La dépression est l’un des troubles mentaux les plus fréquents de nos jours. C’est une maladie liée en général à un déficit en neurotransmetteurs monoaminergiques (sérotonine, noradrénaline et dopamine). Les antidépresseurs actuels agissant via des mécanismes monoaminergiques présentent de nombreux effets secondaires et peuvent conduire à une accoutumance. L’une des approches impliquerait le ciblage des récepteurs mélatoninergiques afin de resynchroniser les rythmes circadiens qui sont perturbés dans certaines pathologies du système nerveux central, notamment la dépression. La conception de ligands non monoaminergiques et possédant des propriétés chronobiotiques constituerait une stratégie prometteuse.L’agomélatine (Valdoxan®) issue d’une collaboration entre le laboratoire de chimie thérapeutique (EA4481 - GRIIOT) et les laboratoires Servier est commercialisée depuis 2009 pour le traitement de la dépression majeure. Ce bioisoster naphtalénique de la mélatonine possède un mécanisme d’action innovant. Elle se distingue par ses propriétés agoniste non sélectif des récepteurs mélatoninergiques MT1 et MT2 et antagoniste des récepteurs 5-HT2c.L’objectif de ce travail réside dans la conception et la synthèse de nouveaux ligands successeurs de l’agomélatine présentant un profil pharmacologique et pharmacocinétique amélioré. Différentes pharmacomodulations ont été réalisées sur l’agomélatine. Les stratégies de «Drug Design» notamment le principe de bioisostérie ont été appliquées, nous permettant ainsi la synthèse de nouvelles familles de composés présentant des profils pharmacologiques intéressants. / Nowadays, depression related to a deficit in nonoaminergic neurotransmitters, is the most frequent mental illness. Available antidepressive drugs acting through monoamnergic mechanisms possess a lot of side effects and can lead to an addiction. One approach involves targeting melatoninergic receptors to resynchronize circadian rhythms, which are known to be perturbed in some pathology related to nervous central system as depression. So conception of non-monoaminergic ligands with chronobiotic properties would constitute a promising strategy.Agomelatine (Valdoxan®) a novel antidepressant developed by Servier and our laboratory (EA4481 - GRIIOT) was granted marketing authorization in 2009 for the treatment of major depressive disorder. This naphthalen analogous of melatonin possess an innovative mechanism. It acts as a non selective melatoninergic MT1/MT2 receptors agonist and a serotonin 5-HT2c receptor antagonist.The aim of this work is to design and synthesize new potential successors of agomelatine with an improved pharmacological and pharmacokinetic profile. Drug Design strategies such as bioisosterism were applied to allow the elaboration of new series of compounds with interesting pharmacological profiles.

La mélatonine est une neurohormone, principalement synthétisée par la glande pinéale et exerçant ses fonctions physiologiques via ses deux récepteurs MT1 et MT2 couplés aux protéines G. Ces derniers sont principalement exprimés dans le cerveau, la rétine et plusieurs autres tissus périphériques pour réguler une grande variété de fonctions physiologiques telles que les rythmes circadiens et saisonniers, la physiologie de la rétine, l'homéostasie du glucose et les fonctions neuronales et immunitaires. Les récepteurs de la mélatonine modulent plusieurs voies de transduction de signaux intracellulaires et inhibent la production d'AMPc et de GMPc et activent les « Extracellular signal-Regulated Kinases » 1/2 (ERK1/2) et les canaux ioniques. Ce travail met l'accent sur le rôle central de la voie ERK1/2 dans la fonction des récepteurs de la mélatonine en définissant la ou les voies moléculaires impliquées et en étudiant les modifications de cette voie dans les conditions pathologiques. Dans l'article 1, nous décortiquons les voies moléculaires impliquées dans l'activation de la voie ERK1/2 par les récepteurs humains MT1 et MT2 dans des cellules HEK293, un modèle cellulaire pertinent. Nous montrons ainsi que les β-arrestines ne participent pas à l’activation d’ERK1/2 induite par les deux récepteurs. Alors que l'activation d'ERK1/2 par MT1 est exclusivement médiée par des protéines Gi/o en libérant leurs sous-unités Gβy, MT2 est strictement dépendante de l'activation coopérative des protéines Gi/o et Gq/11. Les deux récepteurs activent plus en aval la cascade PI3K/PKCζ/c-Raf/MEK/ERK, avec laquelle ils forment des méga-complexes de signalisation préformés. Le phénomène de coopérativité au niveau des protéines G a également été observé plus en aval au niveau des gènes cibles d’ERK1/2 mais pas pour la voie Gi/cAMP. Ce travail a permis de fournir la première description complète de la voie ERK activée par MT1 et MT2, de mettre en évidence des différences entre les deux récepteurs et décrire un nouveau modèle de coopérativité entre les protéines Gi/o et Gq/11.Les variants naturels d’un récepteur peuvent avoir des propriétés de signalisation et des fonctions physiologiques modifiées. L'évaluation fonctionnelle de tels variants associés à des maladies est extrêmement importante pour établir un lien entre la fonction modifiée et la maladie pour concevoir d’éventuelles nouvelles stratégies thérapeutiques. Dans l'article 2, nous avons établi le profil de signalisation de 40 variants naturels du récepteur MT2 associés au diabète de type 2 (DT2). La voie ERK1/2 a été mesurée en suivant la phosphorylation d’ERK directement dans des lysats cellulaires avec la technologie alpha-screen. En résumé, l'article 2 a confirmé l'association générale entre la perte de fonction du récepteur MT2 et l'augmentation du risque de DT2 et a montré que la voie ERK1/2 ne fait pas partie des principales voies associées au DT2.La génération et l'agrégation des peptides amyloïdes bêta (Aβ) est le principal marqueur moléculaire de la maladie d'Alzheimer (MA). Chez les patients atteints de MA, les deux composants du système mélatoninergique, à savoir la production de mélatonine et la fonction des récepteurs de la mélatonine, sont nettement réduits. Cependant, les mécanismes moléculaires y participant ne sont pas encore bien compris. Dans l'article 3, nous démontrons que l'Aß abolit la synthèse de la mélatonine et diminue les fonctions de MT1 et MT2 telle que l'activation de la voie ERK1/2 par la mélatonine. Ce travail fournit une base mécanistique pour expliquer la diminution de la fonction du système mélatoninergique chez les patients atteints de la MA.En résumé, cette thèse fournit de nouvelles idées sur la façon dont les récepteurs humains de la mélatonine activent la voie ERK1/2 et comment cette activation est modifiée par Aβ dans le contexte de la MA et par des variants de MT2 associés au DT2. / The neurohormone melatonin, primarily synthesized by the pineal gland, exerts its physiological functions by two high-affinity G protein-coupled receptors: MT1 and MT2. Both are widely expressed in the brain, retina and several other peripheral tissues to regulate a wide variety of physiological function such as circadian and seasonal rhythms, retinal physiology, glucose homeostasis and neuronal and immune functions. Melatonin receptors modulate several intracellular signal transduction pathways and inhibit cAMP and cGMP production and activate extracellular signal-regulated kinases 1/2 (ERK1/2) and ion channels. This work focuses on the central role of ERK1/2 signaling in melatonin receptor function by defining the molecular pathway(s) involved and by studying modifications of this pathway under disease conditions. In article 1, we decipher the molecular pathways involved in the activation of the ERK1/2 signaling cascade by human MT1 and MT2 receptors in HEK293 cells, a relevant cellular model system. We show that β-arrestins do not participate in ERK1/2 activation by both receptors. Whereas ERK1/2 activation by MT1 is exclusively mediated though Gi/o proteins by liberating Gβγ subunits, MT2 is strictly dependent on the cooperative activation of Gi/o and Gq/11 proteins. Both receptors activate further downstream the PI3K/PKCζ/c-Raf/MEK/ERK cascade, with which they are forming preformed mega-signaling complexes. G protein cooperativity was also observed further downstream on the ERK1/2 target gene level but not for the Gi/cAMP pathway. This work provides the first full description of the ERK signaling pathway activated by MT1 and MT2, highlights differences between the two receptors and describes a new cooperativity model between Gi/o and G/11 proteins.Naturally occurring receptor variants might have modified signal transduction properties and modified physiological functions. The functional assessment of disease-associated variants is therefore extremely important to establish a link between modified function and disease to eventually design new therapeutic strategies. In article 2, we established the ERK1/2 signaling profile of 40 natural MT2 variants associated with type 2 diabetes (T2D) by measuring ERK phosphorylation directly in cell lysates with the alpha-screen technology. Collectively, article 2 confirmed the general association between loss-of-function of MT2 and increased T2D risk and showed that the ERK1/2 pathway is not among those pathways primarily associated to T2D risk.Generation and aggregation of the amyloid-beta peptides (Aβ) is the main molecular hallmark of Alzheimer’s disease (AD). In AD patients both components of the melatoninergic system, i.e. melatonin production and melatonin receptor function, are markedly reduced. However, the mechanistic basis for that is still poorly understood. In article 3, we demonstrate that Aβ abolishes melatonin synthesis and diminishes MT1- and MT2 functions such as the activation of the ERK1/2 pathway by melatonin. This work provides a mechanistic basis for the diminished responsiveness of the melatoninergic system in AD patients.Collectively, this thesis provides new insights on how human melatonin receptors promote ERK1/2 activation and how this activation is modified by Aβ in the context of AD and by MT2 variants associated with T2D.

Le mélanome uvéal constitue le cancer intraoculaire le plus fréquent chez l’adulte. Il s’agit d’un cancer très agressif puisque plus de 50% des patients développent des métastases principalement localisées au niveau du foie. Dans le but d’identifier des gènes pronostiques de développement métastatique, nous avons comparé le transcriptome de 28 tumeurs de mélanome uvéal issues de patients ayant développé des métastases dans les trois années qui ont suivi l’énucléation et 29 tumeurs issues de patients n’ayant pas développé de métastases ou ayant développé des métastases après 36 mois. Le gène PTP4A3/PRL-3 (protein tyrosine phosphatase type IV member 3/Protein of Regenerating Liver-3) a été identifié comme prédictif de l’apparition de métastases. Il code une phosphatase et sa surexpression dans des cellules de mélanome uvéal augmente leur migration in vitro et leur invasivité in vivo. Les évènements protéolytiques à la surface des cellules sont essentiels pour la migration et l’invasivité durant plusieurs processus physiologiques ou pathologiques tels que le développement de métastases. Ces évènements sont assurés par les métalloprotéases (MMPs) qui sont responsables de la dégradation et du remodelage de la matrice extracellulaire.Dans la première partie de cette thèse, nous avons observé que la métalloprotéase transmembranaire MT1-MMP est enrichie à la surface des cellules de mélanome uvéal OCM-1, des cellules MP41 issues de xénogreffes de tumeurs de mélanome uvéal humaines ou dans des tumeurs primaires de mélanome uvéal, surexprimant PTP4A3. Nous avons aussi observé que cette accumulation de MT1-MMP à la surface des cellules de mélanome uvéal est accompagnée d’une accumulation de la sécrétion de MMP2 dans le milieu extracellulaire des cellules exprimant PTP4A3. De plus, nous avons montré que PTP4A3 et MT1-MMP s’associent physiquement et que le trafic vésiculaire de MT1-MMP est accéléré dans les cellules exprimant PTP4A3 mais pas dans celles exprimant le mutant catalytique inactif PTP4A3(C104S). Enfin, nous avons démontré que l’inhibition de l’expression de MT1-MMP dans les cellules exprimant PTP4A3 diminue leur migration in vitro et leur invasivité in vivo. Pour conclure, nos résultats indiquent que PTP4A3 agit en amont de MT1-MMP à travers une accélération de son trafic vésiculaire et son accumulation à la surface des cellules afin de promouvoir la migration et l’invasivité cellulaires.Dans la deuxième partie de cette thèse, nous nous sommes intéressés au rôle de PTP4A3 pendant le développement embryonnaire. Les mélanocytes, incluant ceux de l’uvée, dérivent de la crête neurale pendant le développement embryonnaire. Nous avons alors supposé que la fonction de PTP4A3 pendant la progression métastatique pourrait refléter un rôle de la phosphatase dans la migration des cellules de crête neurale pendant le développement embryonnaire. Dans cette partie de la thèse, nous avons montré que PTP4A3 joue un rôle important dans la migration des cellules de crête neurale céphalique pendant le développement de l’embryon de Xenopus laevis. La perte de fonction de PTP4A3 provoque une réduction du territoire de la crête neurale, alors que le gain de fonction de cette phosphatase élargit les faisceaux de migration des cellules de crête neurale céphalique. De plus, des expériences d’isogreffes montrent que les explants de crête neurale dépourvus de l’expression de PTP4A3, sont incapables de migrer dans les embryons greffés. Plus encore, l’inhibition pharmacologique de PTP4A3 dans des cellules de crête neurale en culture diminue de façon significative leur vitesse de migration in vitro. Les résultats de cette étude démontrent que PTP4A3 est requise pour la migration des cellules de crête neurale céphalique in vivo pendant le développement embryonnaire de Xenopus laevis. Donc, les effets pro-migratoire et -invasif reliés à l’expression de la protéine PTP4A3 peuvent refléter son rôle durant la migration des cellules de crête neurale. / Uveal melanoma (UM) is the most common intraocular malignancy in adults and is an aggressive tumor since about 50% of patients will develop metastases mostly in the liver. In order to identify metastasis prognostic genes, we compared 28 uveal melanoma tumors from patients who developed metastases within three years after enucleation to 29 tumors from patients who did not develop metastases or who developed metastases after 36 months. The PTP4A3/PRL-3 gene (protein tyrosine phosphatase type IV member of Regenerating Liver 3/Protein-3) was identified as a strong predictor of metastasis occurence. PTP4A3 encodes a dual specificity phosphatase and its expression in UM cells increases their in vitro migration and in vivo invasiveness. Proteolytic events at the cell surface are essential for cell migration and invasiveness during many physiological and pathological processes such as tumor metastasis. MMPs are responsible for the degradation and turnover of the extracellular matrix (ECM). In the first part of this thesis, We found that the membrane anchored MT1-MMP is enriched at the cell surface of OCM-1, xenograft MP41 or primary human uveal melanoma tumors expressing PTP4A3. We also found that membrane accumulation of MT1-MMP in presence of PTP4A3 in OCM-1 cells is accompanied by enhanced secretion of MMP2 in the extracellular medium. Moreover, we demonstrated that PTP4A3 and MT1-MMP physically associate and that the vesicular trafficking of MT1-MMP is accelerated in presence of active PTP4A3 but not in presence of the mutant PTP4A3(C104S). Furthermore, we found that inhibition of MT1-MMP expression in PTP4A3 expressing uveal melanoma cells impairs their migration in vitro and invasiveness in vivo. Collectively, our results indicate that PTP4A3 acts upstream of MT1-MMP through acceleration of its vesicular trafficking and accumulation at the cell surface to enhance cell migration and invasiveness of uveal melanoma cells. In the second part of this thesis, we investigated the role of PTP4A3 during embryonic development. Melanocytes, including uveal melanocytes, are derived from the neural crest during embryonic development. We therefore suggested that PTP4A3 function in uveal melanoma metastasis may be related to an embryonic role during neural crest cell migration. We show that PTP4A3 plays a role in cephalic neural crest development in Xenopus laevis. PTP4A3 loss of function resulted in a reduction of neural crest territory, whilst gain of function experiments increased neural crest territory. Isochronic graft experiments demonstrated that PTP4A3-depleted neural crest explants are unable to migrate in host embryos. Pharmacological inhibition of PTP4A3 on dissected neural crest cells significantly reduced their migration velocity in vitro. Our results demonstrate that PTP4A3 is required for cephalic neural crest migration in vivo during embryonic development.Therefore, the pro-invasive and migratory effects related to the expression of PTP4A3 protein may reflect its role during neural crest migration. Thus, understanding the mechanism of action of PTP4A3 during NC migration may provide insight into PTP4A3 related migratory and invasive phenotypes in human uveal melanoma pathology.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Le cancer du sein triple négatif (TNBC) représente entre 15-20% des cancers du sein et est l'un des types les plus agressifs. De plus, un sous-groupe de ces patientes est résistant à la radiothérapie (RT) et développe fréquemment une récidive hâtive de la maladie. Des études précédentes ont démontré que l’inflammation induite par la RT accélère la progression du cancer et le développement des métastases. Cette hypothèse a donc été validée dans un modèle pré-clinique de TNBC en implantant les cellules de carcinome de souris triple négatives D2A1 dans les glandes mammaires de la souris Balb/c. Premièrement, la tumeur primaire à été irradiée à une dose sous-curative une semaine post-implantation des cellules. En deuxième lieu, le tissu mammaire de la souris a été pré-irradié avant d'implanter les cellules cancéreuses afin de bien discerner l'effet du microenvironnement irradié sur celles-ci. Ces deux modèles ont mené à une augmentation significative des cellules tumorales circulantes ainsi que du nombre de métastases pulmonaires. Plusieurs molécules inflammatoires dont l'interleukine-1 bêta (IL-1β), l'interleukine-6 (IL-6) ou encore la cyclooxygénase 2 (COX-2) ont été identifiées comme facteurs clés impliqués dans la migration radio-induite des cellules cancéreuses du sein. Conséquemment, un inhibiteur large-spectre comme la chloroquine (CQ), entre autres utilisé comme traitement anti-malarien et anti-inflammatoire, a su prévenir ces effets secondaires associés à la RT. Étant donné que l'action de la CQ est peu sélective, une répression de l'expression de l'ARNm de la métalloprotéinase (MMP) de membrane de type 1 (MT1-MMP), une MMP de surface impliquée notamment dans la migration cellulaire, l'invasion tumorale et l'angiogenèse, a été réalisée afin d'éclaircir le mécanisme d'inhibition des métastases radio-induites. Cette répression de la MT1-MMP prévient la formation des métastases pulmonaires radio-induites, démontrant ainsi un des mécanisme important de l'invasion radio-induite. Ce résultat confirme donc l'importance de la MT1-MMP dans ce phénomène et son potentiel comme biomarqueur de prédiction de l'efficacité des traitements de RT, particulièrement chez les patientes atteintes de TNBC.

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No. of bitstreams: 1 castro_tb_dr_sjrp.pdf: 17135465 bytes, checksum: 7e68f8d98ca4ec7d64be1f324b5e48d7 (MD5) Previous issue date: 2017-10-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O câncer de mama é a principal causa de mortes relacionadas ao câncer em mulheres e pesquisas têm sido focadas em identificar e validar biomarcadores que podem ser utilizados para confirmar o diagnóstico e determinar o prognóstico. Mudanças no ritmo circadiano podem contribuir para o desenvolvimento do câncer e sendo assim, a melatonina, um hormônio sintetizado pela glândula pineal à noite, na ausência de luz e seus metabolitos AFMK e AMK são sugeridos como potenciais biomarcadores. A melatonina pode atuar através do receptor MT1 regulando cinases e a expressão de genes específicos relacionados a proliferação, angiogênese, diferenciação celular e transporte de glicose. A expressão elevada de GLUT1 (Glucose Transporter-1) está associada ao prognóstico ruim no câncer. Os objetivos deste estudo foram comparar os níveis de melatonina, AFMK e AMK em mulheres recentemente diagnosticadas com câncer de mama, mulheres em quimioterapia adjuvante, enfermeiras que trabalham à noite em comparação com mulheres saudáveis e hábitos normais e avaliou-se a expressão do receptor MT1 e GLUT1 em tumores de mama e correlacionou com os subtipos moleculares, características patológicas e prognóstico. Foi coletado sangue de 53 mulheres com câncer de mama sendo 47 sem tratamento e 6 em quimioterapia adjuvante, 19 mulheres saudáveis sendo 1 O enfermeiras de turno noturno e 9 mulheres de hábitos normais. Os compostos foram quantificados por espectrometria de massas. Para a expressão de MT1 e GLUT1 foi realizada imunohistoquímica em 42 tumores de mama. Os resultados mostraram que mulheres com câncer de mama tiveram níveis menores de melatonina em comparação com mulheres de hábitos normais (p< 0.01 ), níveis ainda mais baixos em enfermeiras que trabalham no período noturno e em pacientes em quimioterapia adjuvante (p<0.01). Não houve diferença significativa nos níveis de AFMK e AMK entre os grupos (p>0.05). Além disso, pacientes com metástase apresentaram níveis elevados de melatonina e AFMK (p=0.02 e p=0.01, respectivamente) e altos níveis de AFMK estavam presentes quando linfonodos estavam acometidos (p=0.04), pacientes com tumores maiores que 20mm e que dormem com luz à noite (p=0.02 e p=0.007, respectivamente). Na análise imunohistoquímica, alta expressão do MT1 foi associada ao subtipo Luminal A, melhor prognóstico (p<0.05) e em tumores RE+ (p=0.002), enquanto que a expressão elevada de GLUT1 foi associada ao triplo­negativo, pior prognóstico (p<0.05), tumores maiores que 20mm 9p=0.004) e baixa em tumores RE+ (p=0.003). Em conclusão, nossos resultados mostram menores níveis de melatonina em pacientes com câncer de mama e apontam para o benefício da suplementação de melatonina como proteção para mulheres que trabalham à noite o que poderia reduzir o risco de desenvolvimento de câncer de mama. Os resultados da imunohistoquímica indicam que o MT1 e o GLUT1 podem ser utilizados como marcadores prognósticos e são potenciais alvos para o tratamento do câncer de mama. De acordo com os resultados pode­se concluir que a quebra do ciclo circadiano e, portanto, alterações nos níveis de melatonina e a expressão de MT1 e GLUT1 podem estar relacionadas com o risco de desenvolvimento e prognóstico do câncer de mama. / Breast cancer is the Jeading cause of cancer-related deaths in women and researches has been focused on identify and validate biomarkers that can be used to confirm the diagnosis and determine the prognosis. Changes in circadian rhythm may contribute to the development of cancer and thus melatonin, a hormone synthesized by the pineal gland at night, in the absence of light and its metabolites AFMK and AMK are suggested as potential biomarkers. Melatonin can act through the MT1 receptor by regulating kinases and the expression of specific genes related to proliferation, angiogenesis, ce/1 differentiation and glucose transport. High GLUT1 (Glucose Transporter-1) expression is associated with poor prognosis in cancer. The objectives of this study were to compare the leveis of melatonin, AFMK and AMK in women newly diagnosed with breast cancer, women on adjuvant chemotherapy, health nurses which work at night compared to healthy women and normal habits and the expression of MT1 receptor and GLUT1 in breast tumors and correlated with molecular subtypes, pathological features and prognosis. Blood from 53 women with breast cancer was colletcted, 47 of them without treatment and 6 in adjuvant chemotherapy, 19 healthy women, 1 O of them night shift nurses and 9 women of normal habits. Compounds were quantified by mass spectrometry. For the expression of MT1 and GLUT1, immunohistochemistry was performed in 42 breast tumors. The results showed that women with breast cancer had lower leveis of melatonin compared to normal women (p <0.01), even Jower leveis in nurses working at night and in adjuvant chemotherapy (p <0.01). There was no significant difference in AFMK and AMK leveis between groups (p> 0.05). ln addition, patients with metastasis had high leveis of melatonin and AFMK (p = 0.02 and p = 0.01, respectively) and high leveis of AFMK were present when lymph nades were affected (p = 0.04), patients with tumors larger than 20mm and who sleep with light at night (p = 0.02 and p = 0.007, respectively). ln the immunohistochemical analysis, high MT1 expression was associated with the Luminal A subtype, with a better prognosis (p <O.OS) and in ER + tumors (p = 0.002), whereas high GLUT1 expression was associated with tripie negative, worse prognosis (p<0.05), tumors greaterthan 20mm (p= 0.004) and low in ER + tumors (p = 0.003). ln conclusion, our results show Jower leveis of melatonin in breast cancer patients and point to the benefit of melatonin supplementation as protection for women who work at night which could reduce the risk of developing breast cancer. The results of immunohistochemistry indicate that MT1 and GLUT1 can be used as prognostic markers and are potential targets for the treatment of breast cancer. According to the results it can be concluded that the breakdown of the circadian cyc/e and, therefore, changes in melatonin leveis and the expression of MT1 and GLUT1 may be related to the risk of development and prognosis of breast cancer / FAPESP 2015/02935-2

In seasonal mammals the duration of nocturnal melatonin secretion accurately reflects the environmental photoperiod. The endocrine rhythm is decoded by a specialised portion of the pituitary gland (the pars tuberalis, PT) which then relays this information to the pars distalis and hypothalamus, precipitating huge annual changes in physiology and behaviour. However how the PT decodes the melatonin signal is currently unknown. Melatonin influences gene transcription in the ovine PT at its onset and offset, and the phase relationship of these two groups is believed to form the underlying mechanism by which the PT integrates seasonal time. The transcripts induced at melatonin offset are understood to be under the control of a seasonally gated cAMP mechanism. Conversely processes involved in melatonin-mediated gene induction are currently not understood.The work in this thesis ultimately aims to reveal how the seasonal melatonin signal is decoded by the PT. To this end melatonin-mediated gene induction has been characterised through RNAseq, the highly displaced cohorts submitted to bioinformatic promoter analysis and the paradigm tested though in vitro modelling techniques.In this study a 1.5 h infusion with melatonin acutely regulated 219 transcripts in the ovine PT (115 induced, 104 repressed, >1.5 fold change), confirming previous association of several genes (including Cry1, MT1, Gadd45g, Nampt and Npas4) to rapid melatonin control. Gross promoter analysis of these groups indicated that the induced gene cohort was significantly enriched for GC content and CpG islands suggesting the involvement of epigenetic mechanisms of transcriptional control. Further bioinformatic analysis specifically implicated the importance of transcription factors ZFP161 and PAX5 in melatonin-mediated gene induction in the PT. Several immortalised cell lines were screened for the presence of a functional melatonin receptor. Two strains (MCF7 oMT1 and NES2Y) exhibited significant attenuation of forskolin-mediated cAMP accumulation when co-treated with melatonin, a hallmark of melatonin Gαi-coupled protein receptor signalling. These lines were subsequently evaluated as models of melatonin-mediated gene induction of the sheep PT through ovine promoter reporter assays of Cry1, Nampt, NeuroD1 and Npas4. However, treatment with melatonin failed to evoke a reporter response suggesting that the cell line models were inadequately equipped to reflect PT biology. Subsequently a protocol was established to culture ovine PT explants culture which faithfully recapitulated melatonin mediated transcriptional dynamics in vitro, providing a possible tool for the future investigation of the PT. Lastly, previous work has shown the transcriptional profile of Npas4 to peak highly and transiently, pre-empting the expression of other melatonin-induced genes. Using a COS7 cell line model, heterologously-expressed NPAS4 was shown to form functional heterodimeric partnerships with ARNT and ARNTL and transactivate both Cry1 and Nampt promoter reporters through novel binding sites. Collectively these data indicated NPAS4 to act as an immediate activator of melatonin regulated circuits

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Melatonin is an indoleamine hormone with neuromodulatory and neuroprotective properties. It mediates many of its effects by its two G protein-coupled receptors, MT1 and MT2. We have shown that valproic acid (VPA) induces melatonin receptor expression in cultured rat C6 glioma cells, and in the rat hippocampus. VPA is known to affect gene expression through several mechanisms, including the modulation of intracellular kinase pathways and/or transcription factors, as well as the inhibition of histone deacetylase (HDAC) activity. In this study, we show that HDAC inhibitors of distinct chemical classifications, including suberanilohydroxamic acid (SAHA) and 4-(dimethylamino)-n-[7-(hydroxyamino)-7-oxoheptyl] benzamide (M344), parallel the effects of VPA on MT1 induction in vitro. However valpromide, a VPA analogue that lacks the ability to inhibit HDAC activity, does not. The observed increase in MT1 expression by VPA is matched by an increase in global histone H3 acetylation. More importantly, an enrichment of histone H3 acetylation occurs along the rat MT1 promoter following treatment with VPA, indicating that histone acetylation and chromatin remodelling are a primary mechanism underlying this induction. Independent of VPA, the rat MT1 gene may be regulated by a number of intracellular kinase pathways and transcription factors, which are also targeted by VPA. KG501-mediated CREB inhibition did not block MT1 upregulation by VPA. Blockade experiments targeting the PKC, PI3K/AKT, or GSK3β signaling pathways suggest that VPA induces melatonin receptor expression independent of these intracellular signaling cascades as well. The relevance of melatonin receptor upregulation was assessed using in vivo VPA and melatonin combination treatments on neuroprotective gene expression. The results of this study provide evidence that expression of the melatonin receptor is epigenetically induced by VPA by means of promoter histone acetylation. Melatonin receptor upregulation by VPA, or other HDAC inhibitors, may represent a therapeutic strategy for the management of several nervous system disorders. / Dissertation / Doctor of Philosophy (PhD)

<p> The MT1 receptor is involved in the oncostatic action of melatonin and valproic acid (VPA) in human MCF-7 breast cancer cells and VPA can upregulate this receptor in C6 glioma cells. Therefore, the effect of VPA on the expression of the MT1 was examined in MCF-7 cells. Treatment of MCF-7 cells in low serum conditions with VPA (0.5 or 1mM) for 24 or 72 h caused a significant increase in MT1 receptor expression, as shown by reverse transcription-polymerase chain reaction analysis (RT-PCR). MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays performed in high serum conditions revealed a significant concentration-dependent inhibition of MCF-7 cell proliferation by VPA (0.5 - 5 mM), whereas melatonin (1 or 10 nM) showed modest effects alone. However, a combination of VPA and melatonin produced a marked synergistic inhibition of cell proliferation. In subsequent experiments, under high serum conditions, VPA treatment for 24h on these cells resulted in a significant decline of MT1 mRNA while the protein levels were still increasing, as seen by RT-PCR and western blotting respectively. The involvement of multiple biochemical events, such as: induction of the p53 tumor suppressor gene and repression of the estrogen receptor (ER)-alpha might be responsible for the synergistic inhibition of these cells after the simultaneous exposure to VPA and melatonin. These results indicate that clinically relevant concentrations of VPA upregulate melatonin MT1 receptor expression in human breast cancer cells. Moreover, the enhanced antiproliferative effect observed with a combination of VPA and melatonin suggests that a similar therapeutic approach may be beneficial in human breast cancer.</p> / Thesis / Master of Science (MSc)