Dissertations / Theses on the topic 'Genetic Epigenesis'

Orientador: Maria Luiza Silveira Mello
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Envelhecimento pode ser definido como as mudanças sofridas por um organismo ao longo do tempo. Esse processo, em biologia, é denominado senescência. A senescência celular é um fenômeno observado em células isoladas, e tem sido estudada tipicamente em células em cultura. Sua ocorrência in vivo foi observada em alguns tecidos de mamíferos. As mudanças na estrutura e organização da cromatina que ocorrem em células senescentes incluem, aumento na resistência da cromatina à digestão por nucleases e acúmulo de modificações de histonas e proteínas associadas à heterocromatina. Embora nem todas as células em um organismo envelhecido estejam em estado de senescência, espera-se que mudanças na estrutura e organização da cromatina ocorram. A restrição calórica é a única intervenção conhecida que tem a capacidade de estender o tempo de vida em mamíferos. Após uma dieta de restrição calórica ou jejum muitos genes, cuja expressão encontra-se alterada em animais idosos, têm sua expressão restabelecida aos níveis observados em animais jovens. Acredita-se que mudanças na cromatina também possam ocorrer durante o jejum, e que induzam mudanças no nível de expressão de diversos genes. No presente trabalho, buscando-se alterações na organização da cromatina em hepatócitos de camundongo ao longo do envelhecimento ou submetidos ao jejum, observou-se um aumento das propriedades viscoelásticas da cromatina ao longo do envelhecimento, de acordo com as mudanças na habilidade dessa cromatina em formar fibras estendidas de cromatina. Essas diferenças foram acompanhadas por um desempacotamento da cromatina. Observou-se também que essa viscoelasticidade da cromatina era dependente principalmente de interações desta com a matriz nuclear, e que cópias de genes cuja atividade transcricional não é mais requerida, ou requerida em um nível menor em animais idosos, podem desligar-se temporariamente da matriz nuclear. Mudanças nas propriedades viscoelásticas da cromatina e no seu grau de compactação já haviam sido observadas previamente em animais em jejum. Apesar disso, no presente trabalho, nenhuma diferença com relação à interação dos genes rDNA com a matriz nuclear foi encontrada em animais em jejum. Contudo, independente da condição fisiológica, o DNA aderido à matriz nuclear parece ser rico em genes, enquanto as seqüências heterocromáticas, pobres em genes, geralmente são encontradas tanto associadas com a matriz nuclear quanto dissociadas desta (cuidado com essa conclusão. Está forte). Em hepatócitos de animais idosos foi observado acúmulo de marcadores heterocromáticos (modificações de histonas) e de outras proteínas (proteínas formadoras de heterocromatina e glicoproteínas presentes principalmente nos cromocentros), assim como diminuição das modificações de histonas associadas com transcrição ativa. Todas essas modificações estão relacionadas com alterações na síntese de RNA já relatadas para animais idosos, e são uma evidência de que o controle da expressão gênica, a organização e a composição da cromatina estão intimamente relacionados. Em um outro tipo celular como espermatozóides de camundongo, uma diferente organização nuclear levou a propriedades diferenciadas de sua cromatina com relação às suas propriedades viscoelásticas (aumentadas). Tais diferenças possivelmente estejam relacionadas com um padrão modificado de expressão gênica, uma vez que em espermatozóides, a atividade transcricional é nula ou quase ausente
Abstract: Aging may be defined as the changes that take place in an organism with time. This process, in biology, is called senescence. Cellular senescence is observed in isolated cells, and has been studied typically in cultured cells, but its occurrence in vivo has been shown only in some mammalian tissues. Chromatin changes that take place with cellular senescence include increase in the resistance of chromatin to nuclease digestion and accumulation of histone modifications and non-histone proteins associated with heterochromatin. Although not all cells in an aged organism are subjected to cellular senescence, it is expected that changes in the chromatin structure and organization still occur. Caloric restriction is the only intervention known to extend life span in mammals. It has been shown that many genes whose expression pattern is altered in aged animals can be reverted to the levels observed in young animals after a caloric restriction diet or complete food withdrawal. Changes in chromatin structure may occur during the starvation period to induce changes in the expression level of several genes. With the aim of screening for alterations in the chromatin organization in mouse hepatocyte nuclei with aging or following starvation, we observed an increase in the viscoelastic properties of chromatin with aging, in terms of changes in the ability of this chromatin to form extended chromatin fibers after a lysis treatment in liver imprints on histological slides. These differences were accompanied by chromatin unpackage. Most of the viscoelasticity of the chromatin were dependent on its interactions with the nuclear matrix, and copies of genes whose transcription are no longer required in aged animals, tended to detach from the nuclear matrix. Changes in the viscoelastic properties and packing degree of chromatin had been shown previously in starved animals. However, no differences regarding this feature were seen in the present work. Nevertheless, regardless the physiological condition, DNA attached to the nuclear matrix seems to be gene-rich, while heterochromatic gene-poor regions were found both attached and detached from the nuclear matrix. We observed accumulation of heterochromatic marks (histone modifications) and non-histone proteins (heterochromatin proteins and glycoproteins present mainly in the chromocenters), as well as decreased histone modifications associated with transcription in hepatocyte nuclei of aged mice. All these changes are related to altered RNA synthesis observed in aged animals and are an evidence of the strong relationship between chromatin organization, composition, and control of gene expression. In another cell type, mouse sperm cells, its nuclear organization lead to different chromatin properties regarding its viscoelastic properties (increased). These differences are possibly related to a modified pattern of gene expression since gene transcription is almost or completely absent in sperm cells
Doutorado
Biologia Celular
Doutor em Biologia Celular e Estrutural

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007.
Additional advisors: Vithal K. Ghanta, J. Michael Ruppert, Theresa V. Strong, R. Douglas Watson. Description based on contents viewed Oct. 3, 2008; title from PDF t.p. Includes bibliographical references.

Methionine dependence, the inability of cells to grow when the amino acid methionine is replaced in culture medium by its metabolic precursor homocysteine, is characteristic of many cancer cell lines. Most cells proliferate normally under these conditions. The methionine dependent tumorigenic human melanoma cell line MeWo-LC1 was derived from the methionine independent non-tumorigenic line MeWo. The MeWo-LC1 cell line has been shown to have a cellular phenotype similar to that of cells from patients with the cblC inborn error of cobalamin metabolism, with decreased synthesis of cobalamin coenzymes and decreased activity of the cobalamin dependent enzymes methionine synthase and methylmalonyl-CoA mutase. Inability of cblC cells to complement the defect in cobalamin metabolism in MeWo-LC1 suggested that the defect was caused by decreased activity of the MMACHC gene product. However, no potentially disease causing mutations could be detected in the coding sequence of MMACHC in MeWo-LC1. No MMACHC expression could be detected in MeWo-LC1, and there was virtually complete methylation of a CpG island at the 5' end of the MMACHC gene in MeWo-LC1, consistent with inactivation of the gene by methylation; the CpG island was partially methylated in MeWo and only lightly methylated in control fibroblasts. Transfection of MeWo-LC1 with wild type MMACHC with a constitutive promoter resulted in correction of the defect in cobalamin metabolism and restoration of the ability of cells to grow in medium containing homocysteine. We conclude that epigenetic inactivation of the MMACHC gene is responsible for methionine dependence in MeWo-LC1.

Schizophrenia is a neurodevelopmental disorder affecting 1% of the general population. Dysfunction of the prefrontal cortex (PFC) is associated with the etiology of schizophrenia. Moreover, a substantial deficit of GAD1mRNA in schizophrenic PFC has been reported by different groups. However, the underlying molecular mechanisms are still unclear. Interestingly, epigenetic factors such as histone modifications and DNA methylation could be involved in the pathogenesis of schizophrenia during the maturation of the PFC. In my work, I identified potential epigenetic changes in schizophrenic PFC and developmental changes of epigenetic marks in normal human PFC. Furthermore, mouse and neuronal precursor cell models were used to confirm and investigate the molecular mechanisms of the epigenetic changes in human PFC. My initial work examined whether chromatin immunoprecipitation can be applied to human postmortem brain. I used micrococcal nuclease (MNase)-digested chromatin instead of cross-linked and sonicated chromatin for further immunoprecipitation with specific anti-methyl histone antibodies. Surprisingly, the integrity of mono-nucleosomes was still maintained at least 30 hrs after death. Moreover, differences of histone methylation at different genomic loci were detectable and were preserved within a wide range of autolysis times and tissue pH values. Interestingly, MNase-treated chromatin is more efficient for subsequent immunoprecipitation than crosslinked and sonicated chromatin. During the second part of my dissertation work, I profiled histone methylation at GABAergic gene loci during human prefrontal development. Moreover, a microarray analysis was used to screen which histone methyltransferase (HMT) could be involved in histone methylation during human prefrontal development. Mixed-lineage leukemia 1 (MLL1), an HMT for methylation at histone H3 lysine 4 (H3K4), appears to be the best candidate after interpreting microarray results. Indeed, decreased methylation of histone H3 lysine 4 at a subset of GABAergic gene loci occurred in Mll1 mutant mice. Interestingly, clozapine, but not haloperidol, increased levels of trimethyl H3K4 (H3K4me3) and Mll1 occupancy at the GAD1 promoter. I profiled histone methylation and gene expression for GAD1 in schizophrenics and their matched controls. Interestingly, there are deficits of GAD1 mRNA levels and GAD1 H3K4me3 in female schizophrenics. Furthermore, I was also interested in whether the changes of GAD1 chromatin structure could contribute to cortical pathology in schizophrenics with GAD1 SNPs. Remarkably, homozygous risk alleles for schizophrenia at the 5’ end of the GAD1 gene are associated with a deficit of GAD1 mRNA levels together with decreased GAD1 H3K4me3 and increased GAD1H3K27me3 in schizophrenics. Finally, I shifted focus on whether DNA methylation at the GAD1 promoter could contribute to a deficit of GAD1 mRNA in schizophrenia. However, no reproducible techniques are available for extracting genomic DNA specifically from GABAergic neurons in human brain. Therefore, I used an alternative approach that is based on immunoprecipitation of mononucleosomes with anti-methyl-histone antibodies differentiating between sites of active and silenced gene expression. The methylation frequencies of CpG dinucleotides at the GAD1 proximal promoter and intron 2 were determined from two chromatin fractions (H3K4me3 and H3K27me3) separately. Consistently, the proximal promoter region of GAD1 is more resistant to methylation in comparison to intron 2 of GAD1 in either open or repressive chromatin fractions. Interestingly, overall higher levels of DNA methylation were seen in repressive chromatin than in open chromatin. Surprisingly, schizophrenic subjects showed a significant decrease of DNA methylation at the GAD1proximal promoter from repressive chromatin. Taken together, my work has advanced our knowledge of epigenetic mechanisms in human prefrontal development and the potential link to the etiology of schizophrenia. It could eventually provide a new approach for the treatment of schizophrenia, especially in the regulation of methylation at histone H3 lysine 4.

Different cell types in multi-cellular organisms heritably maintain different gene expression patterns despite carrying the same genome; a phenomenon termed epigenetics. It is widely believed that the packaging state of the genome, known as chromatin structure, carries epigenetic information. How chromatin states are inherited and how chromatin structure changes during development, moreover how different epigenomes, such as chromatin and DNA modifications communicate with each other during these processes are important questions. Accordingly, understanding the mechanisms that govern pluripotency and differentiation requires details of chromatin dynamics. The major goal of my doctoral thesis was to understand the genome wide view of chromatin dynamics in embryonic stem cells. My studies centered on two aspects of chromatin dynamics in mouse embryonic stem cells—localization and function of two antagonistic chromatin regulators and genome-wide histone variant dynamics. In the first part, we examined the roles of several chromatin regulators whose loss affects the pluripotent state of ES cells. We found that two such regulators, Mbd3 and Brg1, control a large number of genes in ES cells via antagonistic effects on promoter nucleosome occupancy. Moreover, we found that both Mbd3 and Brg1 play key roles in the biology of 5-hydroxymethylcytosine (5hmC), a newly identified DNA modification. Mbd3, which was named by homology to known cytosine methyl binding domains, yet does not bind methylcytosine in vitro, co-localized in ES cells with 5hmC. Furthermore, Mbd3 localization was lost in knockdown cells lacking the major 5mC hydroxylase, Tet1. Our results suggest, contrary to current dogma, that 5hmC is more than just an intermediate in cytosine demethylation pathways, that it may regulate genes via the Mbd3/NuRD complex. Finally, we showed that both Mbd3 and Brg1 are themselves required for normal levels of 5hmC in vivo, identifying a feedback loop between 5hmC and Mbd3. Together, our results identified a possible effector for 5hmC, thereby suggesting a functional role for this DNA modification. Moreover, Brg1 and Mbd3 can now be added to the growing list of regulators with opposite effects on ES cell gene expression, suggesting that pairs of antagonistic chromatin binding proteins may be a common phenomenon in ES cell transcription regulation (Yildirim et al., Cell 2011). The second part of my dissertation concerns the dynamics of several histone variants. Seminal studies in the Henikoff lab showed that certain histone variants are replaced throughout the cell cycle, in contrast to the predominant replication-coupled mode of histone assembly. Work in yeast and flies showed that rapid histone turnover occurs at epigenetically-regulated genomic regions, such as chromatin boundary elements or Polycomb/Trithorax binding sites. Notably, promoter regions of actively transcribed genes exhibit rapid turnover, suggesting that histone turnover may have an important role in gene regulation, as higher histone turnover rate would provide higher probability of DNA element exposure and faster erasure of chromatin marks of the replaced histones. In order to extend such studies to a model for pluripotency and differentiation, we developed a system for measuring histone replacement in mouse ES cells. To be able to carry out turnover experiments in ES cells, we generated stable ES cell lines that can be induced to express epitope-tagged histone variants. Our results confirmed that histone turnover patterns are conserved from yeast to mammals and that turnover profiles are histone variant specific. Murine H3.3 turnover is similar to H3.3 turnover in flies, with peaks at the promoters of highly transcribed genes. MacroH2A2, a variant generally linked to gene repression, had a more complex turnover profile. Surprisingly, we found rapid exchange of macroH2A2 occurring around transcription start sites of a number of highly expressed genes. At poorly expressed genes, on the other hand, macroH2A2 localizes upstream or downstream of transcription start sites and is incorporated slowly, either via slow turnover or via replication-coupled incorporation. Finally, we have used those inducible ES cell lines to generate mice, which will enable future studies on tissue-specific histone replacement in vivo. In summary, my thesis work not only significantly extends our understanding of chromatin regulation in general but also provides a more detailed landscape of chromatin structure and regulation in ES cells. Extending these analyses to differentiating cells and in vivo tissue specific dynamics should provide us with a better understanding not only of cell type specific chromatin organization but also improve our ability to program and re-program genomic landscapes in vitro.

Orientador: Marcelo Rocha Marques
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba.
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Resumo: A periodontite crônica é uma doença inflamatória que leva à perda de inserção de elementos dentários, e é desencadeada e mantida por um biofilme subgengival periodontopatogênico. A presença de alguns tipos de lipopolissacarídeos (LPS), derivados de bactérias no sítio periodontal doente, pode iniciar uma sinalização por meio das células do tecido gengival, que culminará com um microambiente com diferentes células do sistema imune e com uma alteração no padrão de expressão de citocinas inflamatórias. Já foi evidenciado que no tecido gengival de pacientes com periodontite crônica, genes que codificam receptores celulares para o LPS, podem sofrer alterações epigenéticas. O objetivo deste estudo foi avaliar se a periodontite crônica e o LPS bacteriano derivado de P. gingivalis podem modular a expressão gênica de alguns fatores reguladores de eventos epigenéticos. Biópsias de tecido gengival inflamado e sem inflamação foram coleados de pacientes com periodontite crônica e de pacientes saudáveis respectivamente, o RNA total foi extraído e a expressão dos genes DNMT1 (DNA metiltransferase 1), DNAMT3a (DNA metiltransferase 3a), histona demetilase JMJD3 e histona demetilase UTX foram analisadas por meio de RT-PCR quantitativo. Fibroblastos gengivais humanos derivados de cultura primária, e queratinócitos (HaCaT) foram expostos a LPS de P. gingivalis ou ao veículo do LPS, e foram avaliadas a viabilidade celular por meio do teste MTT e a expressão gênica de DNMT1, DNMT3a, JMJD3 e UTX por meio de RT-PCR quantitativo. As análises dos resultados demonstraram que nem a periodontite e nem o LPS exposto a fibroblastos gengivais foram capazes de modular a expressão dos genes estudados. Contudo, o LPS promoveu a diminuição da expressão de DNMT1, DNMT3a e JMJD3 nas células HaCaT. Pode-se concluir que LPS derivado P. gingivalis pode modular, em queratinócitos, a expressão gênica de algumas enzimas promotoras de eventos epigenéticos
Abstract: The aim of this study was to assess whether P. gingivalis LPS can modulate, in culture of the human keratinocytes and human gingival fibroblasts, gene expression levels of the some enzymes that promote epigenetic events. In addition, the same enzymes were evaluated in sample from healthy and periodontitis affected individuals. Primary gingival fibroblast culture and keratinocytes (HaCaT) were treated with medium containing P. gingivalis LPS or P. gingivalis LPS vehicle for 24hs. After this period, cell viability were assessed by MTT test, and total RNA were extracted to evaluate gene expression levels of the enzymes: DNMT1 (DNA methyltransferase 1), DNMT3a (DNA methyltransferase 3a), histone demethylases JMJD3 and UTX, by qRT-PCR. To evaluate the gene expression in healthy and periodontitis affected individuals, total RNA was extracted from biopsies of gingival tissue from sites with (periodontitis) or without periodontitis (healthy), and gene expression of DNMT1, DNAMT3a, JMJD3 and UTX were evaluated by qRT-PCR. No significant differences were found in the gene expression analysis between healthy gingival tissues and gingival tissue from periodontitis sites. The results showed that LPS downregulated DNMT1 (p<0.05), DNMT3a (p<0.05) and JMJD3 (p<0.01) gene expression in HaCaT cells, but no modulation was found to gingival fibroblasts. P. gingivalis LPS exposure to keratinocytes, downregulates gene expression of the enzymes that promote epigenetic events
Mestrado
Histologia e Embriologia
Mestre em Biologia Buco-Dental

Cellular senescence is thought to play an essential role in many biological functions including tumor suppression and organismal aging. Senescent cells, which are permanently removed from the cell cycle, can be found both in vivo in many different tissue types and in vitro within cultures of non-immortalized cells. Despite their inability to proliferate, these cells persist and remain metabolically active for indefinite periods of time. This physiologic process occurs in response to a variety of cellular insults including oxidative stress, shortened telomeres, constitutive oncogene expression, and DNA damage, and can be initiated by upregulation of one of the two known senescent pathways, involving p16/Rb or p53/p21. The senescent cell phenotype is also characterized by changes to cell and nuclear morphology and to the secretory profile of the cell. Related to changes in nuclear morphology, epigenetic modifications to the packaging of DNA are thought to be key to the initiation and maintenance of the senescence program. While a large number of earlier studies focused on the findings that senescent cells gain regions of condensed heterochromatin, often in the form of Senescent Associated Heterochromatin Foci (SAHF), this thesis work shows that there is a marked loss of heterochromatin in the peri/centromeric regions of the genome. In fact, both α-satellite and satellite II sequences across the genome distend in a striking and unanticipated fashion; this can be readily visualized by fluorescence in situ hybridization (FISH) as their structure changes from a condensed spot to highly elongated and fine thread-like signals. We have termed this exceptional decondensation of constitutive heterochromatin Senescence Associated Distension of Satellites (SADS). Importantly, a series of experiments shows that SADS is both a consistent and an early event in the cell senescence process, which occurs as a result of every senescence induction method examined. We also observed that this distension was characteristic of both human and murine cells and in vivo in human benign Prostatic Intraepithelial Neoplasia (PIN) tissue. Furthermore, unlike SAHF formation, SADS can occur due to the activation of either of the two senescence pathways, p16/Rb or p53/p21. Additionally, the cytological dimensions of the thread-like satellite signals indicates that SADS represents “unraveling” of DNA on an unprecedented scale. Thus, it was surprising that this event was not facilitated by changes to several canonical histone modifications associated with condensed heterochromatin, namely H3K9Me3, H3K27Me3, or H3K4Me3, nor is it caused by loss of DNA methylation. Consequently, we believe that this marked distension of satellite DNA is due to changes in higher-order folding of the chromatin fiber. This is important for understanding fundamental events in the cell senescence process, but also provides a unique system for study of chromatin packaging that may provide new insights into the organization of DNA well beyond nucleosome packaging and the ten nanometer fiber. In fact, initial super resolution images of SADS suggest that the satellite sequences may be organized into domains or “globules”. Hence, we suggest that the changes to satellite sequence packaging may be facilitated by changes to higher-order nuclear structural proteins, such as LaminB1, which is reduced in senescent cells. Finally, this work provides analysis of the literature and preliminary experiments to consider the possibility that there are increased levels of cell senescence in Down syndrome (trisomy 21) cells. As individuals with Down syndrome (DS) experience many manifestations of premature aging (including early-onset Alzheimer’s Disease), have a resistance to solid tumor formation, are more susceptible to oxidative stress, and are trisomic for several genes implicated in causing senescence, our analysis provides plausibility for the hypothesis that accelerated rates of senescence may play a significant role in DS physiology. We also provide results of preliminary studies and outline the next steps for experimentation, using DS fibroblasts and a unique genetically engineered DS iPS cell system. As a final note, the quantification of cell senescence in trisomic versus disomic cells for these experiments relies substantially on the new single-cell marker of senescence discovered and established by this theses work, the Senescence-Associated Distension of Satellites.

Cellular senescence is thought to play an essential role in many biological functions including tumor suppression and organismal aging. Senescent cells, which are permanently removed from the cell cycle, can be found both in vivo in many different tissue types and in vitro within cultures of non-immortalized cells. Despite their inability to proliferate, these cells persist and remain metabolically active for indefinite periods of time. This physiologic process occurs in response to a variety of cellular insults including oxidative stress, shortened telomeres, constitutive oncogene expression, and DNA damage, and can be initiated by upregulation of one of the two known senescent pathways, involving p16/Rb or p53/p21. The senescent cell phenotype is also characterized by changes to cell and nuclear morphology and to the secretory profile of the cell. Related to changes in nuclear morphology, epigenetic modifications to the packaging of DNA are thought to be key to the initiation and maintenance of the senescence program. While a large number of earlier studies focused on the findings that senescent cells gain regions of condensed heterochromatin, often in the form of Senescent Associated Heterochromatin Foci (SAHF), this thesis work shows that there is a marked loss of heterochromatin in the peri/centromeric regions of the genome. In fact, both α-satellite and satellite II sequences across the genome distend in a striking and unanticipated fashion; this can be readily visualized by fluorescence in situ hybridization (FISH) as their structure changes from a condensed spot to highly elongated and fine thread-like signals. We have termed this exceptional decondensation of constitutive heterochromatin Senescence Associated Distension of Satellites (SADS). Importantly, a series of experiments shows that SADS is both a consistent and an early event in the cell senescence process, which occurs as a result of every senescence induction method examined. We also observed that this distension was characteristic of both human and murine cells and in vivo in human benign Prostatic Intraepithelial Neoplasia (PIN) tissue. Furthermore, unlike SAHF formation, SADS can occur due to the activation of either of the two senescence pathways, p16/Rb or p53/p21. Additionally, the cytological dimensions of the thread-like satellite signals indicates that SADS represents “unraveling” of DNA on an unprecedented scale. Thus, it was surprising that this event was not facilitated by changes to several canonical histone modifications associated with condensed heterochromatin, namely H3K9Me3, H3K27Me3, or H3K4Me3, nor is it caused by loss of DNA methylation. Consequently, we believe that this marked distension of satellite DNA is due to changes in higher-order folding of the chromatin fiber. This is important for understanding fundamental events in the cell senescence process, but also provides a unique system for study of chromatin packaging that may provide new insights into the organization of DNA well beyond nucleosome packaging and the ten nanometer fiber. In fact, initial super resolution images of SADS suggest that the satellite sequences may be organized into domains or “globules”. Hence, we suggest that the changes to satellite sequence packaging may be facilitated by changes to higher-order nuclear structural proteins, such as LaminB1, which is reduced in senescent cells. Finally, this work provides analysis of the literature and preliminary experiments to consider the possibility that there are increased levels of cell senescence in Down syndrome (trisomy 21) cells. As individuals with Down syndrome (DS) experience many manifestations of premature aging (including early-onset Alzheimer’s Disease), have a resistance to solid tumor formation, are more susceptible to oxidative stress, and are trisomic for several genes implicated in causing senescence, our analysis provides plausibility for the hypothesis that accelerated rates of senescence may play a significant role in DS physiology. We also provide results of preliminary studies and outline the next steps for experimentation, using DS fibroblasts and a unique genetically engineered DS iPS cell system. As a final note, the quantification of cell senescence in trisomic versus disomic cells for these experiments relies substantially on the new single-cell marker of senescence discovered and established by this theses work, the Senescence-Associated Distension of Satellites.

During each life cycle, gametes must preserve and pass on both genetic and epigenetic information, making the germline both immortal and totipotent. In the male germline the dramatic morphological transformation of a germ cell through meiosis, into a sperm competent for fertilization, while retaining this information is an incredible example of cellular differentiation. This process of spermatogenesis is inherently thermosensitive in numerous metazoa ranging from worms to man. Here, I describe the role of two redundant AGO-class paralogs, ALG-3/4, and their small RNA cofactors, in promoting thermotolerant male fertility in Caenorhabditis elegans. alg-3/4 double mutants exhibit temperature dependent sterility resulting from defective spermiogenesis, the postmeiotic differentiation of haploid spermatids into spermatozoa competent for fertilization. The essential Argonaute CSR-1 functions with ALG-3/4 to positively regulate target genes required for spermiogenesis by promoting transcription via a small RNA positive feedback loop. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal loaded into CSR-1 to maintain transcriptionally active chromatin at genes required for spermiogenesis and to provide an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. The ALG-3/4 small RNA pathway also initiates silencing small RNA signals loaded into WAGO vii Argonautes, which function to posttranscripitonally silence their target mRNAs. Silencing WAGO/small RNA-complexes are present in sperm and presumably transmitted to offspring upon fertilization. Together these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic activating and silencing signals in the form of Argonaute/small-RNA complexes, constituting a memory of gene expression in preceding generations.

During each life cycle, gametes must preserve and pass on both genetic and epigenetic information, making the germline both immortal and totipotent. In the male germline the dramatic morphological transformation of a germ cell through meiosis, into a sperm competent for fertilization, while retaining this information is an incredible example of cellular differentiation. This process of spermatogenesis is inherently thermosensitive in numerous metazoa ranging from worms to man. Here, I describe the role of two redundant AGO-class paralogs, ALG-3/4, and their small RNA cofactors, in promoting thermotolerant male fertility in Caenorhabditis elegans. alg-3/4 double mutants exhibit temperature dependent sterility resulting from defective spermiogenesis, the postmeiotic differentiation of haploid spermatids into spermatozoa competent for fertilization. The essential Argonaute CSR-1 functions with ALG-3/4 to positively regulate target genes required for spermiogenesis by promoting transcription via a small RNA positive feedback loop. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal loaded into CSR-1 to maintain transcriptionally active chromatin at genes required for spermiogenesis and to provide an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. The ALG-3/4 small RNA pathway also initiates silencing small RNA signals loaded into WAGO vii Argonautes, which function to posttranscripitonally silence their target mRNAs. Silencing WAGO/small RNA-complexes are present in sperm and presumably transmitted to offspring upon fertilization. Together these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic activating and silencing signals in the form of Argonaute/small-RNA complexes, constituting a memory of gene expression in preceding generations.

Orientador: Maria Luiza Silveira Mello
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A cromatina pode existir em núcleos interfásicos em dois estados distintos: como eucromatina e como heterocromatina, podendo ser esta constitutiva ou facultativa. Em células somáticas do final da fase ninfal dos hemípteros reduviídeos Triatoma infestans e Panstrongylus megistus há núcleos grandes, poliploides, nos quais a heterocromatina apresenta-se como corpos conspícuos (cromocentros), daí tais células apresentarem-se como um bom modelo para investigação de características morfológicas e funcionais da cromatina. Em estudos sobre a constituição cromatínica, a composição em bases do DNA é algo muito explorado, dado o conteúdo informativo dos achados. Já quando se objetiva a investigação da funcionalidade da cromatina, mais recentemente, tem-se feito uso da abordagem epigenética. Neste trabalho buscou-se investigar a composição em bases do DNA destas células, associando-a aos domínios cromatínicos aí existentes e também à presença das NORs. Por meio de colorações fluorescentes com Cromomicina A3 (CMA3)/Distamicina e 4',6-diamidino-2-fenilindol (DAPI)/Actinomicina D concluiuse que o DNA dos cromocentros de T. infestans e P. megistus são ricos em sequências AT e pobres em GC. Isto foi ainda confirmado por imunodetecção de 5-metilcitosina, que ocorreu somente na eucromatina, e tratamento de ninfas de T. infestans com 5-aza-2'-deoxicitidina (agente demetilante), seguido da análise dos fenótipos nucleares e análise de imagem, em que se observou expansão somente da área eucromática. Com o método de AgNOR evidenciou-se que a região rica em bases GC ao redor do cromocentro coincide com um acúmulo de proteínas argirofílicas, o que sugere associação com NORs. A presença de modificações epigenéticas nas caudas das histonas na cromatina destes insetos foi investigada por meio do uso de anticorpos contra marcadores epigenéticos específicos, permitindo identificar a participação diferencial dos mesmos na composição e na estrutura dos territórios heterocromáticos. Assim, observou-se hipoacetilação e hipermetilação de histonas na região do corpo heterocromático, o que indicaria uma possível ação da modificação de histonas na manutenção da estrutura heterocromática nas células somáticas de ambas as espécies de reduviídeos. Por meio da avaliação da ação de drogas inibidoras de deacetilases de histonas sobre a cromatina dos insetos percebeu-se que, quando ninfas de T. infestans e P. megistus foram tratadas com as drogas, houve aumento na frequência de necroses e, no caso específico do tratamento com tricostatina A (TSA) e butirato de sódio (NaBt), ocorreu descompactação da heterocromatina. Sugere-se que o tratamento com TSA e NaBt afete o processo de deacetilação de histonas, o qual seria, então, um fator importante na estruturação dos cromocentros. A observação da ocorrência de mudas e da sobrevivência de ninfas de T. infestans, realizada a fim de se avaliar a ação do ácido valproico (VPA) sobre o desenvolvimento dos insetos, mostrou que a droga, assim como a injeção de solução salina, reduziu seu período de sobrevivência, além de afetar a ocorrência de mudas
Abstract: Chromatin in interphase cell nuclei can be present in two distinct states: euchromatin and heterochromatin, which may be constitutive or facultative. In somatic cells at the end of the nymphal stage of Triatoma infestans and Panstrongylus megistus there are large nuclei, in which heterochromatin is presented as conspicuous bodies (chromocenters). These cells are an appropriate model for investigation of morphological and functional characteristics of the chromatin. In studies about chromatin constitution, the base DNA composition is explored due to the informational content of the findings. If the objective is to investigate the chromatin functionality, recently has been used the epigenetic approach. In the current study, the aim was to investigate the DNA base composition in these cells, associating with the chromatin domains therein and also the presence of NORs. Through fluorescent stains with Chromomycin A3 (CMA3)/Distamycin and 4',6-diamidino-2-fenilindole (DAPI)/Actinomycin D was found that the chromocenters DNA of T. infestans and P. megistus were AT-rich and GC-poor. This was also confirmed by immunodetection of 5-methylcytidine, which occurred only in the euchromatin, and by T. infestans nymphs treatment with 5-aza-2'- deoxycytidine (demethylating agent), followed by nuclear phenotypes analysis and image analysis, in which expansion was observed only in the euchromatic area. AgNOR test evidenced that the GC-rich region around the chromocenter coincides with an accumulation of argyrophilic proteins, suggesting association with NORs. Epigenetic modifications on histone tails in chromatin of these insects were investigated by using antibodies against specific epigenetic markers, in order to identify their differential participation in the composition and structure of these heterochromatic regions. It was observed hypoacetylation and hypermethylation in heterochromatic body area, suggesting a possible action of histones modification in the maintenance of the heterochromatic structure in somatic cells of both species of reduviids. Through evaluation of histones deacetylases inhibitors action on the chromatin, it was observed that when T. infestans and P. megistus nymphs were treated with these drugs there was an increase in the frequencies of necrosis, and in the specific case of Trichostatin A (TSA) and sodium butyrate (NaBt), occurred heterochromatin decondensation. It is suggested that treatments with TSA and NaBt could affect the histones deacetilation process, which would be an important factor in chromocenters structuring. Observations of the molting occurrence and survival of T. infestans nymphs, carried out in order to evaluate the action of valproic acid (VPA) on the development of insects, showed that this drug, as well as injection of saline, reduced the survival period, besides affecting the molting occurrence
Mestrado
Biologia Celular
Mestre em Biologia Celular e Estrutural

Several aspects of Drosophila telomere biology indicate that telomere protection can be regulated by an epigenetic mechanism. First, terminally deleted chromosomes can be stably inherited and do not induce damage responses such as apoptosis or cell cycle arrest. Second, the telomere protection proteins HP1 and HOAP localize normally to these chromosomes and protect them from fusions. Third, unprotected telomeres still contain HeT-A sequences at sites of fusions. Taken together these observations support a model in which an epigenetic mechanism mediated by DNA damage response proteins protects Drosophilatelomeres from fusion. Work presented in this thesis demonstrates that the Drosophila proteins ATM and Nbs are required for the regulation of DNA damage responses similar to their yeast and mammalian counterparts. This work also establishes a role for the ATM and ATR DNA damage response pathways in the protection of both normal and terminally deleted chromosomes. Mutations that disrupt both pathways result in a severe telomere fusion phenotype, similar to HP1 and HOAP mutants. Consistent with this phenotype, HOAP localization at atm,atr double mutant telomeres is completely eliminated. Furthermore, telomeric sequences are still present, even at the sites of fusions. These results support a model in which an epigenetic mechanism mediated by DNA damage response proteins protects Drosophila telomeres from fusion.

Les altérations transcriptionnelles présentes dans le tissu cérébral et dans les leucocytes sanguins des patients souffrant de trouble dépressif majeur (TDM) ou de troubles schizophréniques (TSZ) pourraient être dues à des altérations de la machinerie épigénétique. Ce travail de thèse (i) a recherché des altérations transcriptionnelles des gènes codant pour la machinerie épigénétique dans le tissu cérébral et les leucocytes sanguins de patients souffrant de TDM ou de TSZ, (ii) a confronté les altérations transcriptionnelles périphériques aux altérations cérébrales afin d'évaluer leur significativité neurobiologique. Chez des patients avec TDM, une première étude utilisant la PCR en temps réel a identifié une surexpression des gènes codant pour des enzymes responsables de la mise en place de marques épigénétiques répressives vis-à-vis de l'expression génique dans le cortex préfrontal dorsolatéral (DLPFC), le cortex cingulaire et les leucocytes sanguins. Chez des patients avec TSZ, une deuxième étude de data-mining a identifié une surexpression des gènes codant pour la machinerie de biogénèse des microARNs dans le DLPFC et le cervelet, suggérant une augmentation de la production des microARNs dans ces deux régions cérébrales. Des altérations transcriptionnelles distinctes ont été observées dans les cellules sanguines mononucléées périphériques et l'épithélium olfactif. Une troisième étude utilisant la PCR en temps réel a identifié des réseaux de co-expression génique distincts dans les leucocytes et le tissu cérébral. Certains réseaux, exclusivement retrouvés chez les patients souffrant de TDM, pourraient participer à la constitution du phénotype pathologique. Dans le DLPFC et les leucocytes sanguins, le TDM est associé à une modification de la régulation transcriptionnelle des gènes intervenant dans les processus de stress et de sénescence cellulaires. Au total, ce travail retrouve une altération transcriptionnelle de la machinerie épigénétique dans le TDM et les TSZ. Dans le tissu cérébral, ces anomalies pourraient conduire à une répression de l'expression génique et participer aux altérations transcriptionnelles retrouvées dans le TDM et les TSZ. Dans les leucocytes périphériques, les altérations transcriptionnelles ne reflètent que partiellement les altérations centrales, suggérant que le TDM et les TSZ sont des affections systémiques. L'expression sanguine d'HDAC2 et celle de DICERl pourraient constituer de potentiels biomarqueurs périphériques, respectivement pour le TDM et les TSZ
Transcriptional abnormalities in brain tissue and blood leucocytes of patients with major depressive disorder (MDD) or schizophrenic disorders (SZ) may be due to alterations in the epigenetic machinery. This thesis work aimed to (i) identify altered expression of genes encoding the epigenetic machinery in brain and blood tissues of patients with MDD or SZ, (ii) and compare the peripheral transcriptional alterations to the cerebral ones in order to evaluate their neurobiological relevance.In MDD patients, a first study using real-time PCR identified overexpression of genes encoding enzymes which transfer repressive transcriptional marks in the dorsolateral prefrontal cortex (DLPFC), the cingulate cortex and the blood leucocytes. In SZ patients, a second study using a data-mining software identified an overexpression of genes encoding the microRNA biogenesis machinery in the DLPFC and the cerebellum, suggesting an increase in microRNA production in these two brain regions. Distinct transcriptional alterations were observed in peripheral mononuclear blood cells and the olfactory epithelium. In MDD patients, a third study using real-time PCR identified MDD-specific, distinct co-expression networks of stress- and senescence-related genes in the DLPFC and blood leucocytes. This suggests that MDD is associated with a transcriptional regulatory shift in the DLPFC and blood leucocytes.Altogether, these studies report transcriptional alterations of the epigenetic machinery coding genes in the brain tissues and blood leucocytes of MDD and SZ patients. In the brain tissues, aberrant expression of the epigenetic machinery coding genes may lead to the repression of gene expression and participate in the transcriptional abnormalities associated with MDD and SZ. In peripheral leucocytes, transcriptional alterations only partially reflect central ones, emphasizing that MDD and SZ are systemic disorders. Finally, HDAC2 and DICER1 blood expression may represent clinically useful peripheral biomarkers for MDD and SZ, respectively

The following work demonstrates that paternal diet controls medically important metabolic phenotypes in offspring. We observe transmission of dietary information to the zygote via sperm, and this information evades reprogramming that typically occurs after fertilization. Cytosine methylation is implicated as a major contributor to meiotic epigenetic inheritance in several transgenerational phenomena. Our extensive characterization of the sperm methylome reveals that diet does not significantly affect methylation patterns. However, we find that extensive epivariability in the sperm epigenome makes important contributions to offspring variation. Importantly, coordinate cytosine methylation and copy number changes over the ribosomal DNA locus contributes to variation in offspring metabolism. Thus, rDNA variability acts independently of postadolescent paternal diet to influence offspring metabolism. Therefore, at least two mechanisms exist for epigenetically controlling offspring metabolism: stochastic epivariation and diet acting by an unknown mechanism to further modulate metabolism. This work argues that an offspring's phenotype can no longer be viewed solely as the result of genetic interactions with the developmental environment - the additional influences of paternal environment and inherited epigenetic variability must also be considered. These findings reveal novel contributions to metabolism that could revolutionize how we think about the risk factors for human health and disease.

The following work demonstrates that paternal diet controls medically important metabolic phenotypes in offspring. We observe transmission of dietary information to the zygote via sperm, and this information evades reprogramming that typically occurs after fertilization. Cytosine methylation is implicated as a major contributor to meiotic epigenetic inheritance in several transgenerational phenomena. Our extensive characterization of the sperm methylome reveals that diet does not significantly affect methylation patterns. However, we find that extensive epivariability in the sperm epigenome makes important contributions to offspring variation. Importantly, coordinate cytosine methylation and copy number changes over the ribosomal DNA locus contributes to variation in offspring metabolism. Thus, rDNA variability acts independently of postadolescent paternal diet to influence offspring metabolism. Therefore, at least two mechanisms exist for epigenetically controlling offspring metabolism: stochastic epivariation and diet acting by an unknown mechanism to further modulate metabolism. This work argues that an offspring's phenotype can no longer be viewed solely as the result of genetic interactions with the developmental environment - the additional influences of paternal environment and inherited epigenetic variability must also be considered. These findings reveal novel contributions to metabolism that could revolutionize how we think about the risk factors for human health and disease.

It is increasingly recognized by the scientific community that the field of epigenetics is a key step for a better understanding of human biology in both normal and pathological states. Its implication in cancer, and in particular in breast cancer, is now well accepted. Breast cancer, responsible for more than 450,000 deaths worldwide yearly, is a heterogenous disease at the histological and clinical levels as well as at the molecular level. Despite considerable efforts to develop new treatments and improve patient management, patients with a same “profile” of breast cancer can respond differently to therapies and have completely different clinical outcomes. There is therefore a critical need to improve our understanding of breast cancer biology and diversity, in order to find new markers that should provide a better management of patients and the development of new therapies. An increasing number of biologists, pathologists as well as clinicians are currently working towards these goals. During my PhD, we have conducted two studies in order to gain new insights into the contribution of epigenetics in breast cancer biology.

In the first study, by performing large genome-scale DNA methylation profiling of numerous breast tumors as well as of normal breast tissues, we first revealed the existence of six groups of breast tumors based on their DNA methylation profiles. Three of these groups showed a strong association with the basal-like, HER2 and luminal A breast cancer subtypes, previously identified by gene expression profiling. Interestingly, the three other groups were found to be a mixture of several gene expression-based subtypes, thus revealing the capacity of DNA methylation profiling to improve breast tumor taxonomy. Second, our study suggests that the establishment of DNA methylation patterns of breast tumors might help to determine their cell type of origin. Finally, we also showed that DNA methylation profiling can reflect the cell type composition of the tumor microenvironment and that a signature of T cell tumoral infiltration is associated with a good prognosis in particular categories of breast cancer patients.

In the second study, we revealed the clinical relevance of the KDM5 histone demethylases in breast cancer. The expression of these histone demethylases was deregulated in the analyzed breast tumors as well as in the pre-invasive samples as compared to normal breast samples. This suggests that KDM5 enzymes might be good markers for early diagnosis of breast cancer. Moreover, we showed a prognostic value of the KDM5C histone demethylase.

In conclusion, the above data should provide a better understanding of breast cancer biology and diversity, and this should bring new insights to improve breast cancer patient management.

Doctorat en Sciences biomédicales et pharmaceutiques
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La régulation transcriptionnelle des gènes constitue une étape clef de la biologie cellulaire. Parmi les mécanismes impliqués dans la répression génique, les modifications épigénétiques jouent un rôle fondamental. Deux machineries épigénétiques, la méthylation de l’ADN et les protéines du groupe Polycomb, établissent des profils moléculaires qui permettent de distinguer les formes active et inactive de la chromatine. L’établissement et la maintenance de la répression épigénétique des gènes interviennent dans de nombreux processus liés au développement tant biologiques (inactivation du chromosome X chez les mammifères femelles, empreinte génomique ou encore l’expression de gènes tissus-spécifiques) que pathologiques (cancers).

Au cours de notre thèse de doctorat, nous nous sommes attachés à l’étude des mécanismes par lesquels la méthylation de l’ADN est ciblée en des régions génomiques précises et participe à la répression de l’expression des gènes. La méthylation de l’ADN est catalysée par des enzymes, appelées méthyltransférases de l’ADN (DNMTs), qui transfèrent des résidus méthyls sur les cytosines. Cette modification chimique covalente constitue un niveau de contrôle transcriptionnel important :il existe une corrélation entre méthylation de l’ADN et répression de l’expression génique au niveau de sites génomiques spécifiques. En outre, il semble de plus en plus clair qu’une méthylation aberrante de l’ADN participe au processus de cancérogenèse. A l’heure actuelle, les mécanismes moléculaires par lesquels la méthylation contribue au développement, à la différenciation et à la répression génique restent peu connus. Les données de la littérature suggèrent l’existence d’un lien étroit entre la méthylation de l’ADN et la structure de la chromatine. Celle-ci est notamment régulée par des modifications post-traductionnelles des histones. Il apparaît de plus en plus évident que la méthylation de l’ADN et les modifications des histones prennent part à une «boucle de répression» assurant le maintien et la propagation d’états épigénétiques répressifs. L’étude des mécanismes de la répression médiée par les DNMTs s’avère donc étroitement liée à celle de la structure de la chromatine.

Dans ce contexte, notre travail de thèse est basé sur l’hypothèse selon laquelle les deux principaux systèmes épigénétiques, la méthylation de l’ADN et les protéines Polycomb, agiraient de concert. Les protéines Polycomb participent au système de mémoire cellulaire, régulent l’expression et la différenciation, agissent sous forme de complexes multimériques associés à la chromatine et interviennent dans le contrôle de la prolifération cellulaire. Au cours de notre travail, nous nous sommes particulièrement intéressé à la protéine Polycomb EZH2 (Enhancer of Zeste) parce qu’elle possède une activité méthyltransférase d’histone sur les 27 de l’histone H3, impliquée dans la répression transcriptionnelle.

Dans un premier temps, nous avons mis en évidence un lien mécanistique entre les deux machineries épigénétiques principales, méthylation de l’ADN et protéines du groupe Polycomb. Nous avons montré qu’EZH2 interagit in vivo avec les DNMTs et purifie une activité méthyltransférase de l’ADN in vitro. Des expériences d’immunoprécipitation de la chromatine indiquent que les DNMTs fixent les régions promotrices de gènes cibles de EZH2 et que cette liaison est dépendante de la présence d’EZH2. Par ailleurs, l’analyse des promoteurs cibles d’EZH2 par séquençage au bisulfite suggère qu’EZH2 semble également requise pour la méthylation de l’ADN de ces séquences. Nos résultats permettent l’ébauche d’un modèle où EZH2 agit comme une plateforme de recrutement pour les DNMTs (Viré et al. Nature 2006).

Dans la deuxième partie de notre travail, nous avons investigué le rôle de MeCP2 dans ce modèle. MeCP2 est une protéine à domaine MBD (methyl-binding domain) qui se fixe sélectivement aux cytosines méthylées. Le recrutement de MeCP2 représente un mécanisme majeur par lequel la méthylation de l’ADN réprime la transcription. Nos données montrent que MeCP2 interagit avec EZH2 in vitro et in vivo et que ces protéines fixent des régions promotrices communes. De plus, le niveau de méthylation des cytosines semble prérequis à la présence d’EZH2. Ce travail suggère que MeCP2 puisse recruter EZH2 à la chromatine et renforcer un état réprimé de la chromatine en agissant comme un pont entre deux modifications épigénétiques essentielles, la méthylation de l’ADN et les proteins Polycomb (Viré et al. soumis).

En conclusion, notre travail de doctorat devrait permettre un meilleure compréhension des mécanismes moléculaires de l’épigénétique et plus particulièrement de cerner comment la méthylation de l’ADN est intimement connectée au remodelage de la chromatine, participe à la répression transcriptionnelle, est spécifiquement ciblée au sein du génome et contribue au développement et à la cancérogenèse.

Doctorat en Sciences biomédicales et pharmaceutiques
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La méthylation des cytosines de l’ADN est un niveau de contrôle essentiel de la transcription génique. Elle joue un rôle primordial dans plusieurs étapes du développement comme l’inactivation du chromosome X et l’empreinte génomique. De plus, il est de plus en plus évident que la méthylation de l’ADN participe à la cancérogenèse.

Actuellement, le monde de la méthylation de l’ADN n’en est encore qu’à l'aube de son histoire. En effet, les mécanismes moléculaires la gouvernant sont encore peu connus. La méthylation de l’ADN est caractérisée par deux concept clés :le verrouillage de la transcription des gènes et le ciblage en des régions spécifiques du génome. Au cours de notre travail de thèse de doctorat, nous avons poursuivi les avancées réalisées dans ces deux domaines.

Dans un premier temps, nous nous sommes attachés à l’étude de la répression transcriptionnelle entraînée par la méthylation de l’ADN. Grâce à plusieurs études récentes, il paraît de plus en plus clair que la méthylation agit de paire avec la structure de la chromatine. Nous avons donc concentré nos recherches sur l’interconnexion de celle-ci avec deux machineries impliquées dans la régulation de son degré de compaction :la désacétylation et la méthylation des histones. Par diverses expérimentations, nous avons démontré un lien étroit entre ces machineries répressives pour l’imposition d’un état silencieux de la transcription.

Dans la deuxième partie de ce travail, nous avons dirigé notre attention sur le ciblage des Dnmt. Pour cela, nous avons mené deux stratégies de front. La première est une approche ciblée et consiste en l’étude de l’association des Dnmt avec l’oncoprotéine bien connue, Myc. La seconde approche est plus large. Grâce à l’utilisation de la technique du double hybride en levure, nous avons identifié de nouveaux partenaires des Dnmt, dont un qui pourrait s’avéré particulièrement intéressant :le protéine Cart1 (cartilage homeoproteine 1) impliquée dans le développement du système nerveux central.

En conclusion, notre travail de doctorat devrait permettre une meilleure compréhension des mécanismes moléculaires de la méthylation de l’ADN ainsi que son implication dans les divers processus physiologiques mais aussi pathologiques auxquels elle participe.

Doctorat en sciences biomédicales
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The solid childhood cancer neuroblastoma arises from cells of the sympathetic nervous system. Neuroblastoma is the most common childhood cancer in children younger than one year old worldwide and accounts for 28% of all cancers diagnosed in Europe and the U.S. in infants. 25% of neuroblastomas bear an amplification of the proto-oncogene MYCN; in addition to this genetic alteration, epigenetic modifications such as transcriptional silencing of tumour suppressor genes by promoter DNA hypermethylation and histone modifications can occur. Previous studies have shown an interaction between MYC and DNA hypermethylation, through recruitment of de novo DNA methyltransferases to the promoter of MYC target genes, in order to repress their transcription. We hypothesised that MYCN might be similarly capable of repressing its target genes. Our results revealed that MYCN interacts with the maintenance and de novo methyltransferase DNMT1 and DNMT3A, respectively in MYCN amplified neuroblastoma cell lines and all three bind the promoter region of the hypermethylated gene RASSF1A. However, only minor changes in DNA methylation of RASSF1A were shown by pyrosequencing analysis upon MYCN knock-down. This probably indicates only a small involvement of MYCN in epigenetically-induced gene silencing by DNA hypermethylation. The important role of MYCN in gene activation was highlighted from DNA methylation microarray studies on neuroblastoma cell lines compared to human neural crest cells. 73% of all hit genes showed hypomethylation, which led to the conclusion that a more open chromatin configuration and thus increased gene expression is favoured in high stage neuroblastoma cell lines, where MYCN and MYC compensate each others protein levels. The same study also led to the discovery of a novel methylated gene MEGF10 in neuroblastoma. The importance of epigenetic silencing in neuroblastoma was also highlighted by the ability of DNA demethylating agent 5-Aza-2'-deoxycytidine to re-sensitise chemotherapeutic resistant neuroblastoma cells to commonly used cytotoxic drugs.

This mini-review investigates and compiles the latest knowledge regarding epigenetic changes on the mammalian mitochondrial DNA and its proteins. Methylation of the DNA, acetylation of the proteins and silencing of genes by short non-coding RNAs are the main epigenetic changes known today to affect mitochondrial DNA, mostly leading to repression. Methylation mainly occurs at non-CpG sites in the main non-coding region called the D-loop, with methylation patterns being cell type specific. Acetylation of proteins are mainly controlled by the deacetylase SIRT3, with its function being correlated to longevity. On the other hand, mitochondrial dysfunction is directly associated with a plethora of diseases, such as neurodegenerative disorders and heart disorders. The mitochondrion and nucleus are immensely dependent on each other and exchange vital proteins and RNAs, with epigenetic changes on one potentially affecting the other. Recent research shows that heteroplasmy is a proven cause of mitochondrial malfunction and that paternal inheritance is possible. The mitochondrial haplotype also shows different vulnerability to certain diets and diseases, leading to the conclusion that the mitochondrial haplotype can be used to more than just tracing human origins, such as to predicting and preventing diseases.

Epigenetic modification of DNA plays an important role in gene regulation. During gametogenesis and early embryogenesis epigenetic states are reset to ensure embryonic-specific gene expression patterns after fertilization. However, certain genomic regions may resist epigenetic reprogramming. This may result in transgenerational epigenetic inheritance. Earlier, a grandparental origin dependent (GPO) transmission ratio distortion (TRD) of alleles in the distal region of mouse chromosome 12 had been found (Croteau et al ., 2002). The distorted region overlaps with the imprinted region of chromosome 12. The mechanism underlying this TRD is unknown, and we hypothesized that it was due to failure to reset imprints in the imprinted region in a proportion of germ cells. Such an imprint resetting failure would represent a particular case of transgenerational epigenetic inheritance. DNA (Cytosine-5) methyltransferase 1 (DNMT1) plays a key role in the maintenance of epigenetic states in mammalian genomes. To test the role of DNA methylation and DNMT1 in the genesis of TRD and its relationship to epigenetic inheritance we investigated the effect of Dnmt1 loss-of-function mutations using two mouse models: GPO (grandparental origin dependent)-TRD (transmission ratio distortion) and epigenetic inheritance at the agouti locus. Here, we report that Dnmt1 mutations have a modifying parental effect on the transmission of grandparental chromosome 12 alleles. However, the same Dnmt1 mutation did not affect the agouti coat color inheritance patterns in mice that inherited the Avy (agouti viable yellow) mutant allele from the father. Our results suggest that Dnmt1 is a trans-acting modifier of allelic transmission and support the role of epigenetic states in the genesis of TRD.

Primary malignant bone tumours are rare cancers that are characterised by different genetic and epigenetic alterations. A functional epigenomic approach was combined with the Illumina HumanHT-12.v4-BeadChip expression microarray in three Ewing Sarcoma (ES) cell lines to identify genome-wide functional methylation changes in these cells and ES primary samples. This study revealed eight frequently methylated genes in ES patients’ samples, where NPTX2 and PHF11 promoter methylation was associated with poor patient prognosis. The second methylation study involved genome-wide DNA methylation profiling of chordoma samples using the Infinium-HumanMethylation450-BeadChip microarray. This study identified a list of 8,819 loci which were differentially methylated between chordomas and controls and eight genes which were differentially methylated between recurrent and non-recurrent chordoma samples. RNA sequencing (RNA-seq) analysis of primitive small blue round cell tumour (SBRCT) samples was also carried out in order to identify gene fusions in this type of cancer. Three different somatic gene fusions in SBRCT samples were identified using RNA-Seq (CRTC1-SS18;BCR-UPB1 and KHDRBS2-CIC). Moreover, two other gene fusions were identified in unpaired SBCRT samples. Overall, this study used high-throughput technologies to identify novel genetically and epigenetically altered genes in different types of bone sarcoma which may, therefore, provide unique insight into bone sarcoma tumorigenesis.

Medulloblastoma is the most common malignant brain tumour of childhood. Transcriptomic profiling has revealed the existence of four core molecular subgroups (SHH, WNT, Group 3 and Group 4) with distinct clinical, pathologic and molecular characteristics. However, the specific molecular events associated with tumour development in these groups are poorly understood. DNA methylation plays a key role in epigenetic transcriptional regulation, and promoter hypermethylation leading to gene silencing is a common feature of medulloblastoma. DNA methylation profiling has identified distinct methylomic profiles associated with the four subgroups of medulloblastoma, and the wider role of DNA methylation in medulloblastoma now requires investigation. Using two high-throughput screening approaches, this project therefore undertook a comprehensive investigation into the potential role of specific DNA methylation events in the development of the distinct subgroups of medulloblastoma. Using DNA methylation profiles, which were generated for 216 medulloblastomas using the GoldenGate methylation array, the first approach identified 73 CpG methylation markers (encompassing 63 genes) which significantly distinguished Group 3 and/or Group 4 medulloblastomas. Subgroup-specific differential gene expression analysis showed that, for the majority of the methylation markers identified, there was no clear inverse association between methylation and gene expression. One gene (RHOH) was identified which showed strong evidence of epigenetic dysregulation in medulloblastomas. RHOH methylation represented a potential epigenetic event in Group 4 tumours; 51% of Group 4 medulloblastomas showed aberrant hypomethylation of multiple RHOH promoter-associated CpG residues, which was associated with upregulated RHOH expression in Group 4 tumours. RHOH was re-expressed in 4 out of 6 methylated cell lines following treatment with the demethylating agent 5’-aza-2’-deoxycytidine (5-azaCdR). This study has thus identified a novel putative oncogenic role for RHOH in Group 4 medulloblastoma development. In the second approach, a functional epigenomics screen identified 283 genes which were upregulated in 2 or more cell lines investigated (n=10) following 5-azaCdR treatment. Assessment of DNA methylation using the Illumina 450K methylation array identified 160 CpG residues (encompassing 21 of the 283 genes) whose methylation status was consistent with expression alterations observed after 5-azaCDR, and methylation-dependent gene regulation, in cell lines. 9/160 CpG residues (6%) showed evidence of subgroup-specific differential methylation which was concordant with differential gene expression and potential epigenetic gene regulation in medulloblastoma subgroups. These 9 sites represented 5 candidate genes (ACTC1, ANXA2, FAM46A, PRPH and S100A4). Aberrant hypermethylation of multiple gene body CpG residues was associated with FAM46A silencing in non-SHH tumours, while aberrant hypermethylation of multiple promoter-associated residues was associated with ACTC1 silencing in Group 3 and Group 4 medulloblastomas. Single site v hypomethylation events associated with upregulated expression in WNT tumours were identified for ANXA2, PRPH and S100A4. This study has identified six genes with putative oncogenic or tumour suppressor roles in the development of distinct subgroups of medulloblastoma through their epigenetic dysregulation. Further work is now required to validate these findings and to assess their functional significance in medulloblastoma subgroups, as well as their potential relevance in medulloblastoma sub-classification and prognostication.

Microfluidics has revolutionized how molecular biology studies are conducted. It permits profiling of genomic and epigenomic features for a wide range of applications. Microfluidics has been proven to be highly complementary to NGS technology with its unique capabilities for handling small volumes of samples and providing platforms for automation, integration, and multiplexing. In this thesis, we focus on three projects (diffusion-based PCR, MID-RRBS, and SurfaceChIP-seq), which improved the sensitivities of conventional assays by coupling with microfluidic technology. MID-RRBS and SurfaceChIP-seq projects were designed to profiling genome-wide DNA methylation and histone modifications, respectively. These assays dramatically improved the sensitivities of conventional approaches over 1000 times without compromising genomic coverages. We applied these assays to examine the neuronal/glial nuclei isolated from mouse brain tissues. We successfully identified the distinctive epigenomic signatures from neurons and glia. Another focus of this thesis is applying electrical field to investigate the intracellular contents. We report two projects, drug delivery to encapsulated bacteria and mRNA extraction under ultra-high electrical field intensity. We envision rapid growth in these directions, driven by the needs for testing scarce primary cells samples from patients in the context of precision medicine.
Ph. D.

Multiple computational algorithms were developed for analyzing ChIP-seq datasets of histone modifications. For basic ChIP-seq data processing, the problems of ambiguous short sequence read mapping and broad peak calling of diffuse ChIP-seq signals were solved by novel statistical methods. Their performance was systematically evaluated compared with existing approaches. The potential utility of finding meaningful biological information was demonstrated by the applications on real datasets. For biological question driven data mining, several important topics were selected for algorithm developments, including hypothesis-driven insulator prediction, unbiased chromatin boundary element discovery and combinatorial histone modification signature inference. The integrative computational pipeline for insulator prediction not only produced a list of putative insulators but also recovered specific associated chromatin and functional features. Selected predictions have been experimentally validated. The unbiased chromatin boundary element prediction algorithm was feature-free and had the capability to discover novel types of boundary elements. The predictions found a set of chromatin features and provided the first report of tRNA-derived boundary elements in the human genome. The combinatorial chromatin signature algorithm employed chromatin profile alignments for unsupervised inferences of histone modification patterns. The signatures were associated with various regulatory elements and functional activities. Both the computational advantages and the biological discoveries were discussed.

Cancer is a result of multiple genetic and epigenetic alterations that are acquired during tumour formation and progression. The development of cost-effective high-throughput technologies has positively contributed to the field of cancer research to identify cancerassociated genes, in addition to diagnostic and prognostic markers. Using the most recent Illumina Infinium HumanMethyaltion450K BeadChip, this work has identified 23 genes that were significantly and differentially hypermethylated in short-term survivor (STS) glioblastoma patients and showed an association with poor prognosis. Interestingly, \(NR2F2\) showed hypermethylation across multiple CpG loci and was significantly correlated with poor survival. In addition, the work has identified a subset of long-term survivors (LTS) that showed high DNA hypermethylation across many CpG loci indicating the presence of CIMP\(^+\). The other methylation study has identified 7 genes (\(EOMES\), \(WDR69\), \(MIR125B1\), \(DZIP1\), \(SOX1\), \(PHOX2B\) and \(PRDM13\)) that were differentially hypermethylated in breast cancer brain metastasis (BCBM) tumours compared to non-metastatic breast tumours. These genes also showed hypermethylation in the paired breast tumours and BCBM samples, signifying the importance of the 7 genes in the development of brain metastasis in the early stages. Loss of expression of \(DZIP1\), \(PRDM13\) and \(PHOX2B\) was significantly associated with poor patient survival, indicating their potential as prognostic markers for BCBM patients. Whole exome sequencing (WES) analysis of sporadic non-\(NF2\) vestibular schwannoma was disappointing and was not able to identify somatic mutations in the novel genes involved in schwannoma formation. However, the identification of novel deleterious mutations in the \(NF2\) gene confirmed the validation of WES to identify cancer-associated variants. This study has used the most recent and developed high-throughput technologies to identify candidate genes that were genetically or epigenetically altered in a range of CNS tumours.

Osteoarthritis (OA) is a common degenerative disease of synovial joints that principally affects older individuals. The genetic architecture of OA is highly complex, with heterogeneous pathological pathways converging on a common end phenotype characterised by the painful loss of articular cartilage. In this study, I investigated both genetic and epigenetic aspects of the OA disease process. The genetic investigation focused on the functional analysis of a female hip OA association signal that was discovered in the arcOGEN study, marked by the single nucleotide polymorphism (SNP) rs4836732. The signal surpasses the genome-wide significance threshold, with p = 6.11x10-10. The signal encompasses only two other SNPs that have a high degree of linkage disequilibrium (LD) with rs4836732. Functional studies using luciferase reporter assays, electrophoretic mobility shift assays (EMSAs) and a range of transformed cell lines did not identify any differential allelic activity between SNP alleles, but did identify differential EMSA banding patterns for the C allele of rs4836732 when compared to the T allele, suggestive of differential protein complex binding dependent on the allele present at rs4836732. Subsequent investigations focused on three genes in the immediate vicinity of the association signal: ASTN2, PAPPA and TRIM32. All three genes were expressed in cartilage, synovium and fat pad from OA patients, though none displayed differential allelic expression correlating with rs4836732 genotype in these tissues. This lack of positive evidence in end-stage disease tissue may be indicative of the association signal mediating risk during joint development or growth. Of the three genes, PAPPA was deemed the most likely candidate to have a functional affect in joint development. Knock-down of the gene in mesenchymal stem cells revealed that PAPPA is required for osteogenic and chondrogenic differentiation, providing support ii for further investigation of this gene. My second line of investigation focused on OA epigenetics, specifically on subgroups of hip and knee OA patients who displayed altered cartilage methylation of inflammatory genes when analysed by high-density CpG methylation array. As part of this study, I developed pyrosequencing assays to determine the inflammatory subgroup of new patient cartilage samples, negating the need for a high-density array. I also further interrogated the inflammatory status of the patients, analysing serum markers of systemic inflammation and gathering biometric data to ascertain the primary cause of inflammation, with a focus on the metabolic status of patients in relation to body fat. Interestingly, no systemic differences in inflammation, circulating leptin or BMI were observed. This suggests the elevated inflammatory status may be local to the synovial joint, as opposed to being secondary to increased systemic inflammation. Overall, the two investigation routes that I have pursued further emphasise the heterogeneous nature of OA. They also highlight the importance of patient stratification for OA research and provide platforms to improve our comprehension of this debilitating disease.

Mestrado em Biomedicina Molecular
Laryngeal carcinomas belong to a bigger family of tumours known as Head and Neck Cancer (HNC). HNC is the sixth most malignant type of cancer in the world and it can arise from several anatomical sites. Among them, the larynx is the second most common affect organ. The incidence of laryngeal carcinoma is 1,9% worldwide and it presents a high mortality rate (1,6%). Despite technological advances in diagnosis and treatment fields, the 5 year-survival rate did not improved significantly. The low survival rates are mainly explained by a late diagnosis, tumour aggressiveness and the fact that laryngeal carcinoma metastasize easily. Taking this into consideration, it is essential to identify biomarkers with significant diagnostic and prognostic value in order to anticipate the detection of laryngeal carcinoma in an early stage. This study arises mainly for characterize the genetic and epigenetic profile of laryngeal squamous cell carcinoma (LSCC). Eight LSCC samples and seven non-tumour samples contralateral to the primary tumour were collected upon resection surgery and characterized by MLPA, MS-MLPA and array CGH. The results showed that gain of genetic material was mainly present in chromosomes 3q, 8q, 11q, 14q13.1, Xp22.31 and Xq21.1 while genetic loss occurred mainly in chromosomes 3p, 9p23.1 and Y. Gain of MYC and TNFRSF1A was the most common event among the tumour samples included in this study. Regarding the methylation profile, the genes CDKN2A, CHFR, RARβ e RASSF1 were the only ones which were methylated in this samples. In conclusion, this study allowed to identify genetic alterations associated with LSCC that have already been reported in scientific papers as well as alterations that have been associated with tumour development and progression. In addition, a few genetic alterations which have never been reported as being associated with human cancer before were identified. Nevertheless, new studies must be carried out, with a higher number of samples. Ultimately, the main goal would be to identify genetic alterations significantly associated with LSCC progression and establish a correlation with clinicopathological data.
O carcinoma da laringe pertence a uma grande família de tumores conhecida como Cancro da Cabeça e do Pescoço que é considerado o sexto tipo de tumor mais maligno em todo o mundo. Dentro desta família, os tumores podem ter origem em diversos locais anatómicos, sendo a laringe o segundo órgão mais comummente afetado. O cancro da laringe apresenta uma incidência mundial de 1,9% e uma taxa de mortalidade elevada de 1,6%. Apesar dos avanços tecnológicos na área do diagnóstico e da terapêutica, a taxa de sobrevivência ao fim de 5 anos não apresentou melhorias significativas. As baixas taxas de sobrevivências são explicadas essencialmente pelo diagnóstico tardio, pela agressividade do tumor e pela sua propensão a desenvolver metástases. Desta forma, torna-se essencial a identificação de biomarcadores com valor de diagnóstico e prognóstico a fim de detetar a presença do tumor numa fase mais precoce. Este estudo surge com o objetivo principal de caracterizar o perfil genético e epigenéticos do carcinoma das células escamosas da laringe com recurso às técnicas de MLPA, MS-MLPA e array CGH, usando oito amostras tumorais e sete amostras não-tumorais contra laterais ao tumor, ambas coletadas após cirurgia A análise genética revelou uma maior taxa de ganho de material genético nos cromossomas 3q, 8q, 11q, 14q13.1, Xp22.31, Xq21.1 e perda de material genético nos cromossomas 3p, 9p23.1 e Y. O ganho dos genes MYC e TNFRSF1A revelou ser o evento mais comum entre as amostras analisadas. Relativamente ao perfil epigenético, observou-se que os genes CDKN2A, CHFR, RARβ e RASSF1 se encontravam metilados nas amostras em estudo. Em suma, este trabalho permitiu identificar algumas alterações genéticas e epigenéticas descritas na literatura como estando associadas ao CCEL, assim como alterações associadas ao desenvolvimento tumoral. Foram ainda identificadas alterações que ainda não foram reportadas como estando associadas ao cancro. Desta forma, este estudo piloto permitiu dar início ao estudo de potenciais biomarcadores associados ao CCEL. Porém, novos estudos devem ser realizados, com um número de amostras superior, de forma a identificar alterações genéticas significativas no desenvolvimento e progressão do CCEL e associa-las às características clinico patológicas dos doentes.

Die molekulare Charakterisierung von genetischen Veränderungen der Akuten Myeloischen Leukämie (AML) wurde vor allem bei jungen Patienten vorangebracht, hingegen fehlen Analysen für die AML bei älteren Patienten. Entsprechend ergab sich die Rationale, genetische und epigenetische Alterationen bei älteren AML Patienten zu untersuchen, um spezifische Signaturen zu identifizieren. Wir untersuchten ältere 93 AML Patienten aus dem Study Alliance Leukemia (SAL) Register. Es wurden 555 Gene auf der Illumina HiSeq2000 Plattform sequenziert und DNA Methylierungsprofile mittels dem Illumina 450K Array untersucht. Insgesamt wurden 814 molekulare Alterationen in 281 Genen detektiert. Besonders hohe Mutationsfrequenzen wurden in den Genen DNMT3A (33%), TET2 (24%), SRSF2 (23%) und ASXL1 (21%) notiert. Alterationen in epigenetischen Regulatoren (85%) und in Genen, die in Splicing involviert sind (38%), wurden gehäuft beobachtet. Ältere AML Patienten mit Mutationen in DNMT3A oder DNA Reparaturgenen wiesen eine geringere Lebenserwartung im Vergleich zu der restlichen Kohorte auf. Wir verglichen die Meythlierungsdaten aus der SAL mit denen der TCGA Kohorte, umso die Methylierungsmuster von älteren mit denen von jüngeren Patienten zu differenzieren. Es konnte ein distinktes Methylierungsprofil in den DNA Proben von älteren AML Patienten nachgewiesen werden. Die im Vergleich zu jüngeren AML Patienten unterschiedlich methylierten Regionen bei älteren AML Patienten überlappten mit Genen verschiedener Signalwege, die Hallmarks von Alterungsprozessen und Krebs entsprechen. Zusammenfassend konnten wir für die Kohorte älterer AML Patienten genetische Alterationen nachweisen und mit spezifischen Profilen von Mutationen in epigenetischen Regulatoren korrelieren. Diese molekulare Kategorisierung unterstreicht distinkte biologische Mechanismen in älteren AML Patienten und die Notwendigkeit von spezifischen Therapieansätzen für diese Kohorte mit ungünstiger Prognose.
Despite advances in the characterization of molecular alterations in younger acute myeloid leukemia (AML) patients, comprehensive studies in elderly AML are lacking. Thus, we investigated genetic and epigenetic alterations and probed for specific signatures to understand the unfavorable outcomes of elderly AML. We studied 93 AML patients (65 to 90 years old), enrolled in the Study Alliance Leukemia registry (SAL). To capture a broad spectrum of alterations, we sequenced 555 genes on an Illumina HiSeq2000 platform and investigated DNA methylation profiles using the Illumina 450K array. Overall, we detected 814 molecular alterations in 281 genes, with a median of 7 genes mutated per patient. Particularly high mutation frequencies were identified for DNMT3A (33%), TET2 (24%), SRSF2 (23%) and ASXL1 (21%). We observed frequent alterations in epigenetic regulators (85%) and in splicing factors (38%). Notably, SAL elderly AML patients with mutations in DNMT3A or DNA repair genes (in absence of mutations in NPM1 or splicing factors) had an inferior survival of only 9 months (compared to 17 months for the remaining patients). In addition, for the analysis of elderly AML DNA methylation, we integrated the SAL cohort with TCGA methylation data for comparisons of methylation levels to younger patients. A distinct DNA methylation profile was observed in older AML patients, which correlated with the presence of mutations in IDH1/2, RUNX1 and ASXL1 and epigenetic similarities with TP53/Complex samples. The differential methylated regions of elderly AML (compared to younger AML samples) were shown to overlap genes from several pathways that are hallmarks of both age and cancer. In conclusion, we unraveled distinct patterns of genetic alterations and correlated specific epigenetic profiles of elderly AML to high rate mutated epigenetic regulators. This molecular categorization underscored the distinct biology and the need for specific therapeutic approaches in elderly AML.

L’aborto spontaneo (AS) è la perdita della gravidanza che porta alla morte del feto prima delle 20-28 settimane, secondo l’Organizzazione Mondiale della Sanità (OMS). Circa il 10-15% di tutte le gravidanze clinicamente riconosciute esitano in aborto spontaneo. La perdita di gravidanza ri-corrente (RPL) è definita come due o più aborti spontanei consecutivi. RPL colpisce almeno l’1-2% delle donne in età riproduttiva. La maggior parte degli aborti si verifica durante il primo trimestre di gestazione. Le principali cause conosciute di RPL sono anomalie cromosomiche, anomalie anatomiche, malattie autoimmuni, malattie da infezione, et a materne e paterna avanzata e agenti ambientali. Nel 50 % dei casi, le cause di RPL sono ancora sconosciute. La diagnosi ha luogo solo dopo la morte dell’embrione e solo in pochi casi ci sono follow-up per comprendere le cause genetiche, con tecniche che permettono di discriminare le aneuploidie. Lo scopo di questa tesi è capire se il profilo genetico ed epigenetico nelle donne in gravidanza e, per la prima volta, sul prodotto del concepimento, svolgono un ruolo nel determinare il rischio di perdita della gravidanza(RPL). In questo studio sono stati analizzati 156 campioni di interruzioni volontari di gravidanza (VTP) e 91 di RPL e 40 di abortività spontanea (FPL), per un totale di 131 campioni, utilizzati come casi. I campioni sono stati collezionati presso l’Unita di Ostetricia e Ginecologia all’Ospedale-Universitario Sant’Anna di Ferrara, Italia, dal 2016 al 2020. Le analisi svolte in questa tesi mi hanno permesso di osservare che l’allelec.677T dell’SNP rs1801133 nel gene MTHFR` è associato, nel gruppo delle madri, ad un significativo aumento del rischio di RPL (OR = 1,72, p= 0,0018), seguendo un modello co-dominante. L’aumento del rischio osservato di RPL è aumentato di quasi 3 volte nelle donne omozigoti T/T (OR = 2,64, IC 95 % = 1,33-5,22, p = 0,004). Negli eterozigoti si osserva una leggera diminuzione di tale rischio (OR = 1,61, IC 95% = 0,95-1,77, p = 0,075). Negli embrioni, il genotipo G/G sul SNPrs2617170 nel geneNKG2Dmostra un effetto protettivo per RPL (OR= 0,27, IC 95 % = 0,10-0,72, p = 0,0067). L’analisi della metilazione del DNA non mostra alcuna differenza significativa nella metilazione dellaLINE-1 tra gDNA da RPL e VTP nel sangue materno. Tuttavia, un significativo aumento del livello di metilazione può essere osservata nei casi RPL rispetto ai casi VTP se si considera il gDNA proveniente dai villi corionici (p-value 0.0010). Un effetto significativo si osserva quando si considera l’esposizione della supplementazione periconcezionale con acido folico (400 g/day). Infatti, la metilazione di LINE-1 tra i casi RPL in CV è significativamente più alta rispetto ai casi RPL senza supplementazione con acido folico (p-value = 0.030). Questo studio `e importante perché ́e per la prima volta ho esaminato il gDNA presente nel sangue delle madri soggette ad abortività spontanea che dai villi coriali provenienti dal proprio prodotto di concepimento. Fino ad ora, la maggior parte degli studi si sono concentrati solo sul sangue della madre e/o sullo sperma dei padri, ma nessuno di questi ha incluso analisi anche sui villi coriali a causa della difficoltà del loro reperimento dal materiale materno. Questo studio dimostra l’importanza di concentrarsi sul trio (madre, padre e feto) durante la gravidanza, al fine di prevenire l’abortività ricorrente spontanea. In particolare, può essere rilevante agire direttamente sullo stile di vita materno erroneo che può provocare un esito negativo della gravidanza. Questi risultati gettano le basi per identificare un protocollo efficace per il trattamento delle donne con aborto spontaneo ricorrente senza causa apparente, che attualmente `e assente.
Pregnancy loss(PL) is the loss of pregnancy resulting in fetal death before 20-28 weeks, according to the World Health Organization (WHO). Roughly 10-15% of all clinically recognized pregnancies result in miscarriage. Recurrent pregnancy loss (RPL) is specified as two or more consecutive spontaneous loss of pregnancy. RPL affects at least 1-2 %of women in reproductive age. Most of the miscarriage happens during the first trimester of gestation. The main known causes of RPL are chromosomal anomalies, anatomical abnormalities, autoimmune diseases, infection diseases, advanced maternal and paternal age and environmental agents. In 50% of the cases, the causes of RPL are still unknown. The diagnosis takes place only after the death of the embryo, and only a few cases there are follow-up to understand the genetic causes with techniques that can discriminate aneuploidies. The aims of this dissertation is to understand if the genetic and epigenetic profile in the women and, for the first time, on the product of conception play a role in determining the risk of Recurrent Pregnancy Loss (RPL). This study analyzes 156 Volunteer termination pregnancy(VTP) and 91 recurrent pregnancy loss (RPL), 40 first pregnancy loss (FPL) in a total of 131 cases. The samples were provided by the Unit of Obstetrics and Gynecology at The Sant’Anna University Hospital of Ferrara, Italy, from 2016 to 2020. I found that the c.677T allele of the SNP rs1801133 in the MTHFR gene associates in mothers with significant increased risk of RPL (p=0.0018) under a co-dominant model, with an almost 3-fold increased risk in T/T homozygote women (p=0.004) and a slight decrease in heterozygotes (p=0.075). In embryos the genotype G/G at the SNP rs2617170 in the gene NKG2D shows a protective effect from RPL (p=0.0067). The anaylsis of methylation of DNA, show no significant LINE-1 methylation difference between gDNA from RPL and VTP in mother’s white blood cells (WBC). However, a significant increase of 5% higher methylation level (p-value 0.0010) can be seen in RPL cases compared to VTP cases when considering gDNA from chorionic villi. A significative effect was observed when considering the exposure to periconceptional supplementation with folic acid (FA) (400 μg/day). In fact, the LINE-1 DNA methylation among RPL cases in CV is significantly higher in cases not supplemented with FA (p-value = 0.030). This study is important because for the first time I examined the gDNA both in mothers and chorionic villi from miscarriage. Until now, the majority of study focus only on mother's blood and sperm from fathers, but no one include also chorionic villi due to difficult to obtained it. This study shows the importance to focus on the trios (mother, father and fetus) during pregnancy in order to prevent miscarriage. Acting on mother life style where this are not suitable for good result of pregnancy. This results lay the foundations for identifying an effective protocol for the treatment of women with recurrent spontaneous abortion without apparent cause, that currently is absent.

Mechanisms underlying congenital malformations are largely unknown despite its high incidence, affecting 2-3% of liveborn infants. A broader knowledge about the causes of birth defects would provide valuable information regarding the outcome and prognosis of the anomaly, the development and establishment of diagnostic protocols, the design of therapeutic strategies and genetic counseling to the family. Different approaches have been used in the present thesis regarding technologies and model diseases to elucidate the contribution of genetic and epigenetic alterations in the etiopathogenesis of congenital malformations. Copy number variations, methylation patterns, as well as point mutations have been explored. Moreover, a study to analyze genetic counseling in relation to one of the new molecular techniques used has been performed. Obtained data reveal a relevant role of genetic and epigenetic alterations in congenital malformations, in some cases as a unique cause to explain the disease and in others as part of an oligogenic or multifactorial model.
Els mecanismes causants de les malformacions congènites són poc coneguts malgrat l’elevada incidència d’aquestes patologies, que afecten el 2-3% de recent nascuts. Un coneixement més ampli de les causes de les anomalies congènites proporcionaria informació rellevant pel que fa al pronòstic de l’anomalia, el desenvolupament i establiment de protocols diagnòstics, el disseny d’estratègies terapèutiques, així com l’assessorament genètic a la família. En la tesi que es presenta s’han utilitzat diferents estratègies, pel que fa a tecnologies i models de malalties, amb l’objectiu d’esbrinar la contribució d’alteracions genètiques i epigenètiques en l’etiopatogènia de les malformacions congènites. S’han analitzat variacions en número de còpia, patrons de metilació, així com mutacions puntuals. D’altra banda, també s’ha realitzat un estudi per aprofundir en l’assessorament genètic en relació a una de les noves tècniques moleculars utilitzades. Els resultats obtinguts indiquen que les altercacions genètiques i epigenètiques tenen una contribució molt rellevant en l’etiologia de les malformacions congènites, en alguns casos com a causa única de la malaltia i en altres com a component d’un model oligogènic o multifactorial.

In multicellular organisms all cells in one individual have an identical genotype, and yet their bodies consist of many and very different tissues and thus many different cell types. Somehow there must be a difference in how genes are interpreted. So, there must be signals that tell the genes when and where to be active and inactive, respectively. In some instances a specific an expression pattern (active or inactive) is epigenetic; it is established and maintained throughout multiple rounds of cell divisions. In the developing Drosophila embryo, the proper expression pattern of e.g. the homeotic genes Abd-B and Ubx is to be kept active in the posterior part and silenced in the anterior. Properly silenced homeotic genes are crucial for the correct segmentation pattern of the fly and the Polycomb group (Pc-G) proteins are vital for maintaining this type of stable repression.

As part of this thesis, Suppressor of zeste 12 (Su(z)12) is characterized as a Drosophila Pc-G gene. Mutations in the gene cause widespread misexpression of several homeotic genes in embryos and larvae. Results show that the silencing of the homeotic genes Abd-B and Ubx, probably is mediated via physical binding of SU(Z)12 to Polycomb Response Elements in the BX-C. Su(z)12 mutations are strong suppressors of position-effect-variegation and the SU(Z)12 protein binds weakly to the heterochromatic centromeric region. These results indicate that SU(Z)12 has a function in heterochromatin-mediated repression, which is an unusual feature for a Pc-G protein. The structure of the Su(z)12 gene was determined and the deduced protein contains a C2-H2 zinc finger domain, several nuclear localization signals, and a region, the VEFS box, with high homology to mammalian and plant homologues. Su(z)12 was originally isolated in a screen for modifiers of the zeste-white interaction and I present results that suggests that this effect is mediated through an interaction between Su(z)12 and zeste. I also show that Su(z)12 interact genetically with other Pc-G mutants and that the SU(Z)12 protein binds more than 100 euchromatic bands on polytene chromosomes. I also present results showing that SU(Z)12 is a subunit of two different E(Z)/ESC embryonic silencing complexes, one 1MDa and one 600 kDa complex, where the larger complex also contains PCL and RPD3.

In conclusion, results presented in this thesis show that the recently identified Pc-G gene, Su(z)12, is of vital importance for correct maintenance of silencing of the developmentally important homeotic genes.

Alcoholic liver disease (ALD) is a significant and growing global health problem, responsible for over 10000 deaths per year in the UK alone. Clinical liver failure can result from gradual, chronic depletion of the hepatocyte pool and replacement with fibrous tissue in cirrhosis or from rapid, acute hepatocellular dysfunction secondary to inflammation in acute alcoholic hepatitis (AAH) which carries a mortality of up to 35% on first presentation. Corticosteroid therapy has shown some benefit in AAH but its utility is limited by uncertainty in patient selection and poor clinical response in a proportion of cases. Our current understanding of AAH pathogenesis attributes hepatocellular dysfunction to the action of supra-physiological concentrations of proinflammatory cytokines. Evidence from animal and human studies suggests that the major source of cytokine release is the hepatic macrophage or Kupffer cell responding to an increased concentration of bacterial endotoxin in portal blood following an ethanol-mediated increase in gut permeability. However, this enhanced and sustained inflammatory response is at odds with the normal response in the liver in which endotoxin tolerance allows bacterial components to be cleared from the blood without an inflammatory response. This study set out to investigate factors that determine the enhanced inflammatory response in AAH and its response to therapy. Genetic analysis revealed a single nucleotide polymorphism in a component of the endotoxin response pathway (the Toll-like receptor adapter molecule MAL) associated weakly with advanced disease in both ALD and the related condition non-alcoholic steatohepatitis. Different alleles associated with advanced disease in the two conditions, suggesting divergent importance of signalling pathways in their pathogenesis. Assays in AAH patients demonstrated that their lymphocyte steroid sensitivity was impaired relative to normal controls, correlated with clinical markers of steroid responsiveness, improved in recovery and could be improved by ex vivo supplementation with theophylline, a known recruiter of histone deacetylases. The role of histone modifications in the enhancement of inflammatory responses in ethanol was investigated in a human macrophage cell-line model which revealed increased histone acetylation at pro-inflammatory cytokine gene promoter regions associated with potentiated cytokine responses to endotoxin after culture in ethanol or its metabolite acetate. This effect was abrogated by knockdown of acetyl-coA synthetases, suggesting that increased synthesis of acetyl-coA from acetate is crucial for histone acetylation and consequent increased cytokine production after ethanol exposure. These findings suggest that while genetic predisposition may have some effect on innate immune responses in the pathogenesis of alcoholic liver disease, the more significant contribution is likely to come from gene-environment interactions such as modulation of histone acetylation by products of ethanol metabolism. This epigenetic relationship between metabolism and gene expression in inflammation, mediated by histone deacetylases such as the sirtuin proteins, may be a novel therapeutic target in ALD and potentially also in other inflammatory conditions.

The aim of this study was to characterise the genetic and epigenetic profiles of rare forms of sporadic renal cancers (RCC) and identify differential patterns of DNA methylation or somatic mutations that may permit distinction between different subtypes of RCC and could facilitate disease prognosis or identify molecular pathways that could be targeted therapeutically. Illumina Infinium HumanMethylation 450K BeadChip permitted the comparison of the epigenome of the malignant chromophobe RCC and the benign renal oncocytoma. This study identified several genes to be differentially hypermethylated in chromophobe RCC, and renal oncocytoma showing that although both visually and pathologically similar, both tumours have a distinct methylation pattern. Whole exome sequencing (WES) of renal oncocytoma samples identified somatic mutations in eighteen genes involved in a variety of cellular functions. Sanger sequencing was then used to confirm the mutations identified, followed by further screening by Sanger in a cohort of additional renal oncocytoma samples to identify if the somatic mutations are recurrent. Modern high throughput and quantitative techniques have permitted further characterisation of these rare renal cancers and have enabled unique insights into their molecular genetics, findings that may hopefully be of clinical benefit in the future.

Common, complex disorders are polygenic and multifactorial traits representing interactions between environmental, genetic and epigenetic risk factors. More often than not, contributions of these risk factors have been studied individually and this is especially true for complex reproductive traits where application of genomic technologies has been challenging and slow to progress. This thesis explores the potential of genetic and epigenetic components contributing to a better understanding of the biological pathways underlying disease risk in two specific female complex reproductive traits - polycystic ovary syndrome (PCOS) and preterm premature rupture of membranes (PPROM). The PCOS projects focus on characterization of a gene, DENND1A, whose association to PCOS has been established by Genome Wide Association Studies (GWAS) and is known to contribute to PCOS steroidogenic phenotype. In addition, differential microRNAs expression contributing to DENND1A expression regulation in PCOS theca cells was identified. The studies on PPROM utilize a Whole Exome Sequencing approach to identify rare variants in fetal genes contributing to extracellular matrix composition and synthesis contributing to PPROM risk. The results suggest that fetal contribution to PPROM is polygenic and is driven by a significant genetic burden of potentially damaging rare variants in genes contributing to fetal membrane strength and integrity. Tissue and location specific expression patterns of the Chromosome 21 miRNA cluster (miR-99a, miR-125b, let-7c) in fetal membranes from term pregnancies with spontaneous rupture were investigated. The results suggest that these miRNAs play potential roles in fetal membrane rupture and fetal membrane defects associated with T21.

Exposure assessment in cancer epidemiological studies relies on measurable intermediate molecular biomarkers with high sensitivity and specificity in order to prevent common problems due to misclassification of exposure. Studies on the early stages of carcinogenesis have helped to identify molecular changes that are detectable in pre-cancerous lesions and that are thought to occur as the result of specific exposures such as tobacco smoking. More recently, in vitro evidence started to support the potential cancer-protective role of various micronutrients acting through epigenetic and genetic mechanisms. Somatic mutations in “master” cancer genes and modifications of epigenetic patterns in the promoter region of specific genes involved in cell cycle, apoptosis or DNA repair may prove good candidates of carcinogenic and dietary exposure even if the evidence that these changes may be present and detectable in “normal” tissue are still scarce (due in part to the practical and ethical difficulty to conduct experimental prospective studies in healthy individuals). In this thesis, I have developed two projects exploring the application of TP53, KRAS, EGFR mutations and of DNA methylation changes as biomarkers of exposure to tobacco smoking, in experimental and observational study designs. Somatic mutations were analysed by dHPLC, ME-PCR, RFLP and sequencing and DNA-methylation analysis was performed by pyrosequencing. Moreover, somatic mutations were analysed in a prospective context of lung cancer recurrence; also the capacity of dietary polyphenols and isothiocyanates to modify methylation patterns in smokers was assessed in an intervention trial. The results show that somatic mutations are good markers of different forms of tobacco-related lung cancers but have limited short-term prognostic value, with the exception of KRAS mutations in adenocarcinoma. Methylation data suggested that a specific short-term dietary intervention may stabilize global epigenetic (LINE1 DNA methylation) patterns in peripheral white blood cells.

L’artrite reumatoide (AR) è una patologia autoimmune infiammatoria cronica che colpisce prevalentemente le articolazioni ed è potenzialmente invalidante. L’AR presenta manifestazioni eterogenee e colpisce lo 0.5-1.0% della popolazione caucasica. Nello sviluppo di tale patologia concorrono diversi fattori di rischio, come quelli genetici, legati al sesso e fattori ambientali come l’esposizione al silice, agenti infettivi, fumo di sigaretta, carenza di vitamina D, obesità e alterazioni del microbiota umano. I fattori genetici giocano un ruolo chiave nello sviluppo e mantenimento dell’artrite reumatoide nel circa 50-60% dei casi. I geni coinvolti sono classificati come i geni per l’Antigene Leucocitario Umano (HLA) e geni non-HLA. Il Methotrexate (MTX) è uno dei farmaci più utilizzati per la terapia dell’AR e viene considerato il gold standard per il trattamento di tale patologia. Può essere utilizzato in combinazione con altre terapie. L’epigenetica sta rappresentando un ambito promettente per l’AR, poiché può contribuire non solo nell’arrestare il decorso di tale patologia, ma può aiutare nel migliorare le terapie e quindi la sua gestione. Scopo: Lo studio presentato in questa tesi ha lo scopo di: a) valutare l’associazione di polimorfismi a singolo nucleotide (SNP) noti, dei geni HLA e geni non-HLA, con lo sviluppo dell’artrite precoce (ERA) tra pazienti a cui è stata diagnosticata artrite indifferenziata; b) valutare la risposta alla terapia nei pazienti ERA in base al risultato genetico basato sugli SNP coinvolti nella via MTX/ciclo dei folati, c) studiare lo stato di metilazione dei Long Interspersed Transposable Element 1 (LINE-1) nei controlli sani e nei casi di ERA e RA. Inoltre si vuole studiare una eventuale modificazione nello stato di metilazione tra i pazienti ERA sottoposti al trattamento con MTX . Materiali e Metodi: I pazienti totali con artrite indifferenziata reclutati e analizzati in questo studio sono in totale 471. Il DNA genomico è stato estratto delle cellule bianche del sangue (WBC). La genotipizzazione è stata effettuata su 7 polimorfismi tra i gene HLA, come: rs1233334, rs1063320, 14 bp Insertion/deletion, rs660895, rs6910071, rs9275595 e rs10807113, mentre per i geni non-HLA sono: PTPN22 rs2476601, PADI4 rs2240340, STAT4 rs7574865, CTLA4 rs231775, TRAF1 rs3761847, IL-10 rs1800871 e IL-6 rs1800795. Per quanto riguarda lo studio della risposta alla terapia MTX/ciclo dei folati sono stati genotipizzati i seguenti geni: ATIC rs2372536, MTRR rs1801394, SHMT1 rs1979277, SLC19A1 rs1051266, MTHFR rs1801133 e MTHFR rs1801131. Le analisi epigenetiche hanno visto lo studio dello stato di metilazione di LINE-1 nelle cellule sinoviali e WBC. Lo stato di metilazione è stato valutato e comparato in 50 pazienti sani, 50 pazienti con ERA e 30 con AR. Inoltre è stato valutato lo stato di metilazione di LINE-1 nei pazienti ERA in base alla risposta alla terapia a cui sono stati sottoposti. Sono state condotte analisi statistiche per lo studio di associazione genetica, interazione gene-gene e lo studio di metilazione. Risultati: L’associazione delle varianti HLA coinvolte nello sviluppo di ERA non ha mostrato alcun risultato significativo. Anche per quanto riguarda gli SNP non-HLA, non si è riscontrata alcuna associazione significativa con lo sviluppo di ERA. Dopo la stratificazione in base al sesso, si è osservata un’associazione significativa con il gene TRAF1 rs3761847 per il genotipo eterozigote G/A nei maschi (p<0.05), secondo il modello dominante. Le analisi Multifactorial Dimensionality
Undifferentiated arthritis (UA) is a common inflammatory type of arthritis which is known by joint swelling, pain, and stiffness and is not classified as a specific rheumatologic disease. It is estimated that 32% of UA patients will develop rheumatoid arthritis (RA) that is a chronic, inflammatory, autoimmune disease which could cause joint damage and unchangeable disability. RA has heterogeneous presentations and affects 0.5 to 1.0% in white populations. Several risk factors have been reported for RA development. These include genetic, sex, and environmental factors such as slilica exposure, infectious agents, smoking, deficiency of vitamin D, obesity, and microbiota changes. Genetic factors play a significant role in rheumatoid arthritis development, and probably account for 50–60% of disease susceptibility which are classified as Human Leukocyte Antigen (HLA) and non-HLA genes. Methotrexate (MTX) is a first-line treatment in rheumatoid arthritis management. It is the most important drug in combination therapies, and is considered as a gold standard among RA therapies. Epigenetics as a promising and growing research field in rheumatoid arthritis not only contributes to the RA development but also could be involved in disease management and therapy. Objectives: The current study aimed in a) evaluating the association of well-known single nucleotide polymorphisms (SNPs) belonging to human leukocyte antigen (HLA) and non-HLA genes with early rheumatoid arthritis (ERA) development among UA diagnosed patients; b) assessing the response to therapy of ERA patients according to genetic background based on SNPs involved in MTX/folate pathway, and c) studying long interspersed transposable element 1 (LINE1) methylation status among healthy control, ERA and RA cases and also its methylation changes in response to MTX therapy among ERA patients. Subjects, Material and Methods: A total of 471 patients with UA have been recruited. Genomic DNA from white blood cells (WBC) was extracted and genotyping for 7 polymorphisms belonging to HLA genes including rs1233334, rs1063320, 14 bp Insertion/deletion, rs660895, rs6910071, rs9275595 and rs10807113 and another 7 SNPs belonging to non-HLA genes including PTPN22 rs2476601, PADI4 rs2240340, STAT4 rs7574865, CTLA4 rs231775, TRAF1 rs3761847, IL-10 rs1800871 and IL-6 rs1800795 have been performed. Regarding response to the therapy, 6 SNPs involving in MTX/Folate pathway including ATIC rs2372536, MTRR rs1801394, SHMT1 rs1979277, SLC19A1 rs1051266, MTHFR rs1801133 and MTHFR rs1801131 had been genotyped. In respect of epigenetics, LINE1 methylation status of synovial cells and WBC was evaluated. Besides, the methylation status of 50 healthy controls, 50 ERA and 30 RA patients were compared. Finally, LINE1 methylation of ERA patients according to the response to therapy have been investigated. Statistical analyses for genetic association, gene-gene interaction and methylation level has been carried out. Results: Regarding the association of HLA variants with ERA development, no significant association was found. With respect to non-HLA SNPs there was not significant association with ERA development, too. After stratification according to sex, there was a significant association of TRAF1 rs3761847 GA heterozygous in males (p<0.05). The association was under dominant model. Multifactorial Dimensionality Reduction (MDR) analysis also revealed that there was an association between smoking and anti-Citrullinated Protein Antibody (ACPA) with ERA development. Regarding association of MTX/folate-related SNPs, there was a significant association of CC homozygous genotype of SLC19A1 rs1051266 in ACPA-positive patients with response to the therapy (p<0.02).

Thesis (Ph. D.)--University of California, Riverside, 2009.
Includes abstract. Includes bibliographical references (leaves 105-122). Issued in print and online. Available via ProQuest Digital Dissertations.