A leucoplasia é a mais comum das lesões potencialmente malignas (LPM) da mucosa bucal acometendo cerca de 2% da população. A via PI3K- AKT-mTOR, tem papel importante na carcinogênese em diversos tumores, incluindo o câncer de cabeça e pescoço e estudos têm apontado os inibidores do mTOR como promissores agentes de avaliação terapêutica. Desta forma, no presente estudo avaliamos a expressão imuno-histoquímica de lesões bucais diagnosticadas clinicamente como leucoplasias, comparando-as com carcinomas espinocelulares (CEC), hiperqueratoses irritativas (HI) e mucosa normal (MN). Foram avaliados 186 casos, divididos em 5 grupos tais como displasia epitelial de alto risco (DAR), displasia epitelial de baixo risco (DBR), CEC, HI e MN. Os casos foram retirados do arquivo do Serviço de Patologia Cirúrgica da Disciplina de Patologia Bucal da Faculdade de Odontologia da Universidade de São Paulo. As informações clínicas, relativas ao sexo e idade dos pacientes e localização das lesões foram compiladas. Todas as lesões selecionadas foram observadas ao HE por dois patologistas, ao microscópio de luz para confirmação dos diagnósticos. As proteínas pesquisadas incluiram: pAKT, pmTOR, pS6, e p4EBP1. A marcação dos diversos anticorpos foi quantificada com o auxílio da aquisição de imagens realizada com o uso de um fotomicroscópio. Na imagem capturada (aumento de 400x), foi observada a presença ou ausência de células marcadas, bem como, numa segunda análise, a quantidade percentual das mesmas. Na primeira análise, o padrão de marcação foi classificado em positivo e negativo e, na segunda, foi classificado em graus de 0 a 3. As variáveis do estudo foram avaliadas pelos testes Qui-quadrado e o teste F, ANOVA e posteriormente foi realizada uma regressão logística univariada. Entre todos os casos de mucosa oral normal, foi encontrada positividade somente para o anticorpo pS6, em 50%dos casos; nas HI houve marcações positivas para a pS6 (em 54,8% dos casos) e p4EBP1 (em 22,6% dos casos). Nas DBR foi observada a imunorreatividade aos anticorpos pS6 (em 67,4% dos casos), pAKT(em 56,2% dos casos), p4EBP1(em 41,7%dos casos) e pmTOR (em 29,2% dos casos), já nas DAR houve positividade para a pS6 (em 74% dos casos), pAKT (em 68% dos casos), p4EBP1 (em 44% dos casos) e pmTOR (em 28% dos casos). Os CECs expressaram pAKT ( em 83,3% dos casos), pS6 (em 77,4% dos casos), p4EBP1(em 50% dos casos) e pmTOR (em 50%dos casos). Quando se considerou o resultado da marcação positiva ou negativa, houve diferença estatisticamente significante, ente os grupos, em relação ao número de casos que expressaram as proteínas pAKT, pmTOR e p4EBP1, sendo que o grupo dos CECS foi o que apresentou maior frequência de imunorreatividade para todos os anticorpos estudados. Comparando-se apenas as lesões de CEC e DEO, observou-se que mais casos de CEC foram positivos para pAKT e pmTOR e não observou-se diferença na expressão do p4EBP1. Logo, podemos concluir que todas as proteínas estudadas, exceto a pS6, representam bons biomarcadores no que se concerne à diferenciação entre MN, HI, DEO e CEC. Entretanto somente as proteínas pAKT e pmTOR podem ser relacionadas à carcinogênese oral . / Leukoplakia is the most common potentially malignant lesion (PML) of the oral cavity affecting approximately 2% of the population. PI3K-AKT-mTOR pathway plays an important role in carcinogenesis in many tumors, including head and neck cancer, and several studies have showed mTOR inhibitors as promising therapeutic agents. In this study we evaluated the immunohistochemical expression of oral lesions diagnosed clinically as leukoplakia and squamous cell carcinoma (SCC), comparing them to normal mucosa (NM) and frictional hyperkeratosis (FR). We evaluated 186 cases, divided into 5 groups including high risk dysplasia (HRD), low risk dysplasia (LRD), SCC, NM and FR. The cases were obtained from the archives of the Oral Pathology Laboratory of the School of Dentistry of the University of São Paulo. Clinical information regarding sex, age of the patient and location of the lesions were compiled. The slides (HE staining) were observed by two pathologists and all cases were re-evaluated under light microscope. The proteins investigated were: pAKT, pmTOR, PS6, and p4EBP1. The staining pattern of the antibodies was quantified by acquiring images using a photomicroscope. In the captured image (400x magnification), the total of counted labeled cells, were divided by the total number of cells present in the field captured. The staining pattern was classified into positive and negative and also divided into degrees starting from 0 to 3. The study variables were evaluated by chi-square test, ANOVA F and univariate logistic regression analysis. Among all the cases of MN, positivity was found only for pS6 (50% of cases); in FH cases immunoreactivity was observed in pS6 (54.8% of cases) and 4EBP1 (22.6% of cases). In LRD immunoreactivity was observed in pS6 (67.4% of cases), pAKT (56.2% of cases), p4EBP1 (41.7% of cases) and pmTOR (29.2% of cases), while for HRD cases positivity was found for pS6 (74% of cases), pAKT (68% of cases), p4EBP1 (44% of cases) and pmTOR (28% of cases). In SCCs cases positivity was found for pAKT (83.3% of cases), PS6 (77.4% of cases), p4EBP1 (50% of cases) and pmTOR (50% of cases). Statistically significant differences were observed in all positive study groups for the proteins pAKT, pmTOR and p4EBP. After the evaluation of SCC and the oral dysplasia groups, there were statistically significant differences for the study groups that showed imunorretivity for the proteins pAKT and pmTOR. Therefore, we conclude that all the proteins of the study are good biomarkers to differentiate normal tissue from OD and SCC, but only pAKT and pmTOR proteins could be related to carcinogenesis.

mTOR (mammalian target of rapamycin) é uma proteína com papel central no crescimento, na proliferação e na manutenção das células, que participa da formação de dois complexos, mTORC1 e mTORC2. Diversos estudos associam menor atividade de mTOR, em especial o complexo 1, com efeitos protetores contra o envelhecimento e mesmo aumento da expectativa de vida máxima. Alterações no DNA têm sido propostas desde cedo na história dos estudos bioquímicos sobre o envelhecimento como um fator causar da perda de função dos organismos com a idade. Muitos estudos já foram realizados tentando analisar diversos aspectos do acúmulo de alterações no DNA e da capacidade de reparo com a idade. No entanto, a possível relação entre mTOR e reparo de DNA foi muito pouco explorada, em especial em relação ao DNA mitocondrial. Este estudo teve como objetivo avaliar o papel de mTOR na regulação dos níveis de reparo de DNA, em especial da via de reparo por excisão de bases (BER). Os resultados demonstraram que, aparentemente, mTOR surte algum efeito na regulação de duas enzimas da via BER (APE1 e Polγ), além de TFAM, diminuído os níveis das três, tanto no núcleo quanto nas mitocôndrias. No entanto, a atividade de incisão de oligonucleotídeos de APE1 não demonstrou alteração, e indução de apoptose por indução de estresse oxidativo revelou que células com menor expressão de mTOR se encontravam mais resistentes. Adicionalmente, a inibição de mTOR pareceu não alterar o número decópias de DNA mitocondrial e a massa mitocondrial, sugerindo que as células com knockdown de mTOR possuem uma maior reserva respiratória. Em conjunto, os resultados sugerem um possível envolvimento de mTOR na regulação de BER, mesmo que indiretamente, embora não estaja claro por qual via, ou por qual complexo de mTOR / mTOR (mammalian target of rapamycin) is a central protein in the regulation of cell growth, proliferation and maintenance, that participates in the formation of two complexes, mTORC1 and mTORC2. Several studies associate a lower activity of mTOR, especially complex 1, with beneficial effects against aging, and even increased maximum lifespan. DNA alterations have been proposed since the beginnings of the history of the biochemical studies on aging to be a cause of the loss of function that in observed in organisms with age. Several studies have been carried out to analyze several aspects of DNA alterations and DNA repair with age. However, the possible relationship between mTOR and DNA repair has not been explored satisfactorily, especially in relation to mitochondrial DNA. This study had the objective of evaluating the role of mTOR in the regulation of the levels of DNA repair, especially the base excision repair (BER) pathway. The results showed that, apparently, mTOR has some effect in the regulation of two enzymes of the BER pathway (APE1 and Polγ), as well as TFAM, decreasing their levels, both in the nucleus and in the mitochondria. However, APE1 oligonucleotide incision activity was not diminished, and apoptosis induction by methylene blue treatment revealed that cells with mTOR knockdown were more resistant. Addicionally, mTOR inhibition didnt seem to alter mitochondrial DNA copy number and mitochondrial mass, suggesting that mTOR knockdown cells have more respiratory reserve. Takentogether, these results suggest a possible role for mTOR in the regulation of BER, even if indirectly, although it is not clear through which pathway, or which mTOR complex

A leucoplasia é a mais comum das lesões potencialmente malignas (LPM) da mucosa bucal acometendo cerca de 2% da população. A via PI3K- AKT-mTOR, tem papel importante na carcinogênese em diversos tumores, incluindo o câncer de cabeça e pescoço e estudos têm apontado os inibidores do mTOR como promissores agentes de avaliação terapêutica. Desta forma, no presente estudo avaliamos a expressão imuno-histoquímica de lesões bucais diagnosticadas clinicamente como leucoplasias, comparando-as com carcinomas espinocelulares (CEC), hiperqueratoses irritativas (HI) e mucosa normal (MN). Foram avaliados 186 casos, divididos em 5 grupos tais como displasia epitelial de alto risco (DAR), displasia epitelial de baixo risco (DBR), CEC, HI e MN. Os casos foram retirados do arquivo do Serviço de Patologia Cirúrgica da Disciplina de Patologia Bucal da Faculdade de Odontologia da Universidade de São Paulo. As informações clínicas, relativas ao sexo e idade dos pacientes e localização das lesões foram compiladas. Todas as lesões selecionadas foram observadas ao HE por dois patologistas, ao microscópio de luz para confirmação dos diagnósticos. As proteínas pesquisadas incluiram: pAKT, pmTOR, pS6, e p4EBP1. A marcação dos diversos anticorpos foi quantificada com o auxílio da aquisição de imagens realizada com o uso de um fotomicroscópio. Na imagem capturada (aumento de 400x), foi observada a presença ou ausência de células marcadas, bem como, numa segunda análise, a quantidade percentual das mesmas. Na primeira análise, o padrão de marcação foi classificado em positivo e negativo e, na segunda, foi classificado em graus de 0 a 3. As variáveis do estudo foram avaliadas pelos testes Qui-quadrado e o teste F, ANOVA e posteriormente foi realizada uma regressão logística univariada. Entre todos os casos de mucosa oral normal, foi encontrada positividade somente para o anticorpo pS6, em 50%dos casos; nas HI houve marcações positivas para a pS6 (em 54,8% dos casos) e p4EBP1 (em 22,6% dos casos). Nas DBR foi observada a imunorreatividade aos anticorpos pS6 (em 67,4% dos casos), pAKT(em 56,2% dos casos), p4EBP1(em 41,7%dos casos) e pmTOR (em 29,2% dos casos), já nas DAR houve positividade para a pS6 (em 74% dos casos), pAKT (em 68% dos casos), p4EBP1 (em 44% dos casos) e pmTOR (em 28% dos casos). Os CECs expressaram pAKT ( em 83,3% dos casos), pS6 (em 77,4% dos casos), p4EBP1(em 50% dos casos) e pmTOR (em 50%dos casos). Quando se considerou o resultado da marcação positiva ou negativa, houve diferença estatisticamente significante, ente os grupos, em relação ao número de casos que expressaram as proteínas pAKT, pmTOR e p4EBP1, sendo que o grupo dos CECS foi o que apresentou maior frequência de imunorreatividade para todos os anticorpos estudados. Comparando-se apenas as lesões de CEC e DEO, observou-se que mais casos de CEC foram positivos para pAKT e pmTOR e não observou-se diferença na expressão do p4EBP1. Logo, podemos concluir que todas as proteínas estudadas, exceto a pS6, representam bons biomarcadores no que se concerne à diferenciação entre MN, HI, DEO e CEC. Entretanto somente as proteínas pAKT e pmTOR podem ser relacionadas à carcinogênese oral . / Leukoplakia is the most common potentially malignant lesion (PML) of the oral cavity affecting approximately 2% of the population. PI3K-AKT-mTOR pathway plays an important role in carcinogenesis in many tumors, including head and neck cancer, and several studies have showed mTOR inhibitors as promising therapeutic agents. In this study we evaluated the immunohistochemical expression of oral lesions diagnosed clinically as leukoplakia and squamous cell carcinoma (SCC), comparing them to normal mucosa (NM) and frictional hyperkeratosis (FR). We evaluated 186 cases, divided into 5 groups including high risk dysplasia (HRD), low risk dysplasia (LRD), SCC, NM and FR. The cases were obtained from the archives of the Oral Pathology Laboratory of the School of Dentistry of the University of São Paulo. Clinical information regarding sex, age of the patient and location of the lesions were compiled. The slides (HE staining) were observed by two pathologists and all cases were re-evaluated under light microscope. The proteins investigated were: pAKT, pmTOR, PS6, and p4EBP1. The staining pattern of the antibodies was quantified by acquiring images using a photomicroscope. In the captured image (400x magnification), the total of counted labeled cells, were divided by the total number of cells present in the field captured. The staining pattern was classified into positive and negative and also divided into degrees starting from 0 to 3. The study variables were evaluated by chi-square test, ANOVA F and univariate logistic regression analysis. Among all the cases of MN, positivity was found only for pS6 (50% of cases); in FH cases immunoreactivity was observed in pS6 (54.8% of cases) and 4EBP1 (22.6% of cases). In LRD immunoreactivity was observed in pS6 (67.4% of cases), pAKT (56.2% of cases), p4EBP1 (41.7% of cases) and pmTOR (29.2% of cases), while for HRD cases positivity was found for pS6 (74% of cases), pAKT (68% of cases), p4EBP1 (44% of cases) and pmTOR (28% of cases). In SCCs cases positivity was found for pAKT (83.3% of cases), PS6 (77.4% of cases), p4EBP1 (50% of cases) and pmTOR (50% of cases). Statistically significant differences were observed in all positive study groups for the proteins pAKT, pmTOR and p4EBP. After the evaluation of SCC and the oral dysplasia groups, there were statistically significant differences for the study groups that showed imunorretivity for the proteins pAKT and pmTOR. Therefore, we conclude that all the proteins of the study are good biomarkers to differentiate normal tissue from OD and SCC, but only pAKT and pmTOR proteins could be related to carcinogenesis.

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.

As tiazolidinedionas (TZDs), ligantes sintéticos dos receptores nucleares PPARγ, têm sido amplamente utilizadas no tratamento da resistência à insulina, dislipidemias e síndrome metabólica. Estas drogas melhoram a homeostase da glicose promovendo redistribuição de gordura dos estoques viscerais para o subcutâneos, aumento da secreção de adiponectina, redução da lipemia, lipotoxicidade e da inflamação do tecido adiposo. Um estudo recente mostrou que o tratamento de ratos com a TZD rosiglitazona (RSG) induz um aumento na atividade dos complexos 1 e 2 da mTOR, que desempenham função importante no controle do metabolismo lipídico, adiposidade e função endócrina do tecido adiposo. Assim, o presente estudo teve como objetivo central elucidar o envolvimento especificamente do complexo 1 da mTOR de adipócitos nas alterações morfológicas, metabólicas e secretórias do tecido adiposo branco e marrom induzidas pela ativação farmacológica de PPARγ em camundongos. Para isto, camundongos com deleção de raptor (mTORC1) exclusivamente em adipócitos alimentados com dieta hiperlipídica foram tratados ou não com RSG (30 mg/kg/dia) por 8 semanas. Nossos dados mostraram que tanto o mTORC1 quanto o agonista de PPARγ são importantes reguladores da adiposidade, onde observamos que a deleção genética de mTORC1 em adipócitos conteve o aumento de adiposidade. Além disso, RSG mostrou-se eficente em reduzir a massa dos depósitos viscerais retroperitoneal a epididimal sem alterar o depósito subcutâneo inguinal. RSG aumentou significativamente a massa do tecido adiposo marrom, efeito esse que foi completamente abolido pela deficiência do complexo 1 da mTOR. Deficiência de mTORC1 em adipócitos promoveu aumento no conteúdo de UCP1 (expressão gênica e proteica), efeito este que não foi alterado pelo tratamento com RSG. Outros efeitos de RSG mostraram-se dependentes de mTORC1 como o aumento de frequência de adipócitos de menor área, aumento dos níveis de adiponectina e redução dos níveis de BCAA séricos, além da expressão gênica de CD36 e PEPCK, lipídeos mitocondriais como a cardiolipina e fosfatidiletanolamina e mediadores lipídicos como as ceramidas de cadeia longa no tecido adiposo branco. Por outro lado encontramos efeitos de RSG independentes de mTORC1, como a redução nos níveis séricos de TAG, redução de expressão gênica de fatores inflamatórios, tais como IL1 e TNF, NLRP3, DUSP6, além de PGC1 e FAS, insulina plasmática, melhora na homeostase glicêmica. Concluímos assim que mTORC1 em adipócitos é importante mediador de ações de agonista de PPARγ. / Thiazolidinediones (TZDs), synthetic ligands of nuclear receptors PPARγ, have been widely used in the treatment of insulin resistance, dyslipidemia and metabolic syndrome. These drugs improve glucose homeostasis by promoting redistribution of fat from visceral to subcutaneous depots, increasing adiponectin secretion, reducing lipemia, lipotoxicity, and inflammation of adipose tissue. A recent study showed that the treatment of rats with TZD rosiglitazone (RSG) induces an increase in the activity of mTOR complexes 1 and 2, which play an important role in the control of lipid metabolism, adiposity and endocrine function of adipose tissue. Thus, we investigated herein the specific involvement of adipocyte mTOR complex 1 in the morphological, metabolic and secretory alterations of white and brown adipose tissue induced by pharmacological activation of PPARγ in mice. For this, mice with raptor deletion (mTORC1) exclusively in adipocytes and littermate controls were fed a hyperlipidic diet and treated or not with RSG (30 mg/ kg/ day) for 8 weeks. Our data showed that both mTORC1 and PPARγ agonist are important adiposity regulators, where we observed that the genetic deletion of mTORC1 in adipocytes prevented the increase in adiposity. In addition, RSG was effective in reducing the masses of visceral fat depots retroperitoneal and epididymal without altering the mass of the subcutaneous fat depot inguinal. RSG induced an expressive increase in brown adipose tissue mass, such, an effect that was blocked by mTOR 1 complex deficiency. mTORC1 ablation in adipocytes increased UCP1 content (gene and protein expression). Interesting, RSG lost its ability to reduce the percentage of smaller adipocytes, to increase serum levels of adiponectin and reduce those of BCAA, as well as to increase mRNA levels of CD36 and PEPCK, mitochondrial lipids such as cardiolipin and phosphatidylethanolamine, lipid mediators as long chain ceramides in white adipose tissue. Other effects of RSG such as reducing serum TAG and insulin levels, adipose tissue inflammation, such as IL1 and TNF, and improving glucose homeostasis were not affected by mTOR complex 1 deficiency. We conclude thus that mTORC1 is important mediator of some actions of PPARγ agonism.

Devant l’incidence de la suractivation de la voie PI3K/Akt/mTOR sur les cancers, nous avons choisi d’inhiber cette voie de signalisation. Etant donné la forte analogie structurale qui existe entre les enzymes PI3K et mTOR, nous avons conçu des inhibiteurs doubles ciblant deux kinases majeures de la voie. Ces inhibiteurs possèdent un noyau original pyrido[3,2-d]pyrimidinique. Afin d’apporter de la diversité fonctionnelle et d’engendrer un effet thérapeutique, les sommets C-4, C-2 et C-7 furent fonctionnalisé séquentiellement selon l’ordre suivant. Tout d’abord, la position C-4 fut fonctionnalisée par des hétérocycles par substitution nucléophile aromatique. Puis divers cycles hétéroaromatiques furent introduits en position C-2 par couplage de type Suzuki-Miyaura. Enfin, des groupements variés furent insérés en position C-7 par différentes réactions. Dans l’étude de l’influence du squelette, l’isomère de position pyrido[2,3-d]pyrimidine fut également synthétisé et fonctionnalisé. Afin de tester ces inhibiteurs originaux, une plateforme de tests d’activités in vitro a été mise en place où cinq kits enzymatiques ont été optimisés sur la kinase PI3K, et un test sur mTOR. Ces tests exploitant la méthode de TR-FRET et de bioluminescence ont été validés avec des inhibiteurs de référence basés sur 4 facteurs : la corrélation entre IC50(littérature) et IC50(mesurée), Z’, R², et S/B.Au final, plus de soixante produits finaux ont été évalués in vitro sur PI3K et mTOR. La moitié présentent une IC50 inférieure à 100 nM et 5 ont des IC50 inférieures à 10 nM. Dans le cadre des échanges du Cancéropôle Grand Ouest, les produits ont été testés sur 6 lignées de cellules cancéreuses. / Considering the impact of overactivation of the PI3K/Akt/mTOR pathway in cancer, we chose to inhibit this signaling pathway. Given the high structural similarity between the PI3K and mTOR enzymes, we designed dual inhibitors targeting two of the three major kinases of the pathway. These inhibitors posses an original pyrido[3,2-d] pyrimidine scaffold. In order to provide a functional diversity and generate a therapeutic effect, the peaks C-4, C-2 and C-7 were functionalized sequentially in the following order. Position C-4 was first functionalized with aliphatic heterocycles by nucleophilic aromatic substitution. Then, various heteroaromatic rings were introduced at the C-2 position by Suzuki-Miyaura coupling. Finally, different functions were inserted at the C-7 peak by different reactions. In order to study the influence of the scaffold, the pyrido[2,3-d]pyrimidine isomer was also synthesized and functionalized. To test these original inhibitors a platform testing in vitro activities was set up in which five assay kits were optimized for the kinase PI3K, and one kit for mTOR. These tests exploit the TR-FRET and bioluminescence methods and were validated with commercially available inhibitors based on four factors: the correlation between IC50(literature) and IC50(measured), Z’, R², and S/B. In the end, more than sixty final products were evaluated in vitro on PI3K and mTOR. Half present an IC50 below 100 nM and 5 of them show an IC50 under 10 nM. As part of collaboration within the Cancéropôle Grand Ouest, the products were also tested on six cancer cell lines.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In the first study, we characterized an in vitro culture system able to delaying meiosis resumption of bovine oocytes. Firstly, we demonstrated that the use of an EGFR inhibitor (AG1478; 5μM) in a culture system with follicular hemisections (FHS) was effective to maintain 89.3% of the oocytes in germinal vesicle stage (GV) during 15 h. This blocking effect was dependent on the FHS, since in its absence only 40% of the oocytes remain in GV stage. The meiosis blockage was totally reversible, since the oocytes reached matured stages after an additional 18 and 20 h maturation period and were able to support the embryonic development after in vitro fertilization. Regarding the molecular profile of the cells involved in the blocking system, we did not observe treatment effect on mRNA expression of the genes evaluated in oocyte. However, in cumulus cells, whereas the expression of EGR-1, TNFAIP6 and HAS2 was inhibited by AG1478 treatment, the expression of CX43 and IMPDH1 was decreased by FHS influence. Moreover, in the granulosa cells we observed a downregulation in the expression levels of PGR and ADAMTS1 by AG1478 treatment. The Western blot data revealed that the treatment with AG1478 plus FHS induces a downregulation in p-ERK1/2 protein abundance. In the next experiment, we verified that the AngII or PGE2 and PGF2α did not reverse the inhibitory effect of AG1478 plus FHS on meiosis resumption. In conclusion, findings from this study revealed an effective and reversible system to prevent meiosis resumption of bovine oocytes. In the second study, we investigate the role of mTOR system and its relation with LH regulated genes during preovulatory period in cattle. Using an in vivo model, we demonstrated mTOR kinase activity in granulosa cells 3 and 6 h after induction of ovulation with GnRH. In the similar moments (3 h after GnRH), we observed an increase in p-ERK1/2, STAR and EGR1 protein abundance. The inhibition of mTOR kinase activity by intrafollicular injection of rapamycin did not alter the ovulation rate. However, the treatment of granulosa cells in vitro with rapamycin interrupted the LH-induced increase in EREG mRNA levels. Moreover, the effect of rapamycin in culture was proved by inhibiting the p-P70S6K protein levels. In the same Western blot analysis, we verified that rapamycin may be inducing AKT activity and did not alter Phospho-ERK1/2 status and EGR1 protein abundance. These results provided the first evidence in cattle that mTOR system is upregulated by LH at time points similar to p-ERK1/2, STAR and EGR1. In addition, the mTOR inhibition data contribute to suggest an AKT dependent pathway during ovulation process, in which occurs ERK1/2 activation in a pathway independent of EREG, AREG and PTGS2 mRNA levels. / O primeiro estudo caracterizou um modelo in vitro de bloqueio do reinício da meiose de oócitos bovinos. Em um primeiro momento, demonstramos que o uso de um inibidor dos EGFR (AG1478; 5μM) em um sistema de cultivo com metades foliculares (FHS) foi eficiente para manter 89,3% dos oócitos em vesícula germinativa durante 15 h. Esse efeito de bloqueio foi dependente das FHS uma vez que na sua ausência apenas 40% dos oócitos permanecem em vesícula germinativa. O sistema de bloqueio foi totalmente reversível, tendo em vista que os oócitos completaram a maturação após um período adicional de 18 e 20 h, e suportaram o desenvolvimento embrionário subsequente após fertilização in vitro. Quanto ao perfil molecular das células envolvidas no bloqueio, no oócito não foi verificado efeito do tratamento na expressão dos genes avaliados. Entretanto, nas células do cumulus, enquanto a expressão de EGR1, TNFAIP6 e HAS2 foi diminuída pelo tratamento com AG1478, a expressão de CX43 e IMPDH1 foi diminuída pela influência das FHS. Além disso, nas células da granulosa observamos uma diminuição nos níveis de expressão de PGR e ADAMTS1 pelo tratamento com AG1478. Os dados de Western blot nos mostraram que a abundância de p-ERK1/2 diminui em decorrência do tratamento com AG1478 associado as FHS. Posteriormente, verificamos que o efeito inibitório do AG1478 juntamente com as FHS não foi revertido pelo tratamento com AngII ou PGs. Em conclusão, este estudo propõem um modelo efetivo e reversível para o bloqueio do reinício da meiose de oócitos bovinos. Em um segundo estudo, investigamos o papel do sistema mTOR e sua relação com genes regulados pelo LH durante o período pré-ovulatório em bovinos. Utilizando um modelo in vivo, demonstramos que ocorre um aumento na atividade do mTOR em células da granulosa 3 e 6 h após indução da ovulação com GnRH. Em momentos similares (3 h após GnRH) ocorreu maior abundância proteica para p-ERK1/2, STAR e EGR1. Ao injetar rapamicina no ambiente intrafolicular in vivo, não foram observadas alterações nas taxas de ovulação. Entretanto, o uso da rapamicina em cultivo in vitro de células da granulosa inibiu a expressão de RNAm para EREG induzida pelo LH. Além disso, os dados de cultivo comprovaram o efeito da rapamicina em bloquear a atividade do mTOR, caracterizada pela abundância proteica de p-P70S6K, induzir um provável aumento na abundância de p-AKT e não alterar os níveis de p-ERK1/2 e EGR1. Esses resultados fornecem a primeira evidência em bovinos que o sistema mTOR é regulado positivamente pelo LH em momentos similares a p-ERK1/2, STAR e EGR1. Além disso, os dados de inibição do mTOR contribuem para sugerir uma outra rota para a ovulação controlada pela p-AKT, na qual ocorre ativação de ERK1/2 em uma via independente dos níveis de expressão de EREG, AREG e PTGS2.

Mon travail de thèse porte sur les épilepsies focales avec ou sans malformations cérébrales de type dysplasie corticale focale. Il s'articule autour de (1) une étude fonctionnelle et génétique sur tissu cérébral postopératoire humain et (2) la caractérisation d'un nouveau modèle génétique chez l'animal. Tout d'abord, des mutations germinales hétérozygotes perte de fonction ont été identifiées dans DEPDC5, NPRL2 et NPRL3 qui codent pour le complexe GATOR1, un inhibiteur du complexe 1 de mTOR (mTORC1). Par la suite, des mutations somatiques cérébrales gain de fonction ont été identifiées dans MTOR. Nous avons émis l'hypothèse que ces mutations entrainent une hyperactivité de mTORC1, responsable des malformations cérébrales et de l'épilepsie des patients. J'ai observé une hyperactivité de mTORC1 dans les cellules cytomégaliques obtenues à partir de tissu cérébral post-opératoire de patients porteurs de mutations dans les gènes de GATOR1 ou MTOR. En parallèle, la caractérisation du premier modèle KO de Depdc5 a montré que les rats Depdc5+/- présentent des anomalies corticales rappelant celles des patients : délamination des couches corticales et cellules cytomégaliques avec une hyperactivité de mTORC1. Ce phénotype est prévenu par l'injection de rapamycine, un inhibiteur spécifique de mTORC1. Une susceptibilité accrue aux crises épileptiques induites par le pentylènetétrazole ainsi qu'un défaut des propriétés neuronales passives et actives ont été rapportés chez les rats Depdc5+/-. En conclusion, mes travaux de thèse ont contribué à mettre en évidence l'implication de la voie GATOR1-mTORC1 dans les épilepsies et dysplasies corticales focales. / In my PhD thesis work, I investigated focal epilepsies with and without brain malformations such as focal cortical dysplasia. I focused on two complementary aspects: (1) genetics and functional studies on human tissue samples and (2) characterization of a novel genetic animal model. First, germline heterozygous loss-of-function mutations were identified in DEPDC5, NPRL2 and NPRL3 genes that encode proteins which together form the GATOR1 complex, a repressor of the mTOR complex 1 (mTORC1). Additionally, brain somatic gain-of- function mutations were identified in MTOR gene that encodes mTOR itself. Both types of mutations are thought to lead to mTORC1 hyperactivity, and cause brain malformation and epilepsy in patients. To test this hypothesis, mTORC1 activity was monitored on post-operative brain tissue from patients carrying GATOR1 or mTOR genes mutations. Cytomegalic cells with mTORC1 hyperactivity were observed. Besides, the characterization of the first Depdc5 KO model revealed that Depdc5+/- rats present cortical structural abnormalities reminiscent of patient histopathology hallmarks: cortical layer dyslamination and cytomegalic cells with increased mTORC1 activity. This phenotype was prevented by rapamycin injection, a specific mTORC1 inhibitor. An increased susceptibility to pentylenetetrazol-induced epileptic seizures, as well as impaired passive and active neuronal properties were observed in Depdc5+/- rats compared to Depdc5+/+ rats. In conclusion, my PhD work largely contributed to emphasize the prominent role of the GATOR1-mTORC1 pathway in focal cortical dysplasia and epilepsies.

Submitted by LUIS GABRIEL RIVERA CALDERON (lgriveramvz@gmail.com) on 2018-05-02T17:50:21Z No. of bitstreams: 1 Tese_Luis_Gabriel_Rivera_Calderon.pdf: 12272198 bytes, checksum: 78d4519bb4d6661c6d6284b28f290374 (MD5) / Rejected by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br), reason: Solicitamos que sejam feitas as correções listadas abaixo: O arquivo PDF submetido no repositório deve conter ficha catalográfica e certificado de aprovação (documentos obrigatórios). Favor inserir os mesmos no arquivo PDF e fazer novamente a submissão. Agradecemos a compreensão. on 2018-05-02T18:50:24Z (GMT) / Submitted by LUIS GABRIEL RIVERA CALDERON (lgriveramvz@gmail.com) on 2018-05-03T11:50:35Z No. of bitstreams: 1 Tese Luis Gabriel Rivera.pdf: 12908129 bytes, checksum: a2bb73ad103cbc2de776d2b460919a3c (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-05-04T18:04:24Z (GMT) No. of bitstreams: 1 riveracalderon_lg_dr_jabo.pdf: 12908129 bytes, checksum: a2bb73ad103cbc2de776d2b460919a3c (MD5) / Made available in DSpace on 2018-05-04T18:04:24Z (GMT). No. of bitstreams: 1 riveracalderon_lg_dr_jabo.pdf: 12908129 bytes, checksum: a2bb73ad103cbc2de776d2b460919a3c (MD5) Previous issue date: 2018-03-26 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O câncer é uma doença complexa que precisa de um microambiente favorável para seu crescimento e progressão. Esse microambiente tumoral esta constituido por células neoplásicas, vasos sanguíneos, células imunes, fibroblastos e a matriz extracellular (MEC). Geralmente, as células neoplásicas apresentam modificações nas suas vias de sinalização. No homem, a via mTOR/4E-BP1/eIF4E foi descrita como alterada em diferentes tumores, incluindo o câncer de próstata (CP).Além do homem, o cão é espécie doméstica que desenvolve com mais frequência o CP, sendo considerada um potencial modelo para estudos na área de Oncologia Comparada. Devido à limitada informação sobre a via mTOR/4E-BP1/eIF4E e os componentes da MEC nos CP caninos, o objetivo deste estudo foi avaliar a expressão gênica e protéica de mTOR, 4E-BP-1 e eIF4E neste tipo de tumor. Outrossim, avaliar a expressão dos colágenos (C-I e C-III) pela técnica Picrosirius (PSR) e imuno-histoquímica no tecido prostático normal e neoplásico. Foram utilizadas 35 amostras de tecido prostático caninos. Identificou-se alta expressão protéica de p-mTOR e eIF4E nos CP caninos com alto GS (≥ 8), assim como, correlação positiva entre essas proteínas. Nos colágenos não foi observada diferença de expressão quando comparadas amostras de próstata normal e CP canino. De forma similar com o CP humano, estes resultados sugerem que p-mTOR e eIF4E podem ser bons marcadores para o processo carcinogênico prostático canino e estão correlacionados com alto GS. Além disso, a distribuição de colágenos foi similar no tecido prostático normal e neoplásico. / Cancer is a complex disease that needs a favorable microenvironment for its growth and progression. This tumor microenvironment consists of neoplastic cells, blood vessels, immune cells, fibroblasts and extracellular matrix (ECM). Generally, neoplastic cells show modification in their signaling pathways. In men, the mTOR/4EBP1/eIF4E pathway has been described as altered in different tumors, including prostate cancer (PC). Apart from men, the dog is the only species that develops with high frequency the PC, being considered a potential model for comparative oncology initiatives. Due to limited information on this pathway and ECM components in canine tumors, this study aimed to investigate mTOR, 4E-BP1 and eIF4E gene and protein expression in canine PC. Additionally, to evaluate the expression of collagens (C-I and C-III) by Picrosirius Red and Immunohistochemistry in normal samples and canine PC. Were used a total of 35 formalin-fixed paraffin-embedded (FFPE) samples from canine prostatic tissue. We identified higher p-mTOR and eIF4E protein levels in the canine PC with higher GS (≥ 8), as well as, significant positive correlation between these proteins. No difference statistical was observed in the collagen expression between normal samples and canine PC. Similar to human PC; our data suggested that p-mTOR and eIF4E good markers for canine prostatic carcinogenic process and are correlated with higher GS. Also, the distribution and collagen levels are similar in normal and neoplastic canine prostate. / 443884/2014-5

Les léiomyosarcomes (LMS) sont des tumeurs d’origine mésenchymateuse caractérisées par une différenciation musculaire lisse. La voie de signalisation PI3K/mTOR (qui contrôle la prolifération et la survie cellulaire) joue un rôle majeur dans le développement de ces tumeurs. De nos jours, cette voie est devenue une cible thérapeutique majeure en oncologie. Cette étude est la première qui évalue le bénéfice thérapeutique de l’inhibition de la voie PI3K/mTOR pour des patients atteint de LMS. Nous avons mis en évidence qu’une double inhibition de PI3K et mTOR est associée à une activité antitumorale supérieure à celle observée avec une inhibition de PI3K ou mTOR seule. Nous avons également montré que l’inhibition de la voie PI3K/mTOR est associée à une activation paradoxale de la voie MAPK et qu’un ciblage concomitant de cette voie est associé à une synergie antitumorale in vitro et in vivo. Afin de caractériser les mécanismes de résistance secondaire à l’inhibition de la voie PI3K/mTOR, nous avons développé in vitro et in vivo un modèle de résistance secondaire à l’inhibiteur double cible PI3K/mTOR. Nous avons notamment détecté une sous-population de cellules résistantes à l’inhibiteur et ayant des caractéristiques proches de celles des cellules souches. Nous avons mis en évidence que l’inhibition pharmacologique d’EZH2, une protéine cruciale du complexe Polycomb, permet de restaurer la sensibilité des modèles résistants. Ces résultats apportent de nouvelles perspectives thérapeutiques pour les patients atteints de LMS. / Leiomyosarcomas (LMS) are tumors of mesenchymal origin characterized by a smooth cell differentiation. The PI3K/mTOR pathway has been shown to play a crucial role in the tumorigenesis of LMS. Several agents targeting this pathway are under clinical development for the treatment of solid tumors and hematological malignancies. We report here the first study evaluating its potential therapeutic benefit for patients with LMS. We have demonstrated that dual inhibition of PI3K and mTOR is associated with more effective antitumor activity than agents targeting PI3K or mTOR only. We have also shown that PI3K and mTOR inhibition is associated with a paradoxal activation of the MAPK pathway and that combined treatment with MEK inhibitor resulted in synergistic antitumor activity in vitro and in vivo. Moreover, we developed in vitro and in vivo resistant model to dual PI3K/mTOR inhibitor. Interestingly, we have found that a cancer stem cell-like subpopulation may be involved in treatment resistance. We have shown that pharmacological inhibition of EZH2, a crucial protein of the Polycomb complex, is able to reverse dual PI3K/mTOR inhibitor resistance in vitro and in vivo. These results provide new therapeutic strategies for patients with LMS.