La spermatogenèse est un processus complexe qui dépend de la coopération de nombreux gènes. Son produit final le spermatozoïde, est un sujet d’étude idéal car il renferme à la fois des indices d’événements passés ainsi que des informations qui seront transmises à l'ovocyte lors de la fécondation. L'identification de nouveaux acteurs de la spermatogenèse, des modifications spécifiques de l'ADN du sperme ou la présence de transcrits spécifiques pourraient servir comme biomarqueurs dans le diagnostic de l’infertilité. Cette thèse avait pour but d’analyser le génome, le transcriptome et l’épigénome de spermatozoïdes dans le contexte de l'infertilité masculine. Nous avons identifié de nouvelles causes génétiques et confirmé la présence d'anomalies de méthylation dans le sperme d'hommes infertiles. Nous avons découvert 20 mutations dans le gène SOX8, chez des patients atteints de trouble du développement sexuel ou d'infertilité masculine ou féminine, qui apparaît comme un régulateur du développement et de la fonction gonadique. Par séquençage d’exome, une mutation dans le gène ATAD2 modeleur de la chromatine spécifique de la lignée germinale mâle fut également identifiée. Par RNA-seq et MeDIP-chIP du sperme d’hommes fertiles et infertiles, nous avons caractérisé la signature transcriptionnelle du sperme. La majorité des ARNs spermatiques humain est remarquablement conservée chez les mammifères placentaires suggérant des fonctions ancestrales importantes. Enfin, nos données transcriptomiques et épigénétiques tendent à indiquer qu’une expression et une régulation adéquates des gènes impliqués dans le remodelage de la chromatine constituent un facteur clé pour la fertilité masculine. / Spermatogenesis is a complex process which depends on the cooperation of many genes. The end-product, the spermatozoon, is an ideal subject for study since it carries both clues of the past events and information which will be transmitted to the oocyte at fertilization. The identification of main actors of spermatogenesis, specific modifications of sperm DNAs or sperm specific isoforms could improve our understanding of a such complex mechanism and could serve as a determination of biomarkers or diagnostic tools for fertility. The aim of the project was to go further three omes: genome, epigenome and transcriptome of mature human sperm in the context of male infertility. We identified new genetic causes of male infertility and confirmed the presence of methylation abnormalities in sperm cells of infertile men. Firstly, SOX8 gene was found mutated in a cohort of 20 patients with disorder of sex development and male or female infertility. Similarly, to NR5A1, SOX8 appears to be a novel regulator of gonadal development and function. Then by exome-sequencing, we identified a homozygous nonsense mutation in the male germline-specific chromatin modeler ATAD2. Furthermore, RNA-seq and MeDIP-chIP of sperm from fertile and infertile men along with bioinformatics analyzes of the generated data, enabled us to characterize more deeply the normal sperm transcriptional signature. We also found that the majority of human sperm RNAs are remarkably preserved in placental mammals suggesting crucial ancestral functions. Finally, proper expression and regulation of chromatin remodelers seem to be critical for male fertility, as revealed by both the transcriptomic and the epigenetic data.

Les protéines du groupe Polycomb (PcG) sont des facteurs épigénétiques dont le rôle est de maintenir la répression de leurs gènes cibles au niveau de la chromatine via la modification des protéines histones. EZH2 est une protéine clé dans les mécanismes de régulation puisqu’elle catalyse la mise en place de la marque répressive H3K27me3. Dans le cadre de ma thèse, je me suis intéressée au modèle des leucémies aiguës myéloïdes (LAM) dans lesquelles, contrairement à d’autres pathologies myéloïdes, des mutations affectant EZH2 ou des membres Polycomb ne sont retrouvées que très rarement (˂1%). Des études ont montré que dans ce type de leucémies, de nombreux gènes cibles d’EZH2 sont dérégulés bien que son activité répressive soit toujours présente, mettant en évidence d’éventuels défauts de recrutement de cette protéine. Parmi les facteurs de transcription susceptibles de réguler l’association des protéines PcG à la chromatine, se trouve PLZF qui est un candidat intéressant. En effet, le laboratoire a mis en évidence une interaction entre PLZF et la protéine Polycomb BMI-1 et a montré que la distribution génomique de PLZF concorde avec celle de certains composants des Polycomb. L’objectif de mon travail de thèse a donc été de déterminer dans quelle mesure PLZF intervient dans le recrutement ou l’activité d’EZH2. / Polycomb group (PcG) proteins are epigenetic factors which play a major role in maintaining epigenetic silencing via histone modifications at the chromatin level. EZH2 is a key regulator that catalyzes the trimethylation of H3K27, which is a repressive mark. During my PhD, I was interested in the acute myeloid leukemia (AML) model in which, unlike other myeloid malignancies, EZH2 or other PcG protein mutations are very rare (˂1%). Studies have shown that in this type of leukemia, many of EZH2 target genes are deregulated although its repressive activity is still present highlighting possible EZH2 recruitment defects. Among the transcription factors that regulate the association of PcG proteins to chromatin, the transcription factor PLZF is an interesting candidate. Indeed, the laboratory has demonstrated an interaction between PLZF and the Polycomb protein BMI -1 and showed that the genomic distribution of PLZF is consistent with that of some Polycomb components. The aim of my thesis was therefore to determine in which extent PLZF is involved in the recruitment or activity of EZH2.

The human GI tract has evolved to simultaneously absorb nutrients and be the frontline in host defence. These seemingly mutually exclusive goals are achieved by a single cell thick epithelial barrier, and a complex resident immune system which lives in symbiosis with the intestinal microflora and is also able to rapidly respond to invading pathogens. An immunological balance is therefore required to permit tolerance to the normal intestinal microflora, but also prevent the dissemination of pathogenic micro-organisms to the rest of the host. Inappropriate immune responses in genetically susceptible individuals are the hallmark of human inflammatory bowel disease (IBD) and are thus targeting effector immune cells and their cytokines remains the mainstay of treatment. However despite vigorous efforts to delineate the genetic contribution to IBD disease susceptibility using large multinational cohorts, the majority of disease heritability remains unknown. Epigenetics describes heritable changes in chromatin that are not conferred by DNA sequence. These incorporate changes to histones, chromatin structure and DNA methylation, which confer changes to gene transcription and thus gene expression and cellular function. Methylbinding proteins (MBD) have the ability to bind to methylated DNA and recruit large chromatin remodeling complexes that underpin a variety of epigenetic modifications. Methyl- CpG-binding domain protein 2 (MBD2) is one such MBD that is required for appropriate innate (dendritic cell) and adaptive (T cell) immune function, though its role has not been investigated in the GI tract. We hypothesized that alterations in chromatin are central to the reprogramming of normal gene expression that occurs in disease states. By defining the phenotype of immune cells in the absence of MBDs we hope to understand the mechanisms of chromatin-dysregulation that lead to immune-mediated diseases such as IBD. We therefore aimed to assess the role of MBD2 in colon immune cells in the steady state and in murine models of GI tract inflammation, thereafter identifying the culprit cell types and genes responsible for any observed changes. We envisaged that investigating heritable, epigenetic changes in gene expression that are inherently more amenable to environmental manipulation than our DNA code, may provide novel insight to a poorly understood mechanism of disease predisposition. In addition identifying the cellular and gene targets of Mbd2 mediated changes to immune homeostasis that may provide exciting and novel approaches to therapeutic modulation of pathological inflammatory responses. In chapter 3 we assessed the expression of Mbd2/MBD2 in the murine/human GI tract. Consistent with existing mouse data, levels of Mbd2 mRNA increased between anatomical divisions of small (duodenum, ileum, terminal ileum) and large intestine (caecum, colon, rectum). In addition MBD2 mRNA was greater in the rectum versus ileum, with active IBD associated with lower rectal MBD2 mRNA compared to quiescent IBD controls. Thus we sought to understand the role of Mbd2 in the colon, where mRNA levels were the highest in the GI tract and where appropriate immune function is central to prevent damaging inflammation. To address these aims required the development of existing methods of cell surface marker expression analysis using flow cytometry techniques to simultaneously identify multiple innate and adaptive immune populations. Using naïve Mbd2 deficient mice (Mbd2-/-) we observed CD11b+ CD103+ DCs were significantly reduced in number in Mbd2 deficiency. To understand the role of Mbd2 in colonic inflammation we employed a mouse model of chemical (DSS) and infectious (T. gondii) colitis comparing Mbd2-/- and littermate controls (WT). Mbd2-/- were extremely sensitive to DSS and T. gondii mediated colonic inflammation, characterized by increased symptom score, weight loss and histological score of tissue inflammation (DSS) and increased antibody specific cytokine responses (T. gondii) in Mbd2 deficient animals. Flow cytometry analysis of colon LP cells in both infectious and chemical colitis revealed significant accumulation of monocytes and neutrophils in Mbd2-/-. Indeed monocytes and neutrophils were the principal myeloid sources of IL-1b and TNF in DSS colitis and the number of IL-1b/TNF+ monocytes/neutrophils was significantly greater in Mbd2-/-. Lastly we employed our colon LP isolation techniques to analyse immune populations in active and quiescent IBD and healthy controls, using endoscopically acquired biopsy samples. Analysis revealed that as in murine colitis, active human IBD is characterized by the accumulation of CD14High monocyte-like cells, with an associated increased ratio of macrophage:monocyte-like cells. In Chapter 4 we sought to understand the cellular sources of Mbd2 that may explain the predisposition of Mbd2-/- to colitis. Firstly we restricted Mbd2 deficiency to haematopoietic cells using grafting Mbd2-/- bone marrow (BM) into lethally irradiated WT mice. These animals treated with DSS displayed increased weight loss, symptom score, neutrophil accumulation and histopathology score compared to mice irradiated and grafted with WT BM. Given the accumulation of monocytes in Mbd2-/- DSS treated mice, and existing literature supporting a pathogenic role in this model, we then investigated the role of Mbd2 in monocyte function. Colon monocytes sorted from Mbd2-/- and WT DSS treated mice displayed similar expression for many pro-inflammatory genes (Il6, Il1a, Il1b, Tnf), but demonstrated significantly dysregulated expression for some others (Regb, Lyz1, Ido1, C4a). To investigate this in a more refined model, we lethally irradiated WT mice and repopulated them with a WT:Mbd2-/- BM mix. This enabled the analysis of WT and Mbd2-/- haematopoietic cells in the same animal. Colon WT and Mbd2-/- monocyte recruitment and cytokine production in DSS treated mixed BM chimeras was equivalent between genotypes suggesting that Mbd2 deficiency in monocytes alone did not explain the increased susceptibility of Mbd2-/- to DSS colitis. We then restricted Mbd2 deficiency to CD11c expressing cells, given the known role for Mbd2 in their function, and for CD11c+ cells in DSS, using a CD11cCreMbd2Fl/Fl system. DSS treated mice with Mbd2 deficient CD11c+ cells demonstrated increased weight loss, symptoms score, histolopathology score, monocyte and neutrophil colon accumulation compared to controls. To further explore the role of Mbd2 in colon CD11c+ cells, macrophage and DCs from DSS treated WT and Mbd2-/- mice were purified and their gene expression analysed. Mbd2-/- versus WT macrophages demonstrated significantly altered expression of both pro- (Il1a, C6, Ido1, Trem2) and antiinflammatory (Tgfbi, Retnla) pathways that we hypothesized was a method for attempted host control of excessive colon damage in Mbd2-/- mice. DC gene expression analysis was hampered by small sample size, but demonstrated a large number of small expression changes, including IL-12/IL-23 (Jak2) and autophagy (Lrrk2) pathways. Lastly levels of costimualtory molecules (CD40/CD80) were increased in Mbd2-/- but not CD11cΔMbd2 colon LP DCs/macrophages suggesting that non-CD11c+ cellular sources of Mbd2 were required to produce increased activation phenotype in these cells. Finally in Chapter 5 we explored the role for Mbd2 in non-haematopoietic cells, namely the colonic epithelium. Here we first developed a novel method for identifying and purifying these cells using flow cytometry. Mbd2 deficient colonic epithelium demonstrated increased expression of activation markers MHC II and LY6A/E in the steady state and in DSS / T. muris mediated colonic inflammation. Indeed FACS purified colon epithelial cells from naive and DSS treated, Mbd2-/- and WT mice revealed conserved dysregulated gene expression independent of inflammation: Both naïve and inflamed Mbd2 deficient epithelium displayed significantly increased expression of genes responsible for antigen processing/presentation (MHC I, MHC II, immunoproteasome) and decreased expression of genes involved in cell-cell adhesion (Cldn1, Cldn4). Lastly we investigated whether the observed differences in Mbd2-/- cell types conferred alterations in the makeup of the intestinal microflora. Interestingly independent of co-housing of Mbd2-/- and WT animals, Mbd2 deficiency consistently predicted the microbial composition, with increased levels of Clostridales and decreased levels of Parabacteroides bacteria. Collectively we have identified CD11c+ cells, monocytes and colon epithelial cells as key cell types for Mbd2 mediated changes in gene expression that affect mucosal immune responses. These data thus identify Mbd2 gene targets within these cell types as exciting new areas for investigation and therapeutic modulation to limit damaging GI tract inflammation.

Brain tumours account for the most frequent type of solid tumours among children. Despite advances in surgery and chemotherapy, brain tumours are still the main cause of cancer deaths in children. Furthermore, little is known about DNA methylation changes in paediatric astrocytoma. Recent investigations suggest that many tumours are initiated not only by genetic abnormalities, but also caused by epigenetic changes. DNA methylation is a key epigenetic mechanism that controls the regulation of gene expression. Interestingly, unlike DNA mutations, epigenetic abnormalities are reversible. The reversibility of epigenetic abnormalities upon pharmacological unmasking has prompted interest in developing epigenetic therapy with the crucial goal of restoring the expression of aberrantly silenced genes. The focus of this study was to utilise a combination of different microarray strategies to develop an integrative candidate gene approach to identify several novel frequently methylated genes in a cohort of paediatric HGA (High grade glioma) samples. In addition, to investigate the potential of therapeutic efficacy of a DNA methyltransferase inhibitor, 5-Aza-dC in paediatric HGA. There were 147 genes commonly identified to be potentially methylated in IN699 cells using the two different array strategies integration; re-expression array and Illumina Infinium Human Methylation 450k array. Furthermore, using two complementary microarray strategies including methylation 450k array and expression array, this work identified 55 genes that were both methylated and under-expressed in these HGA cultures. Following validation with CoBRA and RT-PCR coupled with the response of hypermethylated promoters to the demethylating agent 5-Aza-dC, six novel genes (CXCL14, PRR5L, ELTD1, ITGA2, KRT8 and NTM) that are frequently silenced in paediatric astrocytoma were identified. This study suggests that re-expression of ii CXCL14 inhibited the colony formation and cell growth and reduces the migration rate significantly in IN699 short term culture and likely have functional significance in the development of paediatric HGA and an excellent candidate gene for further analysis. In parallel, the efficacy of 5-Aza-dC treatment was examined in paediatric HGA aiming to introduce this epigenetic therapy as a potential mechanism in management of this tumours. This study demonstrated that, relatively low dose of 5-Aza-dC sharply reduced the colony formation and inhibited proliferation and not through the apoptotic effect. It is likely that this reduction in proliferation without cell death is due to using relatively low doses that do not acutely kill cells, thus, allow the sustained alterations in both gene expression patterns and appearance of a new phenotype to emerge. Taken together, this work contributes to a more detailed understanding of the effect of epigenetic silencing on paediatric HGA. This investigation also demonstrated the use of epigenetic drug, 5-aza-dC to reverse the gene silencing for the potential treatment of paediatric HGA.

The cell differentiation process is associated with activation and repression of different genes, whereby the formation of heterochromatin is mediated by spreading of repressor proteins along large chromatin domains. Some of these proteins are methyltransferases, including GLP and G9a that are implicated in the addition of mono- and dimethyl groups to lysine 9 at Histone 3. Despite extensive research the exact mechanism of binding and spreading of G9a and GLP is unclear. To better understand the molecular mechanisms through which G9a and GLP bind to chromatin we tested the in vivo binding of a mutant G9a that is unable to bind to H3K9me2 histone marks via its Ankyrin domain. Murine erythroleukemia (MEL) cell line with expression of mutant G9a was generated using recombinant DNA technologies; G9a binding targets genome-wide were detected by the analysis of ChIP-sequencing data. We validated ChIP-sequencing data providing a reliable tool to visualize G9a targets in MEL cells. We also found that G9a Ankyrin mutant bound to all tested regions suggesting that the Ankyrin domain is not the only factor that contributes to the binding of G9a on chromatin in vivo.

Le striatum est une région du cerveau impliquée dans d'importantes fonctions physiologiques telles que l'apprentissage par renforcement ou le contrôle du mouvement, mais aussi dans des pathologies comme l'addiction. Le fonctionnement de ce système s'appuie sur deux types de neurones de projection, appelés " neurones épineux de taille moyenne ". Les uns expriment le récepteur de la dopamine de type 1 (D1R) et les autres expriment le récepteur de type 2 (D2R). L'objectif de cette thèse est de caractériser ces deux types de neurones au niveau épigénétique, en conditions basales et après traitement à la cocaïne. Il a été nécessaire de développer de nouvelles méthodes utilisant la cytométrie de flux pour distinguer les populations de neurones exprimant D1R ou D2R. La première méthode utilise des souris transgéniques L10a-GFP et du tissu non fixé, la seconde répond aux limitations de la précédente et utilise du tissu fixé. Nous avons montré que la cocaïne régule de nombreuses modifications post-traductionnelles d'histones, de façon spécifique de populations neuronales. Par ailleurs, nous avons identifié plus d'une centaine de gènes différemment méthylés ou hydroxyméthylés entre les deux types neuronaux. Certains gènes sont déjà connus pour avoir un rôle fonctionnel important dans l'une des populations. La comparaison des neurones exprimant D1R ou D2R est un bon modèle pour explorer les liens entre méthylation de l'ADN, hydroxyméthylation et transcription. Par exemple, nous observons une association très claire entre l'augmentation de la méthylation de l'ADN et la répression de la transcription, ainsi qu'une corrélation entre modifications de méthylation et d'hydroxyméthylation. / The striatum is a brain region implicated in physiological functions such as reinforcement learning or movement selection but also in pathologies such as addiction or Parkinson’s disease. It relies on two types of projecting neurons, named “medium spiny neurons” because of their morphology. They are very similar but have a complementary and opposite role. One type expresses the dopamine receptor type 1 (D1R) and the other type expresses the dopamine receptor type 2 (D2R). The aim of this work was to characterize this two neuronal types epigenetically, in basal conditions and after cocaine treatment. We have developed new flow cytometry techniques to be able to distinguish the two cell types. The first method uses transgenic L10-eGFP mice and fresh tissue, the second one goes beyond the limitations of the first one and uses fixed tissue. We have shown that cocaine regulates many post-translational histone modifications, dynamically, and differently between the two populations. Moreover, we have identified more than 100 genes differentially methylated or hydroxymethylated between the two neuronal types. Some of these genes are already known for having a functional role in one of the populations. The comparison between D1R and D2R neurons is a good model to explore the links between DNA methylation, hydroxymethylation and transcription. For example, we have observed a strong association between an increase in DNA methylation and a transcriptional repression, as well as a correlation between DNA methylation and hydroxymethylation.

Epidemiological studies show that offspring subjected to maternal undernutrition during early pregnancy are prone to developing obesity and other diseases in adulthood. The hypothalamic energy regulating pathway may be altered in these offspring, with epigenetic changes as a core mechanism. Therefore, this thesis aimed to determine if epigenetic changes are present in this pathway in the hypothalami from offspring subjected to maternal undernutrition. The investigations are focused on the glucocorticoid receptor (GR) as an inhibitor of the anorexigenic neuropeptide pro-opiomelanocortin (POMC), with potential modifications leading to increased food intake and the development of obesity. To achieve this, an established sheep model developed by our collaborators was used, during which maternal ewes were undernourished periconceptionally to produce a 10-15% decrease in body weight. We found that hypothalami from fetal offspring had greater epigenetic modifications when this reduction in maternal body weight was maintained from 60 days before conception until 30 days into pregnancy, with lower levels of POMC and GR promoter methylation. This was associated with increased GR mRNA expression. Other regions of the brain that also express POMC and GR, did not exhibit these epigenetic modifications. This study revealed that maternal undernutrition induces tissue specific epigenetic changes in fetal hypothalami which may contribute to disease in later life. Twins have been shown to have similar phenotypic characteristics as maternally undernourished offspring and therefore it has been suggested that they may also be programmed, but by intrauterine growth restriction. Consequently, extensive methylation and histone analysis of GR and POMC promoter regions was carried out in twin fetal hypothalami and compared to maternally undernourished groups. Interestingly, the decreased POMC and GR methylation of our amplicons in the maternally undernourished fetal hypothalami was also observed in twin fetal hypothalamic. This was concomitant with histone modifications and alterations in overall DNA methyltransferase activity. However, it was found that there were no changes in the POMC and GR mRNA expression levels in twin fetuses, but we postulate that this may occur later in life. To determine if changes in the fetal epigenetic status of hypothalamic GR and POMC impacted the adult progeny, tissues were obtained from adult offspring of maternally undernourished ewes. Epigenetic changes in the hypothalamic GR promoter observed in the fetal group persisted into adulthood, with concurrent increases in GR mRNA and GR protein expression. Of these groups the undernourished adult male offspring had decreased hypothalamic POMC expression and increased fat mass, changes that are consistent with an obese phenotype. The epigenetic and expression status of GR in the hippocampus and pituitary were modified, but in a tissue and sex specific manner. POMC epigenetic changes in the brain were complex, with various levels of epigenetic and expression changes. Overall periconceptional undernutrition induces hypothalamic specific changes in the epigenetic status of the GR gene which is known to regulate energy balance. Hypothalamic changes were persistent from the fetal stage into adulthood, with modifications in other tissues occurring after birth. These adaptations have the potential to increase the offspring’s propensity to develop obesity and altered stress regulation in later life.

Orientador: Ana Paula de Souza Pardo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-27T07:52:53Z (GMT). No. of bitstreams: 1 Portinho_Danielle_D.pdf: 3218538 bytes, checksum: 09966eb0179669b7c0b2ceee93e7f695 (MD5) Previous issue date: 2015 / Resumo: A associação entre inflamação e câncer vem sendo há tempo estudada, havendo atualmente fortes evidências que mostram o microambiente inflamatório como fator de risco para tumorigênese. Dentre os tipos de câncer mais frequentes, o câncer de boca ocupa a sexta colocação como maior causa de óbitos em todo o mundo. Os fatores que levam ao aparecimento do câncer oral são variados, entretanto existe dependência de um acúmulo de mutações as quais levam a alterações genéticas e epigenéticas das células afetadas, sendo que estas alterações influenciam diretamente no prognóstico da doença. Nos últimos anos, o estudo das alterações epigenéticas relacionadas aos diversos tipos de câncers vem se destacando, havendo marcadores epigenéticos já descritos para diferentes tipos de tumores. Dentre as alterações epigenéticas mais estudadas está a metilação do DNA que acontecem normalmente em regiões com grandes concentrações de citocina que precedem guanina (CpG). Estas regiões ricas em dinucleotídeos CpGs são denominadas ilhas CG. Ilhas CG são frequentes em regiões promotoras dos genes, sendo a metilação um mecanismo de controle do nível transcricional. Ilhas CGs metiladas na região promotora são esperadas em genes pouco expressos, podendo haver até mesmo o completo silenciamento da expressão do gene. No caso de genes supressores de tumor, pode provocar o aparecimento e o descontrole no crescimento tumoral. Assim, o objetivo deste estudo foi analisar o padrão de metilação do DNA em sequências LINE1 e nos genes supressores tumorais: SFRP1, SFRP2 e TP73, em amostras de gengiva coletadas de pacientes, pacientes com periodontite crônica e pacientes diagnosticados com carcioma espinocelular (CEC). A metilação do DNA foi analisada utilizando-se a metodologia COmbined Bisulfite Restriction Analysis (COBRA) em tecidos homogeneizados e microdissecados. A análise de expressão gênica dos genes foi realizada pela técnica qPCR amostras de tecido gengival e CEC. Os resultados obtidos pelas análises COBRA evidenciaram um desequilíbrio no estado de metilação todo DNA anto nos tecidos inflamados quanto nos tumorais. Entretanto, as regiões CpG analisadas não demonstraram estar diretamente relacionadas com o nível de expressão gênica. Adicionalmente, os resultados das análises da metilação global (LINE1) do tecido homogeneizado em comparação com as amostras microdissecadas indicam que os dados obtidos podem ser contraditórios aos reais podendo interferir no prognóstico do paciente. Esta contradição possivelmente se deve a contaminação do tecido tumoral por tecidos adjacentes a ele. As análises estatísticas dos resultados foram realizadas pelos testes Kruskall-Wallis e correlação de Pearson ao nível de significância de 5%. Alterações no padrão de metilação do DNA dos genes estudados são encontradas tanto em tecidos inflamados quanto em amostras de câncer bucal. A metodologia de escolha para análise destes tecidos pode influenciar nos resultados obtidos, gerando falsos negativos e consequentemente comprometendo o tratamento da doença / Abstract: The association between inflammation and cancer has always been sought by researchers. Nowadays, there are evidences of the role of the inflammatory microenvironment in tumorigenesis. Among the most common types of cancer, oral cancer is the sixth type that causes more deaths worldwide. The factors that lead to the development of an oral cancer are varied, depending on an accumulation of mutations that lead to genetic and epigenetic alterations of the affected cells, and these changes directly influence the prognosis of the disease. In recent years epigenetic have been widely studied in this field of researchs, and several genes have been described as markers for different types of tumors. Among the most studied epigenetic changes there is the DNA methylation in areas with large concentrations of the cytokine that precedes guanine (CpG), these regions are called CpG islands. GpC islands are fairly common in the promoter regions of the genes, being the methylation a control mechanism of gene level transcripts and it can even silence the expression of a gene. In the case of tumor suppressor genes this silencing can lead to uncontrolled tumor growth. The aim of this study was to analyse the DNA methylation status of LINE1 sequences and specific genes (SFRP1, SFRP2 e TP73) from human tissue collected from gingival normal tissue from patients underwent aesthetic surgery, patients with cronic periodontitis and patients with oral squamous cell carcinoma (OSCC). The extracted DNA was analysed by Combined Bisulfite Restriction Analysis (COBRA) submitting the DNA to bisulfite conversion by MethylSEQrBisulfiteConversion Kit from homogenized and microdissected tissues. Through qPCR technique, samples from gingival tissue and OSCC were subjected to RNA expression analysis of candidate genes. The results of COBRA analysis of genes exhibited an imbalance in methylation state of inflamed and tumor tissues. However, the CpG regions analyzed have not shown to be directly related to the level of expression of these genes. The results of the analysis of the global methylation (LINE1) of the homogenized tissue compared with microdissected samples showed that the obtained data may be contradictory to the real one and may interfere in the prognosis of the patient. This inconsistency is probably due to contamination of tumor tissue by adjacent tissues. Statistical analysis of results was performed by Kruskal-Wallis test and Pearson correlation at the 5% confidence level. Changes in DNA methylation pattern of the genes studied are found in both inflamed tissues as oral cancer samples. The choice of methodology for the analysis of these tissues can influence results, and consequently generating false negative results affecting the treatment of disease / Doutorado / Histologia e Embriologia / Doutora em Biologia Buco-Dental

Orientadores: Adriana Souza Torsoni, Marciane Milanski / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Aplicadas / Made available in DSpace on 2018-08-27T16:45:13Z (GMT). No. of bitstreams: 1 Fante_Thaisde_M.pdf: 3029616 bytes, checksum: 5e68ab17d3076784e4f20c127d76838f (MD5) Previous issue date: 2015 / Resumo: O estilo de vida moderno tem levado ao aumento na prevalência de obesidade e suas co-morbidades em gestantes e na população cada vez mais jovem. Muitos dos efeitos do consumo direto de dieta hiperlipídica (DH) no metabolismo de glicose e lipídios já são bem estabelecidos. No entanto, considera-se importante avaliar se o consumo de DH durante períodos críticos do desenvolvimento seria capaz de ativar mecanismos epigenéticos, perpetuando mudanças no metabolismo da prole e criando um ciclo vicioso que não poderia ser interrompido. O objetivo desse estudo foi avaliar o efeito potencial da programação metabólica em prejudicar a sinalização de insulina na prole recém desmamada de mães alimentadas com dieta hiperlipídica durante a gestação e lactação. Além disso, investigamos se a exposição precoce a um ambiente obesogênico seria capaz de exacerbar o prejuízo no metabolismo de glicose na vida adulta de animais reexpostos à dieta hiperlipídica. Para isso, camundongos fêmeas da linhagem Swiss foram alimentados com dieta controle ou DH durante os períodos de adaptação, gestação e lactação, e os tecidos da prole macho foram analisados nos dias 28 e 82. Os resultados mostram que a prole de mães obesas (HC-O) apresentou maior ganho de peso, adiposidade e ingestão alimentar que a prole de mães controle (CC-O). Além do mais, apresentou prejuízos na sinalização de insulina em tecidos periféricos como fígado, adiposo e músculo, e centrais, como o hipotálamo, provavelmente devido à maior ativação de vias inflamatórias. A reexposição à DH parece agir como um fator agravante para o desenvolvimento do fenótipo obeso, levando a resistência sistêmica à insulina e hiperleptinemia. É válido ressaltar que o tecido adiposo parece ser o tecido mais afetado na prole adulta após a reexposição da dieta (HH-O), o que pode contribuir para a desregulação metabólica observada. Em conjunto, nossos resultados sugerem que o consumo materno de dieta hiperlipídica durante a gestação e lactação pode ocasionar alterações no metabolismo glicídico da prole tanto em animais recém desmamados quanto adultos. Por fim, a obesidade materna leva à maior susceptibilidade ao desenvolvimento de obesidade e prejuízos na sinalização de insulina na prole que não podem ser revertidos pelo consumo de uma dieta controle, no entanto, podem ser agravados especialmente quando os animais são reexpostos à DH / Abstract: Modern lifestyle has resulted in an increase in the prevalence of obesity and its comorbidities in pregnancy and young population. Many effects from direct consumption of a high-fat diet (HFD) on glucose and lipid metabolism are well established. However, it is important to assess whether maternal consumption of HFD during critical periods of development can trigger epigenetic mechanisms, perpetuating changes in offspring metabolism and creating a vicious circle that cannot be broken. This study evaluated the potential effect of metabolic programming in impairing the insulin signaling in recently weaned offspring of obese dams. In addition, we investigated if early exposure to obesogenic environment is able to exacerbate the impairment of glucose metabolism in adult life in response to a high-fat diet. For this, Swiss female mice were fed with Stardard chow (SC) or HFD before and during mating, gestation and lactation. Tissues from male offspring were obtained at d28 and d82 to analyze activation of key proteins of inflammatory and insulin signaling pathways by Western Blot. Offspring of obese dams (HC-O) showed greater weight gain, adiposity and food intake than offspring of control dams (CC-O). Furthermore, they showed impairment in insulin signaling in central and peripheral tissues, associated to increased activation of inflammatory pathways. The HFD re-exposure seems to be an aggravating factor in development of obese phenotype leading to systemic insulin resistance and hyperleptinaemia. Moreover, adipose tissue was ultimately the most affected tissue in adult offspring after HFD rechallenged (HH-O) which may have contributed to the metabolic deregulation observed. Together our results suggest that maternal consumption of high-fat diet during pregnancy and lactation can cause changes in glucose metabolism of offspring in both weaned and adult animals. Additionally, maternal obesity leads to increase susceptibility to the development of obesity and impairment in insulin signaling in offspring that cannot be reversed by SC consumption, but can be aggravated especially when re-exposed to HFD / Mestrado / Metabolismo e Biologia Molecular / Mestra em Ciências da Nutrição e do Esporte e Metabolismo

A descondensação da cromatina exerce um papel central nas diversas etapas do processo de carcinogênese abrindo o genoma para a ação de fatores de transcrição, exercendo papel na progressão e resistência tumoral. Bromodomínios e proteínas com terminal extra, como o BRD4, são leitores epigenéticos que regulam a expressão gênica e, portanto, também estão envolvidos na patogênese do câncer. O objetivo do presente estudo foi estudar o efeito da inibição do BRD4 no carcinoma espinocelular de cabeça e pescoço (CECP). Para esse propósito, foi utilizado JQ1, inibidor de BRD4, em concentração de 1uM, nas linhagens de carcinoma de cabeça e pescoço HN6, HN12 e HN13. Foi analisado os níveis de BRD4, H4 acetilada e SIRT1 fosforilado através de reações de imunofluorecência e p16ink4 por imunohistoquímica. Foi realizado western blot para checar os níveis de p53 e p53 acetilado. Ensaio de formação de colônias e câmera de invasão foram realizados para testar o efeito do inibidor na proliferação e invasão celular. Através da citometria de fluxo foi analisado o efeito da apoptose com a marcação de caspase-3 clivada, do ciclo celular através da reação por iodeto de propídio e ainda da população de células tronco tumorais pela análise de ALDH e CD44. Por fim, foi realizado modelo xenográfico subcutâneo para analisar o efeito do JQ1. Os resultados mostraram diminuição significativa da expressão de BRD4 e H4ac após tratamento com JQ1. As linhagens celulares mostraram redução na capacidade de invasão e de formação de colônias quando submetidas ao JQ1. Não foram encontradas diferenças em relação ao número de células caspase-3 clivada positivas. Por outro lado, foi encontrado um maior número de células na fase G1 do ciclo celular após o uso do inibidor estudado. As células tratadas com JQ1 mostraram menor expressão de p-SIRT1 o que levou a uma diminuição da acetilação do p53 e um aumento na expressão de p16ink4. Paralelamente, foi encontrado uma diminuição na população de células positivas para ALDH e CD44. Houve diminuição do crescimento do tumor no modelo xenográfico tratado com JQ1 quando comparado ao veículo. Nos tecidos derivados do ensaio in vivo, houve uma diminuição nos marcadores p16ink4, pSIRT1 além de acúmulo de H2AX. Conclui-se que o uso de JQ1 resulta na disrupção do crescimento do CECP associado a ativação de senescência, indução de dano de DNA além de reduzir a população de células tronco tumorais. Esses novos achados indicam que o BRD4 é um importante modificador epigenético nos CECP sendo um viável alvo terapêutico. / Chromatin descondensation plays a central step in the various stages of the carcinogenesis process opening the genome for transcription factors playing a role in tumor progress and resistance. Bromodomains and extra terminal family, as BRD4, are epigenetics readers that regulate gene expression thus they are also involved in cancer pathogenesis. The objective of this project was studied the effect of BRD4 in head and neck squamous cell carcinoma (HNSCC). For this purpose, JQ1, a BRD4 inhibitor, was used in 1uM concentration, in HN6, HN12 and HN13 head and neck carcinoma cell lines. The levels of BRD4, acetylates h4 and phosphorylated SIRT1 were analyzed by immunofluorescence and p16ink4 labeling by immunohistochemistry. Western blot was performed to check the levels of p53 and acetylated p53. Colony assay and invasion chamber were performed to test the inhibitory effect on cell proliferation and invasion. The effect of apoptosis with the cleaved caspase-3 labeling, the cell cycle by propidium iodide and of the population of tumor stem cells by the analysis of ALDH and CD44 was analyzed through flow cytometry. Finally, a subcutaneous xerographic model was performed to analyze the effect of JQ1. A significant decrease in the expression of BRD4 and H4ac was found after application of JQ1. The cell lines results showed a reduction in the capacity of invasion and also formation of colonies when submitted to JQ1. No differences were found in relation to the number of cells caspase-3 cleaved positives. On the other hand, a large number of cells were found in G1 arrest of cell cycle after use of the BRD4 inhibitor studied. Cells treated with JQ1 showed lower expression of p-SIRT1 which led to a decrease in p53 acetylation and an increase in p16ink4 expression. In parallel, a decrease of ALDH and CD44 positive cells population was found. A decrease in tumor growth was discovered when treated by JQ1 if compared to the vehicle. In tissues samples derived from the in vivo assay, there was a decrease in p16ink4, pSIRT1 markers in addition to -H2Ax accumulation. In conclusion JQ1 causes HNSSC tumor growth disruption associated a senescence activation, DNA damage and a reduce number of cancer stem cells. These new findings indicate that BRD4 is an important genetic modifier in HNSSC and is a viable therapeutic target.