed near the origin of the internal carotid artery.20,21 Both common carotid arteries were then exposed and clamped for 90 minutes. Restoration of carotid blood flow was directly observed in all cases, after removal of the clamps. Then, the animals were infused with (1,2-13C2) acetate solution (32 ␮mol 䡠 min–1 䡠 100 g body wt–1, pH 7.2) for 60 minutes, either immediately after carotid blood flow was reestablished (1-hour group, n⫽5) or 23 hours later (24-hour group, n⫽5). The right femoral artery was cannulated during surgery and subsequent infusion, allowing continuous monitoring of arterial pressure. At the end of the infusion, 0.3-mL blood samples were drained from both jugular veins and from the femoral artery to determine arterial and

May 1998

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venous blood gas values, pH, and glucose. Body temperature was maintained at 37⫾0.5°C during the experiment with a servo-controlled rectal probe heating pad. An additional thermistor probe was placed in the region of the middle cerebral artery under the temporal muscle and over the cerebral cortex, while brain temperature was maintained at 36⫾0.5°C with a tungsten lamp located over the head. At the end of the infusion, the head of the animals was funnel-frozen with liquid nitrogen. The ipsilateral and contralateral hemispheres were dissected separately while still at low temperature, powdered under liquid nitrogen, and extracted separately with methanol/HCl/perchloric acid.22 Extracts were lyophilized to dryness and resuspended in D2O (99.9%D) before high 13 resolution C and 1H NMR.

High Resolution 13C and 1H NMR Spectroscopy of Brain Extracts High resolution 13C NMR spectra (90.56 MHz, 22°C, pH 7.4) of neutralized perchloric acid extracts of individual hemispheres, were obtained at 8.4 T with a Bruker AM-360 NMR spectrometer (Bruker Analytik GMBH). Acquisition conditions were as follows: 60o pulses, 18.5-KHz sweep width, 64K word computer memory (1.769second acquisition time), total recycle time of 5.7-seconds, and approximately 22 000 scans. Broad band de-coupling (1 watt average forward power) was applied only during the acquisition. Chemical shifts were calibrated with the resonance of a 10% (vol/vol) dioxane solution (67.4 ppm) placed in a coaxial capillary. High-resolution 1H NMR spectra (360.1 MHz, 22°C) of the extracts used for 13C NMR spectroscopy were acquired using the following: 90o pulses, 3968 Hz spectral width, 16K computer memory (1.3-second acquisition time), 128 transients for each spectrum, and 20-second relaxation delay. The residual water signal was attenuated with a 3-second presaturating pulse applied with the decoupler on the water resonance. Chemical shifts were referenced to internal TSP. 1H and 13C NMR assignments were made by comparison with published values.13,23

Computer-Assisted Simulation of 13C NMR Spectra From (1,2-13C2) Acetate Metabolism in Both Hemispheres of Rat Brain After FCI Computer-assisted interpretation of the 13C NMR spectra from extracts of each cerebral hemisphere, in both time groups, was performed using the METASIM program version 2.1.14 METASIM simulates the 13C NMR spectrum produced by the metabolism of a 13 given C-labeled substrate in a user-defined metabolic network consisting of metabolite pool sizes and interconnecting fluxes (Figure 1). The network consists of two main cerebral compartments, namely neuronal and glial, with extracellular spaces for plasma and interstitial fluid. The neuronal compartment groups both glutamatergic and GABAergic neurons, whereas the glial compartment contains both astroglia and oligodendroglia. Under steady state conditions the METASIM program calculates the probability of formation (or disappearance) of individual 13C isotopomers of the metabolites located in the network nodes, using an algorithm based on the recursive definition of input-output equations.24 Calculated 13C isotopomer populations are used to compute and display the resulting 13C NMR spectrum as the weighted sum of the individual 13C isotopomer contributions, using predefined chemical shifts and 13C-13C coupling constants for each carbon. The METASIM curve fits an experimental spectrum by automatically optimizing the values of pool sizes and relative metabolic fluxes converging on the nodes of the specified network using an iterative, nonlinear least-squares procedure. Briefly, the following procedure was followed for flux and pool size optimization. Simulations of the 13 C multiplet structures of all the observable carbons of glutamate, glutamine, and GABA in both cerebral hemispheres were performed with METASIM, starting with the values for pool sizes and relative fluxes previously validated in the normoxic brain.14,15 Progressive modifications of these starting values generated 13C NMR spectra that were virtually identical to the experimental ones (c.f. Figure 5). Finally, the set of refined parameter values was automatically optimized by curve fitting the 13C multiplets of the experimental

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Figure 1. Metabolic network used to simulate cerebral metabolism of (1,2-13C2) acetate in both hemispheres of rat brain after FCI. The network consists of fluxes (circled numbers) and metabolite nodes (abbreviated names). Two metabolic compartments associated with the glial (left) or neuronal (right) environments exist in the brain. Infused (1,2-13C2) acetate (flux 1) is activated to (1,2-13C2) acetyl-CoA only in the glial compartment, where it is diluted with unlabeled acetyl-CoA from unlabeled glucose (flux 2), originating the glial acetylCoA pool (Node AcCoAg). AcCoAg enters the glial cycle (flux 3) labeling the small glutamate pool (Node Glug) and the glutamine pool (Glng). The glial compartment also metabolizes glutamate (flux 6) and GABA (flux 7) produced in the neurons (Nodes Glug and SUCCg, respectively). The neuronal tricarboxylic acid cycle uses unlabeled glucose (flux 8) and some glial glutamine (flux 5) as substrates (Nodes AcCoAn and Glun, respectively). AcCoAn enters the neuronal cycle (flux 9) labeling the large neuronal glutamate pool (Node Glun), which is the main precursor of GABA. Only the glial compartment contains glutamine synthase activity.11 The neuronal compartment contains the glutamate decarboxylase and most of the glutaminase activity as well as the pyruvate recycling system (flux 10). The METASIM program was used for the simulations.14,15

spectra to those of the simulated ones. More detailed descriptions of METASIM have been provided.14,25,26

Other Determinations MABP was recorded in the femoral artery during the infusion using a pressure transducer (Schiller). Once the acetate solution had been infused, PO2, PCO2, and pH were determined in samples from arterial and venous blood taken from the femoral artery and jugular veins, respectively, using a clinical automatic analyzer (Nova Biomedical Analyzer). Glucose was determined spectrophotometrically with an NADP-coupled assay.27 Total amino acid concentrations were measured by reverse-phase HPLC.28 The Student’s t test for unpaired values was used to assess statistical significance (P⬍0.05, P⬍0.01, and P⬍0.001) between the groups.

Materials (1,2-13C2) acetate (99.9% 13C) and D2O (99.9% D) were purchased from ISOTEC Inc. The rest of the reagents were of the highest purity available from Sigma Chemical Co.

Results Physiological Parameters of Arterial and Venous Blood After FCI Table 1 shows physiological parameters measured in arterial and venous blood in the course of FCI. Measurements

performed in the right and left jugular veins gave very similar results, and the mean value was taken as representative of venous blood in every animal. MABP remained within normal values before and during the carotid occlusion period, but decreased slightly in the post-occlusion period. After the infusion of (1,2-13C2) acetate, arteriovenous differences in glucose were larger in the 1-hour group (1.2⫾0.12 mmol/L) than in the 24-hour group (0.7⫾0.07 mmol/L). Venous pH was lower in the 1-hour group than in the 24-hour group. Similar metabolic changes have been reported previously.29

Effect of FCI on the 1H NMR Spectra of Extracts From Both Hemispheres of Rat Brain After (1,2-13C2) Acetate Infusion Figure 2 shows representative 1H NMR spectra obtained from extracts of the contralateral (left) and ipsilateral (right) cerebral hemispheres from rats infused with (1,2-13C2) acetate in the 1-hour (top) and 24-hour (bottom) groups. Spectra obtained from the ipsilateral hemispheres showed higher Lac/NAA ratios (1.32⫾0.12 and 0.78⫾0.02) than the contralateral hemispheres (0.27⫾0.055 and 0.20⫾0.03). The latter Lac/NAA ratios are similar to those previously reported

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TABLE 1. Physiological Parameters in Arterial and Venous Blood of Rats in the Course of FCI Followed by Infusion of (1,2-13C2) Acetate 1-Hour Group Parameter

24-Hour Group

Femoral

Jugular

MABP pre-carotid occlusion, mm Hg

95.4⫾4.7

MABP during carotid occlusion, mm Hg

98.6⫾5.5

MABP post-carotid, occlusion, mm Hg

84.1⫾3.9

Glucose, mmol/L

9.8⫾0.8

Femoral

Jugular

䡠䡠䡠

99.7⫾4.0

䡠䡠䡠

䡠䡠䡠

96.48⫾3.7

䡠䡠䡠

䡠䡠䡠 8.6⫾1.05

88.1⫾3.8 12.3⫾1.5

䡠䡠䡠 11.5⫾1.6

PO2, mm Hg

97.2⫾8.7

50.5⫾4.8

99.0⫾8.4

55.7⫾3.5

PCO2, mm Hg

46.0⫾3.9

52.7⫾2.9

47.5⫾1.9

51.9⫾1.8

pH

7.4⫾0.03

7.35⫾0.04

7.4⫾0.05

7.4⫾0.03

MABP was determined during the occlusion of both common carotid arteries. The remaining measurements were performed in samples from arterial and venous blood at the end of the infusion with (1,2-13C2) acetate.

for normoxic brains.30 1H NMR spectra from the ipsilateral hemispheres also showed more intense signals for the acetate methyl group (resonance 2) than those from the contralateral hemispheres, a situation that was specially apparent in the 24-hour group. In this case, 13C satellites corresponding to the methyl signals of (1,2-13C2) acetate were clearly visible (arrows). This observation indicates that (1,2-13C2) acetate accumulates in the ipsilateral hemispheres, probably because of reduced oxidation in the tricarboxylic acid cycle. No other significant alterations could be detected in the remaining metabolites observable by 1H NMR. Amino acid analysis revealed no significant difference in the total concentrations

of glutamate, glutamine, and GABA between the ipsilateral and contralateral hemispheres of the 1-hour and 24-hour groups (not shown).

Effects of FCI on the 13C NMR Spectra of Extracts From Both Hemispheres of Rat Brain After (1,2-13C2) Acetate Infusion Figure 3 depicts the aliphatic portion of representative proton-decoupled 13C NMR spectra of extracts from both cerebral hemispheres of rats infused with (1,2-13C2) acetate, either 1 hour after FCI (top) or 24 hours later (bottom). Resonances from the C2, C3, and C4 carbons of glutamate and glutamine

Figure 2. Representative 1H NMR spectra (360.13 MHz) of extracts from both hemispheres of rat brain infused with (1,2-13C2) acetate, 1 hour or 24 hours after FCI. Permanent occlusion of the middle cerebral artery was performed in the right (ipsilateral) hemisphere. Only the aliphatic portion of the spectra is shown. 1 indicates lactate H3; 2, Ac H2 (12C bonded); 3, NAA H6; 4, Glu-Gln H3, H3⬘; 5, Glu H4, H4⬘; 7, NAA H3, H3⬘; 8, Cr (PCr) methyl; 9, trimethyl ammonium groups of choline derivatives; 10, Ino H1, H3; 11, Cr (PCr) methylene; and 12, Ino H2. Arrows indicate 13C satellites of (1,2-13C2) acetate H2.

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Figure 3. Representative proton decoupled 13C NMR spectra (90.55 MHz) of extracts from both hemispheres of rat brain infused with (1,2-13C2) acetate, 1 hour and 24 hours after FCI. Permanent occlusion of the middle cerebral artery was performed in the right (ipsilateral) hemisphere. Only the aliphatic portion of the spectra is shown. 14 indicates lactate C3; 15, Gln C3; 16, Glu C3; 17, Gln C4; 18, Glu C4; 19, GABA C2; 20, Gln C2; and 21, Glu C2.

and the C2 carbon of GABA were clearly detected in all spectra (see Figure legend for assignments). Even though infusion conditions were identical, intensities of the 13C signals were lower in the 1-hour group than in the 24-hour group. In most cases, these 13C resonances depicted an apparent triplet structure produced by the superposition of a singlet and a doublet. Singlets are derived from metabolites containing the observed 13C bonded to12C neighbors, while doublets, and doublets of doublets, are derived from molecules containing the observed 13C bonded to one or two 13C neighbors, respectively.13,14 The multiplet structure of individual 13C resonances is a direct consequence of the metabolic pathways that produce and degrade the observed metabolite carbon.13–15,26 The main pathways affecting the relative intensities of the singlets, doublets, and multiplets observed in the resonances of glutamate, glutamine, and GABA carbons are as follows: the relative amounts of labeled and unlabeled acetyl-CoA entering the neuronal and glial tricarboxylic acid cycles and the relative exchanges of glutamate, glutamine, and GABA between the neuronal and glial compartments (c.f. Figure 1). A comparison of the 13C NMR spectra shown in Figure 3 reveals that the relative proportions of singlets, doublets, or multiplets corresponding to the same carbon resonance are different in the ipsilateral and contralateral hemispheres, irrespective of the time elapsed after FCI. These results reveal differences in substrate selection between the two hemispheres. Of particular interest is the ratio of singlets to doublets in the glutamate C4 resonance (number 18 in Figure 3). This ratio provides an estimate of the relative flux through the pyruvate recycling system, with larger values of the ratio corresponding to higher relative fluxes through the recycling pathway.13,14 It is possible to obtain a more quantitative interpretation of the changes observed in the multiplet structures of Figure 3 in

terms of metabolic fluxes using the METASIM program. Figure 4 illustrates this procedure with a comparison of representative experimental spectra (top) from the ipsilateral (right panels) and contralateral (left panels) hemispheres, with the METASIM simulations (bottom) obtained using the parameters indicated in the legend. Similar simulations were performed with five animals of the 1-hour group and five animals of the 24-hour group. Values of pool size and relative metabolic flux that best fit the model described in Figure 1 in these animals are summarized in Figure 5. In the 1-hour group, the glial compartment of the ipsilateral hemispheres showed decreased acetate oxidation (flux 3) and increased oxidation of the (13C) glutamate (flux 6) and (13C) GABA (flux 7) imported from the neurons. The neuronal compartment showed increased oxidation of unlabeled glucose (flux 8) and decreased contribution of the pyruvate recycling system to the neuronal cycle (flux 10). The glial glutamate pool size decreased. There was a trend for a decreased glial glutamine pool size and increased neuronal glutamate pool size. These results are consistent with astrocytic swelling. Twenty-four hours later, acetate oxidation augmented (flux 1) in the glial compartment of the ipsilateral hemisphere and increased (13C) glutamate oxidation (flux 6) was maintained. The enhanced glucose oxidation relative to pyruvate recycling seen in the ipsilateral neuronal compartment of the 1-hour group was not seen at 24 hours. Glutamine oxidation (flux 5) in the neuronal compartments of both hemispheres increased as compared with the 1-hour group. It should be mentioned that morphological studies indicate that the effects of focal cerebral ischemia are heterogeneous in the ipsilateral hemisphere. Heterogeneity includes the presence of a zone of well-perfused healthy tissue and a necrotic core surrounded by a penumbral zone with increasingly better perfusion.18 –20 Notably, heterogeneity has also been reported in the contralateral hemisphere.18,19 The relative flux values

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Figure 4. Representative experimental (top) and simulated (bottom) 13C NMR spectra from extracts of each cerebral hemisphere 24 hours after FCI. Simulations were performed using the METASIM program configured to calculate the 13C NMR spectra resulting from (1,2-13C2) acetate metabolism in the network described in Figure 1. Fifteen iterations were used with an artificial line broadening of 1 Hz and a simulated signal/noise ratio of 35/1. Pool size (␮mol/g wet weight) and relative flux values used for the depicted simulations of ipsilateral (contralateral) hemispheres are Glug 0.66 (0.16); Glun 5.0 (5.5); Glutamine 7.5 (6.1); GABA 1.2 (1.2); flux 1 0.4 (0.45); flux 2 0.29 (0.53); flux 3 0.69 (0.98); flux 4 0.67 (0.69); flux 5 0.11(0.36); flux 6 0.30 (0.02); flux 7 0.33 (0.30); flux 8 0.24 (0.45); flux 9 0.89 (0.66); and flux 10 0.65 (0.19).

and pool size measurements presented in Figure 5 represent the weighted averages of the value of each parameter over the complete ipsilateral or contralateral hemispheres.

Discussion Cerebral ischemia is the consequence of the reduction in blood flow below a critical threshold and the concomitant limitation in the supplies of primary cerebral substrates like glucose and oxygen. Under these limiting circumstances the ability of neurons and glial cells to obtain energy from substrates other than glucose contributes importantly to cellular survival. The amino acids glutamate, GABA, and glutamine are available as substrates in the extracellular fluid, and may be used as alternative fuels by astrocytes or neurons under conditions of glucose deprivation. Although it is known that these amino acids are used as substrates by enriched cultures of neurons or glial cells,31 evidence on the physiological competence of this protective mechanism during glucose deprivation has been missing in the adult brain in vivo. Metabolic utilization of glutamate, glutamine, and GABA as fuel alternatives or complementary to glucose is determined by their relative availability in the extracellular fluid as well as by the kinetics of their corresponding transport systems. The extracellular concentration of these amino acids in normoxic and ischemic brain tissues32,33 and the kinetics of

their uptake by cultures of cortical neurons or astrocytes have been addressed previously.34 –38 These studies allow us to calculate values for the availability of different substrates for the neuronal and glial metabolisms using in vitro experiments (Table 2) and to compare the results with those found in the present study for the in vivo brain (Figure 6). Glucose is the main substrate for the adult brain under physiological conditions. The rates of glucose utilization as measured by the deoxyglucose method are approximately, 3.7 and 2.1 nmol glucose 䡠 min–1 䡠 mg protein–1 in cultivated astrocytes and cortical neurons, respectively.39 Glutamate, GABA and glutamine are known to be transported to astrocytes and neurons by cell specific transporters.34 –38 Given physiological extracellular concentrations of glutamate, GABA and glutamine of approximately 1, 3 and 94 ␮mol/L respectively,32,33 the amounts of glutamate and GABA transported to the astrocyte can be calculated as 0.07 and 0.0042 nmol 䡠 min–1 䡠 mg protein–1, respectively, and the amount calculated for glutamine transported to the neuron is 0.85 nmol 䡠 min–1 䡠 mg protein–1. On the other hand, tricarboxylic acid cycle rates of approximately 13 and 3 nmol min–1 䡠 mg protein–1 have been proposed for the neuronal and glial compartments in vivo.10 Thus, under normoxic conditions complete oxidation of glutamate in the tricarboxylic acid cycle of astrocytes would represent at most 2% of their tricarboxylic cycle flux, whereas complete oxidation of glu-

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Figure 5. Pool sizes and relative flux values obtained from computer-assisted interpretations of 13C NMR spectra from extracts of both hemispheres of rat brain infused with (1,2-13C2) acetate, 1 hour and 24 hours after FCI. Values were optimized iteratively using METASIM as indicated in “Materials and Methods” (c.f. References 14 and 15) and illustrated in Figure 4. Results represent the mean⫾SD of the fittings performed on four different 13C NMR spectra of extracts from the ipsilateral (dark bars) and contralateral (light bars) hemispheres of the 1-hour and 24-hour groups, respectively. Circled numbers and node names refer to the nomenclature of Figure 1. Subscripts “g” or “n” refer to the glial or neuronal glutamate pools. Relative flux refers to the probability (Pi) of occurrence of a flux (0 ⬍ Pi ⬍ 1), as compared with the added probability of occurrence of all fluxes converging on the same node taken arbitrarily as one (⌺ Pi⫽1). *P⬍0.05, §P⬍0.01, #P⬍0.001.

lular levels of glutamate, glutamine, and GABA to rise to approximately 200, 250, and 20 ␮mol/L, respectively.32,33 These concentrations saturate the corresponding amino acid transport systems of neurons or astrocytes, transiently increasing the metabolic availability of glutamine or glutamate and GABA, in the respective intracellular environments (Table 2). The calculated rates of amino acid transport under ischemic conditions represent an important increase in the intracellular metabolic availability of glutamate and GABA or of glutamine for oxidation. The results obtained in the

tamine would contribute at most 6% of the tricarboxylic acid cycle flux in neurons. These results match well with those obtained in the present study, indicating that the contribution of glutamate oxidation to the energetics of the astrocytes (or glutamine to that of neurons) is minor under physiological conditions. This situation is observed in the contralateral hemisphere of the 1-hour group. Under ischemic conditions the availability of extracellular substrates for neurons and astrocytes undergoes a drastic change. Glucose deprivation and hypoxia cause the extracel-

TABLE 2. Calculated Availability of Glutamate, Glutamine, and GABA as Oxidative Substrates in Cultivated Neurons and Glial Cells Under Normoxic or Ischemic Conditions Neuron, nmol 䡠 min⫺1 䡠 mg protein⫺1 Substrate Availability (v)/Condition

Normoxia a

Glutamate

0.9

20.5 e

Glutamine

0.85

g

GABA

0.09

Tricarboxylic acid cycle ratei

13j

Ischemia a

Extracellular Fluid Concentration EFC ␮mol/L Normoxia 1

2.17

e

0.15

g

b,c

94 3

b,c

b,c

Astrocyte, nmol 䡠 min⫺1 䡠 mg protein⫺1

Ischemia b,c

200

b,c

250 20

b,c

Normoxia 0.07 1.4

d

4.2d

f

0.0042

Ischemia 3.5f

h

0.0083h

3j NA 䡠䡠䡠 䡠䡠䡠 Intracellular substrate availability (v) was calculated using the expression v⫽Vmax 䡠 EFC/(Km⫹EFC) where Km (␮mol/L) and Vmax (nmols 䡠 min⫺1 䡠 mg protein⫺1) refer to the kinetic constants of the transport system in enriched cultures of neurons or glial cells and EFC is the concentration of the substrate (␮mol/L) in the interstitial fluid of the brain. Vmax/Km or EFC values used to calculate v were a 23/24 (34), b(32), c(33), d6.1/91 (35), 328.2/3000 (36), f50.2/3300 (36), g0.171/2.8 (37), h0.01/4.1 (37), inmol acetyl-CoA 䡠 min⫺1 䡠 mg protein⫺1, j(10). Kinetic constants and EFC values were taken from the references indicated in parentheses. A value of 100 mg protein 䡠 g⫺1 wet weight was used to transform measurements carried out in the whole brain (␮mol⫺1 䡠 min⫺1 䡠 g wet weight) into nmol 䡠 min⫺1 䡠 mg protein⫺1; NA, not applicable. NA

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Figure 6. Summary of neuronal-glial interactions in both hemispheres of rat brain infused with (1,2-13C2) acetate, 1 and 24 hours after FCI. Primary cerebral substrates and metabolic end products in plasma are shown schematically in the blood vessels located outside the hemispheres. Exchanges of glutamate, glutamine, and GABA between the tricarboxylic acid cycles of neurons and glial cells are shown by arrows inside the hemispheres. The relative size of the arrows indicates approximately the relative contributions of the fluxes. Black arrows indicate ipsilateral fluxes; white arrows, contralateral fluxes. Numbers indicate the contribution (%) of the corresponding flux, taken from Figure 5.

present study (Figure 6) confirm this prediction showing increased oxidations of (13C)* glutamate and (13C) GABA in the glial tricarboxylic acid cycle and (13C) glutamine in the neuronal tricarboxylic acid cycle of the adult brain in vivo, respectively. The detection of increased glutamate and GABA oxidation in the glial compartment agrees with previous observations in astrocyte cultures30 and shows the important role of glial metabolism in regulating the extracellular concentrations of these amino acids in the ischemic brain tissue in situ. Notably, an increased contribution of the previously described pyruvate recycling system13 is observed in ipsilateral hemisphere of the 24-hour group as compared with the 1-hour group. Neuroprotective actions of this pathway may include providing the neurons with (1) an increased NADPH generation through mitochondrial and synaptosomal malic enzymes40 and (2) a mechanism to generate acetyl-CoA from glial glutamine under conditions of reduced glucose oxidation.14 Indeed, our current results show that the relative contribution of the pyruvate recycling system to the neuronal cycle increases in the ipsilateral hemisphere during the late phases of ischemic insult when glucose oxidation is reduced and neuronal glutamine oxidation is increased. Interestingly, recently bicarbonate has been shown to modulate glutamine synthesis in cultivated astrocytes.41 Thus, a reduction in tissue bicarbonate caused by ischemic acidosis could contribute to the reduced neuronal glutamine oxidation and pyruvate recycling observed in the 1-hour group as compared with the 24-hour group. An alternative mechanism in which an astrocytic pyruvate recycling would provide the neurons with recycled lactate, rather than with glutamine, has been proposed, using astrocyte cultures prepared from neonatal brain tissues.42,43 Recently, enzymes of this pathway have been shown to experience important changes in activity and cellular localization during ontogenic development, and malic *We use the term (13C) X to denote the total 13C accumulated in metabolite X during the infusion of a 13C-labeled substrate.

enzyme and phosphoenolpyruvatecarboxykinase activities have been reported in neuronal mitochondria and synaptosomes prepared from adult brain tissues.40,44 It should also be mentioned here that the use of lower doses of acetate could decrease the reported contributions of recycling. In summary, our current results are consistent with important time-dependent changes in substrate selection during the development of ischemic brain damage. Glutamate and GABA or glutamine appear to be efficiently oxidized as alternative substrates to glucose by the glial or neuronal compartments of the adult brain in situ. Investigation of the effects of ischemic damage in the different perfusion zones surrounding the necrotic foci constitutes an attractive topic for future research in this field.

Acknowledgments This work was supported by grants PB-94011, PB96 – 0864-C02, and AC-105/96 to Dr Cerdan and grant FISss 1177/96 to Dr Carceller. The authors wish to thank Dr. B. Ku¨nnecke for providing a copy of the METASIM v2.1 program, Dr O. Herreras for helpful comments, Mr. J. Pe´rez for assistance with the illustrations, and Mrs. C.F. Warren for editorial processing of the manuscript.

References 1. Sochocka E, Juurlink BHJ, Code WE, Hertz V, Peng L, Hertz L. Cell death in primary cultures of mouse neurons and astrocytes during exposure to and recovery from hypoxia, substrate deprivation and simulated ischemia. Brain Res. 1994;638:21–24. 2. Huang R, Hertz L. Effect of anoxia on glutamate formation from glutamine in cultured neurons: dependence on neuronal subtype. Brain Res. 1994;660:129 –137. 3. Huang R, Shuaib A, Hertz L. Glutamate uptake and glutamate content in primary cultures of mouse astrocytes during anoxia, substrate deprivation and simulated ischemia under normothermic and hypothermic conditions. Brain Res. 1993;618:346 –351. 4. Kimmelberg HK, Rutledge E, Goderie S, Charniga C. Astrocytic swelling due to hypotonic or high K⫹ medium causes inhibition of glutamate and aspartate uptake and increases their release. J Cereb Blood Flow Metab. 1995;15:409 – 416. 5. Badar-Goffer RS, Bachelard HS, Morris PG. Neuronal-glial metabolism under depolarizing conditions: a 13C NMR study. Biochem J. 1992;282: 225–230.

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6. Cox DWG, Morris PG, Bachelard HS. Kinetic analysis of the cerebral creatine kinase reaction under hypoxic and hypoglycemic conditions in vitro. Biochem J. 1988;255:523–527. 7. Bachelard H, Badar-Goffer R. NMR spectroscopy in neurochemistry. J Neurochem. 1993;61:412– 429. 8. Berl S, Clarke DD. The metabolic compartmentation concept. In: Hertz L, Kvame E, Mc Geer G, Schousboe A, eds. Glutamate, Glutamine and GABA in the Central Nervous System. New York, NY: Alan R. Liss Inc; 1983:205–217. 9. Van den Berg CJ, Krzalic LJ, Mela P, Waelch H. Compartmentation of glutamate in brain: evidence for the existence of two different tricarboxylic acid cycles in brain. Biochem J. 1969;113:281–289. 10. Van den Berg CJ, Garfinkel D. A simulation study of brain compartments: metabolism of glutamate and related substances in mouse brain. Biochem J. 1971;123:211–218. 11. Erecinska M, Silver I. Metabolism and role of glutamate in mammalian brain. Prog Neurobiol. 1990;35:245–296. 12. Tsacopoulos M, Magistretti PJ. Metabolic coupling between glia and neurons. J Neurosci. 1996;16:877– 885. 13. Cerda´n S, Ku¨nnecke B, Seelig J. Cerebral metabolism of (1,2-13C2) acetate as detected by in vivo and in vitro 13C NMR. J Biol Chem. 1990;265:12916 –12926. 14. Ku¨nnecke B, Cerdan S, Seelig J. Cerebral metabolism of (1,2-13C2) glucose and (U-13C4) ␤-hydroxybutyrate as detected by in vivo and in vitro 13C NMR. NMR in Biomed. 1993;6:264 –277. 15. Chapa F, Ku¨nnecke B, Calvo R, Escobar del Rey F, Morreale de Escobar G, Cerda´n S Adult onset hypothyroidism and the cerebral metabolism of (1,2-13C2) acetate as detected by. 13C NMR. Endocrinology. 1995;136: 296 –305. 16. Chen ST, Hsu CY, Hogan EL, Maricq MD, Balentine JD. A model of focal ischemic stroke in the rat: reproducible extensive cortical infarction. Stroke. 1986;17:738 –743. 17. Liu TH, Beckman JS, Freeman BA, Hogan EL, Hsu CY. Polyethylene glycol-conjugated superoxide dismutase and catalase reduce ischemic brain injury. Am J Physiol. 1989;256:H589 –H593. 18. Avendan˜o C, Roda JM, Carceller F, Dı´ez-Tejedor E. Morphometric study of focal cerebral ischemia in rats: a stereological evaluation. Brain Res. 1995;673:83–92. 19. Roda JM, Carceller F, Dı´ez-Tejedor E, Avendan˜o C. Reduction of infarct size by intra-arterial nimodipine administration at reperfusion in a rat model of partially reversible focal ischemia. Stroke. 1995;26:1888 –1892. 20. Roda JM, Carceller F, Pajares RL, Dı´ez-Tejedor E. Prevention of cerebral ischaemic reperfusion by intraarterial administration of superoxide dismutase in the rat. Neurol Res. 1991;13:160 –163. 21. Carceller F, Roda JM, Gutie´rrez-Molina M, Lo´pez-Pajares R, Albarin˜o AR, Dı´ez-Tejedor E. Can nimodipine prevent ischemic reperfusion in the rat brain? Neurol Res. 1993;15:395– 400. 22. Preece NE, Cerdan S. Metabolic precursors and compartmentation of cerebral GABA in vigabratin treated rats. J Neurochem. 1996;67: 1718 –1725. 23. Cerda´n S, Parrilla R, Santoro J, Rico M. 1H NMR detection of cerebral myo-inositol. FEBS Lett. 1985;187:167–172. 24. Malloy CR, Sherry AD, Jeffrey FMH. Analysis of the tricarboxylic acid cycle of the heart using 13C isotope isomers. Am J Physiol. 1990;259: H987–H995. 25. Ku¨nnecke B. Computer assisted simulation and interpretation of metabolic data derived from 13C NMR spin coupling patterns. PhD Thesis. University of Basel, Switzerland, 1991:155–327. 26. Ku¨nnecke B. Application of 13C NMR spectroscopy to metabolic studies in animals. In: Beckman N, ed. Carbon-13 NMR Spectroscopy of Biological Systems. New York, NY: Academic Press; 1995:159 –267.

27. Ku¨nst A, Draeger B, Ziegenhorn J. D-glucose:UV methods with hexokinase and Glucose 6-phosphate dehydrogenase. In: Bergmeyer HU, Bergmeyer J, Grassl M, eds. Methods of Enzymatic Analysis. Weinheim, Germany: Verlag-Chemie; 1984;6:163–172. 28. Chang JY, Knecht R, Brown DG. Amino acid analysis in the picomole range by precolumn derivatization and HPLC. Methods Enzymol. 1981; 91:41– 48. 29. Mies G, Paschen W, Hossman KA. Cerebral blood flow, glucose utilization, regional glucose and ATP content during the maturation period of delayed ischemic injury in gerbil brain. J Cereb Blood Flow Metab. 1990;10:638 – 645. 30. Gadian DG, Williams SR, Bates TE, Kauppinnen RA. NMR spectroscopy: current status and future possibilities. Acta Neurochir. 1993; 57:1– 8. 31. McKenna MC, Sonnewald U, Huang X, Stevenson J, Zielke HR. Exogenous glutamate concentration regulates the metabolic fate of glutamate in astrocytes. J Neurochem. 1996;66:386 –393. 32. Hagberg H, Lehmenn A, Sandberg M, Nystro¨m B, Jacobson I, Hamberger A. Ischemia induced shift of inhibitory and excitatory amino acids from intra to extracellular compartments. J Cereb Blood Flow Metab. 1985;5: 413– 419. 33. Takagi K, Ginsberg M, Globus MYT., Dietrich D, Martinez E, Kraydieh S, Busto R. Changes in amino acid neurotransmitters and cerebral blood flow in the ischemic penumbral region following middle cerebral artery occlusion in the rat: correlation with histopathology. J Cereb Blood Flow Metab. 1993;13:575–585. 34. Yu ACH, Hertz, L. Uptake of glutamate, GABA and glutamine into a predominantly GABAergic and a predominantly glutamatergic nerve cell population. J Neurosci Res. 1982;7:23–35. 35. Garlin AB, Sinor A, Sinor JD, Jee SH, Grinspan JB, Robinson MB. Pharmacology of sodium dependent high affinity L-[3H]glutamate transport in glial cultures. J Neurochem. 1995;64:2572–2580. 36. Schousboe A, Svenneby G, Hertz L Uptake and metabolism of glutamate in astrocytes cultured from dissociated mouse brain hemispheres. J Neurochem. 1977;29:999 –1005. 37. Lewin L, Mattsson MO, Rassin DK, Sellstrom A. On the activity of ␥-aminobutyric acid and glutamate transporters in chick embryonic neurons and rat synaptosomes. Neurochem Res. 1992;17:333–337. 38. Nilsson M, Hansson E, Ro¨nnback L. Interactions between valproate, glutamate, aspartate and GABA with respect to uptake in astroglial primary cultures. Neurochem Res. 1992;17:327–332. 39. Peng L, Zhang X, Hertz L. High extracellular potassium concentrations stimulate oxidative metabolism in a glutamatergic neuronal culture and glycolysis in cultured astrocytes but have no stimulatory effect in GABAergic neuronal culture. Brain Res. 1994;663:168 –172. 40. Cruz F, Scott R, Barroso I, Santisteban P, Cerda´n S. Ontogeny and cellular localization of the pyruvate recycling system in rat brain. J Neurochem. 1998 (in press). 41. Gamberino WC, Berkich DA, Lynch CJ, Xu B, La Noue K. Role of pyruvate carboxylase in facilitation of synthesis of glutamate and glutamine in cultured astrocytes. J. Neurochem. 1997;69:2312–2325. 42. Sonnewald U, Westegard N, Jones P, Taylor A, Bachelard H, Schousboe A. Metabolism of (U-13C) glutamine in cultured astrocytes studied by NMR spectroscopy: first evidence of astrocytic pyruvate recycling. J Neurochem. 1996;67:2566 –2572. 43. Hassel B, Sonnewald U. Glial formation of pyruvate and lactate from TCA cycle intermediates: implications for the inactivation of transmitter amino acids. J Neurochem. 1995;65:2227–2234. 44. Vogel R, Hamprecht B. Enzymatic and immunocytochemical studies for localizing mitochondrial malic enzyme in brain. J Neurochem. 1997; 69(suppl):S170B. Abstract.

Editorial Comment In the mature brain, glucose is the predominant energy substrate with small contributions from plasma-derived lactate, ␤-hydroxybutyrate, and acetoacetate. In addition, components of the amino acid pool can exchange with intermediates of the tricarboxylic acid cycle (TCA) and thereby provide alternative energy substrates when

glycolysis is reduced. During prolonged focal cerebral ischemia, glucose availability and acidosis may limit the activity of glycolytic and other energy-associated enzymes. Whether amino acids such as glutamate and glutamine, which are normally thought to cycle between neuronal and astrocytic compartments, undergo increased

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Pascual et al oxidation as alternative energy substrates during ischemia in vivo is not well-established. In the study of Pascual et al, the authors use an innovative approach of carbon-13 NMR spectroscopy to track changes of carbon-13 incorporation into key energy substrates and to allow computer simulation of metabolic pathways that can predict changes in glutamate, glutamine, and GABA oxidation during focal ischemia in the rat. Ischemia was induced by permanent middle cerebral artery occlusion plus bilateral carotid occlusion for 90 minutes. The critical feature of the experiment is the use of carbon-13 in both the one and two carbon positions of acetate. When these two adjacent carbons are incorporated into the TCA cycle, they provide both singlet and multiplet signatures on the NMR spectra for glutamate, glutamine, GABA, and lactate carbons. Labeled carbon positions depend on the number of turns of the TCA cycle and on a pyruvate cycling pathway. By assuming two metabolic compartment pools based in neurons and glia, the authors conclude (1) that oxidation of neuronally derived glutamate and GABA increases in the glial compartment after 90 minutes of focal ischemia and (2) that oxidation of glialderived glutamine increases in the neuronal compartment in conjunction with an increase in activity of the pyruvate recycling pathway after 24 hours of focal ischemia. The latter effect of increased glutamine oxidation also occurred in cortex contralateral to the middle cerebral artery occlusion on the day after bilateral carotid occlusion. These results are consistent with the notions (1) that glia increase their utilization of carbon skeletons derived from amino acids for energy metabolism during the early period of focal ischemia and (2) that neurons outside the ischemic core and surviving the moderate ischemia associated with bilateral carotid occlusion increase their utilization of carbon skeletons derived from amino acids for energy metabolism 1 day after the insult. In performing the computer simulation of the metabolic fluxes, the authors used the most simplified model consistent with the information content of their data. Reducing the model to the simplest elements is often necessary for computer iterations to converge on a solution. However, when applying these models to the in vivo situation with multiple neuronal and glial subtypes and subcellular compartments, some of which will be selectively altered by the ischemic insult, one needs to critically assess the simplifying assumptions of the model. The main assumptions of the model are that (1) all of the labeled acetate is metabolized in the glial compartment, (2) all of the glutamine is synthesized in the glial compartment,

May 1998

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and (3) all of the pyruvate recycling occurs in the neuronal compartment. These assumptions, which are based on reports from the literature and previous work from this laboratory, are reasonable first approximations. However, future studies need to be directed at validating these assumptions during ischemia and at further refining the model to include additional pathways. For example, blood-brain barrier injury during ischemia may increase intracellular availability of 13C-acetate relative to glucose, particularly in neurons if 13C-acetate can more readily diffuse past astrocyte foot processes. Because 13CNMR is relatively insensitive, a 1.5 millimolar plasma concentration of labeled acetate was used rather than a tracer concentration. Thus, it is possible that substantial changes in intracellular acetate availability during ischemia could occur and influence the rate of glycolysis. The increased signal intensity across all peaks of the 24-hour spectra might be due to increased acetate transport across the barrier. Another consideration is the gliosis and increased glial fibrillary acidic protein–staining typically seen 1 day after ischemia. Such phenotypic changes in astrocytes may be accompanied by an altered expression of metabolic enzymes. Thus, pyruvate recycling enzymes present in neonatal glial cultures could become prominent in postischemic astrocytes in vivo and modify the assumption of selective neuronal localization. Because the observed changes in the ratio of singlet-to-doublet resonances in C4 glutamate are presumed to reflect changes in pyruvate recycling activity, changes in cell localization of this pathway during ischemia will have a considerable impact on the data interpretation. Moreover, an increased fraction of the glutamine synthesized in glia may be metabolized by glutaminase in glial mitochondria rather than in neuronal mitochondria when neurons are selectively injured. Finally, the model does not consider the potential transport of carbon between astrocytes and neurons in the form of lactate, which is known to increase during ischemia. This study breaks new ground in demonstrating increased use of alternative energy substrates during ischemia. However, further work is needed to validate the assumptions of the model in postischemic tissue, to expand the number of metabolic compartments and pathways, and to use other carbon-labeled substrates such as 13C-glucose to complement the information derived from 13C2-acetate. Raymond C. Koehler, PhD, Guest Editor Anesthesiology/Critical Care Medicine The Johns Hopkins University Baltimore, Maryland

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Glutamate, Glutamine, and GABA as Substrates for the Neuronal and Glial Compartments After Focal Cerebral Ischemia in Rats José M. Pascual, Fernando Carceller, José M. Roda and Sebastián Cerdán Stroke. 1998;29:1048-1057 doi: 10.1161/01.STR.29.5.1048 Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1998 American Heart Association, Inc. All rights reserved. Print ISSN: 0039-2499. Online ISSN: 1524-4628

The online version of this article, along with updated information and services, is located on the World Wide Web at: http://stroke.ahajournals.org/content/29/5/1048

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CURRÍCULUM NORMALIZADO COMPLETO (CVNC)

García-Martín, M.L., Ballesteros, P., S. Cerdán Dynamics and metabolism of water in cells and tissues as detected by Magnetic Resonance Methods Progr. NMR Spectroscopy (2001) 39, 41-77. I.F.: 6.162 (8/113 Q1 Chemistry, Physical , 1/31 Q1 Physics, Atomic Molecular) ISSN: 0079-6565

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                        Š     Œ  Š  ˆ   ‡‹­             Š   —Ÿ‰ ¨†˜     Š     ƒ      ¥    –      Œ   „……™ ‹…… ¦•     Š       –   Š  ¢       Š    ¨  ƒ          Š     ,‹ £ ‡… 2‡…  ˆ         Š        –     ƒ Š     Š  † Š   ŠŠ       †    Š             –Š      ¢   ¤   Š     Š        –        ª„‡„„„š‰„«  ƒ  œ„  –    Šƒ     

    œ„ ƒ         ¦ —“… 2 t 2 ‡Ÿ…‰ 2 t Œ ˜  ™ ™  ™ — “… 2 t 2 —‡Ÿ…‰ 2 t ˜Œ ˜  ª‰‹‰‹Ž« ¤  Š ™ Š     œ„

’“’ ”  ‡  ‡  ’  •    Š           ‰ –… ‹‚„„—Œ —˜™™

‰š

       ƒ    –        Š          œ‡—„˜ ƒ        –   œ‡—„˜    ƒ      ‘     ¨        Š           –         Š  ©       œ‡—„˜    ƒ        –    X ‡=œ‡…„† ˆ   =œ‡…„† …Ž† 

¤  ‹ —œ— Š ¡ ›   ›   œ‡  Š  Š  ’ ¦•        ¢     –      ¢    Œ    ƒ   Œ Š     Š     ¢       ™  –Š         Š ƒ Š      Š  ƒ „…… ¦• n   ¥–  Š       ¢  ƒ   ‡›œ‡

  ƒ  ¨ ‘Š    ª„„«  Š   ” ƒ  € 

Œ  ž ‡=œ„ ˆ  0 …‹t 1 Žt =…‡ 1 v„ t„ † 1 „t =…‡ 1 ‰v„ t„ †† …‹†

  0 ˆ ‹=Ÿ……m… =‰p†„ g„ „ =…‰p„  ’ †: ‘    ¤  „  –Š       œ‡        …vt p ‡†: ¦ ƒ         œ„      ƒ  œ‡

           

   –   œ„  Š  œ‡    

  œ„ , œ‡ :       ¢  

     Š  Œ ƒ  v         – Š    ƒ  ƒ    v ƒ   ˆ      –       ƒ  ‘     –    œ‡—„˜‘  œ‡—„˜   ƒ œ‡—„˜    ƒ       ‡=œ‡…„† ˆ  ‘ =œ‡…„†‘ 1   =œ‡…„†

…‰†

  ‘    Š     ƒ 

œ„      Š  ƒ    – ƒ                 Š           ƒ 

      Š  ª‰‹™‰Ÿ«     œ„         Š  Š        „ ª‰“«  Š œ„     

     ŠŠ–   ‡‰…       –       Š   —ŠŠ– ‡…  ˜             – ¦ ƒ œ„  Š    ƒ   — „  ˜     „  –         — ‡…  ˜             œ„ ƒ   Š   

   —œ„˜  –   ƒ      ƒ ©          œ„  –      – Š       •  Š       ƒ    –    

            

    –    –         –   Š         –Š   ‡=œ ˆ ‡=œ„ 2 ‡=œ„

…’†

 œ  ŠŠ –   œ„   ƒ  –       Š —   ˜       Š  Š   „  œ„  œ„      ƒ         Š –Š    Š  Š  –’—’—’‚’ œ    ‰  ’    

‰Ÿ

’“’ ”  ‡  ‡  ’  •    Š           ‰ –… ‹‚„„—Œ —˜™™

¤  ‰ ž       ‰        ‰ š •  ¢  ”    € š ‹•¢”€Œ  š£  ‘ž ¤€” Œ d ž     D ž   Š   ž      ž ‹’…‡‹ ¦• ‡¦  Š   Š  ƒ    Š   —   ˜   —ŠŠ ˜  Š   ‘  ž ˆ‘ ¦„ —Ÿ‡Ž ŠŠ˜ ˆ‘ ¦‰ ¦Ž —š„… ŠŠ˜  —‰šŸ ŠŠ˜    —¦‹˜‹ 1 —‹„… ŠŠ˜   ¦‹ —‡‹Ž ŠŠ˜ ˆ ž ”   Š         ž € 

    …=… †       ƒ  —       ˜       ˆ‘—¦‰ ¦Ž˜       Š      Š     ¢     ”Œ —š˜ †ž        ˆ‘      ‘Š    ª‡„Ž«  Š   ‘   

             ¤     €Š  ” —¤€”˜  Œ     ŠŠ   €¬ ¨   ªŽ…Ž‡«    –     Š Š    ªŽ„™Ž’«                  ƒ          –  ŠŠ    ¢    ƒ            ªŽšŽŸ«  ¥  ¦ Š  

–Š  Š       Š      ¢       Š  —”˜      —d ˜           p Š  —¤  ‰‘˜  –Š                ƒ ¯    °   ƒ  —D 2 d ›‹˜  D                Š      d      ¢   

’“’ ”  ‡  ‡  ’  •    Š           ‰ –… ‹‚„„—Œ —˜™™

    Œ       Š     Š   ‘   p Š                        ƒ  ƒ              ƒ                   ƒ  ¢        Œ                  Š  —˜    —…˜   ŠŠ    ”      ŠŠ     ¢  —‘†˜   

    –Š ž  =… ˆ –Š‰2g„ ”„ d„ …D 2 d=‹†‘†Š ˆ –Š‰2‘†Š …š†

                     ”  ‡¦     g —„’šŽ £ ‡… Ÿ  2‡  2‡˜      ”   Š     ‘†       –Š     ¢      ƒ   ›…   

   ƒ   ”Œ —š˜ —¤  ‰˜    Š ‘      ƒ    •‘  •      ¢  ‘    Š    Š       ƒ ›…      ¤€” –Š 

 Š –         ¢    ƒ    ƒ     

   Š t ‘  t  ªŽ“«       –Š   =… ˆ •‡ –Š…2‡ D† 1 •„ –Š…2„ D†

…Ÿ†

 ‡…„† ˆ ‡=„‰‘ 1  1 ‡=…‡=t‘ 1 ‡=t † ^ …… 2 ‘ 1 ‡=……‡=t 2 ‡=t‘ ††„ 1 ‰ „ t‘ t ††‡=„ Š •„ ˆ ‡=…„ 2 ‡ †‰…•‘ ‘ 1 •  2 ‡ †Š •‡ ˆ ‡ 2 • „  ˆ g„ d„ „  –Š  ƒ   Š ¢         ž — ˜  – ƒ   … t  p ‡†      –Š    ˆ •‘ ‘ 1 •   —

˜  – ƒ  … t  q ‡†; ‡ ˆ ‘ ; „ ˆ  ; •‡ ˆ

‰“

•‘  •„ ˆ • : ˆ     ƒ   t             ˆ           Š   ƒ 

t ˆ …‘ 2 ‡ …‡ 1 ‡=Š† 1  =Š†=…‡ …‘ 1  2 ‡ † 2 ‘ 

—“˜

 Š ˆ •‘ =• : ˆ     

     ¢            –   Š  p ‘ ; –Š  —“˜ Š ¢ ž

t ˆ ‘ 2 ‡ …‡ 1 ‡=Š†=…‡ …‘ 2 ‡ †  t         ¨  ƒ   ‘   –   Š    ‡     ƒ       –   ƒ   ˆ     Š  Š         –   ƒ     ¢  ‘†    ƒ        Š ‘† ž X ‘† ˆ   ‘†

…‡…†

     Š     ƒ   Š    ƒ   ƒ    Š  ‘ Š  Š  ¤€” –Š                           ˆ         ‘† Š    ¦ ƒ            

          ‘† ƒ  Š   ªŽ‰’…« ˆ  ƒ  ƒ     ‘† ƒ  –Š  ŠŠ  ‘†   Š        Š 

  ¢       ƒ        ‘      Š     ¢     ƒ           ƒ 

   ¢  ¨   Š    ¢    ƒ   ‘†    ƒ    ƒ      –Š   Š             •      Š          Š             ƒ   ƒ       Š 

Ž…

’“’ ”  ‡  ‡  ’  •    Š           ‰ –… ‹‚„„—Œ —˜™™

   =… ƒ  —¤  ‰†˜  •   Š             ƒ            ƒ      ¨     ƒ §    ŠŠ     ¢  

         ƒ     Š  kl          kl ˆ …„‘† †‡=„

…‡‡†

  Š –Š    Š    ‘†   Š         Š   –Š  Š       ‘† ˆ           ƒ   ‘†

Š       Š                 ‘† —„Ž8˜  „… £ ‡… Ž  „ 2‡ ƒ ’‹„ m  ‡  „ m ±  ‡ Š        ¨

 ‡   …’ ‘ – —š‡„…… ˜       ¢  ‹… m „ 2‡ ƒ   š m  ‡  …„‰ m  ±  ‡ Š         ‡   ……š ‘    Š      ƒ                Š     ˆ             ƒ   ƒ   ƒ   Š            Š       ƒ      Š     ƒ            ƒ ƒ    –            Š  Š        ¢   •  ¤    Š  Š    

 Š        Š   Š      ŠŠ           ƒ      ƒ  ŠŠ            ¢      ª’‡’„«      Š    ƒ    ƒ ƒ      Š ªŽŽ’‹™’’«  ‘† ƒ        

 Š     ƒ      ŠŠ       Š            Šƒ  ƒ                Š     Š               ƒ ƒ ˆ       

 Š        ¨ – Š   ƒ ƒ     ª’š« –’—’‚’                  ŠŠ        

Š    – ª„« —    ‡˜ ‡ ¦    ‡¦         Š         Š       –         ƒ ¦ ƒ „¦ ‡¦  ‡š — ‡Ÿ   ˜ ƒ      ª’Ÿ™ š‡«     ŒŠ   –          „¦      –Š         Š   Š     Š  ‡¦ —     ’‡‰Ž˜ ªš„« ¨              ƒ        „¦   Š  ‡¦  —  ¤  “˜       „¦  „¦„  – ŠŠ–          ‡¦  ‡¦„        ˆ „¦„      ŒŠ        Š       „¦„  –        t    ™ „¦   „¦„      ¨ œ‡   „¦  „¦„  Š

     ƒ    œ‡  ‡¦  ¦„       ƒ  ª„« ˆ Š   „¦          Š 

 ªš‹« ‹ ¦  Š  ‡›„          ‡¦           Œ    ƒ          ¢  ªš‰™šš« ¦ ƒ ‹¦    ƒ    ƒ    ‹¦    Š    Š       

  ƒ      ‡¦  ŠŠ          ƒ      ‡š                ƒ           Š       ƒ    ‡š      ƒ   ‡š  ©    ŠŠ    ‡š        ŒŠ              Š   ŠŠ–   ‡……… ŠŠ             –         Œ Š    

   Š ¢    Š   

’“’ ”  ‡  ‡  ’  •    Š           ‰ –… ‹‚„„—Œ —˜™™

¤  Ž ‚¤˜ —–          š›      …„’¥¥ ¤‘ —– Š    ‘ž ŠŠ  ž ƒ     ™”ž             €     Š    Š windaysy       Š   ‡ ¦‡‹ „ ¦ ˆ „…:…’ ¦•;   Š    D‡ ˆ 2„„:Ÿ“ ¦•=; ƒ  

Š    D„ ˆ 2“:Ÿ“ ¦•=:

    Š ªšŸš“« ‡š¦„ ‡¦  –    ŠŠ–           ‡’¦„ ¨    – Š             –    ¦ ƒ ‡š     ¢           –   Š ™ Š  Š   ‡š     ¢    ƒ   Š ‘   ŠŠ      Š    ‡š¦„      ‡š ¦„            ‡š„       ¢  ŠŠ            ƒ  ªŸ…Ÿ‡« ‘         ‡ ¦ „¦ –        ‡‹  Š

Ž‡

Š ªŸ„«           Š   ‡‹                ‡‹      Š  Š  Š 

Š     ŠŠ   ‡‹       ƒ         ªšŸ« ¤  Ž  Š  Š  Š  Š 

Š       ‡‹    Š              ƒ              ‡‹      Š          ‡ž‡ž‡  Š    Š¢  Š     ŠŠ–   2…„Ž ŠŠ €                     Œ   Š    ƒ Š     2…Ž  2…šŽ ŠŠ ”ƒ    ƒ      ƒ ‡‹ Š ƒ   Š     ŠŠ–   2…‡Ž ŠŠ    Š   ‡‹      ƒ   Š    „ ¦™ ‡‹       ƒ     ‡‹ Š      Š               ‡‹         ƒ ŠŠ   ‡¦™ ‡‹  „¦™ ‡‹ Š    „      ƒ „¦™ ‡‹ Š     Š             Š      Š  ƒ ƒ    — „¦ ‡¦ ‡‹ ˜   Š      ¤  ‡‡ –’‚’ ˆš   š †        ƒ      ŠŠ    ƒ          ¢   Œ                         –   Š    Œ ƒ      ŠŠ      ƒ      ƒ    Š  ¢               ƒ Š       –     ²       Š Š            ƒ ƒ    ”  ¥  ŠŠ  ‘    ƒ  ƒ     ª‡š« ˆ             ƒ  ¥      Š    ƒ

           

Ž„

’“’ ”  ‡  ‡  ’  •    Š           ‰ –… ‹‚„„—Œ —˜™™

    ƒ      Š  ŠŠ         ”  ŠŠ            ƒ ‡¦„              ƒ       

        Š                    ªŸ‹™Ÿš«    ƒ     ƒ        ƒ      b       † ƒ

           Š   ƒ  Š  Š     Š    ƒ Š   Š         Š  •      –Š     ƒ    ¨  Š        ‡¦    

   Š ¢           —  „¦„˜         Š  ƒ           ªŸŸ«  –Š     „¦„

    –Š   ‡¦„     „¦„ Š     ŠŠ   Š    ªŸŸ™“‡«              ƒ     ‡¦„  „¦„           Š         Š   ƒ ƒ    ªŸŸ« ¤        ƒ –Š   Š ƒ Š      ª“„™“’«         ¥      ¥     Š   –  ¤    ƒ     

  ƒ        ƒ ¥    Š ª“š™““«              ¤   ƒ ‘     —¤‘˜  Œ  ŠŠ      ¥     ¥        • — „™‰ m˜     Š          ¥  ƒ     Š  •   ¤‘ ƒ      Šƒ               ¢    Š      ˆŠ             ƒ   Š              ¤‘ ¤   ƒ    ƒ  ¥  Š Šƒ       Š     

  ¢           –   Š   Š         ‘ Š ƒ          ƒ               ƒ           –     ƒ         Š  ŠŠ            ƒ        ž — ˜  ¤‘                —

˜   ¢       –   Š         ¨       —

˜  Š       Š Š    Š        Š            Š     —  €  ‹‡‡„˜      ¨   ¢   Š       ¤‘   –   ¥      Š                                ¥   

  Š                    ¥   Š   Œ ª“Ž‡……‡…‡«          Š   ¥      Š   –      Š   Š     –      Š     Šƒ                Š  Š Š    ƒ   ƒ  ¤‘  Š   ŠŠ                      Œ      Š  Š   ƒ   Š   Š  ŠŠ         ƒ  ƒ                   ’—’ ž     ”–     Š      Š          ž      Š Š Š                Š Š  ª‡…„« †       Š  Š Š    Œ     ƒ     

’“’ ”  ‡  ‡  ’  •    Š           ‰ –… ‹‚„„—Œ —˜™™

Ž‹

  „      ‡‹™ „¦ Š     „¦  Š     ‡‹ Š   Š            ‡¦ Š  ‡‹  Š  ƒ – —  ‡¦ Š  ‡‹  Š —‡Ž…‡‹ ¦• „„8˜     –  ƒ Š    —‹ ‡‹˜     §        Ž…­ „¦„         ¤  “   ƒ     ˆ  2 ˆ    ˆ  Š  Œ ˆ Œ   ˆ   —          € Š   ƒ  ˜˜ — ‡‹ „ ‡¦˜ ˆ Š›  ƒ 

    —ŠŠ˜ — Š    —ŠŠ˜˜

  Š  

—‹ ‡‹˜  Š ¦„¦—¦‹1˜ ¦„ „—¦‹1˜  „¦—¦‹1˜  „ „¦ —¦‹1˜

‹š‹ —2……Ÿ˜ —2…„Ÿ˜ —2…‹’˜

 

‡“„‡ ‡“„‡

—„ ‡‹˜  Š ¦„¦—¦‹1˜ ¦„ „—¦‹1˜  „¦—¦‹1˜  „ „—¦‹1˜  „„¦—¦‹1˜

Ž„“ —2……’˜ —2…‡„˜ —2…„Ÿ˜ —2…‹‰˜

 

„„ „„

—‰ ‡‹˜   ¦„¦„¦ —¦‹1˜ ¦„¦„ „—¦‹1˜ ¦„¦„¦ —¦‹1˜ ¦„¦„ „—¦‹1˜ ¦„¦ „¦ —¦‹1˜ ¦„ „ „—¦‹1˜ ¦„ „„¦ —¦‹1˜ ¦„ „„ „—¦‹1˜

‹‰„ —2……‹˜ —2………˜ —2……‹˜ —2……“˜ —2…‡„˜ —2…‡Ÿ˜ —2…„‡˜

 

—‹ ‡‹˜   ¦„¦„¦ —¦‹1˜ ¦„¦„¦ —¦‹1˜ ¦„¦„ „—¦‹1˜ ¦„¦„ „—¦‹1˜ ¦ „¦„¦ —¦‹1˜ ¦ „¦„ „—¦‹1˜  „„¦„¦ —¦‹1˜  „„¦„ „—¦‹1˜

„šš —2………˜ —2…‡…˜ —2…‡…˜ —2……š˜ —2…‡š˜ —2…‡‰˜ —2…„‰˜

 

—„ ‡‹˜   ¦„¦„¦ —¦‹1˜ ¦„¦ „¦ —¦‹1˜ ¦„ „„¦ —¦‹1˜ ¦„¦„ „—¦‹1˜ ¦ „¦† „—¦‹1˜ ¦ „ „„ „—¦‹1˜

ŽŽ‹š —2……š˜ —2…‡‰˜ —2…‹„˜ —2…‹“˜ —2…‰’˜

  

„„ „„ „„

—‹ ‡‹˜    ¦‹¦—¦‹1˜ ¦‹ „—¦‹1˜ ¦„ „¦—¦‹1˜ ¦„ „ „—¦‹1˜ ¦ „„¦—¦‹1˜

‡’ŸŸ —2…‡‡˜ —2…„Ž˜ —2…‹’˜

 

„… „…

¦  Š    ‡ ¦ ‡‹„¦ —¦•˜

Ž‰

’“’ ”  ‡  ‡  ’  •    Š           ‰ –… ‹‚„„—Œ —˜™™

  „ —  ˜ — ‡‹ „ ‡¦˜ ˆ Š›  ƒ 

    —ŠŠ˜ — Š    —ŠŠ˜˜

  Š  

¦  Š    ‡ ¦ ‡‹„¦ —¦•˜

¦ „„ „—¦‹1˜

—2…Ž…˜ —2…’‡˜

Œ Œ

„… „…

„…ŸŽ —2…‡‡˜ —2…‹‹˜ —2…‰‰˜ —2…’’˜ —2…šš˜

  Œ Œ

‡“Ž ‡“Ž ‡“Ž ‡“Ž

—‹ ‡‹˜  ¦‹¦—¦˜ ¦‹ „—¦˜ ¦„ „¦—¦˜ ¦„ „ „—¦˜ ¦ „„¦—¦˜ ¦ „„ „—¦˜

 ƒ  —ŠŠ– ‹…™‰… ¨®  2‡˜     Š       „™Ž… £ ‡… 2‰  2‡ ¤               ƒ  —ŠŠ– ‡’™„‰ ¨®  2‡˜              Šƒ    Š   Š  „…… £ ‡… 2‰  2‡   Š        Š  

                Š       ŠŠ    —‰ £ ‡… 2‰  2‡˜        Š     Š     Š         Š  Š ˆ        Š  ƒ      ƒ                         Š      Š        Š         ŠŠ           ƒ        ƒ     €     Š        Š       Š            

    Š     €        Š      Š      –   Š        ¨  ˆ       Š    Š       Š¦    Š  Š ƒ    ¬       ª¦« 1     Š    ’—’—’ œ   š šš  ‰            

     Š           Š Š Š    —ŠŠ–   ‡… 2Ÿ  „ 2‡˜     ¦³  ™ ƒ ™ ³   ª‡…‹«            Š ƒ     Š œ       Œ       ©       Š                –Š ‹…™‰…­     ƒ     ¨  Š      ¢ 

       –   Š          Š  ƒ   ƒ ƒ         Š    Š      ¨                Š Š Š      ™™ ¨ ¨     Š         ƒ        Š  ™™ ¨ ¨   –    ŠŠ         Š     ª‡…‰«      Š         ª‡…Ž™‡…š« ’—’‚’ œ  š š    š        ƒ     Š      Š–     ƒ     ƒ            ŠŠ  Š     –  

 Š      ‰…  ª‡…Ÿ‡…“«    Š    ¢  

      Š   ¢ 

    ¦ „       

’“’ ”  ‡  ‡  ’  •    Š           ‰ –… ‹‚„„—Œ —˜™™

¢      Š             Š ˆ       ¨   –   ¨  Š ¢         ƒ Š —¦‹ 1˜    Š     Š Š       ƒ   ŒŠ  —‘£˜ ª‡…“«   –                 ¬    Š      —ˆ˜     Š     —¦ˆ„Ÿ˜    ƒ      ŠŠ         

 ¢  –Š     ¦ˆ„Ÿ  –Š   §        

 Š    ƒ     ¦ˆ„Ÿ  ˆ  ¨     ‘£‡  ‘£…  Š ƒ  ©Š  ‡…      ‘£   ƒ        €    ‘£ ƒ ¨ – ŒŠ    Š

    —‘£… ‘£‡ ‘£„ ‘£‰ ‘£Ž ‘£’  ‘£Ÿ˜     Œ Š    Š          ¨     —‘£‹ ‘£š ‘£“˜ € 

   ¨  ‘£‡  ¨¨   ¨     ‘£‡  ‘£‰ ƒ  Š ª‡‡…‡‡‡«    ŒŠ  ƒ     •   ª‡…“«    ŒŠ   Š       Š Š¢       ±   ƒ      Š Š  ‹Ž ‘     ª‡‡„‡‡‹« ‘£‡      ƒ  –     Š  a 

                ŒŠ  Š  ƒ      —¤  ’˜    ŠŠ     Š    ¨        ŒŠ     ª‡…’« ”       Š    

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  Š        ƒ Šƒ  ŠŠ–  ƒ   “…­ Š ˆ ‡…­ Š ˆˆ  …‡­ Š ˆˆˆ Š    Š  Š        ¨  ª‡š« ƒ Š        Œ Š  Š  ƒ  ŠŠ      Š   Šƒ 

    Š     Š  ª‡‰Ÿ« ¦ ƒ  Š  ¨        ƒ   ŠŠ    ¯ °

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        §         ‰    Š  ˆ ª‡ŽŽ™‡Ž“« ˆ          ¥      –     ¤‘ 

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•        ‘† Š        ‘† Š Š        • –  ‹      ª‡Ÿ‰« ˆ         –  ƒ         ‹     –

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       ƒ      „  2¦‹       1 „ 1 ¦„  $ ¦2 ‹ 1¦

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          ¬      ƒ    ª„…‰„…Ž«     Š  

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ƒ    Š     Š    ƒ     ‡‹   Š         Š     Š   ¤  “  Š  ƒ ‡¦  „¦  Š   ƒ –         Š    „¦„      „¦  Š  Š     ƒ   ƒ 

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’“’ ”  ‡  ‡  ’  •    Š           ‰ –… ‹‚„„—Œ —˜™™

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ŠŠ                     ƒ     Š           –  Š ¦„  ¦‹    Š —  €  ‰„˜ ˆ      „¦™ ‡‹ Š   „¦  Š     ƒ   ƒ    ‡¦™ „¦ – •  Š

 Š   ‡‹           ƒ  –Š    —    ‰˜ ªŸ„„‡‰«

š„

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€    ‚ƒ             –       Š 

       Š

          —  ¤  ‡˜      ¨        

 –         –     Š     

      ˆ      ƒ        –      ¨  ƒ Š   Š       Š                 —€  ‰„‡™‰„‰  ‰‹‡˜        ¨  ƒ Š           •    – —€  ‰‹‡ ‰‹„˜ ‘

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   ŠŠ–   ‡„ m  2‡ – 

         ‡………ž‡            Š       —€  ‰‹‡˜ ¤      Š     Œ –              

   Š   Š Š Š              Œ              ƒ    œ‡  œ„                •    – ŠŠ  

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ƒ ƒ     –      Š      ¨  ƒ                •    –               Š         Š    ƒ      ƒ  ˆ

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 ƒ  ¤       Šƒ                 Š       –   

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   ŠŠ       Š                    ƒ         Œ ¢      ƒ •     Š                  ƒ       •                       ƒ ƒ     ¢     ¢  Š                

š‰

’“’ ”  ‡  ‡  ’  •    Š           ‰ –… ‹‚„„—Œ —˜™™

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CURRÍCULUM NORMALIZADO COMPLETO (CVNC)

García-Martín,M.L., García-Espinosa,M.A., Ballesteros,P. Bruix,M., S. Cerdán. TITULO: Hydrogen turnover and subcellular compartmentation of hepatic (2-13C) glutamate and (3-13C) aspartate as detected by high field 13C NMR. J. Biol. Chem. (2002) 277, 7799-7807 I.F.: 5.520 (41/276 Biochemistry and Molecular Biology) ISSN:0014-5793123

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METABOLISM AND BIOENERGETICS: Hydrogen Turnover and Subcellular Compartmentation of Hepatic [2- 13 C]Glutamate and [3-13C]Aspartate as Detected by 13C NMR

J. Biol. Chem. 2002, 277:7799-7807. doi: 10.1074/jbc.M107501200 originally published online December 14, 2001

Access the most updated version of this article at doi: 10.1074/jbc.M107501200 Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites. Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 47 references, 16 of which can be accessed free at http://www.jbc.org/content/277/10/7799.full.html#ref-list-1

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Mari?a L. Garci?a-Marti?n, Mari?a A. Garci?a-Espinosa, Paloma Ballesteros, Marta Bruix and Sebastián Cerdán

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 277, No. 10, Issue of March 8, pp. 7799 –7807, 2002 Printed in U.S.A.

Hydrogen Turnover and Subcellular Compartmentation of Hepatic [2-13C]Glutamate and [3-13C]Aspartate as Detected by 13C NMR* Received for publication, August 6, 2001, and in revised form, December 13, 2001 Published, JBC Papers in Press, December 14, 2001, DOI 10.1074/jbc.M107501200

Marı´a L. Garcı´a-Martı´n‡, Marı´a A. Garcı´a-Espinosa§, Paloma Ballesteros¶, Marta Bruix储, and Sebastia´n Cerda´n** From the Instituto de Investigaciones Biome´dicas C.S.I.C., c/Arturo Duperier 4, E-28029 Madrid, Spain, the ¶Department of Organic Chemistry and Biology UNED, c/Senda del Rey 9 E-28040 Madrid, Spain, and the 储Instituto de Estructura de la Materia C.S.I.C., c/Serrano 119, E-28006 Madrid, Spain

13

Hydrogen turnover in hepatic metabolites begins with the transport of water across the plasma membrane (1, 2). Cytosolic water is then distributed rapidly among different subcellu-

* This work was supported in part by Grants SAF 2001–2245 from the Spanish Ministry of Science and Technology (MCT), Grants 08.1/ 0023/97 and 08.1/0046/98 from the Community of Madrid (to S. C. and P. B.), and strategic group Grant 2000 –3 from the Community of Madrid (to P. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Supported by a predoctoral fellowship from the MCT. § Received a predoctoral fellowship from Consejo Superior de Investigaciones Cientı´ficas. ** To whom correspondence and reprint requests should be addressed. Tel.: 34-91-585-4633; Fax: 34-91-585-4587; E-mail: [email protected] uam.es. This paper is available on line at http://www.jbc.org

lar organelles (3, 4) experiencing a variety of enzyme catalyzed exchange reactions which replace stereospecifically, the preexisting metabolite hydrogens with those from subcellular water (5, 6). The turnover completes when replaced metabolite hydrogens return to intracellular water through the same exchange reactions, eventually leaving the organelle and the cell. Classical studies analyzed the exchange between specific hydrogens of metabolites and those of the solvent, providing well established tools to investigate the kinetic mechanisms of enzymes in vitro (6, 7). Similarly, tritiated or deuterated water were used to probe metabolic turnover of fatty acids and carbohydrates (8, 9). However, the exchange of specific hydrogens from hepatic amino acids, like glutamate or aspartate, with hydrogens from intracellular water or its potential metabolic implications remained less explored. 13 C NMR techniques have been used routinely in the last decades to investigate the turnover of individual metabolite carbons in vivo or in vitro in animals and humans by administering precursors selectively enriched in 13C (10, 11). Notably, most metabolite carbons are attached to one or more hydrogen atoms, a circumstance which should allow to extend the use of 13 C NMR to analyze hydrogen turnover. To this end, we proposed earlier a double isotope labeling strategy which combined the administration of a 13C-labeled substrate and 2H2O, monitoring hydrogen-deuterium exchange at steady state through the analysis of the geminal and vicinal1 isotopic shifts or 2H-13C couplings detected in observable 13C resonances by high resolution 13C NMR (5, 12). The present study reports on the kinetics of enzyme-catalyzed exchange of metabolite hydrogens with deuterons from intracellular heavy water with an emphasis on the exchanges occurring in the H2 and H3 hydrogens of hepatic [2-13C]glutamate and [3-13C]aspartate. The kinetics of 1H-2H exchange in the H2 and H3 hydrogens of glutamate and aspartate depicted very rapid components, occurring significantly faster than previously reported time courses of 13C labeling in the neighboring carbons (13–17). The quicker time scale allowed to resolve in time, faster processes than previously accessible to conventional experiments of 13C turnover. In particular it became possible to observe directly ␣-ketoglurate/glutamate exchange and oxalacetate/ aspartate exchange as well as to follow in a time resolved manner, the sequence of events involved in the traffic of these metabolites through mitochondria and cytosol ex vivo. Preliminary accounts of this work have been reported (18, 19).

1 Geminal or vicinal locations relative to an observed 13C refer to hydrogen or deuterium atoms located at one or two bonds distance from the observed 13C.

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C NMR monitored the dynamics of exchange from specific hydrogens of hepatic [2-13C]glutamate and [3-13C]aspartate with deuterons from intracellular heavy water providing information on ␣-ketoglutarate/ glutamate exchange and subcellular compartmentation. Mouse livers were perfused with [3-13C]alanine in buffer containing or not 50% 2H2O for increasing periods of time (1 min < t < 30 min). Liver extracts prepared at the end of the perfusions were analyzed by high resolution 13 C NMR (150.13 MHz) with 1H decoupling only and with simultaneous 1H and 2H decoupling. 13C-2H couplings and 2H-induced isotopic shifts observed in the glutamate C2 resonance, allowed to estimate the apparent rate constants (forward, reverse; minⴚ1) for (i) the reversible exchange of [2-13C]glutamate H2 as catalyzed mainly by aspartate aminotransferase (0.32, 0.56), (ii) the reversible exchange of [2-13C]glutamate H3proS as catalyzed by NAD(P) isocitrate dehydrogenase (0.1, 0.05), and (iii) the irreversible exchanges of glutamate H3proR and H3proS as catalyzed by the sequential activities of mitochondrial aconitase and NAD isocitrate dehydrogenase of the tricarboxylic acid cycle (0.035), respectively. A similar approach allowed to determine the rates of 1H-2H exchange for the H2 (0.4, 0.5) or H3proR (0.3, 0.2) or the H2 and H3proS hydrogens (0.20, 0.23) of [3-13C]aspartate isotopomers. The ubiquitous subcellular localization of 1H-2H exchange enzymes and the exclusive mitochondrial localization of pyruvate carboxylase and the tricarboxylic acid cycle resulted in distinctive kinetics of deuteration in the H2 and either or both H3 hydrogens of [2-13C]glutamate and [3-13C]aspartate, allowing to follow glutamate and aspartate trafficking through cytosol and mitochondria.

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Hydrogen Turnover and Subcellular Compartmentation EXPERIMENTAL PROCEDURES

resonance (cf. Figs. 3 and 4); (i) the reversible exchange of H2 originating [2-13C, 2-2H]glutamate with rate constants k1, k⫺1, catalyzed mainly by aspartate aminotransferases and revealed by a 1:1:1 triplet (t); (ii) the reversible exchange of the H3proS hydrogen originating [2-13C, 3-2HS]glutamate with rate constants k2, k⫺2, catalyzed by cytosolic isocitrate dehydrogenase (NADP) revealed by the shifted singlet (ss); (iii) the reversible exchange of hydrogens H3proR and H2 originating [2-13C, 2-2H, 3-2HR]glutamate with rate constants k3, k⫺3, caused by the combined activities of aconitase and aspartate aminotransferase, revealed by shifted triplet (st); and (iv) the irreversible deuterations of both H3 hydrogens originating [2-13C, 3-2H2]glutamate with rate constant k4 caused by the sequential activities of mitochondrial aconitase and isocitrate dehydrogenase in the tricarboxylic acid cycle, revealed by the doubly shifted singlet (dss). In the case of [3-13C]aspartate the model considers, observing the C3 resonance (cf. Figs. 3 and 4); (v) the reversible exchange of H2 originating [3-13C, 2-2H]aspartate as catalyzed by aspartate aminotransferase (k5, k⫺5) and detected in the shifted singlet (ss); (vi) the reversible exchange of H3proR originating [3-13C, 3-2HR]aspartate as catalyzed by fumarase (k6, k⫺6), and detected in the triplet (t); and (vii) the occurrence of both H2 and H3proS exchanges on the same [3-13C]aspartate isotopomer originating [3-13C, 2-2H, 3-2HS]aspartate as catalyzed by aspartate aminotransferase and the tricarboxylic acid cycle enzymes succinate dehydrogenase, fumarase, and malate dehydrogenase (k7, k⫺7), detected in the corresponding shifted triplet (st). The following differential equations apply, d[[2-13C]Glu]/dt⫽ [[2-13C,2-2H]Glu]k⫺1⫹[[2-13C,3-2HS]Glu]k⫺2⫹[[2-13C,2-2H,3-2HR]Glu]k⫺3 ⫺[[2-13C]Glu]k1⫺[[2-13C]Glu]k2⫺[2-13C]Glu]k3 ⫺[[2-13C]Glu]k4⫹f1⫹f2⫹f3⫹f4 (Eq. 1) d[[2-13C,2-2H]Glu]/dt⫽ k1 [[2-13C]Glu]⫺k⫺1 [[2-13C,2-2H]Glu]⫺f1

(Eq. 2)

d[[2-13C,3-2HS]Glu]/dt⫽ k2 [[2-13C]Glu]⫺k⫺2 [[2-13C,3-2HS]Glu]⫺f2 13

2

(Eq. 3)

2

d[[2- C,2- H,3- HR]Glu]/dt⫽ k3 [[2-13C]Glu]⫺k⫺3 [[2-13C,2-2H,3-2HR]Glu]⫺f3

(Eq. 4)

d[[2-13C,3-2H2]Glu]/dt⫽k4 [[2-13C]Glu]⫺f4

(Eq. 5)

d[[3-13C]Asp]/dt⫽ k⫺5[[3-13C,2-2H]Asp]⫹k⫺6 [[3-13C,3-2HR]Asp]⫹k⫺7[3-13C,[2-2H,3-2HS]Asp] ⫺ k5[[3-13C]Asp]⫺k6 [[3-13C]Asp]⫺k7 [[3-13C]Asp]⫹f5⫹f6⫹f7 13

(Eq. 6)

2

d[[3- C,2- H]Asp]/dt⫽ k5 [[3-13C]Asp]⫺k⫺5 [[3-13C,2-2H]Asp]⫺f5

(Eq. 7)

d[[3-13C,3-2HR]Asp]/dt⫽ k6 [[3-13C]Asp]⫺k⫺6 [[3-13C,3-2HR]Asp]⫺f7 13

2

(Eq. 8)

2

d[[3- C,2- H,3- HS]Asp]/dt⫽ k7 [[3-13C]Asp]⫺k⫺7 [[3-13C,2-2H,3-2HS]Asp]⫺f6 1

2

13

(Eq. 9)

where the concentration of the different ( H, H, C) isotopomers is given as a relative contribution to the total C2 glutamate or C3 aspartate resonances observed as indicated in Fig. 3; k1(⫺1) ⫺ k7(⫺7) refer to the apparent pseudo-first order rate constants of the reactions depicted in Fig. 1 and f1-f7 denote the relative input-output fluxes required to maintain steady state conditions in the system, respectively. Optimal values for these parameters were estimated by nonlinear fitting of the kinetics of deuteration in the different (1H, 2H) isotopomers of [2-13C]glutamate and [3-13C]aspartate (cf. Fig. 5). Briefly, the system of differential equations was provided with an initial set of estimated parameters which was successively refined iteratively by minimizing the squared difference between calculated and mean experimental values of relative deuteration for every investigated isotopomer in each time point. For this purpose, we used the Stella package for simulations

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Perfusion Techniques and Experimental Design—Male Swiss albino mice (30 ⫾ 5 g) starved for 24 h but having access to drinking water ad libitum were used as donors. Mouse livers (2.5 ⫾ 0.5 g) were isolated and perfused outside the magnet as described previously (12, 13, 20). Briefly, animals were anesthetized with Nembutal (50 mg kg⫺1 body weight), the abdomen opened and the portal vein cannulated. During cannulation and subsequent surgery, livers were pre-perfused in the flow-through mode with Krebs-Ringer bicarbonate (KRB) buffer (119 mM NaCl, 24 mM NaHCO3, 4,7 mM KCl, 1,2 mM MgSO4, 1,2 mM KHPO4, 1,3 mM Ca2Cl, 95% O2, 5% CO2, pH 7.4, ⫾0.04; 37 °C; 6 ml/min). After isolation of the liver from the animal carcass, the perfusion medium was changed to 6 mM [3-13C]alanine (99.9% 13C, Isotec Inc., Miamisburgh, OH) in KRB under recirculating conditions for 30 min. This time was sufficient to achieve steady state 13C labeling in glutamate, aspartate, and alanine carbons (12, 13, 21). After this equilibration period the perfusion medium was changed to 6 mM [3-13C]alanine in KRB containing 50% (v/v) 2H2O (99.9% 2H, Appollo Scientific Ltd., Stockport, UK) maintaining the recirculation for 1, 3, 5, 7, 10, 15, or 30 min. During this perfusion period with 50% 2H2O mouse livers maintained appropriate bioenergetics and metabolism (21). At the end of the perfusion, livers were freeze-clamped with aluminum tongs precooled in liquid nitrogen and stored at ⫺70 °C until extraction. Perchloric acid extracts from these livers were prepared, neutralized with KOH, lyophilized, and resuspended in 2H2O (99.9% 2H) prior to high resolution 13C NMR analysis (12). Total contents of glutamate, aspartate, and alanine of liver extracts obtained at the end of the perfusions were determined by automatic ionic exchange chromatography after ninhidrin derivatization (22). Hepatic oxygen consumption due to alanine oxidation was measured polarographicaly using Clark oxygen electrodes located before and after the liver in the perfusion circuit, correcting for the basal rate of endogenous oxygen consumption (23). High Resolution 13C NMR—Extracts from livers were analyzed (pH 7.2, 22 °C) by 13C NMR at 14.09 T (150.9 MHz) using a Bruker AMX-600 NMR spectrometer (Bruker Analystische Messtechnik, Rheinsteten, Germany). Acquisition conditions (150.9 MHz) were: ␲/3 pulses, 33.3 KHz sweep width, 128 K data table (1.97 acquisition time), 7.97-s recycle time, and ⬃600 scans. To minimize nuclear Overhauser effects in quantitation, broad band proton decoupling was applied only during the acquisition. In addition, 1H-decoupled and 2H-decoupled 13C NMR spectra were obtained from the same samples at 11.9 Tesla with a Bruker AVANCE 500 NMR Spectrometer operating at 125.76 MHz for 13 C, equipped with a commercial crioprobe refrigerated with liquid nitrogen. Conditions were: ␲/3 pulses, 25.06 KHz, 64 K data table, 6-s total cycle time, and 4096 scans. 2H decoupling was performed simultaneously with 1H decoupling only during the acquisition, using the lock channel and a software driven lock switch. Chemical shifts were referred to that of a 10% dioxane solution (67.4 ppm) placed in a concentric coaxial capillary. At least four extracts from different livers were analyzed for every time point. Determination of Relative (1H, 2H, 13C) Isotopomer Populations— Covalent binding of one or more 2H atoms to 13C results in the formation of 2H-13C couplings and 2H-induced isotopic shifts which split and shift the perprotonated 13C resonance into characteristic 2H-13C multiplets (5, 12, 24). Replacement of one geminal hydrogen by one deuterium splits the original 13C singlet into a 1:1:1 triplet (19.21 ⬍ 1JCH ⬍ 22 Hz) inducing a geminal isotopic shift (⫺0.25 ⬍ ⌬1 ⬍ ⫺0.33 ppm). Multiple replacements of geminal hydrogens with two or three deuterons result in additive isotopic shifts and 2H-13C coupling patterns of five or seven line multiplets, respectively. Smaller and additive isotopic shifts are also observed upon deuterium substitution of one or more vicinal hydrogens (⫺0.03 ⬍ ⌬2 ⬍ ⫺ 0.11 ppm). Vicinally shifted resonances maintain the multiplicity caused by the geminal couplings since vicinal couplings to 2H are too small to be resolved. Thus high resolution 13 C NMR allows to determine the number of deuterium replacements, their relative contributions, and their geminal or vicinal location with respect to the observed 13C carbon of specific 13C isotopomers through the analysis of the shifted and unshifted (1H,2H)13C multiplets (5, 12). This is conveniently accomplished using the simulation program WINDAISY (Bruker Analystische Messtechnik, Rheinstetten, Germany) which allows the quantitative determination of the relative contributions of the individual (1H,2H)13C multiplets to the analyzed resonance. Model of 1H-2H Exchange in [2-13C]Glutamate and [3-13C]Aspartate Isotopomers—Fig. 1 depicts the model used for the analysis of the exchange of the H2 and H3 hydrogens from [2-13C]glutamate or [3-13C]aspartate with intracellular heavy water deuterons. In the case of [2-13C]glutamate the model considers, observing the C2

Hydrogen Turnover and Subcellular Compartmentation

7801

of system dynamics (High Performance Systems, Hanover, NH) as implemented in a desktop Pentium III platform and the BMDP package (BMDP Statistical Software Inc., Los Angeles, CA) as implemented on a Alpha 2100 mainframe computer (Digital Corp., San Diego, CA). RESULTS 1

2

H- H Exchange in Hepatic Metabolites as Detected by 13C NMR—Fig. 2 shows illustrative examples of the time courses of 1 H-2H exchange as detected in the (1H,2H)13C multiplets of the C2 carbon of [2-13C]glutamate (top) and the C3 carbon of [3-13C]aspartate (bottom) from extracts obtained at increasing times of perfusion with [3-13C]alanine in KRB containing 50% 2 H2O. Similar spectra were obtained for the C2 carbon of [2-13C]aspartate, the C3 carbon of [3-13C]lactate, the C3 carbon of [3-13C]glutamate and, with significantly smaller intensity, for the C4 carbon of [4-13C]glutamate (not shown). All these resonances depicted evident (1H,2H)13C multiplet patterns revealing characteristic and time-dependent increases in deuteration. These spectra were obtained at 14.09 Tesla, a field which improved considerably earlier results both in terms of sensitivity and resolution (12). However, despite the high field used, more quantitative determinations of the kinetics of 1H-2H exchange required deconvolution of the observed (1H,2H)13C mul-

tiplets into their individual components. Fig. 3 shows representative deconvolutions of the C2 carbon resonance of [2-13C]glutamate and C3 carbon resonance from [3-13C]aspartate, respectively. Deconvolution of the C2 carbon resonance revealed contributions from five different [1H, 2H, 13 C]glutamate isotopomers: (i) [2-13C]glutamate as the unshifted singlet (55.50 ppm); (ii) [2-13C, 2-2H]glutamate as a triplet (1J13C2H ⫽ 19,2 Hz) shifted ⫺0.32 ppm; (iii) [2-13C, 3-2H]glutamate as an upfield shifted singlet (⌬1⫽ ⫺0.07 ppm); (iv) [2-13C, 2-2H, 3-2H⬘]glutamate as an upfield shifted triplet (⌬1⫽ ⫺0.39 ppm, 1J13C2H ⫽ 19.2 Hz); and (v) [2-13C, 3,3⬘2 H2]glutamate as a doubly shifted singlet (⌬1 ⫽ ⫺0.14 ppm). Similarly, deconvolution of the C3 resonance from [3-13C]aspartate revealed the relative contributions of four different (13C, 1H, 2H) isotopomers: (i) [3-13C]aspartate, as an unshifted singlet (37.30 ppm); (ii) [3-13C, 2-2H]aspartate, as a singlet isotopically shifted ⫺0.08 ppm; (iii) [3-13C, 3-2H]aspartate, as a 1:1:1 triplet (1J13C2H ⫽ 19,21 Hz) shifted ⫺0.28 ppm; and (iv) [3-13C, 2,3-2H2]aspartate, as a 1:1:1 triplet shifted ⫺0.36 ppm. These assignments were confirmed in independent experiments, in which simultaneous 1H and 2H decoupling were applied to the same samples during 13C NMR acquisition. The use of simultaneous 1H and 2H decoupling caused the

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FIG. 1. Model of 1H-2H exchange in the H2 and H3 hydrogens of [2-13C]glutamate (A) and [3-13C]aspartate (B). Exchange processes are characterized by rate constants (k) and input-ouput fluxes (f). Multiplicities for the individual (1H,2H)13C isotopomers detected by 13C NMR in the C2 carbon resonance of [2-13C]glutamate or the C3 carbon resonance of [3-13C]aspartate are abbreviated as: s, singlet; t, triplet; ss, shifted singlet; st, shifted triplet; dss, doubly shifted singlet (see Figs. 3 and 4).

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multiplets derived from 2H-13C couplings to collapse into the corresponding 2H-decoupled singlets, thus reducing the complexity of the (1H,2H)13C spectrum and increasing the sensitivity for 2H-13C isotopomer detection. This triple resonance experiment became particularly useful in the determination of the relative contributions of 2H-13C triplets or higher order multiplets appearing many times with relatively low intensity in 13C specta decoupled only for 1H. Fig. 4 illustrates this aspect by showing a representative comparison of 1H decoupled only (upper panels) and simultaneously 1H- and 2H-decoupled 13 C NMR spectra (lower panels) of the C2 glutamate and C3 aspartate resonances. The 2H- and 1H-decoupled spectra depict more clearly resolved and with increased intensity, the singlet resonances corresponding to the 2H-13C triplets observed the proton decoupled spectrum of [2-13C, 2-2H]- and [2-13C, 3-2H, 2-2H]glutamate (t and st, left panel) or [3-13C, 3-2H]- and [3-13C, 3-2H, 2-2H]aspartate (t and st, right panel), respectively. The concentrations of glutamate and aspartate measured in the extracts were 24.7 ⫾ 4.5 ␮mol g⫺1 dry weight and 8.85 ⫾ 2.3 ␮mol g⫺1 dry weight (n ⫽ 6), respectively, and did not vary appreciably during the entire 2H2O perfusion period. Oxygen consumption due to alanine oxidation under these conditions was 2.3 ⫾ 0.3 ␮mol min⫺1 g⫺1 dry weight (n ⫽ 4). Turnover of Specific Hydrogens from [2-13C]Glutamate and [3-13C]Aspartate—The relative contributions of specific (13C, 2 H, 1H) isotopomers of [2-13C]glutamate and [3-13C]aspartate was determined as indicated in Figs. 3 and 4, in liver extracts prepared at increasing times of perfusion with 50% deuterated KRB and [3-13C]alanine (Fig. 5). In the case of glutamate, [2-13C, 2-2H]glutamate production (t) was the fastest process, [2-13C, 3-2H]glutamate was produced slower (ss) and [2-13C; 3-2H⬘, 2-2H]glutamate or [2-13C, 3-2H2]glutamates (st or dss) were produced even slower but at similar rates. Concerning deuteration of [3-13C]aspartate isotopomers, the fastest process was the deuteration of H2 to

produce [3-13C, 2-2H]aspartate (ss), while the formation of [3-13C, 3-2H]- and [3-13C, 3-2H⬘, 2-2H]aspartates (t and st) was slower. Taken together these results indicate that [2-13C]glutamate and [3-13C]aspartate are deuterated first to their (2-2H) isotopomers, the corresponding (3-2H), (3-2H⬘, 2-2H), or (3-2H2) isotopomers being produced successively later. We performed a more quantitative interpretation of the kinetics of 2H incorporation into [2-13C]glutamate and [3-13C]aspartate by simulating the experimental data of Fig. 5 with the model shown in Fig. 1. This model describes the dynamics of exchange of the H2 and H3 hydrogens of [2-13C]glutamate or [3-13C]aspartate by intracellular heavy water deuterons, starting from the perprotonated molecules. The best fits for the kinetics of individual (13C, 1H, 2H) isotopomers, obtained as described under “Experimental Procedures,” are illustrated by the different lines in Fig. 5. A good agreement between calculated and experimentally measured values can be observed in every case. The apparent rate constants and input-output fluxes optimized in this way are summarized in Table I. Notably, the kinetics for the exchange of some of the hydrogens shown in Table I are significantly faster than previously measured values of turnover from the neighboring 13C carbon, indicating that these hydrogens turn over several times during a single 13C turnover (13, 15, 25, 26). This is the case of the H2 hydrogens of glutamate and aspartate. In previous experiments of mouse liver perfusion performed under the same conditions, [3-13C]alanine-labeled the C2 and C3 carbons of glutamate with apparent rate constants of 0.07 ⫾ 0.02 and 0.06 ⫾ 0.01 min⫺1 and aspartate C2 and C3 with apparent rate constants of 0.15 ⫾ 0.02 and 0.033 ⫾ 0.003 min⫺1, respectively (13).2 Steady state 13C labeling of the glutamate carbons under these conditions is achieved after approximately 30 min of

2

M. Garcı´a-Martı´n and S. Cerda´n, unpublished observations.

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FIG. 2. Time course of 1H-2H exchange in the C2 carbon resonance from [2-13C]glutamate (A) and the C3 carbon resonance of [3-13C]aspartate (B) as observed by 13C NMR. Livers were perfused with 6 mM [3-13C]alanine in Krebs-Ringer buffer containing 50% 2H2O for increasing periods of time. Extracts were prepared at the end of the perfusions and analyzed by 13C NMR (150.90 MHz, 22 °C, pH 7.2). Results show representative spectra obtained after the combination of extracts from three livers perfused under identical conditions.

Hydrogen Turnover and Subcellular Compartmentation

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perfusion. However, the steady-state deuteration of these carbons is reached after 10 –15 min of perfusion only (Fig. 5). As indicated in Table I, the exchange of H2 glutamate occurs with a rate constant of the order of 0.3 min⫺1, approximately five times faster than the turnover of the geminally attached 13C2 carbon. Moreover, in addition to the fast exchange of glutamate H2, it is possible to measure also a slower exchange process of the H2 glutamate hydrogen, occurring only in those glutamate molecules deuterated in H3⬘ (see [2-13C, 2-2H, 3-2H⬘]glutamate, st in Figs. 3–5). This slower H2 exchange occurs essentially at the same rate than the exchange of both H3 hydrogens by deuterium (see [2-13C, 3,3⬘-2H2]glutamate, dss in Figs. 3–5). A similar situation occurs with the H2 hydrogen of [3-13C]aspartate which exchanges approximately five times faster than the attached 13C3 carbon and presents additionally a slower exchange, occurring after one H3⬘ deuteration (see the production of [2-13C, 2-2H]- and [2-13C, 2-2H, 3-2H⬘]aspartate, ss and st in Figs. 3–5). DISCUSSION

During the course of metabolism hydrogen atoms from hepatic glutamate and aspartate are exchanged with those of intracellular water by distinct enzymes (5, 12, 27, 28). The H2 hydrogens of aspartate or glutamate are exchanged mainly by aspartate and alanine aminotransferases and glutamate dehydrogenase (28 –31). The H3proS hydrogen of glutamate (in the ␣-ketoglutarate skeleton) may be exchanged either reversibly by cytosolic isocitrate dehydrogenase (NADP) or irreversibly by

mitochondrial isocitrate dehydrogenase (NAD) (32). The glutamate H3proR hydrogen is exchanged by cytosolic and mitochondrial aconitases (6, 33–35) and glutamate H4 hydrogens are exchanged by citrate synthase (36). In the case of aspartate, the H3proR hydrogen is exchanged by both cytosolic and mitochondrial fumarases while the H3proS hydrogen can be traced with equal probablity to the H2 or H3 hydrogens of fumarate and succinate or to the H3 and H4 hydrogens of ␣-ketoglutarate (34, 37). Present results show that the kinetics of the 1H-2H exchange of the H2 and H3 hydrogens of glutamate and aspartate can be conveniently followed by high field 13C NMR and occur in some cases significantly faster than the previously measured time courses of 13C enrichment (13, 25, 26). The next sections show that the faster timescale and ubiquitous location of 1H-2H exchange enzymes, together with the exclusive mitochondrial location the tricarboxylic acid cycle, allows to investigate the exchange of ␣-ketoglutarate/glutamate or oxalacetate/aspartate between mitochondrial and cytosolic compartments with more detail than previously possible with classical experiments of 13C turnover. 1 H-2H Exchange and Subcellular Compartmentation during 13 [3- C]Alanine Metabolism—The hepatic metabolism of alanine and [3-13C]alanine has been covered in several classical studies (38 – 41). [3-13C]Alanine is transported to the cytosol and transaminated to [3-13C]pyruvate. In the fasted liver, [3-13C]pyruvate enters the mitochondrial tricarboxylic acid cycle mainly through pyruvate carboxylase, originating

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FIG. 3. Representative deconvolutions of the C2 carbon resonances from [2-13C]glutamate (left panels) and the C3 carbon resonances from [3-13C]aspartate (right panels) into the contributions of individual (1H,2H)13C multiplets. 13C NMR spectra (150.90 MHz, 22 °C, pH 7.2) were obtained from the extracts of single livers perfused with 6 mM [3-13C]alanine in 50% 2H2O for 15 min (C2 glutamate) or 10 min (C3 aspartate). Relative contributions of specific isotopomers are given as the fractional contribution of the corresponding multiplet to the total area of the analyzed resonance taken arbitrarily as one. Simulations were performed with the WINDAISY program. Multiplicities of individual components are abbreviated as described in the legend to Fig. 1.

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[3-13C]oxalacetate and [2-13C]␣-ketoglutarate in the first turn. Further metabolism of [2-13C]␣-ketoglutarate in the cycle yields an approximately equimolar mixture of [1-13C]- and [4-13C]succinate and oxalacetate molecules, which loose subsequently the 13C label by decarboxylation. Equilibration of mitochondrial [3-13C]oxalacetate by malate dehydrogenase and fumarase, originates an equimolar mixture of [2-13C]- or [3-13C]oxalacetate and, after condensation with new acetylCoA molecules in the tricarboxylic acid cycle, a mixture of [3-13C]- or [2-13C]␣-ketoglutarate. Mitochondrial [13C]oxalacetate and [13C]␣-ketoglutarate are then rapidly transaminated to the corresponding [13C]aspartate and [13C]glutamate. Under steady-state conditions, a continuous cycling of [13C]aspartate and [13C]glutamate occurs between mitochondria and cytosol, associated mainly to the malate aspartate shuttle as described in Fig. 6. Briefly, [13C]glutamate enters the mitochondrial matrix through the aspartate/glutamate exchanger, returning later to the cytosol as [13C]␣-ketoglutarate through the dicarboxylate carrier after tricarboxylic acid cycle metabolism (40, 42, 43). Coupled to the exit of [13C]␣-ketoglutarate, [13C]malate enters the matrix though the dicarboxylate carrier, leaving it later as [13C]aspartate through aspartate/ glutamate exchanger (42). If under steady-state conditions of 13 C metabolite cycling, intracellular water is substituted partially by 2H2O, deuterons from heavy water will become almost instantly available for exchange with the H2 and H3 hydrogens from both mitochondrial and cytosolic [13C]glutamates and aspartates or the corresponding ketoacid precursors. This is so because 2H2O transport to cytosol or mitochondria proceeds in the millisecond or microsecond range, a much faster rate than that measured in this work for the 1H-2H exchange reactions (cf. Fig. 5) (1, 3, 5). However, the exclusive mitochondrial location of pyruvate carboxylase, succinate dehydrogenase, and citrate synthase will make those irreversible 1H-2H exchanges

and 13C exchanges associated to the operation of the tricarboxylic acid cycle to occur only in the mitochondria. Hydrogen-Deuterium Exchange of H2 and H3 Glutamate— The time courses of deuteration of the H2 and H3 hydrogens of [2-13C]glutamate, as detected by 13C NMR (Fig. 5) support the exchange of ␣-ketoglutarate/glutamate through cytosol and mitochondria as depicted in Fig. 6. A crucial observation is that the H2 and one of the H3 hydrogens from [2-13C]glutamate are shown to be replaced successively at two different rates, fast and slow (Fig. 5). This indicates necessarily that H2 and H3 deuterons incorporated initially in the fast processes (noted with f in Fig. 6) must be removed before the slow deuteron from the solvent (noted with s in Fig. 6) occupies, at a later stage, the same position in the carbon skeleton of ␣-ketoglutarate/glutamate. In the case of the H2 deuterations of [2-13C]␣-ketoglutarate, the formation of [2-13C, 2-2H]glutamate occurs first while the slower H2 deuteration occurs always after one (or both) H3 hydrogens of [2-13C]ketoglutarate have been replaced by deuterium (cf. Fig. 5). For H3 deuterations, the first deuteration originates [2-13C, 3-2H]glutamate fast, the subsequent H3 deuterations producing [2-13C, 3-2H⬘, 2-2H]- and [2-13C, 3-2H2]glutamate much slower and at similar rates. This sequence of events agrees well with the dynamics of ␣-ketoglutarate/glutamate exchange between cytosol and mitochondria and with the topology of the tricarboxylic acid cycle. First, [2-13C, 2-2H]- or [2-13C, 3-2H]glutamates are produced rapidly in the cytosol from ␣-ketoglutarate by cytosolic aspartate aminotransferase, NADP isocitrate dehydrogenase, or aconitase (Fig. 6, f, Pre). These molecules are transferred to the mitochondria where the cytosolic 2H2 deuteron is lost by mitochondrial transamination originating mitochondrial [2-13C]- or [2-13C, 3-2H]␣-ketoglutarate which can enter then the tricarboxylic acid cycle. [2-13C]␣-Ketoglutarate will necessarily be decarboxylated during tricarboxylic acid metabolism to origi-

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FIG. 4. Effects of simultaneous 1H and 2H decoupling on the multiplet structure of the C2 resonance from [2-13C]glutamate (left) and the C3 resonance from [3-13C]aspartate (right). 13C NMR spectra (125.76 MHz, 22 °C, pH 7.2) were acquired from the extract of a liver perfused with [3-13C]alanine in KRB containing 50% 2H2O during 15 min as indicated under “Experimental Procedures.” Upper panels, broad band 1 H decoupling only. Lower panels, broad band 1H and 2H decoupling. Increased sensitivity and resolution in the lower panels allows detection of multiplets difficult to observe with 1H decoupling only (q and sq). Multiplicities of individual components are abbreviated as indicated in Fig. 1. q, quintet; sq, shifted quintet.

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TABLE I Rate constants for 1H-2H exchange and steady state input/output fluxes of individual (13C, 1H,2H) isotopomers determined from the experimental data of Fig. 5 using the model of Fig. 1 Apparent pseudo-first order rate constants and input/output fluxes were determined as described under “Experimental Procedures.” Rate constant (min⫺1) or input/output flux

k1,k⫺1 k2,k⫺2 k3,k⫺3 k4 k5,k⫺5 k6,k⫺6 k7,k⫺7 f1 f2 f3 f4 f5 f6 f7

[2-13C]Glutamate

[3-13C]Aspartate

0.32 ⫾ 0.035, 0.56 ⫾ 0.062 0.1 ⫾ 0.011, 0.05 ⫾ 0.006 0.035 ⫾ 0.004, 0.06 ⫾ 0.007 0.035 ⫾ 0.004 0.4 ⫾ 0.017, 0.5 ⫾ 0.021 0.3 ⫾ 0.013, 0.2 ⫾ 0.008 0.2 ⫾ 0.008, 0.23 ⫾ 0.010 0.008 0.010 0.002 0.008

⫾ ⫾ ⫾ ⫾

0.0009 0.0011 0.0002 0.0009 0.01 ⫾ 0.0004 0.005 ⫾ 0.0002 0.02 ⫾ 0.0008

nate [1-13C]- or [4-13C]succinate, fumarate, malate, and oxalacetate and loosing later the 13C label by decarboxylation. [2-13C, 3-2H]␣-Ketoglutarates will produce [1-13C, 2-2H]succinate and fumarate, [1-13C, 2-2H]- or [1-13C, 3-2H]malate, and eventually [1-13C, 3-2H]oxalacetate, loosing both 13C and 2H labels in the next turn of the cycle. Therefore, both H3 hydrogens (or deuterons) from the original cytosolic ␣-ketoglutarate are removed in the mitochondria during the first turn (and in

every turn) of the tricarboxylic acid cycle. Simultaneously, new [3-13C]oxalacetate molecules are produced in the mitochondria each time [3-13C]pyruvate enters the cycle through pyruvate carboxylase, generating citrate, isocitrate, and newly formed molecules of [2-13C]␣-ketoglutarate. The new mitochondrial [2-13C]␣-ketoglutarate molecules synthesized by the cycle must incorporate the slower mitochondrial deuterium labeling (Fig. 6, s) in either or both of its H3 hydrogens through mitochondrial aconitase and NAD-isocitrate dehydrogenase, respectively. These newly synthesized [2-13C, 3-2H]- or [2-13C, 3-2H2]␣-ketoglutarate molecules can receive finally the slow deuteron in H2 either by transamination or by reductive amination through glutamate dehydrogenase (Fig. 6, s, Post). The reductive amination process of ␣-ketoglutarate requires previously the formation of mitochondrial [4-2HS]NAD(P), resulting probably in a slower and smaller contribution to H2 deuteration of glutamate than transamination (28, 44). Stereospecific Exchange of the H3,H3⬘ Hydrogens of Glutamate—13C NMR is not able to resolve directly the stereochemistry of the single deuterations of the proR and proS H3 hydrogens of [2-13C]glutamate from the vicinal and geminal isotopic shifts observed in the C2 resonance (cf. Figs. 4 and 5). However, it is possible to infer the stereospecificity because the presence of substituents in the 3-R or 3-S configuration of the amino acid is known to have important consequences on the aminotransferase catalyzed exchange of H2 (28, 45). H3 substituents in the erythro-configuration with the amino group inhibit transamination probably because of a steric hinderance, while H3 substituents in the threo-configuration have no significant effect

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FIG. 5. Kinetics of deuteration of the H2 and H3 hydrogens from [2-13C]glutamate (A) or [3-13C]aspartate (B) during perfusions with [3-13C]alanine in KRB containing 50% 2 H2O. Fractional contributions of individual isotopomers in each time point were determined as described in the legend to Fig. 3 and are indicated by specific symbols (insets). Lines represent the best fit to the model of Fig. 1 obtained with the rate constants and fluxes depicted in Table I. Multiplicities are abbreviated as indicated in Fig. 1 corresponding to the following (13C,2H,1H) isotopomers A; s, [2-13C]glutamate; t, [2-13C, 2-2H]glutamate; ss, [2-13C, 3-2H]glutamate; st, 2-13C, 2-2H, 3-2H⬘]glutamate; dss, [2-13C, 3,3⬘2 H2]glutamate. B, s, [3-13C]aspartate; ss, [3-13C, 2-2H]aspartate; t, [3-13C, 3-2H]aspartate; st, [3-13C, 3-2H⬘, 2-2H]aspartate.

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(45). This indicates that [3-2HR]␣-ketoglutarate molecules produced by aconitase may incorporate deuterium in H2 normally, while the [3-2HS]␣-ketoglutarate molecules deuterated by NAD- or NADP-isocitrate dehydrogenases will not incorporate appreciably 2H in H2 or will do it very slowly. This agrees well with the situation found experimentally in Fig. 5. In the case of [2-13C]glutamate, the slow H3⬘ deuteration allows deuterium incorporation in H2 and produces [2-13C, 3-2H⬘, 2-2H]glutamate (st in Figs. 4 and 5), suggesting that it corresponds to the 3-R configuration as incorporated by mitochondrial aconitase. In contrast, substitution of the other H3 hydrogen either fast, as in [2-13C, 3-2H]glutamate (ss in Figs. 4 and 5), or slow as in [2-13C, 3-2H2]glutamate (dss in Figs. 4 and 5), appears to preclude 2H incorporation in H2, suggesting that it corresponds to the 3-S configuration as exchanged by isocitrate dehydrogenases. The fast 2H3S exchange occurs most probably on preexisting cytosolic [2-13C]␣-ketoglutarate while the slow one uses mitochondrial [2-13C]␣-ketoglutarate newly formed in the

tricarboxylic acid cycle. In the latter case, the slow 2H3S deuteration is limited by the previous incorporation of the 2H3R through aconitase. In agreement with this, 2H3R or 2H3R and 2 H3S deuterations are found experimentally to proceed at the same rate, since both are limited by mitochondrial transport and the tricarboxylic acid cycle activity. In this respect it is worthwhile mentioning that the value of 1.15 ⫾ 0.2 ␮mol min⫺1 g⫺1 for the tricarboxylic acid cycle flux calculated from oxygen consumption measurements,3 matches well the value for the tricarboxylic acid cycle flux of 0.9 ⫾ 0.2 ␮mol min⫺1 g⫺1 calculated as the product of the glutamate concentration times the slow rate constant for double deuteration in H3 (k4). Hydrogen-Deuterium Exchange of H2 and H3 Aspartate— The sequence of deuterations observed in the H2 and H3 hydrogens of aspartate follow a similar trend to those described 3 Note that two oxygen molecules are used for every acetyl-CoA oxidized in the cycle.

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FIG. 6. Subcellular compartmentation of ␣-ketoglutarate/glutamate and oxalacetate/aspartate exchange as reflected in the kinetics of deuteration of the H2 and H3 hydrogens from [2-13C]glutamate and [3-13C]aspartate. [2-2H]- or [3-2HS]glutamate (t and ss in Fig. 5A) are produced fast (f) in the cytosol and transported to the mitochondria (a). In the mitochondrial space, glutamate is transaminated to ␣-ketoglutarate loosing the cytosolic H2 deuteron (b). The cytosolic [3-2H3S]deuteron is lost later in the tricarboxylic acic cycle where [2-13C]- and [2-13C, 3-2HS]␣-ketoglutarate produce a mixture of undeuterated and deuterated [1-13C]- and [4-13C]succinate, fumarate, malate (c), and oxalacetate molecules which eventually condense with incoming acetyl-CoA. Mitochondrial H3R (u) and H3S (‰) deuterons are incorporated successively slower (s) into newly formed [2-13C]- or [3-13C]␣-ketoglutarate molecules produced from [3-13C]alanine entering the cycle through pyruvate carboxylase (d). Mitochondrial [2-13C]␣-ketoglutarate molecules deuterated in H3 can finally receive the slowest deuteron (or hydrogen) in H2(s), with transamination kinetics limited by mitochondrial transport and/or the tricarboxylic acid cycle activity (e, st, and dss in Fig. 5A). Further metabolism of H3 deuterated [2-13C]- or [3-13C]␣-ketoglurate in the cycle originates a mixture of deuterated [2-13C]- and [3-13C]succinate, fumarate, and malate (g, scrambled 13C molecules of succinate and fumarate are illustrated over the same carbon skeleton). Cytosolic [3-13C]oxalacetate may be deuterated fast (f) in the cytosol to [3-13C, 2-2H]aspartate through AAT (h) or to [3-13C, 3-2HR]aspartate through fumarase (ss and t in Fig. 5B), a process which appears to inhibit H2 deuteration (i). However, [3-13C, 3-2HS]oxalacetate or [2-13C, 3-2HS]oxalacetate molecules produced during tricarboxylic acid cycle metabolism of [13C]␣-ketoglutarate molecules deuterated in H3 or H4 (d), may become deuterated in H2 originating the slowest (3-13C, 3-2HS, 2-2H) (or 2-13C, 3-2HS, 2-2H) aspartate components (j and st in Fig. 5B). Acetyl-CoA entering the cycle is assumed to be perprotonated. A, deuterated [2-13C]glutamate isotopomers observed in Fig. 5A. B, deuterated [3-13C]aspartate isotopomers observed in Fig. 5B. Pre, before TCA cycle metabolism; Post, after TCA cycle metabolism; C, aspartate/glutamate exchanger; D, dicarboxylate carrier; AAT, aspartate aminotransferase; CS, citrate synthase; AC, aconitase (NAD or NADP); ICDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; FM, fumarase; ␣-KG, ␣-ketoglutarate; Glu, glutamate; Asp, aspartate; Mal, malate; Fum, fumarate. ●, 13C; U, scrambled 13C; E, 12 C, 2H (Š, , u). Black or gray symbols indicate cytosolic or mitochondrial deuterations, respectively. Left or right location of deuterons in the carbon skeleton indicates S or R configuration of the attached carbon. Subscripts c or m indicate cytosolic or mitochondrial. f, fast; s, slow. 13C multiplicities are indicated in parentheses and abbreviated as in Fig. 5.

Hydrogen Turnover and Subcellular Compartmentation

Acknowledgments—We are deeply indebted to Sylvain Meguellatni, Bruker Analytische Messtechnik, Rheinstetten (Germany), and Dr. Detlev Moskau, Bruker AG, Fallanden (Switzerland), for performing the triple resonance, simultaneous proton, and deuterium decoupling experiments.

REFERENCES 1. Yano, M., Marinelli, R. A., Roberts, S. K., Balan, V., Pham, L., Tarara, J. E., de Groen, P. C., and LaRusso, N. F. (1996) J. Biol. Chem. 271, 6702– 6707 2. Koyama, Y., Yamamoto, T., Kondo, D., Funaki, H., Yaoita, E., Kawasaki, K., Sato, N., Hatakeyama, K., and Kihara, I. (1997) J. Biol. Chem. 272, 30329 –30333 3. Lopez-Beltra´n, E. A., Mate, M. J., and Cerda´n, S. (1996) J. Biol. Chem. 271, 10648 –10653 4. Garcı´a-Perez, A. I., Lopez-Beltran, E. A., Kluner, P., Luque, J., Ballesteros, P., and Cerda´n, S. (1999) Arch. Biochem. Biophys. 362, 329 –338 5. Garcı´a-Martı´n, M. L., Ballesteros, P., and Cerda´n, S. (2001) Progr. NMR. Spec. 39, 41–77 6. Popja´ck, G. (ed) (1970) in The Enzymes (Boyer, P. D., ed) Vol. 2, pp. 116 –215, Academic Press, New York 7. Schowen, K. B., and Schowen, R. L. (1982) Methods Enzymol. 87, 551– 606 8. Lowenstein, J. M. (1971) J. Biol. Chem. 246, 629 – 632 9. Rognstad, R., Clark, G., and Katz, J. (1974) Eur. J. Biochem. 47, 383–388 10. Ku¨nnecke, B. (1995) in Carbon-13 NMR Spectroscopy of Biological Systems (Beckman, N., ed) pp. 155–267, Academic Press, New York 11. Shulman, R. G., and Rothman, D. L. (2001) Annu. Rev. Physiol. 63, 15– 48 12. Moldes, M., Cerda´n, S., Erhard, P., and Seelig, J. (1994) NMR in Biomed. 7, 249 –262 13. Moldes, M., Cruz, F., Chapa, F., and Cerda´n, S. (1995) Quart. Mag. Res. Med. Biol. 2, 5–17 14. Mason, G. F., Gruetter, R., Rothman, D. L., Behar, K. L., Shulman, R. G., and Novotny, E. J. (1995) J. Cereb. Blood Flow Metab. 15, 12–25 15. Fitzpatrick, S. M., Hetherington, H. P., Behar, K. L., and Shulman, R. G. (1990) J. Cereb. Blood Flow Metab. 10, 170 –179 16. Chance, E. M., Seeholzer, S. H., Kobayashi, K., and Williamson, J. R. (1983) J. Biol. Chem. 258, 13785–13794 17. Chatham, J. C., Forder, J. R., Glickson, J. D., and Chance, E. M. (1995) J. Biol. Chem. 270, 7999 – 8008 18. Garcı´a-Martı´n, M. L., Garcı´a-Espinosa, M. A., Cruz, F., and Cerda´n, S. (1999) Magn. Reson. Mat. Sci (MAGMA) 8, Suppl. 1, 113 19. Garcı´a-Martı´n, M. L., Garcı´a-Espinosa, M. A., Ballesteros, P., Bailey, L., Bruix, M., and Cerda´n, S. (1999) Proc. Int. Soc. Magn. Res. Med. 7, 37 20. Meijer, D. K. F., Keulemans, K., and Mu¨lder, G. K. (1981) Methods Enzymol. 77, 81–129 21. Moldes, M., Cruz, F., Garcı´a-Martı´n, M. L., Garcı´a-Espinosa, M. A., Alvarez, J., and Cerda´n, S. (1997) Cell. Mol. Biol. 43, 731–740 22. Speckman, D. H., Stein, W. H., and Moore, S. (1957) Anal. Chem. 30, 1190 –1206 23. Misra, H. P., and Fridovich, I. (1976) Anal. Biochem. 70, 632– 634 24. Hansen, P. E. (1988) Progr. NMR Spec. 20, 207–225 25. Sherry, A. D., Zhao, P., Wiethoff, A. J., Jeffrey, F. M., and Malloy, C. R. (1998) Am. J. Physiol. 274, H591–599 26. Jucker, B. M., Lee, J. Y., and Shulman, R. G. (1998) J. Biol. Chem. 273, 12187–12194 27. Mahler, H. K., and Cordes, E. H. (1967) Biological Chemistry, Harper and Row and John Weatherhill, New York 28. Walsh, C. (1979) Enzymatic Reaction Mechanisms, W. H. Freeman, San Francisco, CA 29. Julin, D. A., and Kirch, J. F. (1989) Biochemistry 28, 3825–3833 30. Julin, D. A., Wiesinger, H., Toney, M. D., and Kirch, J. F. (1989) Biochemistry 28, 3815–3821 31. Rife, J. E., and Cleland, W. W. (1980) Biochemistry 19, 2321–2328 32. Rose, Z. B. (1966) J. Biol. Chem. 241, 2311–2313 33. Pickworth-Glusker, J. (1971) in The Enzymes (Boyer, P. D., ed) Vol. 5, pp. 413– 439, Academic Press, New York 34. Englard, S. (1960) J. Biol. Chem. 235, 1510 –1516 35. Schloss, J. V., Emptage, M. H., and Cleland, W. W. (1984) Biochemistry 23, 4572– 4580 36. Kosicki, G. W., and Srere, P. A. (1961) J. Biol. Chem. 263, 2566 –2570 37. Donnelly, D. F., O’Hare, M. C., and Doonan, S. (1985) Int. J. Biochem. 17, 279 –282 38. Cohen, S. M. (1983) J. Biol. Chem. 258, 14294 –14308 39. Cohen, S. M. (1987) Biochemistry 26, 563–572 40. Williamson, J. R., Scholz, R., and Browning, E. T. (1969) J. Biol. Chem. 244, 4617– 4627 41. Groen, A. K., Sips, H. J., Verboorn, R. C., and Tager, J. M. (1982) Eur. J. Biochem. 122, 87–93 42. LaNoue, K. F., and Schoolwerth, A. C. (1979) Annu. Rev. Biochem. 48, 871–922 43. Duszynski, J., Mueller, G., and LaNoue, K. (1978) J. Biol. Chem. 253, 6149 – 6157 44. You, K. S., Allison, W. S., and Kaplan, N. O. (1978) Trends Biochem. Sci. 3, 265–268 45. Jenkins, W. T. (1961) J. Biol. Chem. 236, 1121–1125 46. Mason, G. F., Rothman, D. L., Behar, K. L., and Shulman, R. G. (1992) J. Cereb. Blood Flow Metab. 12, 434 – 447 47. Griffin, J. L., O’Donnell, J. M., White, L. T., Hajjar, R. J., and Lewandowski, E. D. (2000) Am. J. Physiol. Cell Physiol. 279, C1704 –1709 48. Yu, X., White, L. T., Doumen, C., Damico, L. A., LaNoue, K. F., Alpert, N. M., and Lewandowski, E. D. (1995) Biophys. J. 69, 2090 –2102 49. Yu, X., White, L. T., Alpert, N. M., and Lewandowski, E. D. (1996) Biochemistry 35, 6963– 6968 50. Yu, X., Alpert, N. M., and Lewandowski, E. D. (1997) Am. J. Physiol. 272, C2037–2048 51. Weiss, R. G., Stern, M. D., De Alburquerque, C. P., Vandegaer, K., Chacko, V. P., and Gerstenblith, G. (1995) Biochim. Biophys. Acta 1243, 543–548

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for glutamate. The fast deuteration of H2 occurs first originating [3-13C, 2-2H]aspartate, followed by slower deuterations originating [3-13C, 3-2H]aspartate and [3-13C, 2-2H, 3-2H⬘]aspartate, respectively. Similar reasonings to those described for glutamate allow to infer the stereospecificity of H3 deuterations in [3-13C]aspartate. The slow H3⬘ deuteration, originating [3-13C, 3-2H⬘]oxalacetate and malate should be assigned the 2 H3S configuration since it allows H2 deuteration to produce [2-13C, 2-2H, 3-2HS]aspartate. The fast H3 deuteration originating [3-13C, 3-2H]aspartate may then correspond to the [2H3R]aspartate diasteroisomer. Therefore the fast 2H3R deuteration appears to be caused by cysolic fumarase while the slow 2H3S should be incorporated through mitochondrial tricarboxylic acid cycle activity. Concluding Remarks—The results described in this study may present more general implications. Classical 13C NMR approaches to cerebral, cardiac, and hepatic metabolisms used 13 C turnover of glutamate carbons to determine flux through the tricarboxylic acid cycle and associated anaplerotic reactions. Rate-limiting roles of citrate synthase (vtca) and a fast and complete equilibration of all intracellular ␣-ketoglutarate/ glutamate pools by aspartate aminotransferases (vx) and across the mitochondrial membrane were initially assumed in brain, heart, and liver (14 –16, 26, 46). Model calculations provided in these cases values for vx in the range from 1 to 72 times faster than vtca. More recently, further modeling approaches proposed glutamate/oxoglutarate exchange and transport across the mitochondrial membrane as the rate-limiting step of [13C]glutamate turnover in perfused heart (47–51) with vx values closer to unity or even smaller (17, 48). The hydrogen turnover experiments reported here provide direct information on the relative rates of aspartate aminotransferases and the tricarboxylic acid cycle through the specific deuteration reactions observed in the H2 and H3 hydrogens of [13C]glutamate. Our results show that intracellular ␣-ketoglutarate/glutamate exchange can now be resolved at least in two kinetically different components. A fast component involves H2 exchange of the ␣-ketoglutarate/glutamate couple in the cytosol, proceeding approximately five times faster than the tricarboxylic acid cycle rate (vxc ⬇ 5 vtca). A slower component involves deuteration of the H3proR or H3proR and H3proS hydrogens of glutamate requiring ␣-ketoglutarate/glutamate transport through the mitochondrial membrane and the operation of the tricarboxylic acid cycle (vxm ⬇ 1). Some kinetic limitation precludes the equilibration of the mitochondrially produced 2H3R and [3-2H2]glutamates with the complete cellular glutamate pool. However, it is not possible at present to determine which process, tricarboxylic acid cycle, exchange through the mitochondrial membrane, or other, is limiting the formation of the slow components of [13C,2H]glutamate labeling. In summary, this study shows that careful analysis of 2H-13C couplings and 2H-induced isotopic shifts observed by 13C NMR in the C2 glutamate and C3 aspartate carbon resonances reveals accurately the exchange by deuterium of the neighboring H2 and H3 hydrogens. This provides a wealth of information on hepatic ␣-ketoglutarate/glutamate and oxalacetate/aspartate exchange and their subcellular compartmentation. The proposed (1H, 2H, 13C) approach may be easily extended to alternative metabolites and additional carbons, other metabolic pathways or to different tissues.

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CURRÍCULUM NORMALIZADO COMPLETO (CVNC)

Delgado,T.C., Violante,I.R., Nieto-Charques, L., Cerdán, S. Neuroglial metabolic compartmentation underlying leptin deficiency in the obese ob/ob mice as detected by magnetic resonance imaging and spectroscopy methods Journal of Cereb Blood Flow and Metabolism (2011), 31 :2257-2266 I.F.: 5.46 (29/231 Q1 Neurosciences) ISSN: 1559-7016, doi: 10.1038/jcbfm.2011.134

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Feature Article

Neuroglial metabolic compartmentation underlying leptin deficiency in the obese ob/ob mice as detected by magnetic resonance imaging and spectroscopy methods Teresa C Delgado, Ineˆs R Violante, Laura Nieto-Charques and Sebastia´n Cerda´n Laboratory for Imaging and Spectroscopy by Magnetic Resonance (LISMAR), Instituto de Investigaciones Biome´dicas de Madrid ‘Alberto Sols’ C.S.I.C./U.A.M., Madrid, Spain

Manganese-Enhanced Magnetic Resonance Imaging (MEMRI), 1H and 13C High-Resolution-Magic Angle Spinning (HR-MAS) Spectroscopy, and genomic approaches were used to compare cerebral activation and neuronal and glial oxidative metabolism in ad libitum fed C57BL6/J leptin-deficient, genetically obese ob/ob mice. T1-weighted Magnetic Resonance Images across the hypothalamic Arcuate and the Ventromedial nuclei were acquired kinetically after manganese infusion. Neuroglial compartmentation was investigated in hypothalamic biopsies after intraperitoneal injections of [1-13C]glucose or [2-13C]acetate. Total RNA was extracted to determine the effects of leptin deficiency in the expression of representative genes coding for regulatory enzymes of hypothalamic energy pathways and glutamatergic neurotransmission. Manganese-Enhanced Magnetic Resonance Imaging revealed enhanced cerebral activation in the hypothalamic Arcuate and Ventromedial nuclei of the ob/ob mice. 13C HR-MAS analysis showed increased 13C accumulation in the hypothalamic glutamate and glutamine carbons of ob/ob mice after the administration of [1-13C]glucose, a primarily neuronal substrate. Hypothalamic expression of the genes coding for glucokinase, phosphofructokinase, pyruvate dehydrogenase, and glutamine synthase was not significantly altered while pyruvate kinase expression was slightly upregulated. In conclusion, leptin deficiency associated with obesity led to increased cerebral activation in the hypothalamic Arcuate and Ventromedial nuclei, concomitant with significant increases in neuronal oxidative metabolism and glutamatergic neurotransmission. Journal of Cerebral Blood Flow & Metabolism (2011) 31, 2257–2266; doi:10.1038/jcbfm.2011.134; published online 5 October 2011 Keywords: brain imaging; diet; energy metabolism; magnetic resonance; MRI; neuronal–glial interactions

Introduction Obesity is a pandemic syndrome associated with the most prevalent and morbid pathologies in developed countries including heart disease, atherosclerosis, diabetes, and cancer (Das, 2010). The syndrome is thought to arise from disturbances in the highly elaborated mechanisms controlling food intake and Correspondence: Professor S Cerda´n, Laboratory for Imaging and Spectroscopy by Magnetic Resonance (LISMAR), Instituto de Investigaciones Biome´dicas de Madrid ‘Alberto Sols’ C.S.I.C./ U.A.M., C/ Arturo Duperier 4, 28029 Madrid, Spain. E-mail: [email protected] This work was supported in part by grants SAF 2008-01327 and S-BIO-2006-0170 to SC. TCD held a fellowship from the Fundac ¸a˜o para a Cieˆncia e Tecnologia, Portugal (SFRH/BD/17010/2004) and IRV was a fellow from Coimbra University under the Erasmus Program. Received 24 February 2011; revised 26 July 2011; accepted 4 August 2011; published online 5 October 2011

energy expenditure involving both systemic and cerebral signalling systems. Body adiposity is currently believed to be regulated systemically through an endocrine ‘adiposity’-negative feedback loop, mainly through the hormones insulin and leptin (Morton et al, 2006). After a meal, increased circulating leptin levels induce an anorexigenic response consisting of a reduction in food intake and an increase in energy expenditure, whereas in fasting periods, decreased plasma levels of leptin promote increased food intake and decreased energy consumption (Coll et al, 2007). Disruptions in the leptin signalling systems are often associated with obesity. For example, obese humans normally present ‘leptin resistance’, a state of reduced responsiveness to plasma leptin, hampering the inhibition of food intake (Lee et al, 2001). Likewise, the leptin-null ob/ob mice exhibit decreased energy expenditure, hyperphagia, and obesity. The administration of exogenous leptin is known to correct the

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above-mentioned defects in the ob/ob mouse (Pelleymounter et al, 1995). Abundant evidence indicates that the Central Nervous System is the site for leptin’s antiobesity activity, the hypothalamus being the primary cerebral structure supporting leptin performance (Luquet and Magman, 2009). Leptin receptors are expressed at high levels in several hypothalamic regions, mainly in the Arcuate and Ventromedial nuclei both in neurons and in astrocytes (Hakansson et al, 1998; Hsuchou et al, 2009). Indeed by rescuing leptin receptors in the hypothalamus of mice otherwise lacking them, blood glucose and insulin levels are normalized (Coppari et al, 2005). In addition, mice with hypothalamic lesions are resistant to leptin treatment (Koyama et al, 1998). After a meal, leptin released from adipose tissue binds to leptin receptors in the hypothalamus. On leptin binding, the orexigenic neurons releasing neuropeptide Y (NPY) and agouti-related peptide (AgRP) are inhibited and the activity of the anorexigenic neurons expressing pro-opiomelanocortin and the cocaine–amphetamineregulated transcript is increased (Elmquist et al, 2005). Both orexigenic and anorexigenic neurons project to the Paraventricular nuclei and other hypothalamic sites, to modulate the activity of the melanocortin pathway controlling food intake and energy expenditure (Ghamari-Langroudi et al, 2011). In addition to the production of orexigenic or anorexigenic neuropeptides, hypothalamic neurons release excitatory and inhibitory amino-acid neurotransmitters as glutamate and gamma-aminobutyric acid (GABA) from glutamate–glutamine and GABA transcellular cycles. However, the neuroglial interactions underlying hypothalamic leptin-dependent activation remain poorly understood. Recently, it was shown that the anorexigenic pro-opiomelanocortin and the orexigenic NPY/AgRP neurons in the Arcuate nucleus of hypothalamus display GABAergic activity (Hentges et al, 2004) whereas other studies indicated that key neurons involved in the regulation of energy balance are glutamatergic and/or densely innervated by glutamatergic nerve terminals (Collin et al, 2003). A variety of Magnetic Resonance Imaging (MRI) and Magnetic Resonance Spectroscopy methods have been shown recently to provide comprehensive information on cerebral activation and the underlying metabolic coupling mechanisms operating between neurons and glial cells. In particular, ManganeseEnhanced MRI (MEMRI) approaches have revealed the activation of individual hypothalamic nuclei through the localized increased intensity of T1-weighted MRI (Kuo et al, 2007). Similarly, 13C Nuclear Magnetic Resonance investigations of the cerebral metabolism of [1-13C]glucose or [2-13C]acetate contributed comprehensive information on the operation of the neuronal and glial tricarboxylic acid (TCA) cycles and the transcellular exchanges of glutamate– glutamine or GABA between neurons and glia of the whole brain (Gruetter et al, 2002; Garcia-Espinosa Journal of Cerebral Blood Flow & Metabolism (2011) 31, 2257–2266

et al, 2004). More recently, we showed that it is possible to improve the spatial resolution and investigate directly hypothalamic metabolism using 13C HighResolution-Magic Angle Spinning (HR-MAS), a novel technique yielding high quality spectra from very small hypothalamic biopsies (Violante et al, 2009). On these grounds, we investigate here the substrate dependence and neuroglial compartmentation underlying hypothalamic function in control and leptin-deficient, genetically obese ob/ob mice, combining the use of MEMRI and 13C HR-MAS. Our results show that leptin deficiency is associated with an increased cerebral activation in the hypothalamic Arcuate and Ventromedial nuclei, as well as with enhanced hypothalamic neuronal oxidative capacity and glutamatergic neurotransmission. These changes do not appear to involve pleiotropic effects of leptin deficiency on gene expression of regulatory enzymes from energy metabolism or glutamatergic neurotransmission, reflecting mainly a relatively enhanced activity of NPY/AgRP hypothalamic pathway in leptin-deficient animals.

Materials and methods Animals and Research Design Experimental design and protocols were approved by the Institutional Animal Care and Use Committee and met the guidelines of the appropriate government agency. Male control C57BL6/J and leptin-deficient B6.V-Lepob/J ob/ob mice (8- to 12-weeks old) were obtained from the Jackson Laboratories (Bar Harbor, ME, USA). Animals were housed under constant temperature (221C to 251C) and maintained on a 12-hour light/dark cycle with ad libitum access to standard chow food and water. In the morning of the study, blood glucose levels were assessed using a standard glucometer. Plasma insulin or leptin levels were determined using commercially available ELISA kits (Millipore Corporation, Billerica, MA, USA or LINCO Research, St Charles, MO, USA). Brain volumes were determined in each animal from quantitative volumetry studies of T1-weighted MRI sections as described below. The effects of leptin deficiency on hypothalamic function were assessed noninvasively, after the hypothalamic uptake of infused manganese (II) chloride (MnCl2) (Sigma-Aldrich, St Louis, MO, USA) by T1-weighted MRI in control (n = 6) and ob/ob (n = 5) mice fed ad libitum. Anesthesia was induced with a mixture of oxygen/ isofluorane (96/4%) and maintained by administering oxygen/isofluorane (99/1%). Anesthetized animals received a 30-minute constant (0.2 mL/h) infusion of 0.033 mmol/kg body weight of MnCl2 in the tail vein, a nontoxic dose (Silva et al, 2004). Manganese (Mn2 + ) infusion was started after two initial baseline acquisitions, after manganese accumulation in the hypothalamus by successive T1-weighted MRI acquisitions for a total experimental time B2 hours. 13 C HR-MAS studies were performed in a parallel cohort of animals. In these experiments, control (n = 10) and ob/ob

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(n = 9) mice, fed ad libitum, received either an intraperitoneal saline injection of [1-13C]glucose (20 mmol/g body weight, 99.9% 13C, Cambridge Isotope Laboratories, Andover, MA, USA) or [2-13C]acetate (60 mmol/g body weight, 99% 13C, Cambridge Isotope Laboratories). Fifteen minutes after, cerebral metabolism in animals under terminal anesthetic was arrested within 0.8 seconds using a highpower (5 kW) microwave fixation system (TMW-6402C, Muromachi Kikai Co. Ltd., Tokyo, Japan). The hypothalamic region was dissected from the fixed brains and kept frozen at 801C until 1H and 13C HR-MAS spectroscopy analysis. Finally, to investigate the genetic basis of the observed effects we prepared biopsies from the hypothalamus of ad libitum fed control (n = 4) and ob/ob (n = 5) mice and determined the expression of genes involved in the regulation of the glycolysis or TCA and glutamate– glutamine cycles.

Magnetic Resonance Imaging We used T1-weighted MRI to monitor kinetically Mn2 + accumulation under high-spatial resolution conditions and T2-weighted MRI to determine total cerebral volume in vivo. Magnetic Resonance Imaging was performed using a 7.0-T horizontal-bore (160 mm) superconducting magnet (PharmaScan, Bruker BioSpin Gmbh, Ettlingen, Germany) equipped with a 1H selective birdcage resonator of 38 mm. Spin-echo multi-slice T1-weighted imaging of sagittal sections across the mouse brain (field of view 22 mm, 256  256 matrix size, 1 mm slice thickness, 5 slices, 85 m2 in plane resolution) was acquired successively using repetition time = 500 ms, echo time = 11.8 ms and three averages, resulting in a total acquisition time of 6 minutes 24 seconds for each scan. In each scan, regions of interest corresponding to the hypothalamic Arcuate and Ventromedial nuclei, and the fourth ventricle were selected and analyzed, after alignment and regionalization with a standard mouse brain atlas (Paxinos and Franklin, 2001). Signal intensity changes, were assessed using a standard image-processing software (NIH ImageJ 1.37v, http://rsbweb.nih.gov/ij/index.html), normalizing the values of each animal to those obtained before Mn2 + infusion. T2-weighted MRI axial images were acquired under identical resolution conditions with repetition time = 3,000 ms, echo time = 60 ms and three averages. T2 sections swept the entire mouse brain and cerebral areas in each slice were quantified by using the freely accessible Image-processing software (NIH ImageJ 1.37v). The brain volume of each slice was obtained by multiplying the MRI determined brain area by the slice thickness, the sum of all slices providing the brain volume.

1

H-decoupled 13C HR-MAS spectra were acquired using a pulse-acquire sequence with WALTZ-16 1H decoupling applied during the acquisition and relaxation delay periods. Conditions were p/4 pulse width, 64 k data points acquired in 8 k scans with 5 seconds recycle delay with a total acquisition time B16 hours. The chemical shifts of the 13C resonances were referred to those of the lactate C3 peak (21.9 parts per million (p.p.m.)) in the glucose infusion experiments or to the acetate C2 peak (24.5 p.p.m.) in the acetate infusion experiments. Spectral deconvolution was performed using a PC-based Nuclear Magnetic Resonance analysis program, NUTS (Acorn, Freemont, CA, USA). 1 H HR-MAS spectra were acquired at the beginning and after the overnight 13C acquisition for quantification of total metabolite concentrations and verification of sample stability. Acquisition parameters for the 1H HR-MAS sequence were 5 seconds water presaturation, echo time = 144 ms, t = 1 ms, n = 100, 10 kHz spectral width, 32 k data points, and 128 scans. Total metabolite concentrations were quantified by using a modified version of the LCModel processing software programme (Linear Combination of Model Spectra, http://s-provencher.com/pages/schtm; Provencher, 1993) with a customized set of spectra from cerebral metabolites (Righi et al, 2009).

Gene Expression Assays Total RNA was prepared from hypothalamic biopsies using the RNAspin Mini RNA Isolation kit (Amersham Biosciences, GE Healthcare, Piscataway, NJ, USA) and retrotranscribed to cDNA using the high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). The expression of the genes for the main enzymes regulating glycolysis, TCA cycle, and glutamate–glutamine cycling was assayed by Real-Time PCR (RT-PCR) assays using the individual probes for each gene commercialized as TaqMan1 (Applied Biosystems). The following genes (gene symbol, TaqMan1 probe) were investigated: GCK, Glucokinase (mCG21380, Mn00439129_m1); PFK, Phosphofructokinase (mCG8414, Mm00445461_m1); PDH, Pyruvate Dehydrogenase (mCG14551, Mm00468675_m1); PK, Pyruvate Kinase (mCG17567, Mm00443090_m1); and GS, Glutamate-ammonia Ligase or Glutamine Synthetase (mCG20133, Mm00725701_s1).

Statistical Analysis Results are shown as mean±s.e.m. and unpaired Student’s t-test or two-way analysis of variance was used to compare control and ob/ob mice. Comparisons with P < 0.05 were considered statistically significant.

1 H and 13C High-Resolution-Magic Angle Spinning Magnetic Resonance Spectroscopy 1 H and 13C HR-MAS spectra were acquired at 11.7 T with a Bruker AVANCE wide bore spectrometer equipped with a magic angle spinning accessory (Bruker Biospin, Rheinstetten, Germany). Biopsies from the hypothalamus were placed in 50 mL HR-MAS Zirconium Oxide rotors, transferred to the HR-MAS probe and spun at 4 kHz (277 K).

Results Physiological and Endocrine Variables

Ob/ob mice are obese when compared with control animals (53±1 versus 26±1 g, P < 0.01). As expected, in the fed state, ob/ob mice glycemia was slightly lower Journal of Cerebral Blood Flow & Metabolism (2011) 31, 2257–2266

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than in controls (9.7±0.6 versus 12.5±0.8 mmol/L, P < 0.05) (probably reflecting the hyperinsulinemia observed, 7.7±1.6 versus 0.6±0.2 ng/mL, P < 0.01, in agreement with previous results; Hinoi et al, 2008). Elevated blood glucose levels detected in either ob/ob or control mice reflect the fact that animals were under heavy anesthesia. After overnight feeding, leptin secretion was virtually undetectable in ob/ob mice relative to control animals (0.1±0.1 versus 2.5±1.0 ng/ mL, P < 0.05). Analysis of T2-weighted MRI scans (data not shown) revealed that the cerebral volume was significantly reduced in the ob/ob mice relative to their counterpart controls (428±18 versus 475±7 mm3, P < 0.05), in agreement with a recent report where obesity was associated with detectable brain volume deficits in cognitively normal elderly subjects (Raji et al, 2009). Manganese-Enhanced Magnetic Resonance Imaging of Leptin Deficiency

Figure 1 shows a representative T1-weighted image of the mouse brain after Mn2 + uptake (upper panel) and the time course of normalized signal intensity changes in the hypothalamic Arcuate and Ventromedial nuclei as well as in the fourth ventricle of control and ob/ob mice during Mn2 + infusion (lower panel), localized as described in Materials and methods. The enhancement of signal intensity in the forth ventricle (V4) was similar in both groups of animals. However, normalized signal intensities in the hypothalamic Arcuate (Arc) and Ventromedial (VMH) nuclei during Mn2 + infusion were consistently higher in the ob/ob mice than in the controls.

The average five-fold increase in MEMRI signal observed in the nuclei of leptin-deficient animals is consistent with significantly enhanced hypothalamic activation.

Glutamatergic and GABAergic Neurotransmission Under Leptin Deficiency 1

H HR-MAS spectra from representative hypothalamic tissue biopsies after [1-13C]glucose or [2-13C]acetate administration are illustrated in Figures 2A and 2B, respectively. Comparisons between the 1H spectra acquired before and after each 13C spectrum (not shown) showed that samples did not suffer significant degradation during the B16-hour acquisition period necessary to obtain the 13C HR-MAS spectra (Violante et al, 2009). Hypothalamic metabolite concentrations for control and ob/ob mice (relative to N-acetyl aspartate), after the administration either of [1-13C]glucose or of [2-13C]acetate are presented in Figure 3. After the administration of [1-13C]glucose, ob//ob mice showed reduced hypothalamic glucose concentrations relative to control animals whereas the relative concentrations of GABA, glutamate, and glutamine were similar in both groups. After the intraperitoneal injection of [2-13C]acetate, no significant differences were observed between hypothalamic metabolite concentrations of control and ob/ob mice. However, in control animals the hypothalamic myo-inositol concentrations were lower than those determined in the animals receiving [1-13C]glucose infusions and did not change appreciably after the [2-13C]acetate infusion.

Figure 1 Representative T1-weighted image of the mouse brain (sagittal section) after intravenous infusion of Manganese (Mn2 + ) in a control mouse (upper panel). A coronal section of the area of interest is also represented in the upper left insert. The change in normalized signal intensities with time is shown in the lower panels for the hypothalamic Arcuate nucleus (Arc), hypothalamic Ventromedial nucleus (VMH) and the forth ventricle (V4) in ob/ob mice, n = 5 (black squares) and in control animals, n = 6 (gray squares). Data are shown as average±s.e.m., *P < 0.05 relative to controls. Journal of Cerebral Blood Flow & Metabolism (2011) 31, 2257–2266

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Figure 2 1H High-Resolution-Magic Angle Spinning (HR-MAS) spectra (500 MHz, 277 K, 4 KHz) from representative hypothalamic tissue biopsies of a control mouse after (A) [1-13C]glucose or (B) [2-13C]acetate administrations. Assignments: Ac, acetate; GABA, gamma-aminobutyric acid; Glu, glutamate; Gln, glutamine; Glx, glutamate and/or glutamine; Lac, lactate; NAA, N-acetyl aspartate; p.p.m., parts per million.

Figure 3 Relative concentrations of acetate, gamma-aminobutyric acid (GABA), glucose, glutamine, glutamate, lactate, and myoinositol with respect to N-acetyl aspartate (NAA) in the hypothalamus of control and ob/ob mice after the administration of either [1-13C]glucose or [2-13C]acetate. Data are represented as average±s.e.m., *P < 0.05 is indicated. White columns correspond to controls infused with [2-13C]acetate (n = 5), light gray columns to controls infused with [1-13C]glucose (n = 5), gray columns to ob/ob mice infused with [2-13C]acetate (n = 4), and finally dark gray columns to ob/ob mice infused with [1-13C]glucose (n = 5). Acetate/NAA ratios are shown multiplied by 10.

Representative 13C HR-MAS spectra from hypothalamic biopsies after [1-13C]glucose or [2-13C]acetate administration are depicted in Figures 4A and 4B, respectively. In the experiments with [1-13C] glucose, the areas of 13C HR-MAS resonances were normalized to the area of the myo-inositol (mI) C1, C3 (73.2 p.p.m.) resonance (Cerda´n et al, 1985). The incorporation of 13C from the labelled precursor in the different 13C HR-MAS detectable carbons revealed that [1-13C]glucose and [2-13C]acetate were efficiently used as energy substrates in the

Figure 4 13C High-Resolution-Magic Angle Spinning (HR-MAS) spectra (125 MHz, 277 K, 4 KHz rotation) from representative hypothalamic biopsies of a control mouse after (A) [1-13C]glucose or (B) [2-13C]acetate administration. Ac, acetate; GABA, gamma-aminobutyric acid; Glu, glutamate; Gln, glutamine; Lac, lactate; p.p.m., parts per million.

hypothalamus of both types of animals. Moreover, the different labelling patterns observed in the hypothalamus parallel those previously described in the whole brain, with higher 13C labelling obtained after the [1-13C]glucose infusions (Cerda´n et al, 1990; Chapa et al, 2000). The lower incorporation detected after [2-13C]acetate administration reflects the smaller incorporation of 13C label from labelled acetate into hypothalamic metabolites, suggesting a relatively lower contribution of oxidative glial metabolism to hypothalamic labelling. Figure 5 compares 13C incorporation from [1-13C]glucose or [2-13C]acetate in individual carbons of metabolites from the hypothalamus of control and ob/ob mice. In Figure 5A, the administration of [1-13C]glucose resulted in significantly increased 13 C incorporation (ca. three-fold) in glutamate and glutamine carbons of ob/ob mice without a concomitant increase in the total concentration of these metabolites (see Figure 3). This suggests that the leptin-null ob/ob mice depict net increases in neuronal oxidative capacity and glutamatergic neurotransmission together with elevated glutamate–glutamine cycling. Since the cerebral glucose concentration decreases slightly in ob/ob animals (Figure 3) and its 13 C content increases (Figure 5A), the large increases in neuronal oxidative capacity and glutamine cycling reported here, may include a small contribution derived from increased 13C enrichment in the cerebral glucose precursor. With labelled acetate, the smaller concentrations of myo-inositol in control and ob/ob mice (Figure 3), made the natural abundance myo-inositol C1,C3 13C resonances not detectable in these animals, precluding their absolute quantification. As described for the whole brain, the glutamate/ Journal of Cerebral Blood Flow & Metabolism (2011) 31, 2257–2266

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Figure 5 (A) 13C concentration of relevant hypothalamic metabolite carbons relative to the C1,C3 myo-inositol resonance (73.2 p.p.m.) after administration of [1-13C]glucose for control (n = 5) and ob/ob mice (n = 5). (B) Hypothalamic GluC4/ GlnC4 ratios obtained after the administration of [1-13C]glucose or [2-13C]acetate. White columns correspond to controls infused with [2-13C]acetate (n = 5), light gray columns to controls infused with [1-13C]glucose (n = 5), gray columns to ob/ob mice infused with [2-13C]acetate (n = 4), and finally dark gray columns to ob/ob mice infused with [1-13C]glucose (n = 5). Assignments: GABA, gamma-aminobutyric acid; Glc, glucose and its respective anomers a and b; Glu, glutamate; Gln, glutamine; Lac, lactate. Data are represented as average±s.e.m., *P < 0.05 and **P < 0.01 relative to controls are indicated.

glutamine labelling ratio was higher with [1-13C]glucose as substrate and smaller with [2-13C]acetate as substrate (Figure 5B), a similar metabolic compartmentation effect to that described in the whole brain (Cerda´n et al, 1990; Chapa et al, 2000). No significant difference in the glutamate C4/glutamine C4 ratio of control and ob/ob mice infused with [2-13C]acetate was measured while a significant increase in the glutamate C4/glutamine C4 ratio was detected, as expected, in the [1-13C]glucose-infused animals. These findings suggest that glial oxidative metabolism and the glutamine cycle are not significantly modified during leptin-dependent hypothalamic appetite regulation in the [2-13C]acetate-infused animals. Taken together, our results disclose a significantly increased neuronal oxidative capacity and glutamate–glutamine cycling in the hypothalamus of ob/ob mice.

Gene Expression Studies in Control and ob/ob Mice Hypothalamus

To investigate if the metabolic and glutamate– glutamine cycling differences observed between control and ob/ob mice were due to genetic determinants, we investigated the expression of the genes coding for the main regulatory enzymes of energy metabolism including glycolysis, the TCA cycle, and glutamateric neurotransmission in ob/ob mice relative to controls. In particular, we assayed the expression of GCK, PFK, PDH, PK, and GS (Figure 6). Journal of Cerebral Blood Flow & Metabolism (2011) 31, 2257–2266

Figure 6 Relative expression of the genes coding for the main regulatory enzymes of glycolysis, tricarboxylic acid (TCA) cycle metabolism, and glutamate–glutamine cycling, in ob/ob mice (n = 5) (dark gray columns) relative to controls (n = 4). Data are represented as average±s.e.m. and *P < 0.05 is indicated. GCK, glucokinase; PFK, phosphofructokinase; PDH, pyruvate dehydrogenase; PK, pyruvate kinase; and GS, glutamateammonia ligase (glutamine synthetase).

No significant differences were observed in the bulk expression of the main regulatory enzymes of glycolysis (GCK and PFK) and glutamate–glutamine cycle (GS). However, gene expression of PK was significantly upregulated in the ob/ob mice hypothalamus.

Discussion In the fed state, leptin is secreted from adipose tissue and binds to the hypothalamic leptin receptor inhibiting appetite. In the leptin-deficient ob/ob mice, hypothalamic leptin signalling is drastically reduced and hyperphagia develops leading to obesity. In this study, MEMRI was used to compare hypothalamic activation in ad libitum fed control and in the obese leptin-deficient ob/ob mice. It is a valuable tool for assessing noninvasively regions of cerebral activity since it combines two important properties of manganese: (1) as an indicator of calcium (Ca2 + ) influx entering the neurons during activation and (2) as a paramagnetic label, its intracellular accumulation yielding positive MRI contrast in activated areas (Silva et al, 2004). Calcium is an essential second messenger necessary for brain function as neuronal depolarization leads to a rise in intracellular Ca2 + levels derived from increased transport across the plasma membrane through ligand-gated or voltage-gated Ca2 + channels. Intracellular Ca2 + rise is essential for neurotransmitter release involved in neuronal signal transduction (Nicholls et al, 1992). Since Mn2 + competes effectively with Ca2 + transport, the MEMRI signal detected is thought to reflect increased Ca2 + uptake during cerebral activation and can be used to interrogate whole-brain neuronal tracts (Chuang and Koretsky, 2006). Indeed, MEMRI was previously used successfully to investigate alterations of

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cerebral activity in key appetite-regulatory regions of the hypothalamus after peripheral injection of orexigenic or anorexigenic peptides in healthy control mice (Kuo et al, 2007). Our results show increased Mn2 + accumulation concomitant with enhanced cerebral activity in the hypothalamic Arcuate and Ventromedial nuclei of the leptin-null obese ob/ob mice as compared with controls. It is well known that homeostatic levels of leptin inhibit the orexigenic NPY/AgRP neurons and stimulate the anorexigenic neurons expressing pro-opiomelanocortin and cocaine–amphetamine-regulated transcript in the hypothalamic Arcuate nucleus (Elmquist et al, 2005). On fasting, reduced circulating leptin levels release the inhibition of the NPY/AgRP neurons, resulting in increased orexigenic activity (Takahashi and Cone, 2005). In the leptin-deficient ob/ob mice, the inhibition of NPY/AgRP neurons is almost completely eliminated, driving the orexigenic response even higher. This is supported by previous results where a higher excitatory tone onto NPY neurons and a marked net increase in inhibitory tone onto the pro-opiomelanocortin neurons was observed in hypothalamic Arcuate nucleus slices of the ob/ob mice (Pinto et al, 2004). In contrast with the hypothalamic Arcuate nucleus, the hypothalamic Ventromedial nucleus does not produce large amounts of orexigenic or anorexigenic neuropeptides. However, a vesicular glutamate transporter protein, a marker for glutamatergic neurons, is expressed in the vast majority of these neurons suggesting that glutamate, most probably through glutamate/glutamine neuroglial cycling, may have an important role integrating the metabolic information in the hypothalamic Ventromedial nucleus (Collin et al, 2003). Early studies on the regulation of appetite and obesity emphasized the role of peptide hormones, neuropeptides, and monoamines acting on membrane receptors to trigger intracellular signalling cascades, with relatively little attention dedicated to the role of amino-acid neurotransmission and associated neuroglial interactions (Morton et al, 2006). The view that amino-acid neurotransmitters are present in key hypothalamic neurons that regulate appetite and energy status modified this perception (Meister, 2007). In vivo and in vitro 1 H and 13C Nuclear Magnetic Resonance approaches have been successfully used in the study of neurotransmitter metabolism and the associated neuroglial interactions during sensory or motor activation (Sappery-Marinier et al, 1992; Hyder et al, 1996). However, the relatively large voxel size used in the acquisition of in vivo 13C Magnetic Resonance spectra precludes its use for studying the relatively small appetite-controlling hypothalamic area of small rodents. Likewise, the relatively large amounts of cerebral tissue needed to prepare brain extracts for high-resolution 13C Nuclear Magnetic Resonance constituted an important limitation. Recently, a new methodology combining 13C labelled substrates with 13C HR-MAS Spectroscopy was proposed to

provide further insights into hypothalamic neurotransmitter metabolism (Violante et al, 2009). 13C HRMAS Spectroscopy offers the advantage of providing information on cerebral metabolic pathways by allowing for the use of high-resolution spectroscopy in smaller tissue biopsies. 13 C HR-MAS Spectroscopy analysis indicates that the leptin-dependent hypothalamic activation involves mainly increases in neuronal oxidation and glutamatergic neurotransmission. Indeed, it has been previously reported that the injection of glutamate in the hypothalamus rapidly elicits an intense feeding response in satiated healthy rats (Stanley et al, 1993). In contrast with other cerebral activation paradigms (e.g., motor and sensorial) where increased glycolytic responses are observed as lactate increases (Ueki et al, 1988), the neuronal oxidative response appears to be predominant in leptin-dependent appetite regulation. Increased neuronal oxidative metabolism in ob/ob mice involves necessarily increased neuronal pyruvate availability, consistent with the bulk PK gene upregulation as measured by RT-PCR. Hypothalamic PK expression appears to be associated with the prandial state as in fasting (low leptin levels). PK is overexpressed relative to the fed state (high leptin) (Poplawski et al, 2010). In this sense, leptin deficiency may further account for the overexpression of the hypothalamic PK gene in the ob/ob mice as detected in the present study. This finding appears to contrast with the modelling comments by Jolivet et al (2010) where neurons are proposed to be unable to upregulate glycolytic flux. However, this discrepancy may only be apparent. Our gene expression assays measure the net balance in hypothalamic gene expression, making the results compatible with opposite changes in the neuronal or glial compartments. In this sense, it is possible that the net increase in hypothalamic PK expression reflects the balance between a more marked increase in astrocytic PK expression and a decreased neuronal PK expression. In any case, the increased PK expression found did not result in increased lactate levels, suggesting that the ultimate metabolic impact of hypothalamic PK upregulation may be limited by posttranslational events. It should be mentioned in this respect here, however, that independent work reported also that neurons are able to increase their oxidative metabolism in parallel with an increase in pyruvate availability as generated by neuronal glycolysis during sustained checkerboard stimulation of human visual cortex in vivo (Gjedde and Marrett, 2001). The elevated incorporation of 13C from [1-13C]glucose into hypothalamic glutamine in ob/ob mice reveals increased glutamine and glutamate cycling as associated with increased glutamatergic activity (Sibson et al, 1997), providing also additional information on the origin of increased neuronal pyruvate. Our results reveal that the increased [4-13C]glutamine labelling is produced in ob/ob mice under conditions where glial oxidative metabolism, as monitored by the oxidation of [2-13C]acetate, is not Journal of Cerebral Blood Flow & Metabolism (2011) 31, 2257–2266

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significantly enhanced. This suggests that the ATP required for augmented glutamine synthesis, may be mainly derived from astrocytic anaerobic glycolysis, supporting a glial origin for the additional pyruvate required to fuel the increased neuronal oxidation detected. Notably, we found no significant change in the lactate 1H or 13C HR-MAS resonances in ob/ob mice, implying that the increased lactate produced to support the augmented glutamine synthesis, is eventually consumed, most probably as pyruvate to fuel the neuronal cycle. Furthermore, since leptin deficiency stimulates NPY/AgRP neurons and inhibits POMPCT/ cocaine–amphetamine-regulated transcript neurons, it is most probable that the increased oxidative activity and glutamatergic neurotransmission detected by 13C HR-MAS reflect these circumstances, as visualized by the corresponding increases in MEMRI signal intensities in the hypothalamic Arcuate and Ventromedial nuclei. It should be mentioned, however, that these results do not exclude the operation of additional

signalling molecules and mechanisms regulating appetite and obesity, including ghrelin, peptide YY, fatty acids, insulin, and glucose itself (Morton et al, 2006). To the best of our knowledge, this is the first time that a positive correlation is established between increased cerebral oxidative activity and glutamatergic neurotransmission and the elevated MEMRI signal observed during cerebral activation. These concepts are summarized in Figures 7A and 7B in the frame of previous neuroendocrine concepts (Morton et al, 2006; Coll et al, 2007). In conclusion, the data presented in this work show that leptin deficiency associated with obesity leads to increased cerebral activation in the hypothalamic Arcuate and Ventromedial nuclei. These changes involve enhanced neuronal oxidative capacity and glutamatergic neurotransmission whereas pleiotropic effects of leptin deficiency in gene expression appear to be much less pronounced under these conditions.

Figure 7 Summary of the effects of leptin in the hypothalamus of control (A) and ob/ob mice (B), as revealed by ManganeseEnhanced Magnetic Resonance Imaging (MEMRI) and 13C High-Resolution-Magic Angle Spinning (HR-MAS). In the Arcuate nucleus of control animals, leptin (gray triangles, red arrow up) binds to the leptin receptors (inverted Y) inhibiting (negative red circles) the orexigenic Agouti-related peptide/Neuropeptide Y (AgRP/NPY) neurons and stimulating (positive red circles) the anorexigenic proopiomelanocortin/cocaine- and amphetamine-regulated transcript (POMC/CART) neurons. In the genetically obese, leptin-null ob/ob mice (red arrow down), the physiological inhibition of the AgRP/NPY neurons in the Arcuate and downstream Paraventricular (PVN) connections is almost fully released (positive red circles), resulting in augmented neuronal oxidative capacity (darker TCAn), enhanced glutamatergic neurotransmission and neuroglial glutamate/glutamine cycling (darker transcellular arrows) without appreciable effects in the astrocytic oxidative capacity (TCAg). Together, these changes lead to relative increases in MEMRI signal intensities in the Arcuate (MEMRI red circles + ) and 13C labelling from [1-13C]glucose in glutamate/glutamine in AgRP/NPY neurons and downstream paraventicular (PVN) connections and melanocortin (MC) pathway, resulting in an uncompensated orexigenic response and obesity. A, Astrocyte; ARC, Arcuate; GLU, glutamate; MC4, MC3, melanocortin receptors; N, neuron; TCA, tricarboxylic acid cycle; PVN, Paraventricular. Journal of Cerebral Blood Flow & Metabolism (2011) 31, 2257–2266

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The possibility to visualize noninvasively hypothalamic leptin activity by MEMRI and evaluate the underlying neuroglial interactions by 13C HRMAS opens a new avenue in the molecular imaging of signalling cascades and may become instrumental for the development of novel therapeutic strategies for obesity and other feeding disorders.

Acknowledgements The authors are indebted to Mr Javier Pe´rez CSIC for careful drafting of the illustrations. The authors would also like to acknowledge Santiago Canals CSIC for helpful discussion regarding MEMRI experimental protocols and Jesu´s Pacheco-Torres CSIC for insights of the LC-Model software program.

Disclosure/conflict of interest The authors declare no conflict of interest.

References Cerda´n S, Kunnecke B, Seelig J (1990) Cerebral metabolism of [1,2-13C2]acetate as detected by in vivo and in vitro 13C NMR. J Biol Chem 265:12916–26 Cerda´n S, Parrilla R, Santoro J, Rico M (1985) 1H NMR detection of cerebral myo-inositol. FEBS Lett 187:167–72 Chapa F, Cruz F, Garcia-Martin ML, Garcia-Espinosa MA, Cerda´n S (2000) Metabolism of (1-13C)glucose and (2-13C, 2-2H3)acetate in the neuronal and glial compartments of the adult rat brain as detected by 13C, 2H NMR Spectroscopy. Neurochem Int 37:217–28 Chuang KH, Koretsky A (2006) Improved neuronal tract tracing using manganese enhanced magnetic resonance imaging with fast T(1) mapping. Magn Reson Med 55:604–11 Coll AP, Farooqi S, O’Rahilly S (2007) The hormonal control of food intake. Cell 129:251–62 Collin M, Backberg M, Ovesjo M, Fisone G, Edwards R, Fujiyama F, Meister B (2003) Plasma membrane and vesicular glutamate transporter mRNAs/proteins in hypothalamic neurons that regulate body weight. Eur J Neurosci 18:1265–78 Coppari R, Ichinose M, Lee CE, Pullen AE, Kenny CD, McGovern RA, Tang V, Liu SM, Ludwig T, Chua Jr SC, Lowell BB, Elmquist JK (2005) The hypothalamic arcuate nucleus: a key site for mediating leptin’s effects on glucose homeostasis and locomotor activity. Cell Metab 1:63–72 Das UN (2010) Obesity: genes, brain, gut, and environment. Nutrition 26:459–73 Elmquist J, Coppari R, Balthasar N, Ichinose M, Lowell B (2005) Identifying hypothalamic pathways controlling food intake, body weight, and glucose homeostasis. J Comp Neurol 493:63–71 Garcia-Espinosa MA, Rodrigues TB, Sierra A, Benito M, Fonseca C, Gray HL, Bartnik BL, Garcia-Martin ML, Ballesteros P, Cerda´n S (2004) Cerebral glucose metabolism and the glutamine cycle as detected by in vivo and in vitro 13C NMR spectroscopy. Neurochem Int 45:297–303

Ghamari-Langroudi M, Srisai D, Cone RD (2011) Multinodal regulation of the arcuate/paraventricular nucleus circuit by leptin. Proc Natl Acad Sci USA 108:355–60 Gjedde A, Marrett S (2001) Glycolysis in neurons, not astrocytes, delays oxidative metabolism of human visual cortex during sustained checkerboard stimulation in vivo. J Cereb Blood Flow Metab 21:1384–92 Gruetter R, Seaquist ER, Ugurbil K (2002) A mathematical model of compartmentalized neurotransmitter metabolism in the human brain. Am J Physiol Endocrinol Metab 281:E100–12 Hakansson ML, Brown H, Ghilardi N, Skoda RC, Meister B (1998) Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus. J Neurosci 8:559–72 Hentges ST, Nishiyama M, Overstreet LS, Stenzel-Poore M, Williams JT, Low MJ (2004) GABA release from proopiomelanocortin neurons. J Neurosci 24:1578–83 Hinoi E, Gao N, Jung DY, Yadav V, Yoshizawa T, Myers MG, Chua Jr SC, Kim JK, Kaestner KH, Karsenty G (2008) The sympathetic tone mediates leptin’s inhibition of insulin secretion by modulating osteocalcin bioactivity. J Cell Biol 183:1235–42 Hsuchou H, Pan W, Barnes MJ, Kastin AJ (2009) Leptin receptor mRNA in rat brain astrocytes. Peptides 30: 2275–80 Hyder F, Chase JR, Behar KL, Mason GF, Siddee KM, Rothman DL, Shulman RG (1996) Increased tricarboxylic acid flux in rat brain during forepaw stimulation detected with 1H, 13C NMR. Proc Natl Acad Sci USA 93:7612–7 Jolivet R, Allaman I, Pellerin L, Magistretti PJ, Weber B (2010) Comment on recent modelling studies of astrocyte-neuron metabolic interactions. J Cereb Blood Flow Metab 30:1982–6 Koyama K, Shimabukuro M, Chen G, Wang MY, Lee Y, Kalra PS, Dube MG, Kalra SP, Newgard CB, Unger RH (1998) Resistance to adenovirally induced hyperleptinemia in rats. Comparison of ventromedial hypothalamic lesions and mutated leptin receptors. J Clin Invest 102:728–33 Kuo YT, Parkinson JR, Chaudhri OB, Herlihy AH, So PW, Dhillo WS, Small CJ, Bloom SR, Bell JD (2007) The temporal sequence of gut peptide CNS interactions tracked in vivo by magnetic resonance imaging. J Neurosci 27: 12341–8 Lee J, Reed D, Price R (2001) Leptin resistance is associated with extreme obesity and aggregates in families. Int J Obes Relat Metab Disord 25:1471–3 Luquet S, Magman C (2009) The central nervous system at the core of the regulation of energy homeostasis. Front Biosci 1:448–65 Meister B (2007) Neurotransmitters in key neurons of the hypothalamus that regulate feeding behaviour and body weight. Physiol Behav 92:263–71 Morton G, Cummings E, Baskin G, Barsh S, Schwartz W (2006) Central nervous system control of food intake and body weight. Nature 443:289–95 Nicholls JG, Martin R, Wallace BG, Fuchs PA (eds) (1992) From Neuron to Brain, 4th edn., Sinnauer Associates: Sunderland, MA, USA Paxinos G, Franklin K (eds) (2001) The Mouse Brain in Stereotaxic Coordinates. Academic Press, Inc.: New York Pelleymounter M, Cullen M, Baker M, Hecht R, Winters D, Boone T, Collins F (1995) Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269:540–3

2265

Journal of Cerebral Blood Flow & Metabolism (2011) 31, 2257–2266

Neuroglial compartmentation during leptin deficiency TC Delgado et al 2266

Pinto S, Roseberry AG, Liu H, Diano S, Shanabrough M, Cai X, Friedman JM, Horvath TL (2004) Rapid rewiring of arcuate nucleus feeding circuits by leptin. Science 304:110–5 Poplawski MM, Mastaitis JW, Yang XJ, Mobbs CV (2010) Hypothalamic responses to fasting indicate metabolic reprogramming away from glycolysis toward lipid oxidation. Endocrinology 151:5206–17 Provencher S (1993) Estimation of metabolite concentrations from localized in vivo proton NMR spectra. Magn Reson Med 30:672–9 Raji C, Ho A, Parikshak N, Becker J, Lopez O, Kuller L, Hua X, Leow AD, Toga AW, Thompson PM (2009) Brain structure and obesity. Hum Brain Mapp 73:1899–905 Righi V, Roda J, Paz J, Mucci A, Tugnoli V, RodriguezTarduch G, Barrios L, Schenetti L, Cerda´n S, Garcı´aMartı´n ML (2009) 1H HR-MAS and genomic analysis of human tumor biopsies discriminate between high and low grade astrocytomas. NMR Biomed 22:629–37 Sappery-Marinier D, Calabrese G, Fein G, Hugg JW, Biggins C, Weiner MW (1992) Effects of photic stimulation on human visual cortex lactate and phosphates using 1H and 31P Magnetic Resonance Spectroscopy. J Cereb Blood Flow Metab 12:618–23 Sibson NR, Dhankhar A, Mason GF, Behar KL, Rothman DL, Shulman RG (1997) In vivo 13C NMR measurements

Journal of Cerebral Blood Flow & Metabolism (2011) 31, 2257–2266

of cerebral glutamine synthesis as evidence for glutamate-glutamine cycling. Proc Natl Acad Sci USA 94:2699–704 Silva A, Lee J, Aoki I, Koretsky A (2004) Manganeseenhanced magnetic resonance imaging (MEMRI): methodological and practical considerations. NMR Biomed 17:532–43 Stanley B, Ha L, Spears L, Dee M (1993) Lateral hypothalamic injections of glutamate, kainic acid, D,Lalpha-amino-3-hydroxy-5-methyl-isoxazole propionic acid or N-methyl-D-aspartic acid rapidly elicit intense transient eating in rats. Brain Res 613:88–95 Takahashi KA, Cone RD (2005) Fasting induces a large, leptin-dependent increase in the intrinsic action potential frequency of orexigenic arcuate nucleus neuropeptide Y/agouti-related protein neurons. Endocrinology 146:1043–7 Ueki M, Linn F, Hossmann KA (1988) Functional activation of cerebral blood flow and metabolism before and after global ischemia of rat brain. J Cereb Blood Flow Metab 8:486–94 Violante IR, Anastasovska J, Sanchez-Canon GJ, Rodrigues TB, Righi V, Nieto-Charques L, Parkinson JR, Bloom SR, Bell JD, Cerda´n S (2009) Cerebral activation by fasting induces lactate accumulation in the hypothalamus. Magn Reson Med 62:279–83

CURRÍCULUM NORMALIZADO COMPLETO (CVNC)

Lizarbe,B., Benitez, A., Sanchez-Montañes, M., Lago-Fernández,L., Garcia-Martín, M.L., Lopez-Larrubia,P., Cerdan, S. Imaging Hypothalamic Activity using Diffusion Weighted Magnetic Resonance Imaging in the mouse and human brains. Neuroimage (2013) 64 : 448-57. I.F. : 5.895 (1/14 Neuroimaging Q1) ISSN :1662-6247, doi : 10.1016/j.neuroimage.2012.09.033

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NeuroImage journal homepage: www.elsevier.com/locate/ynimg

Imaging hypothalamic activity using diffusion weighted magnetic resonance imaging in the mouse and human brain ☆ Blanca Lizarbe a, Ania Benítez a, b, Manuel Sánchez-Montañés b, Luis F. Lago-Fernández b, María L. Garcia-Martin c, d, Pilar López-Larrubia a, Sebastián Cerdán a,⁎ a

Instituto Investigaciones Biomédicas “Alberto Sols” CSIC-UAM, c/Arturo Duperier 4, Madrid 28029, Spain Departamento de Ingeniería Informática, Escuela Politécnica Superior, Universidad Autónoma de Madrid, Cantoblanco, Madrid 28049, Spain Resonancia Magnética Nuestra Señora del Rosario, Príncipe de Vergara 56, 28006 Madrid, Spain d Andalusian Centre for Nanomedicine and Biotechnology (BIONAND), Málaga, Spain b c

a r t i c l e

i n f o

Article history: Accepted 11 September 2012 Available online 19 September 2012 Keywords: Appetite regulation Functional imaging Cerebral activation Diffusion weighted MRI Image analysis Biexponential diffusion

a b s t r a c t Hypothalamic appetite regulation is a vital homeostatic process underlying global energy balance in animals and humans, its disturbances resulting in feeding disorders with high morbidity and mortality. The objective evaluation of appetite remains difficult, very often restricted to indirect measurements of food intake and body weight. We report here, the direct, non‐invasive visualization of hypothalamic activation by fasting using diffusion weighted magnetic resonance imaging, in the mouse brain as well as in a preliminary study in the human brain. The brain of fed or fasted mice or humans were imaged at 7 or 1.5 Tesla, respectively, by diffusion weighted magnetic resonance imaging using a complete range of b values (10b bb 2000 s.mm−2). The diffusion weighted image data sets were registered and analyzed pixel by pixel using a biexponential model of diffusion, or a model-free Linear Discriminant Analysis approach. Biexponential fittings revealed statistically significant increases in the slow diffusion parameters of the model, consistent with a neurocellular swelling response in the fasted hypothalamus. Increased resolution approaches allowed the detection of increases in the diffusion parameters within the Arcuate Nucleus, Ventromedial Nucleus and Dorsomedial Nucleus. Independently, Linear Discriminant Analysis was able to classify successfully the diffusion data sets from mice and humans between fed and fasted states. Present results are consistent with increased glutamatergic neurotransmission during orexigenic firing, a process resulting in increased ionic accumulation and concomitant osmotic neurocellular swelling. This swelling response is spatially extendable through surrounding astrocytic networks until it becomes MRI detectable. Present findings open new avenues for the direct, non‐invasive, evaluation of appetite disorders and other hypothalamic pathologies helping potentially in the development of the corresponding therapies. © 2012 Elsevier Inc. All rights reserved.

Introduction The appetite impulse originates in the brain from an imbalance in the systemic and intrahypothalamic mechanisms controlling food intake and energy expenditure (Morton et al., 2006). Following a meal, increased insulin and leptin levels induce an anorexigenic response consisting in a reduction in food intake and an increase in energy expenditure, whereas in fasting periods decreased plasma levels of leptin and insulin promote increased food intake and energy expenditure. Abbreviations: Dslow, Slow diffusion coefficient; Dfast, Fast diffusion coefficient; FDP, Fast diffusion phase; LDA, Linear Discriminant Analysis; SDP, Slow diffusion phase. ☆ This work was supported in part by grants SAF-2008-01327 and SAF2011-23622 to SC, grant CTQ-2010-20960-C02-02 to PLL, grants S-BIO-2006-0170 and S2010/BMD-2349 to SC, PLL and MSM and grant CAM/UAM (CCG10-UAM/TIC-5864) to LFLF. BL and ABSDC held predoctoral fellowships from the Spanish Ministry of Science and Technology (BES 2009-027615) and the Spanish Agency for International Cooperation and Development. ⁎ Corresponding author. Fax: +34 91 585 4401. E-mail address: [email protected] (S. Cerdán). 1053-8119/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neuroimage.2012.09.033

These systemic and intrahypothalamic neuroendocrine signals interact mainly in the Arcuate Nucleus (ARC), the Ventromedial Nucleus (VMN), Dorsomedial Nucleus (DMN) and the Paraventricular Nucleus (PVN), modifying the balance between the activity of orexigenic neurons, releasing Agouti Related Peptide (AgRP) or Neuropeptide Y (NPY), and anorexigenic neurons releasing Proomileanocortin (POMPCT) or Cocaine and Amphetamine Regulated Transcript (CART) (Coll et al., 2007). In addition to neuroendocrine signaling, appetite stimulation is known to involve hypothalamic increases in glutamatergic and gabaergic neurotransmissions (Delgado et al., 2011; van den Pol et al., 1990). Moreover, it has been recently reported (Spanswick et al., 2012) that the excitatory synaptic control of AgRP neurons is regulated by fasting and hormones, and that increased glutamatergic activity is a necessary requirement for a physiological response to fasting (Liu et al., 2012; Yang et al., 2011). Despite the progress obtained in the interpretation of the molecular events underlying the fasting response, the direct evaluation of appetite regulation in the brain still remains difficult, very often limited to

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indirect measurements of food intake and body weight. On these grounds, the implementation of non‐invasive methodologies for the evaluation of appetite entails considerable relevance. A variety of neuroimaging tools have been proposed including mainly positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) methods (Carnell et al., 2011). PET studies provide information on cerebral activation by detecting the emitted positrons derived from the increased uptake of 18F-deoxiglucose, an event revealing the metabolic coupling between glucose uptake, blood flow and neuronal activity in the hypothalamus during feeding-related stimuli (Gautier et al., 2000; Tataranni et al., 1999). Blood Oxygenation Level Dependent (BOLD) fMRI, infers regional neuronal activity from the changes in magnetic susceptibility that take place during the transition between oxygenated hemoglobin and deoxygenated hemoglobin, occurring after increased oxygen delivery to activated neurons in the hypothalamus of rats and humans (Mahankali et al., 2000; Matsuda et al., 1999). Finally, manganese enhanced magnetic resonance imaging (MEMRI), uses manganese ion accumulation as a surrogate marker of the increased calcium movements occurring during neuronal activation. MEMRI has revealed the time course of hypothalamic activation as a response to the systemic administration of different orexigenic or anorexigenic peptides (Parkinson et al., 2009). Notably, these previous approaches are not devoid of limitations to investigate hypothalamic physiology in animals and man, mainly derived from their reduced spatial and temporal resolution in the PET and BOLD fMRI approaches, and the potential neurotoxicity of Mn2+, in the MEMRI technique. To overcome these limitations, we propose here the use of functional diffusion weighted imaging fDWI (Le Bihan, 2003) a novel functional approach improving the spatial and temporal resolution provided earlier by PET or BOLD and avoiding the use of the potentially toxic doses of manganese precluding the use of MEMRI in humans. Materials and methods Experimental models The experimental protocols used in this study were approved by the appropriate institutional committees and met the guidelines of the appropriate government agency. Experiments with animals were carried out using healthy adult male C57BL/6 mice (n= 12) aged nine weeks. Each animal was investigated in two successive experimental conditions both receiving drinking water ad libitum; “fed”; receiving normal mice chow diet (A04 http://www.safe-diets.com/eng/home/home. html, SAFE Augy, France, 2900 kcal/kg), and “fasted”; following either 48 h (group 1, n = 6, 25± 4 g) or 16 h (group 2, n = 6, 26± 2 g) after complete food removal. In all small animal imaging experiments, anesthesia was initiated in a plexiglass induction box (Isofluorane 2%/99.9% O2, 1 mL/min) and maintained during the imaging time with a nose mask (Isofluorane 1%/O2 99.9% mixture, 1 mL/min). Anesthetized animals were placed in a water heated probe, which maintained the core body temperature at approximately 37 °C during scanning. The physiological state of the animal during the imaging process was monitored by the respiratory rate and body temperature using a Biotrig physiological monitor (Bruker Biospin, Ettlingen, Germany). We performed a pilot study with human subjects to illustrate the potentialities of the proposed methodology in a routine clinical environment. Human participants in the study were six healthy male volunteers, aged 24–33. Conditions for the participation were: (1) healthy clinical trajectory without familiar history of obesity, diabetes or other endocrine disorders; (2) Body Mass Index (BMI) of 18.5–25, corresponding to normal body weight; and (3) volunteers were required to follow a balanced diet (2000–2500 cal/kg) during seven days prior to the basal image acquisitions, with no drinks other than water ad libitum, no medication or abnormal exercise. Specific instructions to follow a balanced diet were provided for each individual at the beginning of the study and the degree of compliance with these obtained

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individually before the imaging sessions. All individuals adhered correctly to the outlined protocol. Each volunteer was imaged in two successive conditions; first, “fed”, after one week of a balanced diet and second “fasted”, 24 h after food deprivation. Blood samples from the median cubital vein were drained before the “fed” and “fasted” image acquisitions and analyzed for routine biochemical parameters T3, T4, TSH and insulin levels. MRI sequences The magnetic resonance imaging (MRI) experiments with mice were performed on a 7.0-T horizontal-bore (16 cm) superconducting magnet equipped with a 1H selective birdcage resonator of 23 mm and a 90 mm diameter gradient insert (36 gauss/cm). Imaging data were acquired using Hewlett-Packard console running Paravision 4.0 software (Bruker Medical Gmbh, Ettlingen, Germany) operating on a Linux environment. Fig. 1 provides an overview of the acquisition and image analysis approaches implemented in this study and the regions of interest investigated, as illustrated for the mouse brain. A collection of diffusion weighted images of the fed and fasted mouse brain was obtained (left panel) and analyzed using either a biexponential diffusion model (upper right panels) or a model-independent Linear Discriminant Analysis approach (lower right panels). The set of DWI was acquired with the conditions indicated below, across an axial plane containing the hypothalamus (Paxinos and Franklin, 2001) with the diffusion gradient oriented along three orthogonal directions; Left–Right (L–R), Antero-Posterior (A–P) and Head–Feet (H–F). This structure was localized using a sagittal section showing the pituitary gland and selecting after the first axial section rostral to it, as indicated in Fig. 2A. This section is located anatomically Bregma −1.46 mm. In the six mice of group 1, the acquisition parameters were (Bruker Pharmascan, Bruker Medical Gmbh, Ettlingen, Germany): repetition time (TR) = 3000 ms, echo time (TE)= 51 ms, four shot EPI readout, averages (Av) = 3, Δ = 20 ms, δ= 4 ms, field of view (FOV)= 35 mm, acquisition matrix= 128 × 128, corresponding to an in-plane resolution of 296 × 296 μm 2, slice thickness of 1.5 mm, number of slices = 3, using a collection of five low b values (10 b b b 100 s.mm−2) and six high b values (200b b b 1800 s.mm−2). The six mice of group 2, were investigated using nine diffusion weighted images (DWI) acquired along the axial plane containing the hypothalamus, with b values (300b b b 2000 s.mm−2). Acquisition parameters were (Bruker AVANCE III, Bruker Medical Gmbh, Ettlingen, Germany): repetition time (TR) =3000 ms, echo time (TE) = 31 ms, four shot EPI readout, averages (Av) = 3, ∆ = 20 ms, δ = 4 ms, field of view (FOV) = 21 mm, acquisition matrix = 128 × 128, corresponding to an in-plane resolution of 164 × 164 μm 2, slice thickness of 1.25 mm and number of slices = 3. T2-weighted (T2w) spin echo anatomical images were acquired previous to the DWI with improved resolution to localize and resolve precisely the hypothalamic region in every mouse. Axial T2w images across the section containing the hypothalamus were acquired using the rapid acquisition with relaxation enhancement (RARE) sequence with the following parameters: TR=3200 ms, TE= 60 ms, RARE factor = 8, Av = 3, FOV = 38 mm (Pharmascan console), 21 mm (AVANCE III console), acquisition matrix= 256× 256 corresponding to an in-plane resolution of 148×148 μm2 (Pharmascan console) or 80×80 μm2 (AVANCE III console), number of slices=3 and slice thickness=1.5 mm (Pharmascan console), or 1.25 mm (AVANCE III console). The magnetic resonance imaging (MRI) experiments with human volunteers were performed in the Magnetic Resonance Unit of the Hospital Nuestra Señora del Rosario (Madrid, Spain), using a GE Medical Systems 1.5-T horizontal-bore superconducting magnet, equipped with a 1H quadrature head resonator. Prior to the imaging experiments, volunteers signed up an informed consent and confidentiality document. Image acquisitions were medically supervised by the neuroradiology

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Fig. 1. Overview of the methodology. Diffusion weighted images (10 b b b 2000 s.mm−2) are acquired consecutively in the same slice, in three diffusion directions A–P, L–R and H–F. The diffusion data set is analyzed with two independent approaches: a biexponential model fit (upper panels) and a Fisher LDA (lower panels). From the biphasic model, colored maps of the independent parameters fitted are obtained, (SDP, FDP, Dslow, Dfast). Investigated areas were the complete hypothalamus (circled dark blue, excluding the third ventricle) and its substructures and the somatosensory cortex (circled in green). Individual hypothalamic nuclei investigated are depicted in light blue (Arcuate Nucleus), yellow (Ventromedial Nucleus) and red (Dorsomedial Nucleus). Using Fisher LDA, the entire diffusion imaging data set from the fed and fasted brains, can be classified between the two feeding sates: with the most different pixels representing the fed (blue) or fasted (red) pixels. The overview is illustrated with the mouse brain data.

and clinical laboratory staff of the clinic. Multi b-value diffusion weighted images were acquired using 6 b values (200b bb 1200 s.mm−2). T2weighted (T2W) spin echo anatomical images were acquired in every subject in the same plane as the DWI, using a rapid acquisition with Fast Relaxation Fast Spin Echo (FRFSE) sequence in coronal orientations. Acquisition parameters were: TR=3200 ms, TE=90 ms, Echo number= 1, Echo Train Length=8, Av=4, FOV=240 mm, acquisition matrix= 512×512, corresponding to an in-plane resolution of 468.7×468.7 μm2, and slice thickness=3 mm. Image analysis Images acquired with the small animal or human scanners were transformed in DICOM format and exported to external HP Z-400 Workstations. Image analysis was performed as described below, using Statistical Parametric Mapping Software (SPM, http://www.fil. ion.ucl.ac.uk/spm/software) and a collection of in‐house developed programs (Matlab v7, The Mathworks, Nattick, MA, USA) for non‐ linear model fitting and classification of diffusion weighted images using Linear Discriminant Analysis. Selection of ROIs In the mouse brain, the cortical area, the hypothalamus and the hypothalamic nuclei ARC, DMN and VMN, were selected manually based on the anatomical descriptions given by the mouse brain atlas (Paxinos and Franklin, 2001). Fig. 1 (leftmost panels) illustrates the localization of the different ROIs in a representative mouse brain. In the human images, hypothalamic and cortical ROIs were selected with the assistance of experienced clinical neuroradiologists (Fig. 4A). Diffusion model We used the biexponential model of attenuation of the DWI signal (Niendorf et al., 1996), as described by the expression; SðbÞ=Sð0Þ ¼ SDP ⋅ expð−b ⋅Dslow Þ þ FDP ⋅ expð−b ⋅ Dfast Þ

ð1Þ

where, S(b) and S(0) represent the individual pixel intensities in the presence and absence of diffusion gradient, b indicates the diffusion weighting factor (s.mm −2), SDP represents the slow diffusion phase

containing water molecules moving with a slow diffusion coefficient Dslow, and FDP refers to the fast diffusion phase containing the water molecules moving with a fast diffusion coefficient Dfast. The addition of diffusion phases represents the total water molecules contributing to the signal decay (SDP + FDP = 1).

Parameter fitting and statistical analysis Parameters of the biexponential model were determined independently for each voxel and direction in the two feeding states, using the non‐linear least-squares fitting Trust-Region algorithm, customized to limit parameter ranges and optimize the goodness of fit. In particular, the goodness of fit (r2) was restricted to be higher than 0.9 to guarantee an optimal fitting. Parameters are presented as mean±standard deviation within the considered ROIs in each animal or human. Statistical comparisons between the fed and fasted conditions were made constructing a cumulative, representative ROI that grouped the contributions of the individual ROIs selected from each mouse or human. The statistical analysis was made first using unpaired Student's t test, comparing each pair of fed and fasted cumulative ROIs. Further differences were assessed using a multiple linear regression model with the generalized estimating equation (GEE), a process that uses robust standard error estimates taking into account within-subjects correlations (Burton et al., 1998). All statistical analyses were performed using SPSS (IBM Statistical Package for the Social Sciences, 2000, http://www.spss.com).

Model-free DWI analyses We also implemented a model-free classification algorithm, based on Fisher Linear Discriminant Analysis (Fisher LDA, Hastie et al., 2001). This model-free approach, allowed to investigate whether the DWI images could be automatically classified into the “fed” and “fasted” groups, without constrains imposed by the biexponential model. LDA constructs a projection of the original data onto a low dimensional subspace trying to maximize the separation amongst the different classes. When there are only two classes, as in the present case (fed/fasted), this subspace is one-dimensional. Hence, the projection may be interpreted as an Appetite Index that can be used to discriminate between the two conditions (see Fig. 1).

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Fig. 2. Imaging appetite by fDWI in the mouse brain. A: Axial and coronal MRI sections containing the hypothalamus in a representative mouse brain model and in a brain atlas (inset). B: Hypothalamic A–P SDP color maps obtained from the DWI of fed or fasted animals (upper panels), superimposed to the corresponding T2-weighted (T2w) anatomical brain images. The hypothalamic region is shown enlarged in the lower panels. C: Values of hypothalamic SDP, Dslow and Dfast parameters (mean±SD) in the L–R, A–P and H–F directions. The number of pixels (N) used in the comparisons was: Nfed =120, Nfasted =141 in L–R; Nfed =95, Nfasted =82 in L–R and Nfed =201, Nfasted =176 in H–F, respectively. D: Cortical areas investigated from a representative mouse brain, in the fed and fasted states (upper images). The A–P SDP values are shown superimposed to anatomical T2w images. The corresponding enlargements of the cortical areas are shown below. E: SDP, Dslow and Dfast values (mean±sd) of six mice in the fed and fasted conditions, and statistical differences (*pb 0.05, **pb 0.005, ***pb 0.001, Student's t test). Nfed =7, Nfasted =21 in L–R; Nfed =31, Nfasted =32 in A–P; and Nfed =57, Nfasted =71 in H–F. F: SPM analysis of the areas that best differentiate the fed and fasted hypothalamus in a representative mouse brain. Note the hypothalamic (yellow arrow) and vascular (blue arrow) activations. G: “Appetite Index” histogram of the fed (blue) and fasted (red) pixels as projected over the calculated LDA vector. Note the almost complete resolution of fed and fasted histograms. H: Panels that illustrate the localization in the mouse brain image of the 5% lowest (fed) and highest (fasted) points located at each end of the Appetite Index histogram.

Results The effects of fasting in the mouse brain using DWI Fig. 2A shows the anatomical localization of MRI sections used to localize the hypothalamus in the mouse brain and the directions of diffusion measurements. Hypothalamic activation after 48 h of fasting is conveniently detected by fDWI as a shift to red in the fasted condition, in the diffusion maps shown in Fig. 2B. The upper panels show the hypothalamic SDP maps (A–P direction) in the fed and fasted conditions, superimposed on the corresponding anatomical T2w images from the whole brain. These hypothalamic SDP maps are shown enlarged in the corresponding lower panels. The shift from blue to orange and red, revealing increased SDP, is clearly detected in the fasted condition. Artworks in Fig. 2C summarize the mean values, standard deviations and comparative statistical significances of the diffusion parameters (SDP, Dslow and Dfast), from six mice under the fed and fasted conditions, investigated in three orthogonal directions (L–R, A–P and H–F). SDP and Dslow increased significantly with fasting (pb 0.005 and p b 0.001) in

the L–R and A–P directions, whereas Dfast decreased in the A–P and H–F orientations (pb 0.005). The impact of this fasting paradigm was investigated additionally in the cerebral somatosensory cortex of the same animals, obtaining a different, albeit significant, diffusion response (Fig. 2D). In particular, subsidiary effects of fasting become significant as reflected by the red-shift in the A–P SDP parameter of fasted animals (2D, upper right panels), reflecting most probably, motor activations related to the feeding impulse. Enlarged SDP maps with improved resolution in this region are shown below (2D, lower panels). Mean values, standard deviations and statistical significance of the different diffusion parameters are shown in the corresponding bar graphs of the panels in Fig. 2E. SDP and Dslow increase significantly (pb 0.05) in the A–P direction and Dfast decreases (pb 0.05) in the L–R measurements. Notably, the model independent analysis of the diffusion weighted data sets using Linear Discriminant Analysis (LDA) (Figs. 2F–H) was also able to classify successfully all mice investigated between the fed and fasted states. This indicates that the intrinsic differences between fed and fasted brains may be detected even automatically, independently

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of the diffusion model used. Fig. 2F shows how SPM processing detected, as activated areas by the fasting paradigm, an area including the hypothalamus (yellow arrow) and an area of subsidiary vascular activation with the carotid arteries and the jugular veins (blue arrow). Taken together this evidence reveals a highly complex cerebrovascular response to the fasting paradigm involving not only a hypothalamic activation but also an additional vascular adaptation. LDA was applied to detect the most different pixels between the fed and fasted image data sets of every mouse. Results from a representative mouse are illustrated in Figs. 2G and H. The Fisher LDA projection (Appetite Index, Fig. 2G) separates optimally the pixels of the fed (blue) and fasted (red) states. The pixels that are most representative of each state, located at the edges of the histogram (b5% or >95%), are shown in Fig. 2H, superimposed on the corresponding T2w images. Using a leave one out cross validation strategy, it was possible to classify correctly, as fed or fasted, the image data sets from all mice investigated, when using the diffusion data sets acquired with the whole range of b values (six high b and five low b). However, using only the six highest b value data sets, only five mice out of the six mice investigated were classified correctly between fed and fasted states. This suggests that the microvascular contributions emphasized by the low b values are needed to obtain the optimal discriminant power required to classify correctly the images of every mouse between the fed and fasted states. We investigated then the possibilities to resolve the effects of fasting in individual hypothalamic nuclei with shorter fasting times (16 h) and increased image resolution obtainable after a console upgrade. Fig. 3A shows the anatomical localization of MRI sections used to identify the hypothalamus in the mouse brain and the directions of diffusion measurements. The inset on the top shows the anatomical localization of the hypothalamic Dorsomedial Nucleus (DMN, red), Ventromedial Nucleus (VMN, yellow) and Arcuate Nucleus (ARC, light blue). Hypothalamic activation after 16 h of fasting is revealed by fDWI as an increment of Dslow coefficients in the fasted condition depicted in the diffusion maps and bar graphs of Figs. 3B and C. The upper panels of Fig. 3B show the hypothalamic Dslow maps (H–F direction) in the fed and fasted conditions of a representative mouse, superimposed on the corresponding anatomical T2w images from the whole brain. The lower panels in Fig. 3B show the hypothalamic Dslow maps with augmented resolution, depicting clearly a shift to the red that reveals an increase in Dslow. Fig. 3C summarizes the mean values, standard deviations and comparative statistical significances of the slow diffusion parameters in the L–R, A–P and H–F directions from the six mice under the fed and fasted conditions. Dslow increased significantly with fasting in the A–P (pb 0.01) and H–F (pb 0.001) directions. Figs. 3D–I show the effects of the 16 h fasting in the Arcuate Nucleus, the Ventromedial Nucleus and the Dorsomedial Nucleus, respectively. The upper panels of Figs. 3D, F and H depict the Dslow maps (H–F direction) in the fed and fasted conditions of a representative mouse, superimposed on co‐localized T2w images. The lower panels depict enlarged corresponding Dslow maps showing very significant increases in the H–F direction (pb 0.001). The bar graphs of Figs. 3E–I show the mean± sd of Dslow values from the six mice of group 2 for each investigated nucleus. Significant increases (pb 0.001) in Dslow were detected in all nuclei in the H–F direction. The DMN and VMN nuclei showed also significantly increased SDP (L–R, p b 0.001; H–F, p b 0.05,) and (A–P; p b 0.05), respectively. The effects of fasting in the human brain using DWI Table 1 shows the results for the blood samples taken from the cubital vein of the human volunteers before the “fed” and “fasted” image acquisitions. Blood samples were analyzed for routine biochemical and endocrine parameters including; glucose, cholesterol, triglycerides, HDL, LDL, T3, T4, TSH and insulin. Values of these parameters fell within the normal clinical range both in the “fed” and “fasted” conditions for every individual.

Fig. 4A shows the localization of MRI sections containing the hypothalamus in a human brain model and the directions of our DWI measurements. The diffusion maps shown in Fig. 4B show that fDWI visibly detects the hypothalamic activation induced by 24 h of fasting. The upper images show the hypothalamic SDP maps (H–F direction) in the fed and fasted conditions, superimposed on the corresponding anatomical T2w images from the whole brain. These hypothalamic diffusion maps are shown enlarged in the corresponding lower images. The fasted condition is clearly detected by a shift to orange and red of the SDP color map, revealing a significant increase in SDP. Bar graphs in Fig. 4C summarize the mean values, standard deviations and comparative statistical significances of the diffusion parameters (SDP, Dslow and Dfast) fitted as described in the Materials and methods section. We investigated six healthy volunteers under the fed and fasted conditions, monitoring the diffusion of water molecules in three orthogonal directions (L–R, A–P and H–F). As in the mouse brain, we found a significant increase of SDP (A–P; p b 0.05, H–F: p b 0.001) and Dslow (H–F; p b 0.05) with fasting. However, the human brain did not show a significant cortical activation associated with the fasting paradigm (Figs. 4D and E). The resolution of individual hypothalamic substructures was not possible when using the clinical 1.5 T scanner. Even under these unfavorable field conditions, our results show that the detection of hypothalamic activation is feasible by DWI, becoming most probably much improved at higher magnetic fields of 3 T and 7 T. LDA of the human hypothalamic data set was also able to separate successfully the diffusion weighted images from the fasted and fed conditions (Figs. 4F and G), implying again that the intrinsic differences between fDWI data sets in the two feeding conditions, are independent of the diffusion model used. A distribution of the fed and fasted projections from the hypothalamus of a representative subject is illustrated in the histogram (Appetite Index, Fig. 4F). In Fig. 4G, the upper images show enlarged hypothalamic areas that best discriminate the fed (blue pixels) and fasted (red pixels) groups and the corresponding localizations in the anatomical T2w images of the human brain (lower panels). An evaluation of the accuracy of classifications between fed and fasted states, as performed with the leave one out strategy, classified correctly the imaging data sets of five out of the six subjects, the same percentage obtained in the mouse brain images acquired with six b values under similar conditions. Discussion We report here that fasting results in significant changes in the diffusion parameters of cerebral water in mice and humans providing a novel approach for the objective, non-invasive, fully translational evaluation of the feeding impulse in vivo. Our data are consistent with previous applications of fDWI to cerebral activation studies, in particular the visual activation in humans (Le Bihan et al., 2006) and with in vitro DWI measurements of induced neuronal activity (Flint et al., 2009). In these cases, neuronal activation was associated with increments of the slow diffusion parameters in the activated areas, a finding proposed to reveal a relative redistribution of water molecules between the fast (FDP) and slow (SDP) diffusion phases, most probably reflecting, activation-induced increases in neurocellular volume (swelling) (Le Bihan et al., 2006). The interpretation of the physiological or pathological changes in water diffusion parameters in the in vivo brain has been a matter of debate in the last decades. Early physiological interpretations described the existence of two dynamically different diffusion phases, fast and slow, attributed initially to the extracellular and intracellular environments, respectively. Further studies showed that the volume fractions of the intra- and extracellular phases predicted by DWI did not match those determined histologically (Niendorf et al., 1996; Sehy et al., 2002), implying that factors additional to morphological compartmentalization contributed appreciably to the diffusion environments detected by MRI in the brain (Garcia-Martin et al., 2001). More recently, the slow diffusion phase (SDP), containing the water

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Fig. 3. Imaging appetite by fDWI in the hypothalamic nuclei of the mouse brain. A: Axial MRI section containing the hypothalamus in a representative mouse brain and in a brain atlas (inset) showing the localization of the main periventricular hypothalamic nuclei: Dorsomedial Nucleus (DMN, red), Ventromedial Nucleus (VMN, yellow) and Arcuate Nucleus (Arc, light blue). B: Hypothalamic H–F Dslow color maps from fed or fasted mice, superimposed to T2w images. The hypothalamic region is depicted enlarged in the lower panels. C: Values of hypothalamic Dslow (mean ± SD) in the L–R, A–P and H–F directions for 6 mice in the fed state and after 16 h of fasting, and statistical differences (*p b 0.05, **pb 0.005, ***pb 0.001, Student's t test). The number of pixels (N) used in the comparison was; Nfed =255, Nfasted =215 in L–R; Nfed =150, Nfasted =130 in A–P and Nfed =306, Nfasted = 463 in H–F measurements. D: H–F Dslow parameter maps of the Arcuate Nucleus in the fed (left) or fasted (right) states in a representative mouse, superimposed to T2w images. The region is shown enlarged in the bottom panels. E: Dslow values (mean±SD) of the ARC of six mice in the fed state and after 16 h of fasting in the three directions of measurement and statistical differences between states (pb 0.001, using GEE statistical methods) in the H–F direction. Nfed =11, Nfasted =13 in L–R, Nfed =3, Nfasted =2 in A–P and Nfed =4, Nfasted =13 in H–F. F: H–F Dslow parameter maps of VMN in the fed (left) or fasted (right) states for a representative mouse, superimposed to T2w images. The hypothalamic region is shown enlarged in the bottom panels. G: Dslow values (mean±SD) of the VMN of six mice in the fed state and after 16 h of fasting, in the three directions of measurement, and statistical differences between states (H–F direction pb 0.001, Student's t test). Nfed =25, Nfasted =17 in L–R; Nfed =12, Nfasted =5 in A–P and Nfed =22, Nfasted =29 in H–F. H: H–F Dslow parameter maps of the DMN of a representative mouse, superimposed to T2w images of the mouse brain (upper panels) and with the hypothalamic area enlarged (bottom panels). I: Dslow values (mean±SD) of the DMN of six mice in the fed or fasted states, in the three directions (L–R, A–P, H–F) with statistical differences (pb 0.001) in the H–F direction. Nfed =9, Nfasted =8 in L–R, Nfed =15, Nfasted =11 in the A–P direction and Nfed =19,Nfasted =23 in the H–F direction.

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Table 1 Blood concentrations of relevant biochemical parameters in each of the six human volunteers, determined before each imaging session. Volunteer/blood concentrationa (units)

1

Condition

Fed

Fasted

Fed

Fasted

Fed

Fasted

Fed

Fasted

Fed

Fasted

Fed

Fasted

Fed

Fasted

Glucose (mg/dL) Colesterol (mg/dL) Triglycerides (mg/dL) HDL‐cholesterol (mg/dL) LDL-cholesterol (mg/dL) T3 (ng/mL) T4 (mcg/dL) TSH (mcIU/mL) Insulin (mcIU/mL)

98 190 97 63 108 1.1 6.3 1.4 6.2

88 175 47 58 108 1 7.6 0.6 3.1

87 161 63 42 106 1 7.9 0.7 5.5

79 162 45 43 110 0.9 8.9 0.6 b3

90 112 32 40 66 1.2 7.8 2 3.7

79 126 41 43 75 1.1 9.3 1.9 b3

95 131 51 48 73 1. 7.6 1.6 b3

77 125 47 46 70 0.9 7.7 0.9 b3

88 155 67 36 106 1 7.6 0.7 3

83 162 35 39 116 1 7.7 0.4 b3

86 219 129 36 157 1.3 7.6 1.2 3.6

80 227 87 37 173 1.2 7.8 0.8 b3

90.7 161.3 73.2 44.2 102.7 1.1 7.5 1.3 4.4

81 162.8 50.3 44.3 108.7 1 8.2 0.9 b3

2

3

4

5

6

Mean volunteer valuesb

Reference valuesb

65–105 130–240 35–155 > 32 b 180 0.75–1.78 6.09–12.23 0.34–5.6 b25

a Determinations were performed using clinically validated protocols by the Analytical Biochemistry Laboratory Services of the Clinica Nuestra Sra. Del Rosario (Madrid). Briefly, glucose was measured using the glucose oxidase method (One touch Ultra, LifeScan, Johnson and Johnson, Issy-les-Moulineaux, FR). Total serum cholesterol and triglycerides were measured enzymatically using a CHOD-PAP test (Boehringer-Mannheim, DE); T3, T4 and TSH determinations used the Accubind-ELISA kit and insulin with the insulin ELISA kit (Millipore, Billerica, MA, USA). b Mean values of the parameters in both feeding conditions are also provided, as well as reference values for healthy adult conditions.

molecules with slow diffusion coefficient (Dslow), has been suggested to represent dynamically restricted water molecules associated with membranes and cytoskeleton structures. Conversely, the fast diffusion phase (FDP), containing the water molecules with a fast diffusion coefficient (Dfast), has been proposed to represent the remaining, freely moving, solvent water molecules (Niendorf et al., 1996) (Fig. 4,

upper panels). On these grounds, increases in SDP detected by MRI upon neural activation (Le Bihan, 2007) reveal a relative increment of water molecules diffusing in the vicinity of the membrane and cytoskeleton structures. This is consistent with the large increments in monovalent and divalent cation transports occurring during the action potentials, thought to be localized initially in the vicinity of the neural

Fig. 4. Imaging appetite in the human brain. A: Coronal and sagittal sections containing the hypothalamus in a representative human brain. B: Parameter maps showing the H–F SDP pixel values from six volunteers in the fed and fasted conditions, superimposed to anatomical T2w images (upper panels). The lower panels show enlarged hypothalamic maps. C: Bar graphs of SDP, Dslow and Dfast parameters (mean±SD) of six human subjects in the fed and fasted conditions. The number of pixels (N) used in the comparisons is: Nfed =7, Nfasted =4 in L–R; Nfed = 13, Nfasted =12 in A–P and Nfed =24, Nfasted =16 in H–F, with significant increases in A–P (pb 0.05) and H–F (pb 0.001) SDP and H–F (pb 0.05) Dslow, (GEE protocol). D: Cortical areas analyzed in the human brain, in the fed and fasted conditions (upper panels), with H–F SDP values superimposed to anatomical T2w images. The corresponding enlargements of the cortical areas are shown below. E: Bar graphs of SDP, Dslow and Dfast values (mean±SD) in the fed and fasted states. Nfed =55, Nfasted =114 in L–R; Nfed =104, Nfasted =106 in A–P and Nfed =102, Nfasted =83 in H–F. F: “Appetite Index” histogram for the fed (blue) and fasted (red) human hypothalamus calculated using LDA. G: Identification of the areas representing the position of the 5% of the points located in the lower (fed) and upper (fasted) extremes of the Appetite Index histogram.

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Fig. 5. Neuroglial metabolic coupling and astrocytic volume changes underlie the orexigenic hypothalamic activation as detected by fDWI. The upper panels illustrate the effects of volume changes in the phase distribution (SDP) and dynamics (Dslow) of water molecules around the plasma membrane, under the fed (left) and fasted (right) conditions. Water molecules close to the plasma membrane (dark blue) experience more restricted diffusional motions than those far apart (light blue). Increases in astrocytic volume (right) produce; (i) an increase in the available membrane surface, augmenting the contribution of diffusionally restricted water molecules (increased SDP, more water molecules in the dark blue area) and (ii) a decrease in the average obstructions allowing faster average water diffusion (increased Dslow, faster diffusion in the light blue area). The lower panels illustrate the neuroglial metabolic coupling mechanisms and associated volume responses in the fed (left) and fasted (right) hypothalamus. During orexigenic neurotransmission, excess glutamate released to the synaptic cleft by AGRP/NPYY neurons (orange) is recaptured by surrounding astrocytes (blue), together with 3Na+, through the astrocytic glutamate cotransporters GLAST (gray). Intracellular sodium ions thus incorporated are extruded to the extracellular space, through the electrogenic Na+/K+ ATPase (yellow), incorporating two intracellular K+ ions. Astrocytic glutamate produces glutamine, through glutamine synthase, which is later recaptured by the neurons to operate the glutamine cycle. Astrocytic ATP molecules required for glutamine synthesis and the Na/K+ ATPase, are generated from plasma glucose, after transport through GLUT1 (white), by anaerobic glycolysis and the tricarboxylic acid cycle. Lactate produced is extruded through the monocarboxylate transporter (MCT1, green) and potentially oxidized by surrounding neurons. Additional K+ ions may be incorporated to the astrocyte through the Na+K+2Cl− cotransporter (purple), resulting in increased intracellular K+ concentrations and thus triggering, an osmotically driven, aquaporin 4 (AQP4) mediated (blue channel), water transport and astrocytic swelling. Under fasting conditions (lower right), orexigenic firing is increased (darker lines) as well as glutamatergic neurotransmission (increased glutamate in the synaptic cleft), resulting in augmented ionic and water trafficking (darker arrows, more water influx through AQP4). The accumulated K+ in one astrocyte may be transferred to the neighboring astroglia, through gap junctions (inverted brackets), providing a spreading mechanism for the swelling response through the astrocyte syncithium. The central color coded bar graphs, represent variations of SDP (increase as dark blue) and Dslow (increase as light blue), as detected by fDWI. A: Astrocyte, ARC: Arcuate Nucleus, PVN: Paraventricular Nucleus, MC4, MC3: Melanocortin receptors, N: Neuron, AGRP/NPY: Agouti Related Protein/NeuroPeptideY, POMPCT/CART: Proopiomielanocortin Transcript/Cocaine and Amphetamine Regulated Transcript. Mechanisms are shown only in one astrocyte for simplicity.

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membranes. The redistribution may also reflect the concomitant increment of glial volume reported to occur during neuronal activation (Andrew and MacVicar, 1994; Hertz et al., 2007; Le Bihan, 2007). In our case, the increase in diffusion parameters detected during fasting, might reveal the osmotic swelling of glial cells in the vicinity of stimulated orexigenic synapses, as expected to occur during increased glutamatergic neurotransmission (Hansson et al., 2000; Simard and Nedergaard, 2004) (Fig. 5, lower panels). Importantly, these changes become detectable even after 16 h of fasting in mice and may be even localized within the subhypothalamic regions as the Arcuate Nucleus, the Ventromedial Nucleus and the Dorsomedial Nucleus. More specifically, under fasting conditions, excess glutamate released to the orexigenic cleft is recaptured by surrounding astrocytes, by Na + dependent cotransport mainly through the GLAST/EAAT1 and GLT-1/EAAT2 transporters, with a 3Na + per glutamate stoichiometry (Anderson and Swanson, 2000). The three sodium ions incorporated in this way, are extruded to the extracellular space, in exchange with two potassium ions through the Na +/K + ATPase. Astrocytic ATP required for the operation of the Na +/K + ATP-ase and glutamine synthesis during activation by fasting, is thought to be derived from increased glucose consumption and metabolism by oxidative and glycolytic pathways (Violante et al., 2009). This increased metabolic demand results in an increased hypothalamic microvascular blood flow, a circumstance consistent with the changes detected here by SPM and LDA under low b weightings (Fig. 2D). Additional K + ions accumulated in the extracellular space during orexigenic firing, may enter the astrocyte by stimulation of the Na +/K +/2Cl − cotransporter (Hertz et al., 2007). Indeed, increased K + concentrations are known to be tightly coupled to neuronal activation, and have been detected using metallographic microscopic imaging approaches (Goldschmidt et al., 2004). Taken together, these processes lead to intracellular potassium accumulation during activation, a circumstance triggering a concomitant water influx and volume increase of the astrocytes, mainly mediated through the highly abundant aquaporin AQP-4 (Badaut et al., 2002). The osmotic swelling response associated with orexigenic stimulation, is proposed to occur initially in few astrocytes, those surrounding the orexigenic clefts, but can be rapidly extended to a plethora of neighboring astrocytes, through the numerous interconnecting gap junctions of the network arrangement (Halassa and Haydon, 2010). This increases significantly the spatial distribution of the orexigenic activation and makes it MRI detectable. The osmotic swelling response associated with hypothalamic fasting appears not to be isotropic, suggesting smaller anatomical restrictions to volume increase in the directions where the increase in diffusion parameters is observed. The fDWI approach to the fasting paradigm explored here is not devoid of limitations. In particular, a higher anatomical resolution is desirable, but limited at this field by the S/N obtainable, particularly at high b values. This may be improved by increasing the number of averages, at the compromise of augmenting linearly the image acquisition time. In this respect, the use of single shot EPI readout may help to decrease the acquisition time. However, this is a very demanding technique at high fields, leading frequently to distorted hypothalamic geometry, making difficult in our hands an adequate pixel by pixel analysis of the hypothalamus and its substructures. Additional limitations may derive from the constrains imposed by the biexponential model, in particular by the potential influence of microvascular flow on the diffusion coefficient measurements (Autio et al., 2011; Miller et al., 2007). On these grounds, the implementation of integral dynamic model considering diffusion and flow would represent a considerable improvement (Le Bihan, 2008). In any case, model imposed limitations may be overcome through the use of a model-free approach, as the LDA strategy proposed here. Finally, despite its inherent limitations, fDWI presents valuable advantages over other non‐invasive methods of investigating hypothalamic physiology. fDWI provides improved anatomical resolution over the PET FDG method, highlights diffusion

alterations, an event not detectable by fMRI BOLD and avoids the use of potentially toxic Mn2+ doses required by MEMRI, becoming then fully translational to the clinic. Conclusion In summary, our results show that hypothalamic activation by fasting in mice or humans can be detected non-invasively by diffusion weighted magnetic resonance imaging through changes in the water diffusion parameters. These findings may prove useful in the diagnostic imaging of appetite disorders and can be extended easily to visualize other hypothalamic activation dysfunctions. Acknowledgments Authors are indebted to Dr. Diana Quiñones Tapia for expert neuroradiological assistance during the human studies and to Mr. Javier Pérez CSIC, for careful, professional drafting of the illustrations. References Anderson, C.M., Swanson, R.A., 2000. Astrocyte glutamate transport: review of properties, regulation, and physiological functions. Glia 32, 1–14. Andrew, R.D., MacVicar, B.A., 1994. Imaging cell volume changes and neuronal excitation in the hippocampal slice. Neuroscience 62, 371–383. Autio, J.A., Kershaw, J., Shibata, S., Obata, T., Kanno, I., Aoki, I., 2011. High b-value diffusionweighted fMRI in a rat forepaw electrostimulation model at 7 T. NeuroImage 57, 140–148. Badaut, J., Lasbennes, F., Magistretti, P.J., Regli, L., 2002. Aquaporins in brain: distribution, physiology, and pathophysiology. J. Cereb. Blood Flow Metab. 22, 367–378. Burton, P., Gurrin, L., Sly, P., 1998. Extending the simple linear regression model to account for correlated responses: an introduction to generalized estimating equations and multi-level mixed modelling. Stat. Med. 17, 1261–1291. Carnell, S., Gibson, C., Benson, L., Ochner, C.N., Geliebter, A., 2011. Neuroimaging and obesity: current knowledge and future directions. Obes. Rev. 13, 43–56. Coll, A.P., Farooqi, I.S., O'Rahilly, S., 2007. The hormonal control of food intake. Cell 129, 251–262. Delgado, T.C., Violante, I.R., Nieto-Charques, L., Cerdan, S., 2011. Neuroglial metabolic compartmentation underlying leptin deficiency in the obese ob/ob mice as detected by magnetic resonance imaging and spectroscopy methods. J. Cereb. Blood Flow Metab. 31, 2257–2266. Flint, J., Hansen, B., Vestergaard-Poulsen, P., Blackband, S.J., 2009. Diffusion weighted magnetic resonance imaging of neuronal activity in the hippocampal slice model. NeuroImage 46, 411–418. Garcia-Martin, M.L., Ballesteros, P., Cerdan, S., 2001. The metabolism of water in cells and tissues as detected by NMR methods. Prog. Nucl. Magn. Reson. Spectrosc. 39, 41–77. Gautier, J.F., Chen, K., Salbe, A.D., Bandy, D., Pratley, R.E., Heiman, M., Ravussin, E., Reiman, E.M., Tataranni, P.A., 2000. Differential brain responses to satiation in obese and lean men. Diabetes 49, 838–846. Goldschmidt, J., Zuschratter, W., Scheich, H., 2004. High-resolution mapping of neuronal activity by thallium autometallography. NeuroImage 23, 638–647. Halassa, M.M., Haydon, P.G., 2010. Integrated brain circuits: astrocytic networks modulate neuronal activity and behavior. Annu. Rev. Physiol. 72, 335–355. Hansson, E., Muyderman, H., Leonova, J., Allansson, L., Sinclair, J., Blomstrand, F., Thorlin, T., Nilsson, M., Ronnback, L., 2000. Astroglia and glutamate in physiology and pathology: aspects on glutamate transport, glutamate-induced cell swelling and gap-junction communication. Neurochem. Int. 37, 317–329. Hastie, T., Tibshirani, R., Friedman, J., 2001. The elements of statistical learning: data mining, inference and prediction. Springer Series in Statistics. New York. Hertz, L., Peng, L., Dienel, G.A., 2007. Energy metabolism in astrocytes: high rate of oxidative metabolism and spatiotemporal dependence on glycolysis/glycogenolysis. J. Cereb. Blood Flow Metab. 27, 219–249. Le Bihan, D., 2003. Looking into the functional architecture of the brain with diffusion MRI. Nat. Rev. Neurosci. 4, 469–480. Le Bihan, D., 2007. The ‘wet mind’: water and functional neuroimaging. Phys. Med. Biol. 52, R57–R90. Le Bihan, D., 2008. Intravoxel incoherent motion perfusion MR imaging: a wake-up call. Radiology 249, 748–752. Le Bihan, D., Urayama, S., Aso, T., Hanakawa, T., Fukuyama, H., 2006. Direct and fast detection of neuronal activation in the human brain with diffusion MRI. Proc. Natl. Acad. Sci. U. S. A. 103, 8263–8268. Liu, T., Kong, D., Shah, B.P., Ye, C., Koda, S., Saunders, A., Ding, J.B., Yang, Z., Sabatini, B.L., Lowell, B.B., 2012. Fasting activation of AgRP neurons requires NMDA Receptors and involves spinogenesis and increased excitatory tone. Neuron 73, 511–522. Mahankali, S., Liu, Y., Pu, Y., Wang, J., Chen, C.W., Fox, P.T., Gao, J.H., 2000. In vivo fMRI demonstration of hypothalamic function following intraperitoneal glucose administration in a rat model. Magn. Reson. Med. 43, 155–159. Matsuda, M., Liu, Y., Mahankali, S., Pu, Y., Mahankali, A., Wang, J., DeFronzo, R.A., Fox, P.T., Gao, J.H., 1999. Altered hypothalamic function in response to glucose ingestion in obese humans. Diabetes 48, 1801–1806.

B. Lizarbe et al. / NeuroImage 64 (2013) 448–457 Miller, K.L., Bulte, D.P., Devlin, H., Robson, M.D., Wise, R.G., Woolrich, M.W., Jezzard, P., Behrens, T.E., 2007. Evidence for a vascular contribution to diffusion FMRI at high b value. Proc. Natl. Acad. Sci. U. S. A. 104, 20967–20972. Morton, G.J., Cummings, D.E., Baskin, D.G., Barsh, G.S., Schwartz, M.W., 2006. Central nervous system control of food intake and body weight. Nature 443, 289–295. Niendorf, T., Dijkhuizen, R.M., Norris, D.G., van Lookeren Campagne, M., Nicolay, K., 1996. Biexponential diffusion attenuation in various states of brain tissue: implications for diffusion-weighted imaging. Magn. Reson. Med. 36, 847–857. Parkinson, J.R., Chaudhri, O.B., Bell, J.D., 2009. Imaging appetite-regulating pathways in the central nervous system using manganese-enhanced magnetic resonance imaging. Neuroendocrinology 89, 121–130. Paxinos, G., Franklin, K.B.J., 2001. The Mouse Brain in Stereotaxic Coordinates. Academic Press, New York. Sehy, J.V., Ackerman, J.J., Neil, J.J., 2002. Evidence that both fast and slow water ADC components arise from intracellular space. Magn. Reson. Med. 48, 765–770.

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Simard, M., Nedergaard, M., 2004. The neurobiology of glia in the context of water and ion homeostasis. Neuroscience 129, 877–896. Spanswick, D.C., Simonds, S.E., Cowley, M.A., 2012. Transmitter time: synaptic plasticity and metabolic memory in the hypothalamus. Cell Metab. 15, 275–276. Tataranni, P.A., Gautier, J.F., Chen, K., Uecker, A., Bandy, D., Salbe, A.D., Pratley, R.E., Lawson, M., Reiman, E.M., Ravussin, E., 1999. Neuroanatomical correlates of hunger and satiation in humans using positron emission tomography. Proc. Natl. Acad. Sci. U. S. A. 96, 4569–4574. van den Pol, A.N., Wuarin, J.P., Dudek, F.E., 1990. Glutamate, the dominant excitatory transmitter in neuroendocrine regulation. Science 250, 1276–1278. Violante, I.R., Anastasovska, J., Sanchez-Canon, G.J., Rodrigues, T.B., Righi, V., Nieto-Charques, L., Parkinson, J.R., Bloom, S.R., Bell, J.D., Cerdan, S., 2009. Cerebral activation by fasting induces lactate accumulation in the hypothalamus. Magn. Reson. Med. 62, 279–283. Yang, Y., Atasoy, D., Su, H.H., Sternson, S.M., 2011. Hunger states switch a flip-flop memory circuit via a synaptic AMPK-dependent positive feedback loop. Cell 146, 992–1003.

CURRÍCULUM NORMALIZADO COMPLETO (CVNC)

Calle, D., Negri,V., Ballesteros,P., Cerdán,S. Magnetoliposomes loaded with Poly-unsaturated Fatty Acids as Novel Theranostic AntiInflammatory Formulations Theranostics (2015), 5, 489-503 I.F. 7,827 (9/124 Medicine, Research&Experimental Q1) ISSN:1838-7640

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Ivyspring

Theranostics

International Publisher

2015; 5(5): 489-503. doi: 10.7150/thno.10069

Research Paper

Magnetoliposomes Loaded with Poly-Unsaturated Fatty Acids as Novel Theranostic Anti-Inflammatory Formulations Daniel Calle1, Viviana Negri1,2, Paloma Ballesteros2 and Sebastián Cerdán1 1. 2.

Instituto de Investigaciones Biomédicas Alberto Sols CSIC/UAM, c/Arturo Duperier 4, Madrid 28029, Spain. Laboratorio de Síntesis Orgánica e Imagen Molecular por Resonancia Magnética, Facultad de Ciencias, UNED, Unidad Asociada al CSIC, c/ Paseo Senda del Rey 9, Madrid 28040, Spain.

 Corresponding author: Prof. Sebastian Cerdan, Instituto de Investigaciones Biomédicas Alberto Sols CSIC/UAM c/ Arturo Duperier 4, Madrid 28029, Spain. Phone: 0034-91-585-4444 Fax: 0034-91-585-4401 Email: [email protected] © 2015 Ivyspring International Publisher. Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. Please see http://ivyspring.com/terms for terms and conditions.

Received: 2014.07.09; Accepted: 2014.12.18; Published: 2015.02.15

Abstract We describe the preparation, physico-chemical characterization and anti-inflammatory properties of liposomes containing the superparamagnetic nanoparticle Nanotex, the fluorescent dye Rhodamine-100 and omega-3 polyunsaturated fatty acid ethyl ester (ω-3 PUFA-EE), as theranostic anti-inflammatory agents. Liposomes were prepared after drying chloroform suspensions of egg phosphatidylcholine, hydration of the lipid film with aqueous phases containing or not Nanotex, Rhodamine-100 dye or ω-3 PUFA-EE, and eleven extrusion steps through nanometric membrane filters. This resulted in uniform preparations of liposomes of approximately 200 nm diameter. Extraliposomal contents were removed from the preparation by gel filtration chromatography. High Resolution Magic Angle Spinning 1H NMR Spectroscopy of the liposomal preparations containing ω-3 PUFA-EE revealed well resolved 1H resonances from highly mobile ω-3 PUFA-EE, suggesting the formation of very small (ca. 10 nm) ω-3 PUFA-EE nanogoticules, tumbling fast in the NMR timescale. Chloroform extraction of the liposomal preparations revealed additionally the incorporation of ω-3 PUFA-EE within the membrane domain. Water diffusion weighted spectra, indicated that the goticules of ω-3 PUFA-EE or its insertion in the membrane did not affect the average translational diffusion coefficient of water, suggesting an intraliposomal localization, that was confirmed by ultrafiltration. The therapeutic efficacy of these preparations was tested in two different models of inflammatory disease as inflammatory colitis or the inflammatory component associated to glioma development. Results indicate that the magnetoliposomes loaded with ω-3 PUFA-EE allowed MRI visualization in vivo and improved the outcome of inflammatory disease in both animal models, decreasing significantly colonic inflammation and delaying, or even reversing, glioma development. Together, our results indicate that magnetoliposomes loaded with ω-3 PUFA-EE may become useful anti-inflammatory agents for image guided drug delivery. Key words: Magnetoliposomes, ω-3 poly-unsaturated fatty acid ethyl ester, Superparamagnetic nanoparticle, MRI, Image guided drug delivery.

Introduction Inflammatory lesions are associated to the most prevalent and morbid pathologies in developed countries including atherosclerosis [1, 2], neuro-

degeneration [3, 4] diabetes or obesity [5, 6] and cancer [7-9], among others. Decreases in the inflammatory phenotype are known to reduce disease progreshttp://www.thno.org

Theranostics 2015, Vol. 5, Issue 5 sion and improve patient´s quality of life, as well as to enhance recovery [7, 9]. With this aim, a variety of anti-inflammatory drugs have been proposed as adjuvant therapies for the different diseases, including mainly steroidal or non-steroidal treatments [10-12]. However, anti-inflammatory therapies demand the use of relatively large doses of the free drugs and inevitably end up developing, a collection of adverse secondary effects that preclude continuation of the treatment [13, 14]. On these grounds, methods to improve the efficiency of anti-inflammatory drug delivery to the lesion, decreasing the dose and increasing its selectivity and efficacy, entail considerable importance in a wide spectrum of diseases. Liposomes have been proposed previously as novel nanotechnology formulations to improve drug delivery to a variety of inflammatory diseases [15-17]. Targeting of the inflammatory region may be achieved using, either active or passive approaches. Active targeting involves the use vectorial reagents embedded in the liposomal membrane that recognize epitopes of the target lesion [18]. Passive targeting refers to the passive accumulation of the liposomes in the inflamed regions because of the Enhanced Permeability Retention (EPR), an effect derived from their relatively increased capillary permeability and limited clearance [19]. In both targeting strategies, it becomes difficult to visualize non-invasively if the liposomal preparation has arrived to the target tissue and many times, only indirect measurements of the inflammation size or volume provide an index of the anti-inflammatory effect. It would become then very useful, to be able to locate directly and non-invasively the presence of the drug loaded liposomes in the lesion. Liposomes are optimal structures for this purpose, since they can be prepared to contain in their lumen, in addition to the anti-inflammatory drug, a variety of imaging probes including radioactive, fluorescent or superparamagnetic agents, among others [20-22]. The liposomal lumen can be loaded with water-soluble drugs, maintained in solution in the lumen, or lipid-soluble drugs, thought to incorporate spontaneously in the bilayer membrane [23, 24]. This approach is known as image guided drug delivery, representing currently a powerful strategy to diagnose and treat inflammatory diseases [25-28]. Important anti-inflammatory effects have been reported using preparations of omega-3 fatty acids [29-35], a kind of long chain polyunsaturated fatty acids (PUFAs) mainly present in fish oils and used as dietary supplement. Omega-3 PUFAs allow prolonged anti-inflammatory treatments without the appearance of the deleterious secondary effects of alternative steroidal or non-steroidal drugs [14, 36], operating simultaneously on a variety of targets of the

490 inflammatory cascade. In this work we report on a successful protocol to encapsulate ω-3 PUFA [37] containing in addition, either or both, the novel Nanotex superparamagnetic nanoparticle [38] or the Rhodamine-100 dye, thus allowing in vivo monitorization using magnetic resonance imaging or fluorescence. These advanced theranostic preparations maintain completely the therapeutic potential of free ω-3 PUFA, potentiated additionally with highly versatile multimodal imaging capabilities. We demonstrate here the anti-inflammatory effects of these preparations in vivo, in animal models of colonic and oncologic inflammation.

Materials and Methods Preparation of Nanotex Nanotex is a superparamagnetic nanoparticle containing a magnetite (Fe3O4) core with poly(acrylic) acid coating. Magnetite (Fe3O4) nanoparticles were prepared by the co-precipitation of Fe3+ and Fe2+ ions (molar ratio 2:1) at 25 °C and a concentration of 0.3 M iron ions with ammonia solution (29.6%) at pH 10 in an inert atmosphere, followed by hydrothermal treatment at 80 °C for 30 min. For the binding of poly(acrylic acid) (PAA), 100 mg of Fe3O4 nanoparticles were first mixed with 2 mL of buffer A (0.003 M phosphate, pH 6) and 0.5 mL of carbodiimide solution (0.025 g/mL in buffer A). After being sonicated for 10 min, 2.5 mL of PAA solution (60 mg/mL in buffer A) was added and the reaction mixture was sonicated for another 30 min. Finally, the PAA-coated Fe3O4 nanoparticles were recovered magnetically, washed with water twice and dialyzed against a buffer saline solution. Nanotex preparations were highly water soluble, stable for several months, and depicted uniform diameters smaller than 15 nm [38].

Preparation and characterization of liposomes Lipid film hydration and extrusion method Liposomes were prepared essentially using the film hydration method [17, 39, 40]. Briefly, 20 mg (26 µmol) of L-α-phosphatidylcholine (PtdCho, Avanti Polar Lipids Inc. 840051P, Alabaster, Alabama, USA) were dissolved in 2 mL of chloroform (Merck, Darmstad, DE K36897545). The solution was placed in a round bottom flask and subjected to rotary evaporation (Heindolph Instruments, Schwabach, DE) for sixty minutes (280 rpm, 474 mBar, 40 °C). The lipid film formed after chloroform evaporation was further rotated under the same conditions to remove chloroform traces. Then, the dried lecithin film was rehydrated with 5 mL of water (containing or not the active principle and imaging probes) and rotated 60 minutes at atmospheric pressure and 50 °C. This http://www.thno.org

Theranostics 2015, Vol. 5, Issue 5 process generates a heterogeneous suspension of liposomes of different sizes containing or not the active principles and imaging probes in their lumen or outside as delivered with the hydration buffer. To homogenize the size distribution, the liposomal suspension was extruded eleven times (Northen Lipids, Burnaby, CA) under nitrogen pressure through a 200 nm membrane (Whatman, GE Healthcare, Fairfield, Connecticut, US) maintaining the extrusor temperature at 50 °C. The resulting suspension containing homogenous liposomes, loaded or not with the selected imaging agent and ω-3 PUFA ethyl esther (ω-3 PUFA-EE), was stored at 4 °C until further utilization.

491 Figure 1A provides a schematic description of the process. We prepared liposomes encapsulating either suspensions of (i) buffer alone, (ii) Nanotex superparamagnetic nanoparticle (0.5 mg Fe/mL, Solutex S.L., Alcobendas, ES) as MRI detectable probe, (iii) Nanotex and ω-3 PUFA-EE (0.1% v/v final volume, 0.75 mM, 95% w/v eicosapentanoic acid ethyl ester, Solutex S.L., Alcobendas, ES) as theranostic anti-inflammatory agents detectable by MRI, or (iv) Nanotex with Rhodamine-100 chloride (10 mg/mL Sigma Aldrich, Barcelona, ES) as a novel hybrid imaging agent detectable by MRI and fluorescence.

Figure 1. Preparation and characterization of liposomes. A:Liposomes are prepared from egg yolk lecithin, containing or not ω-3 PUFA-EE, Nano