Optimizing culture conditions that reduce genomic instability in human pluripotent stem cells (hPSCs) is an unmet challenge in the field. Results from our lab and numerous research groups demonstrate that hPSCs are prone to genomic aberrations and single-cell passaging increases the rate of genomic alterations. However, single-cell based passaging maintains advantages for scale-up and standardizing differentiation protocols. In this study, we investigated the problem of genomic instability in hPSC cultures with the goal towards identifying and characterizing candidate genes that could contribute to generation and survival of abnormal hPSCs. Based on microarray analysis, we identify ARHGDIA, located on 17q25, as a candidate gene conferring selective advantage to trisomy 17 hPSCs. Using lentiviral approaches to overexpress ARHGDIA in hPSCs, [hPSC (Arg)], we functionally validate that in enzymatically passaged co-cultures, hPSC (Arg) lines exhibit competitive advantage against wild type hPSCs, [hPSC (WT)]. Additionally, hPSC (Arg) lines exhibit increased single-cell survival at low density plating. In co-cultures with hPSC (WT), ROCKi exposure attenuated the competitive advantage of hPSC (Arg) subpopulations. For the first time, this work demonstrates that increased expression of a gene on 17q25 confers selective advantage to hPSCs. In parallel studies, using medium devoid of bFGF containing LIF plus two inhibitors, MEK inhibitor (PD0325901) and p38 inhibitor (SB203580), we demonstrate that hPSCs are LIF responsive and can be stably maintained in naive pluripotent culture conditions. Based on their clonal viability, we propose that naive hPSCs are a more genetically stable population than primed hPSCs, when passaged as single- cells. These studies will aid the long-term goal of hPSC scale-up while promoting stable propagation of genomically normal hPSCs.

Investigating the biological basis of ageing is both fascinating and medically relevant, as we strive to understand both how organisms age, and how our knowledge might be put to good use in an increasingly long-lived human population. Despite the complexity of ageing biology, it is very striking that longevity, in a wide variety of organisms, can be modified by manipulating single genes. In this thesis, I investigate phenotypes associated with mutations in C. elegans homologues of human WRN, the gene mutated in the progeroid Werner syndrome (WS). Mutant phenotypes in the worm recapitulate aspects of the pathophysiology observed in WS patients, including premature ageing, genomic instability, and sensitivity to DNA damaging agents. wrn-1 overexpression, on the other hand, appears to enhance longevity, suggesting that wrn-1 acts as a bona fide anti-gerontogene. The combination of wrn-1 mutations with mutation in the worm p53 homologue, cep-1, unexpectedly triggers a novel and very striking enhanced lifespan and healthspan phenotype, termed synthetic super-viability (SSV). The SSV phenotype is modulated by various environmental inputs such as temperature stress. The data presented here can be incorporated into a model in which stress sensing (involving p53) is the crucial determinant of longevity outcomes. Several theories of ageing incorporate the idea that 'that which does not kill us, makes us stronger' - encapsulated in a biological sense in the idea of hormesis, a physiological shift in response to stress. Here, this hypothesis is expanded to include the notion that intrinsic <strong>responses</strong> to stress may themselves act to limit lifespan - too much of a good thing can be bad.

Un enjeu majeur de la génétique évolutive est de comprendre comment l'adaptation locale se développe en population naturelle, et comment les différentes forces évolutives y contribuent. Les études expérimentales d'adaptation locale utilisent couramment les gradients altitudinaux présentant une variation spatiale marquée des conditions environnementales. Dans ces conditions, on s'attend à ce que la différentiation génétique pour les caractères (traditionnellement mesurée par QST) et pour les gènes déterminant ces caractères (traditionnellement mesurée par FSTq) le long du gradient soit gouvernée de façon prédominante par la sélection et les flux de gènes, et peu influencée en revanche par la dérive génétique et la mutation. En particulier, des études théoriques ont montré un découplage entre QST et FST lorsque que les flux de gènes sont forts et/ou que la sélection est récente. Dans cette étude, nous avons testé cette hypothèse en combinant une approche de génomique expérimentale et des simulations dans des populations naturelles de hêtre commun (F. sylvatica) séparées de ~trois kilomètres et soumis à des environnements contrastés.Pour l'approche expérimentale, nous avons échantillonné 4 populations sur deux gradients altitudinaux sur le Mont Ventoux (avec une population à haute altitude et une à basse altitude sur chaque gradient). Cinquante huit gènes potentiellement impliqué dans la réponse aux stress abiotiques et dans le débourrement ont été séquencés sur un total de quatre-vingt seize individus, révélant 581 SNPs (Single Nucleotide Polymorphisms). Différentes approches ont été utilisées pour identifier les SNP outlier, présentant une différentiation plus forte qu'attendu sous un modèle neutre sans sélection. Le nombre de SNPs outlier identifié comme étant sous sélection s'est révélé être grandement dépendant de la méthode utilisé. La méthode fréquentiste a détecté de nombreux outliers alors que l'approche bayésienne n'a pu permettre de détecter des SNPs sous sélection. Par ailleurs, nous avons utilisé un modèle mécaniste individu-centré pour simuler les patrons de diversité phénotypique et génétique attendus le long du gradient pour la phénologie du débourrement végétatif, un caractère généralement adaptatif dans la réponse aux variations de température. Les résultats des simulations confirment que la différentiation génétique observée pour le caractère (QST) est généralement plus forte que celle observée au gène (FSTq), et que cette différentiation génétique au trait intervient dès la première génération. Toutefois, les tests d'outlier conduits sur le le modèle simulé ont révélé que plus de 95% des SNPs outlier sont des faux positifs. Comme dans l'approche expérimentale, l'approche Bayésienne ne s'est pas révélé suffisamment fiable pour détecter des QTLs dans des populations spatialement proche et génétiquement faiblement différentiée. Néanmoins une approche multi-locus basée sur un estimateur peu utilisé en génétique (le Zg) a révélé la forte corrélation inter-populations inter-gènes des QTLs confirmant les attendus théoriques. Toutefois, cette approche ne permet pas de détecter précisément les QTLs sans connaissance a priori sur les QTLs. En conclusion, les travaux de cette thèse mettent en évidence la rapidité des changements génétique qui interviennent en moins de 5 générations pendant la modification du climat, et la difficulté de détecter les gènes codant pour des traits complexes. / A major challenge in population genetics is to understand the local adaptation process in natural population and so to disentangle the various evolution forces contributing to local adaptation. The experimental studies on local adaption generally resort to altitudinal gradients that are characterized by strong environmental changes across short spatial scales. Under such condition, the genetic differentiation of the functional trait (measured by the Qst) as well as the genes coding for trait (measured by Fstq) are expected to be mainly driven by selection and gene flow. Genetic drift and mutation are expected to have minor effect. Theoretic studies showed a decoupling between Qst and Fst under strong gene flow and / or recent selection. In this study, I tested this hypothesis by combining experimental and modelling genomic approach in natural population of Fagus sylvatica separated by ~3 kilometres and under contrasted environments.Sampling was conducted in south-eastern France, a region known to have been recently colonised by F.sylvatica. Four naturally-originated populations were sampled at both high and low elevations along two altitudinal gradients. Populations along the altitudinal gradients are expected to be subjected to contrasting climatic conditions. Fifty eight candidate genes were chosen from a databank of 35,000 ESTs according to their putative functional roles in response to drought, cold stress and leaf phenology and sequenced for 96 individuals from four populations that revealed 581 SNPs. Classical tests of departure of site frequency spectra from expectation and outlier detection tests that accounted for the complex demographic history of the populations were used. In contrast with the mono-locus tests, an approach for detecting selection at the multi-locus scale have been tested.The results from experimental approaches were highly contrasted according the method highlighting the limits of those method for population loosely differentiated and spatially close. The modelling approach confirmed the results from the experimental data but revealed that up to 95% of the SNPs detected as outliers were false positive. The multi-locus approach revealed that the markers coding for the trait are differentially correlated compared to the neutral SNPs. But this approach failed to detect accurately the markers coding for the trait if no a priori knowledge is known about them. The modelling approach revealed that genetic changes may occur across very few generation. But while this genetic adaptation is measurable at the trait level, the available method for detecting genetic adaptation at the molecular level appeared to be greatly inaccurate. However, the multi-locus approach provided much more promise for understanding the genetic basis of local adaptation from standing genetic variation of forest trees in response to climate change.

D'importants progrès ont été réalisés dans les domaines du génotypage et du séquençage, ce qui permet de mieux comprendre la relation génotype/phénotype. Il est possible d'analyser l'architecture génétique des caractères (génétique d'association, GA), ou de prédire la valeur génétique des candidats à la sélection (sélection génomique, SG). L'objectif de cette thèse était de développer des outils pour mener ces stratégies de manière optimale. Nous avons d'abord dérivé analytiquement la puissance du modèle mixte de GA, et montré que la puissance était plus faible pour les marqueurs présentant une faible diversité, une forte différentiation entre sous groupes et une forte corrélation avec les marqueurs utilisés pour estimer l'apparentement (K). Nous avons donc considéré deux estimateurs alternatifs de K. Des simulations ont montré qu'ils sont aussi efficaces que la méthode classique pour contrôler les faux positifs et augmentent la puissance. Ces résultats ont été confirmés sur les panels corné et denté du programme Cornfed, avec une augmentation de 40% du nombre de SNP détectés. Ces panels, génotypés avec une puce 50k SNP et phénotypés pour leur précocité et leur biomasse ont permis de décrire la diversité de ces groupes et de détecter des QTL. En SG, des études ont montré l'importance de la composition du jeu de calibration sur la fiabilité des prédictions. Nous avons proposé un algorithme d'échantillonnage dérivé de la théorie du G-BLUP permettant de maximiser la fiabilité des prédictions. Par rapport à un échantillon aléatoire, il permettrait de diminuer de moitié l'effort de phénotypage pour atteindre une même fiabilité de prédiction sur les panels Cornfed. / Major progresses have been achieved in genotyping technologies, which makes it easier to decipher the relationship between genotype and phenotype. This contributed to the understanding of the genetic architecture of traits (Genome Wide Association Studies, GWAS), and to better predictions of genetic value to improve breeding efficiency (Genomic Selection, GS). The objective of this thesis was to define efficient ways of leading these approaches. We first derived analytically the power from classical GWAS mixed model and showed that it was lower for markers with a small minimum allele frequency, a strong differentiation among population subgroups and that are strongly correlated with markers used for estimating the kinship matrix K. We considered therefore two alternative estimators of K. Simulations showed that these were as efficient as classical estimators to control false positive and provided more power. We confirmed these results on true datasets collected on two maize panels, and could increase by up to 40% the number of detected associations. These panels, genotyped with a 50k SNP-array and phenotyped for flowering and biomass traits, were used to characterize the diversity of Dent and Flint groups and detect QTLs. In GS, studies highlighted the importance of relationship between the calibration set (CS) and the predicted set on the accuracy of predictions. Considering low present genotyping cost, we proposed a sampling algorithm of the CS based on the G-BLUP model, which resulted in higher accuracies than other sampling strategies for all the traits considered. It could reach the same accuracy than a randomly sampled CS with half of the phenotyping effort.

Genomic selection (GS) works by simultaneously selecting hundreds or thousands of markers covering the genome so that the majority of quantitative trait loci are in linkage disequilibrium (LD) with such markers. Thus, markers associated with QTLs, regardless of the significance of their effects, are used to explain the genetic variation of a trait. Simulation and empirical results have shown that genomic prediction presents sufficient accuracy to help success in breeding programs, in contrast to traditional phenotypic analysis. For this end, an important step addresses the use of statistical genetic models able to predict the phenotypic performance for important traits. Although some crops have benefited from this approach, studies in the genus Coffea are still in their infancy. Until now, there have been no studies of how predictive models work across populations and environments or, even, their performance for different complex traits. Therefore, the main objective of this research is investigating important aspects related to statistical modeling in order to enable a more comprehensive understanding of what makes a robust prediction model and, as consequence, apply it in practical breeding programs. Real data from two experimental populations of Coffea canephora, evaluated in two brazilian locations and SNPs identified by Genotyping-by-Sequencing (GBS) were considered to investigate the genotype-phenotype relationship. In terms of statistical modelling, two classes of models were considered: i) Mixed models, based on genomic relationship matrix to define the (co)variance between relatives (called GBLUP model); and ii) Multilocus association models, which thousands of markers are modeled simultaneously and the marker effects are summed, in order to compute the genetic merit of individuals. Both approaches were considered in separated chapters. Chapter entitled \"A mixed model to multiplicative harvest-location trial applied to genomic prediction in Coffea canephora\" addressed an expansion of the traditional GBLUP to accommodate interaction effects (Genotype × Local and Genotype × Harvest). For this end, we have tested appropriate (co)variance structures for modeling heterogeneity and correlation of genetic effects and residual effects. The proposed model, called MET.GBLUP, showed the best goodness of fit and higher predictive ability, when compared to other methods. Chapter in the sequence was entitled \"Comparison of statistical methods and reliability of genomic prediction in Coffea canephora population\" and addressed the use of different modelling assumptions considering multilocos association models. The usual assumption of marker effects drawn from a normal distribution was relaxed, in order to seek for a possible dependency between predictive performance and trait, conditional on the genetic architecture. Although the competitor models are conceptually different, a minimal difference in predictive accuracy was observed in the comparative analysis. In terms of computational demand, Bayesian models showed higher time of analysis. Results discussed in both chapters have supported the potential of genomic selection to reshape traditional breeding programs. In practice, compared to traditional phenotypic evaluation, it is expected to accelerate the breeding cycle in recurrent selection programs, maintain genetic diversity and increase the genetic gain per unit of time. / Seleção Genômica pode ser definida como a seleção simultânea de centenas ou milhares de marcadores moleculares, os quais cobrem o genoma de forma densa, de modo que locos de caracteres quantitativos (QTL) estejam em desequilíbrio de ligação com uma parte desses marcadores. Assim, marcadores associados a QTLs, independentemente da significância dos seus efeitos, são utilizados na predição do mérito genético de um indivíduo para um determinado caráter. Simulações e estudos empíricos mostram que essa abordagem apresenta acurácia suficiente para garantir o sucesso em programas de melhoramento genético, quando comparado com os métodos tradicionais de seleção fenotípica. Para tanto, uma das etapas requeridas é o uso de modelos genético-estatísticos que contemplem a predição fidedigna da performance fenotípica da população sob estudo. Apesar da relevância, o número de estudos no gênero Coffea ainda são reduzidos, não havendo relatos sobre o desempenho desses modelos em diferentes populações e ambientes, ou mesmo, a sua performance para diferentes caracteres agronômicos do cafeeiro. Dessa forma, este estudo tem como finalidade investigar aspectos relacionados a modelagem estatística, a fim de compreender quais são os fatores que tornam os modelos preditivos mais acurados e utiliza-los em programas aplicados de melhoramento genético. Dados reais de duas populações de seleção recorrente de Coffea canephora, avaliados em dois ambientes e genotipados pela tecnologia de genotipagem por sequenciamento (GBS, do inglês Genotyping-by-Sequencing) foram considerados para o estudo da relação entre genótipo-fenótipo. Em termos de modelagem estatística, duas classes de modelos foram considerados: i) Modelos mistos, baseados no cálculo da matriz de parentesco realizado como medida de (co)variância genética entre indivíduos (modelo GBLUP); e ii) Modelos de associação multilocos, no qual milhares de marcadores moleculares são modelados simultaneamente e os efeitos estimados dos marcadores são somados, a fim de computar o mérito genético dos indivíduos. Ambas estratégias foram descritas em capítulos separados no formato de artigo científico. O capítulo intitulado \"A mixed model to multiplicative harvest-location trial applied to genomic prediction in Coffea canephora\" abordou uma expansão do modelo GBLUP de modo a contemplar efeitos de interações entre Genótipo × Colheita e Genótipo × Local. Para tanto, apropriadas estruturas de variância e covariância para modelagem da heterogeneidade e correlação dos efeitos genéticos e residuais foram testadas. O modelo proposto, denominado de MET.GBLUP, apresentou melhor qualidade de ajuste e capacidade preditiva, quando comparado com outros métodos. O capítulo em sequência, intitulado de \"Comparison of statistical methods and reliability of genomic prediction in Coffea canephora population\" investigou a capacidade preditiva de diferentes modelos de associação multilocos. A suposição usual de efeitos dos marcadores amostrados de uma distribuição normal foi relaxada, a fim de testar métodos alternativos que pudessem melhor descrever o fenômeno biológico e, consequentemente, resultar em maior capacidade preditiva. Embora os modelos testados sejam conceitualmente distintos, diferenças mínimas nos valores de acurácia de predição foram observadas nos cenários testados. Em termos de demanda computacional, modelos Bayesianos apresentaram maior tempo de análise. Os resultados descritos em ambos os capítulos apoiam o potencial do uso da seleção genômica em programas de melhoramento assistido de café. Em termos práticos, comparado com métodos tradicionais de avaliação fenotípica, é esperado que a implementação desses conceitos em programas de seleção recorrente possam acelerar o ciclo de melhoramento, manter a diversidade genética e, sobretudo, aumentar o ganho genético por unidade de tempo.

La réplication de l’ADN est un phénomène physiologique essentiel à la transmission du patrimoine génétique mais est aussi une source importante de stress endogène. Le stress réplicatif peut conduire à une instabilité génomique et a été mis en évidence à une étape très précoce du développement tumoral et de la sénescence. La recombinaison homologue (RH) est un processus de réparation qui permet la prise en prise en charge du stress réplicatif. De ce fait, un défaut de RH devrait permettre de révéler les stress réplicatifs endogènes. Ainsi, une progression ralentie des fourches de réplication a été observée dans des cellules déficientes pour la RH (RH-), et ce en absence de tout traitement exogène (Daboussi 2008). De plus, de nombreux travaux ont mis en évidence la présence de défauts mitotiques dans les cellules RH-, en absence de tout traitement exogène (Griffin 2000; Kraakman-van der Zwet 2002; Bertrand 2003; Daboussi 2005; Laulier 2011; Rodrigue 2013). L’origine de ces défauts mitotiques spontanés reste peu claire. En effet, la RH étant un processus préférentiellement actif au cours des phases S et G2, le lien avec la mitose reste à éclaircir. Cette thèse a pour but de comprendre l’impact du stress réplicatif très faible ou endogène sur les phases post-réplicatives du cycle cellulaire. Dans un premier temps, je me suis intéressée à l’impact de ce stress sur la mitose. Les résultats obtenus montrent que le traitement des cellules contrôle à de très faibles doses d’hydroxyurée (HU) n’affecte pas la progression dans le cycle cellulaire mais induit cependant une diminution de la vitesse de réplication, comparable à celle observée dans les cellules RH-. De plus le traitement des cellules contrôle à des faibles doses d’HU induit l’apparition de défauts mitotiques, notamment des centrosomes surnuméraires, à la même fréquence que dans les cellules RH- non traitées. Inversement, l’ajout de précurseurs de nucléotides dans les cellules RH- permet de supprimer la diminution de la vitesse de réplication ainsi que les centrosomes mitotiques surnuméraires. Ainsi, un stress réplicatif subtil, qui n’impacte pas de façon détectable la progression dans les phases S et G2 du cycle cellulaire, ni l’entrée en mitose, cause cependant des défauts mitotiques sévères. De façon importante, les centrosomes mitotiques surnuméraires peuvent entrainer des mitoses multipolaires, impactant ainsi l’ensemble du génome. Ces données mettent en évidence la connexion qui existe entre la réplication des chromosomes et leur ségrégation. Dans un second temps, j’ai étudié l’impact du stress réplicatif faible ou endogène en phase G2. Cette étude a été réalisée en utilisant des cellules RH-, ainsi qu’un modèle d’induction de faible stress réplicatif après traitement à très faible dose d’HU. La présence de foyers pRPA-Ser33 en phase G2 a été observée dans ces deux modèles, mettant en évidence des zones de stress réplicatif. Après traitement à très faible dose d’HU, nous observons également la présence en phase G2 de foyers 53BP1 et RAD51 qui colocalisent partiellement avec les foyers pRPA-Ser33. L’analyse en spectrométrie de masse après co-immunoprécipitation de la protéine 53BP1 en phase G2 a permis d’établir un lien avec des protéines impliquées dans le contrôle de l’assemblage du fuseau mitotique ainsi que dans le points de contrôle mitotique, étayant ainsi le lien entre le stress réplicatif et les défauts mitotiques. Pour finir, l’immunoprécipitation de la chromatine liée à la protéine pRPA-Ser33 en phase G2, suivie d’un séquençage (ChIPseq), a permis de révéler l’absence d’enrichissement au niveau des sites fragiles communs et de mettre en évidence un enrichissement au niveau des régions promotrices de certains gènes, notamment de gènes impliqués dans la régulation du cycle cellulaire et de la mort cellulaire. Ces résultats soulignent le lien entre le stress réplicatif très faible ou endogène et l’instabilité chromosomique, qui peut mener à l’initiation tumorale. / DNA replication is a physiological process, essential for genetic information transmission but DNA replication is also an important source of endogenous stress. Replicative stress can lead to genomic instability and has been reported in early-stage malignancies and senescence. Homologous recombination is a repair process which can handle replicative stress. Therefore, a defect in homologous recombination could reveal endogenous replicative stresses. Consistently, a slow down in replication fork progression has been observed in homologous recombination deficient (HR-) cells, in absence of any exogenous treatment (Daboussi et al. 2008). In addition, several studies have shown the presence of mitotic defects in HR- cells, in absence of any exogenous treatment (Griffin 2000; Kraakman-van der Zwet 2002; Bertrand 2003; Daboussi 2005; Laulier et al. 2011; Rodrigue 2013). The origin of these spontaneous mitotic defects is still unclear. Indeed, homologous recombination is preferentially active in S and G2 phases thus, the link with mitosis remains to be elucidated. The aim of this thesis is to understand the impact of a low or endogenous replicative stress on post-replicative phases. First, I studied the impact of a low or endogenous replicative stress on mitosis. Control cells were treated with very low hydroxyurea doses, that did not affected cell cycle progression but did slow down the replication fork progression to the same level than unchallenged HR- cells. Importanntly, exposure of the control cells to these low hydroxyurea doses generated the same mitotic defects, notably extra centrosomes, and to the same extent than in untreated HR- cells. Reciprocally, supplying nucleotide precursors to HR- cells suppressed both their replication deceleration and mitotic extra centrosome phenotypes. Therefore, subtle replication stress that does not impact S and G2 phase progression nor the entry in mitosis, nevertheless causes severe mitotic defects. Importantly, mitotic extra centrosome can lead to multipolar mitosis and then impact the whole genome stability. These data highlight the crosstalk between chromosome replication and segregation. Secondly, I studied the impact of low or endogenous replicative stress on G2 phase. This study was done using HR- cells as well as control cells treated with very low HU doses to induce a very low replicative stress. In both of these models, the presence of pRPA-Ser33 foci was observed in G2 phase, highlighting replicative stress regions. After very low HU treatement, we observed 53BP1 and RAD51 foci in G2 phase. These foci partially colocalized with pRPA-Ser33 foci in G2 phase. Mass spectrometry analyse after 53BP1 coimmunoprecipitation allowed to etablish a link between proteins involved in mitotic spindle assembly control and in mitotic checkpoint. These data support the link between replicative stress and mitotic defects. Lastly, the immmunoprecipitation of the chromatin interacting with pRPA-Ser33 in G2 phase, followed by sequencing (ChIPseq) allowed to reveal the absence of common fragile site enrichment and to highlight an enrichment at promoter regions of genes involved in cell cycle and cell death regulation. These data underline the link between very low or endogenous replicative stress and chromosomal instability, which can lead to tumorigenesis.

O imprinting genômico, processo regulado epigeneticamente segundo o qual os genes se expressam de acordo com sua origem parental, está envolvido no crescimento e desenvolvimento placentário. Na região 11p15.5 encontram-se vários genes regulados por duas regiões controladoras de imprinting (ICR1 e ICR2), onde se encontram as regiões diferencialmente metiladas H19DMR e KvDMR1. Acredita-se que o padrão de imprinting seja dinamicamente regulado durante o desenvolvimento da placenta. Em humanos, há poucas informações sobre imprinting genômico e desenvolvimento placentário, principalmente para estágios precoces do desenvolvimento devido às dificuldades técnicas de obtenção dessas placentas. A descrição de mosaicismo do padrão de metilação restrito a placenta ou entre a placenta e o feto evidencia um perfil epigenético único deste órgão. A 5-hidroximetilação, a qual não tem um papel de silenciamento gênico, pode ser confundida com a metilação do DNA nas análises moleculares. O objetivo principal do presente estudo foi o de verificar a influência da idade gestacional (IG) no perfil de metilação do DNA das ICRs 1 e 2 em vilosidade coriônica, bem como a existência de mosaicismo do perfil de metilação intra-placentário. Neste trabalho também foi investigada a presença de hidroximetilação na KvDMR1. Foram coletadas amostras de tecido placentário, sendo 25 de vilosidades coriônicas (VC) (15 de 3° trimestre gestacional e 10 do 1° trimestre) e nove de cordão umbilical (UC) de 1° trimestre (pareadas com a VC). Quatro placentas de 3° trimestre foram analisadas em separado para o estudo de mosaicismo. O perfil de metilação do DNA das regiões foi verificado por PCR Específica para a Metilação (MS-PCR), Análise Combinada de Bissulfito e Restrição Enzimática (COBRA) e Método de Digestão Enzimática Sensível à Metilação Associada à PCR em Tempo Real (DESM-RT), além do ensaio para hidroximetilação na KvDMR1. Com os ensaios qualitativos (MS-PCR e COBRA) foi observado um perfil de metilação monoalélico, sendo que na H19DMR foi identificada a presença de CpGs diferentemente metilados. Para a H19DMR foram observadas médias de 0,43 de metilação em VC e 0,31 em UC de 1° trimestre, e de 0,41 em VC de 3° trimestre. Para a KvDMR1, foram encontradas médias de 0,47 em VC e 0,57 em UC de 1° trimestre, e de 0,41 em VC de 3° trimestre. A presença de hidroximetilação na KvDMR1 foi excluída. Não foram observadas diferenças significativas entre as médias das diferentes IGs ou entre tecidos pelos testes t e F para ambas as regiões. Não foi observada correlação positiva no perfil de metilação para H19DMR e KvDMR1 entre os tecidos. Em relação ao mosaicismo, não houve diferenças significativas no perfil de metilação entre os diferentes cotilédones amostrados numa mesma placenta. Os resultados demonstram uma discordância entre tecido embrionário (UC) e extraembrionário (VC). Apesar de não serem observadas alterações significantes nos perfis de metilação da H19DMR e KvDMR1 em diferentes IGs, as informações apresentadas são importantes para as pesquisas sobre a dinâmica do fenômeno de imprinting genômico ao longo da gestação, para os estudos de mosaicismo intraplacentário bem como o perfil epigenético da placenta em relação a outros tecidos. / Genomic imprinting, an epigenetically regulated process by which genes are expressed accordingly to their parental origin, is involved in placental growth and development. In 11p15.5 region, there are many genes regulated by two Imprinting Control Regions (ICR1 and ICR2), in which are found two Differentially Methylated Regions, H19DMR and KvDMR1, respectively. Imprinting patterns seem to be adjusted during placenta development. In humans, there is little information on genomic imprinting and placental development, especially for early stages of development due to technical difficulties in obtaining these placentas. The description of mosaicism in methylation pattern restricted to placenta or between placenta and fetus shows a unique epigenetic profile of this organ. The 5-hidroxymethylation, which has no role in gene silencing, can be confused with DNA methylation in molecular analysis. The main aim of our study was to verify the influence of gestational age (GA) in DNA methylation profile of ICRs 1 and 2 in chorionic villi, as well as the existence of intra-placental methylation profile mosaicism. The presence of hydroximethylation in the KvDMR1 was also investigated. Samples were collected from placentas, 25 from chorionic villi (CV) (15 of the 3rd gestational trimester and 10 of the 1st trimester) and nine from umbilical cord (UC) in 1st trimester (paired with the CV samples). Four 3rd trimester placentas were separately analyzed for mosaicism. DNA methylation profile was verified by Methylation Specific PCR (MS-PCR), and Combined Bisulfite Restriction Analysis (COBRA) and Methylation-Sensitive Enzyme Digestion Method associated with Real-Time PCR (DESM-RT), in addition to hydroximethylation test in the KvDMR1 region. With qualitative assays (MS-PCR and COBRA), it was observed a monoallelic methylation pattern, and, only for the H19DMR, differently methylated CpGs were observed. For the H19DMR, we observed methylation means of 0.43 in CV and 0.31 in UC of 1st trimester, and 0.41 in CV of 3rd trimester. For KvDMR1, we observed means of 0.47 in CV and 0.57 in UC of 1st trimester, and 0.41 in CV of 3rd trimester. No hydroximethylation in the KvDMR1 was observed. There were no significant differences between the means of different GAs or between tissues by F and t tests for both regions. No positive correlation was found on methylation profile for H19DMR and KvDMR1 between tissues. In relation to mosaicism, there were no significant differences in methylation profile between different cotyledons sampled in the same placenta. The results showed a discrepancy between embryonic (UC) and extra-embryonic (CV) tissues. Although it was not observed significant changes in methylation profiles of H19DMR and KvDMR1 in different GAs, the presented results are important to research on dynamics of genomic imprinting phenomenon during pregnancy, studies of intra-placental mosaicism and placenta epigenetic profile in relation to other tissues.

A endometriose é uma doença ginecológica benigna comum, mas agressiva, caracterizada pela presença de tecido endometrial ectópico. A teoria mais aceita para explicá-la é a teoria de Sampson, na qual o tecido endometrial descamado durante a menstruação sofre refluxo através das tubas uterinas, adere e se prolifera em sítios ectópicos da cavidade peritoneal. Por outro lado, apenas o refluxo tubário não é capaz de estabelecer a doença e vários estudos sugerem uma etiologia multidimensional incluindo fatores hereditários, hormonais e imunológicos. Várias metodologias têm sido propostas com o objetivo de identificar genes candidatos para a endometriose. A hibridação genômica comparativa (CGH) é uma técnica que permite que o genoma inteiro seja analisado em um só experimento, sem a necessidade de cromossomos metafásicos obtidos por cultura celular. Nossa proposta foi avaliar, por CGH, amostras de endometriomas ovarianos e de tecido endometrial eutópico de dez pacientes com diagnóstico firmado de endometriose, para screening do genoma. No grupo eutópico, 6/10 amostras apresentaram alterações caracterizadas por perdas ou ganhos de regiões cromossômicas e no grupo ectópico foram encontradas alterações em 7/10 casos. A presença de perdas e ganhos de regiões cromossômicas no endométrio eutópico, histologicamente normal, de mulheres com endometriose ovariana, pode ser considerada como alteração primária ao desenvolvimento da doença. A metodologia de CGH permitiu a detecção das regiões cromossômicas 11q12.3-q13.1, 17p11.1-p12 e 17q25.3-qter como regiões críticas, direcionando investigações futuras para identificação de genes associados à endometriose. / Endometriosis is a common benign gynecological disease, very aggressive, characterized by the presence of ectopic endometrial tissue. The most accepted theory to explain it is Sampson\'s implantation theory, which says that the endometrial tissue exfoliated during menstruation undergoes reflux through the uterine tubes, adheres and proliferates in ectopic sites of the peritoneal cavity. On the other hand, only reflux is not enough to the establishment of the disease and a number of studies suggest a multidimensional etiology including hereditary, hormonal and immunological factors. Several methodologies have been proposed for the identification of candidate genes for endometriosis. The comparative genomic hybridization (CGH) is a versatile technique that allows the entire genome to be analyzed in only one experiment without the necessity of metaphasic chromosomes from the sample, excluding the cell culture. We aimed to evaluate, by CGH, ovarian endometriomas and eutopic endometrial tissue samples from 10 patients with confirmed diagnosis of endometriosis, for a genomic screening. In the eutopic group, 6/10 samples presented genomic imbalances and 7/10 cases showed alterations in the ectopic group. The presence of losses and gains of chromosomic regions in the histologically normal eutopic endometrium from women with ovarian endometriosis can be considered as a primary alteration in the development of the disease. The CGH methodology allowed the detection of chromosomic regions 11q12.3-q13.1, 17p11.1-p12 and 17q25.3-qter as critical regions, leading to future investigations for the identification of genes associated to endometriosis.

A síndrome de Silver-Russell (SRS) é caracterizada principalmente por grave retardo de crescimento intrauterino e pós-natal e face típica, pequena e triangular, entre outras características variáveis. A SRS é geneticamente heterogênea, ocorrendo em geral de forma esporádica. Mutações genéticas e epigenéticas em regiões sujeitas a imprinting genômico nos cromossomos 7 e 11 são detectadas em cerca de 50% dos pacientes. Mais frequentemente, a SRS é causada pela alteração da expressão gênica na região 11p15 devido à hipometilação do centro de imprinting telomérico (ICR1) que ocorre em pelo menos 40% dos afetados. Duplicações cromossômicas de origem materna incluindo o centro de imprinting centromérico (ICR2) estão presentes em 1-2% dos casos. A dissomia uniparental materna do cromossomo 7 (matUPD7) é responsável por 5-10% dos casos. Mais recentemente microdeleções e microduplicações cromossômicas foram detectadas em um grupo pequeno de pacientes, algumas delas se mostrando com possível efeito patogênico. Com a identificação da hipometilação de ICR1 em 11p15, matUPD(7) e desequilíbrios (sub)microscópicos, a confirmação molecular para o diagnóstico clínico da SRS tornou-se possível em ~50% dos pacientes, o que deixa metade dos casos sem causa genética determinada. A amostra foi constituída por 64 pacientes brasileiros não aparentados, com suspeita clínica da síndrome de Silver-Russell. O número de cópias de DNA e o padrão de metilação do cromossomo 11p15 foram investigados em 49 pacientes utilizando MS-MLPA, e 21 (43%) deles apresentaram hipometilação de ICR1. Em um desses pacientes (2%), ambos os centros, ICR1 e ICR2, estavam hipometilados, alteração complexa que já foi relatada em ~4% dos pacientes com SRS que apresentavam hipometilação de ICR1. Em outro paciente (2%), foi detectada uma microduplicação de origem materna que incluía o domínio ICR2, mas não ICR1. Essa microduplicação segrega em três gerações de uma família e a manifestação da síndrome depende da transmissão via materna: houve quatro casos de transmissões paternas da microduplicação de um único homem uniformemente resultando em prole normal, e cinco transmissões maternas, de duas irmãs clinicamente normais, com todas as crianças apresentando SRS. Outra microduplicação de origem materna restrita ao domínio ICR2 e associada com SRS em um menino foi descrita anteriormente. Entre os genes duplicados nos dois casos, CDKN1C aparece como candidato para o fenótipo da SRS, uma vez que codifica para um inibidor de quinase dependente de ciclina que regula negativamente o crescimento celular e tem papel crucial no desenvolvimento fetal humano. Esse novo caso familial vem confirmar que a duplicação restrita ao domínio ICR2, de herança materna, está causalmente associada com a SRS; mostra também que nenhuma alteração fenotípica aparente está presente, quando a duplicação é herdada via paterna. Entre os 64 pacientes da amostra, três (4,7%) foram identificados apresentando matUPD(7), pela genotipagem de microssatélites do cromossomo 7. As frequências de hipometilação de ICR1 (43%) e matUPD(7) (4,7%) entre os nossos pacientes, concordantes com o de outros estudos semelhantes, apontam para a seleção adequada dos pacientes com SRS, do ponto de vista clínico. A investigação de microrrearranjos cromossômicos por a-CGH foi realizada em 19 pacientes, que previamente tiveram afastadas alterações (epi)genéticas em 11p15 e a matUPD(7). A maioria dos pacientes não apresentou alterações (n=7) ou possuía apenas CNV frequentes em indivíduos normais da população e consideradas polimorfismos (n=8). Quatro microdeleções potencialmente patogênicas foram detectadas, em 2p23.3 (~320 Kb), 13q24 (~94,3 Kb), 15q11.2 (~320 Kb) e 16p13.11 (~95,8 Kb). Em nenhum dos casos foi possível estabelecer relação direta com o fenótipo da SRS, porque não foi possível investigar ambos os genitores ou a alteração estava presente em um genitor clinicamente normal ou já tinha sido relatada em indivíduo normal da população, não havendo, entretanto, indicação de ser polimórfica. A penetrância incompleta ou a manifestação de alelo recessivo patogênico no cromossomo homólogo são duas possíveis explicações para o efeito patogênico das microdeleções herdadas de genitor clinicamente normal. Três estudos recentes que utilizaram microarrays na busca de genes ou regiões cromossômicas associadas com a SRS, em que a causa genética era desconhecida, detectaram microduplicações e microdeleções, algumas potencialmente patogênicas: uma microdeleção em 15q26.3, incluindo o gene IGF1R, foi identificada em dois pacientes; outras microdeleções incluíam os genes IGF2BP3 em 7p15, GPC5 em 13q31.3, o MAPK1 em 22q11.2 e o HMGA2 em 12q14, considerados candidatos, possivelmente influenciando o crescimento. Esse conjunto de resultados indica que a investigação de microrrearranjos deve estender-se a um número maior de pacientes com SRS, na busca regiões cromossômicas e genes que possam estar causalmente associados com a síndrome. Em 30 pacientes com SRS, buscamos mutações no gene CDKAL1, por sequenciamento direto das regiões codificadoras. Esse gene foi considerado candidato para a síndrome, após ter sido interrompido em um dos nossos pacientes com SRS, portador de uma translocação t(5;6). Nenhuma alteração patogênica foi detectada, indicando que mutações de ponto na região codificadora do gene CDKAL1 não é causa comum da SRS. Em 18 dos 30 pacientes, investigamos a presença de microdeleções e microduplicações por a-CGH e não encontramos alteração que incluísse esse gene. Considerando o pequeno tamanho amostral, não podemos excluir definitivamente a possibilidade de que alterações no gene CDKAL1 possam contribuir para a etiologia da SRS. / Silver Russell syndrome (SRS) is characterized by severe intrauterine and postnatal growth retardation in association with a typical small triangular face and other variable features. Most cases are sporadic. Genetic and epigenetic disturbances on imprinted regions at chromosomes 7 and 11 are detected in about 50% of the patients. Most frequently, SRS is caused by altered gene expression on chromosome 11p15 due to hypomethylation of the telomeric imprinting center (ICR1) that is present in at least 40% of the patients. Maternally inherited duplications encompassing the centromic imprinting center (ICR2) domains at 11p15 are present in about 1-2% of cases. Maternal uniparental disomy of chromosome 7 (mUPD7) is identified in 5-10% of patients. More recently, chromosomal microdeletions and microduplications were detected in a small group of SRS patients, some of them with possible pathogenic effect. This leaves approximately half of the SRS cases without a genetic cause determined. Our cohort consisted of 64 unrelated Brazilian patients with clinical diagnosis of SRS. DNA copy number changes and the methylation pattern on chromosome 11p15 were investigated in 49 patients by MS-MLPA, and 21 (43%) presented with hypomethylation of ICR1. In one patient (2%), both centers (ICR1 and ICR2) were hypomethylated, a complex alteration that has been reported in ~4% of SRS patients that shows hypomethylation of ICR1. In a further patient (2%), we detected a ~1.6 Mb microduplication encompassing the whole ICR2 domain, but not the ICR1. This microduplication was shown to segregate in a three-generation family, and was associated with SRS whenever maternally transmitted: there were four instances of paternal transmissions of the microduplication from a single male uniformly resulting in normal offspring, and five maternal transmissions, via two clinically normal sisters, with all the children exhibiting SRS. A maternally inherited microduplication also restricted to the ICR2 domain and associated with SRS in a boy was described previously. Among the duplicated genes in both cases, CDKN1C is a likely candidate for the SRS phenotype, because it encodes a cyclin-dependent kinase inhibitor that negatively regulates cell proliferation and growth, and plays a crucial role in human fetal development. This new case brings confirmatory evidence that microduplications restricted to the ICR2 domain result in SRS when maternally transmitted. It also shows that no apparent phenotypic change is present when ICR2 duplication is paternally inherited. By genotyping chromosome 7 microsatellites, we identified three patients (4.7%) with mUPD(7), in the cohort of 64 patients. The frequencies of hypomethylation of ICR1 (43%) and mUPD(7) (4.7%) among our patients are in accordance with the literature, and point to a proper selection of patients with SRS, from the clinical point of view. The investigation of submicroscopic chromosomal imbalances by a-CGH was performed in 19 patients in whom (epi)genetic mutations at 11p15 and mUPD(7) had been excluded. Most patients showed no changes (n = 7) or had only CNV considered to be polymorphic (n = 8). Four potentially pathogenic microdeletions were detected, on chomosomes 2p23.3 (~320 Kb), 13q24 (~94.3 Kb), 15q11.2 (~320 Kb) and 16p13.11 (~95.8 Kb). In neither case we could establish a direct relationship between the imbalance and the phenotype, because it was not possible to investigate both parents or the change was present in a clinically normal parent or it had been reported in normal individuals, without, however, indication of being polymorphic. Incomplete penetrance or unmasking of a pathogenic recessive allele on the homologous chromosome are two possible explanations to the pathogenic effect of a microdeletion inherited from a clinically normal parent. Three recent studies that used microarrays to identify genes or chromosomal regions associated with SRS, wherein the genetic cause was unknown, detected microdeletions and microduplications, some of them potentially pathogenic: a microdeletion at 15q26.3, including the IGF1R gene, was identified in two patients; other microdeletions included the IGF2BP3 gene at 7p15, GPC5 gene at 13q31.3, MAPK1 gene at 22q11.2 and HMGA2 gene at 12q14, which were considered candidates, possibly influencing growth. This set of results, including ours, indicates that the investigation of submicroscopic chromosomal imbalances should be extended to a larger cohort of SRS patients, in the search for chromosomal regions and genes that may be causally associated with the syndrome. In 30 SRS patients, we searched for point mutations in the CDKAL1 gene by direct sequencing of coding regions. This gene was considered a candidate for SRS, after being disrupted in one of our SRS patients with a t(5;6). No pathogenic mutation was detected and, therefore, point mutations in the coding region of CDKAL1 do not appear to be a common cause of SRS. In 18 of the 30 patients, we investigated the presence of microdeletions and microduplications by a-CGH and found no changes encompassing CDKAL1 gene. Considering the small cohort size, we cannot definitely exclude the possibility that changes in CDKAL1 gene may contribute to the etiology of SRS.

Gracilariaceae is a worldwide distributed family that includes numerous economically important species. Currently, five genera are recognized in the Gracilariaceae: Gracilariophila (parasitic), Curdiea, Melanthalia, Gracilaria, and Gracilariopsis. Some species of Gracilaria were taxonomically transferred to Hydropuntia. However, this genus is quite controversial. High-throughput sequencing (HTS) techniques has led to an increase in studies using complete organellar genomes, which have been used to infer phylogenetic relationships in Rhodophyta and the investigation of other aspects of red algal genomes, including gene synteny and horizontal gene transfers (HGT). HTS also facilitated the search for extrachromosomal plasmids and its influence in the organellar genomes by HGT. We applied HTS to assemble and annotate organellar genomes (mitochondria and chloroplast) from seven species of Gracilariaceae using Illumina HiSeq 2500 platform. We also received raw reads of 31 samples of Gracilariaceae from Dr. Goia Lyra that were analysed and included in our work. We used these data, combined with published genomes, to infer phylogenies and compare the genome architecture of these species representing the main lineages in Gracilariaceae. The mitochondrial and chloroplast genomes were highly conserved in gene synteny among the species, and variation mainly occurred in regions where insertions of plasmid-derived sequences (PDSs) were found, which were similar to known red algae extrachromosomal plasmids. In mitochondrial genomes, the PDS insertions were in two regions where the transcription direction changes: between cob and trnL genes, and trnA and trnN genes. PDS insertions in chloroplast genome were in different positions, but generally found between psdD and rrs genes. The bacterial leuC/leuD operon was found in Gracilaria tenuistipitata, G. chilensis, M. intermedia chloroplasts genomes, and also in G. vermiculophylla extrachromosomal plasmid. Phylogenetic trees show two different origins of leuC/leuD: genes found in chloroplasts and plasmids were close to proteobacteria, and genes encoded in the nucleus are close to Viridiplantae and cyanobacteria. Gracilariaceae may be a good model to study the impact of PDS in genome evolution due to the frequent presence of these sequences inserted in organellar genomes. Our phylogenetic analyses demonstrated similar evolutionary histories between the chloroplast and mitochondrial genomes. However, chloroplast phylogeny was better resolved with full support. Our taxonomical sampling supports the presence of three main lineages: Melanthalia/Curdiea, Gracilariopsis and Gracilaria. Melanthalia intermedia was sister to a monophyletic clade including Gracilaria and Gracilariopsis, which were resolved as monophyletic genera. Furthermore, the characteristics of organellar genome architecture, Gracilariopsis and Gracilaria genera are also supported by the loss of the plastid gene petP in Gracilaria and the rearrangement position of the gene trnH in the mitochondrial genome. Beside this, we found no support for the genus Hydropuntia as originally proposed / A família Gracilariaceae está globalmente distribuída e inclui várias espécies economicamente importantes. Atualmente, cinco gêneros são reconhecidos em Gracilariaceae: Gracilariophila (parasita), Curdiea, Melanthalia, Gracilaria e Gracilariopsis. Algumas espécies de Gracilaria foram taxonomicamente transferidas para Hydropuntia. Entretanto, esse gênero é bastante controverso. Técnicas de sequenciamento de alta performance (HTS) levaram a um aumento de estudos usando genomas organelares completos, que têm sido usados para inferir relações filogenéticas em Rhodophyta e na investigação de outros aspectos dos genomas de algas vermelhas, incluindo sintenia gênica e transferências horizontal de genes (HGT). O HTS também facilitou a busca por plasmídeos extracromossômicos e sua influência nos genomas organelares por HGT. Nós utilizamos HTS para montar e anotar genomas organelares (mitocôndrias e cloroplastos) de sete espécies de Gracilariaceae usando a plataforma Illumina HiSeq 2500 e recebemos sequências de 31 amostras Gracilariaceae da Dr. Goia Lyra que foram montadas, anotadas e incluídas em nossas análises. Utilizamos esses dados, combinados com genomas publicados, para inferir filogenias e comparar a arquitetura do genoma dessas espécies representando as principais linhagens em Gracilariaceae. Os genomas mitocondrial e plastidial são altamente conservados na sintenia gênica e a variação ocorreu principalmente em regiões onde foram encontradas inserções de sequências derivadas de plasmídeos (PDS), similares aos plasmídeos extracromossômicos conhecidos de algas vermelhas. Nos genomas mitocondriais, as inserções de PDS estavam em duas regiões onde a direção da transcrição muda: entre os genes cob e trnL e os genes trnA e trnN. As inserções de PDS no genoma do cloroplasto estavam em posições diferentes, mas geralmente encontradas entre os genes psdD e rrs. O operon bacteriano leu/leuD foi encontrado nos genomas dos cloroplastos de Gracilaria tenuistipitata, G. chilensis, M. intermedia e também no plasmídeo de G. vermiculophylla. As árvores filogenéticas mostram duas origens diferentes de leuC/leuD: os genes encontrados no cloroplasto e no plasmídeo estavam próximos de proteobactérias, e os genes codificados no núcleo estavam próximos de Viridiplantae e cianobactérias. Gracilariaceae pode ser um bom modelo para estudar o impacto de PDS na evolução de genomas devido à presença frequente de inserções PDS em genomas organelares. Nossas análises filogenéticas demonstraram histórias evolutivas similares entre cloroplasto e mitocondria. No entanto, a filogenia de cloroplasto foi melhor resolvida com valores máximos de Bootstrap em todos os ramos. Nossa amostragem taxonômica corrobora a presença de três linhagens principais: Melanthalia/Curdiea, Gracilariopsis e Gracilaria. Melanthalia intermedia aparece como grupo-irmão do clado monofilético incluindo Gracilaria e Gracilariopsis, que foram resolvidos como gêneros monofiléticos. Além disso, baseado nas características da arquitetura do genoma organelar, os gêneros Gracilariopsis e Gracilaria se distinguem pela perda do gene plastidial petP em Gracilaria e pela posição de rearranjo do gene trnH no genoma mitocondrial. Nós não encontramos evidências para a permanencia o gênero Hydropuntia como originalmente proposto