L'objectiu principal de la present tesis doctoral ha estat l'estudi de la possible contribució de l'Amino Oxidasa Sensible a Semicarbazida (SSAO) en el dany vascular, la seva implicació en l'Angiopatia Cerebral Amiloide (CAA) i la Malaltia d'Alzheimer (AD). La SSAO és un enzim multifuncional, al qual se li han atribuït diferents papers biològics depenent del teixit on es localitzi. La seva expressió es troba amplament distribuïda en mamífers, especialment en teixits altament vascularitzats, associat a cèl·lules endotelial i de múscul llis. Així mateix, existeix una forma de SSAO soluble present en el plasma de totes les espècies de mamífers estudiades. En el present treball, en primer lloc, es va estudiar la possible alteració de l'enzim en el desordre neurodegeneratiu tipus AD associat a CAA. Els resultats obtinguts van permetre demostrar, per una part, una sobreexpressió SSAO a nivell cerebrovascular humà en pacients amb AD-CAA. Per altra banda, es va determinar l'activitat SSAO plasmàtica de pacients amb demència tipus AD esporàdic i es va establir una correlació positiva entre els valors d'activitat SSAO soluble present en plasma i la severitat de la demència.Donat que els productes generats de l'acció catalítica de la SSAO són considerats altament reactius i amb propietats potencialment tòxiques, es va estudiar l'efecte de l'oxidació del substrat fisiològic de la SSAO, la metilamina, sobre cèl·lules vasculars en cultiu. Els resultats van permetre confirmar que l'oxidació de la metilamina, per part tant de la SSAO soluble com la SSAO tissular, era capaç d'induir apoptosis en cèl·lules de múscul llis. En resum, en situacions patològiques, on els nivells de SSAO cel·lular i SSAO soluble es troben incrementats, la seva acció catalítica podria contribuir al dany vascular en desordenes cerebrovasculars com l'Angiopatia Cerebral Amiloide associada a la Malaltia d'Alzheimer.

La Ciclosporina A (CsA) és un immunosupressor àmpliament utilitzat per evitar el rebuig de l'òrgan trasplantat. Malgrat tot, el seu ús s'ha vist limitat pels seus marcats efectes nefrotòxics. En el ronyó s'observa una diferent sensibilitat a la CsA segons el tipus cel·lular, sent les cèl·lules dels túbuls proximals les més sensibles. En aquest sentit, diversos treballs anteriors demostren la capacitat de la CsA d'induir efectes tòxics directament sobre la cèl·lula tubular en cultiu. L'objectiu d'aquesta tesi ha estat estudiar els efectes tòxics de la CsA en cèl·lules derivades del túbul proximal de ratolins i intentar determinar els mecanismes moleculars implicats en la toxicitat per CsA. Per a aquest fi s'ha analitzat els efectes del tractament amb CsA en la viabilitat cel·lular i sobre dues importants vies de senyalització implicades en mecanismes de supervivència i mort com són les vies ERK 1/2 i PI3K. Els nostres resultats mostren que el tractament amb CsA indueix toxicitat i pèrdua de la viabilitat cel·lular de manera dosis i temps depenent. El tractament amb CsA també indueix l'activació d'ambdues vies, si bé amb diferents conseqüències fisiològiques. Així, l'activació d'ERK no participa en els efectes tòxics de la CsA mentre que el bloqueig d'algunes isoformes de PI3K resulta en un important rescat de la citotoxicitat. En analitzar els mecanismes que condueixen a aquests efectes, hem observat que els efectes de la CsA sobre la viabilitat i l'activació d'aquestes vies de senyalització venen mediades per la transactivació del receptor de EGF (EGFR). L'activació del EGFR depèn, al seu torn, de la secreció d'un o més factors en resposta a CsA i que actuarien per un mecanisme autocrí sobre la pròpia cèl·lula. En el procés d'activació de EGFR estarien implicades també les metal·loproteïnases de matriu (MMPs). Els nostres resultats mostren que aquest component secretat en resposta a CsA és un factor d'unió a heparina que actuaria a través de la unió a proteoglicans de tipus heparan sulfats (HSPGs) de la superfície cel·lular. En l'intent d'identificar aquest factor hem pogut descartar la implicació del TGF-β, a priori un bon candidat, i hem comprovat la implicació al menys indirecta de la Ciclofilina B (CypB), el receptor intracel·lular de la CsA. En conclusió, els nostres resultats demostren que la CsA és capaç d'induir diferents respostes en les cèl·lules PCT3, on part d'aquest efectes vindrien mediats per un "loop" autocrí en la que es secretaria un factor d'unió a heparina que activaria EGFR i desencadenaria l'activació de ERK, PI3K i la toxicitat cel·lular. En un futur, la identificació dels factors secretats en resposta a CsA podria aportar una valuosa informació en el tractament de la nefrotoxicitat crònica per CsA. / The immunosuppressor cyclosporine A (CsA) has been widely used to prevent organ transplant rejection. The beneficial effects of CsA are restricted by its toxic side effects, with nephrotoxicity being the most remarkable one. In the kidney, there is a site-selective action of CsA on the cells of the proximal tubular region of the nephron, preferentially in epithelial cells of the S3 segment. In addition, recent reports have concluded that CsA exerts a direct toxic effect on cultured proximal tubular cells. The aim oh the present thesis was to study the toxic effects of CsA on the PKSV-PCT (PCT3) cell line, derived from the convoluted portion of the proximal tubule of kidneys from transgenic mice. For this purpose we have analyzed the effects of CsA treatment on cell viability and on ERK 1/2 and PI3K pathways, two important signalling pathways involved in cell survival and death processes. Our results show that CsA treatment affected cell viability and induces activation of both ERK and PI3K pathways in a dose and time-dependent manner. ERK activation was not involved in CsA-induced toxic effects while inhibition of some PI3K isoforms resulted in a significant decrease of CsA-induced cytotoxicity. We have observed that CsA triggered effects on cell viability and ERK and PI3K pathways activation where mediated by matrix metalloproteinases (MMPs) and EGF receptor (EGFR) transactivation. EGFR activation in response to CsA was also dependent of the secretion of an heparin binding factor that would actuate through autocrine signalling. Cell surface proteoglycans of the heparin sulphate family (HSPGs) were also implicated in CsA triggered effects. To further explore the underlying mechanism of CsA triggered effects, we tried to identify the secreted factor responsible of CsA effects. Although TGF-β was a suitable candidate, our results show that it was not involved. Moreover, our results show that Cyclophilin B, the CsA intracellular receptor, was probably implicated in CsA effects. In conclusion, our results show that CsA is able to trigger different cell responses in PCT3 cells with different physiological outcomes. These effects would be mediated by an autocrine loop where an heparin binding factor would be secreted and would activate EGFR thus leading to ERK and PI3K activation and cell toxicity. In a future, the identification of the secreted factors in response to CsA could give us valuable information in the treatment of CsA-induced chronic nephrotoxicity.

La activación del Receptor de Muerte Fas, también llamado APO-1 o CD95, da lugar a la formación del Death Inducing Signaling Complex (DISC). Este complejo proteico contiene FADD y caspasa-8, entre otras proteínas, y causa el corte y activación de caspasa-8, que finalmente da lugar a la apoptosis. Existen dos vías de señalización diferenciadas en la apoptosis inducida por Fas. En las células tipo I, la activación de Fas da lugar a la formación de altos niveles de DISC, y los niveles de caspasa-8 son suficientes para cortar directamente la caspasa-3 ejecutora, que desencadena la apoptosis. En las células tipo II, los niveles de formación de DISC son más bajos y caspasa-3 no es cortada directamente por caspasa-8. En vez de ello, caspasa-8 corta la proteína BH3- only Bid, dando lugar a su forma truncada tBid, que transloca a la mitocondria e induce su permeabilización. Esto conlleva la salida de factores apoptogénicos de la mitocondria al citosol, como citocromo c, que da lugar a la activación de caspasa-3 a través del apoptosoma. Así, las células tipo II necesitan un paso de amplificación de la señal a través de la mitocondria. Los Antagonistas de los Receptores de Muerte son proteínas que son capaces de modular la actividad de los Receptores de Muerte. Entre ellos, Lifeguard (LFG), también llamado NMP35 o FAIM2, es un antagonista de Fas altamente expresado en el sistema nervioso. Esta proteína ha sido caracterizada como un inhibidor de la apoptosis inducida por Fas, y se ha comprobado su localización en los sitios postsinápticos y en las dendritas. Además, se ha comprobado que interacciona directamente con el receptor Fas en los rafts, e inhibe la actividad de las caspasas-8 y -3 tras la activación de Fas. Sin embargo, su mecanismo de acción aún no ha sido descrito. En este trabajo tratamos de arrojar algo de luz sobre este problema. Primero, resultados previos en nuestro laboratorio mostraron que LFG interacciona con varias proteínas del sistema ubiquitina. Confirmamos que LFG está ubiquitinado, y también mostramos que esta ubiquitinización no induce su degradación. Además, nuestros resultados sugieren que la ubiquitinización de LFG es no-canónica. Por otro lado, llevamos a cabo un extenso estudio para elucidar la localización subcelular de LFG. Demostramos que LFG localiza en las membranas del RE y del Golgi, y en menor medida, también en los endosomas. Dado que LFG es miembro de la familia de proteínas TMBIM, que son capaces de modular la actividad de las proteínas de la familia Bcl-2, investigamos su relación con ellas, y encontramos que interacciona con Bcl-xL y Bcl-2 a través de su región C-terminal. Además, demostramos que LFG solamente protege a las células tipo II de la muerte inducida por Fas, y esta protección es dependiente de la expresión endógena de Bcl-xL. Para finalizar, nuestros resultados revelan un paso no descrito hasta el momento en la vía de señalización de las células tipo II. La movilización de calcio del RE se ha comprobado que es relevante en la vía de señalización apoptótica inducida por Fas. Aquí, demostramos que LFG modula la salida de calcio del RE tras la estimulación de Fas, e inhibe la apoptosis inducida por Fas en células tipo II. Basándonos en estas observaciones, proponemos que LFG protege de la apoptosis inducida por Fas mediante la modulación de la salida de calcio del RE. / Activation of the Death Receptor Fas, also called APO-1 or CD95, leads to the formation of the Death Inducing Signaling Complex (DISC). This protein complex comprises FADD and caspase-8, among other proteins, and it causes the cleavage and activation of caspase-8, that ultimately leads to apoptosis. There are two differentiated pathways in the Fas induced apoptosis. In type I cells, high levels of DISC are formed upon Fas activation, and caspase-8 levels are sufficient to directly cleave the effector caspase-3, which will trigger apoptosis. In type II cells, DISC formation levels are lower and caspase-3 is not directly cleaved by caspase-8. Instead, caspase-8 cleaves the BH3-only protein Bid, generating its truncated form tBid, which translocates to the mitochondria and induce its permeabilization. This will result in release of apoptogenic factors from the mitochondria to the cytosol, such as cytochrome c, which will activate caspase-3 through the apoptosome. Thus, type II cells need a signal amplification step through the mitochondria. Death Receptor Antagonists are proteins that are able to modulate Death Receptor activity. Among them, Lifeguard (LFG), also called NMP35 or FAIM2, is a Fas antagonist highly expressed in the nervous system. This protein has been characterized as a Fas-induced apoptosis inhibitor, and it has been shown that localizes at postsynaptic sites and dendrites. Moreover, it has been reported to interact directly with Fas receptor in the lipid rafts and inhibit caspase-8 and caspase-3 activity upon Fas activation. However, its mechanism of action has remained elusive. In this work, we try to shed some light on this problem. First, previous results in our lab have shown that LFG interacts with several proteins from the ubiquitin system. We confirmed that LFG is ubiquitinated, and we also show that this ubiquitination does not induce its degradation. In addition, we present data that suggest that LFG ubiquitination is done in a non-canonycal way. On the other hand, we make an extensive study to elucidate LFG subcellular localization. We demonstrate that LFG localizes to ER and Golgi membranes, and to a lesser extent, to endosomes. Since LFG is member of the TMBIM family proteins, that are able to modulate Bcl-2 family activity, we investigated its relationship with them, and found that it interacts with Bcl-xL and Bcl-2, through its C-terminal region. Moreover, we prove that LFG protects only type II cells from Fasinduced apoptosis, and this protection is dependant on Bcl-xL endogenous expression. Finally, our results reveal a hitherto undescribed step in the signaling pathway in type II cells. Calcium mobilization from the ER has been shown to be relevant in Fas apoptotic signaling. We demonstrate that LFG modulates calcium release from the ER after Fas stimulation and inhibits Fasinduced apoptosis in type II cells. On the basis of our observations, we propose that LFG protects against Fas-induced apoptosis by modulating calcium release from the ER.

La retinosis pigmentària (RP) es una enfermedad hereditaria que provoca una degeneración progresiva de la retina y en la mayoría de los casos conduce a la ceguera.El trabajo realizado ha permitido clasificar genéticamente a los pacientes afectados de RP, atendiendo a su patrón de herencia.Uno de los objetivos ha sido la determinación del origen molecular de la RP en los casos de retinosis pigmentaria autosómico dominante (RPAD) en la población española así como en casos esporádicos o aislados de RP (SRP). Así, se ha realizado el estudio genético directo de los genes de expresión específica de retina asociados a retinosis pigmentària autosómica dominante (RHO, RDS-periferina, ROM-1, RP1, NRL y CRX) y la caracterización de las mutaciones detectadas. Se recogen también la expresión clínica de las mutaciones descritas y, en los casos que ha sido posible, se ha procurado establecer una co-relación genotipo-fenotipo.Así mismo, se han clonado los genes NRL y CRX, factores de transcripción de genes específicos de retina asociados a RPAD. A partir del gen NRL clonado y, mediante mutagénesis dirigida por PCR, se han obtenido in vitro dos mutantes del gen NRL detectados en la población estudiada. Estudios in vitro de expresión génica con estos mutantes muestran una regulación diferencial del promotor del gen de la rodopsina con respecto la proteína salvaje, produciendo una sobre expresión del gen regulado. Se postula que una sobre alteración de la dosis génica puede ser un mecanismo patológico que desencadene la enfermedad. / The retinosis pigmentària (RP) is a hereditary disease that provokes a progressive degeneracy of the retina and in the majority of the cases he(she) drives to the blindness.The realized work has allowed to classify genetically the affected patients of RP, attending to his(her,your) boss of inheritance(heredity).One of the aims(lenses) has been the determination of the molecular origin of the RP in the cases of retinosis pigmentaria autosómico dominant (RPAD) in the Spanish population as well as in cases sporadic or isolated of RP (SRP). This way, there has been realized the genetic direct study of the genes of specific expression of retina associated to retinosis pigmentària autosómica dominant (RHO, RDS-periferina, ROM-1, RP1, NRL and CRX) and the characterization of detected mutations.There is gathered also the clinical expression of the described mutations and, in the cases that it(he,she) has been possible, a co-relation has tried(got) to establish genotype - fenotipo.Likewise, there are clonado the genes NRL and CRX, factors of transcription of specific genes of retina associated with RPAD. From the gene NRL clonado and, by means of mutagénesis directed by PCR, there have been obtained in vitro two mutants of the gene NRL detected in the studied population. In vitro studies of gene expression with these mutants show a differential regulation of the promoter of the gene of the rodopsina with respect the wild protein, producing one on expression of the regular gene. There is postulated that one on alteration of the dose génica can be a pathological mechanism that unleashes the disease.

El càncer de pròstata és el segon tumor més freqüent i la segona causa de mort per malaltia oncològica en els homes del món occidental. Per a créixer, les cèl·lules epitelials del tumor necessiten els andrògens, les accions dels quals són mediades pel receptor d’andrògens, l’AR, un factor de transcripció que pertany a la família dels receptors hormonals esteroïdals. L’AR és clau en el desenvolupament del tumor de pròstata i per això és la major diana terapèutica en el tractament amb deprivació hormonal en aquesta patologia. La teràpia és efectiva durant un cert període de temps, però després els tumors esdevenen resistents a la castració, augmentant la seva agressivitat. La diabetis melitus (DM) s’ha associat amb un risc augmentat de patir nombrosos càncers. No obstant, diversos estudis demostren que la DM comporta menys risc de patir càncer de pròstata. Amb tots aquests antecedents en la literatura vam voler investigar quin paper jugava l’AR en el procés de l’hormonoresistència i si era un factor que influïa en el menor risc de patir càncer de pròstata en els pacients diabètics. Els resultats obtinguts en la tesi (gràcies a l’ús de models cel·lulars i un model animal xenograft de càncer de pròstata) demostren que hi ha un procés de metilació durant la supressió hormonal, però aquest procés només té lloc en el promotor de l’AR quan la supressió és constant i prolongada en el temps. A més a més s’ha comprovat que en absència d’hormones l’AR es localitza majoritàriament a la mitocòndria. Hem demostrat l’existència de 5 unitats de transcripció de l’AR, 3 de les quals no havien estat descrites anteriorment, que no presenten illes CpG i que podrien jugar un paper important en el càncer de pròstata i el seu progrés. Finalment vam comprovar que la hiperglicèmia redueix els nivells d’AR, a través de l’activació de NF-kB, per tant, els pacients diabètics tindrien menys risc de patir càncer de pròstata degut al silenciament de l’AR canònic. De tos els resultats obtinguts en la tesi podem concloure que el receptor d’andrògens té una gran importància en el desenvolupament i la progressió del càncer de pròstata i que és una molt bona diana per desenvolupar nous fàrmacs. / Prostate cancer is the second most common tumour and the second cause of cancer death in men in western world. To grow, tumour epithelial cells need androgens whose actions are mediated by androgen receptor, a transcription factor that belong to the family of hormonal receptors. AR is essential in the development of prostate cancer and for this reason is the main target in most of the therapies of the tumour. Androgen depletion is effective for a certain period of time but at the end the tumors become resistant to castration, increasing their aggressiveness. Diabetes mellitus (DM) is associated with an increased risk of developing many cancers. However, several studies show that the DM carries less risk of prostate cancer. With all this background in literature we wanted to investigate the role played by AR in the process of hormone resistance and if it AR was a key factor in the reduced risk of prostate cancer in diabetic patients. Our results show that there is a process of methylation during hormonal suppression but it only occurs in AR promoter when the androgen ablation is prolonged in time. In addition, it was found that in the absence of hormones AR is located basically in mitochondria. We have demonstrated the existence of 5 transcription units of the AR, 3 of which had not been previously described, with no CpG islands. These new transcription units could play an important role in prostate cancer and its progression. Finally we found that hyperglycemia reduces AR levels through the activation of NF-kB, so diabetic patients have less risk of prostate cancer due to silencing of canonical AR. As a summary, our results indicate that the androgen receptor has a important role in the development and progression of prostate cancer and is a key target for developing new drugs.

La búsqueda de procesos de síntesis de moléculas orgánicas altamente eficientes y selectivos es uno de los retos de la química. En los últimos años, las enzimas se han presentado como una seria alternativa a los catalizadores homogéneos tradicionales debido a su alta eficiencia y selectividad natural. Desafortunadamente, las reacciones que pueden catalizar estas especies se suelen restringir a aquellas que tienen importancia para el huésped que las alberga, limitando su aplicación en el ámbito químico/industrial. Para poder solventar estas limitaciones se han desarrollado las llamadas metaloenzimas artificiales. Estos híbridos se obtienen a partir de la inserción de un catalizador homogéneo en una proteína. De este modo el receptor protege el cofactor inorgánico y aporta un entorno quiral (enantioselectivo) mientras que el metal es responsable de la reactividad del sistema. Pero el diseño de estos híbridos representa todo un reto: la proteína no ha sido diseñada evolutivamente para reconocer el fragmento inorgánico y el complejo resultante puede no reconocer de forma eficiente el sustrato. Todo esto provoca que la enzima diseñada presente deficiencias en cuanto a la eficiencia catalítica y/o selectividad. Las técnicas de modelización molecular pueden resultar de gran ayuda en el proceso de diseño y de optimización de las metaloenzimas artificiales. Sin embargo, su aplicación en este tipo de sistemas no es trivial. Necesitamos técnicas que permiten una amplia y rápida búsqueda tanto del espacio conformacional como del químico (mecánica molecular) así cómo técnicas que permitan una descripción precisa del metal y sus propiedad electrónicas (mecánica cuántica). Lamentablemente, estas últimas son demasiado costosas a nivel computacional cómo para poder tratar sistemas de estas dimensiones. En esta tesis nos hemos basado en la combinación de diferentes técnicas de modelización molecular, basadas tanto en aproximaciones mecanoclásicas como mecanocuánticas, para poder solventar estas limitaciones. Este enfoque ha sido aplicado al diseño y optimización de metaloenzimas artificiales. En primer lugar hemos estudiado la unión entre el cofactor inorgánico y la proteína, obteniendo una gran correlación entre los modelos teóricos y los resultados experimentales. A continuación las hemos aplicado al estudio de la reactividad del sistema, tanto a la unión del sustrato al complejo proteína-cofactor como a la caracterización de los estados de transición más probables. En este último caso hemos podido comprobar que estos protocolos integrativos son capaces de ofrecer un nivel muy aceptable de predictibilidad de la enantioselectividad del sistema. Aunque en esta tesis hemos demostrado la gran utilidad que pueden tener los protocolos que integran diversas técnicas de modelización molecular en el estudio de complejos biometálicos, su desarrollo no es trivial. La mayoría de programas de modelización molecular no han sido diseñados para ser utilizados en tándem y todos ellos tienen su propia forma de tratar la información molecular. Para solventar estas incompatibilidades se necesitaría una plataforma integrativa que englobara todas estas técnicas y regulara el flujo de información entre ellas. A nivel comercial estas plataformas ya existen, pero su uso es altamente restrictivo ya que son muy caras y son de código cerrado, dejando muy poco margen para su adaptabilidad a cada problema molecular. Por este motivo en esta tesis nos hemos centrado en el desarrollo de una plataforma integrativa de código abierto accesible a toda la comunidad de modelizadores. Hasta el momento, esta plataforma incluye interfaces para realizar cálculos de modos normales y dinámicas moleculares, así como permitir el análisis de cálculos cuánticos con Gaussian y cálculos de docking proteína-ligando con GOLD. Se espera incorporar muchas mas técnicas en un futuro próximo. / The search of highly efficient and selective processes for the synthesis of organic molecules is one of the challenges of chemistry. In the last years, enzymes have positioned as a clear alternative to traditional homogeneous catalysts due to their high natural efficiency and selectivity. Unfortunately, their range of different reactions is restricted to those that are useful for their host. To solve this limitation the so-called artificial metalloenzymes have been developed. These hybrids are based on the insertion of an homogeneous catalyst into a protein scaffold. This way, the receptor protects the inorganic cofactor and provides with a chiral (enantioselective) environment while the metal is responsible for the reactivity of the system. However, their design is highly challenging: the protein has not been optimized to bind such inorganic compounds and the resulting complex may not efficiently recognize the substrate. Altogether, these could mean that the artificial enzyme has several deficiencies in terms of catalytic efficiency and/or enantioselectivity. Molecular modelling techniques could aid in the design and optimization of artificial metalloenzymes. However, their application is not straightforward due to the complexity of these hybrids. We need both techniques allowing wide and fast conformational and chemical space searches (molecular mechanics) and techniques offering an accurate description of the metal and its electronic states (quantum mechanics). Unfortunately, the later ones are too computational intensive to treat systems of a huge size such as enzymes. In this Ph. D. thesis we combined several molecular modeling techniques, based either in classical or quantum mechanics approximations, to solve those limitations. This approach has been applied on the design and optimization of artificial metalloenzymes. First, we have studied the binding of the inorfanic cofactor in the protein scaffold, obtaining very good agreement between the theoretical models and the experimental results. Afterwards, we applied them to the study of the reactivity of the system, including the binding of the substrate to the cofactor-protein complex and the characterization of the most-likely transition states. In this last case we demonstrated that these kind of integrative protocols are able to offer high predictive profiles of the enantioselectivity of the system. Even though in Ph. D. thesis we have demonstrated the great utility that integrative approaches encompassing several different molecular modeling tehcniques could have in the study of biometallic complexes, their development is far from easy. The major part of modeling softwares have not been designed to be part of this kind of integrative protocols and each one of them has their own way to treat the molecular data. To solve those incompatibilities, it is necessary an integrative platform encompassing all those techniques that regulates the flux of information between them. This kind of platforms are already available at a commercial level, but they are rather expensive and are based on a black-box approach, thus the user cannot adapt them to its own molecular problem. For this reason, in this thesis we have developed an integrative platform, which is open-code and freely-available to all the members of the modeling community. At the moment, it includes interfaces to perform Normal Modes Analysis and Molecular Dynamics simulations. Additionally, it is also able to analyze the results of quantum calculations performed with Gaussian and protein-ligand dockings performed with GOLD. More implementations are still under development.

1- ROLE OF SIRT1 IN THE REGULATION OF FATTY ACID OXIDATION AND KETOGENESIS UNDER DIFFERENT NUTRIONAL CHANGES. The homolog of the yeast silencing information regulator2 (SIRT1) has been implicated in several aspects of food limitation and caloric restriction in mammals. We have observed that there were no important changes, between wild type (WT) and SIRT1 liver-specific knockout (LKO) mice subjected to either CR or high fat diet (HFD), in the mRNA expression of Cpt1a, Cpt2 and Hmgcs2. SIRT1 had been shown to control hepatic glyconeogenic/glycolytic pathways in response to nutrients (Rodgers et al., 2005). So, we have hypothesized that SIRT1 could have a role in the metabolic adaptation to the changes of nutrient of weaning, when milk is replaced by the adult diet which contains less fat and more carbohydrate. Neither fatty acid oxidation (Cpt1a), ketogenesis (Hmgcs2), nor gluconeogenic (Pck1) liver pathways were significantly affected by the liver-specific knockdown of SIRT1, in both suckling and post-weaning conditions. If SIRT1 is involved in the response to aging, old LKO mice might be more susceptible to age-associated diseases as obesity, type 2 diabetes, hypertension, etc. To test this hypothesis we first weight and performed a glucose tolerance test in 7 months-old mice. There were no differences in weight neither in glucose tolerance between WT and LKO mice. Using a cell system, PPARα induced the expression of its known target genes CPT1A, HMGCS2, and FGF21 in HepG2 cells. SIRT1 overexpression by itself had almost no effect, although it increased PPARα induction of its target genes. We have interfered SIRT1 in these cells, and PPARα-induced expression of PEPCK and FGF21 was SIRT1-dependent. We have fasted WT and LKO mice for 15h and we found that the expression of Pck1 in liver was moderately but significantly induced in SIRT1-LKO mice after fasting, consistent with an increase in glucose levels. However, neither G6pase nor Pgc1α mRNA levels were affected. As expected, liver mRNA levels of Cpt1a, Hmgcs2, and Fgf21 were also induced upon fasting. However, Cpt1a and Hmgcs2 mRNA transcripts were comparable in fasted WT versus LKO mice, while Fgf21 expression was reduced around 40% in LKO mice liver. This result was consistent with the fact that fasting induction of FGF21 serum levels was also impaired in LKO mice. 2- ROLE OF SIRT1 IN THE HMGCS2 REGULATION OF FGF21 EXPRESSION. (Article 1: Human HMGCS2 regulates fatty acid oxidation and FGF21 expression in HepG2 cells. Vilà-Brau et al., 2011) Recently, our group has seen that HMGCS2 expression stimulates FGF21 expression and that these events are dependent on HMGCS2 activity. A catalytic dead mutant (C166A) failed to induce either fatty acid β-oxidation or FGF21 expression, whereas acetoacetate (an oxidized form of ketone bodies) could stimulate FGF21 mRNA expression in a dose-dependent manner. Because ketone bodies production implies the reduction of acetoacetate to β- hydroxybutyrate with the concomitant generation of NAD+ (Hegardt et al, 1999), and SIRT1 is a NAD+-dependent deacetylase enzyme, this specificity could explain why FGF21 fasting induction was affected in LKO-SIRT1 mice liver, while other PPARα target genes were not. We have treated HepG2 cells with the oxidizing (acetoacetate) partner of ketone bodies, and endogenous SIRT1 was knockdown by a specific siRNA. FGF21 induction was dependent on SIRT1 expression, since knocking down impaired acetoacetate response. 3- ACTIVATING TRANSCRIPTION FACTOR 4-DEPENDENT INDUCTION OF FGF21 DURING AMINO ACID DEPRIVTION (Article 2: De Sousa-Coelho et al., 2012) Considering the central role of PPARα in the regulation of metabolic homeostasis we sought to investigate how the turnover of PPARα affected the expression of its target genes. HepG2 cells were infected with PPARα and exposed to DMSO or to the 26S proteasome inhibitor MG132. As expected, MG132-treatment blocked the PPARα-dependent expression of HMGCS2, indicating that the transcriptional activity of PPARα is increased by protein degradation (Blanquart et al, 2004). Contrary to what we had predicted, the expression of FGF21 was strongly increased by the MG132 treatment. We hypothesized that proteasome inhibition in HepG2 could decrease the pool of free amino acids. We treated HepG2 cells with histidinol (HisOH) a potent and reversible inhibitor of protein synthesis, (Hansen et al, 1972). Amino acid deprivation produced a time-dependent induction of FGF21 mRNA. To test whether this induction was due to an increase in the FGF21 gene transcription, we measured the FGF21 primary transcript (hnRNA) levels; HisOH treatment clearly induced FGF21 hnRNA levels in a time-dependent manner. As expected, HisOH induced an increase in the ATF4 protein levels after 2h treatment. By analyzing the sequence of the 5’-flanking region of the human FGF21 gene, we found two putative ATF4 response elements (AARE) starting at positions -152 and -610 upstream of the transcription start site. HepG2 cells were transfected with pGL3b-hFGF21 promoter-luciferase constructs and an expression vector for human ATF4. The expression of ATF4 induced the WT reporter in a concentration dependent manner. This induction was totally obliterated either when the AARE1 was mutated or when both elements were deleted. Induction was diminished when AARE2 was mutated. To further analyze the functionality of this sequence we tested the binding of ATF4 by an EMSA, where ATF4 bound as a C/EBPβ heterodimer to both AARE sequence elements. We also confirmed the in vivo binding by ChIP experiments. The chromatin binding of ATF4 was greatly increased in both ATF4 responsive sequences in HisOH treated cells. To confirm if the induction of FGF21 produced by amino acid starvation was mediated by ATF4, we treated siCtl and siATF4 HepG2 cells with HisOH. FGF21 mRNA levels after HisOH treatment were significantly lower when ATF4 was depleted. To analyze the effect of amino acid deprivation on FGF21 expression in vivo, we fed mice with a leucine-deficient [(-)leu] diet or a control (Ctl, nutrionally complete) diet for 7 days. Fgf21 mRNA levels were greatly increased in liver from mice fed a (-)leu diet compared to control. The circulating FGF21 levels were also increased in the serum of leucine deprived animals, paralleling hepatic gene expression. 4- LEUCINE DEPRIVATION SIGNALING UNDER FASTING CONDITIONS. We were interested to know whether FGF21 induction by a (-)leu diet would affect, or be affected by, the adaptive fasting response. We have fed mice for 7 days within a Ctl or (-)leu diet. Weights and food intake were recorded daily. Then, mice were randomly separated in a total of 4 groups, where 2 groups (one from each diet) were fasted overnight. Leucine deprivation affected the levels of free fatty acids and ketone bodies in serum in the fed state, while it does not upon fasting. Although no changes between groups were observed in the fed state, after fasting the β-oxidation, ketogenesis and gluconeogenesis keygenes were further up-regulated in the (-)leu diet group compared to control. The highest induction was seen in the Pgc1α gene, a known coactivator on these processes. The fasting activation of FGF21 was impaired in mice fed with (-)leu diet, underlying a crosstalk between the fasting and amino acid deprivation signalling. 5- ROLE OF FGF21 IN THE LEUCINE DEPRIVATION PHENOTYPE IN MICE (Article 3: De Sousa-Coelho et al., in preparation). According with our previously reported results (De Sousa-Coelho et al., 2012) mice maintained on a leucine-deficient [(-)leu] diet show a dramatic increase in FGF21 circulating levels. To check its origin we analyzed the Fgf21 gene expression in liver, where Fgf21 mRNA levels paralleled those in serum; brown adipose tissue (BAT), where it were unchanged; and in epididymal white adipose tissue (eWAT), where unexpectedly it were significantly decreased in wild type mice maintained in (-)leu diet. Because upon (-)leu diet, mice undergo rapid weight loss (Cheng et al., 2010), we wanted to investigate whether this phenotype is FGF21-dependent. For this purpose, WT and FGF21-KO mice were fed a Ctl or (-)leu diet for 7 days. Weight loss was diminished in FGF21-KO, while food intake decrease by (-)leu was unchanged between genotypes. Histological analysis of WAT showed that leucine deprivation resulted in a reduction in adipocyte volume compared with mice fed a control diet, while it was only slightly reduced in (-)leu FGF21-KO mice. It has been previously described that leucine deprivation increases lipolysis in WAT (Cheng et al., 2010). Consistent with changes in body weight, lack of FGF21 significantly decreased levels of phosphorylated (P)-HSL in WAT, indicating an impaired lipolysis. Gene expression analysis revealed reduction in the mRNA levels of the lipogenic genes Fas, Srebp1c and Acc1 in the WAT of mice maintained in (-)leu diet. These changes were impaired in FGF21-KO. Consistent with previous results (Cheng et al., 2010), leucine deprivation increased levels of Ucp1 mRNA in WT mice BAT. This increase was not observed in the FGF21-KO mice. mRNA levels of Pgc1α, which regulates the expression of Ucp1 (Handschin and Spiegelman, 2006), were also increased, although did not differ between WT and FGF21-KO mice under either control or (-)leu diet conditions. It has been recently proposed a link between FGF21 and SREBP1c during lipogenesis in HepG2 cells (Zhang et al., 2011). We examined levels of Fas, Srebp1c and Acc1 mRNA in liver of WT and FGF21-KO. As expected (Guo and Cavener, 2007), lipogenic program was decreased upon (-)leu diet, but this reduction was blocked in FGF21-KO mice. However, the amino acid response program was correctly initiated in these mice as shown by the increased levels of ATF4 protein and the increase in mRNA levels of Asns, a prototypical ATF4 target gene. The liver staining showed a decreased lipid accumulation under (-)leu in WT animals that is not produced in the FGF21-KO mice. These results demonstrate an important role of FGF21 in the regulation of lipid metabolism during amino acid starvation. References: Blanquart C, Mansouri R, Fruchart JC, Staels B, & Glineur C (2004) Different ways to regulate the PPARalpha stability. Biochem Biophys Res Commun 319, 663-70. Cheng, Y., Meng, Q., Wang, C., Li, H., Huang, Z., Chen, S., Xiao, F., and Guo, F. (2010). Leucine deprivation decreases fat mass by stimulation of lipolysis in white adipose tissue and upregulation of uncoupling protein 1 (UCP1) in brown adipose tissue. Diabetes 59, 17-25. Guo, F., and Cavener, D.R. (2007). The GCN2 eIF2alpha kinase regulates fatty-acid homeostasis in the liver during deprivation of an essential amino acid. Cell Metab 5, 103-14. Handschin, C., and Spiegelman, B.M. (2006). Peroxisome proliferator-activated receptor gamma coactivator 1 coactivators, energy homeostasis, and metabolism. Endocr Rev 27, 728-735. Hansen BS, Vaughan MH, & Wang L (1972) Reversible inhibition by histidinol of protein synthesis in human cells at the activation of histidine. J Biol Chem 247, 3854-3857. Hegardt FG (1999) Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase: a control enzyme in ketogenesis. Biochem J. 338, 569-582. Hotta, Y., Nakamura, H., Konishi, M., Murata, Y., Takagi, H., Matsumura, S., Inoue, K., Fushiki, T., and Itoh, N. (2009). Fibroblast growth factor 21 regulates lipolysis in white adipose tissue but is not required for ketogenesis and triglyceride clearance in liver. Endocrinology 150, 4625-4633. Rodgers JT, Lerin C, Haas W, Gygi SP, Spiegelman BM, Puigserver P (2005) Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1. Nature 434, 113-8 Zhang, Y., Lei, T., Huang, J.F., Wang, S.B., Zhou, L.L., Yang, Z.Q., and Chen, X.D. (2011). The link between fibroblast growth factor 21 and sterol regulatory element binding protein 1c during lipogenesis in hepatocytes. Mol Cell Endocrinol 342, 41-47. / Durante su vida un individuo se somete a diversos cambios nutricionales. La capacidad de detectar la disponibilidad de nutrientes y regular la homeostasis energética es un proceso fundamental. SIRT1 es un regulador clave en el metabolismo energético. SIRT1 puede modular la expresión génica en tejidos metabólicamente activos en respuesta a la restricción calórica o el ayuno. La dependencia de la actividad deacetilasa de SIRT1 en los niveles de NAD+ constituye un vínculo fundamental entre el estado metabólico celular y la regulación de genes. FGF21 es una hormona que se induce en el ayuno y que afecta al metabolismo de los carbohidratos y de los lípidos. Recientemente se han demostrado sus efectos benéficos en la protección de la obesidad inducida por la dieta y en la mejoría en la resistencia a la insulina. En este trabajo hemos demostrado que en células hepáticas en cultivo, SIRT1 desempeña un papel importante en la activación por PPARα de la expresión de FGF21, CPT1A, HMGCS2, y PEPCK. También, que la actividad de SIRT1 regula los niveles de glicemia y la expresión de PCK1 en hígado, en la respuesta al ayuno. Aún así, hemos visto que la actividad de SIRT1 no afecta la expresión de los genes de la oxidación de los ácidos grasos o la cetogénesis en el hígado, en respuesta a diferentes cambios nutricionales, como la restricción calórica, la transición de la lactancia/destete, y el ayuno. Adicionalmente, hemos demostrado que la activación de FGF21 por SIRT1 depende de la actividad HMGCS2. También hemos descrito que FGF21 se induce por la privación de aminoácidos, de manera dependiente de ATF4, y hemos identificado dos elementos de respuesta funcionales en la región promotora del gene humano, altamente conservados entre las especies. Además, hemos demostrado que FGF21 interviene en la regulación del metabolismo de los lípidos en el hígado y el tejido adiposo blanco, y de la termogénesis en el tejido adiposo marrón, durante la privación de aminoácidos. De todas formas, hemos visto que la privación de leucina afecta a los niveles de ácidos grasos libres y cuerpos cetónicos en suero en el estado de alimentación, mientras que no lo hace en el ayuno; y la activación de FGF21 en el ayuno está afectada en los ratones alimentados con esta dieta, desvelando un “crosstalk” entre la señalización del ayuno y la privación de aminoácidos.

Els pacients diabètics de tipus 1 necessiten teràpia substitutòria amb insulina per poder sobreviure, però tot i això, no sempre s’aconsegueix una regulació adequada de la seva glucèmia. La hiperglucèmia crònica porta al desenvolupament d’importants complicacions secundàries que estan associades amb una elevada morbilitat i mortalitat. El desenvolupament de complicacions secundàries es pot enlentir mitjançant un control estricte de la glucèmia, però això resulta difícil degut a que la teràpia amb insulina exògena està associada amb un risc sever d’episodis d’hipoglucèmia. Per tant, la regulació precisa de l’homeostasi de la glucosa és un repte en el tractament de la diabetis. La manipulació genètica del múscul esquelètic representa una estratègia atractiva per tal de tractar la diabetis. El múscul esquelètic és el responsable de la captació de més d’un 70% de la glucosa postpandrial. A més a més, el múscul esquelètic és fàcilment manipulable, és capaç de secretar proteïnes a torrent sanguini i és transduible per varis vectors terapèutics. Al nostre laboratori prèviament s’ha demonstrat que és possible generar al múscul esquelètic un “sensor de la glucosa” mitjançant la coexpressió de glucoquinasa (Gck) i de nivells constitutius i basals d’insulina (Ins). En aquest sistema, el flux de glucosa cap al múscul esquelètic és regulat pels nivells de glucosa circulants, permetent un increment en la captació de glucosa en presència d’hiperglucèmia, però evitant-se possibles episodis d’hipoglucèmia. Per tal de comprovar aquesta hipòtesi, ratolins diabètics van ser administrats intramuscularment amb 2 vectors adenoassociats de tipus 1 (AAV1) que expressaven els gens de la Ins i la Gck. Després del tractament els ratolins diabètics van corregir la malaltia. Els vectors AAV han estat els vectors d’elecció per moltes aplicacions de teràpia gènica in vivo degut al seu excel·lent perfil de seguretat i eficàcia. Estudis preclínics han mostrat que la transferència genètica mitjançant vectors AAV permet en models animals petits i grans de malalties l’expressió gènica a llarg plaç. Recentment vàries d’aquestes aproximacions de teràpia gènica han estat traslladades a humans amb resultats prometedors. Tot i això, la gran majoria d’assajos preclínics exitosos en ratolins han fracassat al ser traslladats a models animals grans o en humans. Encara no s’ha aconseguit traslladar amb èxit a animals grans cap aproximació de teràpia gènica o cel·lular per la diabetis. En aquest estudi, l’administració en gossos diabètics una única vegada de vectors AAV1 que codifiquen pels gens de l’Ins i de la Gck va resultar en la normalització de la glucèmia en dejú, de la tolerància a la glucosa després d’un bolus oral de glucosa i en l’absència d’episodis d’hipoglucèmia en exercici durant >4 anys després de la transferència gènica. Això va estar associat amb una recuperació del pes corporal, una normalització dels nivells de proteïnes glicosilades en plasma i una supervivència a llarg plaç sense el desenvolupament de complicacions secundàries. En canvi, el tractament amb insulina exògena o la transferència gènica únicament d’Ins o Gck va ser incapaç d’aconseguir una correcció completa de la diabetis, demonstrant-se que l’acció conjunta de la Ins i la Gck és necessària per tal d’obtenir un efecte terapèutic complet. Aquesta demonstració de la correcció a llarg plaç de la hiperglucèmia diabètica en gossos diabètics representa el pimer èxit en un model animal gran de una teràpia gènica o cel·lular pel tractament de la diabetis y senta les bases pel futur assaig d’aquesta aproximació terapèutica en humans. / Type 1 diabetes patients need insulin replacement therapy to survive, but glycemia is not always regulated in a physiologic manner. Chronic hyperglycemia leads to the development of severe secondary complications that are associated with significant morbidity and mortality. The development of secondary complications can be delayed by tight control of glycemia, however, this is difficult to achieve with exogenous insulin administration because of the associated risk of severe hypoglycemic episodes. Thus, precise regulation of glucose homeostasis is a major challenge in diabetes management. Genetic engineering of skeletal muscle to counteract hyperglycemia is an attractive strategy to correct diabetes. Skeletal muscle is responsible for the disposal of most (70%) of the circulating glucose after a meal. In addition, skeletal muscle is easily manipulated and is able to secrete proteins into the circulation as well as being transducible by a number of therapeutic vectors. We previously demonstrated in our laboratory that it is possible to generate a “glucose sensor” in the skeletal muscle through co-expression of glucokinase (Gck) and constitutive basal levels of insulin (Ins). In such a system, glucose flux into skeletal muscle is regulated by circulating glucose levels, allowing increased glucose uptake whenever hyperglycemia is present, but avoiding hpyoglycemia. To test this hypothesis, two adeno-associated viral vectors of serotype 1 (AAV1) expressing Ins and Gck were delivered intramuscularly to diabetic mice, showing correction of the disease. AAV vectors are the vector of choice for many in vivo gene therapy approaches due to their excellent safety and efficacy profile. Pre-clinical studies have shown that AAV vector-mediated gene transfer results in long-term gene expression in small and large animal models of disease. Recently, some of this preclinical data have been translated into humans with encouraging results. However, for the vast majority of successful proof-of-concept studies in mice, scale-up and long-term efficacy in large animal models or humans has been problematic or disappointing. Scale-up has yet to be demonstrated in large animal models of diabetes with either gene or cell therapy approaches. In this study, a one-time intramuscular administration of AAV1 vectors encoding for Gck and Ins in diabetic dogs resulted in normalization of fasting glycemia, normalized disposal of glucose after oral challenge, and no episodes of hypoglycemia during exercise for >4 years after gene transfer. This was associated with recovery of body weight, normal glycosylated plasma proteins levels, and long-term survival without secondary complications. Conversely, exogenous insulin or gene transfer for Ins or Gck alone failed to achieve complete correction of diabetes, indicating that the synergistic action of Ins and Gck are needed for full therapeutic effect. This demonstration of long-term correction of diabetic hyperglycemia has provided the first proof-of-concept in a large animal model for a gene transfer approach to treat diabetes and lays the foundations for the future translation of this approach to the clinic.

Aquesta Tesi Doctoral se centra en el desenvolupament de noves metodologies analítiques en biosensors òptics com a solucions alternatives per a la diagnosi o la monitorització terapèutica de diferents malalties, com ara l’al·lèrgia, la celiaquia o el càncer. En particular, es proposa l’ús de biosensors nanoplasmònics per a la detecció de biomarcardors presents en fluids humans de manera ràpida, sensible i que no requereixi d’amplificació de senyal o de l’ús d’etiquetes. Tant el ja ben establert biosensor de Ressonància de Plasmó Superficial (SPR) com un innovador biosensor nanoplasmonic basat en nanodiscs d’or han estat avaluats per a la seva aplicació real en l’àrea clínica. Les distintes metodologies biosensores presentades estan basades en l’ús d’anticossos, tant com a elements de bioreconeixement o com a biomarcadors específics de malalties. Primer, es presenta un estudi en profunditat de dues estratègies d’immobilització orientada d’anticossos per tal d’obtenir immunoassaigs en format directe de biomarcadors proteics en fluids biològics. En segon lloc, es proposa una nova estratègia immunosensora per a la detecció de pèptids derivats del gluten directament en orina com a tècnica ràpida i no invasiva per al control dietètic de pacients celíacs. A més, s’han desenvolupat dues metodologies utilitzant el biosensor nanoplasmònic per a detectar anticossos circulants en sang com a biomarcadors de malalties. Per una banda, s’ha dissenyat una estratègia alternativa per a la diagnosi d’al·lèrgia als medicaments (en particular a l’antibiòtic amoxicil·lina) basada en uns receptors dendrimèrics per a la detecció directa d’anticossos tipus IgE en sèrum. Finalment, s’ha avaluat una nova estratègia biosensora per a quantificar específicament autoanticossos tumorals per a la diagnosi precoç de càncer colorectal. El treball d’aquesta Tesi combina l’experiència del grup de recerca en el disseny i fabricació de tecnologia biosensora avançada i innovadora amb el desenvolupament de tècniques bioanalítiques i de química de superfície per tal de superar els reptes actuals relacionats amb el cost i el temps requerit per a les anàlisis clíniques. A més, l’àmplia experiència del grup de recerca en transferència tecnològica i les col·laboracions establertes durant la tesi doctoral amb empreses com Biomedal S.L. o Protein Alternatives S.L. obren oportunitats interesants de cara a facilitar el procés de transferència tecnològica per a la implementació real de biosensors tipus Point-of-Care. / This Doctoral Thesis focuses on the development of novel analytical methodologies in optical biosensors as alternative solutions for diagnosis or therapy monitoring of relevant diseases, such as allergy, celiac disease or cancer. In particular, we propose the use of nanoplasmonic biosensors for a rapid, sensitive and label-free detection of biomarkers present in human fluids. Both the well-known Surface Plasmon Resonance (SPR) biosensor and an innovative nanoplasmonic biosensor based on gold nanodisks surfaces have been evaluated for their real application in the clinical field. The different biosensor methodologies make use of antibodies, either as biorecognition elements in immunoassays or as specific disease biomarkers for diagnostics. First, an in-depth study of two site-directed antibody immobilization strategies is presented for the direct immunoassay of protein biomarkers in biological fluids. In second place, a novel immunosensing strategy is proposed for the detection of gluten-derivative peptides in urine as a rapid and non-invasive technique for dietary control in celiac patients. On the other hand, two assays have been developed employing the nanoplasmonic biosensor to detect blood circulating antibodies as disease biomarkers. First, we have designed an alternative approach for drug allergy diagnosis (in particular for amoxicillin) based on dendrimer-based receptors, which enable the detection IgE antibodies directly in serum. And second, a new biosensing strategy is assessed to quantify specific tumor-related autoantibodies for the early diagnosis of colorectal cancer. The work in this Thesis combines the wide knowledge of the research group in the design and fabrication of powerful biosensor technology with the development of surface activation chemistry and bioanalytical techniques to overcome current challenges related to costly and time-consuming clinical analysis. Besides, the strong experience of our research group in technological transfer and the established collaborations during this doctoral work with companies as Biomedal S.L. or Protein Alternatives S.L. open up interesting opportunities to facilitate the technology-transfer process for the real implementation of Point-of-Care biosensors.

Los tumores cerebrales constituyen menos del 2% de los tumores primarios, pero son uno de los peores tipos de cáncer en cuanto a “años de vida perdidos”. Los gliomas son los tumores cerebrales más prevalentes, con una esperanza de vida inferior a 15 meses para los casos de alto grado, como los glioblastomas (GBM). El método no invasivo más utlilizado para diagnóstico y seguimiento de la respuesta a la terapia en tumores cerebrales es la Resonancia Magnética (MR), en forma de imagen (MRI) y espectroscopía (MRS) o imagen espectroscópica (MRSI). Sin embargo, debido a restricciones éticas relacionadas a la participación de pacientes humanos en investigación, la optimización de los métodos de diagnóstico y seguimiento de la terapia requieren modelos preclínicos que reproduzcan las patologías humanas. Para este tipo de estudios, se utilizan principalmente modelos murinos, que pueden dividirse en modelos genéticamente modificados (GEM) con desarrollo espontáneo de tumor y los modelos de generación de tumores por inyección estereotáxica de líneas celulares. En la presente tesis, se llevó a cabo una detallada caracterización de dos colonias GEM, S100β-v-erbB/inK4a-Arf (+/-) y GFAP-V12 HA-ras B8. Se observó una baja penetrancia de desarrollo de tumores (16% y 1% respectivamente), haciendo de éstos una herramienta poco útil para estudios de respuesta a la terapia. La escasez de modelos preclínicos de grado bajo/intermedio sirvió de motivación para desarrollar un modelo de tumor glial transplantable con esas características, por disgregación de tumores provenientes de la colonia GEM. Ello nos debería permitir obtener una mayor incidencia tumoral en comparación con las colonias GEM. Se generaron gliosferas a partir de tumores GEM de grado III y se logró más de un 60% de penetrancia cuando estas células fueron inyectadas estereotácticamente en el estriado de ratones C57BL/6. Sin embargo, la aplicación de protocolos de descongelación y cultivo a éstas células ocasionó una progresión a grado IV (GBM), lo que sugiere que el modelo generado constituye, potencialmente, un modelo murino de GBM secundario. Además, este modelo transplantable fue ampliamente caracterizado por métodos de MRI/MRSI, así como por métodos de perturbación del patrón espectral con MRSI (PE-MRSI) para una posible aplicación en el desarrollo de clasificadores de respuesta a la terapia en tumores. También se llevó a cabo una evaluación genética restringida de los modelos murinos seleccionados (p.e. tumores GL261, línea celular GL261, GEM y tumores derivados de GEM), utilizando el método de secuenciación de Sanger para comprobar la presencia de mutaciones normalmente presentes en gliomas (en los genes IDH1, IDH2 y p53). Finalmente, esta tesis describe la estrategia desarrollada para estudios longitudinales de detección de respuesta temprana a la terapia/recidiva en tumores preclínicos, utilizando métodos de imagen molecular basados en MRSI. Así ratones implantados con tumores GL261 (glioblastoma) recibieron tratamiento con temozolamida (TMZ), basándonos en protocolos previamente establecidos. La detención del crecimiento tumoral (respuesta a la terapia) fue detectada por MRI. Tanto animales tratados como animales control fueron estudiados por MRSI y técnicas de reconocimiento de patrones (extracción de fuentes en sistema semi-supervisado). Las fuentes extraídas de regiones de interés fueron capaces de distinguir entre tumores GL261 proliferando activamente y tumores respondiendo a la terapia, basándose en cambios de patrón del metaboloma registrado por MRSI. Se obtuvieron mapas nosológicos codificados en tipo e intensidad de color durante y después de la terapia, lo que permitió un seguimiento de la respuesta, así como la detección de heterogeneidad intratumoral en dicha respuesta, siendo capaces de detectar la detención del crecimiento tumoral y la recidiva antes de los cambios observados en el volumen tumoral por MRI. Esta metodología fue ratificada por análisis histopatológico y cálculo de tasas de proliferación, apoptosis y de índice mitótico. / Brain tumours account for less than 2% of all primary tumours, but are one of the most lethal cancers when “life lost” years are considered. Gliomas are the most prevalent type with a median life expectancy below 15 months for the high grade ones, such as glioblastomas (GBM). The most common non-invasive medical technique used for tumour diagnosis and therapy monitoring of brain tumours patients is Magnetic Resonance (MR), in the form of imaging (MRI) and spectroscopy (MRS) or spectroscopic imaging (MRSI). However, due to the ethical restrictions regarding the use of human patients for research study, the improvement of diagnostic and therapy follow-up protocols requires reliable models that mimic human disease. In this regard, mainly murine models are used and can be divided into the genetically engineered model (GEM) of spontaneous tumour development and the engrafted tumour model. In this thesis, a comprehensive MR characterization of two GEM colonies, namely S100β-v-erbB / inK4a-Arf (+/-) and GFAP-V12 HA-ras B8, was carried out. A low tumour penetrance found (16% and 1%, respectively) together with stochastic onset of GEM tumours, made them impractical for use in therapy response studies. The latter and the scarcity of low/intermediate grade brain tumour preclinical models motivated us to attempt to develop a transplantable glial tumour model of low/intermediate grade by disaggregation of a tumour mass from GEM. This should allow us to obtain an increased tumour incidence rate in comparison to GEM animals. Gliospheres from a grade III GEM tumour were successfully generated and displayed more than 60% penetrance, when stereotactically injected into the striatum of C57BL/6 mice. However, the application of freezing and cell culture protocols produced a progression to grade IV GBM, which made the developed transplantable model qualify as potential secondary GBM model in mice. Additionally, this transplantable model was widely characterized using MRI/MRS methods, as well as perturbation-enhanced MRSI (PE-MRSI) for a possible application in the future in therapy strategies and development of tumour therapy response detection classifiers. A restricted genetic evaluation of selected murine tumour models (i.e. GL261 tumours, GL261 cell line, GEM and GEM-derived tumours) was carried out using the Sanger method to check for a possible presence of particular driver mutations commonly occurring in gliomas (IDH1, IDH2 and p53). Finally, the work describes the strategy followed for longitudinal therapy studies follow-up and early response/relapse detection in preclinical brain tumours, through molecular imaging methods based in MRSI. GL261 (glioblastoma) tumour bearing mice were treated with temozolomide (TMZ), based on previously established protocols. The expected transient growth arrest (response to therapy) was detected by MRI. Animals subjected to therapy and control animals were followed up by MRSI and pattern recognition techniques (semi-supervised source extraction) were applied. The sources extracted from the region of interest were able to discriminate between GL261 tumours actively proliferating and tumours responding to therapy, based on their metabolome pattern changes recorded by MRSI. Colour-coded nosological images produced throughout and after the course of therapy allowed convenient tracking of response changes and differentiated the intratumoural heterogeneity of response, hinting the growth arrest and relapse, before changes in tumour volume were observed by MRI. The methodology was validated with histopathological analysis and calculation of proliferation and apoptotic rates and mitotic index.