Les corticosurrénalomes (CS), bien que rares, sont des tumeurs malignes du cortex surrénalien très agressives. Environ 30% des patients atteints de cancer surrénalien présentent des métastases au diagnostic et leur survie à 5 ans est inférieure à 20%. Les mécanismes à l’origine de la progression cancéreuse ne sont pas complètement élucidés. Leur compréhension est pourtant un préalable à la mise au point de traitements adaptés. Les mutations du gène P53 font parties des altérations génétiques les plus fréquentes dans les CS. Dans ce contexte, il est légitime d'étudier l'effet de l'inactivation de P53 spécifiquement dans les surrénales de souris. L'antigène T du virus SV40 est un oncogène qui se lie et inhibe P53 et RB. Le laboratoire dispose de souris transgéniques (modèle AdTAg) exprimant l’antigène T de SV40 dans le cortex surrénal qui développent des tumeursévolutives. L’objectif de ce travail était de caractériser l’ontogenèse de ces tumeurs et d’explorer les modifications cellulaires et moléculaires qui accompagnent leur progression maligne notamment en lien avec les signalisations β-caténine et IGF2/mTOR. Les souris AdTAg développent des tumeurs surrénaliennes récapitulant l’ensemble des caractéristiques décrites pour les CS humains. En effet, elles présentent une surmortalité à partir de 22 semaines associée à la survenue de métastases pulmonaires et hépatiques. Les tumeurs sont à l'origine d'une hypercorticostéronémie témoignant de leur différenciation stéroïdogénique. L'analyse du score de Weiss à différents stades montre une évolution de la bénignité vers la malignité. Cette progression tumorale s’accompagne d’une activation précoce de la voie mTOR et tardive de la voie Wnt/β-caténine. Ces deux voies de signalisation pourraient donc constituer des cibles thérapeutiques intéressantes. La deuxième partie du projet visait à utiliser ce modèle murin pour tester une thérapie anticancéreuse applicable au carcinome surrénalien. La rapamycine, un inhibiteur de mTOR, inhibe la prolifération cellulaire et induit une apoptose des cellules tumorales. Après 3 mois de traitement, une réduction significative du volume tumoral est constatée ainsi que la normalisation des taux de corticostérone. Nous avons également évalué l'effet antitumoral d'inhibiteurs de la voie Wnt/β-caténine: la quercetine et le PRI-724. La quercetine stoppe la progression tumorale en inhibant la prolifération cellulaire. Elle prolonge significativement la survie des souris AdTAg. Cependant, nous n'avons pas de preuve moléculaire d'inhibition de la voie Wnt/β-caténine dans les surrénales AdTAg et les mécanismes d'action de la molécule restent à élucider. A l'inverse, le PRI-724 semble être un inhibiteur spécifique de la voieWnt/β-caténine capable de bloquer l'interaction CBP/β-caténine. Un traitement de 2 mois permet une réduction significative du volume tumoral chez les souris AdTAg. La baisse d'expression de certains gènes cibles de l'interaction CBP/β-caténine témoigne d'une inhibition de la voie. Les résultats obtenus avec les inhibiteurs des voies mTOR et Wnt/β-caténine dans le modèle murin de CS sont prometteurs. L'utilisation de ces molécules pourrait donc être envisagée dans le traitement du CS. / Adrenocortical carcinoma (ACC) is a rare aggressive malignant tumor of adrenal cortex. 30% of patients have metastatic disease at diagnosis and the 5 year-survival rate is obtained inonly 20%. Unfortunately, the mechanisms of tumorigenesis are not well identified. Understanding these mechanisms could offer perspectives for new targeted therapies improving the survival in these patients. P53 inactivation in the adrenal cortex seems a good target to study its role in the tumorigenesis. Large T antigen of SV40 virus is an oncogene that fixes and inhibits P53 and RB. Our laboratory has mouse models expressing this antigen (AdTAg mouse model) in the adrenal cortex and developping progressive adrenal tumors. The initial objective was to characterize the ontogeny of these tumors, studying their molecular characteristics, especially β-catenin and IGF2/mTOR signaling, during the malignant progression. AdTAg mouse models develop adrenocortical tumors with characteristics that are identical to human ACC. They present pulmonary and liver metastases that lead to increased mortality rate from 22 weeks old. These tumors lead to hypercorticism that suggest their steroidogenic differentiation. Weiss score analyses indifferent ages show that these tumors progress from benign to malignant ones, associated with a precocious activation of mTOR pathway and tardive activation of Wnt/β-catenin pathway. These pathways are thus interesting therapeutic targets. The second part of this thesis was concentrated on the anti-cancer treatment trials. Rapamycin, an mTOR inhibitor inhibits cell proliferation and increases cell apoptosis in these tumors. After 3 months of treatment, the tumor burden was significantly reduced and corticosterone levels were normalized. We have also evaluated effects of Wnt/ β-catenininhibitors, Quercetin and PRI-742, in our mouse models. Quercetin inhibits tumor proliferation and progression and it extends the survival rate of AdTAg mice. Surprisingly, this effect was independent of Wnt/β-catenin activity and the molecular mechanisms remain to be elucidated. Inversely, PRI-724 seems to be a specific inhibitor of this pathway, blocking the interaction between CBP and β-catenin. A treatment of 2 months reduced significantly the tumor volume in AdTAg mice. This effect was through the inhibition of CBP and β-catenininteraction and signaling. These results encourage using the inhibitors of mTOR and Wnt/β-catenin pathway offering promising targets to improve the survival in patients with ACC.

Biological information processing in living systems like cells, tissues and organs critically depends on the physical interactions of molecular signaling components in time and space. How endocytic transport of signaling molecules contributes to the regulation of developmental signaling in the complex in vivo environment of a developing organism is not well understood. In a previously performed genome-wide screen on endocytosis, several genes have been identified, that selectively regulate transport of signaling molecules to different types of endosomes, without disrupting endocytosis. My PhD thesis work provides the first functional in vivo characterization of one of these candidate genes, the novel, highly conserved Rab5 effector protein P95 (PPP1R21). Cell culture studies suggest that P95 is a novel endocytic protein important to maintain the balance of distinct endosomal sub-populations and potentially regulates the sorting of signaling molecules between them (unpublished work, Zerial lab). The scientific evidence presented in this study demonstrates that zebrafish P95 is essential for early zebrafish embryogenesis. Both, knockdown and overexpression of zebrafish P95 compromise accurate morphogenetic movements and patterning of the zebrafish gastrula, showing that P95 functions during zebrafish gastrulation. P95 is functionally required to maintain signaling activity of signaling pathways that control embryonic patterning, in particular for WNT/β-catenin signaling activity. Knockdown of zebrafish P95 amplifies the recruitment of β-catenin to early endosomes, which correlates with the limitation of β-catenin to translocate to the nucleus and function as transcriptional activator. The obtained results suggest that zebrafish P95 modulates the cytoplasmic pools of β-catenin in vivo, via endosomal transport of β-catenin. In conclusion, the data presented in this thesis work provides evidence that the cytoplasm-to-nucleus shuttling of β-catenin is modulated by endocytic trafficking of β-catenin in vivo. We propose the endocytic modulation of β-catenin cytoplasm-to-nucleus trafficking as potential new mechanism to fine-tune the functional output of WNT/β-catenin signaling during vertebrate gastrulation.

Comprendre comment les facteurs génétiques et environnementaux interagissent au cours du développement est une question fondamentale en biologie. Je me suis intéressée à cette question en utilisant le réseau de développement de la vulve du nématode C. elegans comme système modèle. L’objectif de mon projet était une étude quantitative de la modulation par l’environnement des voies de signalisation impliquées dans ce processus telles que, Ras, Delta-Notch et Wnt. J’ai tout d’abord analysé comment un facteur environnemental spécifique (la carence nutritionnelle) modifie les activités et les interactions entre les voies de signalisation sous-jacentes au développement vulvaire chez C. elegans. J’ai ainsi mis en évidence que l’augmentation de l’induction vulvaire par la carence passe par une augmentation de l’activité de la voie Ras et est indépendante de la voie Wnt. Cet effet de l’environnement est assuré par la détection de la diminution de l’apport en nutriments, probablement par l’action de la voie TOR, et affecte l’induction vulvaire en parallèle ou en amont du récepteur à l’EGF. J’ai ensuite examiné la sensibilité environnementale du système de développement de la vulve de Caenorhabditis dans une perspective évolutive et ce, grâce à l'analyse comparative de différents isolats. J’ai pu observer que l’exposition à des températures extrêmes induit des variants et des défauts de manière fortement dépendante de la souche et de l’espèce. L’occurrence de certains défauts développementaux induits par la température révèlent en outre que certaines cellules précurseurs de la vulve et les voies de signalisation associées présentent une sensibilité environnementale différente. / How genetic and environmental factors interact during development is a key question in current biology, yet little is known about how molecular and cellular processes integrate environmental information. In my PhD research I aimed to address this problem using the network of C. elegans vulval signalling pathways as a model system. The principal objective of my project was to quantitatively examine how involved major signalling pathways, EGF-Ras-MAPK, Wnt and Delta-Notch, are modulated by specific environmental signals. First, I analysed how a specific environmental factor (starvation) alters activities and interplay of signalling pathways underlying C. elegans vulval cell fate patterning. I found that starvation consistently increased vulval induction through upregulation of the EGF-Ras-MAPK pathway activity independent of the Wnt pathway. This environmental effect is mediated by internal sensing of nutrient deprivation, likely acting through the TOR pathway, and affects vulval induction at the level or upstream of the EGF receptor. Second, I examined the environmental sensitivity of the Caenorhabditis vulval developmental system from an evolutionary perspective through comparative analysis of different C. elegans and C. briggsae isolates. I found that extreme temperature induced diverse developmental variants and defects, which were strongly genotype- and species-dependent. The occurrence of certain developmental defects induced by temperature extremes further revealed that vulval precursor cells and associated fates differ in temperature sensitivity, and this cell-specific sensitivity shows evolutionary variation.

Les tumeurs cortico-surrénaliennes bénignes et malignes sont associées à une morbidité élevée résultant de l’hypersécrétion des hormones cortico-surrénaliennes, retrouvée chez près de 60% des patients. Au delà des perturbations endocrines, les carcinomes cortico-surrénaliens (CCS) sont des tumeurs de mauvais pronostic avec 16 à 38% de survie à 5 ans. Cette agressivité résulte à la fois de la présence de métastases chez de nombreux patients, au moment du diagnostic (30 à 40% des cas) et de l’absence d’approches thérapeutiques, au delà de la résection chirurgicale de la tumeur primaire. Au début de ma thèse, les mécanismes moléculaires impliqués dans le développement des tumeurs bénignes et malignes de la cortico-surrénale, étaient largement méconnus. L’activation anormale de la voie de signalisation Wnt/ßcaténine dans 48% des tumeurs bénignes et 37% des tumeurs malignes, suggérait que cette voie pouvait, comme dans d’autres tissus, participer au développement tumoral dans la cortico-surrénale. Afin de confirmer cette hypothèse, nous avons développé et caractérisé un modèle de souris transgéniques dans lesquelles la ßcaténine est constitutivement activée, spécifiquement dans le cortex surrénalien (souris ∆Cat). Grâce à ces souris, nous avons démontré pour la première fois que la ßcaténine agit comme un oncogène dans la cortico-surrénale, mais que son activation constitutive ne suffit pas à déclencher systématiquement le développement de tumeurs malignes. Chez plus de 90% des patients, la formation des CCS est associée à la surexpression du facteur de croissance IGF2. Grâce à des modèles de souris transgéniques qui surexpriment Igf2, nous avons pu montrer que cette surexpression n’a que peu d’effet sur l’initiation ou la progression tumorale, suggérant que d’autres altérations sont requises pour favoriser la transition maligne. Des résultats préliminaires encourageants suggèrent que la surexpression de l’histone méthyl-transférase EZH2 et les altérations épigénétiques résultantes, pourraient être la clé du développement des CCS. Parallèlement, nous avons montré que l’activation constitutive de la ßcaténine conduit au développement d’un hyperaldostéronisme primaire chez les souris ∆Cat, suggérant que l’activation de la voie Wnt/ßcaténine pourrait participer à la formation d’adénomes surrénaliens producteurs d’aldostérone (APA) chez les patients. Effectivement, nous avons mis en évidence que l’activation constitutive de la ßcaténine est l’altération moléculaire la plus fréquente dans les APA, avec une prévalence de 68%. Des analyses in vitro m’ont permis de montrer que la ßcaténine stimule la production d’aldostérone en contrôlant directement et indirectement l’expression de deux enzymes clés de la synthèse d’aldostérone – CYP21 et CYP11B2 – et du récepteur à l’angiotensine II (le sécrétagogue naturel de l’aldostérone), AT1R. Nous avons par ailleurs montré que la production excessive d’aldostérone chez les souris ∆Cat, pouvait être maîtrisée par un régime enrichi en quercétine, un inhibiteur naturel de l’activité transcriptionnelle de la ßcaténine. L’ensemble de ces résultats démontre l’importance de la voie Wnt/ßcaténine dans la tumorigenèse surrénalienne et dans l’hypersécrétion d’aldostérone ce qui fait d’elle une nouvelle cible thérapeutique potentielle. / Benign and malignant adrenocortical tumours are associated with a high morbidity caused by the hypersecretion of adrenocortical hormones found in approximately 60% of patients. Moreover, adrenocortical carcinomas (ACC) have poor prognosis with a 5 years survival rate of 16 to 38%. This aggressiveness results from both the presence of metastases at diagnosis in most patients (30 to 40% of cases) and the absence of therapeutic approaches apart from surgical resection of primary tumours. At the start of my thesis, the molecular mechanisms involved in the development of benign and malignant adrenocortical tumours were largely unknown. Abnormal activation of the Wnt/ßcatenin pathway found in 48% of benign tumours and 37% of malignant tumours suggests that as in other tissues, this pathway could participate in tumour development in the adrenal cortex. To confirm this hypothesis, we developed and characterized a transgenic mouse model with constitutive activation of ßcatenin, specifically in the adrenal cortex (∆Cat mice). With this model, we demonstrated for the first time that ßcatenin acted as an adrenocortical oncogene but that this activation was insufficient to systematically induce the development of adrenocortical carcinomas. In almost 90% of patients, CCS formation is associated with the overexpression of the growth factor IGF2. However, the development of a model of Igf2 overexpression in transgenic mice, allowed us to demonstrate that this overexpression could not initiate tumour formation and that it had a mild effect on tumour progression. This suggested that other alterations were necessary for malignant progression. Our encouraging preliminary results suggest that upregulation of the histone methyltransferase EZH2 and the resulting epigenetic defects could be the cause of ACC development. In parallel, we demonstrated that constitutive ßcatenin activation induced primary hyperaldosteronism development in ∆Cat mice suggesting that aberrant activation of the Wnt pathway could be involved in formation of aldosterone-producing adenomas (APA) in patients. Indeed, we showed that constitutive activation of ßcatenin was the most frequent molecular alteration in APA with a prevalence of 68%. In vitro analysis allowed us to demonstrate that ßcatenin stimulates aldosterone production by controlling directly and indirectly the expression of two key enzymes of aldosterone synthesis –CYP21 and CYP11B2- and of the angiotensin II receptor, AT1R. Furthermore, we showed that excessive aldosterone production in ∆Cat mice could be controlled by a diet enriched in quercetin, a natural inhibitor of the transcriptional activity of ßcatenin. Altogether these results demonstrate the essential role of the Wnt/ßcatenin pathway in adrenocortical tumorigenesis and aldosterone secretion. Consequently, this pathway could be a new potential therapeutic target for the treatment of most adrenal tumours.

INTRODUÇÃO: A molécula HIG2 pode atuar como agonista da via Wnt/beta-catenina, pois se liga ao receptor Frizzled 10 e induz a expressão de genes da mesma. Dados recentes do nosso grupo mostraram expressão diferencial do gene HIG2 em células mononucleares do sangue periférico e em especial linfócitos T CD4+ naïve, mas não em células diferenciadas de memória em indivíduos sadios. Também observamos in vitro em linfócitos T CD4+ de indivíduos saudáveis que o peptídeo sintético HIG2 induziu a ativação da via Wnt/beta-catenina, produção de HIG2 e outros produtos da via, além da proliferação de células T CD4+ naïve sugerindo um papel do HIG2 na proliferação homeostática de linfócitos T CD4+. HIPÓTESE: Como as células T CD4+ naïve são diretamente exportadas pelo timo, os níveis aumentados de HIG2 neste tipo celular sejam decorrentes da ativação da via Wnt/?-catenina nos estágios tardios da diferenciação de timócitos. Portanto, as células T CD4+ naïve e timócitos simples positivos para CD4 (SP CD4) apresentariam perfil semelhante de expressão de HIG2 e genes da via Wnt/beta-catenina, incluindo receptores, fatores de transcrição, genes estruturais da via e alvos quando comparadas as demais populações celulares. OBJETIVO: Avaliar a expressão de HIG2 e outros genes da via Wnt/beta-catenina em timócitos e linfócitos T CD4+ naïve e memória de camundongos. MÉTODOS: Isolamos timócitos duplo negativos (DN), timócitos duplo positivos (DP), simples positivos para CD4 e CD8 (SP CD4 e SP CD8) de timo e também células T CD4+ naïve e memória do baço dos mesmos camundongos pelo procedimento de citometria de fluxo. Analisamos a expressão de vários genes da via Wnt/beta-catenina por PCR em tempo real. RESULTADOS: Em timócitos DN há expressão significativa dos genes que codificam para Frizzled 6, LRP5, TCF-1 e TCF-4 em relação as outras populações celulares. Nos timócitos DP há maior expressão dos genes que codificam para LRP5, LRP6, beta-catenina, GSK-3beta, TCF-1 e Bcl-XL em relação às demais populações. Em timócitos SP CD4 foi detectada expressão diferencial de genes que codificam para Frizzled 10, LRP6, beta-catenina, LEF-1 e HIG2 enquanto que na população de timócitos SP CD8 não observamos expressão significativa de nenhum gene da via Wnt/beta-catenina. Nas células T CD4+ naïve há expressão significativa de Frizzled 5 e Frizzled 10 quando comparadas a timócitos SP CD8 e células T CD4+ de memória . Já nos linfócitos T CD4+ de memória, detectamos maior expressão de Frizzled 6, TCF-4, Bcl-XL e ciclina D1 em relação as demais populações. CONCLUSÃO: Cada população apresenta um perfil distinto de expressão gênica. As maiores semelhanças ocorrem entre os timócitos DN e DP onde as principais diferenças são a expressão de Frizzled 6 e Ciclina D1.Os timócitos SP CD4 e as células T CD4+ naïve não apresentaram níveis semelhantes de expressão gênica de elementos da via Wnt canônica, o que não corrobora a hipótese de que o perfil transcripcional de timócitos SP CD4 e linfócitos T CD4+ naïve é semelhante. Ainda, não observamos expressão aumentada de HIG2 em linfócitos T CD4+ naïve comparados aos de memória, o que contrasta com os resultados obtidos anteriormente por nosso grupo com amostras humanas sugerindo que camundongos não regulam a expressão de HIG2 em linfócitos T CD4+ como os seres humanos / INTRODUCTION: HIG2 molecule can act as an agonist of Wnt/?-catenin pathway, because it able to bind to Frizzled 10 receptor and induce the expression of the genes related to this pathway. Recent data from our group have shown differential expression of the HIG2 gene in peripheral blood mononuclear cells, and particularly in naive CD4 + T cells, but not in memory T cells in healthy individuals. We have also observed that inducing the CD4 + T lymphocytes from healthy individuals with HIG2 synthetic peptide in vitro, led to the activation of Wnt/beta-catenin pathway, HIG2 production and expression of other target genes of this pathway and the proliferation of naïve CD4 + T cells, suggesting that HIG2 may play a role in homeostatic proliferation of CD4+ T cells. HYPOTHESIS: As naïve CD4 + T cells are directly exported from the thymus, we have hypothesized that increased levels of HIG2 in this cell type is due to the activation of Wnt/beta-catenin pathway in the later stages of thymocyte differentiation. Therefore, naïve CD4 + T cells and CD4 single-positive thymocytes (CD4 SP) may share a similar pattern of gene expression of HIG2 and Wnt/beta-catenin genes (genes that encodes receptors and co-receptors, transcription factors, structural and target genes) when compared to other cell populations. AIM: our major aim is to evaluate the expression of HIG2 and other genes of the Wnt/beta-catenin in thymocytes, naïve CD4 + T lymphocytes and memory CD4+ T cells from mice. METHODS: We have isolated thymocytes double negative (DN) T cells, positive double positive (DP) T cells, CD4 and CD8 single-positive thymocytes (CD4 SP and CD8 SP) of thymus from BALB/c mice and we have also isolated naïve CD4 + T cells and memory CD4+ T cells of the spleen from the same mice we have used the thymus. We have analysed the expression of several genes of Wnt/beta-catenin by real time PCR RESULTS: In DN cells there was expression of the Frizzled 6, LRP5, TCF-1 and TCF-4 genes compared to other cell populations. In DP thymocytes it could be observed a greater expression of LRP5, LRP6, beta-catenin, GSK-3beta, TCF-1 and Bcl-XL genes compared to other populations. In CD4 SP thymocytes, it was detected differential expression of the Frizzled 10, LRP6, beta-catenin, LEF-1 HIG2 genes and in CD8 SP cells we could not observe significant expression of any gene of Wnt/?-catenin pathway. In naïve CD4 + T cells there was a significant expression of Frizzled5 and Frizzled 10 genes when compared to all the samples. In memory CD4 + T cells, we have detected higher expression of Frizzled 6, TCF-4, Bcl-XL and cyclin D1 genes than in any other populations. CONCLUSION: Each population has a distinct gene expression pattern. The biggest similarities occur between DN and DP thymocytes where the main differences are the expression of Frizzled 6 and cyclin D1.However, the pattern of gene expression in SP thymocytes is not similar to those presented by naïve CD4+ T cells. Moreover, we have not observed increased expression of HIG2 in naïve CD4 + lymphocytes compared to memory CD4+ T cells, which contrasts the results obtained previously by our group with human samples suggesting that mice might not regulate the HIG2 expression in CD4 + T lymphocytes as human beings do

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-09-19T13:13:44Z No. of bitstreams: 2 Tese - Kelly Deyse Segati - 2017.pdf: 1820249 bytes, checksum: 36ea5524af9118dfeefed599a8c8ef16 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-09-19T13:14:09Z (GMT) No. of bitstreams: 2 Tese - Kelly Deyse Segati - 2017.pdf: 1820249 bytes, checksum: 36ea5524af9118dfeefed599a8c8ef16 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-09-19T13:14:09Z (GMT). No. of bitstreams: 2 Tese - Kelly Deyse Segati - 2017.pdf: 1820249 bytes, checksum: 36ea5524af9118dfeefed599a8c8ef16 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-08-08 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Cervical cancer is caused by persistent high-risk HPV infection. In addition, genetic and epigenetic changes such as the silencing of Wnt signaling pathway inhibitor genes appear to be essential for the development and progression of the disease. This study aimed to analyze the prevalence of HPV infections in cervical cancer and to verify the associations between age, histological type, degree of tumor differentiation and the presence of methylation DKK3, SOX17 and SFRP2. This is a cross-sectional study including cases of cervical cancer, distributed in diagnoses of squamous cell carcinomas and adenocarcinomas. The samples were assayed for 25 HPV genotypes using the INNOLipa® kit, then performed M-PCR to identify the presence of methylation in the promoter region of the genes DKK3, SOX17 and SFRP2. The results of the research showed that the age is significantly lower for women with cervical adenocarcinomas compared to those with a diagnosis of squamous cell carcinoma. Infections with genotypes 18 and 45 were associated with the diagnosis of adenocarcinomas in women younger than 50 years. Methylation of inhibitors of the Wnt signaling pathway and HPV infections 16, 18 and 45 are frequent events during multistage carcinogenesis, however, only a significant association with SFRP2 methylation was observed. The methylation of gene promoter SOX17 was related to lower cervical cancer severity but not to HPV types. Adenocarcinomas were significantlyassociated with HPV infections 16, 18 and 45, and demonstrated a borderline association with DKK3 and SOX17 methylation. In summary, the results of the present study contribute to a better understanding of carcinogenesis of the cervix in the Center-West of Brazil. / O câncer de colo uterino é causado pela infecção persistente por HPV de alto risco oncogênico. Em adição, as alterações genéticas e epigenéticas como o silenciamento dos genes inibidoresda via de sinalização Wnt parecem ser essenciais para o desenvolvimento e progressão da doença. Esse estudo teve como objetivo analisar a prevalência de infecções por genótipos específicos de HPV em câncer de colo uterino e verificar as associações entre a idade, tipo histológico, o grau de diferenciação tumoral e a presença da metilação na região promotora dos genes DKK3, SOX17 e SFRP2. Trata-se de um estudo de corte transversal incluindo casos de câncer de colo uterino, distribuídos em diagnósticos de carcinomas de células escamosas e adenocarcinomas. As amostras foram testadas para 25 genótipos de HPV utilizando o kit INNOLipa®, em seguida foram submetidas a M-PCR para identificar a presença da metilação na região promotora dos genes DKK3, SOX17 e SFRP2. Os resultados da pesquisa mostraram que a idade é significativamente menor em mulheres com adenocarcinomas cervicais comparadas com aquelas com diagnóstico de carcinoma de células escamosas. A infecção por genótipos 18 e 45 foram positivamente associadas ao diagnóstico de adenocarcinomas em mulheres com idade menor que 50 anos. A metilação dos inibidores da via de sinalização Wnt e as infecções por HPV 16, 18 e 45 são eventos frequentes durante a carcinogênese em várias etapas, no entanto, apenas foi observada associação significativa com a metilação de SFRP2. A metilação da região promotora de SOX17 foi relacionada com menor gravidade do câncer de colo uterino, mas não com tipos de HPV. Os adenocarcinomas apresentaram associação significativa com infecções por HPV 16, 18 e 45, além de demonstrar uma associação limítrofe com a metilação de DKK3 e SOX17. Em resumo, resultados do presente estudo contribuem para o melhor entendimento da carcinogêneses do colo uterino na região Centro-Oeste do Brasil.

Orientador: Lúcia Elvira Alvares / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T17:37:00Z (GMT). No. of bitstreams: 1 Pedrosa_AngelicaVasconcelos_M.pdf: 1992620 bytes, checksum: ed7d02568860e3ccf1e489cf2928818e (MD5) Previous issue date: 2013 / Resumo: A correta padronização do corpo do embrião requer a atividade de diferentes vias de sinalização. Dentre elas, uma que se destaca é via de sinalização Wnt de polaridade celular planar (Wnt/PCP), que é responsável pelo controle da polaridade celular e pela organização celular de diversos tecidos nos animais. Uma vez interrompida, a via Wnt/PCP pode causar falhas no fechamento do tubo neural, provocando defeitos congênitos. Em seres humanos, mutações em componentes-chave da via Wnt/PCP como as proteínas codificadas pelos genes Vangl1 e Vangl2 têm sido associadas à graves malformações geradas por falhas no fechamento do tubo neural. Estruturalmente, ambos os genes Vangl1 e Vangl2 codificam proteínas de superfície transmembranares, essenciais para o desenvolvimento apropriado do embrião. O presente trabalho teve como objetivo a caracterização do padrão de expressão dos genes Vangl1 e Vangl2 durante a embriogênese de Gallus gallus. Ensaios de hibridação in situ em embrião inteiro (whole mount) e cortes em vibratómo foram realizados com a finalidade de estabelecer temporal e espacialmente o padrão de expressão dos genes Vangl1 e Vangl2. Como resultado, observou-se que estes genes são expressos durante as etapas de gastrulação, neurulação e no início da organogênese do desenvolvimento embrionário de Gallus gallus. No início da gastrulação, os genes Vangl1 e Vangl2 possuem domínios de expressão comuns nos embriões de galinha, uma vez que ambos são expressos na linha primitiva, nódulo de Hensen e crescente cardiogênico. Contudo, nossos dados revelaram particularidades na expressão destes genes, uma vez que há uma predominância dos transcritos de Vangl1 na região posterior da linha primitiva, enquanto Vangl2 apresenta uma expressão uniforme ao longo desta estrutura. Em adição, enquanto Vangl1 é expresso na notocorda e em toda a extensão do nódulo de Hensen, Vangl2 é expresso no entorno desta estrutura. Ao longo da neurulação e na organogênese inicial, ambos os genes Vangl são expressos de maneira similar, em domínios que abrangem a placa, as pregas e o tubo neural. Outros importantes domínios de expressão dos Vangl correspondem às vesículas ópticas e óticas, às vesículas encefálicas particularmente na região das flexuras encefálicas, aos diferentes tipos de mesoderma (paraxial, intermediário e lateral) e ao assoalho da faringe. Ao comparar os resultados obtidos por hibridação in situ em galinha ao um levantamento bibliográfico sobre outros vertebrados, observou-se uma sobreposição dos domínios-chave de expressão nos diferentes organismos, demonstrando a conservação filogenética da atividade destes genes e sugerindo uma possível conservação funcional. Desta forma, nossos dados sugerem que os genes Vangl desempenham um importante papel no desenvolvimento embrionário de aves, possivelmente coordenando os movimentos morfogenéticos durante a gastrulação, bem como a formação da placa neural e posterior dobramento e fechamento do tubo neural, além de outros processos da embriogênese de aves / Abstract: The correct patterning of the embryo's body requires the activity of different signaling pathways. Among them, one that stands out is the Wnt Planar Cell Polarity Signaling Pathway (Wnt/PCP), which is responsible for controlling the cell polarity and cellular organization of many tissues in animals. Failures in the Wnt/PCP signaling can cause neural tube birth defects. In humans, mutations in key components of the Wnt/PCP as the Vangl1 and Vangl2 molecules were identified in patients with neural tube defects. Structurally, both Vangl1 and Vangl2 genes encode transmembrane surface proteins similar, which are essential to proper development. The present study aimed to characterize the expression pattern of Vangl1 and Vangl2 genes during embryogenesis in Gallus gallus. Whole-mount in situ hybridization assays and vibratome sectioning of embryos were conducted in order to establish the spatial and temporal expression pattern of Vangl1 and Vangl2 genes. Our results showed that these genes are expressed during gastrulation, neurulation and early organogenesis in Gallus gallus. At the onset of Gastrulation, Vangl1 and Vangl2 genes have common areas of expression in chicken embryos, since both are expressed in the primitive streak, Hensen's node and cardiogenic crescent. However, our data showed particularities in the expression of these genes, since there is a predominance of Vangl1 transcripts in the posterior region of the primitive streak while Vangl2 has a uniform expression throughout that structure. In addition, while Vangl1 is expressed in the notochord and in the full length of the Hensen's node, Vangl2 is expressed only around this structure. Throughout neurulation and early organogenesis, both Vangl genes are expressed in a similar manner on the neural plate, neural groove, neural folds and in the neural tube. Other important areas of Vangl expression correspond to optical and otic vesicles, the brain vesicles, the different types of mesoderm (paraxial, intermediate and lateral) and the floor of the pharynx. By comparing the chicken expression of Vangl genes with other vertebrates, we notice that there are overlapping expression patterns among key areas among different organisms, showing a phylogenetic conservation of expression domains and suggesting a possible functional conservation. Overall, our data suggests that Vangl genes play an important role in embryonic development of bird, possibly by coordinating the morphogenetic movements during gastrulation, as well as the formation of neural tube, among other processes during the birds embriogenesis / Mestrado / Biologia Celular / Mestra em Biologia Celular e Estrutural

Orientadores: Lúcia Elvira Alvares, José Xavier Neto / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T10:52:38Z (GMT). No. of bitstreams: 1 Sobreira_DeboraRodrigues_D.pdf: 9430834 bytes, checksum: 1c730ee238256c4ea0f303311fa3283b (MD5) Previous issue date: 2013 / Resumo: Os genes da família Dapper (Dpr) codificam proteínas adaptadoras capazes de ligar-se fisicamente a diferentes moléculas e modular as vias de sinalização Wnt e TGF-?. Diferentes análises funcionais revelaram que os Dpr atuam na especificação do eixo corporal e do tecido neural, nos movimentos morfogenéticos, no desenvolvimento do olho, na indução da cardiogênese, adipogênse e cicatrização. Diversos estudos foram realizados a fim de compreender o papel desempenhado pelos Dpr durante a embriogenêse dos vertebrados e na homeostase de tecidos adultos. Contudo, muitas questões ainda necessitam ser elucidadas. Este projeto de Doutorado teve como objetivo (1) descrever os sítios de expressão da família gênica Dpr durante a ontogênese dos membros de galinha, associando-a as vias de sinalização Wnt e TGF-? e (2) investigar a origem e a evolução desses genes durante a filogenia dos metazoários. Nossos resultados confirmaram que os genes Dpr são dinamicamente expressos durante o desenvolvimento dos membros de galinha, provavelmente, modulando os sinais Wnt e TGF-?. Os genes Dpr são encontrados no mesênquima indiferenciado dos membros em formação e em células progenitoras de condrócitos, pericôndrio e tendões. Esses resultados sugerem as moléculas Dpr como um novo grupo de marcadores do desenvolvimento dos membros em galinha. Já nossas análises filogenéticas revelaram que os Dprs surgiram durante a evolução dos organismos deuterostômios e um novo ortólogo dessa família de proteínas, denominado Dpr4, foi descrito. Acreditamos que o nosso trabalho irá fornecer bases para estudos moleculares com o intuito de estabelecer a função individual de cada membro da família Dpr, bem como auxiliar no entendimento sobre como estas proteínas podem interagir e cooperar entre si para modular diferentes vias de sinalização molecular em diferentes contextos celulares / Abstract: The Dapper (Dpr) genes form a small gene family of adaptor proteins important to several processes of vertebrates development, such as the specification of the body axis and neural tissue, morphogenetic movements, eye development, induction of cardiogenesis, adipogenesis and wound healing, by modulating the Wnt and TGF-? signaling pathways using specific conserved domains/motifs. Three Dpr genes have been identified in human and mouse, two in chicken, one in frog and two in zebrafish genome. Since the discovery of Dpr proteins, several assays have been performed in order to understand the role of this family during embryogenesis, although many questions still need to be elucidated. Thus, this PhD project aimed to (1) describe the possible role of Dpr genes during ontogeny of chicken regarding the regulation of Wnt and TGF-? signaling pathways and (2) investigate the origin and evolution of Dpr family over the course of metazoan evolution. Our results demonstrated that Dpr genes are involved in chicken limb development, probably, by modulating Wnt and TGF-? signals. Dpr genes were found in the undifferentiated limb mesenchyme, progenitor of chondrocytes, perichondrium and tendons. These results suggest that Dpr genes are good candidates to a new set of markers in chicken limb development. Furthermore, our phylogenetic analysis revealed that the Dprs arose late during the deuterostomes evolution and allowed the identification of a new Dpr paralog (Dpr4), meaning that a repertoire of four Dact genes is found in vertebrates. Thus, our work will provide the basis for molecular studies in order to establish the role of each individual member of this family as well as how the set of Dpr proteins can interact and cooperate to modulate different molecular signaling pathways in different cellular contexts / Doutorado / Biologia Tecidual / Doutora em Biologia Celular e Estrutural

Le carcinome hépatocellulaire (CHC) présente des mutations génétiques qui altèrent les principales voies de signalisation, notamment la voie Wnt/β-caténine. En absence de mutation génétique, certains CHC peuvent montrer une activité Wnt exacerbée suite à une inactivation épigénétique d’inhibiteurs ou à une surexpression de ligands Wnts ou de ses récepteurs. De plus, le remodelage de la matrice extracellulaire favorise la progression du CHC. Nous avons montré une association entre l’activation du signal Wnt et le remodelage de la matrice extracellulaire (MEC) dans les cirrhoses et le CHC. Puis modélisé in vitro, les effets des stimuli Wnt extracellulaires sur le phénotype de cellules hépatiques, en absence de mutation de la β-caténine. En effet, les cellules HepaRG ne présentent pas de mutations de la β-caténine, de l’axine et de p53. Ainsi, la stimulation Wnt3a des cellules HepaRG induisait la formation de palissades de cellules fusiformes. De plus, les cellules traitées exprimaient des taux élevés de αSMA, COLIV, c-MYC, CK19 et LGR5 suggérant un phénotype myofibroblastique, en accord avec l’expression des marqueurs de transition épithélio-mésenchymateuse (TEM), SNAIL et TWIST. Ces données sont en faveur du rôle déterminant du microenvironnement Wnt activé dans la progression du CHC entrainant les cellules vers un phénotype progéniteur plus agressif via une TEM. En outre, l'analyse in silico de la signature transcriptomique de l'activation non mutationnelle de la voie Wnt a révélé un réseau de gènes impliqués dans le remodelage de la MEC, la TEM et la différenciation cellulaire. Les résultats suggèrent le rôle de HAPLN1 qui affecterait la migration cellulaire et l'expression des gènes de la MEC. De plus, LGR5 semble favoriser la dédifférenciation des hépatocytes. Au total, 8 gènes marqueurs obtenus in vitro ont été validés in vivo dans une série de 81 CHC humains, par qPCR et immunohistochimie en utilisant des tissus micro-array (78 CHC et 5 foies contrôles). Au total, l'ensemble des données suggère que HAPLN1 a une valeur pronostique sur la récidive et la survie globale du CHC. HAPLN1semble être indépendant du statut mutationnel la β-caténine et des variables cliniques. De plus, sa valeur pronostique est additive avec celle de CK19 + EpCAM et il semble agir en synergie avec NOG. / Hepatocellular carcinoma (HCC) displays signaling pathway disorders, including Wnt/β-catenin. Up-regulation of extracellular Wnt pathway agonists and down-regulation of extracellular Wnt pathway inhibitors result in non-mutational activation of Wnt signaling. In addition, increased extracellular matrix remodeling fosters HCC progression. Thus, we showed that enhanced Wnt signaling is associated with extracellular matrix remodeling in human cirrhosis and cancer. To further investigate non-mutational Wnt pathway activation, we established a model of Wnt activation in HepaRG human HCC progenitor cells carrying wild-type β-catenin, axin and p53. HepaRG progenitor cells treated with Wnt3a became fusiform and grew in palisades with enhanced expression of αSMA, COLIV, CK19, c-MYC, LGR5, SNAIL and TWIST, suggesting that enhanced extracellular Wnt signaling may drive HCC cells toward a more aggressive progenitor and epithelial mesenchymal transition (EMT) phenotype. Moreover, in silico analysis of the transcriptomic signature of non-mutational Wnt activation revealed a gene network involved in ECM remodeling, EMT and cell fate. Results suggest a role of HAPLN1, affecting extracellular matrix gene expression and cell migration and of LGR5 in hepatocyte dedifferentiation. Eight genes among the HepaRG gene expression dataset were validated in vivo in a collection of 81 human HCC samples and controls by qPCR and immunohistochemistry using tissue micro-arrays (78 HCC samples and 5 normal livers) in the light of β-catenin activation and mutational status. In conclusion, data suggest that HAPLN1 has a prognostic value on overall survival and recurrence of HCC. HAPLN1 appears to be independent of clinical features and β-catenin mutationnal status. Moreover, HAPLN1 appears to have an additive prognostic value with CK19 + EpCAM and act synergistically with NOG.

Le but de ce travail était d’accroitre notre connaissance des mécanismes qui gouvernent la formation, la spécification et la différentiation des cellules souches, à l’aide du modèle métazoaire non-bilatérien Clytia hemisphaerica. Clytia possède une population particulière de cellules multipotentes appelées cellules interstitielles (i-cells). Ces cellules, présentes au cours du développement larvaire et chez la méduse adulte, sont capables de donner naissances a des cellules somatiques et aux gamètes. Chez les bilatériens, la signalisation Wnt/β-caténine (Wntβc) régule les processus développementaux fondamentaux ainsi que la prolifération, la spécification et la différenciation des cellules souches. Mon travail s’est porté sur l’implication de la signalisation Wntβc dans les dynamiques des i-cells. Mes résultats suggèrent que la signalisation Wntβc est impliquée dans la dernière étape de la différenciation de certains neurones, mais pas pour la spécification du destin cellulaire. D’autre part, j’ai aussi observé qu’en condition contrôle, la formation des i-cells est Wntβc-indépendante et probablement entraînée par le héritage de plasma germinatif contenant les ARNm localisées au pôle animal. Cependant, suite à des expériences de microdissection, j’ai observe que la reformation des i-cells dans la moitie végétative des embryons requérait l’activation de la voie Wntβc. En conclusion, deux mécanismes distincts peuvent conduire à la formation des i-cell pendant l'embryogenèse. Globalement, les résultats obtenus ont fourni une meilleure idée de la façon dont i- cellules et leurs dérivés se posent lors de l'embryogenèse et le développement larvaire. / The aim of this work was to extend our understanding of the mechanisms regulating stem cell formation, specification and differentiation by studies in the non-bilaterian metazoan model Clytia hemisphaerica. Clytia, like other hydrozoan cnidarians, possess a particular population of multipotent stem cells called interstitial cells (i-cells), present during larval development and in the adult medusa, which are able to give rise both to somatic cell types and to gametes.In bilaterian animals Wnt/β-catenin signalling regulates fundamental developmental processes such primary body axis specification, but also regulates stem cell proliferation, lineage specification and differentiation. I investigated the role of Wnt/β-catenin signalling in i-cell specification and differentiation. The results obtained suggest that Wnt/β-catenin signalling is involved in the last step of differentiation for certain neuronal cell types, but not for somatic cell fate choice. In the second part of my study I investigated the role of Wnt/β-catenin signalling in i-cell formation during embryogenesis. The results indicated that during normal development i-cell formation is Wnt/β-catenin independent and probably driven by inheritance of germ plasm containing localised mRNAs from the egg animal pole. In contrast in embryo re-patterning following embryo bisection, Wnt/β-catenin signalling appears to be necessary for de novo i-cell formation in the absence of germ plasm. Thus two distinct mechanisms can lead to i-cell formation during embryogenesis. Overall the results obtained provided a better picture of how i-cells and their derivatives arise during embryogenesis and larval development.