High Mobility Group AT-hook protein 2 (HMGA2) is a non-histone chromatin binding protein expressed in stem cells, cancer cells but not in normal human somatic cells. The presence of HMGA2 in cancer correlates with advanced neoplastic disease and poor prognosis. HMGA2 plays important roles in Base Excision Repair (BER) and at replication forks. HMGA2 is present at mammalian metaphase telomeres and its loss induces chromosomal aberrations. However, the functional role of HMGA2 at telomeres remains elusive. We hypothesized a protective role of HMGA2 that guards telomeres and modulates DNA damage repair signaling pathways. Employing different HMGA2+ human tumor cell models, we investigated the HMGA2-mediated functions that contribute to chemoresistance in glioblastoma (GB). This study presents a novel interaction of HMGA2 with telomeric protein TRF2 (Telomere Repeat-Binding Factor 2). This interaction retains TRF2 at telomeres, thus capping the telomeres and reducing telomere-dysfunction induced foci despite induced telomere stress. Loss of HMGA2 coincides with increased phosphorylation of TRF2, decreased TRF2 retention at telomeres and increased formation of telomeric aggregates, anaphase bridges and micronuclei. These findings provide new evidence for a unique role of HMGA2 at telomeres as a novel contributor of telomeric integrity. We show that upon DNA damage, HMGA2 causes increased and sustained phosphorylation of Ataxia Telangiectasia and Rad3-related kinase (ATR) and checkpoint kinase 1 (CHK1). Prolonged presence of pCHK1Ser296 coincides with prolonged G2/M block and increased tumor cell survival. The relationship between (ATR)-CHK1 DNA damage response pathway and HMGA2 identifies a novel mechanism by which HMGA2 can alter DNA repair function in cancer cells. We identified HMGA2 as a novel factor contributing to temozolomide (TMZ) resistance in GB. HMGA2 knockdown sensitizes the GB cells to TMZ. We propose a specific combination of FDA-approved drugs, TMZ and Dovitinib (DOV), to increase GB cell death. We show that DOV downregulates key BER proteins, attenuates pSTAT3-coordinated Lin28A and HMGA2 expression. Our results suggest that a sequential therapeutic strategy of pretreating GB cells with DOV followed by a sequence of TMZ and DOV diminishes TMZ resistance and enhances the ability of TMZ to induce GB cell death. Overall, we identified HMGA2 as a multifunctional survival factor in human cancer cells and showed that targeting HMGA2 is a valid strategy to combat HMGA2+ cancer cells. / February 2016

The stability of DNA is a critical factor for several diseases, the most prevalent of which is cancer. Several neurodegenerative and accelerated aging diseases are also characterized by genomic instability. The number and complexity of DNA repair pathways that human cells possess underscores the importance of genomic stability. These pathways ensure that damaged DNA is repaired and that a cells complement of DNA remains stable upon cell division. How one particular type of DNA alteration, a DNA loop, is processed in human cells was the focus of this study. We have employed an in vitro system to study defined DNA loop substrates by human nuclear extracts. The influence of either a 5 or 3 nick, the range of loop sizes processed, and the role of DNA mismatch repair, DNA nucleotide excision repair, and the Werner Syndrome helicase proteins were variables tested. The results indicate tha t DNA loops containing between 5 to 12 nucleotides are processed in a strand - specific manner when either a 5 or 3 nick is present , with repair being targeted solely to the nicked strand . This repair occurs by both mismatch repair dependent and independent pathways. The processing of DNA loops containing 30 nucleotides in length is directed either by a 5 nick, or by the loop itself, but not by a 3 nick. The nick independent pathway results solely in loop removal. The large loop pathway is independent of mismatch repair, nucleotide excision repair, and the WRN helicase/exonuclease protein. Both of the 5 nick directed pathways occur by excision that initiates at the pre- existing nick and proceeds towards the loop along the shortest path between the nick and loop. DNA resynthesis occurs using either DNA polymerase , , or and also initiates at the pre-existing 5 nick. The 3 nick directed intermediate loop repair pathway proceeds in a similar fashion, likely after a nick is made 5 to the loop region on the strand that contained the pre-existing nick. DNA synthesis inhibition has only a minor affect on the nick independent loop removal pathway as only a short tract of DNA surrounding the loop site is processed. In total, the results point to at least 3 novel pathways that process DNA loops that likely contribute to total genomic stability.

Asf1 est une protéine chaperon d’histone, qui participe à l’assemblage et au désassemblage des histones H3/H4 sur l’ADN. Asf1 n’est pas essentiel pour la viabilité cellulaire chez S. cerevisiae, mais les voies de surveillance des dommages à l’ADN sont activées de façon constitutive dans les cellules dépourvues d’Asf1 et celles-ci sont hypersensibles à plusieurs types de stress génotoxiques. Chez S. cerevisiae, Asf1 forme un complexe stable avec Rad53 en absence de stress génotoxique. Nos résultats suggèrent qu’au moins trois surfaces d’interaction sont impliquées dans le complexe Asf1-Rad53. Le domaine FHA1 de Rad53 fixe Asf1 phosphorylé sur T270, l’extrémité C-terminale de Rad53 fixe la même surface d’Asf1 impliquée dans la fixation des co-chaperones HirA/CAF-1, et un troisième site putative est constituée de la surface d’Asf1 impliquée dans la fixation de l’histone H3 avec le domaine kinase de Rad53. Lors des stress génotoxiques, Rad53 est phosphorylée et activée. Mes résultats montrent une dissociation totale du complexe Rad53-Asf1 après traitement HU, mais la préservation du complexe après traitement des cellules avec une gamme de concentration de MMS. Nous pensons que la régulation du complexe traduisent des réponses cellulaires distinctes adaptées à des stress génotoxiques spécifiques. Par ailleurs, grâce à la structure du complexe formé par un peptide C-terminal de Rad53 et le domaine N-terminal d’Asf1, nous avons isolé une mutation rad53_A806R-L808R. Nous avons constaté que cette mutation déstabilise l’interaction entre Asf1 et Rad53 et augmente la viabilité des mutants rad9 et rad24 aux stress génotoxiquex. Ce mutant rad53_A806R-L808R semble retourne plus vite dans le cycle cellulaire et/ou traverse plus vite la phase S par rapport à Rad53-WT, et augmente la réparation de l’ADN ou l’adaptation aux dommages du simple mutant rad24Δ. / Asf1 is a histone chaperone, which participates in the assembly and disassembly of histones H3/H4 on DNA. Asf1 is not essential for cell viability in yeast, but the DNA damage checkpoints are constitutively activated in cells lacking Asf1 and they are hypersensitive to several types of genotoxic stress. In yeast, Asf1 forms a stable complex with Rad53 in the absence of genotoxic stress. Our results suggest that this complex involves at Ieast three interaction surfaces. One site involves the H3-binding surface of Asf1 with an as yet undefined surface of Rad53, probably reside in the kinase domain of Rad53. A second site is formed by the Rad53-FHA1 domain binding to Asf1-T270. The third site involves the C-terminal 21 aa of Rad53 bound to the conserved Asf1 N-terminal domain, where Rad53 competes with histone H3/H4 and co-chaperones HirA/CAF-1 for binding to the same surface of Asf1. Rad53 is phosphorylated and activated upon genotoxic stress. The Asf1-Rad53 complex dissociated when cells were treated with hydroxyurea but not methyl methane sulfonate, suggesting a regulation of the complex as a function of the stress.In addition to these results, we also found that the rad53-A806R+L808R mutation at the C-terminus of Rad53 destabilized the Asf1-Rad53 interaction and increased the viability of rad9 and rad24 mutants to genotoxic stress. The rad53-ALRR mutant also appeared to re-enter the cell cycle and/or traverse S-phase more rapidly than wild type and increased repair or adaptation when combined with the rad24 mutant.

Micronutrientes, vitaminas e minerais, são indispensáveis para as vias de metabolismo do DNA e, além disso, são tão importantes para a manutenção da vida quanto os macronutrientes. Na ausência dos nutrientes adequados, a instabilidade genômica compromete a homeostase, ocasionando doenças crônicas e certos tipos de câncer. Meios de cultura celular tem por finalidade mimetizar o ambiente in vivo, proporcionando aos modelos in vitro condições adequadas para que se avalie a resposta celular aos diferentes estímulos. O artigo de revisão sumariza e discute os micronutrientes usados na suplementação das culturas celulares e sua influência na a viabilidade celular e a estabilidade genômica, focando nos estudos in vitro previamente realizados. Nestes estudos, os meios de cultura celular incluem certas vitaminas e minerais em concentrações distintas das fisiológicas in vivo. Em muitos meios de cultura comumente usados, a única fonte de micronutrientes é o Soro Fetal Bovino (SFB), o qual contribui com 5-10% da composição final do meio. Atenção insuficiente tem sido direcionada à composição de SFB, micronutrientes e culturas celulares como um todo, ou à influência de micronutrientes na viabilidade e genética de culturas celulares. Estudos adicionais avaliando melhor o papel de micronutrientes no nível molecular e a sua influência na estabilidade genômica de células ainda se fazem necessários. O micronutriente foco dessa tese é o Ferro (Fe), que por sua vez é um micronutriente essencial, sendo requerido para o crescimento, desenvolvimento e condições normais de funcionamento das células. Tanto seu excesso quanto a sua deficiência podem causar estresse oxidativo e dano ao DNA. Uma vez que os meios de cultura usualmente utilizados para culturas celulares têm níveis de Fe abaixo das concentrações encontradas no soro fisiológico humano, os objetivos deste estudo foram a avaliação do papel da suplementação com Fe na viabilidade celular, na produção de espécies reativas de oxigênio (ERO), na atividade da catalase, na integridade genômica, na expressão de proteínas de reparo de DNA que contém clusters Fe/S em sua estrutura (TFIIH e MutyH) e na expressão de receptores de absorção de Fe (CD71 e Nramp2). Duas linhagens celulares – MRC5 (fibroblasto pulmanar humano) e HepG2 (hepatocarcinoma) - e dois tipos de suplementação com Fe foram utilizados, holo-Transferrina (h-Tf) e FeSO4. Ambas suplementações foram capazes de aumentar os níveis intracelulares de Fe e a viabilidade genômica. A suplementação com Fe também aumentou a formação de ERO, sem alterar a atividade da catalase. No entanto, este aumento de ERO não foi acompanhado por genotoxicidade. No que se refere à expressão de proteínas de reparo ao DNA, os resultados sugerem que o pré-tratamento com h-Tf ou FeSO4 não exercem influência direta na expressão de TFIIH ou MutyH. Entretanto, na expressão de receptores de Fe, os resultados preliminares indicam que CD71 é uma via prioritária de absorção de Fe, estando relacionada com a homeostase de Fe, enquanto Nramp2 parece ter um papel secundário. Devido à importância fisiológica da h-Tf na homeostase do Fe e o acúmulo de ERO menos pronunciado, sugere-se que h-Tf seja uma melhor forma para a suplementação de Fe nas culturas in vitro. Estudos adicionais se fazem necessários para a melhor elucidação do papel do Fe na viabilidade celular e estabilidade genômica. / Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells’ responses to different stimuli. The review summarizes and discusses studies of cellculture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5-10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of culture cells. Further studies better evaluating micronutrients’ roles at a molecular level and its influence on the genomic stability of cells is still required. The micronutrient focus on this thesis is Iron (Fe), which is an essential micronutrient and is required for growth, development, and normal cellular functioning. Either excess or deficiency of iron can cause oxidative stress and DNA damage Since the cell media commonly used for cell culture has a lower iron concentration than the human serum, this study aimed to evaluate the role of iron supplementation on viability, reactive oxygen species (ROS) production, catalase activity, genome integrity and the expression of iron-bearing DNA repair proteins (TFIIH and MutyH) and proteins associated with iron absorption (CD71 and Nramp2). Two human cell lines – MRC5 (normal lung fibroblast) and HepG2 (hepatocellular carcinoma) and 2 sources of iron - holo-Transferrin (h-Tf) or FeSO4 were used. Both iron supplements were able to increase intracellular iron levels and cell viability. Iron supplementation increased the formation of ROS, but did not alter catalase activity. However, this increase was not accompanied by genotoxicity. Regarding the DNA repair protein expressions, the results suggest that 24h pre-treatment with h-Tf or FeSO4 has no role in the TFIIH or MutyH expressions. Although, in iron receptor proteins expression, the preliminary data could indicate that CD71 is priority related with Fe homeostasis while Nramp2 seems to have a secondary role. Due to h-Tf physiological role in the iron homeostasis and the less pronounced ROS accumulation, h-Tf could be a better iron supplier in vitro. Additional studies are still required to better elucidate the role of Fe in cell viability and genomic stability.

Micronutrientes, vitaminas e minerais, são indispensáveis para as vias de metabolismo do DNA e, além disso, são tão importantes para a manutenção da vida quanto os macronutrientes. Na ausência dos nutrientes adequados, a instabilidade genômica compromete a homeostase, ocasionando doenças crônicas e certos tipos de câncer. Meios de cultura celular tem por finalidade mimetizar o ambiente in vivo, proporcionando aos modelos in vitro condições adequadas para que se avalie a resposta celular aos diferentes estímulos. O artigo de revisão sumariza e discute os micronutrientes usados na suplementação das culturas celulares e sua influência na a viabilidade celular e a estabilidade genômica, focando nos estudos in vitro previamente realizados. Nestes estudos, os meios de cultura celular incluem certas vitaminas e minerais em concentrações distintas das fisiológicas in vivo. Em muitos meios de cultura comumente usados, a única fonte de micronutrientes é o Soro Fetal Bovino (SFB), o qual contribui com 5-10% da composição final do meio. Atenção insuficiente tem sido direcionada à composição de SFB, micronutrientes e culturas celulares como um todo, ou à influência de micronutrientes na viabilidade e genética de culturas celulares. Estudos adicionais avaliando melhor o papel de micronutrientes no nível molecular e a sua influência na estabilidade genômica de células ainda se fazem necessários. O micronutriente foco dessa tese é o Ferro (Fe), que por sua vez é um micronutriente essencial, sendo requerido para o crescimento, desenvolvimento e condições normais de funcionamento das células. Tanto seu excesso quanto a sua deficiência podem causar estresse oxidativo e dano ao DNA. Uma vez que os meios de cultura usualmente utilizados para culturas celulares têm níveis de Fe abaixo das concentrações encontradas no soro fisiológico humano, os objetivos deste estudo foram a avaliação do papel da suplementação com Fe na viabilidade celular, na produção de espécies reativas de oxigênio (ERO), na atividade da catalase, na integridade genômica, na expressão de proteínas de reparo de DNA que contém clusters Fe/S em sua estrutura (TFIIH e MutyH) e na expressão de receptores de absorção de Fe (CD71 e Nramp2). Duas linhagens celulares – MRC5 (fibroblasto pulmanar humano) e HepG2 (hepatocarcinoma) - e dois tipos de suplementação com Fe foram utilizados, holo-Transferrina (h-Tf) e FeSO4. Ambas suplementações foram capazes de aumentar os níveis intracelulares de Fe e a viabilidade genômica. A suplementação com Fe também aumentou a formação de ERO, sem alterar a atividade da catalase. No entanto, este aumento de ERO não foi acompanhado por genotoxicidade. No que se refere à expressão de proteínas de reparo ao DNA, os resultados sugerem que o pré-tratamento com h-Tf ou FeSO4 não exercem influência direta na expressão de TFIIH ou MutyH. Entretanto, na expressão de receptores de Fe, os resultados preliminares indicam que CD71 é uma via prioritária de absorção de Fe, estando relacionada com a homeostase de Fe, enquanto Nramp2 parece ter um papel secundário. Devido à importância fisiológica da h-Tf na homeostase do Fe e o acúmulo de ERO menos pronunciado, sugere-se que h-Tf seja uma melhor forma para a suplementação de Fe nas culturas in vitro. Estudos adicionais se fazem necessários para a melhor elucidação do papel do Fe na viabilidade celular e estabilidade genômica. / Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells’ responses to different stimuli. The review summarizes and discusses studies of cellculture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5-10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of culture cells. Further studies better evaluating micronutrients’ roles at a molecular level and its influence on the genomic stability of cells is still required. The micronutrient focus on this thesis is Iron (Fe), which is an essential micronutrient and is required for growth, development, and normal cellular functioning. Either excess or deficiency of iron can cause oxidative stress and DNA damage Since the cell media commonly used for cell culture has a lower iron concentration than the human serum, this study aimed to evaluate the role of iron supplementation on viability, reactive oxygen species (ROS) production, catalase activity, genome integrity and the expression of iron-bearing DNA repair proteins (TFIIH and MutyH) and proteins associated with iron absorption (CD71 and Nramp2). Two human cell lines – MRC5 (normal lung fibroblast) and HepG2 (hepatocellular carcinoma) and 2 sources of iron - holo-Transferrin (h-Tf) or FeSO4 were used. Both iron supplements were able to increase intracellular iron levels and cell viability. Iron supplementation increased the formation of ROS, but did not alter catalase activity. However, this increase was not accompanied by genotoxicity. Regarding the DNA repair protein expressions, the results suggest that 24h pre-treatment with h-Tf or FeSO4 has no role in the TFIIH or MutyH expressions. Although, in iron receptor proteins expression, the preliminary data could indicate that CD71 is priority related with Fe homeostasis while Nramp2 seems to have a secondary role. Due to h-Tf physiological role in the iron homeostasis and the less pronounced ROS accumulation, h-Tf could be a better iron supplier in vitro. Additional studies are still required to better elucidate the role of Fe in cell viability and genomic stability.

Micronutrientes, vitaminas e minerais, são indispensáveis para as vias de metabolismo do DNA e, além disso, são tão importantes para a manutenção da vida quanto os macronutrientes. Na ausência dos nutrientes adequados, a instabilidade genômica compromete a homeostase, ocasionando doenças crônicas e certos tipos de câncer. Meios de cultura celular tem por finalidade mimetizar o ambiente in vivo, proporcionando aos modelos in vitro condições adequadas para que se avalie a resposta celular aos diferentes estímulos. O artigo de revisão sumariza e discute os micronutrientes usados na suplementação das culturas celulares e sua influência na a viabilidade celular e a estabilidade genômica, focando nos estudos in vitro previamente realizados. Nestes estudos, os meios de cultura celular incluem certas vitaminas e minerais em concentrações distintas das fisiológicas in vivo. Em muitos meios de cultura comumente usados, a única fonte de micronutrientes é o Soro Fetal Bovino (SFB), o qual contribui com 5-10% da composição final do meio. Atenção insuficiente tem sido direcionada à composição de SFB, micronutrientes e culturas celulares como um todo, ou à influência de micronutrientes na viabilidade e genética de culturas celulares. Estudos adicionais avaliando melhor o papel de micronutrientes no nível molecular e a sua influência na estabilidade genômica de células ainda se fazem necessários. O micronutriente foco dessa tese é o Ferro (Fe), que por sua vez é um micronutriente essencial, sendo requerido para o crescimento, desenvolvimento e condições normais de funcionamento das células. Tanto seu excesso quanto a sua deficiência podem causar estresse oxidativo e dano ao DNA. Uma vez que os meios de cultura usualmente utilizados para culturas celulares têm níveis de Fe abaixo das concentrações encontradas no soro fisiológico humano, os objetivos deste estudo foram a avaliação do papel da suplementação com Fe na viabilidade celular, na produção de espécies reativas de oxigênio (ERO), na atividade da catalase, na integridade genômica, na expressão de proteínas de reparo de DNA que contém clusters Fe/S em sua estrutura (TFIIH e MutyH) e na expressão de receptores de absorção de Fe (CD71 e Nramp2). Duas linhagens celulares – MRC5 (fibroblasto pulmanar humano) e HepG2 (hepatocarcinoma) - e dois tipos de suplementação com Fe foram utilizados, holo-Transferrina (h-Tf) e FeSO4. Ambas suplementações foram capazes de aumentar os níveis intracelulares de Fe e a viabilidade genômica. A suplementação com Fe também aumentou a formação de ERO, sem alterar a atividade da catalase. No entanto, este aumento de ERO não foi acompanhado por genotoxicidade. No que se refere à expressão de proteínas de reparo ao DNA, os resultados sugerem que o pré-tratamento com h-Tf ou FeSO4 não exercem influência direta na expressão de TFIIH ou MutyH. Entretanto, na expressão de receptores de Fe, os resultados preliminares indicam que CD71 é uma via prioritária de absorção de Fe, estando relacionada com a homeostase de Fe, enquanto Nramp2 parece ter um papel secundário. Devido à importância fisiológica da h-Tf na homeostase do Fe e o acúmulo de ERO menos pronunciado, sugere-se que h-Tf seja uma melhor forma para a suplementação de Fe nas culturas in vitro. Estudos adicionais se fazem necessários para a melhor elucidação do papel do Fe na viabilidade celular e estabilidade genômica. / Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells’ responses to different stimuli. The review summarizes and discusses studies of cellculture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5-10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of culture cells. Further studies better evaluating micronutrients’ roles at a molecular level and its influence on the genomic stability of cells is still required. The micronutrient focus on this thesis is Iron (Fe), which is an essential micronutrient and is required for growth, development, and normal cellular functioning. Either excess or deficiency of iron can cause oxidative stress and DNA damage Since the cell media commonly used for cell culture has a lower iron concentration than the human serum, this study aimed to evaluate the role of iron supplementation on viability, reactive oxygen species (ROS) production, catalase activity, genome integrity and the expression of iron-bearing DNA repair proteins (TFIIH and MutyH) and proteins associated with iron absorption (CD71 and Nramp2). Two human cell lines – MRC5 (normal lung fibroblast) and HepG2 (hepatocellular carcinoma) and 2 sources of iron - holo-Transferrin (h-Tf) or FeSO4 were used. Both iron supplements were able to increase intracellular iron levels and cell viability. Iron supplementation increased the formation of ROS, but did not alter catalase activity. However, this increase was not accompanied by genotoxicity. Regarding the DNA repair protein expressions, the results suggest that 24h pre-treatment with h-Tf or FeSO4 has no role in the TFIIH or MutyH expressions. Although, in iron receptor proteins expression, the preliminary data could indicate that CD71 is priority related with Fe homeostasis while Nramp2 seems to have a secondary role. Due to h-Tf physiological role in the iron homeostasis and the less pronounced ROS accumulation, h-Tf could be a better iron supplier in vitro. Additional studies are still required to better elucidate the role of Fe in cell viability and genomic stability.

Les sites fragiles communs (SFCs) sont des régions génomiques particulièrement sensibles au stress réplicatif et sont impliqués dans l’initiation et la progression du cancer. L’Anémie de Fanconi (AF) est une maladie génétique rare qui se caractérise principalement par une aplasie médullaire, des malformations congénitales ainsi qu’une forte prédisposition au cancer chez les patients (leucémies myéloïdes et tumeurs solides de la tête et du coup). L’instabilité génomique a été identifiée comme étant une source majeure de prédisposition des patients AF au cancer et les SFCs sont particulièrement sensibles dans cette maladie. L’AF est causée par la mutation de gènes codant des protéines participant à une voie moléculaire appelée voie FANC qui a été décrite dans la réparation des ponts inter-brins. Malgré l’importance de la voie FANC dans le maintien de la stabilité des SFCs, les mécanismes sous-jacents restent à élucider. Au cours de ma thèse, nous avons identifié un nouveau rôle de FANCD2 dans le maintien des SFCs. En effet, nous montrons que FANCD2 atténue l’expression des gènes présents au sein des SFCs maintenant leur stabilité. De plus, nous montrons que la transcription de ces gènes est nécessaire au recrutement et au rôle de FANCD2 au sein de ces régions. Enfin, nous avons identifié le stress métabolique comme étant un signal induisant l’expression des gènes des SFCs et que FANCD2 module cette réponse. La réduction de ce stress pourrait être une piste thérapeutique intéressante afin de prévenir l’instabilité des SFCs dans l’AF. / Common fragile sites (CFSs) are genomic regions prone to form breaks and gaps on metaphase chromosomes after replicative stress and promote genomic instability in the earliest steps of tumor development. Proteins involved in replication/repair of CFSs are necessary to prevent their instability. Among them is FANCD2, a key protein of the FANC pathway necessary to resolve inter-strand crosslinks and defective in Fanconi Anemia (FA). FA is a rare genomic instability disorder characterized by bone marrow failure, congenital abnormalities and predisposition to acute myeloid leukemia and epithelial cancer. Genomic instability in FA is supposed to predispose patients to cancers. Importantly, CFSs are more unstable in FA and chromosome breaks observed in FA cells occur preferentially at CFSs. During my PhD, we identified a new role of FANCD2 in CFS stability maintenance. We show that FANCD2 attenuates transcription of the large genes present at CFSs, preventing their instability. Moreover, we demonstrate that transcription is necessary for FANCD2 recruitment and function at CFSs. Importantly, we identified the metabolic stress as a signal triggering CFS gene expression and FANCD2 is necessary to modulate this response. Reducing this stress is a promising therapeutic issue to prevent CFS and genomic instability in FA.

Les cassures double brin (CDB) de l’ADN sont des lésions dangereuses qui peuventêtre produites par des agents physiologiques et environnementaux. La réparation inefficace desCDB dans les cellules souches adultes (CSA), qui sont au sommet de la hiérarchie cellulaire,peut affecter leur capacité d’auto-renouvellement et également le processus de régénération.Le maintien de la stabilité génomique est fondamental et l’altération de ce processus accélèrele vieillissement et peut engendrer des cancers (cellules souches cancéreuses).Les CSA du muscle squelettique (cellules satellites, CS) sont responsables del’homéostasie et de la régénération musculaire. Après activation, les CS quiescentesprolifèrent, régénèrent les myofibres et reconstituent le pool, en s’auto-renouvelant.Ce projet de thèse a eu pour but d’étudier la réparation des CDB dans les CS et leursdescendants, au cours de la différenciation. Nous avons montré que les CS réparent les CDBplus efficacement et plus fidèlement que les cellules différenciées, avec l’implication du NHEJet de DNA-PK. Cette efficacité dépend plus de l’état de différenciation que de la proliférationet la niche a un impact mineur. De plus, des expériences avec des mutants de réparation,apoptose et différenciation suggèrent un mécanisme spécifique de réparation des CDB dans lesCS, qui pourrait être lié à l’architecture distincte de la chromatine de ces cellules. Ces étudesdevraient aider à comprendre comment le maintien de l’intégrité de l’ADN préserve le pooldes CS, influence la régénération et le vieillissement et protège de la carcinogenèse. / DNA double strand breaks (DSBs) are dangerous DNA lesions that are generated byphysiological and environmental DNA agents. Mismanagement of DSBs in adult stem cellsthat are at the top of the hierarchy generating the differentiated tissue, can affect their selfrenewalcapacity and the fate of their progeny. Maintenance of genome stability throughrobust DNA repair is fundamental for tissue regeneration, and impairment of this processaccelerates aging and may lead to cancers (cancer stem cells).Adult muscle stem cells (satellite cells, SCs) sustain skeletal muscle homeostasis andregeneration. Upon activation, quiescent SCs proliferate thereby regenerating muscle fibersand reconstituting the satellite cell pool by self-renewing.This thesis project aims to study DSB repair in SCs and their progeny, duringdifferentiation. We showed that muscle SCs repair DSBs more efficiently and, surprisingly,more accurately than differentiated cells by implicating NHEJ and DNA-PK. The repairefficiency is more a function of the differentiation status than of the replication status ofmyogenic cells, and the niche has a minor effect on the repair efficiency of SCs. Moreover,experiments with DSB repair, apoptosis and differentiation mutants suggest that SCs repairDSBs through a specific mechanism, that may be linked to the distinct chromatin architectureof these cells. These studies should help understanding how the maintenance of genomestability preserves SCs pool, influence regeneration and aging and protect fromcarcinogenesis.

A via de sinalização da GTPase RhoA atua em diversos processos celulares. Para avaliar o comportamento de RhoA, após estresse causado por radiação ultravioleta, foram gerados clones mutantes que expressam RhoA em seu estado constitutivamente ativo e dominante negativo. Após exposição das linhagens à radiação ultravioleta, observou-se uma maior sensibilidade e um maior tempo de recuperação das linhagens quando a atividade de RhoA é reduzida. Estes prejuízos no reparo prejudicaram a proliferação e sobrevivência celular quando da deficiência na atividade de RhoA. Em linhagens deficientes na via de NER, percebemos que estas linhagens possuem uma capacidade ainda mais reduzida de reparo quando a atividade de RhoA é inibida. / The RhoA GTPase signaling pathway acts on many cellular processes. To evaluate this possible RhoA function after stress caused by ultraviolet radiation, mutant clones expressing RhoA in its constitutively active or dominant negative forms were generated. After exposure of the cells to ultraviolet radiation, cell lines showed a higher sensitivity and a delayed recovery capacity when the RhoA activity is reduced. The impaired repair reduced the cells proliferation and survival under RhoA deficiency. In cell lines deficient in NER pathway, we notice that these cell lines, have a further reduced ability to repair damaged DNA under RhoA inhibition.

Pluripotent cells are unique due to their developmental potential and the possibility to study them is the key step to understand human development. These cells are characterized by their ability to originate all the cellular lineages within an adult organism. Within embryonic milieu, pluripotent cells represent a dynamic fraction of the total cell number. Moreover, their physiological existence is constrained to early stages of embryonic development. In vitro culture of the different types of mammalian pluripotent cells, and singularly embryonic stem cells (ESC), enables the characterization of the pluripotent state. In the four articles included in this thesis we have addressed two different aspects of the molecular characterization of mammalian pluripotent cells. First, we investigated the establishment of the trophectoderm and the inner cell mass in the embryo measuring transcript abundance and protein presence of the transcription factors known to play a role in the earliest cellular differentiation process. In addition we have evaluated of genomic stability of human ESC lines during long-term culture, observing the accumulation of sukaryotypic aberrations such as loss of heterozygosity that affect loci comprising genes involved in genomic stability maintenance. We also checked the genomic status of two human ESC lines derived from embryos that had been diagnosed as abnormal after genetic preimplantation diagnosis (PGD). The molecular analysis of these cells ruled out the hypothesized self-correction of the aneuploidies between the PGD and the establishment of the cell lines. / Les cèl·lules pluripotents són úniques atesa la seva plasticitat durant el desenvolupament i la possibilitat d'estudiar-les és un pas essencial per poder comprendre el desenvolupament embrionari. Aquestes cèl·lules es caracteritzen per la seva habilitat per donar lloc a tots els llinatges cel·lulars de l'organisme. Dins de l'embrió, les cèl·lules pluripotents representen una fracció dinàmica del nombre total de cèl·lules i la seva existència fisiològica està constreta a els estadis més primerencs del desenvolupament embrionari. El cultiu in vitro dels diferents tipus de cèl·lules pluripotents en mamífers, i en especial les cèl·lules mare embrionàries, permet la caracterització d'aquest estat cel·lular. En els quatre capítols inclosos en aquesta tesi, hem tractat dos aspectes diferents de la caracterització molecular de les cèl·lules pluripotents. Primer, hem investigat l'establiment del trofectoderm i de la massa cel·lular interna en l'embrió mesurant l'abundància dels trànscrits i la presència de proteina dels factors de transcripció implicats en el primer process de diferenciació cel·lular conegut. A més, hem avaluat l'estabilitat genòmica de dues línies de cèl·lules mare en cultiu durant més de 40 passis. Com a resultat, hem observat l'acumulació de aberracions genòmiques a nivell subcariotípic, en especial pèrdua d'heterozigositat que afecta a locus que contenen gens implicats en el manteniment de l'estabilitat genòmica. També hem comprovat l'estatus genòmic de dos linies de cèl·lules mare embrionàries humanes derivades a partir d'embrions trobats aneuploids per un diagnòstic genètic preimplantacional. L'anàlisi molecular d'aquestes cèl·lules va descartar la hipòtesi d'una autocorrecció de les aneuploidies detectades entre el diagnòstic preimplantacional i la derivació de les línies a partir d'aquests embrions.