L'axe hypothalamo-hypophyso-surrénalien (HPA) permet de maintenir l'homéostasie de l'organisme face à divers stress. Qu'ils soient de nature psychologique, physique ou inflammatoire/infectieux, les stress provoquent la synthèse et la libération de CRH par l'hypothalamus. Les cellules corticotropes hypophysaires perçoivent ce signal et en réaction, produisent et sécrètent l'ACTH. Ceci induit la synthèse des glucocorticoïdes (Gc) par le cortex surrénalien; ces stéroïdes mettent le système métabolique en état d’alerte pour la réponse au stress et à l’agression. Les Gc ont le rôle essentiel de contrôler les défenses de l'organisme, en plus d'exercer une rétro-inhibition sur l'axe HPA. L'ACTH est une petite hormone peptidique produite par le clivage d'un précurseur: la pro-opiomélanocortine (POMC). À cause de sa position critique dans la normalisation de l'homéostasie, le contrôle transcriptionnel du gène Pomc a fait l'objet d'études approfondies au cours des dernières décennies. Nous savons maintenant que la région promotrice du gène Pomc permet une expression ciblée dans les cellules POMC hypophysaires. L'étude du locus Pomc par des technologies génomiques m'a permis de découvrir un nouvel élément de régulation qui est conservé à travers l'évolution des mammifères. La caractérisation de cet enhancer a démontré qu'il dirige une expression restreinte à l'hypophyse, et plus particulièrement dans les cellules corticotropes. De façon intéressante, l'activité de cet élément dépend d'un nouveau site de liaison recrutant un homodimère du facteur de transcription Tpit, dont l'expression est également limitée aux cellules POMC de l'hypophyse. La découverte de cet enhancer ajoute une toute nouvelle dimension à la régulation de l'expression de POMC. Les cytokines pro-inflammatoires IL6/LIF et les Gc sont connus pour leur antagonisme sur la réaction inflammatoire et sur le promoteur Pomc via l'action des facteurs de transcription Stat3 et GR respectivement. L'analyse génomique des sites liés ii par ces deux facteurs nous a révélé une interrelation complexe et a permis de définir un code transcriptionnel entre ces voies de signalisation. En plus de leur action par interaction directe avec l’ADN au niveau des séquences régulatrices, ces facteurs interagissent directement entre eux avec des résultats transcriptionnels différents. Ainsi, le recrutement de GR par contact protéine:protéine (tethering) sur Stat3 étant lié à l'ADN provoque un antagonisme transcriptionnel. Inversement, le tethering de Stat3 sur GR supporte une action synergique, tout comme leur co-recrutement à l'ADN sur des sites contigus ou composites. Lors d'une activation soutenue, ce synergisme entre les voies IL6/LIF et Gc induit une réponse innée de défense cellulaire. Ainsi lors d'un stress majeur, ce mécanisme de défense est mis en branle dans toutes les cellules et tissus. En somme, les travaux présentés dans cette thèse définissent les mécanismes transcriptionnels engagés dans le combat de l'organisme contre les stress. Plus particulièrement, ces mécanismes ont été décrits au niveau de la réponse globale des corticotropes et du gène Pomc. Il est essentiel pour l'organisme d'induire adéquatement ces mécanismes afin de faire face aux stress et d'éviter des dérèglements comme les maladies inflammatoires et métaboliques. / The hypothalamo-pituitary-adrenal (HPA) axis regulates homeostasis in various conditions of stress contributing to both the stress response and its termination. Psychological, physical or inflammatory/infectious stresses all prompt the synthesis and secretion of hypothalamic CRH. The pituitary corticotrope cells receive this signal and in turn, secrete ACTH which triggers the synthesis of glucocorticoids (Gc) by the adrenal cortex; these steroids induce a general state of alertness in order to fight or flight aggressions and stresses. Glucocorticoids have the critical role to restrict the stress response by exerting a negative feedback on the HPA axis. ACTH is a small peptidic hormone produced after cleavage of a precursor protein: pro-opiomelanocortin (POMC). Due to its critical role in homeostasis, transcriptional control of the Pomc gene has been intensely studied during the last decades. Previous investigations identified a promoter region that is sufficient for expression of Pomc in the appropriate pituitary cells. Genome-wide studies of the Pomc locus led me to discover a novel regulatory element that is conserved throughout mammalian evolution. The activity of this enhancer is restricted to the pituitary, and more precisely to the corticotrope lineage. Interestingly, its activity depends on a novel transcription factor binding motif that binds homodimers of Tpit, a transcription factor that is only found in pituitary POMC cells. The discovery of this enhancer adds a new dimension in the control of pituitary Pomc expression. The IL6/LIF pro-inflammatory cytokines and the glucocorticoids are well known for their antagonism in control of the inflammatory response; at the Pomc promoter, their action is mediated by the transcription factors Stat3 and GR, respectively. The analysis of genomic sites bound by these two factors revealed a complex relationship and led us to define a transcription regulatory code linking these signalling pathways. In addition to their direct DNA interaction with cognate regulatory sequences, these factors iv interact with each other with different outcomes. Thus, the recruitment of GR on DNAbound Stat3 through protein:protein contacts (tethering) results in transcriptional antagonism. Conversely, Stat3 tethering to GR produces synergism; this is also the case when the two factors are co-recruited to DNA on contiguous or composite binding sites. Prolonged activation of the IL6/LIF and Gc pathways elicits a synergistic innate cell defense response in all cells and tissues. In summary, this doctoral work has defined transcriptional mechanisms that mediate and control the stress response. In particular, pituitary components of the stress response were defined at the level of the Pomc gene and as a global response of corticotrope cells. This response is critical for appropriate organism defense during stresses such as those produced in inflammatory and metabolic diseases.

The central nervous system (CNS) melanocortin signaling pathway plays a critical role in the regulation of metabolism. However, the regulatory effects of CNS melanocortin signaling on hepatic lipid metabolism and fatty liver disease have not been well established. Although the activity of the CNS melanocortin system is regulated by metabolic signals, the mechanism for this regulation is not fully understood. Variants of the FTO (fat mass and obesity-associated) gene are associated with obesity and FTO is expressed in the hypothalamic neurons including proopiomelanocortin (POMC) neurons. Therefore, it is hypothesized that hypothalamic FTO plays a role in the regulation of metabolism by mediating the effect of metabolic signals on hypothalamic melanocortinergic neurons, and that impairments in this regulation may cause metabolic impairments including obesity and fatty liver disease. Intracerebroventricular (i.c.v.) treatment with SHU9119, a melanocortin antagonist, increased hepatic lipid accumulation and the expression of genes encoding lipogenic enzymes in lean mice. Conversely, i.c.v. treatment with MTII, a melanocortin agonist, reduced the expression of hepatic lipogenic genes in association with reduction in body weight in ob/ob mice, a mouse model of fatty liver disease. Immunohistochemical analysis demonstrated that Fto is co-expressed in both POMC and agouti-related protein (AgRP) neurons in the mouse hypothalamus. Fto mRNA and protein expression was reduced by fasting and increased by glucose treatment in nutritionally important hypothalamic nuclei. Fasting-induced reduction in hypothalamic Fto expression was observed in both lean wild-type and obese ob/ob mice, while the stimulatory effect of glucose on hypothalamic Fto expression was absent in ob/ob mice. These findings support the hypothesis that central melanocortin signaling regulates hepatic lipid metabolism in part by regulating de novo lipogenesis. Impairments in the central melanocortin signaling lead to the development of hepatic steatosis, while enhanced melanocortin signaling may be beneficial in reversing abnormal hepatic lipid metabolism in fatty liver disease (Poritsanos et al., 2008). These findings also support the hypothesis that Fto is expressed in the hypothalamic melanocortinergic neurons and is regulated by metabolic signals involving changes in CNS glucose availability and/or glucose action. Impairments in this regulation may cause metabolic impairments including obesity and fatty liver disease.

The central nervous system (CNS) melanocortin signaling pathway plays a critical role in the regulation of metabolism. However, the regulatory effects of CNS melanocortin signaling on hepatic lipid metabolism and fatty liver disease have not been well established. Although the activity of the CNS melanocortin system is regulated by metabolic signals, the mechanism for this regulation is not fully understood. Variants of the FTO (fat mass and obesity-associated) gene are associated with obesity and FTO is expressed in the hypothalamic neurons including proopiomelanocortin (POMC) neurons. Therefore, it is hypothesized that hypothalamic FTO plays a role in the regulation of metabolism by mediating the effect of metabolic signals on hypothalamic melanocortinergic neurons, and that impairments in this regulation may cause metabolic impairments including obesity and fatty liver disease. Intracerebroventricular (i.c.v.) treatment with SHU9119, a melanocortin antagonist, increased hepatic lipid accumulation and the expression of genes encoding lipogenic enzymes in lean mice. Conversely, i.c.v. treatment with MTII, a melanocortin agonist, reduced the expression of hepatic lipogenic genes in association with reduction in body weight in ob/ob mice, a mouse model of fatty liver disease. Immunohistochemical analysis demonstrated that Fto is co-expressed in both POMC and agouti-related protein (AgRP) neurons in the mouse hypothalamus. Fto mRNA and protein expression was reduced by fasting and increased by glucose treatment in nutritionally important hypothalamic nuclei. Fasting-induced reduction in hypothalamic Fto expression was observed in both lean wild-type and obese ob/ob mice, while the stimulatory effect of glucose on hypothalamic Fto expression was absent in ob/ob mice. These findings support the hypothesis that central melanocortin signaling regulates hepatic lipid metabolism in part by regulating de novo lipogenesis. Impairments in the central melanocortin signaling lead to the development of hepatic steatosis, while enhanced melanocortin signaling may be beneficial in reversing abnormal hepatic lipid metabolism in fatty liver disease (Poritsanos et al., 2008). These findings also support the hypothesis that Fto is expressed in the hypothalamic melanocortinergic neurons and is regulated by metabolic signals involving changes in CNS glucose availability and/or glucose action. Impairments in this regulation may cause metabolic impairments including obesity and fatty liver disease.

No description available.

La maladie de Cushing est caractérisée par une sécrétion excessive de l’hormone adrénocorticotrope (ACTH) à partir des tumeur corticotropes de l'hypophyse. Un excès d'ACTH entraîne un hypercortisolisme et provoque des symptômes tels que diabète, hypertension, obésité et les maladies cardiovasculaires entraînant un risque accru de mortalité si la maladie n’est pas traitée. Les tumeurs corticotropes sont caractérisées par la perte du rétro-contrôle négatif exercé par les glucocorticoïdes (GCs) sur la proopiomélanocortine (POMC) qui est le précurseur de l’ACTH : c'est la caractéristique majeure de la maladie de Cushing. Les causes de la résistance aux GC dans les adénomes corticotropes sont encore mal connues. Des études récentes ont montré une surexpression du récepteur du facteur de croissance épidermique (EGFR) dans les adénomes corticotropes provoquant une augmentation de l'activité du gène POMC et de la sécrétion d'ACTH. Les principaux objectifs de ce travail étaient de comprendre la relation entre la signalisation dérégulée de EGF et la résistance aux GCs. Dans le présent travail, nous avons identifié la voie JAK/STAT3 comme la principale voie de signalisation EGFR qui active la transcription du gène POMC. De plus, nous montrons que l'activation de la signalisation EGFR entraîne une résistance du promoteur POMC aux GCs et que l’activation de STAT3 est responsable de cette résistance. STAT3 affecte le mécanisme de transrepression de GR sans affecter le recrutement de GR au promoteur POMC. L’utilisation d’un inhibiteur de STAT3 restaure la répression de la transcription du promoteur POMC par les GCs. Nous avons aussi trouvé que 50% des adénomes corticotropes humains montrent une surexpression de la forme active de STAT3. Nous avons aussi étudié les mécanismes sous le contrôle des GCs qui régulent la prolifération cellulaire et qui pourraient être dérégulés dans la maladie de Cushing. CABLES1 est un régulateur négatif du cycle cellulaire et son expression est sous le contrôle des GCs. L’expression de CABLES1 est perdue dans 55 % des adénomes hypophysaires corticotropes, mais la cause de cette perte est encore mal comprise. Dans ce travail, nous avons identifié quatre variants faux-sens dans le gène CABLES1, deux chez de jeunes adultes (c.532G > A, c.718C > T) et deux chez des enfants (c.935G > A, et c.1388A > G) atteints de la maladie de Cushing. Les quatre variants touchent une région de la protéine CABLES1 qui est proche du motif de liaison de la kinase-3 dépendante des cyclines (CDK3). Ces variants ont perdu la capacité d’inhiber la croissance de cellules corticotropes tumorales (AtT20). Les quatre variantes sont donc des mutations de perte de fonction. En résumé, nos travaux révèlent le rôle important de STAT3 dans la résistance aux GC et ainsi, le blocage de l'action de STAT3 peut être une nouvelle stratégie pour le traitement de la maladie de Cushing. Nous avons aussi supporté un rôle de CABLES1 en tant que nouveau gène prédisposant aux tumeurs hypophysaires. / Cushing’s disease (CD) is characterized by excess secretion of adrenocorticotropic hormone (ACTH) from corticotroph tumors of the pituitary gland. Excessive ACTH leads to hypercortisolism that causes disabling symptoms such as diabetes, hypertension, obesity and cardiovascular disease resulting in an increased risk of mortality if it is not treated. Corticotroph tumors are characterized by the loss of glucocorticoid (GC) feedback repression of the proopiomelanocortin (POMC) that encodes the precursor of ACTH: this is the hallmark of CD. The causes of GC resistance in corticotroph adenomas of CD patients remain unknown. Recent findings showed overexpression of epidermal growth factor receptor (EGFR) in corticotroph adenomas causing increased POMC activity and ACTH secretion. The main objectives of this work were to understand the relationship between deregulated EGF signaling and GC resistance in the tumorigenesis of CD. In the present work, we identified the JAK/STAT3 pathway as the main EGFR pathway activating transcription of the POMC gene. We found that sustained activation of EGFR signaling or overactivation of STAT3 causes unresponsiveness of the POMC promoter to GCs and that activated STAT3 is responsible for GC resistance. STAT3 affects the transrepression mechanism of GR without affecting GR recruitment to the POMC promoter. The use of STAT3 inhibitor restores the repressive effect of GC on POMC transcription. Importantly, 50% of human corticotroph adenomas showed overexpression of activated STAT3. We also studied the mechanisms under the control of GCs that regulate cell proliferation and that could be deregulated in CD. CABLES1 is a negative cell cycle regulator, its expression is under the control of GC. CABLES1 expression is lost in 55 % of corticotroph adenomas and the underlying reasons remain unclear. In this work, we identified the presence of four missense variants in CABLES1 gene, two in young adults (c.532G > A, c.718C > T) and two in children (c.935G > A, and c.1388A > G) with CD. The four variants are close to the predicted cyclin-dependent kinase-3 (CDK3)-binding region of the CABLES1 protein. The variants have lost the ability to inhibit growth of corticotropinoma cells (AtT20). The four variants are thus loss of function mutations. In summary, our work revealed the important role of STAT3 in GC resistance and further indicates that inhibition of STAT3 action may be a novel strategy for CD treatment. We also provided evidence for a role of CABLES1 as a novel pituitary tumor-predisposing gene.

Tesis por compendio / [ES] El sistema de melanocortina es una estructura clave en la regulación de una amplia gama de funciones fisiológicas que incluyen la melanogénesis, la respuesta al estrés y el equilibrio energético, mediante la unión a una familia de receptores acoplados a la proteína G específicos (MC1R-MC5R). La sobreexpresión de agonistas inversos, la proteína de señalización agutí (Asip) y la proteína relacionada con agutí (Agrp) da como resultado un aumento de la ingesta de alimentos, de crecimiento lineal y de peso corporal. Asip regula la polaridad de pigmentación dorsoventral a través del MC1R, y la sobreexpresión induce obesidad en ratones al unirse al Mc4r central. La sobreexpresión de asip1 en el pez cebra transgénico (asip1-Tg) mejora el crecimiento, sin afectar la acumulación lipídica (obesidad), incluso cuando se alimentan bajo regímenes inductores severos. Los peces asip1-Tg no necesitan comer más para crecer más y más rápido, lo que sugiere una mayor eficiencia alimentaria. Además, los peces asip1-Tg criados en alta densidad son capaces de crecer mucho más que los peces de tipo salvaje (WT) criados en baja densidad, aunque los peces asip1-Tg parecen ser más sensibles al estrés por hacinamiento que los peces WT. El análisis transcriptómico comparativo del intestino de asip1-Tg refleja una expresión diferencial de transportadores aminoácidicos, monocarboxilatos, transportadores iónicos y de vitaminas. La sobreexpresión reduce la integridad del epitelio intestinal aumentando su permeabilidad paracelular y potencia el transporte electrogénico de aminoácidos. Así, los peces transgénicos poseen mayor capacidad para la absorción de nutrientes y, por extensión una mejora en la eficiencia alimenticia que podría explicar, en parte, ese crecimiento diferencial bajo tasas de ingesta similares. Esta tesis tuvo también como objetivo investigar si los asip1-Tg mantienen un fenotipo dominante asociado con una mayor tasa de alimentación. Los resultados muestran, por el contrario, un carácter reactivo/subordinado en los asip1-Tg que aboga por una participación del sistema de melanocortinas en la regulación del comportamiento de peces. El perfil subordinado de los animales asip1-Tg, junto con una activación del eje del estrés, sugiere que estos animales pueden mostrar un comportamiento de ansiedad. Los resultados indicaron que los peces asip1-Tg muestran un comportamiento de ansiedad que además relacionado con una severa disminución de los niveles centrales de serotonina (5HT) y dopamina y elevación de su recaptación neuronal y degradación. La administración de un inhibidor de la recaptación de 5HT, recupera el fenotipo comportamental salvaje, mitigando el comportamiento de ansiedad en los peces transgénicos y rescatando los niveles de 5HT. Esta ansiedad podría repercutir en una alteración del comportamiento locomotor de los animales, por ello estudiamos los ritmos circadianos de actividad locomotora. Los resultados muestran que los animales asip1-Tg exhiben una disrupción completa del ritmo de actividad, con una actividad muy elevada, especialmente durante la noche. Esta disrupción es concomitante con una desaparición del ritmo diario de serotonina y melatonina. Además, los resultados muestran una pérdida de ritmos de expresión de genes reloj (per1a y clock1a). La incubación, in vitro, de glándulas pineales con Asip1 produjo una inhibición de la secreción de melatonina replicando los resultados obtenidos in vivo y demostrando un efecto directo de Asip1, sobre la fisiología de la pineal. En esta tesis, se utilizó el pez cebra como modelo para investigar los efectos de la obesidad sobre la ansiedad y la memoria. La obesidad no tuvo ningún efecto sobre la ansiedad, pero produjo una disminución de la memoria a corto plazo, estudiada mediante test de condicionamiento aversivo. Este estudio proporciona, un protocolo fiable para evaluar el efecto de las enfermedades metabólica en la función cognitiva y conductual. / [CA] El sistema de melanocortina és una estructura clau en la regulació d'una ampla gamma de funcions fisiològiques que inclouen la melanogènesi, la resposta a l'estrès i l'equilibri energètic, mitjançant la unió a una família de receptors acoblats a la proteïna G específics (MC1R-MC5R). La sobreexpressió d'agonistes inversos, la proteïna de senyalització agutí (Asip) y la proteïna relacionada con agutí (Agrp) dona com a resultat un augment de la ingesta d'aliments, de creixement lineal i de pes corporal. Asip regula la polaritat de pigmentació dors-ventral a través del MC1R, y la sobreexpressió indueix obesitat en ratolins en unir-se al MC4R central. La sobreexpressió de asip1 en el peix zebra transgènic (asip1-Tg) millora el creixement, sense afectar l'acumulació lipídica (obesitat), inclús quan s'alimenten sota règims inductors severs. Los peces asip1-Tg no necessiten menjar més per a créixer més i més ràpid, lo qual suggereix una major eficiència alimentària. A més a més, els peixos asip1-Tg criats en alta densitat són capaces de créixer molt més que els peixos de tipus salvatge (WT) criats en baixa densitat, malgrat que els peixos asip1-Tg semblen ser més sensibles a l'estrès per amuntegament que els peixos WT. L'anàlisi transcriptòmic comparatiu de l'intestí de asip1-Tg reflecteix una expressió diferencial de transportadors aminoacídics, monocarboxilats, transportadors iònics i de vitamines. La sobreexpressió redueix la integritat de l'epiteli intestinal augmentant la seua permeabilitat paracel·lular i potencia el transport electrogènic d'aminoàcids. Per tant, els peixos transgènics posseeixen major capacitat per l'absorció de nutrients i, per extensió una millora en la eficiència alimentària que podria explicar, en part, eixe creixement diferencial sota taxes d'ingesta similars. Aquesta tesi tingué també com a objectiu investigar si els asip1-Tg mantenien un fenotip dominant associat amb una major taxa d'alimentació. Els resultats mostren, pel contrari, un caràcter reactiu/subordinat en los asip1-Tg que advoca per una participació del sistema de melanocortines en la regulació del comportament de peixos. El perfil subordinat dels animals asip1-Tg, junt amb una activació de l'eix de l'estrès, suggereix que aquests animals poden mostrar un comportament d'ansietat. Els resultats indicaren que els peixos asip1-Tg mostren un comportament d'ansietat relacionat amb una severa disminució dels nivells centrals de serotonina (5HT) i dopamina i elevació de la seua recaptació neuronal i degradació. L'administració de un inhibidor de la recaptació de 5HT recupera el fenotip comportamental salvatge, mitigant el comportament d'ansietat en els peixos transgènics i rescatant els nivells centrals de 5HT. Esta ansietat podria repercutir en una alteració del comportament locomotor dels animals, per la qual cosa vam estudiar els ritmes circadians d'activitat locomotora. Els resultats mostren que els animals asip1-Tg exhibeixen una disrupció completa del ritme d'activitat, amb una activitat molt elevada durant tot el cicle diari, especialment durant la nit. Esta disrupció es concomitant amb una desaparició del ritme diari de serotonina i melatonina. A més a més, els resultats mostren una pèrdua de ritmes de expressió de gens rellotge (per1a y clock1a). La incubació, in vitro, de glàndules pineals con Asip1 va produir una inhibició de la secreció de melatonina replicant els resultats obtinguts in vivo y demostrant un efecte directe de Asip1 sobre la fisiologia de la pineal. En esta tesi, se va utilitzar el peix zebra com a model per investigar els efectes de la obesitat sobre la ansietat i la memoria. L'obesitat no va tindre cap efecte sobre l'ansietat, però va produir una disminució de la memòria a curt termini, estudiada mitjançant tests de condicionament aversiu. Aquest estudi proporciona, un protocol fiable per a avaluar l'efecte de les malalties metabòliques en la funció cognitiva i conductual. / [EN] The melanocortin system plays a key role in the regulation of a wide range of physiological functions including melanogenesis, stress response and energy balance, through binding to a family of specific G protein-coupled receptors (MC1R-MC5R). Overexpression of inverse agonists, agouti-signalling protein (Asip) and agouti-related protein (Agrp) results in increased food intake, linear growth and body weight. Asip regulates dorso-ventral pigmentation polarity through MC1R, and over-expression induces obesity in mice by binding to the central MC4R. Overexpression of asip1 in transgenic zebrafish (asip1-Tg) enhances growth, without affecting lipid accumulation (obesity), even when fed under severe inducing regimens. The asip1-Tg fish do not need to eat more to grow bigger and faster, suggesting increased feed efficiency. In addition, asip1-Tg fish reared at high density are able to grow much larger than wild-type (WT) fish reared at low density, although asip1-Tg fish appear to be more sensitive to overcrowding stress than WT fish. Comparative transcriptomic analysis of asip1-Tg gut reflects differential expression of amino acid, monocarboxylate, ionic and vitamin transporters. Overexpression reduces the integrity of the intestinal epithelium by increasing its paracellular permeability and enhances electrogenic amino acid transport. Thus, transgenic fish possess a greater capacity for nutrient absorption and, by extension, an improvement in feed efficiency that could explain, in part, this differential growth under similar intake rates. This thesis also aimed to investigate whether asip1-Tg maintain a dominant phenotype associated with a higher feeding rate. Experimental results show, on the contrary, a reactive/subordinate character in asip1-Tg which argues for an involvement of the melanocortin system in the regulation of fish behaviour. Improving feeding motivation without promoting aggression in fish, thus avoiding the threat to native populations in case of an escape, makes the inhibition of the melanocortin system, through the overexpression of asip1, a feasible target for the development of genetically modified lines. The subordinate profile of the asip1-Tg animals, together with an activation of the stress axis, suggests that these animals may exhibit anxiety-like behaviour. The results indicated that asip1-Tg fish show a behaviour similar to our concept of anxiety related to a severe decrease in central serotonin (5HT) and dopamine levels as well as the elevation of their neuronal reuptake and degradation. The administration of fluoxetine, a serotonin reuptake inhibitor, recovers the wild-type behavioural phenotype, mitigating anxiety behaviour in transgenic fish and restoring central 5HT levels. This anxiety could have repercussions on the locomotor behaviour of the animals, so we studied circadian rhythms of locomotor activity. The results show that asip1-Tg animals exhibit a complete disruption of the activity rhythm, with very high activity levels throughout the daily cycle, especially during the night. This disruption is concomitant with a disappearance of the daily rhythm of serotonin and melatonin. In addition, the results show a loss of clock gene expression rhythms (per1a and clock1a). Incubation, in vitro, of pineal glands with Asip1 produced an inhibition of melatonin secretion replicating the results obtained in vivo and demonstrating a direct effect of Asip1 on pineal physiology. In this PhD thesis, zebrafish was used as a model to investigate the effects of overfeeding-induced obesity on anxiety-like behaviour and memory. Obesity had no effect on anxiety, but produced a decrease in short-term memory, studied by means of aversive conditioning tests. This study also provides a reliable protocol for assessing the effect of metabolic diseases on cognitive and behavioural function, supporting zebrafish as a model for cognitive and behavioural neuroscience. / Esta tesis ha sido realizada a través del programa de ‘Ayudas para la formación de personal investigador’ (FPI) BES‐2017‐082424 de la Agencia Estatal de Investigación, en Instituto de Acuicultura Torre de la Sal (IATS) del Consejo Superior de Investigaciones Científicas (CSIC) en el Grupo de investigación de Control de la Ingesta en Peces dirigido por José Miguel Cerdá Reverter, director de esta tesis. Los trabajos llevados a cabo en esta tesis han sido financiados por Ministerio de Ciencia, Innovación y Universidades (MICIU) a través de los siguientes proyectos: MELANOCONDUCT: Implicación del sistema de melanocortinas en la regulación de los mecanismos temporales y conductuales de peces AGL2016-74857-C3-3-R; Cronopeces: Red temática de cronobiología de peces y sus aplicaciones en acuicultura RED2018-102487-T; MacForFish: Nuevos aspectos homeostáticos y comportamentales de la regulación de la ingesta en peces PID2019-103969RB-C33; FISHTASTE: Implicación de los mecanismos sensoriales del gusto en la regulación de la ingesta de peces - Involvement of taste sensing mechanisms in the regulation of feed intake of fish PID2022-136288OB-C33 / Godino Gimeno, A. (2024). Involvement of the Melanocortin System in the Regulation of Circadian and Behavioural Mechanisms in Zebrafish [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203148 / Compendio

Adipositas ist eine polymorphe chronische Erkrankung mit epidemischer Prävalenz. Im katabolen Leptin-Melanocortin-Signalweg ist das Proopiomelanocortin Gen (POMC) ein zentrales Element, das bei Dysfunktion massive Adipositas bewirken kann. Auch eine kürzlich identifizierte intragenische Methylierungsvariante des POMC wurde mit Adipositas assoziiert und deutet somit auf eine mögliche epigenetische Modulation des Gewichtsphänotyps hin. Zur Aufklärung der Relevanz, Stabilität und Entwicklung dieser epigenetischen Modifikation wurden die Funktionalität, Ontogenese und Phylogenese der POMC DNA-Methylierung untersucht. In vitro Analysen zeigten DNA-Methylierungsabhängige Promoteraktivität beider CpG-Inseln (CGIs) des POMC. Diese hier erstmals beschriebene Transkriptionsaktivität der intragenischen CGI weist auf einen alternativen Promoter des POMC hin. Hinsichtlich der Ontogenese konnten in Mensch und Maus postnatal stabile DNA-Methylierungsmuster mit interindividueller Konservierung für beide CGIs des POMC identifiziert werden. Zusätzlich erwiesen sich Gewebeunabhängigkeit der DNA-Methylierungsmuster und ihre pränatale Ausbildung zwischen dem Blastocystenstadium und der frühen Organogenese in der Maus. Die POMC DNA-Methylierungsmuster upstream des Exon3 unterscheiden sich in Mensch und Maus. Der mögliche Einfluss von primatenspezifischen Alu-Elementen im Intron2 des POMC hierauf wurde in verschiedenen Primatenfamilien analysiert. Die Ergebnisse zeigen eine bedingte Assoziation der Alu-Elemente mit der DNA-Methylierung in der entsprechenden Region, lassen jedoch auch weitere Einflussfaktoren vermuten. Insgesamt zeigt diese Arbeit, dass die POMC DNA-Methylierung artspezifisch konserviert ist und in der frühen Embryogenese, vermutlich Alu-abhängig, ausgebildet wird. Dabei könnten stochastische Variationen der DNA-Methylierung die POMC-Aktivität beeinflussen und somit das Risiko für Adipositas erhöhen. / Obesity is a polymorphic chronic disease with epidemic prevalence. Within the catabolic leptin-melanocortin signaling pathway pre-proopiomelanocortin (POMC) is a pivotal element. Dysfunction of POMC, e.g. due to mutations, can cause severe obesity. Moreover, a recently identified intragenic methylation variant of POMC was found to be associated with obesity. Therefore, this indicates potential epigenetic modulation of the weight phenotype. To gain further insight into the relevance, stability, and origin of this epigenetic modification, the functionality, ontogenesis, and phylogenesis of the POMC DNA methylation patterns were analyzed. In vitro analyses revealed DNA methylation-dependent promoter activity of both CpG islands (CGIs) of POMC. Thereby, the intragenic CGI was identified as a potential alternative promoter of POMC, which has not been described before. Regarding the ontogenesis, postnatally stable POMC DNA methylation patterns with interindividual conservation were detected for both CGIs in humans and mice. In addition, it was observed that the POMC DNA methylation patterns are non-tissue-specific, stable upon long time administration of a high fat diet, and develop prenatally between the blastocystal stage and the early organogenesis. The POMC DNA methylation pattern upstream of exon3 differs in humans and mice. A possible influence of primate-specific Alu elements within the intron2 region of POMC was analyzed in various primate families. Results evince a partial association of the Alu elements with the DNA methylation pattern in this particular region, but also suggest an influence of additional factors. Overall, this work demonstrates that DNA methylation of the POMC locus is species-specific highly conserved, and that it is established during early embryogenesis, possibly Alu-triggered. In the course of this, stochastic variances of the POMC DNA methylation might influence the POMC activity and consequently alter the risk to develop obesity.

Secondary sexual traits play an irreplaceable role in the reproduction of a range of animals and are used as quality and fitness sensors during pairing of individuals. Expression of these traits, ornamentes, can correlated with an individual's personal and behavioral strategies. In the case of melanin ornaments, not only the relationships with personal individuals are considered, but there is the possible connections with stress resistance and the level of stress responses too. This hypothesis is based on the pleiotropic effect of the melanocortin system, which can be used during melanogenesis but also in the production of hormones that contribute to range of stress responses. The aim of this work was to clarify the relationship between an individual's ornaments, his stress response and individuality in the barn swallows (Hirundo rustica rustica). In this work the relationship between selected ornaments and the stress reaction of the organism, stressed glucose levels measured 15 minutes after a stress stimulus, was tested. Both sexes showed a correlation between area of white tail spots and stress response. Only for males a relationship with the length of outermost tail feathers was found and a correlation with the color saturation of feathers on the throat was shown for females. The second part of...