Introduction: At our post-genomic era the studies of epigenetic regulation constitutes one of the tools for understanding the function of genes. Epigenetic regulation can directly control the temporal and spatial gene activity or silencing. The molecular basis of these regulations are DNA bases modifications, chromatin remodeling and RNA interference. At the same time, these mechanisms have a special way of transferring genetic information to subsequent generations called epigenetic inheritance. It has been proven epigenetic deregulation of certain genes as cause for many disease. For this reason, the study of epigenome HLA genes seems particularly important because these genes play a fundamental role in regulating the immune system. Aims: The aim of this work is to create a description of epigenetic modifications within families. It is an analysis of histone modifications and DNA methylation in the promoter region of the gene HLA DQA1. The aim was also to compare the differences in epigenetic modifications between alleles and compared the differences in these modifications between generations. The results will be compared with the analysis of the level of expression of the gene HLA DQA1. Methods: From collected peripheral blood of donors were isolated DNA, RNA, and leukocytes. DNA was used for...

Les protéines à « Methyl-CpG-binding domain » (MBD) jouent un rôle important dans l’interprétationde la méthylation de l’ADN conduisant à la répression transcriptionnelle via le recrutement decomplexes remodelant la chromatine. Dans les cancers, MBD2 jouerait un rôle essentiel dans la perted’expression des gènes hyperméthylés. Ainsi, MBD2 serait une cible potentielle pour rétablir, enpartie au moins, leur expression. Caractériser, à l’échelle du génome, la distribution de MBD2 et sesconséquences sur la répression transcriptionnelle au cours de la cancérogenèse est donc une étapeincontournable. (1) L’impact sur l’expression génique de l’inhibition de MBD2 par interférence àl’ARN, a été étudié en utilisant des puces, dans des cellules normales MRC5. La perte de MBD2n’induit pas de surexpression génique globale et la densité en CpG des promoteurs méthylés sembleêtre une composante importante dans la force de répression par MBD2. (2) Les profils de méthylationde l’ADN, de liaisons de MBD2 et de l’ARN polymérase II dans les cellules HeLa ont été analysés parChIP-on-chip avec des puces promoteurs. Ces mêmes approches couplées à l’analyse de l’acétylationdes histones H3 ont été réalisées dans un modèle cellulaire syngénique de progression tumoralemammaire humain. Dans les modèles étudiés, une forte proportion de gènes silencieux et méthylés estliée par MBD2. Les comparaisons entre cellules immortalisées et transformées ne montrent pas dechangements majeurs de la méthylation de l’ADN ou de la répression transcriptionnelle, par contreune redistribution de MBD2 parmi ces sites est observée, suggérant une redondance entre les protéinesliant l’ADN méthylé. / The Methyl-CpG-Binding Domain (MBD) proteins represent key molecules in the interpretation ofDNA methylation signals leading to gene silencing through recruitment of chromatin remodelingcomplexes. In cancer, a member of this protein family, MBD2, seems to play an important role in theloss of expression of aberrantly methylated genes. Thus, MBD2 may be a potential target toreestablish their expression. Mapping of MBD2 binding sites and the relationship between MBD2binding and transcriptional activity was, therefore, a crucial step. (1) We investigated the impact ofMBD2 inhibition by RNA interference on gene expression, using microarray analysis, in a normalhuman fibroblastic cell line, MRC-5. MBD2 depletion did not induce global gene overexpression andCpG density of the methylated promoters seems to be an important parameter in the strength of thetranscriptional repression mediated by MBD2. (2) Global profiling for different layers of epigeneticmodifications (DNA methylation, MBD2 association) and RNA polymerase II binding sites in HeLacells was analyzed by a ChIP-chip method using human promoter arrays. This approach, combinedwith an analysis of H3 histone acetylation patterns, was performed in a syngenic model of breastcancer progression. In the models analyzed MBD2 appeared to be a true methylation-dependenttranscriptional repressor. Furthermore, MBD2 binds to a high proportion of methylated silent genes.Comparisons between immortalized and transformed cells did not indicate major changes of DNAmethylation or gene silencing, while a redistribution of MBD2 among these sites was observed,suggesting a redundancy between methylated binding proteins.

Chez les plantes et les mammifères, la méthylation de l’ADN est une modification chromatinienne qui joue un rôle pivot dans le maintien de l’intégrité des génomes, notamment au travers de l’extinction épigénétique des éléments transposables (ET). Cependant, dans la mesure où la désamination spontanée des cytosines méthylées, qui peut conduire à des transitions C>T, est plus fréquente que celle des cytosines non méthylées, la méthylation est également intrinsèquement mutagène. Cette mutabilité accrue est de fait très certainement à l’origine de la déplétion en dinucléotides CpG observée dans les génomes de mammifères, naturellement méthylés à ces sites sauf au sein des «îlots CpG». A l’exception de cet effet bien connu, aucune étude à ce jour n’a exploré directement et de façon exhaustive l’impact de la méthylation sur le spectre des mutations spontanées. Dans ce travail, je tire profit d’une population de lignées epiRIL (epigenetic recombinant inbred lines) établie chez la plante Arabidopsis pour évaluer à l’échelle du génome l’impact de la méthylation de l’ADN sur le paysage mutationnel. Les epiRILs dérivent du croisement entre deux parents quasi- isogéniques, l’un sauvage et l’autre porteur d’une mutation conduisant à une réduction de 70% de la méthylation du génome, et il a pu être mis en évidence que des différences parentales de méthylation pouvaient être héritées de façon stable pour >1000 régions le long du génome. Au moyen de données de séquençage disponibles pour >100 epiRIL, j’ai effectué la caractérisation exhaustive des variants ADN (autres qu’ET) uniques à chaque lignée mais également en ségrégation parmi les epiRIL, ce qui constitue à terme une ressource pour les différentes équipes qui utilisent cette population. En analysant le patron de variants uniques, j’ai mis en évidence une réduction spécifique du taux de transitions C>T en lien avec l’hypométhylation stable dans les epiRIL. J’ai aussi pu décrire que si la remobilisation extensive des ET dans cette population a modelé le spectre des insertions et délétions ponctuelles, elle ne se traduit pas pour autant par des réarrangements récurrents. Je présente également les développements méthodologiques mis en place afin d’effectuer la caractérisation de QTL (quantitative trait loci) “épigénétiques” préalablement identifiés dans la population. / In both plants and mammals, DNA methylation plays a pivotal role in ensuring proper genome function and integrity, notably through the epigenetic silencing of transposable elements (TEs). However, as spontaneous deamination of 5- methylcytosine (5mC), which can lead to C>T transitions, is more frequent than that of unmethylated C, DNA methylation is also inherently mutagenic. This higher mutability of 5mC has indeed been proposed to explain the depletion in CpG dinucleotides in mammalian genomes, which are typically methylated at these sites except in socalled CpG islands. Despite this well-characterized effect of DNA methylation, we still lack a comprehensive view of its impact on the whole mutation spectrum in any given organism. Here, I take advantage of a population of so-called epigenetic Recombinant Inbred Lines (epiRILs) established in the flowering plant Arabidopsis thaliana to investigate the impact of DNA methylation on the spectrum of spontaneous mutations genome wide. The epiRIL population derives from a cross between a wild-type individual and a near-isogenic mutation deficient in DNA methylation, and it could be shown that parental differences in DNA methylation are stably inherited for at least 8 generations over >1000 regions across the genome. Building on whole-genome sequencing data available for >100 epiRILs, I performed a thorough characterization of non- TE DNA sequence variants that are either private to one line or segregating in the population, therefore establishing a resource for research groups that make use of the epiRIL population. Based on the pattern of private variants, I show a specific reduction in the rate of C>T transitions in the epiRILs, in line with the heritable hypomethylation in this population. I also describe that the extensive TE remobilisation at play among the epiRILs shapes the spectrum of short insertions and deletions yet does not translate into recurrent large-scale mutation events. On another note, I also present methodological developments aimed towards the identification of causal (epi)variants underlying so-called “epigenetic QTL” (quantitative trait loci) previously described in the epiRIL population.

<p>Mammalian development is orchestrated by global transcriptional changes, which drive cellular differentiation, giving rise to diverse cell types. The mechanisms that mediate the temporal control of early differentiation can be studied using embryonic stem cell (ESCs) and embryonal carcinoma cells (ECCs) as model systems. In these stem cells, differentiation signals induce transcriptional repression of genes that maintain pluripotency (PpG) and activation of genes required for lineage specification. Expression of PpGs is controlled by these genes’ proximal and distal regulatory elements, promoters and enhancers, respectively. Previously published work from our laboratory showed that during differentiation of ESCs, the repression of PpGs is accompanied by enhancer silencing mediated by the Lsd1/Mi2-NuRD-Dnmt3a complex. The enzymes in this complex catalyze histone H3K27Ac deacetylation and H3K4me1/2 demethylation followed by a gain of DNA methylation mediated by the DNA methyltransferase, Dnmt3a. The absence of these chromatin changes at PpG enhancers during ESC differentiation leads to their incomplete repression. In cancer, abnormal expression of PpG is commonly observed. Our studies show that in differentiating F9 embryonal carcinoma cells (F9 ECCs), PpG maintain substantial expression concomitant with an absence of Lsd1-mediated H3K4me1 demethylation at their respective enhancers. The continued presence of H3K4me1 blocks the downstream activity of Dnmt3a, leading to the absence of DNA methylation at these sites. The absence of Lsd1 activity at PpG enhancers establishes a “primed” chromatin state distinguished by the absence of DNA methylation and the presence of H3K4me1. We further established that the activity of Lsd1 in these cells was inhibited by Oct3/4, which was partially repressed post-differentiation. Our data reveal that sustained expression of the pioneer pluripotency factor Oct3/4 disrupts the enhancer silencing mechanism. This generates an aberrant “primed” enhancer state, which is susceptible to activation and supports tumorigenicity. </p> <p>As differentiation proceeds and multiple layers of cells are produced in the early embryo, the inner cells are depleted of O₂, which triggers endothelial cell differentiation. These cells form vascular structures that allow transport of O₂ and nutrients to cells. Using ESC differentiation to endothelial cells as a model system, studies covered in this thesis work elucidated a mechanism by which the transcription factor Vascular endothelial zinc finger 1 (Vezf1) regulates endothelial differentiation and formation of vascular structures. Our data show that Vezf1-deficient ESCs fail to upregulate the expression of pro-angiogenic genes in response to endothelial differentiation induction. This defect was shown to be the result of the elevated expression of the stemness factor Cbp/p300-interacting transactivator 2 (Cited2) at the onset of differentiation. The improper expression of Cited2 sequesters histone acetyltransferase p300 from depositing active histone modifications at the regulatory elements of angiogenesis-specific genes that, in turn, impedes their activation. </p> <p>Besides the discovery of epigenetic mechanisms that regulate gene expression during differentiation, our studies also include development of a sensitive method to identify activities of a specific DNA methyltransferase at genomic regions. In mammals, DNA methylation occurs at the C5 position of cytosine bases. The addition of this chemical modification is catalyzed by a family of enzymes called DNA methyltransferases (Dnmts). Current methodologies, which determine the distribution of Dnmts or DNA methylation levels in genomes, show the combined activity of multiple Dnmts at their target sites. To determine the activity of a particular Dnmt in response to an external stimulus, we developed a method, Transition State Covalent Crosslinking DNA Immunoprecipitation (TSCC-DIP), which traps catalytically active Dnmts at their transition state with the DNA substrate. Our goal is to produce a strategy that would enable the determination of the direct genomic targets of specific Dnmts, creating a valuable tool for studying the dynamic changes in DNA methylation in any biological process.</p>

Obesity is a risk factor for development of cardiovascular disease, diabetes type 2 and some cancers. Newly detected genetic risk factor for body weight is the FTO gene ("fat mass and obesity associated"). The aim of this thesis was determine 1) whether the presence of risk alleles correlate with BMI in Czech population and to determine 2) whether there is an association between variants in the FTO gene and risk of myocardial infarction/ acute coronary syndrome (MI/ ACS), 3) renal failure (ESRD), or 4) incidence of colorectal cancer (CRC). We analyzed polymorphisms rs17817449 (first intron) and rs17818902 (3rd intron) using by PCR-RFLP and then also RT PCR. We found an association of the first intron variant (but not the 3rd one) and BMI in Czech control population. We have detected an association of 1st intron SNP and BMI changes during the intervention study in obese children, but not in obese females. We found a correlation between the risk allele and increased risk of ACS (OR 1.49) in patients with MI. In patients with ESRD was detected association between the risk allele and the risk of disease (OR 1.37). We didn't confirmed the association between rs17817449 and the development of CRC. Representative selected groups of the Czech populations "MONICA" and "HAPPIE" were used as controls. One...

Le cancer, principale cause de mortalité dans le monde, est un problème majeur de santé publique. Malgré les nombreux traitements disponibles, il est nécessaire de développer de nouvelles thérapies plus efficaces et moins envahissantes. Aujourd’hui la connaissance du génome humain a dirigé la recherche vers de nouvelles approches: il est possible de moduler la réponse biologique en contrôlant l'accès aux informations génétiques via la régulation épigénétique.L’épigénétique est l’ensemble des modifications de l’expression des gènes n’entraînant pas de modifications dans la séquence d’ADN, qui mènent à un phénotype héritable et stable. Chez les eucaryotes, la régulation épigénétique implique des modifications covalentes de l'ADN (méthylation) et des histones (acétylation, méthylation…). Ces phénomènes modifient la structure de la chromatine, aboutissant à une configuration "ouverte" ou "fermée" permettant la transcription ou la répression de gènes. Dans une situation cancéreuse, le profil épigénétique est modifié ; la méthylation anormale de l’ADN ou des histones mène à la répression de certains gènes comme des gènes suppresseurs de tumeur, ou à l’expression des oncogènes. Contrairement aux changements génétiques irréversibles, les aberrations épigénétiques sont des modifications chimiques réversibles. Ainsi, des molécules capables de rétablir l'équilibre épigénétique représentent des outils thérapeutiques potentiels contre le cancer.La méthylation et l’acétylation sont les modifications épigénétiques les plus étudiées. La méthylation de l’ADN est catalysée par les ADN méthyltransférases (DNMTs), et la méthylation des histones par les histones méthyltransférases (HMTs).Le sujet de ce projet doctoral est porté sur les HMTs et en particulier sur DOT1L (DOT1 like, disruptor of telomericsilencing), responsable des méthylations du résidu Lys79 de l’histone 3 (H3K79), conduisant à la transcription des oncogènes. En effet, des études ont montré que DOT1L est liée à la leucémie et se révèle être une cible intéressante à inhiber. DOT1L comme les DNMT ont un même cofacteur : le SAM (S-adénosyl-L-méthionine). Certains de leurs inhibiteurs présentent un mécanisme d'inhibition commun : ils entrent en compétition avec SAM.Nous présentons la conception basée sur des études de modélisation moléculaire, et la synthèse multi-étapes des séries des molécules formées par 3 motifs principaux : a) un motif aminopyrimidine, b) un motif de type benzimidazole ou phénylurée, liés par c) un groupement phényle ou hétérocyclique. L’activité des composés synthétisés sur DOT1L a été évaluée et des relations structure-activité (RSA) ont été établies. L’activité sur DNMT et d’autres HMTs a été déterminée également afin d’étudier la spécificité de nos composés.Différents structures ont été identifiées comme point de départ pour aboutir à des inhibiteurs sélectives de DOT1L ou à des inhibiteurs mixtes DOT1L/DNMT. Ces molécules sont considérées comme des outils thérapeutiques intéressants dans le traitement du cancer. / Cancer is a serious issue of public health as it is one of the main causes of mortality worldwide. Despite the multiple available treatments, it is necessary to develop more efficient and less invasive therapies against cancer. The knowledge of the human genome and epigenome has directed research to new cancer treatment approaches: it is possible to modulate the biological outcome by controlling the access to the genetic information by means of the epigenetic regulation.Epigenetics are the changes happening on the genome without modifying its DNA sequence, leading to a heritable andstable phenotype. In the eukaryotic chromatin, epigenetic regulation implies covalent modifications of DNA and histones. These chemical modifications remodel the chromatin structure leading to an “opened” or “closed” configuration, which is related to the expression or repression of genes. The epigenetic landscape is altered in cancers; for example, abnormal methylation leads to the silencing of certain genes (such as tumor suppressor genes), or to the over-expression of oncogenes. Unlike genetic alterations that are irreversible, epigenetic aberrations are reversible. Thus, molecules that can reestablish the epigenetic balance represent potent therapeutic tools for cancer treatment.Methylation and acetylation are the most studied epigenetic modifications. DNA methylation is carried out by the DNAmethyltransferases (DNMTs) and histone methylation by the histone methyltransferases (HMTs).This PhD project was focused on the histone methyltransferase DOT1L (DOT1 like, disruptor of telomeric silencing), responsible of methylation of residue Lys79 of histone 3 (H3K79), which leads to the transcription of some oncogenes. Recent studies have shown that DOT1L is implicated in MLL-rearranged leukemia (MLL-r, Myeloid-Lymphoid Leukemia) thus it is a potent target in cancer. As DOT1L and DNMTs share the same cofactor, S-adenosyl-L-methionine (SAM), DNMT and DOT1L inhibitors can present a common inhibition mechanism by competing with SAM.We present herein the in silico – based design, and the multi-step synthesis of some series of molecules containing 3 main moieties: a) an aminopyrimidine motif and b) a benzimidazole or phenylurea motif, linked by c) a phenyl or heterocycle motif. DOT1L activity was determined for the different compounds synthesized and structure-activity relationships (SAR) were established. The activity on DNMT and other HMTs was determined as well, in other to study the DOT1L specificity of our compounds.Different scaffolds were identified to obtain DOT1L-selective or DOT1L/DNMT dual inhibitors. These molecules are interesting therapeutic tools for cancer treatment.

No description available.

A periodontite está relacionada à genética do hospedeiro, constituição do biofilme dental e fatores ambientais como o hábito de fumar. A metilação do DNA é um mecanismo de expressão genética que pode inibir ou silenciar a expressão do gene. Desta forma, vários pesquisadores têm se dedicado a estudar a influência genética sobre a suscetibilidade e/ou risco aumentado à doença periodontal. Estudos têm relatado associação entre vários biomarcadores epigenéticos com a inflamação periodontal. Considerando a hipótese de que existe associação do tabagismo com a metilação em genes relacionados à doença periodontal, o objetivo deste estudo foi verificar o padrão de metilação do DNA em células do epitélio oral de pacientes com periodontite crônica (CP) no promotor de um gene específico envolvido no controle da inflamação, como supressor da sinalização de citocinas (SOCS-1) em pacientes fumantes e não fumantes. O gene SOCS-1 é localizado no cromossomo 16p13.3, compostos por uma região amino-terminal, um domínio SH2 central e uma caixa SOCS. É um regulador negativo da via JAK / STAT. Inibe os efeitos biológicos de várias citocinas, incluindo IL-2, IL-3, IL-4, IL-6, interferão (INF) - &gamma; e INF- &alpha; / &beta;. Este foi um estudo caso-controle, comparando dois grupos, um grupo (teste) com consumo de 10 cigarros mínimos por dia, com diagnóstico de periodontite crônica e outro grupo controle que foram pacientes não fumantes com periodontite crônica. Para tal, DNA genômico foi purificado de células epiteliais bucais obtidas por meio de enxágue com sacarose 3%, por tempo único de coleta. O DNA foi modificado pelo bissulfito de Sódio e os padrões de metilação do DNA foram analisados com a técnica MS-PCR (Polymerase chain reaction). A análise estatística foi realizada pela plataforma estatística R version 3.3.2 Core Team (2016). Foi realizado Teste t de Student para amostras independentes e teste não paramétrico de Wilcoxon & Mann-Whitney para variáveis qualitativas; teste qui-quadrado e para a variável metilação, foi feito um teste exato de Fisher para testar a associação entre os grupos e a metilação. Os resultados indicaram que, para células epiteliais da mucosa bucal, a frequência de desmetilação no gene SOCS-1 é maior no grupo sem o hábito do fumo, em comparação ao grupo fumante. Foram detectadas diferenças no padrão de metilação entre os dois grupos. Ao estabelecer uma estimativa de risco relativo entre os grupos e a variável metilação, foi observado que pacientes fumantes têm 7,08 vezes (risco relativo) com um intervalo (1,95-51.46) de apresentar doença periodontal crônica, com um padrão de metilação no gene SOCS-1 / Periodontitis is related to host genetics, constitution of the dental biofilm and environmental factors such as smoking. DNA methylation is a mechanism of genetic expression that can inhibit or silence gene expression. In this way several researchers have been dedicated to study the genetic influence on the susceptibility and / or increased risk to periodontal disease. Studies have reported association between several epigenetic biomarkers with periodontal inflammation. Considering the hypothesis that there is an association between smoking and methylation in genes related to periodontal disease, the objective of this study was to verify the DNA methylation pattern in oral epithelial cells of patients with chronic periodontitis (ChP) in the promoter of a specific gene involved in the control of inflammation, as suppressor of cytokine signaling (SOCS-1) in smokers and nonsmokers patients. The SOCS-1 gene is located on chromosome 16p13.3 composed of an amino-terminal region, a central SH2 domain and a SOCS box. It is a negative regulator of the JAK / STAT path. It inhibits the biological effects of various cytokines, including IL-2, IL-3, IL-4, IL-6, interferon (INF) -&gamma; and INF-&alpha; / &beta; . This was an case-control type study, comparing two groups, a group with consumption of 10 minimum cigarettes per day, with a diagnosis of chronic periodontitis and another control group were non-smokers with chronic periodontitis. For this, genomic DNA was purified from oral epithelial cells obtained by rinsing with 3% sucrose, for a single time of collection. The DNA was modified by Sodium bisulfite and the methylation patterns of the DNA were analyzed with the MS-PCR technique (Polymerase chain reaction). Statistical analysis was performed by the statistical platform R version 3.3.2 Core Team (2016), Student\'s t-test was performed for independent samples and Wilcoxon\'s & Mann-Whitney non-parametric test for qualitative variables; chi-square test. For the methylation variable, an exact Fisher\'s test was performed to test the association between the groups and the methylation. The results indicated that, for oral mucosal epithelial cells, the frequency of demethylation in the SOCS-1 gene is higher in the non-smoking group as compared to the smoker group. statistically significant differences were detected in the methylation pattern between the two groups. When establishing an relative risk between the groups and the methylation variable, it was observed that smokers are 7.08 times (relative risk) of having chronic periodontal disease with a methylation pattern in the SOCS-1 gene

Afin de protéger durablement les écosystèmes face aux rejets planifiés ou accidentels de radionucléides dans l’environnement, il est essentiel d’évaluer l’impact de l’exposition des organismes aux radiations ionisantes sur le long terme, à l’échelle de plusieurs générations. Dans ce contexte, ce travail de doctorat vise à améliorer la caractérisation des processus moléculaires et la prédiction des effets transgénérationnels lors d’une exposition aux radiations gamma. Une approche expérimentale concerne l’étude des modifications épigénétiques radio-induites, c’est-à-dire des modifications des mécanismes régulant l’activité des gènes sans modification de la séquence d’ADN elle-même et de leur transmission au fil des générations. Cette étude a permis de mettre en évidence que certaines modifications de la méthylation de l’ADN, l’un des mécanismes épigénétiques les plus étudiés, peuvent être transmises par la lignée germinale aux les générations non-exposées (génération F3) suite à une exposition parentale (génération F0) externe aux radiations gamma (6,5 µGy.h-1 et 41,3 mGy.h-1) pendant 25 jours. Dans une seconde approche, un modèle mécaniste DEBtox (Budget Energétique Dynamique appliqué à la toxicologie) est modifié pour permettre l’analyse des effets des radiations gamma sur la croissance et la reproduction de D. magna à l’échelle de plusieurs générations. Pour ce faire, on utilise des compartiments de dommage, dont le niveau peut être hérité d’une génération à la suivante. Le modèle est ajusté aux données avec des méthodes d’inférence bayésienne afin d’estimer les paramètres tout en tenant compte des incertitudes qui leur sont associées. / In order to durably protect ecosystems facing planned or accidental releases of radionuclides, the long-term impact of organism exposure to ionizing radiation must be studied on a multigenerational scale. The aim of this PhD is to improve the characterization of molecular processes and the prediction of transgenerational effects during a gamma irradiation. First, an experimental approach investigated on radio-induced modifications of epigenetic processes, i.e. changes in mechanisms that regulate gene expression without changing DNA sequence itself and on the transmission of these modifications to subsequent generations. Significant changes in DNA methylation, a well-studied epigenetic mechanism, detected in generation F3 clearly showed that epigenetic modifications could be transmitted to unexposed generations, in response to the exposure of a parental generation (F0) to external gamma radiation (6.5 µGy.h-1 et 41.3 mGy.h-1) for 25 days. Second, a mechanistic modelling approach used a modified version of the DEBtox model (Dynamic Energy Budget model applied to toxicology) in order to analyze effects of gamma radiation on D. magna growth and reproduction over several generations. To that end, damage compartments, with damage levels that were transmitted from one generation to the next, were included. The model was fitted to data using Bayesian inference methods, in order to estimate the parameters while considering their associated uncertainty.

A metilação do DNA é uma alteração epigenética que atua na regulação da expressão gênica. A deficiência de vitaminas (cobalamina, B6 e folato) pode interferir na taxa de metilação. O efeito da deficiência destas vitaminas foi determinado em estudos com cultura de células e em animais. No entanto, são raros os estudos realizados com seres humanos. Os objetivos deste trabalho foram: avaliar a taxa de metilação do DNA de leucócitos na região promotora dos genes Interferon gama (IFN&#947;), Serpin B5 e Stratifin; verificar se existe associação entre as concentrações das vitaminas e dos metabólitos com a taxa de metilação do DNA dos 3 genes; e analisar quais são os fatores de predição para a taxa de metilação do DNA (variável dependente) considerando como variáveis independentes os valores séricos das vitaminas e metabólitos, em mulheres com idades gestacionais de 16, 28 e 36 semanas. Cento e oitenta e três mulheres foram convidadas a participar desse estudo, porém apenas 96 completaram o estudo prospectivo. Foram determinadas as concentrações séricas da cobalamina (Cbl), folato, vitamina B6, S-adenosilmetionina (SAM), S-adenosilhomocisteína (SAH), ácido metilmalônico (MMA), homocisteína total (tHcy) e folato eritrocitário. A taxa de metilação nos 3 genes foi determinada por qMSP (Quantitative Methylation Specific - Polimerase Chain Reaction). Várias mulheres estavam fazendo uso de suplementação com ácido fólico e/ou polivitamínicos. Diante deste fato foram formados 4 subgrupos: Grupo 1 (constituído por mulheres que não usaram suplementação), Grupo 2 (mulheres que usaram suplementação em todas as idades gestacionais estudadas - 16, 28 e 36 semanas), Grupo 3 (mulheres que usaram suplementação no início da gestação até a 16ª semana) e Grupo 4 (mulheres que usaram suplementação na 16ª e 28ª semana gestacional). Não houve diferença entre as taxas de metilação do DNA dos genes IFN&#947;, Serpin B5 e Stratifin durante o período gestacional estudado. As taxas de metilação no gene IFN&#947; do grupo 1 foram maiores, quando comparadas as taxas dos demais grupos. Em modelos de regressão linear múltipla, considerando o período gestacional como um todo, apenas a vitamina B6 e a tHcy foram diretamente associadas aos valores da taxa de metilação do gene IFN&#947;. No entanto, a vitamina B6 foi inversamente associada, enquanto que tHcy esteve diretamente associada aos valores da taxa de metilação do gene Stratifin. A taxa de metilação não sofre alterações durante a gestação; a vitamina B6 e a tHcy foram os fatores de predição para as taxas de metilação do DNA na região promotora dos genes IFN&#947; e Stratifin. / DNA methylation is an epigenetic modification that regulates gene expression. Cobalamin, vitamin B6 and folate deficiencies can impair the DNA methylation index. Studies involving cultured cells and animals have evaluated the effect of vitamin deficiencies in DNA methylation. However, few studies were conducted in humans. The goals of this study were: to evaluate the leukocyte DNA methylation index in the promoter region of interferon gamma (IFN&#947;), Serpin B5 and Stratifin genes; to assess the association between vitamins and metabolites concentrations and DNA methylation index in three genes; and to examine the predictive factors for DNA methylation index using as independent variables: serum folate, serum cobalamin, serum vitamin B6, erythrocyte folate, methylmalonic acid (MMA), total homocysteine (tHcy) in three gestational ages (16th, 28th and 36,th weeks). A hundred eighty three women were included, but only 96 completed the prospective study. The serum concentrations of cobalamin, folate, vitamin B6, S-adenosylmethionine (SAM), Sadenosylhomocysteine (SAH), MMA, tHcy were determined. The erythrocyte folate concentration was also evaluated. The DNA methylation index was determined in three genes by qMSP (Quantitative Methylation Specific - Polymerase Chain Reaction). Several women were taking folic acid supplementation and/or multivitamins. Four groups were formed according to supplementation use: Group 1 (women who take no supplementation), Group 2 (women who took supplements at 16th, 28th and 36th weeks of pregnancy), Group 3 (women who took supplements in early pregnancy and up to 16 weeks) and Group 4 (women who took supplements in the 16th and 28th week of pregnancy). There was no difference between the DNA methylation index in the IFN&#947;, Serpin B5 and Stratifin genes during the gestational periods studied. The DNA methylation index in the IFN&#947; gene in group 1 was higher than those indexes from other groups. In multiple linear regression models considering the gestational periods as a whole, only vitamin B6 and tHcy were directly associated to DNA methylation index in IFN&#947; gene. However, vitamin B6 was inversely associated, whereas tHcy was directly associated with values of DNA methylation in Stratifin. The DNA methylation index does not change during pregnancy, vitamin B6 and tHcy were the predictors of DNA methylation in the promoter region of IFN&#947; and Stratifin genes.