Submitted by Vasti Diniz (vastijpa@hotmail.com) on 2017-09-08T11:33:23Z No. of bitstreams: 1 arquivototal.pdf: 1921817 bytes, checksum: 7ba035d7ef40f1aafd9f93476aabedd6 (MD5) / Made available in DSpace on 2017-09-08T11:33:23Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1921817 bytes, checksum: 7ba035d7ef40f1aafd9f93476aabedd6 (MD5) Previous issue date: 2016-03-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Epigenetics is the study heritable changes of in gene expression without modifications in the primary sequence of DNA. In our study we investigated the influence of sun exposure on global DNA methylation and hydroxymethylation status and at specific sites of the miR-9-1, miR9-3 and MTHFR genes in skin samples of subjects with no history of skin diseases. Skin biopsies were obtained by punch on sun-exposed and sun-protected arm areas from 24 corpses aged 16-89 years old from the Brazilian Service of Death Investigation. Genomic DNA was extracted from skin samples that were ranked according to Fitzpatrick’s criteria as light, moderate and dark brown. Global DNA methylation and hydroxymethylation and DNA methylation at specific sites analyses were performed using an ELISA and MSP, respectively. No significant differences in global DNA methylation and hydroxymethylation levels were found between the skin areas, skin type or age. However, gender-related differences were detected, where women showed higher methylation levels in comparison to those in men. Global DNA methylation levels were higher than hydroxymethylation levels, and the levels of these DNA modifications correlated in skin tissue. For specific sites, it was detected no differences among areas. Additional analyses showed no differences in the methylation status when age, gender and skin type were considered. We conclude that sun exposure does not induce changes in the global DNA methylation and hydroxymethylation status or at specific sites in the miR-9-1, miR-9-3 and MTHFR genes for skin types studied. / A epigenética é o estudo das alterações hereditárias na expressão gênica sem mudanças na sequência primária do DNA. No nosso estudo investigamos a influência da exposição solar sobre o perfil de metilação e hidroximetilação global de DNA e em sítios específicos nos genes miR-9-1, miR-9-3 e MTHFR em amostras de pele humana. Para isso, biópsias foram obtidas por punch circular de área exposta e não exposta ao sol do braço de 24 cadáveres de ambos os sexos, com idade entre 16-89 anos sem histórico de doenças de pele oriundos do Serviço de Verificação de Óbitos da Paraíba (SVO). O DNA foi extraído e a análise de metilação e hidroximetilação global do DNA foi realizada através de Elisa indireto. A análise de metilação nos sítios específicos dos genes miR-9-1, miR-9-3 e MTHFR foi realizada por meio de PCR específica para metilação (MSP) seguida de eletroforese. As análises estatísticas foram realizadas pelo software BioEstat 5.0 ao nível de significância de 5%. Não encontramos diferenças significativas nos níveis de metilação e hidroximetilação global de DNA entre as áreas exposta e não exposta da pele, tipo de pele ou idade. No entanto, foram detectadas diferenças em relação ao gênero, onde as mulheres apresentaram nível de metilação global mais alto em comparação aos homens. O nível de metilação global de DNA foi maior do que o nível de hidroximetilação, sendo estes, correlacionados no tecido da pele. Para sítios específicos, não foi detectada nenhuma diferença entre as áreas. Análises adicionais mostraram não haver diferenças significativas no perfil de metilação quando consideradas a idade, gênero e o tipo de pele. Conclui-se que a exposição ao sol não induz mudanças no perfil de metilação e hidroximetilação global do DNA ou em sítios específicos dos genes miR-9-1, miR-9-3 e MTHFR para os tipos de pele estudado.

Submitted by SAMUEL RODRIGUES LOURENÇO DE MORAIS null (samuelrodrigues@foa.unesp.br) on 2016-10-04T19:15:34Z No. of bitstreams: 1 TESE - SAMUEL R L DE MORAIS.pdf: 2524842 bytes, checksum: 61664d3b0e3fff46188835de7508312b (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-10-05T12:13:50Z (GMT) No. of bitstreams: 1 morais_srl_dr_araca.pdf: 2524842 bytes, checksum: 61664d3b0e3fff46188835de7508312b (MD5) / Made available in DSpace on 2016-10-05T12:13:50Z (GMT). No. of bitstreams: 1 morais_srl_dr_araca.pdf: 2524842 bytes, checksum: 61664d3b0e3fff46188835de7508312b (MD5) Previous issue date: 2016-08-22 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A diminuição das concentrações plasmáticas de estrógeno está intimamente relacionada com o aumento do estresse oxidativo e a diminuição da massa muscular em idosos. A terapêutica hormonal estrogênica (THE) e o treinamento de força (TF) apresentam resultados efetivos sobre a manutenção do tecido muscular em idosos. No entanto, os mecanismos responsáveis pelas melhorias induzias por ambas as intervenções são pouco elucidados. Nesse sentido, avaliamos os efeitos da THE, do TF e a associação sobre a manutenção do tecido muscular esquelético de ratas periestropausadas. Ratas Wistar (18 meses) foram distribuídas em: Grupo não treinado (NT-Veh), Grupo NT tratado com a THE (NT-E2), Grupo TF (TF-Veh) e Grupo TF-E2. Os animais receberam a THE (17β estradiol; 2 x semana; 25 µg/kg/administração) e/ou praticaram TF (3 x semana; 80% sobrecarga) durante 16 semanas. A THE e o TF induzem benefícios ao tecido muscular esquelético de ratas periestropausadas, no entanto, por diferentes maneiras. Enquanto a THE induziu diminuição do estresse oxidativo muscular (Dihidroetidina), o TF resultou em melhoras significativas na função muscular, no sistema antioxidante muscular (Catalase) e na expressão de miRNAs (206, 146b e 133a). Já a interação das intervenções resultou em melhora no estado redox (Sirt1, Sirt3, PGC-1α, COXIV), na responsividade dos receptores estrogênicos (ERα, ERβ e GPR30), e atividade de vias de sinalização do tecido muscular (IGF-1/Akt-1/mTOR). Além disso, as intervenções de maneira isolada ou em associação, levaram ao aumento no percentual de fibras glicolíticas e redução das oxidativas. Sugerimos que a aderências das intervenções (associadas ou não) possam minimizar/atenuar a perda da massa muscular observada em fases tardias durante o processo de envelhecimento. / The decrease of estrogen (E2) circulating levels is strongly related to increased oxidative stress and the loss of muscle mass in elderly. The hormone replacement therapy (HRT) and strength training (ST) are the main effective interventions to prevent the loss of muscle mass, however, the mechanisms involved in interventions-induced benefits are not well elucidated. In this sense we evaluate the effect of HRT, ST and association on skeletal muscle maintenance of periestropaused rats. Female Wistar rats (18 months old) were randomly assigned into: non-exercised and non-treated group (NE-Veh), NE treated group (NE-E2), exercised and non-treated group (ST-Veh) and ST-E2 group. The animals received the HRT (17β estradiol; 2 x week; 50 µg/kg/week) and/or performed ST (3 x week, 80% overload) for 16 weeks. The HRT and ST promoted beneficial effects on skeletal muscle of periestropaused rats, however, by different manners. While HRT treatment leaves the reduction of oxidative stress (Dihidroetidine), the ST resulted in significate improvement on skeletal muscle function, in skeletal muscle antioxidant system (Catalase) and in miRNAs expression (2016, 146b and 133a). Already, the association of interventions resulted in improvement of redox state (Sirt1, Sirt3, PGC-1α, COXIV), in estrogen receptor responsiveness (ERα, ERβ and GPR30) and the activity of skeletal muscle signaling pathways (IGF-1/Akt-1/mTOR). In addition, the interventions, isolated or combinated, leaves an increase of the percentage of glycolytic fibers and reduced percentage of oxidative fibers. We suggest that the adherence to interventions (combinated or not) could minimize/attenuate the loss of skeletal muscle mass observed in later phases of aging process. / 13/18907-2

O câncer de próstata é a segunda causa de morte entre homens no Brasil. É tipo um câncer de crescimento lento, podendo levar anos para o tumor atingir 1 cm3, porém, em alguns casos ele pode se espalhar pelo corpo, sendo o osso o principal sítio de metástase. No estágio de desenvolvimento do câncer conhecido como metástase, o principal tratamento consiste em terapia de restrição andrógena, levando as células prostáticas a pararem de proliferar, uma vez que elas crescem em resposta a presença de hormônios andrógenos, como a diidrotestosterona e testosterona. Porém, em alguns casos, as células proliferam mesmo na ausência de andrógenos e isto se deve a diversos fatores que, em geral, estão associados a mutações no receptor andrógeno e/ou alterações no metabolismo andrógeno. Quando isto acontece, os tratamentos disponíveis são menos efetivos e costumam falhar. Porém, estudos sugerem que o γ-orizanol, um fitoesterol extraído do óleo do farelo do arroz; e extratos amplamente utilizados na medicina popular, como o extrato hidroalcólico de Thuya occidentalis, poderiam atuar inibindo o desenvolvimento e progressão do câncer de próstata. Neste estudo, com o uso de abordagens bioquímicas e de biologia molecular, foi demonstrado que o tratamento com γ-orizanol diminui a viabilidade e biomassa celular em cultura, associado ao aumento da morte celular por apoptose e/ou necrose, em linhagens celulares responsivas (LNCaP) e não-responsivas a andrógenos (PC3 e DU145), além de aumentar a pERK1/2 em células LNCaP e DU145. O γ-orizanol também foi capaz de bloquear o ciclo celular em G2/M nas células PC3 e LNCaP e em G0/G1 nas células DU145. Estes efeitos foram ainda acompanhados por uma redução da expressão do gene e proteína caveolina-1- uma importante molécula envolvida no aumento da agressividade do câncer de próstata, e também, na progressão da doença para o fenótipo andrógeno resistente - nas células não-responsivas a andrógenos, e do gene PCGEM1 - gene específico da próstata regulado por andrógeno - nas células LNCaP e DU145. Ainda, γ-orizanol também mostrou capacidade de regular vários miRNAs - pequenas moléculas de RNA não codificantes de proteínas - envolvidos no controle de funções associadas ao desenvolvimento, progressão e invasão no câncer de próstata, como o miR16-1, miR19b-2, miR24b-1, miR24b-2, miR99a, miR133a-5p, miR182-5p, miR198 e miR222. O extrato hidroalcólico de Thuya occidentalis reduziu a viabilidade e biomassa celular nas linhagens responsiva (LNCaP) e não-responsivas (DU145 e PC3) a andrógenos, além de induzir parada do ciclo celular na fase G0/G1 nas células DU145 e aumentar a morte celular por apoptose e/ou necrose em todas as linhagens. Da mesma forma que o γ-orizanol, este extrato reduziu a expressão da caveolina-1 nas linhagens não-responsivas a andrógenos. Trabalhos anteriores mostram que o monoterpeno α-tujona é o principal composto ativo do extrato de Thuya occidentalis. Por cromatografia gasosa acoplada a detector de massas foi mostrada a existência de 0,0016 μg de α-tujona na dose de extrato usada neste estudo. No entanto, o tratamento com 0,0016μg de α-tujona foi efetivo somente sobre linhagem LNCaP, não tendo efeito sobre as outras linhagens estudadas, reforçando a hipótese da diferença de sensibilidade entre as linhagens responsivas e não responsivas a andrógeno e mostrando a contribuição de outros componentes do extrato nos efeitos observados neste estudo. Concluindo, estes resultados demonstram que tanto γ-orizanol como o extrato de Thuya occidentalis podem vir a ser agentes terapêuticos promissores no tratamento de câncer de próstata, não só por inibirem o crescimento celular, mas também e principalmente pela possibilidade de induzirem a recuperação da sensibilidade a andrógenos, aumentando as possibilidades de tratamento da doença. / Prostate cancer is the second cause of death among men in Brazil. It is a slowgrowing cancer and it may take years for tumor to reach 1 cm3, but in some cases it can spread throughout the body and the bone is the main site of metastasis. At this cancer stage known as metastasis, the principal treatment involves antiandrogen therapy, leading to prostate cells stop proliferating, because they grow in response to presence of androgens such as testosterone and dihydrotestosterone. However, in some cases, the cells can proliferate even in the absence of androgens and this fact occurs due to many factors and they are generally associated with mutations in the androgen receptor and/or alterations in androgen metabolism. In this stage, the treatments available are less effective and usually fail. However, studies suggest that γ-oryzanol, a phytosterol extracted of rice bran oil; and extracts widely used in folk medicine, as Thuya occidentalis hidroalcolic extract, could act inhibiting the development and progression of prostate cancer. In this study, using molecular biology and biochemical approaches we showed that γ- oryzanol treatment was able to decrease cell viability and biomass in culture, and this fact was linked to increased cell death by apoptosis and/or necrosis in androgen responsive (LNCaP) and unresponsive (DU145 and PC3) prostate cancer cell lines, besides increasing pERK1/2 in LNCaP and DU145 cells. γ- oryzanol was also able to cause cell cycle arrest at G2/M phase in LNCaP and PC3 cells and at G0/G1 phase in DU145 cells. These effects were also accompanied by a reduction in caveolin-1 gene and protein expression - an important molecule related to high aggressiveness in prostate cancer and also in the progression of the disease to androgen resistant phenotype - in androgen unresponsive cells, and also PCGEM1 gene - a prostate specific gene regulated by androgens - in LNCaP and DU145 cells. γ-oryzanol also showed ability to regulate several miRNAs - small non-coding RNA molecules - involved in the control of many functions associated with the development, progression and invasion of prostate cancer, such as miR16-1, miR19b-2, miR24b-1, miR24b-2, miR99a, miR133a-5p, miR182-5p, miR198 and miR222. Thuya occidentalis hidroalcolic extract also reduce cell viability and biomass in androgen responsive (LNCaP) and unresponsive (DU145 and PC3) cells, in addition to inducing cell cycle arrest at G0/G1 phase in DU145 cells and to increase apoptosis and/or necrosis cell death in all cell lines. The same way that γ-oryzanol, this extract reduced the caveolin-1 expression in androgen unresponsive prostate cancer cells. Prior studies showed that the monoterpene α-thujone is the main active compound in the T. occidentalis extract. By gas chromatography coupled to mass detector it was showed the existence of 0.0016 μg of α-thujone in extract dose used in this study. However, the treatment with 0.0016 μg of α-thujone was effective only on LNCaP cell line, having no effect on the other studied lines, supporting the hypothesis of difference in sensitivity between responsive and unresponsive cell lines and showing the contribution of other components in the effects caused by the extract, observed it this study. In conclusion, these results demonstrate that both γ-oryzanol as T. occidentalis extract may become promising therapeutic agents in treatment of prostate cancer, not only inhibit cell growth but also and manly by the possibility of inducing the recovery of androgen sensitivity, increasing the treatment chances of treatment this disease.

Introdução: O câncer de próstata (CaP) é o tumor mais comum em homens e a segunda causa de óbito por câncer no Brasil. Com a adoção do rastreamento, a maioria dos pacientes apresenta doença localizada ao diagnóstico, enquanto apenas 4% têm doença metastática. Um dos principais mecanismos responsáveis pela progressão tumoral é a transição epitélio-mesenquimal (TEM). Esse é um programa celular reversível no qual a célula epitelial perde a capacidade de aderência intercelular e assume um fenótipo mesenquimal com potencial invasivo e metastático. A principal característica da TEM é a repressão da E-caderina através de fatores de transcrição que incluem ZEB1, ZEB2, Snail, Slug e Twist1. Os microRNAs também estão envolvidos na regulação do processo, sendo a família do miR-200 uma das mais importantes e uma potente indutora da diferenciação epitelial. Assim sendo, o conhecimento dos fatores envolvidos na TEM no CaP é fundamental para a compreensão do comportamento biológico dessa neoplasia. Objetivos: Analisar a expressão dos genes e microRNAs envolvidos na transição epitélio-mesenquimal em espécimes de câncer de próstata localizado e em linhagens celulares de câncer de próstata metastático. Além disso, correlacionar o perfil de expressão dos genes e microRNAs com parâmetros clínico-patológicos. Material e métodos: O estudo consistiu na análise de espécimes de 51 pacientes com CaP localizado tratados por prostatectomia radical e de linhagens celulares de CaP metastático (LNCaP, DU145 e PC3). O grupo controle foi composto por 10 casos de hiperplasia prostática benigna. A expressão dos genes E-caderina, N-caderina, Vimentina, TGF-beta1, ZEB1, ZEB2, Snail, Slug, Twist1 e PDGF-D e dos microRNAs 200a, 200b, 200c, 429, 141, 203, 205, 183, 373, 21, 9, 1, 495, 29b, 30a, 34a, 155 e 10b foi avaliada através da técnica de PCR em tempo real (qRT-PCR) nos espécimes e nas linhagens. Para correlação com parâmetros clínico-patológicos, os pacientes foram divididos em grupos em relação ao escore de Gleason, estadiamento patológico, PSA pré-operatório, recorrência bioquímica e doença de baixo e alto risco. Para avaliação do tumor metastático, agrupamos as linhagens celulares e comparamo-las com tumores pT3. Resultados: A grande maioria dos casos apresentou superexpressão de E-caderina e Twist1 e subexpressão de N-caderina, Vimentina, TGF-beta1, ZEB1 e Slug. ZEB2, Snail e PDGF-D apresentaram expressão variável. A família do miR-200 e os miRNAs 203, 205, 183, 373 e 21 apresentaram superexpressão, enquanto que os miRNAs 9, 495, 29b e 1 apresentaram subexpressão. Os demais miRNAs apresentaram perfil de expressão variável. Níveis menores de expressão dos miRNAs 200b, 30a e 1 associaram-se significativamente com estadiamento patológico. Os pacientes com expressão reduzida do miR-200b também apresentaram associação com escore de Gleason >= 8 e com menor tempo de sobrevida livre de recorrência bioquímica. Ainda, níveis baixos do miR-30a e níveis elevados de Vimentina e Twist1 associaram-se com grupo de alto risco. As linhagens metastáticas apresentaram níveis de expressão do miR-183 e do Twist1 significativamente maiores quando comparadas ao tumor localizado. Conclusão: O CaP localizado mantém um fenótipo molecular epitelial. Menor expressão dos miRNAs 200b, 30a e 1 está associada com estadiamento mais avançado, sendo que o miR-200b também associou-se com maior escore de Gleason e menor tempo de sobrevida livre de recorrência bioquímica, e o miR-30a, com grupo de alto risco. A maior expressão de Vimentina e Twist1 apresentou associação com o grupo de alto risco. miR-183 e Twist1 apresentam maiores níveis de expressão em linhagens celulares metastáticas em relação ao tumor primário. De acordo com nossos resultados, os miRNAs 200b, 30a, 1 e 183 e os genes Twist1 e Vimentina podem ter um papel importante na progressão do CaP, além de serem potenciais marcadores prognósticos / Introduction: Prostate cancer (PCa) is the most common cancer in men and the second leading cause of cancer-related mortality in Brazil. After the adoption of screening, most patients present with localized disease at the time of diagnosis, while only 4% have metastatic disease. One of the main mechanisms of tumor progression is the epithelial-mesenchymal transition (EMT). This is a reversible cell-biological program in which an epithelial cell loses intercellular adhesion and acquires a mesenchymal phenotype with invasiveness and metastatic potential. The main event in EMT is the repression of E-cadherin by transcriptional factors, including ZEB1, ZEB2, Snail, Slug, and Twist1. microRNAs are also involved in the regulation of this process, and one of the most important ones is the miR-200 family, which is a powerful inducer of epithelial differentiation. Therefore, the knowledge of the factors involved in EMT in PCa is essential to understand the biological behavior of this neoplasia. Objectives: Analysis of gene and miRNA expression involved in epithelial-mesenchymal transition in specimens of localized prostate cancer and metastatic prostate cancer cell lines. Correlation between the gene and miRNA expression profiles and clinicopathological features. Material and Methods: This study consisted in the analysis of specimens from 51 patients with localized PCa treated by radical prostatectomy and of metastatic PCa cell lines (LNCaP, DU145, PC3). The control group was composed by 10 cases of benign prostatic hyperplasia. Gene expression of E-cadherin, N-cadherin, Vimentin, TGF-beta1, ZEB1, ZEB2, Snail, Slug, Twist1, and PDGF-D as well as miRNA expression of 200a, 200b, 200c, 429, 141, 203, 205, 183, 373, 21, 9, 1, 495, 29b, 30a, 34a, 155, and 10b were assessed by Real-Time PCR (qRT-PCR). The patients were divided into groups according to Gleason score, pathological stage, preoperative PSA, biochemical recurrence, and low and high-risk disease. For evaluation of metastatic tumor, the cell lines were grouped and compared to pT3 tumors. Results: The vast majority of the cases showed overexpression of E-cadherin and Twist1 and underexpression of N-cadherin, Vimentin, TGF-beta1, ZEB1, and Slug. ZEB2, Snail, and PDGF-D showed a variable expression pattern. miRNA-200 family and miRNAs 203, 205, 183, 373, and 21 were overexpressed, while miRNAs 9, 495, 29b, and 1 were underexpressed. The remaining miRNAs showed a variable expression pattern. Lower expression levels of miRNAs 200b, 30a, and 1 were significantly associated with pathological stage. Patients with lower expression of miR-200b also showed association with Gleason score >= 8 and biochemical recurrence-free survival (BRFS). Furthermore, low expression levels of miR-30a and high expression levels of Vimentin and Twist1 were associated with high-risk group. The metastatic PCa cell lines showed significantly higher expression levels of miR-183 and Twist1 in comparison to the primary tumor. Conclusion: Localized prostate cancer maintains a molecular epithelial phenotype. Lower expression of miRNAs 200b, 30a, and 1 was associated with higher stage. Low levels of miR-200b were also associated with high Gleason score and shorter BRFS, and miR-30a, with high-risk group. High expression levels of Vimentin and Twist1 were associated with high-risk group. miR-183 and Twist1 levels were higher in metastatic cell lines than in the primary tumor. According to our results, miRNAs 200b, 30a, 1, and 183 and the genes Twist1 and Vimentin might play an important role in the progression of PCa and may turn to be important prognostic markers

O timo é um orgão linfóide primário, no qual ocorre a indução da tolerância imunológica central aos antígenos do próprio que são expressos pelos tecidos periféricos (PTAs). A medula tímica é formada por células tímicas medulares epiteliais (mTECs) que expressam centenas desses PTAs que representam virtualmente todos os órgãos e tecidos do corpo. Esse fenômeno foi denominado de expressão gênica promíscua (PGE) a qual é parcialmente regulada pelo modulador da transcrição Autoimmune regulator (Aire). Os precursores de células T oriundos da medula óssea migram para o timo (agora são denominados de timócitos) e na medula desse órgão, passam pela seleção negativa mediada pelas mTECs. As células sobreviventes evoluem para células T maduras e funcionais que migram para a periferia com capacidade de reconhecimento das moléculas de MHC e tolerantes aos PTAs. Além de controlar a transcrição de genes PTAs, Aire também controla a expressão de microRNAs (miRNAs), relacionados com a integridade e funcionalidade do microambiente tímico. A seleção negativa no timo é um processo essencial para a manutenção da autotolerância imunológica e o desbalanço desse processo está associado com o desenvolvimento de doenças autoimunes como, por exemplo, o diabetes mellitus do tipo 1 (DM1).Tendo em vista essas premissas, nosso trabalho se fundamentou em duas hipóteses: 1) Variações na expressão do gene Aire podem perturbar a expressão de genes PTAs e miRNAs no timo, causando alterações na PGE, 2) A expressão balanceada de genes como Aire e/ou PTAs nas mTECs, é fundamental para a integridade da tolerância central. O desbalanço na expressão desses genes, está associado com a emergência do diabetes mellitus tipo 1 no camundongo. Para testar nossa primeira hipótese efetuamos o silenciamento de Aire (Aire knockdown) por meio de eletrotransfeção de RNA interferente (siRNA) anti-Aire in vivo no timo de camundongos BALB/c. Análises do transcriptoma (mRNAs) e miRNoma (miRNAs) das mTECs, revelaram que silenciamento parcial e transitório de Aire foi suficiente para afetar a expressão de PTAs Aire dependentes bem como a de miRNAs. Redes de interação miRNA-mRNA, revelaram que o controle pós-transcricional da PGE também é afetado pelo silenciamento de Aire. Os resultados encontrados revelam que Aire e miRNAs podem formar uma via essencial durante a indução da tolerância central. Para testar nossa segunda hipótese comparamos o transcriptoma de mTECs de camundongos BALB/c (linhagem não-autoimune) com mTECs de camundongos non-obese diabetic NOD (modelo animal utilizado nos estudos de DM1 autoimune). Nossos resultados revelaram que a expressão transcricional de autoantígenos relacionados ao DM1 está desbalanceada em camundongos NOD já numa fase precoce, quando esses animais ainda não apresentavam a doença clínica (fase pré-diabética). Inesperadamente, os níveis transcrionais de Aire apresentaram-se equivalentes no timo dessas duas linhagens, porém os níveis da proteína AIRE estavam reduzidos no timo da linhagem NOD. Esses resultados sugerem a participação de algum mecanismo de atenuação póstrascricional de Aire nessa linhagem provavelmente envolvendo atuação de miRNAs. Isso poderia explicar o desbalanço de PTAs Aire-dependentes e a repressão autoantígenos relacionados ao DM1. Concluímos que nossos resultados, além de abrir novas perspectivas para pesquisas nesta área, contribuem com melhor compreensão dos mecanismos moleculares desencadeados por Aire e por miRNAs no controle da expressão de autoantígenos no timo o que é importante para a tolerância imunológica central. / The thymus is a primary lymphoid organ, in which occurs in the induction of central immune tolerance to self peripheral tissue antigens (PTAs). The thymic medulla is formed by medullary thymic epithelial cells (mTECs) expressing hundreds of such PTAs representing virtually all organs and tissues of the body. This phenomenon has been termed promiscuous gene expression (PGE), which is partially regulated by the Autoimmune regulator (Aire) gene. The T cell precursors derived from the bone marrow migrate to the thymus (now termed thymocytes). A part of these thymocytes are eliminated by negative selection mediated mTEC cells. The surviving cells to evolve and functional mature T cells that migrate to the periphery and are capable of recognizing MHC molecules and are tolerant to PTAs. In addition to controlling the transcription of PTA genes, Aire also controls the expression of microRNAs (miRNAs). The negative selection in the thymus is a process essential to the maintenance of immunologic self-tolerance and imbalance of this process is associated with the development of autoimmune diseases such as type 1 diabetes mellitus (DM1) . Given these assumptions, our work was based on two hypothesis: 1) Changes in the expression of the Aire gene can disrupt the expression of PTA genes and miRNAs in the thymus, causing changes in PGE, 2) The balanced expression of Aire / or PTA genes in mTECs is fundamental for central tolerance. The imbalance in the expression of these genes is associated with the emergence of type 1 diabetes in mice. To test our first hypothesis we made Aire silencing (Aire knockdown) through electrotransfection of anti - Aire interfering RNA (siRNA) in vivo in the thymus of BALB/c mice. Analysis of the transcriptome (mRNAs) and miRNome (miRNAs) of mTECs revealed that partial and transient silencing of Aire was enough to affect the expression of Aire - dependent PTAs as well as miRNAs. miRNA -mRNA interaction networks revealed that the posttranscriptional control of PGE is also affected by the silencing of Aire. The results show that Aire and can form an miRNA pathway essential for the induction of central tolerance. To test our second hypothesis we compared the transcriptome of mTECs of BALB/c mice (non-autoimmune strain) with mTECs from non - obese diabetic NOD (animal model used in studies of autoimmune DM1) . Our results indicate that the transcriptional expression of DM1-related autoantigens are unbalanced in NOD mice in an very early stage, when these animals have not had clinical disease (pre-diabetic period). Unexpectedly, the transcriptional levels of Aire in the thymus was equivalent in these two strains, but the AIRE protein levels were reduced in thymus of NOD strain. These results suggest that some mechanism of post-transcriptional attenuation of Aire is acting in this lineage probably involving action of miRNAs . This could explain the imbalance of Aire - dependent PTAs and repression autoantigens related to DM1. Our results open perspectives for research in this area, contributing to better understanding the molecular mechanisms triggered by Aire and miRNAs in control of the expression of autoantigens in the thymus, which is important for the central immune tolerance.

Le développement d’un lymphome gastrique du MALT (LGM) émane d’un processus inflammatoire chronique initié par Helicobacter pylori.A partir du matériel issu d’un modèle animal de LGM, préalablement développé au laboratoire, basé sur des infections chez des souris thymectomisées à la naissance, la réponse inflammatoire gastrique favorable à l’émergence de LGM a été étudiée. Une dérégulation de cytokines et chimiokines au stade LGM a été identifiée permettant de recruter, faire proliférer et faire émerger des infiltrats lymphoïdes. La susceptibilité des souris thymectomisées à développer des lymphomes n’est pas liée à un déficit en lymphocytes T régulateurs. Cinq microARNs ont été retrouvés dérégulés au stade lymphome agissant probablement en synergie pour favoriser la prolifération lymphocytaire en particulier via un mécanisme anti-apoptotique. Enfin, nous décrivons un modèle original de LGM basé sur l’utilisation de souris C57BL6 exprimant la chimiokine APRIL humaine au niveau des lymphocytes T infectées par des espèces du genre Helicobacter. Ce modèle est prometteur pour une meilleure compréhension de la lymphomagénèse gastrique. / The development of gastric MALT lymphoma (GML) originates from a chronic inflammatory process initiated by Helicobacter pylori.The gastric inflammatory response was investigated in a mouse model of GML previously described by the laboratory using BALB/c mice thymectomized at day 3 post-birth and infected by H. pylori. A deregulation of numerous cytokines and chemokines at GML stage was identified which explained the recruitment, proliferation and emergence of lymphoid infiltrates. The susceptibility of thymectomized mice to develop lymphoma was not linked to a deficiency in regulatory T cells. A deregulation of 5 microRNAs was observed at lymphoma stage. These microRNAs may be involved in cell survival and lymphocyte proliferation and act in synergy to promote the development of GML. Finally, we described an original model of GML based on infection by Helicobacter species of transgenic C57BL6 mice expressing the human form of the cytokine APRIL in T cells. This model is promising for a better understanding of gastric lymphomagenesis.

Nouveau modèle d’étude de l’hépatoblastome in vivo et identification de microARNs régulateurs de la β-caténineL’hépatoblastome (HBL) est le cancer du foie le plus fréquent chez l’enfant. Au niveau moléculaire, il est caractérisé par des mutations activatrices dans le gène de la β-caténine (CTNNB1) entrainant une accumulation de sa protéine et une activation anormale de la voie de signalisation Wnt, responsables de la transformation maligne des cellules hépatiques. Ainsi, la β-caténine est une cible thérapeutique majeure dans l’HBL.Ce manuscrit décrit les deux parties principales de mon projet de thèse, dont l’objectif était d’étudier le rôle de microARNs régulateurs de la β-caténine sur la carcinogenèse de l’HBL.La première partie présente le développement d’un nouveau modèle d’étude in vivo de l’HBL. Ce travail a consisté à réaliser des xénogreffes de cellules dérivées d’HBL sur la membrane chorioallantoidienne (CAM) de l’embryon de poulet, et à étudier le développement des tumeurs par des approches histologiques et moléculaires. Mes résultats montrent que les cellules d’HBL modifient leur phénotype et activent des mécanismes de survie et de prolifération pour former des nodules tumoraux structurés et vascularisés au sein de la CAM. Le traitement des cellules par le cisplatine conduit à un arrêt net de la progression tumorale.La seconde partie présente les résultats concernant la régulation de la β-caténine et des processus oncogéniques par les microARNs dans les cellules d’HBL. Un crible fonctionnel m’a permis d’identifier plusieurs microARNs ayant un effet inhibiteur sur la β-caténine. Quatre microARNs, sous-exprimés dans les tumeurs de patients, bloquent la prolifération cellulaire et l’activité de la voie Wnt in vitro. Le miARN le plus efficace interagit avec l’ARNm de la β-caténine via un site unique localisé dans sa région 3’ non traduite et bloque la croissance des tumeurs d’HBL sur la CAM.Durant ma thèse j’ai développé un nouveau modèle animal d’étude de l’HBL qui permet de modéliser la croissance tumorale et de tester l’impact de molécules thérapeutiques. Ce travail a également permis d’identifier de nouveaux microARNs sous-exprimés dans les tumeurs d’HBL, qui inhibent l’expression de la β-caténine et agissent comme des gènes suppresseurs de tumeur. Ces microARNs constituent de potentielles nouvelles molécules thérapeutiques dans le traitement de l’HBL et pourraient aussi être utilisés comme des biomarqueurs diagnostiques et pronostiques. / Hepatoblastoma (HBL) is the most common pediatric liver cancer. At molecular level it is characterized by activating mutations in β-catenin gene (CTNNB1) that cause protein accumulation and abnormal Wnt signaling pathway activation leading to malignant transformation of hepatic cells. Therefore, β-catenin is a key therapeutic target in HBL.This manuscript describes two major parts of my thesis project that aimed to understand the role of β-catenin-regulating microRNAs in HBL carcinogenesis.The first part presents the development of a new in vivo HBL model. This work consisted of making HBL-derived cells xenograft on chick embryo chorioallantoic membrane (CAM) and studying tumor development using histological and molecular approaches. My results show that HBL cells implanted on CAM modify their phenotype and activate survival and proliferation mechanisms to form organized and vascularized tumor nodules. Cell treatment with cisplatin leads to tumor progression arrest.The second part presents results concerning the regulation of β-catenin and oncogenic processes by microRNAs in HBL cells. A functional screening allowed me to identify several microRNAs with an inhibitory effect on β-catenin. Four microRNAs down-regulated in patient tumors inhibit cell proliferation and Wnt pathway activity in vitro. The most efficient microRNA directly interacts with β-catenin mRNA through a unique site localized in 3’ untranslated region and blocks HBL tumor growth on CAM.During my thesis I developed a new animal model to study HBL that allows modelling rapid tumor growth and testing the impact of therapeutic molecules. My work also led to the identification of new microRNAs down-regulated in HBL tumors that inhibit β-catenin expression and act as tumor suppressor genes. Those microRNAs represent new potential therapeutic molecules for HBL treatment and could be also used as diagnostic and prognostic biomarkers.

L'ovocyte se développe au sein d'un follicule, en contact étroit avec des cellules d'origine somatique, les cellules de cumulus (CC). Ces deux types cellulaires communiquent entre eux via des jonctions intercellulaires, permettant ainsi la régulation et la coordination du métabolisme pendant le développement et la maturation de l'ovocyte. Notre hypothèse est que l'expression et la régulation des gènes dans les CC joue un rôle crucial dans des fonctions essentielles pour la croissance de l'ovocyte et l'acquisition de sa compétence. Mes travaux de thèse comportent deux parties. Dans la première partie nous avons utilisé le séquençage haut débit pour examiner le répertoire des microARN (communément appelés miRNA) dans les cellules de cumulus et dans l'ovocyte. Les miRNA, séquences d'ARN non codantes dont la longueur varie entre 19 et 25 nucléotides, ont émergé récemment comme régulateurs majeurs de nombreux processus biologiques, dont le vieillissement. Nous avons identifié 32 miRNA spécifiquement dans les cellules de cumulus humains et seulement 3 dans l'ovocyte MII. Dans la seconde partie de nos travaux, nous avons analysé l'impact de l'âge maternel sur l'expression des gènes dans les cellules de cumulus. Alors qu'une baisse de la compétence de l'ovocyte avec l'avancement de l'âge maternel est bien établie, les bases moléculaires de ce phénomène demeurent peu connues. Dans une première étape pour aborder cette question, nous avons utilisé des puces à ADN pour analyser les profils d'expression des gènes des CC en fonction de l'âge maternel. De façon remarquable l'âge maternel impacte significativement l'expression de gènes qui sont critiques pour la maturation de l'ovocyte tels que les gènes impliqués dans l'angiogenèse, les voies de signalisation de TGF-ß et de l'insuline. Par l'utilisation d'outils bioinformatiques, nous avons aussi identifié des miRNA potentiels régulateurs de gènes impliqués dans des processus ou des voies impactés par l'âge ; ils pourraient constituer de nouveaux biomarqueurs pour prédire un vieillissement ovarien prématuré ainsi que la qualité et la compétence de l'ovocyte. / The oocyte develops into a follicle where it is in close contact with cumulus cells (CCs), of somatic origin. The two cell types undergo a bidirectional communication via gap junctions, which results in the regulation and coordination of the metabolism during oocyte development and maturation. We assume that gene expression and regulation in the CCs play a crucial role in functions that are essential for oocyte growth and competence acquisition. The present study may be subdivided in two parts. In the first part we used deep sequencing to investigate the repertoire of miRNAs in the cumulus cells and the oocyte. MicroRNAs that are noncoding RNA sequences whose length is approximately 19-25 nucleotides have emerged as important regulators in many biological processes including aging. Our data showed that 32 miRNAs were specifically expressed in human cumulus cells while only 3 miRNAs were identified in MII human oocyte. The impact of maternal age on gene expression in cumulus cells was addressed in a second part of my thesis work. While the correlation of oocyte competence decline with advancing maternal age is well established, little is known on its molecular basis. In a first attempt to address this issue, we used microarrays to study gene expression profiles of human cumulus cells according to maternal age. Remarkably, maternal age greatly impacted expression of genes that are critical for oocyte maturation such as genes involved in angiogenesis, TGF-β signaling, and insulin signaling pathways. Also, using bioinformatic tools, we identified miRNAs that potentially target some of the genes involved in the aging-impacted processes and pathways; this could candidate them as new biomarkers to predict premature ovarian aging and oocyte quality and competence.

Dans les cancers du sein triple négatif sporadiques, BRCA1 est fréquemment inactivé au niveau transcriptionnel, et il a été rapporté que cette inactivation peut être réalisée par une méthylation du promoteur. Plus récemment, il a été constaté que BRCA1 peut également être régulée au niveau post-transcriptionnel par les microARNs. L'accumulation de preuves indique que les miARNs ont un rôle causal dans la tumorigenèse. Nos travaux se sont axés sur l'étude de l’expression et des fonctions des microARN in vitro, in silico et ex vivo.Basé sur nos résultats de profilage de l'expression, quatre miARN candidats (miR-10b, miR-26a, miR-146a et miR-153) ont été choisis comme étant potentiellement impliqués dans le développement du cancer du sein triple négatif. Des essais d'expression exogènes ont révélé que miR-10b et miR-26a, mais pas miR-146a, peuvent réguler négativement l'expression du gène BRCA1 dans les cellules cancéreuses triple négatif MDA-MB-231 et luminales MCF7, alors que miR-153 pourrait réguler négativement l'expression du gène BRCA1 uniquement dans les cellules MCF7. L'analyse in silico des données de Cancer Genome Atlas (TCGA) a confirmé que miR-146a est significativement plus exprimé dans les tumeurs du sein triple négatif par rapport à d'autres tumeurs (non triple négatif) mammaires. L’étude ex vivo a montré que le niveau élévé d’expression de miR-146a et de miR-26 est associé à l’absence des métastases ganglionnaires dans le cancer du sein triple négatif. Aussi une corrélation entre l’expression de 4 miARNs est révélée permettant l’identification de différentes voies de signalisations impliquées dans le cancer du sein triple negatif.Nos travaux fournissent des preuves de l'implication des miARNs spécifiques comme des biomarqueurs potentiels dans le développement du cancer de sein triple négatif. / In sporadic triple-negative breast cancers BRCA1 is frequently inactivated at the transcriptional level, and it has been reported that this inactivation may be brought about by promoter methylation. More recently, it was found that BRCA1 may also be regulated at the post-transcriptional level by miRNAs. Accumulating evidence indicates that miRNAs have a causal role in tumorigenesis. Our work focused on the study of microRNAs expression and functions in vitro, in silico and ex vivo.Based on our expression profiling results, four candidate miRNAs (miR-10b, miR-26a, miR-146a and miR-153) were selected as being potentially involved in triple-negative breast cancer development. Exogenous expression assays revealed that miR-10b and miR-26a, but not miR-146a, can down-regulate the expression of BRCA1 in both triple-negative MDA-MB-231 and luminal epithelial MCF7 breast cancer-derived cells, whereas miR-153 could down-regulate BRCA1 expression only in MCF7 cells. In silico analysis of The Cancer Genome Atlas (TCGA) data confirmed that miR-146a is significantly higher expressed in triple-negative breast tumors compared to other (non triple-negative) breast tumors. The ex vivo study showed that the high level expression of miR-146a and miR-26 is associated with the absence of lymph node metastasis in triple negative breast cancer. Also a correlation between the expression of the 4 miRNAs was revealed, allowing the identification of different signaling pathways involved in the triple negative breast cancer.Our work provides evidence of the involvement of specific miRNAs as potential biomarkers in breast cancer triple negative development.

Argonaute proteins are the core component of an RNA silencing complex. The human genome encodes four Argonaute paralogs –Ago1, Ago2, Ago3 and Ago4– proteins that are guided to target mRNAs by microRNAs. More than 500 miRNAs are conserved between mammals, and each microRNA can repress hundreds of genes, regulating almost every cellular process. We still do not fully understand the molecular mechanisms by which miRNAs regulate gene expression. Although we understand many aspects of microRNA biogenesis and formation of the RNA-induced silencing complex, much less is known about the subsequent steps leading to target mRNA regulation. Mammalian microRNAs rarely have complete complementarity to their target mRNAs so, instead of endonucleolytic cleavage by Ago2, microRNAs destabilize or repress translation of target mRNAs. Here I explored the functional limits of Argonaute proteins bound to their targets directly and indirectly through microRNAs in mammalian cells. I revealed the different abilities for Argonaute proteins bound at multiple sites in a target to generate cooperativity in silencing based on the extent of pairing between the microRNA and target mRNA. Further, I harnessed the endogenous microRNA silencing mechanism to repress an mRNA that is not a direct target of the microRNA by tethering the RNA-induced silencing complex to the 3´ UTR of an mRNA. This strategy allows tissue-specific gene silencing due to the limited endogenous expression profile of the recruited microRNA. Efforts made herein further our mechanistic knowledge of microRNA-induced gene silencing in mammalian cells and advance microRNA-based strategies toward treating human disease.