Embora a transferência nuclear de células somáticas (TNCS) seja uma ferramenta promissora, seu amplo uso é impedido devido às altas taxas de mortalidade durante o desenvolvimento dos animais clonados. Acredita-se que a reprogramação epigenética anormal seja a principal causa desta baixa eficiência. Nós hipotetizamos que agentes modificadores de cromatina (AMCs) atingindo a acetilação das histonas e a metilação do DNA poderiam alterar a configuração da cromatina e torná-la mais facilmente reprogramável. Deste modo, fibroblastos bovinos foram tratados com 5-aza-2\'-deoxicitidina (AZA) mais tricostatina A (TSA) ou hidralazina (HH) mais ácido valpróico (VPA) enquanto, em outro experimento, zigotos bovinos clonados foram tratados com TSA. O tratamento dos fibroblastos com AZA+TSA ou HH+VPA aumentou a acetilação das histonas, mas não afetou o nível de metilação do DNA. Entretanto, o tratamento com HH+VPA diminuiu a viabilidade/proliferação celular. O uso destas células como doadoras de núcleo não mostrou efeitos positivos sobre o desenvolvimento pré- e pós-implantação. Em relação ao tratamento dos zigotos clonados com TSA, o tratamento destes mostrou um aumento nos padrões de acetilação das histonas, mas não reduziu o nível de metilação do DNA. Além disso, este tratamento não resultou em efeito positivo sobre o desenvolvimento pré- e pósimplantação. Este trabalho fornece evidências de que o tratamento de células doadoras de núcleo ou zigotos clonados com AMCs não tem efeito positive sobre o desenvolvimento pré- e pós-implantação de bovinos clonados. / Although somatic cell nuclear transfer (SCNT) is a promising tool, its potential use is hampered by the high mortality rates during the development to term of cloned offspring. Abnormal epigenetic reprogramming of donor nuclei after SCNT is thought to be the main cause of this low efficiency. We hypothesized that chromatinmodifying agents (CMAs) targeting chromatin acetylation and DNA methylation could alter the chromatin configuration and turn them more amenable to reprogramming. Thus, bovine fibroblasts were treated with 5-aza-2\'-deoxycytidine (AZA) plus trichostatin (TSA) or hydralazine (HH) plus valproic acid (VPA) whereas, in another trial, cloned bovine zygotes were treated with TSA. The treatment of fibroblasts with either AZA+TSA or HH+VPA increased histone acetylation, but did not affect the level of DNA methylation. However, treatment with HH+VPA decreased cellular viability and proliferation. The use of these cells as nuclear donors showed no positive effect on pre- and post-implantation development. Regarding the treatment of cloned zygotes with TSA, treated one-cell embryos showed an increase in the acetylation patterns, but not in the level of DNA methylation. Moreover, this treatment revealed no positive effect on pre- and post-implantation development. This work provides evidence the treatment of either nuclear donor cells or cloned zygotes with CMAs has no positive effect on pre- and post-implantation development of cloned cattle.

Recentes estudos genômicos têm mostrado que vários fungos e bactérias possuem um potencial biossintético superior à quantidade de me tabólitos secundários já isolados desses micro-organismos. A descoberta de produtos naturais inéditos e bioativos é limitada pela impossibilidade dos micro-organismos expressarem to das as suas rotas biossintéticas em laboratório. Assim, estratégias alternativas para i nduzir a produção de produtos naturais microbianos são necessárias. A utilização de cultur as mistas de micro-organismos é uma estratégia que vem sendo recentemente utilizada, na tentativa de mimetizar condições mais naturais de crescimento. Além disso, a adição de mo duladores químicos e epigenéticos às culturas microbianas também pode potencialmente est imular a produção de compostos de interesse, seja por ativar mecanismos celulares em resposta à condição de estresse, ou por alterar a taxa de transcrição de certos genes, em f unção de mudanças no grau de enovelamento da cromatina. Alternativamente, a indu ção de certos genes, e até mesmo a diversificação do metabolismo secundário, podem ser conseguidos através de engenharia genética, pela manipulação direta de genes de inter esse. A linhagem endofítica Alternaria tenuissima SS77, selecionada para os experimentos de modulaçã o química e epigenética, teve seu metabolismo secundário alterado após o tra tamento com diferentes moduladores. Provavelmente, o efeito observado ocorreu em função de uma eliciação inespecífica dos diferentes moduladores. Além disso, o cultivo misto desse fungo com o fungo endofítico Nigrospora sphaerica SS67 , isolada da mesma planta hospedeira ( Smallanthus sonchifolius ), levou ao isolamento de dois novos policetídeos, da classe das perilenequinonas, juntamente com um já relatado na literatura científica. Para r ealizar os cultivos microbianos mistos, envolvendo uma linhagem bacteriana e uma fúngica, t rês linhagens de actinobactérias e cinco de fungos, todos endofíticos da planta Lychnophora ericoides , foram selecionadas. Alterações no perfil metabólico da cultura mista de Phomopsis sp FLe6 com Streptomyces albospinus RLe7 foram as mais evidentes e por isso a maioria das investigações foram focadas nessa cultura mista. Várias condições de cu ltivo foram testadas e diferentes resultados foram obtidos. Em alguns casos, o desenv olvimento da linhagem fúngica foi inibido pela bacteriana, e em outros, foi observado o inverso. Da mesma forma, houve acentuada inibição da produção de alguns metabólito s secundários na presença da linhagem desafiadora, mas também foi verificada a eliciação de outros. Os extratos das culturas simples desses micro-organismos também apresentaram relativas alterações nos perfis metabólicos em função das condições de cultivo. Os metabólitos produzidos pelo fungo Phomopsis sp FLe6 e pela actinobactéria Streptomyces albospinus RLe7 foram isolados e caracterizados. Os resultados mostram que as intera ções entre os micro-organismos endofíticos são bastante complexas, estando sujeita s a ação de diversos fatores externos que muitas vezes não podem ser pré-determinados. Po r isso, estabelecer um cultivo misto adequado, do ponto de vista da eliciação da produçã o de metabólitos secundários, pode requerer uma série de tentativas. Ainda assim, os r esultados almejados podem ser conseguidos utilizando essa estratégia. Diferenteme nte das linhagens endofíticas, manipuladas quimicamente através de diferentes estr atégias, a linhagem sequenciada de Fusarium heterosporum ATCC 74349, foi manipulada geneticamente para a co nstrução de um gene biossintético híbrido pks-nrps , contendo a porção nrps do gene híbrido da equisetina e um pks críptico de Aspergillus fumigatus . Era esperado que a linhagem hibridizada fosse capaz de produzir o metabólito se cundário geneticamente planejado, entretanto, após seu cultivo, esse produto não foi detectado nos extratos, e as possíveis razões são discutidas. Ainda que os resultados espe rados não tenham sido obtidos, estudos que contribuam para a ampliação do entendimento das megassintases fúngicas são de extrema valia. / Recently, genetic studies have shown that several b acteria and fungi hold a greater biosynthetic potential than the amount of secondary metabolites isolated from these microorganisms. The discovery of novel bioactive na tural products is limited by the inability of microorganisms to express all their biosynthetic pa thways in laboratory conditions. Therefore, alternative strategies to induce the production of microbial natural products are required. Mixed cultures of microorganisms are a strategy tha t has been used to mimic more natural conditions of growth. Furthermore, the addition of chemical and epigenetic modulators to the microbial cultures can also stimulate the productio n of compounds by activating cellular mechanisms in response to stress conditions or by c hanging the transcription rate of certain genes, due to changes in the chromatin folding. Alt ernatively, the induction of some genes, and even the diversification of secondary metabolis m, can be achieved by genetic engineering, by manipulating genes of interest. The endophytic strain Alternaria tenuissima SS77, which was selected for the experiments of che mical and epigenetic modulation, had changed its secondary metabolism after treatment wi th different modulators. Probably, the observed effect was due to a nonspecific elicitatio n of those modulators. Moreover, the mixed cultures of this fungus with the endophytic fungus Nigrospora sphaerica SS67, isolated from the same host plant ( Smallanthus sonchifolius ), led to the isolation of two new polyketides, belonging to perylene quinone class, along with ano ther one already reported in the scientific literature. Three strains of actinobacteria and fiv e fungi, all endophytes of Lychnophora ericoides , were selected to grow in microbial mixed cultures comprising one bacteria and one fungus. Changes in the metabolic profile of the mix ed culture of Phomopsis sp. FLe6 with Streptomyces albospinus RLe7 were the most obvious, and then further studi es were focused on this mixed culture. Many culture conditions were analyzed and different results were obtained. In some cases, the development of the fun gal strain was inhibited by bacteria, and in other cases was observed the opposite. Similarly , there was a remarkable inhibition of the production of certain secondary metabolites in the presence of the challenging strain, but the eliciting of others was also observed. The extracts of the single cultures of these microorganisms also showed changes in metabolic pro files due to culture conditions. The metabolites produced by the fungus Phomopsis sp. FLe6 and the actinobacteria S. albospinus RLe7 were isolated and characterized. The results show that interactions between endophytic microorganisms are quite complex and are influenced by various external factors that often can not be previously determined. Theref ore, establishing a suitable mixed culture to elicit the production of secondary metabolites m ay require some attempts. Still, the expected results can be achieved using this strateg y. Unlike the endophytic strains, that was chemically manipulated by different strategies, the sequenced strain Fusarium heterosporum ATCC 74349 was genetically manipulated to construct a hybrid PKS-NRPS biosynthetic gene containing the NRPS portion of the hybrid gene of e quisetin and a cryptic PKS gene of Aspergillus fumigatus . It was expected that hybridized strain could be a ble to produce the secondary metabolite genetically planned, however, after its cultivation, this product was not detected in any extracts, and some possible reasons are discussed. Although the expected results have not been obtained, studies that contri bute to increasing the understanding of fungal megasynthases are extremely valuable

Embora a transferência nuclear de células somáticas (TNCS) seja uma ferramenta promissora, seu amplo uso é impedido devido às altas taxas de mortalidade durante o desenvolvimento dos animais clonados. Acredita-se que a reprogramação epigenética anormal seja a principal causa desta baixa eficiência. Nós hipotetizamos que agentes modificadores de cromatina (AMCs) atingindo a acetilação das histonas e a metilação do DNA poderiam alterar a configuração da cromatina e torná-la mais facilmente reprogramável. Deste modo, fibroblastos bovinos foram tratados com 5-aza-2\'-deoxicitidina (AZA) mais tricostatina A (TSA) ou hidralazina (HH) mais ácido valpróico (VPA) enquanto, em outro experimento, zigotos bovinos clonados foram tratados com TSA. O tratamento dos fibroblastos com AZA+TSA ou HH+VPA aumentou a acetilação das histonas, mas não afetou o nível de metilação do DNA. Entretanto, o tratamento com HH+VPA diminuiu a viabilidade/proliferação celular. O uso destas células como doadoras de núcleo não mostrou efeitos positivos sobre o desenvolvimento pré- e pós-implantação. Em relação ao tratamento dos zigotos clonados com TSA, o tratamento destes mostrou um aumento nos padrões de acetilação das histonas, mas não reduziu o nível de metilação do DNA. Além disso, este tratamento não resultou em efeito positivo sobre o desenvolvimento pré- e pósimplantação. Este trabalho fornece evidências de que o tratamento de células doadoras de núcleo ou zigotos clonados com AMCs não tem efeito positive sobre o desenvolvimento pré- e pós-implantação de bovinos clonados. / Although somatic cell nuclear transfer (SCNT) is a promising tool, its potential use is hampered by the high mortality rates during the development to term of cloned offspring. Abnormal epigenetic reprogramming of donor nuclei after SCNT is thought to be the main cause of this low efficiency. We hypothesized that chromatinmodifying agents (CMAs) targeting chromatin acetylation and DNA methylation could alter the chromatin configuration and turn them more amenable to reprogramming. Thus, bovine fibroblasts were treated with 5-aza-2\'-deoxycytidine (AZA) plus trichostatin (TSA) or hydralazine (HH) plus valproic acid (VPA) whereas, in another trial, cloned bovine zygotes were treated with TSA. The treatment of fibroblasts with either AZA+TSA or HH+VPA increased histone acetylation, but did not affect the level of DNA methylation. However, treatment with HH+VPA decreased cellular viability and proliferation. The use of these cells as nuclear donors showed no positive effect on pre- and post-implantation development. Regarding the treatment of cloned zygotes with TSA, treated one-cell embryos showed an increase in the acetylation patterns, but not in the level of DNA methylation. Moreover, this treatment revealed no positive effect on pre- and post-implantation development. This work provides evidence the treatment of either nuclear donor cells or cloned zygotes with CMAs has no positive effect on pre- and post-implantation development of cloned cattle.

Understanding the ability of species to respond to climate change is essential for prediction of their future distribution. When migration is not adequate, reaction via phenotypic plasticity and/or genetic/epigenetic adaptation is necessary. The main aim of this study is to determine mechanisms of response to climate change in dominant grass species Festuca rubra. The study used reciprocal transplant experiment with growth chambers simulating different climatic conditions. Original localities in western Norway represent factorially crossed gradients of temperatures and precipitations, thus it was possible to study the effect of temperature and moisture separately as well as combined. In first part of the experiment, plastic responses were separated from genetic differentiation. To do this, plants with different genotypes from original localities were transplanted to growth chambers set to simulating temperature and moisture course in the four extreme localities (wettest and driest combined with warmest and coldest). After five months, ecophysiological photosynthetic-related traits were measured. These traits are important for species ability to adapt and maintain high fitness and thus they are essential for plants function. Specifically, it was net photosynthetic rate (PN), fluorescence of...

Pesquisas recentes em medicina genômica sugerem que fatores de risco que incidem desde a concepção de uma criança até o final de sua adolescência podem influenciar no desenvolvimento de doenças crônicas da idade adulta. Artigos científicos com descobertas e estudos inovadores sobre o tema indicam que a epigenética deve ser explorada para prevenir doenças de alta prevalência como doenças cardiovasculares, diabetes e obesidade. A grande quantidade de artigos disponibilizados diariamente dificulta a atualização de profissionais, uma vez que buscas por informação exata se tornam complexas e dispendiosas em relação ao tempo gasto na procura e análise dos resultados. Algumas tecnologias e técnicas computacionais podem apoiar a manipulação dos grandes repositórios de informações biomédicas, assim como a geração de conhecimento. O presente trabalho pesquisa a descoberta automática de artigos científicos que relacionem doenças crônicas e fatores de risco para as mesmas em registros clínicos de pacientes. Este trabalho também apresenta o desenvolvimento de um arcabouço de software para sistemas de vigilância que alertem profissionais de saúde sobre problemas no desenvolvimento humano. A efetiva transformação dos resultados de pesquisas biomédicas em conhecimento possível de ser utilizado para beneficiar a saúde pública tem sido considerada um domínio importante da informática. Este domínio é denominado Bioinformática Translacional (BUTTE,2008). Considerando-se que doenças crônicas são, mundialmente, um problema sério de saúde e lideram as causas de mortalidade com 60% de todas as mortes, o presente trabalho poderá possibilitar o uso direto dos resultados dessas pesquisas na saúde pública e pode ser considerado um trabalho de Bioinformática Translacional. / Genomic medicine has suggested that the exposure to risk factors since conception may influence gene expression and consequently induce the development of chronic diseases in adulthood. Scientific papers bringing up these discoveries indicate that epigenetics must be exploited to prevent diseases of high prevalence, such as cardiovascular diseases, diabetes and obesity. A large amount of scientific information burdens health care professionals interested in being updated, once searches for accurate information become complex and expensive. Some computational techniques might support management of large biomedical information repositories and discovery of knowledge. This study presents a framework to support surveillance systems to alert health professionals about human development problems, retrieving scientific papers that relate chronic diseases to risk factors detected on a patient\'s clinical record. As a contribution, healthcare professionals will be able to create a routine with the family, setting up the best growing conditions. According to Butte, the effective transformation of results from biomedical research into knowledge that actually improves public health has been considered an important domain of informatics and has been called Translational Bioinformatics. Since chronic diseases are a serious health problem worldwide and leads the causes of mortality with 60% of all deaths, this scientific investigation will probably enable results from bioinformatics researches to directly benefit public health.

Un certain nombre d’études épidémiologiques ont montré au cours des cinquante dernières années une augmentation des infertilités, des malformations de l’appareil reproducteur masculin et des cancers testiculaires. Une des hypothèses est que, l’exposition durant la vie foetale ou néonatale à des composés présents dans l’environnement capables d’interférer avec le système hormonal (perturbateurs endocriniens), serait responsable de l’augmentation de l’incidence de ces pathologies. Les molécules qui sont suspectées d’avoir des effets néfastes à long terme sur le tractus génital mâle possèdent des activités de type estrogénique ou antiandrogénique. Parmi les mécanismes impliqués dans l’effet à long terme, un certain nombre d’auteurs mettent en avant l’intervention de mécanismes de type épigénétiques. Dans ce contexte, nous avons utilisé deux types de modèles expérimentaux reposant sur l’exposition développementale de rats à ces composés : un modèle d’exposition néonatale à un estrogène (estradiol benzoate) et un modèle d’exposition foetale à un antiandrogène (flutamide). Les deux modèles expérimentaux induisent chez le rongeur un phénotype d’hypospermatogenèse. Dans le cas de l’exposition néonatale à l’estradiol benzoate nous montrons que l’hypospermatogenèse observée chez les animaux à l’âge adulte est due à l’activation chronique de l’apoptose des cellules germinales testiculaires. Cette apoptose mettrait en jeu, par un mécanisme post-transcriptionnel, la diminution à long terme de l’expression de protéines clés de la machinerie épigénétique de méthylation de l’ADN, les ADN méthyltransférases (DNMT 3A, 3B et 1), et du facteur antiapoptotique, MCL-1. D’un point de vue fonctionnel, la chute d’expression des DNMTs se traduit notamment par l’augmentation d’expression des éléments transposables LINE-1 et du gène Ibtk normalement contrôlés par méthylation de l’ADN. En amont, la chute d’expression des DNMTs et de MCL-1 serait dépendante de l’augmentation d’expression d’autres effecteurs épigénétiques, les microRNAs de la famille miR-29. Dans le cas de l’exposition in utero au flutamide, notre travail indique que l’apoptose chronique des cellules germinales serait liée à la diminution à long terme de l’expression des inhibiteurs d’apoptose cIAP1 et cIAP2, et une augmentation d’expression de leur inhibiteur SMAC/DIABLO. En revanche, l’absence de mort des cellules somatiques testiculaires (Sertoli et Leydig) dans ce modèle serait due à l’augmentation d’expression spécifiquement dans ces cellules des inhibiteurs d’apoptose XIAP et SURVIVIN. Par ailleurs, le phénotype d’apoptose observé à l’âge adulte impliquerait également une altération précoce de l’expression des DNMTs. En conclusion, nous apportons une réponse mécanistique au phénotype de programmation foetale/néonatale d’apoptose des cellules germinales testiculaires adultes. En effet, l’augmentation des miR-29s provoquerait : (1) une chute d’expression des DNMTs altérant ainsi le profil de méthylation des gènes, et (2) une chute d’expression de facteurs protégeant les cellules germinales contre l’apoptose comme le facteur MCL-1. / During the five past decades, a number of epidemiological studies have indicated a higher incidence of infertility problems, male reproductive tract abnormalities and testicular cancers. Among the different hypotheses proposed, fetal or neonatal exposure to environmental compounds that can interfere with endocrine system, termed endocrine disruptors, may be at the origin of the rising incidence of these diseases. The molecules that are supposed to have long term adverse effects on the male genital tract have estrogenic or antiandrogenic properties. Some authors have suggested that the long term effects of endocrine disruptors could be mediated through epigenetic mechanisms. In this context, we have used two types of experimental models based on developmental exposure to these compounds: one model of neonatal exposure to an estrogen (estradiol benzoate) and another model of fetal exposure to an antiandrogen (flutamide). The two experimental models induce a phenotype of hypospermatogenesis in rodents. In the context of neonatal exposure to estradiol benzoate we show that the hypospermatogenesis observed in adult animals is the consequence of a chronic activation of the apoptotic process of testicular germ cells. This cell death process seem to involve the decrease of the key epigenetic effectors of DNA methylation machinery DNA methyltransferases (DNMT3A, 3B & 1), and of the antiapoptotic protein MCL-1 through post transcriptional mechanisms. In term of functional consequences the decrease of DNMTs proteins leads to increased expression of transposable element LINE-1 and Ibtk gene that are normally controlled by DNA methylation. Upstream DNMTs and MCL-1 decrease may be triggered by the increase of other epigenetic factors, the microRNAs belonging to miR-29 family. Concerning in utero exposure to flutamide, our work indicate that the chronic apoptotic process of germ cells may be linked to long term decrease of the inhibitors of apoptosis cIAP1 & 2, and an increase of proapoptotic factors SMAC/DIABLO. On the other hand, the lack of testicular somatic cell death in this model may be the result of higher expression of inhibitors of apoptosis XIAP and SURVIVIN in these cells. Besides, the apoptotic process observed at the adult age may also involve a precocious alteration of DNMTs expression. In summary, our work provides a mechanistic view to the fetal/neonatal programming of adult germ cell death. Indeed, the increased levels of miR-29s may induce: (1) a decrease in DNMTs expression levels that consequently could alter the methylation pattern of some genes, and (2) a decrease in factors that normally prevent germ cells death such as MCL-1.

Un certain nombre d’études épidémiologiques font état d’une augmentation de l’infertilité masculine durant ces cinquante dernières années, en particulier dans les pays industrialisés, mais aussi d’une augmentation des malformations de l’appareil reproducteur masculin telles que la cryptorchidie (absence de migration des testicules dans les bourses) ou l’hypospadias (malformation du pénis), et des cancers testiculaires. Des données expérimentales suggèrent que ces anomalies du tractus génital mâle sont liées. Ces symptômes forment le syndrome de dysgénésie testiculaire. Les causes d’apparition ce syndrome semblent être d’origine environnementale. En effet, les évolutions relativement rapides de ce syndrome suggèrent des facteurs dynamiques, en lien avec le mode de vie ou l’environnement. Une des hypothèses est que, l’exposition durant la vie fœtale ou néonatale à des composés présents dans l’environnement capables d’interférer avec le système hormonal (perturbateurs endocriniens environnementaux, PEEs), serait responsable de l’augmentation de l’incidence de ces pathologies. Au banc des principaux accusés, les molécules qui possèdent des activités de type estrogénique ou antiandrogénique. A ce jour, les mécanismes d’action à l’origine du syndrome de dysgénésie testiculaire sont encore mal connus. Certaines études suggèrent des mécanismes de type épigenétique dans les effets à long terme des PEEs. L’objectif de notre travail était d’identifier et caractériser les mécanismes d’action de type épigenétique impliqué dans l’infertilité mâle. Pour cela, nous avons utilisé un modèle expérimental (rats nouveau-nés) reposant sur une exposition développementale à un estrogène (estradiol benzoate). Ce modèle induit chez le rat adulte un phénotype d’hypospermatogenèse liée à une à apoptose chronique des cellules germinales testiculaires. Nous montrons que ce phénotype est lié à l’altération de deux voies, impliquant en amont des effecteurs épigénétiques. La première voie implique la famille des miR-29s. Ainsi, nous observons une augmentation de l’expression des miR-29a, b, c qui provoque une diminution de deux de ses cibles: la protéine antiapoptotique MCL-1 et les enzymes de méthylation de l’ADN DNMTs. La chute des DNMTs entraine une hypométhylation globale (estimée à travers le gène Line-1) et spécifique du facteur de choc thermique HSF1. Ceci provoque une réexpression de ces facteurs entrainant l’apoptose des cellules germinales adultes. La deuxième voie implique le miR-18a. L’augmentation de son expression provoque une chute de l’expression de sa cible HSF2 qui régule la protéine de choc thermique HSP70/HSPA2. Le faible taux d’HSPA2 est une autre explication de l’apoptose des cellules germinales dans notre modèle. Nous montrons aussi que ce phénotype est irréversible lorsque l’exposition à lieu chez le nouveau-né alors qu’il est réversible quand l’exposition à lieu à l’âge adulte. Ces données suggèrent que l’exposition néonatale à l’estradiol benzoate induit une programmation développementale de l’hypospermatogenèse.Enfin, les anomalies tissulaires d’expression des miRNAs se retrouvent au niveau sanguin, suggérant leur utilisation potentielle comme biomarqueurs. Nous avons validé cet aspect chez l’homme en montrant que l’expression des miR29s et du miR-18a était plus élevée chez les patients oligo- ou azoospermiques que les chez patients normospermiques.En conclusion, nos résultats indiquent que l’hypospermatogenèse due à une apoptose chronique des cellules germinales observée chez l’animal adulte après exposition néonatale à l’EB met en jeu une modification d’expression de plusieurs effecteurs épigénétiques clés: miR-29s, miR-18a et DNMTs. De plus, les miR-29s et miR-18a pourraient être de nouveaux biomarqueurs circulants non invasifs de la stérilité masculine dans le contexte d’une oligo ou azoospermie chez l'homme. / Epidemiological studies have reported an increase in male infertility over the past fifty years, especially in industrialized countries, but also an increase in malformations of the male reproductive tract such as cryptorchidism (no migration of the testes into the scrotum) and hypospadias (malformation of the penis), and testicular cancers. Experimental data suggest that these abnormalities of the male genital tract are related. These symptoms form the testicular dysgenesis syndrome. The causes of the occurrence of this syndrome appear to be environmental in origin. Indeed, the relatively rapid evolution of this syndrome suggests dynamic factors related to lifestyle or environment. One hypothesis is that exposure during fetal or neonatal life to compounds present in the environment can interfere with the hormonal system (environmental endocrine disruptors), would be responsible for the increased incidence of these pathologies. Bench of the main accused, molecules that have estrogenic or anti-androgenic activity types. To date, the mechanisms of action behind the testicular dysgenesis syndrome are poorly understood. Some studies suggest that epigenetic mechanisms are at playThe objective of our work was to identify and characterize the epigenetic mechanisms of action involved in male infertility induced by neonatal exposure to xenoestrogen. For this, we used an experimental model based on a developmental exposure to estrogen (estradiol benzoate). This model induced in adult rats a hypospermatogenesis phenotype due to chronic apoptosis of germ cells.We show that this phenotype is related to an alteration of two pathways, involving upstream effectors epigenetic. The first pathway involves the family of miR- 29s. Thus, we observe an up-regulation of miR -29a, b, c, which causes a decrease in two of his targets: the anti-apoptotic protein MCL- 1 and the enzymes of DNA methylation DNMTs. Falling DNMTs leads to a global hypomethylation (estimated through the Line -1 gene) and to specific hypomethylation of the heat shock factor, HSF1. This causes a re-expression of factors that induce apoptosis in adult germ cells. The second pathway involves up-regulation of miR -18a that causes a down-regulation of its target HSF2 which regulates the heat shock protein HSP70/HSPA2. The down-regulation of HSPA2 is another explanation of germ cell apoptosis in our model. We also show that this phenotype is irreversible when the estrogen exposure takes place in the newborn whereas it is reversible when exposure takes place in adulthood, suggesting that neonatal exposure to estradiol benzoate induced a developmental programming of hypospermatogenesis.Finally, abnormal tissue expressions of miRNAs are found in the blood, suggesting their potential use as biomarkers. We validated this aspect in humans showing that the expression of miR29s and miR-18a was higher in patients with decrease or no sperm counts compared to normal sperm count. In conclusion, our results indicate that hypospermatogenesis due to chronic germ cell apoptosis observed in adult animals after neonatal exposure to EB involves a change in expression of several key epigenetic effectors: miR-29, miR-18a and DNMTs. In addition, miR-29 and miR-18a could be new non invasive circulating biomarkers of men infertility.

Mammals have two orthologs, Mll and Trx2, for the Drososphila protein Trithorax (TRX), which is the founding member of the trithorax group (TrxG) of epigenetic regulators. TrxG proteins are characterized by an evolutionary conserved SET domain. A major function of all SET domain- containing proteins is to modulate gene activity, but the underlying mechanisms are poorly understood. Apparently TRX, Mll and Trx2 are histone H3 lysine 4 specific methyltransferases. So far all evidence points to roles in expression of specific target genes. However, target genes and function of the epigenetic regulator Trx2 were still unknown. Homozygous trx2 mutant embryos arrest in development because of severe and widespread defects {Glaser, 2005 #296}. Thus mouse embryonic stem (ES) cells carrying a null mutation of trx2 were used as an alternative model system to address the implication of Trx2 in differentiation. This study showed that Trx2 is redundant for ES cell self-renewal. Homozygous trx2 knockout ES cells did not exhibit cell cycle defects. However, loss of Trx2 resulted in reduced proliferation and increased apoptosis rates in trx2-/- ES cells. Due to the fact that differentiation requires an appropriate rate of population growth, trx2-/- cells were affected adversely upon in vitro differentiation. Neurogeneic differentiation of trx2 mutant cells generated fewer mature neurons than wild type cells. Moreover a temporal delay in the developmental progression to differentiation became apparent. Cardiac differentiation of trx2-/- cells confirmed the developmental defect and temporal delay. Notably differentiation of trx2-/- cells was merely delayed or impaired but it was not absent, implying that Trx2 is not required for gene expression programs specific for neurons or cardiac myocytes. We propose that differentiation of trx2-/- ES cells is impaired because apoptosis is disturbing differentiation. Apart from analyzing the phenotype of trx2 mutant cells, this work was focused on the identification of Trx2 target genes. Oligonucleotide expression arrays were used to identify genes whose expression levels were affected by the absence of Trx2. In general, loss of Trx2 function resulted in more genes with decreased than increased expression levels. This is consistent with the hypothesis that Trx2 functions as a transcriptional activator. Comparison of gene expression profiles for constitutive and conditional trx2 mutant cells enabled a distinction between direct and indirect target genes for Trx2. As a result Magoh2 was identified as the key candidate target gene for Trx2. Interaction between Trx2 and Magoh2 suggested a potential regulatory role for Trx2 in alternative splicing. Furthermore this work provided evidence that Trx2 could be involved in the maintenance of CpG island promoter gene expression, thus providing a potent regulatory mechanism for ubiquitously expressed genes.

<p>Environmental stress has shown to affect both the exposed individuals and the development of their offspring. Generally, it is thought that the stressed organism responds to stress by trying to adapt to it. This thesis investigates possible evolutionary consequences of cross-generational transmissions of stress, where the parent has been stressed but the offspring has not. In two studies we have exposed chicken parents of different breeds to an unpredictable circadian light rhythm, to investigate the influence of genetic background on the transmission of behaviour and patterns of genome-wide gene expression across generations. In Paper I, we can show that the domesticated chicken, by means of epigenetic factors, transmit their behaviours as well as their gene expression profiles to their offspring to a higher extent than their wild ancestor, the red junglefowl. Furthermore, in Paper II, even though the offspring never experienced the stress or had any contact with their stressed parents, they seemed to have adapted to it, which suggests that the parents might have prepared (or pre-adapted) them for living in the unpredictable environment. Additionally, eggs of stressed hens showed increased levels of estradiol that might have affected gene expression of specific immune genes, which were up-regulated in the offspring of stressed parents. It is possible that the traditional distinction between stress responses and evolutionary adaptation may be reevaluated, since our results indicate that they could be parts of the same evolutionary event.</p>

Malgré les vastes études démontrant le rôle de la conformation génomique dans le contrôle transcriptionnel, de nombreuses questions restent en suspens, et en particulier, comment ces structures chromatiniennes sont formées et maintenues. Pour mieux comprendre les liens entre l’état de la chromatine au niveau des éléments régulateurs, la topologie de la chromatine et la régulation de la transcription, nous utilisons la technique CHi-C basée sur la technologie de capture de la conformation chromosomique (3C). En utilisant deux stratégies de capture ciblant deux différentes structure chromatiniennes (les boucles chromatiniennes et les domaines topologiques), nous avons pu décrypter la structure chromatinienne associée à la différenciation des thymocytes et mettre en évidence des mécanismes de contrôle transcriptionnel de certains gènes. Les expériences futures de l’équipe vont consister à examiner les facteurs (hors transcription) qui peuvent influencer l'architecture de la chromatine, comme la liaison différentielle des CTCF, et comment ces facteurs peuvent être coordonnés par le contrôle de transcription. / Chromosome folding takes place at different hierarchical levels, with various topologies correlated with control of gene expression. Despite the large number of recent studies describing chromatin topologies and their correlations with gene activity, many questions remain, in particular how these topologies are formed and maintained. To understand better the link between epigenetic marks, chromatin topology and transcriptional control, we use CHi-C technique based on the chromosome conformation capture (3C) method. By using two capture strategies targeting two different chromatin structures (chromatin loops and topological domains), we have been able to decipher the chromatin structure associated with thymocyte differentiation and to highlight mechanisms for the transcriptional control of certain genes. Future experiments of the lab will examine mechanisms other than transcription which may influence chromatin architecture, such as differential binding of CTCF, and how these may interplay with transcriptional control and chromatin architecture.