Made available in DSpace on 2017-07-10T14:17:02Z (GMT). No. of bitstreams: 1 kelly Jaque.pdf: 1788989 bytes, checksum: 65d07041b0d9003d6666dd84d7fee873 (MD5) Previous issue date: 2013-08-01 / The growing number of overweight and obesity has led to an increase in the number of patients with insulin resistance and diabetes mellitus type 2. MSG obese rats were glucose intolerant, insulin resistant and theirs pancreatic islets secrete more insulin in response to glucose. Subdiafragmatic vagotomy changes the response of islets to glucose and improves glucose homeostasis, supporting the hypothesis that an unbalance of autonomic nervous system with increased parasympathetic nervous system (PNS) action but a decreased sympathetic nervous system function. Studies showed that the PNS is also involved in β-cell proliferation. Therefore, we investigated of PNS participation, using a subdiafragmatic vagal denervation, upon pancreatic β-cell function and mass regulation, and the body glucose control disruption in MSG-obese rats. For this, Male Wistar rats received during the first five days of life monosodium glutamate (MSG) or saline. Subdiaphragmatic vagotomy was performed at 30 days of life. At 90 days of age, we verified static insulin secretion, pancreas morphometric, ERK expression in islets, glucose homeostasis and lipidis. The MSG treatment caused obesity at 90 days of life. MSG rats presented lower body weight and nasoanal length, increased Lee index and fat depots, normoglycemia, hyperinsulinemia, dyslipidemia, glucose intolerance and insulin resistance when compared to CTL. Vagotomy performed at 30-days of age prevented obesity, fat deposition in the liver and ameliorated glucose tolerance and insulin sensitivity in adult MVAG rats in relation to MSG rats. Islets from MSG rats secreted more insulin at stimulatory glucose concentrations than CTL islets. Histological analysis showed that pancreatic islets from MSG rats were lower with a reduction in β-cell area without modification in α-cell content when compared with CTL. Also, MSG group presented an increased number of pancreatic islets per mm2, with higher number of islets, which may contributes to the higher islet and β-cell relative mass in the MSG pancreas. These effects were associated with enhanced proliferation in MSG group. The number of MVAG pancreatic islet were less than MSG. Vagotomoy performed at 30-days of age, reduced islet and β-cell area in the pancreas from 90-days old CVAG rats. Finally, the relative islet and β-cell mass in MVAG and CVAG rats was similar to CTL. Here we verified if ERK was involved in β-cell replication in MSG rats, but presented no alteration. We demonstrate for the first time that adult MSG rats showed enhanced pancreatic β-cell proliferation which contributes to the higher islet insulin secretion in response to glucose. The vagus nerve is the main factor involved in such a process, since vagotomy performed at 30 days of age prevented islet morphological alterations in adult MVAG rats. Possibly this increase PNS activity in MSG endocrine pancreas is responsible to hyperinsulinemia that enhanced fat storage, damaged glucose homeostasis and insulin action in MSG obesity / O crescente número de pessoas com sobrepeso e obesidade tem levado ao aumento no número de pacientes com resistência à insulina (RI) e portadores do Diabetes mellitus tipo 2. Ratos obesos MSG são intolerantes à glicose (Gli), RI e suas ilhotas pancreáticas secretam mais insulina em resposta à concentrações de Gli. A vagotomia subdiafragamática altera a responsividade das ilhotas à Gli e melhora a homeostase glicêmica nestes animais, sugerindo um desbalanço do sistema nervoso autonômico, com aumento do tônus parassimpático e redução do simpático. Estudos demonstram que o sistema nervoso parassimpático (SNP) possui efeito na proliferação das células β-pancreáticas. Desta forma, investigamos a participação do SNP, através da vagotomia subdiafragmática, no metabolismo energético e na proliferação das ilhotas e de células β-pancreáticas de ratos obesos-MSG. Para isto, ratos Wistar machos receberem durante os cinco primeiros dias de vida glutamato monossódico (grupo MSG) ou salina (grupo CTL). A vagotomia subdiafragmática foi realizada aos 30 dias de vida formando os grupos MVAG e CVAG. Aos 90 dias, verificamos a secreção estática de insulina, homeostase glicêmica e lipídica, morfometria do pâncreas e conteúdo proteico da ERK nas ilhotas. Ratos MSG apresentaram redução do peso corporal e comprimento nasoanal, aumento do índice de Lee e acúmulo de gordura, normoglicêmia, hiperinsulinemia, dislipidemia, intolerância à Gli e RI comparados aos CTL. A vagotomia realizada aos 30 dias de vida preveniu obesidade, acúmulo de gordura no fígado e melhorou a tolerância à Gli e a sensibilidade à insulina em ratos MVAG adultos em relação aos ratos MSG. As ilhotas dos animais MSG secretaram mais insulina quando estimulada pela Gli, em relação aos animais CTL. As análises histológicas mostram que as ilhotas pancreáticas dos animais MSG são menores com redução da área das células β sem alteração nas células α em relação aos CTL. O grupo MSG apresenta um aumento do número das ilhotas por mm2, que pode estar contribuindo com o aumento da massa relativa das ilhotas e das células β. Esse efeito está associado ao aumento da proliferação no grupo MSG. O número de ilhotas foi menor nos MVAG em relação aos MSG. A vagotomia realizada aos 30 dias de vida reduziu a área das ilhotas e das células β aos 90 dias de vida nos animais CVAG. Finalmente, a massa relativa das ilhotas e da células β no MVAG e CVAG foram similares ao CTL. Verificamos se a ERK estava envolvida na proliferação das células β nos ratos MSG, porém não apresentaram alterações desta proteína. Pela primeira vez demonstramos que ratos MSG apresentam aumento da proliferação das células β que contribui com o aumento da secreção de insulina em resposta à Gli. O nervo vago é o principal fator envolvido neste processo, visto que a vagotomia realizada aos 30 dias de vida preveniu as alterações morfológicas das ilhotas nos ratos MVAG adultos

Os genes homeobox são responsáveis por codificar proteínas nucleares que agem como fatores de transcrição durante o desenvolvimento embrionário, regulando proliferação e diferenciação celular. A expressão alterada do gene homeobox PROX1 já foi identificado em diferentes neoplasias, incluindo mama, esôfago, fígado, sistema biliar, linfomas e cavidade bucal. Este trabalho teve como objetivo avaliar os efeitos da superexpressão do gene PROX1 em linhagem celular derivada de carcinoma epidermóide bucal nos mecanismos de proliferação e diferenciação celular, apoptose e perfil global de expressão gênica. Após a superexpressão deste gene na linhagem celular SCC-9, foi realizada a análise de proliferação por meio dos ensaios de curva de proliferação celular, citometria de fluxo, índice de incorporação de BrdU ao DNA e expressão de Ki67. A diferenciação celular foi verificada por meio de reações imunocitoquímicas para as citoqueratinas 1, 10, 13, 14, 16, 18 e 19 e a apoptose foi avaliada por meio de células positivas para anexina-V e iodeto de propídeo. O ensaio de microarray foi realizado para avaliação do perfil global de expressão gênica na linhagem celular SCC9 com superexpressão do gene PROX1. Observou-se que a superexpressão do gene PROX1 promove redução da proliferação celular, bem como reduz a expressão das citoqueratinas 1, 13, 18 e 19. Não houve alteração na taxa de apoptose entre as células com superexpressão do gene PROX1 e controles. Os resultados do microarray revelaram a expressão diferencial significante de genes envolvidos com os processos de desenvolvimento, adesão e invasão celular. Desta maneira, estes resultados são fortemente sugestivos de que o gene PROX1 inibe a proliferação celular e contribui para a diferenciação do carcinoma epidermóide bucal. / Homeobox genes encode transcription factors with an important role during normal development by controlling cellular proliferation and differentiation. Altered expression of PROX1 homeobox gene is related to many cancers, including those of the breast, esophagus, liver, billiary system and lymphomas. The aim of this study was evaluate the effects of PROX1 overexpression, in an oral squamous cell carcinoma cell line, on cellular proliferation and differentiation, apoptosis as well as gene expression prolfile. After overexpression of PROX1 gene in SCC9 cell line, proliferation was assessed by proliferation curve, flow citometry, BrdU incorporation to DNA and Ki67 expression. Cell differentiation was verified by immunocytochemistry to cytokeratins 1, 10, 13, 14, 16, 18 and 19 and apoptosis was measured by annexin V positive cells. Gene expression profile was analyzed by microarray in PROX1-overexpressing cells and control. PROX1-overexpressing cells showed a statistically significant decrease in proliferation as well cytokeratin 1, 13, 18 and 19 expression. No significant differences from controls and PROX1- overexpressing cells were observed in apoptosis. Microarray analyses showed differential expression of genes related to development, cellular adhesion an invasion. Our results strongly suggest that overexpression of PROX1 inhibit cell proliferation and contributes to differentiation of oral squamous cell carcinoma.

Comment la taille d'un organisme est-elle régulée au cours du développement embryonnaire ? Quels sont les mécanismes génétiques à l'origine de l'arrêt de la prolifération pendant la croissance d'un organisme pluricellulaire ? Afin d'identifier des acteurs de la sortie du cycle cellulaire au cours du développement, mon travail s’est orienté sur l’étude de gènes suppresseurs de tumeurs pendant la croissance du toit optique (TO) du médaka Oryzias latipes. Le TO, structure dorsale du cerveau moyen des Vertébrés, est un modèle particulièrement adapté à l’étude de la régulation de la prolifération. Trois zones de la marge vers le centre du TO sont discernables : une zone périphérique de prolifération, une zone intermédiaire de cellules sortant du cycle cellulaire et une zone centrale de cellules différenciées. Un crible d'expression par hybridation In Situ a été réalisé et a permis d'identifier 28 gènes exprimés dans le TO, suggérant leur implication dans le contrôle de la sortie du cycle cellulaire au cours du développement. Dans le but de caractériser in vivo la fonction de gènes issus de ce crible, le gène BTG1 (B-cell Translocation Gene 1) et les membres de sa famille, ont été étudiés au cours du développement du médaka. J’ai mené des expériences fonctionnelles sur BTG1, permettant de mettre en évidence son rôle central pour la morphogenèse du système nerveux central. De plus, une autre partie de mon travail s’est penchée sur l’étude de l’expression des membres de la voie de signalisation Hippo, bien connue et caractérisée chez la drosophile et les Mammifères pour son rôle dans le contrôle de la taille des organes via une régulation de l’arrêt de la prolifération. A l’issu de notre travail, une fonction de la voie de signalisation Hippo dans la formation du TO et des somites a pu être mise en évidence au cours du développement du médaka. / How is an organisms’ size regulated during embryonic development? What are the genetic mechanisms that control the proliferation arrest during multicellular organisms growth? In order to identify a cell cycle exit developmental actor genes, I have analysed the role of tumor suppressor genes (TSGs) in the optic tectum (OT) of the medaka Oryzias latipes. This structure is particularly suited for this kind of studies because, during its morphogenesis, there is a strict correlation between the position of a cell and its degree of differentiation. 3 zones can be easily distinguished from the border to the center: a marginal zone made of proliferative cells, an intermediate zone in which cells exit the cycle, and a central zone made of postmitotic cells. Using this criterium, I have performed an in situ hybridization expression screen on 150 TSGs on medaka embryos. The expression patterns of 28 TSGs in the OT suggest their implication in the OT proliferation arrest mechanisms. I focused my study on the BTG1 gene, implicated in many cancers, and for which few developmental data are available. A functional analysis on developing medaka embryos has been performed and permitted to highlight the essential role of BTG1 in central nervous system morphogenesis. Furthermore, I performed an expression study on Hippo signalling pathway components. Hippo pathway is well caracterised for its organ size control function in drosophila and Mammals. Our results show that this pathway could act in OT formation and somitogenesis in medaka fish.

La neurofibromatose de type 2 (NF2) est une maladie autosomique causée soit par l'inactivation du gène NF2, soit par la perte de la protéine issue due ce gène, Merline. Cela entraîne à son tour la formation de plusieurs tumeurs nerveuse bénignes (non invasives) comme les schwannomes, méningiomes et les épendymomes. De plus, une diminution de l'expression de Merline est observée dans les cancers du sein invasifs, toutefois le rôle de Merline dans ces tumeurs invasives est peu étudié. Merline est la seule protéine ayant un rôle de suppresseur de tumeur dans la famille des ERM (Ezrin / Radixin / Moesin). Nous, ainsi que d'autres groupes, avons montré que la partie C-terminale de Merline est importante pour sa fonction inhibitrice de la croissance cellulaire. Par conséquent, j'ai cherché à mettre en évidence de nouveaux partenaires d'interaction non décrits à ce jour, ainsi que de nouveaux sites de phosphorylation sur l'extrémité C-terminale de Merline qui pourrait expliquer la fonction de suppresseur de tumeur de Merlin. L'utilisation d’expériences d'immunoprécipitation couplées à la spectrométrie de masse nous a permis d’identifier de nouveaux interacteurs ainsi que de nouveaux sites de phosphorylation sur ce domaine C-terminal de Merline. Nous avons analysé l'importance d'un nouvel interacteur, AmotL1, ainsi que d'un nouveau site de phosphorylation sur la threonine 581 (T581), dans la fonction suppresseur de tumeur de Merline. La protéine AmotL1 appartient à la famille des motines, qui sont connues pour être impliquées dans la régulation de la migration cellulaire. A cet égard, nous avons montré qu’AmotL1 est un nouveau partenaire d'interaction de Merline. Nous avons étudié l'importance de cette interaction entre Merline et AmotL1 dans la migration cellulaire et nos données suggèrent fortement que Merlin pourrait inhiber la migration cellulaire médiée par AmotL1 dans les cellules du cancer du sein, via notamment la régulation de son expression et de sa localisation. Enfin, nous avons également identifié plusieurs nouveaux interacteurs de Merline, qui pourraient expliquer comment Merlin pourrait agir comme une protéine d'échafaudage à la membrane plasmique, en interagissant avec des composants essentiels de la voie Hippo, comme AmotL1, Kibra, Lats et YAP, pour réguler la prolifération et la migration cellulaire. Dans la deuxième partie, nous avons identifié un nouveau site de phosphorylation spécifique à l'isoforme 1 de Merline, la T581, et nous avons démontré que la phosphorylation de cette threonine est importante pour la progression en mitose au moment approprié. De plus, dans cette étude, nous avons montré que Merlin est un substrat potentiel de la kinase Aurora A, un oncogène majeur, au cours de la mitose et de l'interphase, dans des lignées cellulaires de cancer du sein. Enfin, nous avons fourni des données préliminaires sur la façon dont Aurora A régule la signalisation Hippo et la fonction de DCAF1 en phosphorylant Merline. En résumé, cette thèse met en évidence deux fonctions importantes de Merline : premièrement comment Merline régule la migration/invasion cellulaire dans des tumeurs non-nerveuses telles que les cancers du sein et deuxièmement, comment Merline est régulé au cours de la mitose et de l'interphase dans des lignées de cancer du sein, en agissant comme un substrat pour la kinase Aurora A qui est surexprimée dans plusieurs cancers comme celui du sein, du côlon et l'HCC. Prise dans son ensemble, notre étude montre le rôle potentiel de Merline dans les tumeurs invasives telles que celles rencontrées dans les cancers du sein. / Neurofibromatosis type 2 (NF2) is an autosomal disorder caused by inactivation of NF2 gene or loss of the NF2 product, Merlin. This in turn results in formation of multiple benign (noninvasive) nerve tumors such as schwannomas, meningiomas and ependymomas. Additionally reduced expression of Merlin is observed in invasive breast cancers however the role of Merlin in these invasive tumors is poorly investigated. Merlin is the only tumor suppressor protein in Ezrin/Radixin/Moesin (ERM) family proteins. Previously we and others have shown that C-terminus of Merlin is important for its growth suppressive function. In this regard, I set out to investigate whether there were undiscovered interacting partners and novel phosphorylation sites on the C-terminus of Merlin that could account for tumor suppressor function of Merlin. Using immunoprecipitation coupled to mass spectrometry we have identified new interactors as well as novel phosphorylation on this C-terminus domain of Merlin. We analyzed importance of new interactor, AmotL1, as well as novel phosphorylation site on T581 in the tumor suppressor function of Merlin. AmotL1 belongs to AMOT family proteins which are known to involve in the regulation of cell migration. In this regard, we have shown that AmotL1 is novel interacting partner of Merlin. We have investigated the importance of Merlin and AmotL1 interactions in cell migration and our data strongly suggest that Merlin might inhibit AmotL1 mediated cell migration in breast cancer cells by regulating its expression and localization. Finally, we have also found several new interactors of Merlin and that could explain how Merlin might acts as scaffolding protein at the plasma membrane by interacting with Hippo core components such as AmotL1, Kibra, Lats and YAP to regulate cell proliferation and migration. In the second part, we have identified a novel phosphorylation site at T581 which is specific to Merlin isoform 1 and demonstrated that phosphorylation of Merlin on T581 is important for the timely mitotic progression. Further in this study, we have shown that Merlin is a potential substrate for major oncogene Aurora kinase A in mitosis as well as in interphasic breast cancer cell lines. Finally we have provided initial clues how Aurora A regulates Hippo signaling and DCAF1 function by phosphorylating Merlin. In the summary, this thesis highlights two important functions of Merlin: firstly how Merlin regulates the cell migration/invasion in non-nerve tumors such as breast cancers and secondly how Merlin is regulated in mitosis and interphasic breast cancer cells by acting as a substrate to Aurora Kinase A which is over expressed in several cancers such as breast, colon and HCC. All together our study indicates the potential role for Merlin in invasive tumors such as breast cancers.

As células glias de Müller são capazes de gerar novos neurônios retinianos em resposta a lesões, atuando como uma possível fonte para regeneração retiniana. Nesse contexto, as GTPases Rho podem ter um papel interessante, visto que regulam múltiplas vias de sinalização que controlam, por exemplo, a transcrição gênica, sobrevivência e proliferação celular. No presente estudo analisamos a participação de um dos membros dessa família (Rac1) na proliferação de células gliais de Müller da retina de galinhas após lesão excitotóxica com N-Metil-D-Aspartato (NMDA). A injeção intraocular de NMDA promoveu extensa proliferação de células gliais de Müller. A inibição de Rac1 com NSC23766 não alterou a quantidade de células que entraram no ciclo celular, mas, provocou um retardo em sua progressão. Esses resultados sugerem um importante papel para a GTPase Rac1 na regulação da proliferação de células gliais de Müller em resposta a lesões retinianas. / Müller glial cells may generate new neurons in response to retinal injury, acting as a potential source for retinal regeneration. In this context, Rho GTPases may have an interesting role, since they regulate multiple signaling pathways that control, for example, gene transcription, cell proliferation and survival. This study analyzed the involvement of a member of this family (Rac1) in the proliferation of Müller glial cells of chick retina after excitotoxic injury with N-methyl-D-aspartate (NMDA). Intraocular injection of NMDA promoted extensive Müller glia proliferation. Rac1 inhibition with NSC23766 did not affect the cell cycle entry, but a delay in cell cycle progression was observed. These results suggest an important role for Rac1 in the regulation of Müller glial cells proliferation in response to retinal injury.

A endometriose, caracterizada por crescimento de tecido endometrial fora da cavidade uterina, é responsável por sintomas álgicos com grande impacto na qualidade de vida da paciente. Várias linhas de medicações têm sido estudadas para essa o tratamento da endometriose, já que a cirurgia não é o tratamento de escolha sempre e, quando realizado, não é um tratamento definitivo. O conhecimento da patogenia da endometriose é fundamental para o estudo de novas classes de medicações. Uma delas, os fatores inibitórios da angiogênese, tem papel fundamental no estabelecimento e crescimento de lesões de endometriose. Neste estudo, buscamos a influência da Bevacizumab, droga anti-fator de crescimento endotelial (anti-VEGF), utilizada em duas dosagens diferentes, em endometriose peritoneal induzida em ratas, modelo animal já bem estabelecido para o estudo de endometriose. As ratas permaneceram sob tratamento durante 4 semanas, após as quais foram sacrificadas, sendo as lesões e o corno uterino remanescente retirados para posterior avaliação. Foi realizada avaliação da área das lesões de cada rata, da presença de tecido endometrial à microscopia, da positividade para o anticorpo antiVEGF na imunohistoquímica e da expressão gênica de PCNA, MMP9, Tp63 e VEGFA. O bevacizumab atuou reduzindo a área das lesões nos grupos que receberam medicação (p=0,002) e reduzindo a expressão gênica para Tp63 nas lesões (p=0,04). Não houve resultado significativo nas outras avaliações / Endometriosis, characterized by growth of endometrial tissue outside the uterine cavity, is responsible for painful symptoms with great impact on the quality of life of women. Several lines of medications have been studied for endometriosis\'s treatment, since surgery is not always the treatment of choice and, when done, it is not a definitive treatment. Knowing the pathogenesis of this disease is fundamental for the study of new classes of medications. One of them, the inhibitory factors of angiogenesis, plays a fundamental role in the establishment and growth of endometriosis lesions. In this study, we sought the influence of Bevacizumab, an anti-endothelial growth factor (anti-VEGF) drug, used in two different dosages, in peritoneal endometriosis induced in rats, an animal model well established for the study of endometriosis. The rats remained under treatment for 4 weeks, after which they were sacrificed, with the remaining lesions and uterine horn being removed for further evaluation. An evaluation of the lesion area of each rat, the presence of endometrial tissue under microscopy, the positivity of the anti-VEGF antibody in immunohistochemistry and the gene expression of PCNA, MMP9, Tp63 and VEGFA were performed. Bevacizumab worked by reducing the area of the lesions in the groups receiving medication (p = 0.002) and reducing the gene expression for Tp63 in the lesions (p = 0.04). There was no significant result in the other evaluations

A expressão do gene Dermicidina (DCD) no câncer de mama promove aumento na proliferação e sobrevivência celular, porém os mecanismos envolvidos são desconhecidos. Através de um ensaio de microarranjos de DNA demonstramos que o DCD desempenha atividade de fator de crescimento pela modulação da via de sinalização EGF/ErbB. Estes dados foram confirmados através de silenciamento gênico, superexpressão e tratamento com a proteína recombinante. O silenciamento diminuiu a expressão de EGFR e seus ligantes, e reduziu a ativação da via de sinalização EGF/ErbB. O tratamento da linhagem celular MDA-MB-361 com DCD recombinante resultou em uma curva de proliferação em forma de sino com concomitante aumentou a expressão de EGFR e seus ligantes. A superexpressão de DCD na linhagem MCF-7 também resultou no aumento da expressão dos receptores EGFR e HER-2 e de seus ligantes, além da ativação de suas vias de sinalização. Este trabalho sugere que o DCD é capaz de modular a expressão e ativação da família EGF/ErbB resultando no aumento do crescimento e sobrevivência celular. / The expression of the gene Dermicidin (DCD) in breast cancer promotes increased in cell growth and survival, however the mechanisms involved are unknown. Using a DNA microarray assay we demonstrated that the DCD activity plays by modulating growth factor signaling phathway of EGF/ErbB. These data were confirmed by gene silencing, overexpression and treatment with the recombinant protein. The silencing decreased the expression of EGFR and its ligands, and reduced the activation of the EGF/ErbB signaling pathways. The treatment of MDA-MB-361 cell line with recombinant DCD resulted in a proliferation curve bell-shaped with a concomitant increased in the expression of the EGFR and its ligands. The overexpression of the DCD in the MCF-7 cell line also resulted in increased expression of the receptors HER-2 and EGFR and its ligands, and activation of theirs signaling pathways. This work suggests that DCD is able to modulate the expression and activation of the EGF/ErbB family resulting in increased of cellular growth and survival.

Les tumeurs épithéliales thymiques (TET) humaines sont rares (250 - 300 cas/an en France). On distingue les thymomes de type A, AB ou B d'évolution lente avec une survie actuarielle > 95% à 5 ans pour les stades précoces et les carcinomes thymiques d'évolution plus sévère avec une survie actuarielle à 5 ans < 20% pour les stades IV. La pierre angulaire du traitement des TET est l'exérèse chirurgicale complète, facteur pronostique le plus significatif identifié à ce jour. Les récidives des TET, essentiellement pleurales pour les thymomes et générales pour les carcinomes thymiques, sont de prise en charge complexe. Les avancées thérapeutiques sont limitées notamment par l'absence de modèles d'étude de la cellule épithéliale thymique tumorale. Dans le cadre d'une prise en charge multidisciplinaire des récidives pleurales métastatiques, nous avons développé la pleurectomie de cytoréduction associée à une chimio hyperthermie (cisplatine/ mitomycine ; 42°C) intra thoracique (CHIT) pour la prise en charge des métastases pleurales de thymome. Chez des patients sélectionnés (n=19), la médiane de survie sans récidive était de 53 mois et les survies actuarielles à 1 an et 5 ans étaient respectivement de 93% et 86%. Cette technique chirurgicale innovante a permis de développer une alternative à la morbide pleuro pneumonectomie. L'efficacité de la CHIT pose des questions sur le rôle de l'hyperthermie et sur le type de chimiothérapie à associer. Avec pour objectif d'améliorer la prise en charge des patients, la connaissance de la biologie tumorale thymique et l'identification de potentielles cibles thérapeutiques sont des voies de recherche importantes pour améliorer la survie des patients. Nous avons développé des cultures de cellules épithéliales thymiques dérivées in vitro de 12 TET (11 thymomes A, AB ou B et un carcinome thymique), caractérisées par leur potentiel prolifératif et leur expression de cytokératine. La voie PI3K / Akt / mTOR joue un rôle clé dans de nombreux cancers ; plusieurs études de phases I / II ont rapporté un effet positif des inhibiteurs de mTOR pour le contrôle de l'évolution du thymome chez les patients. Nous avons mis en évidence l'expression et l'activation des effecteurs mTOR, Akt et P70S6K dans les thymomes et dans les cellules épithéliales thymiques dérivées in vitro. Nous avons montré l'efficacité de la rapamycine, inhibiteur de mTOR, à réduire la prolifération cellulaire (30%) sans induire de mort cellulaire. Nos résultats suggèrent que l'activation de la voie Akt / mTOR participe à la prolifération cellulaire associée à la croissance tumorale. Nous avons établi un nouvel outil permettant l'étude de la dérégulation cellulaire au cours des thymomes. Dans un contexte de tumeurs rares, ces cellules permettront d'aborder des études mécanistiques in vitro et de tester l'efficacité de drogues anti tumorales / Human thymic epithelial tumors (TETs) are rare (250 – 300 cases/ year in France). We distinguish thymomas (A, AB and B subtypes) with indolent evolution (5 years actuarial survival in early stages >95%) and more aggressive thymic carcinomas (5 years actuarial survival <20% in stage IV). Surgical complete resection when feasible is the corner stone of a multimodal therapy and the most significant factor on survival. Relapse of TETs principally in pleura for thymomas (75%) and general for thymic carcinomas are difficult to treat. Therapeutics advances are limited given the lack of studies models of tumoral thymic epithelial cell. In a multidisciplinary approach for the treatment of metastatic pleural relapse of thymomas we developed an innovative surgical technique: cytoreductive pleurectomy associated with hyper thermic intra pleural chemotherapy (Cysplatin/ Mitomycin; 42°C) (ITCH). In selected patient (n=19), ITCH provides an efficient alternative to the morbid pleuro pneumonectomy. The median of free disease survival was 53 months, one year and five years actuarial survival were respectively 93% and 86%. However, the effectiveness of ITCH procedure questions on the played role ok hyperthermia, on the choice of chemotherapy association. With the aim to improve TETs therapies, the knowledge of TETs biology to identify potential target therapies is currently challenging. We developed an in vitro study model of tumoral thymic epithelial cells derived from 12 TETs (11 A, AB and B thymomas and one thymic carcinoma) characterized by their proliferative abilities and the cytokeratin expression. The PIK3 / Akt / mTOR pathway is implicated in numerous cancers. Several phase I, II studies advocate the potential role of mTOR inhibitors in the control of the metastatic disease. We highlighted the expression and the activation of mTOR, Akt and P70S6K effectors in TETs and in thymic epithelial cells in vitro derived. We showed the efficacy of rapamycin (mTOR inhibitor) in the inhibition (-30%) of in vitro cell proliferation without cell death induction. Our results suggest the implication of the PIK3 / Akt / mTOR pathway in the tumoral cell growth. We established a new tool to study cell dysregulation in TETs. In the context of rare tumors, these cells could allow in vitro mechanistic studies and test the efficacy of new anti tumoral therapies

La chaperonine CCT/TriC tient un rôle central dans le maintien de la protéostase cellulaire. Elle compte parmi ses clientes un grand nombre de protéines impliquées de près ou de loin dans la régulation du cycle comme l’actine, la tubuline et la cycline E. afin de mieux comprendre l’implication de la CCT/TriC dans la cancérogenèse, nous avons quantifié, dans 18 lignées cellulaires tumorales, une lignée issue d’un tissu sain et un tissu sain, ses taux d’expression et son activité. Nos résultats indiquent que l’expression de la CCT/TriC n’est pas toujours corrélée à son activité. Nous avons dans un second temps, documenté l’expression de ces partenaires/modulateurs d’activité, préfoldine, PhLP3, Hop/p60, Hsp/c70 et leur influence sur son activité. Nos résultats montrent que l’activité de la CCT/TriC pourrait être régulée à travers les variations des niveaux d’expression de la préfoldine et/ ou Hsp/c 70. Enfin, nous avons montré que dans les cellules qui se divisent le plus lentement, l’activité de la CCT/TRiC est la plus faible. Ces observations montrent que les variations de l’activité de la CCT/TriC pourraient constituer un point de contrôle dans la prolifération cellulaire maligne. / The molecular chaperone CCT/TRiC plays a central role in maintaining cellular proteostasis. It mediates the folding of lot of proteins involved in cell cycle regulation as the major cytoskeletal proteins tubulins and actins and the oncoprotein cyclin E. To assess the involvement of CCT/TRiC in tumor genesis, we quantified its expression levels and activity in 18 cancer, one non-cancer human cell lines and a non-cancer human liver. We show that the expression levels of CCT/TRiC in cancer cell lines are higher than that in normal cells. However, CCT/TRiC activity does not always correlate with its expression levels. We therefore documented the expression levels of CCT/TRiC modulators and partners PhLP3, Hop/P60, prefoldin and Hsp/Hsc70. Our analysis reveals a functional interplay between molecular chaperones that might account for a precise modulation of CCT/TRiC activity in cell proliferation through changes in the cellular levels of prefoldin and/or Hsc/p70 and CCT/TRiC client protein availability. Finally, our study reveals that cells with the longest doubling time host the smallest CCT/TRiC activity suggesting that CCT/TRiC-mediated client protein folding constitutes a bottle-neck in cancer cell proliferation. Our observation and approaches bring novel insights in the role of CCT/TRiC-mediated protein folding machinery in cancer cell development.

Previous studies suggest that the IGF-I stimulates the elastin gene transcription through the unique responsive sequence on the elastin promoter, which is a putative retinoblastoma control element (RCE). This site interacts with (Sp)-family transcription factors whose delivery is mediated by the retinoblastoma protein (Rb). Since Rb (phosphorylated on serine 780) has been implicated in the initiation of the cell cycle, we speculated that a different phosphorylation of Rb might determine Rb involvement in elastogenesis. Obtained results demonstrated that, IGF-I-induced elastogenic signaling pathway in human dermal fibroblasts includes activation of cyclinE/cdk2 causing a site specific phosphorylation of Rb on threonine 821. This permits the sequestration of Sp1 by Rb before it could bind the elastin promoter, thereby allowing the elastin gene transcription. We also found that blocking of H-Ras in Costello syndrome fibroblasts (characterized by heightened proliferation and impaired elastogenesis), selectively down-regulated Rb phosphorylation on serine 780 and normalized impaired elastogenesis.