Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-03-16T14:51:26Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-03-16T14:51:39Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) / Made available in DSpace on 2018-03-16T14:51:39Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) Previous issue date: 2017-11-25 / Introduction: The colloid goiter (CG) is a heterogeneous disorder and represents the main cause of benign thyroid nodule. Prevalent in geographical areas with iodine deficiency, in most cases carried out expectant conduct or surgically. Classified according to the morphological and functional characteristics, it may present as diffuse or nodular, toxic or nontoxic (with or without functional autonomy). Can reach large volume and compression of neck structures or even turn into malignant disease. Dependent on environmental and genetic factors, with iodine deficiency being the main determinant for the impact on incidence, other environmental and hereditary factors are listed, but little is known about the molecular pathogenesis. Objectives: To map the Epigenetic alterations of the five different surgical parts of CG from patients submitted to total thyroidectomy in Manaus, Amazonas, to analyze, compare and describe the specific pattern for this population. Methods: Five specimens of CG with histopathological confirmation were obtained from the “Normal Thyroid Tissue Bank and with Goiter” from the UFAM. Were used to identify the global methylation patterns of DNA and histone 3 in trimethylated lysine 4, 9 and 27 (H3K4me3, H3K9me3 and H3K27me3). Was used an ELISA type specific immunoassay method, with the results compared between the patients, between the different region of the gland in the same individual and between the individuals. Results: there was no evidence there were significant differences (5%) in the mean global DNA methylation patterns between the patients (p = 0.114) or between the thyroid regions (p = 0.843) considered similar. The mean pan-methylation patterns of histones H3K4me3, H3K9me3 and H3K27me3 are significantly different among patients (p <1%), although there were no significant differences between parts in the same individual (p> 5%). Conclusion: The results of the analyzes of this study revealed that CG parts obeyed a pattern of distribution of significant DNA hypermethylation between patients and parts of CG, differing only among patients the pattern of histone methylation. As these findings are more common in cancer, we may suggest that at the epigenetic level CG may have some common etiologic factor that leads to this tissue response or that responds by phenotypic expressions and malignant transformation. There are now no efficient markers that accurately detect the risk of CG malignancy, much less the possibility of defining its tissue progression, it is very likely that multiple genes will participate in this process. Therefore, the findings found in these patients may make way for the future application of the CG epigenetic study, which may imply the viability of new therapeutic or preventive forms of progression, since the methylation status can be reversed, but other studies in the population are needed to understand better those finding. / Introdução: O bócio coloide (BC) constitui numa desordem heterogênea e representa a principal causa de nódulo benigno da tireoide. Prevalente em áreas geográficas com deficiência de iodo, sendo, na maioria dos casos, conduzido de forma expectante ou cirúrgica. Classificado de acordo com as características morfológicas e funcionais, pode se apresentar difuso ou nodular, tóxico ou atóxico (com ou sem autonomia funcional), podendo alcançar grandes volumes e ocasionar compressão de estruturas do pescoço, ou, não muito raramente, sofrer transformação maligna. Dependente de fatores ambientais e genéticos, com a deficiência de iodo sendo o principal fator determinante para o impacto na incidência, outros fatores ambientais e hereditários são arrolados, porém pouco se conhece da patogênese molecular. Objetivos: mapear as alterações epigenéticas de diferentes regiões de 05 espécies cirúrgicas de BC de pacientes submetidos a tireoidectomia total em Manaus, Amazonas, com o objetivo de analisar, comparar e descrever o padrão específico para esta população. Metodologia: Foram utilizados 05 espécimes de BC com confirmação histopatológica, provenientes do “Banco de Tecidos de Tireoide Normal e com Bócio” da UFAM, e para identificar os padrões de metilação global do DNA e da histona 3 na lisina trimetiladas 4, 9 e 27 (H3K4me3, H3K9me3 e H3K27me3) foram utilizados método de imunoensaio específico do tipo ELISA, com os resultados comparados entre as pacientes, entre as diferentes regiões da glândula num mesmo indivíduo e entre os indivíduos. Resultados: não houveram evidências de que existam diferenças significativas (ao nível de 5%) dos padrões médios de metilação global do DNA entre os pacientes (p = 0,114), ou entre as regiões da tireoide (p = 0,843), sendo, portanto, consideradas similares. Os padrões médios de pan-metilação das histonas H3K4me3, H3K9me3 e H3K27me3 são significativamente diferentes entre os pacientes (p < 1%), apesar de não apresentar diferenças significativas entre as regiões num mesmo indivíduo, (p > 5%). Conclusão: Os resultados das análises deste estudo, revelou que os espécimes de BC obedeceram a um padrão de distribuição de hipermetilação do DNA significativa entre os pacientes e as regiões do BC, diferindo somente entre os pacientes o padrão de metilação das histonas. Como estes achados são mais comuns no câncer, podemos sugerir que em nível epigenético, o BC possa ter algum fator etiológico comum que leve a esta resposta tecidual ou que responda pelas expressões fenotípicas e transformação maligna. Como, até o momento, não existem marcadores eficientes que detecte com precisão a existência de risco de malignização do BC, muito menos a possibilidade de definir sua progressão tecidual, é muito provável que participem múltiplos genes neste processo. Portanto, os achados encontrados nestas pacientes podem abrir caminho na aplicação futura do estudo epigenético do BC, podendo implicar na viabilização de novas formas terapêuticas ou preventivas da progressão, uma vez que, o status de metilação pode ser revertido, porém outros estudos ampliados na população em questão são necessários para entender melhor estes achados.

O presente trabalho teve por objetivo compreender e analisar os mecanismos genéticos e epigenéticos da expressão diferencial de regiões organizadoras do nucléolo - RONs através do estudo de dois acessos (CRT-1 e CRT-2) de Crotalaria retusa. O acesso CRT-1 é uma cultivar, enquanto que o acesso CRT-2 é proveniente de uma população periférica da orla marítima de Ilhéus BA. Por serem temporalmente e espacialmente separados, acredita-se que os acessos foram submetidos a pressões seletivas diferentes, resultando em alterações dos padrões epigenéticos, principalmente nas RONs. Para o desenvolvimento deste trabalho foram realizadas medidas cromossômicas e nucleolares a partir de células coradas pelo método de Feulgen e por nitrato de prata, coloração com fluorocromos específicos às regiões cromossômicas ricas em nucleotídeos GC e AT, mapeamento físico dos locos de DNA ribossômico 45S por hibridação in situ fluorescente, análise qualitativa e quantitativa de modificações pós-traducionais de histonas por Western blot e eletroforese bidimensional de extrato protéico radicular com enfoque em proteínas envolvidas nos mecanismos epigenéticos. As análises citológicas demonstraram uma grande semelhança nos cariótipos dos dois acessos, diferindo apenas no tamanho do segmento proximal do braço curto do cromossomo 1. Em ambos os acessos foi observada uma expressão nucleolar diferencial em, aproximadamente, 50% das células; contudo, a expressão diferencial em CRT-2 apresentou-se consideravelmente maior. Além disso, os dois acessos demonstraram diferenças quantitativas nas modificações pós-traducionais de histonas e em proteínas possivelmente envolvidas em mecanismos epigenéticos. Uma vez que as variações epigenéticas podem ser modificadas por fatores ambientais, sugere-se que as diferenças nos padrões de modificações de histonas e nos perfis protéicos encontradas entre os acessos, como também a expressão diferencial mais expressiva em CRT-2, sejam devidas às diferentes pressões seletivas as quais as populações originais foram submetidas. O estudo dos mecanismos genéticos e epigenéticos na dominância nucleolar possibilita uma maior compreensão da ação do remodelamento da cromatina no controle da expressão gênica do rDNA, como também da expressão gênica em geral. / The aim of this present work was to understand and analyze the genetic and epigenetic mechanisms of differential expression of the nucleolus organizer regions - NORs through the study of two accesses (CRT-1 and CRT-2) of Crotalaria retusa. Access CRT-1 is a cultivar, while access CRT-2 is from a peripheral population of the shoreline of Ilhéus BA. Because they are temporally and spatially separated, it is believed that the accesses were submitted to different selective pressures, resulting in changes in epigenetic patterns, primarily in NORs. To develop this work, it was carried out chromosomal and nucleolar measurements from cell stained by Feulgen method and silver nitrate, staining with specific fluorochromes to chromosomal regions rich in GC and AT nucleotides, physical mapping of 45S ribosomal DNA loci by fluorescent in situ hybridization, qualitative and quantitative analysis of post-translational histone modifications by western blot, and two-dimensional electrophoresis of root extract protein focusing on proteins involved in epigenetic mechanisms. The cytological analysis showed a great similarity in karyotypes of two accessions, differing only in size of the proximal segment of the sort arm of chromosome 1. In both accesses, it was observed a differential nucleolar expression in approximately 50% of the cells; however, the differential expression in CRT-2 showed considerably larger. Furthermore, the two accesses showed quantitative differences in the posttranslational histone modifications, and in a protein possibly involved in epigenetic mechanisms. Since epigenetic variations can be modified by environmental factors, it is suggested that differences in patterns of histone modifications and protein profiles found between the accesses, but also the most significant differential expression in CRT-2, are due to different selective pressures to which the original populations were submitted. Studies of the epigenetic mechanisms in nucleolar dominance allows a better understanding of the action of the remodeling of chromatin in controlling the dosage of rRNA genes, but also in the control of gene expression in general.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-08-22T17:01:25Z No. of bitstreams: 1 tamiresschaefferfintoura.pdf: 578082 bytes, checksum: 3c65a5dbd95e0a9f922d872aa60cb3d2 (MD5) / Rejected by Adriana Oliveira (adriana.oliveira@ufjf.edu.br), reason: on 2017-08-24T11:36:03Z (GMT) / Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-08-24T14:11:45Z No. of bitstreams: 0 / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-30T12:41:02Z (GMT) No. of bitstreams: 0 / Made available in DSpace on 2017-08-30T12:41:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-02-15 / A obesidade é um distúrbio multifatorial complexo causada por fatores genéticos e epigenéticos, dentre outros. Um meio intra-uterino adverso, com excesso ou escassez de alimentos pode expor o feto ao desenvolvimento da síndrome metabólica, diabetes e doenças cardiovasculares quando adultos, podendo ser passado transgeracionalmente através de alterações epigenéticas. O exercício físico é uma forma não medicamentosa de prevenção e tratamento da obesidade, melhorando a aptidão cardiorrespiratória e composição corporal, dentre outros fatores. Este estudo avaliou se a dieta rica em sacarose e lipídios dada a fêmeas da geração F0 durante toda sua vida, incluindo gestação e lactação, afetaria os níveis de ansiedade e os padrões de memória transgeracionalmente e se o exercício físico possui efeito protetor sobre os mesmos parâmetros. Ratas da geração F0 foram distribuídas em quatro grupos: controle sedentário (CSed) e exercitado (CEx); dieta sedentário (DSed) e exercitado (DEx). Dos 21 aos 120 dias de vida os animais realizaram treinamento físico em esteira, com intensidade de moderada a intensa, e os animais controle receberam dieta normocalórica e os animais dieta receberam a dieta high sugar/ high fat (HS/HF) no mesmo período. Machos e fêmeas da geração F1 e F2 foram distribuídos em grupos iguais ao da geração materna ao qual pertenciam; porém, consumiram a ração normocalórica e não realizaram treinamento físico. Foi acompanhado o consumo alimentar dos animais e a evolução do peso corporal dos mesmos. Aos 80 dias, foi realizado teste oral de tolerância à glicose (TOTG). Aos 90 dias, todos os animais foram submetidos a testes, para avaliação do nível de ansiedade (labirinto em cruz elevado) memórias de trabalho (30 seg) e longo prazo (24h) (teste de esquiva inibitória). Aos 110 dias, houve a eutanásia e os parâmetros biométricos foram avaliados. A dieta normocalórica e o treinamento físico realizado pela geração F0 promoveram menor acúmulo de gordura nos filhotes machos e fêmeas da geração F1 quando comparados ao CSed1 (gordura retroperitonial: 37,7% nas fêmeas e 27,6% nos machos; gordura perigonadal: 28,16% nos machos). Diferentemente do que aconteceu na geração F1, os machos do grupo DSed2 apresentaram um maior acúmulo de gordura perigonadal, 19,4%, quando comparados ao grupo Csed2. Adicionalmente, o efeito protetor do treinamento físico com relação ao menor acúmulo de gorduras retroperitoniais e perigonadais na geração F2 só aconteceu no grupo DEx2 e nos machos, com redução de 21% e 26,8%, respectivamente, em relação ao grupo DSed2. As fêmeas dos grupos DSed das gerações F1 e F2 não ficaram intolerantes a glicose, contudo houve redução de 11,2% na glicemia das fêmeas do grupo DEx1 quando comparadas ao grupo DSed1. Ao contrário do que aconteceu nas fêmeas da primeira geração, os machos do grupo DSed1 ficaram intolerantes a glicose, com aumento da glicemia em 18,45% quando comparados ao grupo CSed1. O grupo DEx1 e DEx2 apresentaram redução da glicemia em 18,45% e 24,75% quando comparados ao grupo DSed. A dieta HS/HF consumida pela geração F0 reduziu a memória de longo prazo das fêmeas do grupo DSed2 em 88,6% quando comparadas ao grupo CSed2. Também aumentou os níveis de ansiedade dos animais machos da geração F1 do grupo DSed1 em relação aos grupos CSed1 e DEx1. A dieta HS/HF aumentou o nível de ansiedade dos machos da geração F1 e causou prejuízo na memória de longo prazo das fêmeas da geração F2, além de aumentar a glicemia dos filhotes machos do grupo dieta da primeira geração e, causar maior acúmulo de gordura na geração F2. O treinamento físico teve efeito protetor sobre a glicemia da descendência, reduziu a adiposidade dos filhotes machos e fêmeas de F1 (grupo controle). Tal efeito só se manteve na geração F2 nos filhotes machos do grupo dieta. A dieta HS/HF consumida por fêmeas desde o desmame até a lactação altera o metabolismo, a memória e os níveis de ansiedade nas gerações F1 e F2, particularmente entre os machos. O treinamento físico de intensidade moderada a intensa realizada pela geração F0 protege a progênie. / Obesity is a complex multifactorial disorder caused by genetic and epigenetic factors, among others. An adverse intrauterine environment with excess or scarcity of food may expose the fetus to the development of metabolic syndrome, diabetes, and cardiovascular disease in adults, and may be transgenerationally passed through epigenetic changes. Physical exercise is a non-medicated form of prevention and treatment of obesity, improving cardiorespiratory fitness and body composition, among other factors. This study evaluated whether the diet rich in sucrose and lipids given to females of the F0 generation throughout their life, including gestation and lactation, would affect anxiety levels and memory patterns transgenerationally and if physical exercise has protective effect on the same parameters. Rats of the F0 generation were divided into four groups: sedentary (CSed) and exercised (CEx) controls; Sedentary (DSed) and exercise diet (DEx). From 21 to 120 days of age, the animals performed physical training on a treadmill at moderate to high intensity, and the control animals received normocaloric diet and the diet animals received the high sugar / high fat (HS / HF) diet during the same period. Males and females of the F1 and F2 generation were distributed in equalgroupsl to the maternal generation to which they belonged; however, consumed the normocaloric chow and did not perform physical training. The food consumption of the animals and their body weight were monitored. At 80 days, oral glucose tolerance test (OGTT) was performed. At 90 days, all animals were submitted to tests, for evaluation of anxiety level(elevated plus-maze) working memory (30 sec) and long-term (24h) (inhibitory avoidance test). At 110 days, there was euthanasia and the biometric parameters were evaluated. The normocaloric diet and physical training performed by the F0 generation promoted a lower accumulation of fat in the offspring, male and female from F1 generation, compared to CSed1 (retroperitoneal fat: 37.7% in females and 27.6% in males, perigonadal fat 28.16%, in males). Differently from what happened in the F1generation, the males of the DSed2 group presented a higher accumulation of perigonadal fat, 19.4%, when compared to the group Csed2. Additionally, the protective effect of physical training in relation to the lower accumulation of retroperitoneal and perigonadal fats in the F2 generation occurred only in the DEx2 group and in the males, with a reduction of 21% and 26.8%, respectively, in relation to the DSed2 group. Females of the DSed groups of the F1 and F2 generations were not intolerant to glucose, however, there was a 11.2% reduction in the glycemia of the females of the DEx1 group when compared to the DSed1 group. In contrast to what happened in the first-generation females, males from the DSed1 group were intolerant to glucose, with an increase in blood glucose by 18.45% when compared to the CSed1 group. The group DEx1 and DEx2 had a reduction in blood glucose of 18.45% and 24.75% when compared to the DSed group. The HS / HF diet consumed by the F0 generation reduced the long-term memory of the females of the DSed2 group by 88.6% when compared to the CSed2 group. It also increased the anxiety levels of male F1 generation from the DSed1 group in relation to the CSed1 and DEx1 groups. The HS / HF diet increased the anxiety level of the F1 generation males and caused long-term memory injury in females of the F2 generation, in addition to increasing the glycemia of the first-generation diet group males and causing higher fat accumulation In the F2 generation. Physical training had a protective effect on offspring glycemia, reducing the adiposity of male and female F1 pups (control group). This effect was only maintained in the F2 generation in the male offspring of the diet group. The HS / HF diet consumed by females from weaning to lactation alters metabolism, memory and anxiety levels in the F1 and F2 generations, particularly among males. Physical training of moderate to intense intensity performed by the F0 generation protects the progeny.

Les éléments transposable (ET) sont des séquences d'ADN qui ont la capacité de se déplacer au sein des génomes. Pour contrebalancer les effets négatifs liés à l'activité des ET, il existe chez leurs hôtes des mécanismes régulant l'activité de transposition. Une fois qu'un ET est régulé, l'accumulation progressive de mutations dans sa séquence conduit fatalement à la perte définitive de son activité de transposition. J'ai cherché au cours de cette thèse à mieux comprendre le succès et le maintien de ces séquences répétées, avec d'une part l'étude des transferts horizontaux (TH) d'ET, un moyen d'échapper aux mécanismes de régulation , et d'autre part l'étude de leur régulation. Dans la première partie de ma thèse, je me suis intéressé à l'étude des TH entre deux espèces proches de drosophiles. Dans cette étude, j'ai développé une nouvelle méthode bioinformatique permettant la détection de séquences transférées horizontalement entre deux génomes eucaryotes qui m'a permis détecter de nombreux TH d'ET. Ce travail m'a aussi conduit à développé une nouvelle méthode de contrôle du taux de faux positifs moyen applicable aux tests multiples unilatéraux. Dans la deuxième partie de ma thèse, j'ai étudié la régulation des ET par la voie des petits ARN, un mécanisme de l'ARN interférence. Dans cette étude, j'ai analysé des données de séquençage de petits ARN, ainsi que d'ARN totaux issues de différentes populations de D. simulans. Ce travail a conduit au développement d'un pipeline d'analyse permettant d'étudier des différences d'expression entre des séquences répétées ainsi que d'une nouvelle procédure de contrôle qualité de ce type de donnée / Transposable elements (TEs) are repeated DNA sequences that are able to move (transpose) within their host genome. To counteract the negative effects of their TEs, regulation mechanisms of the TE transposition are present in the host genome. Once a TE is regulated, the progressive accumulation of mutations in its sequence will inevitably lead to the definitive loss of its transposition capacity. My work during this thesis is was to better understand the succss and the maintaining of these peculiar repeated sequencest, with the study of horizontal transfers (HTs) of TEs enabling them to escape host regulation mechanisms, and the study of this regulation. The first part of my thesis concerns the study of HTs between two closely related drosophila species. I have developed a new bioinformatic method for the detection of HTs between two eukaryotic genomes. The development of this method brought me to work on the unilateral multiple testing problematic for which I have developed a new procedure to control the expected false discovery rate (FDR). The second part of my thesis focuses on the regulation of TEs by the small RNA pathway, an RNA interference mechanism. For this study, I have analyzed sequencing data of small RNAs and total RNAs. For this work, I have developed an analysis pipeline, to study differences of expression between repeated sequences. Some features of the small RNA dataset required the development of a new procedure to parse them. This procedure was extended and implemented in a software to be used for the quality control of next generation sequencing data

L’altération des marques épigénétiques est de plus en plus reconnue comme une caractéristique fondamentale des cancers. Dans cette thèse, nous avons utilisé des profils de méthylation de l’ADN en vue d’améliorer la classification des patients atteints du cancer du sein grâce à une approche basée sur l’apprentissage automatique. L’objectif à long terme est le développement d’outils cliniques de médecine personnalisée. Les données de méthylation de l’ADN furent acquises à l’aide d’une puce à ADN dédiée à la méthylation, appelée Infinium. Cette technologie est récente comparée, par exemple, aux puces d’expression génique et son prétraitement n’est pas encore standardisé. La première partie de cette thèse fut donc consacrée à l’évaluation des méthodes de normalisation par comparaison des données normalisées avec d’autres technologies (pyroséquençage et RRBS) pour les deux technologies Infinium les plus récentes (450k et 850k). Nous avons également évalué la couverture de régions biologiquement relevantes (promoteurs et amplificateurs) par les deux technologies. Ensuite, nous avons utilisé les données Infinium (correctement prétraitées) pour développer un score, appelé MeTIL score, qui présente une valeur pronostique et prédictive dans les cancers du sein. Nous avons profité de la capacité de la méthylation de l’ADN à refléter la composition cellulaire pour extraire une signature de méthylation (c’est-à-dire un ensemble de positions de l’ADN où la méthylation varie) qui reflète la présence de lymphocytes dans l’échantillon tumoral. Après une sélection de sites présentant une méthylation spécifique aux lymphocytes, nous avons développé une approche basée sur l’apprentissage automatique pour obtenir une signature d’une tailleoptimale réduite à cinq sites permettant potentiellement une utilisation en clinique. Après conversion de cette signature en un score, nous avons montré sa spécificité pour les lymphocytes à l’aide de données externes et de simulations informatiques. Puis, nous avons montré la capacité du MeTIL score à prédire la réponse à la chimiothérapie ainsi que son pouvoir pronostique dans des cohortes indépendantes de cancer du sein et, même, dans d’autres cancers. / Epigenetic alterations are increasingly recognised as an hallmark of cancers. In this thesis, we used a machine-learning-based approach to improve breast cancer patients’ classification using DNA methylation profiling with the long term aim to make treatment more personalised. The DNA methylation data were acquired using a high density DNA methylation array called Infinium. This technology is recent compared to expression arrays and its preprocessing is not yet standardised. So, the first part of this thesis was to evaluate the normalisation methods by comparing normalised data against other technologies (pyrosequencing and RRBS) for the two most recent Infinium arrays (450k and 850k).We also went deeper into the evaluation of these arrays by assessing their coverage of biologically relevant regions like promoters and enhancers. Then, we used accurately preprocessed Infinium data to develop a score, called MeTIL score, which shows prognostic and predictive value in breast cancers. We took advantage that DNA methylation can mirror the cell composition to extract a DNA methylation signature (i.e. a set of DNA methylation sites) that reflects presence of lymphocytes within the tumour. After an initial selection of lymphocyte-specific sites we developed a machine-learning-based framework which reduced the predictive set to an optimal size of five methylation sites making it potentially suitable to use in clinics. After conversion of this signature to a score, we showed its specificity to lymphocytes using external datasets and simulations. Then, we showed its ability predict response to chemotherapy and, finally, its prognostic value in independent breast cancer cohorts and even in other cancers. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Le séquençage de nombreux génomes eucaryotes indique que l’augmentation de la «biocomplexité» au cours de l’évolution n’est pas directement corrélée à l’accroissement du nombre de gènes. En d’autres termes, nous sommes plus que la somme de nos gènes et l’ère post-génomique actuelle promet de cerner de façon plus précise les bases moléculaires de notre identité. A cet égard, il semble de plus en plus clair que l’épigénétique est riche d’une information qui se superpose à celle du code génétique. L’information épigénétique est principalement véhiculée par des modifications de l’ADN et des histones. La modification majeure de l’ADN est la méthylation de la cytosine qui est la marque d’une chromatine transcriptionnellement silencieuse. Quant aux histones, différentes modifications posttraductionnelles ont été décrites, comme l’acétylation, la phosphorylation, la méthylation et l’ubiquitinylation. L’ensemble de ces modifications constituerait un «code histone», dont le décryptage n’en est qu’à ses prémices, permettant d’associer à chaque combinaison de modifications un état particulier de la chromatine, et ainsi de l’expression génique. La méthylation des histones a longtemps été considérée comme irréversible mais l'identification récente de déméthylases des histones spécifiques de certains sites a révélé que cette modification est régulée de façon dynamique et réversible. La découverte de ces enzymes a ouvert de nouveaux axes de recherche dans le domaine de l'épigénétique (Klose and Zhang, 2007). <p><p>Au cours de ma thèse de doctorat, nous nous sommes intéressés à la déméthylase PADI4 (peptidylarginine déiminase 4) qui convertit des résidus arginines des histones H3 et H4, associés à l'activation des gènes, en résidus citrullines, ce qui a pour conséquence d'entraîner une répression transcriptionnelle. Cette réaction porte le nom plus particulier de déimination/citrullination des histones. A l’heure actuelle, il est primordial de cerner comment la déméthylation des histones, et plus précisément la peptidylarginine deiminase 4 (PADI4), réprime la transcription. <p><p>Dans un premier temps, nous avons mis en évidence un lien mécanistique entre la deméthylation et la désacétylation des histones. Nous avons montré que PADI4 interagit avec l’histone désacétylase HDAC1. Cette enzyme est responsable du décrochage des groupements acétyls des histones, conduisant à la fermeture de la chromatine. Des expériences d’immunoprécipitation de la chromatine indiquent que PADI4 et HDAC1 s’associent au promoteur du gène de réponse aux oetrogènes pS2 simultanément et de manière cyclique. L’utilisation d’une construction shRNA dirigée contre la protéine endogène HDAC1 indique que la liaison de PADI4 au promoteur du gène pS2 est dépendante de la présence de HDAC1.<p><p>Dans la deuxième partie de notre travail, un lien mécanistique entre la déméthylation des histones par PADI4 et la méthylation de l’ADN a été mis en évidence. La méthylation de l’ADN est catalysée par des enzymes, appelées méthyltransférases de l’ADN (DNMTs), qui transfèrent des résidus méthyls sur les cytosines. Nous avons montré que la protéine DNMT3A interagit avec PADI4. Nous avons également démontré que l’enzyme PADI4 était capable de citrulliner/déiminer (convertir des résidus arginines en résidus citrullines) la méthyltransférase de l’ADN DNMT3A in vitro et que cette citrullination de la protéine DNMT3A par PADI4 stabiliserait DNMT3A in vivo.<p><p>Enfin, nos récents travaux révèlent une relation mécanistique entre la protéine MeCP2, interprète des signaux de méthylation de l’ADN, et la protéine polycomb EZH2. Celle-ci possède une activité méthyltransférase d’histone sur les lysines 27 de l’histone H3. Nos données montrent que MeCP2 interagit avec EZH2 et que ces protéines fixent des régions promotrices communes. De plus, la déplétion en MeCP2 affecte la présence de EZH2 au niveau de ces régions communes.<p><p>En conclusion, ce travail de thèse devrait permettre une meilleure compréhension des mécanismes moléculaires de l’épigénétique. Plus particulièrement, il devrait aider à mieux cerner comment la première histone déméthylase décrite, la peptidylarginine déiminase 4 ou PADI4, verrouille l’expression génique.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

L’acétylation des histones est associée à une activation transcriptionnelle. Cette acétylation est mise en place par des histone acétyltransférases (HATs) qui sont le plus souvent les sous-unités catalytiques de complexes multiprotéiques. Mon travail concerne plus particulièrement deux complexes contenant l’acétyltransférase Mof, MSL et NSL, ainsi que le complexe HAT Tip60-p400 dans les cellules souches embryonnaires de souris (mESCs). Nos analyses de localistaion sur l’ensemble du génome par ChIP-seq indiquent que MSL, NSL et Tip60-p400 se lient aux gènes activement transcrits et agissent comme des co-activateurs transcriptionnels majeurs dans les mESCs. MSL, NSL et Tip60-p400 ont des rôles à la fois chevauchants mais aussi distincts dans la régulation transcriptionnelle dans les mESCs. Chaque complexe présent un profil distinct de liaison à la chromatine. NSL lie principalement des gènes de ménage. MSL et Tip60-p400 sont également présent les gènes impliqués dans le développement. MSL est directement impliqué dans l’augmentation de l’expression de ces gènes au cours de la différenciation des mESCs. / Histone acetylation is involved in transcriptional activation of genes and is carried out by histone acetyltransferases (HATs), which are part of molecular protein complexes. This study focuses on the genome-wide role of Mof-containing MSL and NSL complexes and the Tip60-p400 complex in mouse embryonic stem cells (mESCs). I have analysed these complexes by ChIP-seq, shRNA knockdown and biochemical approaches. The genome-wide binding studies show that NSL, MSL and Tip60-p400 have a global overlap at promoters, but also bind to specific gene sets. There distinct binding profiles propose distinct roles in transcriptional regulation. MSL is the main H4K16 acetylase in mESCs.NSL binds mainly to housekeeping genes, whereas MSL and Tip60 are also present at developmental genes. Importantly, these developmental genes are directly regulated by MSL during cellular differentiation.

Writer, eraser, and reader proteins are three classes of proteins/enzymes that add, remove, and recognize post-translational modifications (PTMs) on histone tails, respectively. The orchestrated action of these protein classes controls dynamic state of chromatin and influences gene expression. Dysregulation of these proteins are often associated with disease conditions. All three classes are targeted with small molecule inhibitors for various disease conditions. This is a promising area of research to develop therapeutics for various clinical conditions. I worked on a methyllysine reader protein Cbx7, which belong to polycomb group of proteins. Cbx7 is a chromodomain containing protein and it uses its chromodomain to recognize methyllysine partners such as H3K27me3. Aberrant expression of Cbx7 is observed in several cancers including prostate, breast, colon, thyroid, etc. Hence targeting Cbx7 with potent and selective inhibitors would be beneficial for therapeutic intervention for Cbx7 associated diseases. Here I report my work on design, synthesis, and evaluation of Cbx7 inhibitors. In my work, we identified several potent and selective inhibitors for Cbx7 and we published first-in-class antagonists for Cbx7. Few of these inhibitors were tested on cancer stem cell models. Further, I propose future work for targeting Cbx7 and other chromodomain containing proteins. / Graduate / 2018-09-04

Dans les cellules eucaryotes, la réponse aux lésions de l'ADN s'accompagne d'une réorganisation de la chromatine. Cette structure, associant l'ADN aux protéines histones, est porteuse de l'information épigénétique, qui définit l'identité cellulaire. Cependant, nos connaissances concernant les mécanismes impliqués dans la réorganisation de la chromatine dont l'intégrité structurale et fonctionnelle a été menacée par un stress génotoxique sont encore limitées, en particulier dans les cellules humaines. Au cours de ma thèse, je me suis donc intéressée à cette thématique en me concentrant sur l'étude de la dynamique des variants de l'histone H3 et de leurs chaperons associés après dommages UVC. En combinant une technologie innovante de suivi spécifique des histones parentales ou néo-synthétisées à des techniques de pointe d'induction de dommages locaux dans l'ADN, j'ai ainsi mis en évidence que le chaperon HIRA (Histone Regulator A) est recruté tôt aux sites de lésions où il stimule l'incorporation locale de nouveaux variants H3.3 et assure la reprise de la transcription après réparation des dommages UVC. Nous avons aussi démontré que les anciennes histones sont initialement redistribuées dans la chromatine autour des sites de lésions par un mécanisme faisant appel au facteur de détection des dommages DDB2 (DNA Damage Binding protein 2). A plus long terme, des histones parentales " reviennent " dans les régions de chromatine en cours de réparation où elles se mélangent aux nouvelles histones incorporées. Le " retour " d'histones préexistantes contribuerait ainsi au maintien de l'intégrité de l'information épigénétique véhiculée par la chromatine avant stress génotoxique. / In eukaryotic cells, the DNA damage response involves a reorganization of chromatin structure. This structure, in which DNA is associated with histone proteins, conveys the epigenetic information, which is critical for cell identity. However, we are still far from understanding the mechanisms underlying chromatin dynamics in response to DNA damage, which challenges both the structural and functional integrity of chromatin architecture. During my PhD, I thus decided to explore this issue in human cells, by deciphering the dynamics of histone H3 variants and their dedicated chaperones in response to UVC lesions. By combining local UVC irradiation with an innovative technology that allows specific tracking of parental and newly synthesized histones, I revealed that the histone chaperone HIRA (Histone Regulator A) is recruited early to UVC-damaged chromatin regions, where it promotes local deposition of new histone H3.3 variant and facilitates transcription recovery upon repair completion. We also demonstrated that old H3 histones are initially redistributed around the damaged chromatin zone, this conservative redistribution requiring the UVC damage sensor DDB2 (DNA Damage Binding protein 2). Later in the repair process, most parental histones recover and mix with newly deposited histones in repairing chromatin regions. The recovery of pre-existing histones may contribute to preserve the integrity of the epigenetic information conveyed by chromatin before genotoxic stress.

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