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Le xeroderma pigmentosum (XP) est une maladie génétique rare caractérisée par une hypersensibilité aux ultraviolets (UV) et une forte incidence des tumeurs cutanées. Les cellules des patients XP sont incapables d’éliminer les lésions induites dans l’ADN par les UV en raison d’un dysfonctionnement du mécanisme de réparation par excision de nucléotides (NER). Plusieurs groupes de complémentation ont été identifiés dans le syndrome XP, parmi lesquels le groupe XP-C représente la majorité des patients à travers le monde.Au cours de mon travail de thèse, j’ai développé un modèle de correction ciblée par recombinaison homologue (RH) d’une délétion de deux nucléotides au niveau de l’exon 9 du gène XPC aboutissant à l’apparition prématurée d’un codon stop. Afin de stimuler la RH, deux types de nucléases ingéniérées sont utilisées : les méganucléases et les TALENs. J’ai observé que la méthylation de la séquence ciblée pouvait affecter l’activité de celles-ci et donc l’efficacité du ciblage de gène. Cependant, deux approches ont été développées pour résoudre ce problème : l’utilisation d’un agent déméthylant (5-aza-2’-désoxycytidine (5azadC)) ou la création d’une endonucléase insensible à la méthylation. L’utilisation des méganucléases en combinaison avec la 5azadC a permis de stimuler la fréquence de coupure de presque 20 fois dans des fibroblastes XPC et la TALEN modifiée permet une augmentation de 40 fois. Avec ces deux stratégies j’ai obtenu des événements de correction génétique par introduction d’une matrice de réparation dans le locus ciblé avec une fréquence proche de 3%. La caractérisation des clones corrigés avec la TALEN XPC montre la correction génomique des deux nucléotides dans l’exon 9, une restauration de l’expression de la protéine XPC et une résistance cellulaire après irradiation UV traduisant le rétablissement des fonctions de la NER. Cette étude représente la première preuve de correction génétique de cellules déficientes en protéine XPC en utilisant une approche ciblée. / Xeroderma pigmentosum (XP) is a rare inherited genetic disorder characterized by an UV hypersensitivity and a severe predisposition to skin cancers. Cells from XP patients are deficient in nucleotide excision repair (NER) of UV‐induced DNA lesions. Several complementation groups have been identified in the XP syndrome and the XP-C group represents the majority of XP patients around the world. During my PhD work, I developed a model of targeted correction by homologous recombination (HR) in order to correct a deletion of two nucleotides in the ninth exon in XPC gene leading to a premature stop codon. To stimulate HR, I used two types of engineered endonucleases : meganucleases and TALEN. I observed that the target methylation status could affect the endonuclease activities and therefore XPC gene correction. Nervertheless, I developed two approaches to overcome this methylation sensitivity : use of a demethylating agent (5-aza-2-deoxycytidine (5azadC)) or a specific engineering of TALEN. Using 5azadC with meganuclease allowed to stimulate the cutting frequency by nearly 20 fold in XPC fibroblasts and the engineered TALEN allowed a 40 fold-increase in frequency. With both strategies I obtained genetic correction events by repair matrix introduction in the targeted locus with a near 3% frequency. The characterization of corrected clones with the XPC TALEN shows genomic correction in the ninth exon, a restoration of the XPC protein expression and cell survival following UV exposure, thus demonstrating fully recovered normal repair activity by NER. This study represents the first evidence of genetic correction of XPC-deficient cells by a targeted approach.

Bmi-1 é uma proteína do grupo Polycomb capaz de induzir atividade de telomerase, levando à imortalização de células epiteliais. As células, quando imortalizadas, se tronam mais susceptíveis a danos em dupla fita (double-strand breaks (DSB))e a recombinação homóloga é uma das duas vias de reparo dos DSBs. Dentre os genes reguladores da recombinação homóloga temos o BRCA-1, que está envolvido na resposta ao dano associado à proteína RAD51, que por sua vez se acumula rapidamente nos focos de dano ao DNA após a sinalização do H2AX, que têm se mostrado um excelente marcador de dano celular por se acumular rapidamente nos focos de lesão, desencadeando o processo de reparo. Topoisomerase III (TopoIII) remove intermediários da recombinação homóloga antes da segregação de cromossomos, prevenindo danos à estrutura do DNA celular. O papel das proteínas envolvidas na recombinação homóloga, em carcinomas ductais invasores positivos para o BMI-1, necessita ser investigado. Utilizando-se tissue microarrays contendo 239 casos de carcinomas ductais mamários primários, foi analisada a expressão imunoistoquímica de BMI-1, receptor de estrógeno, receptor de progesterona, HER-2, Ki67, p53 e BRCA-1, H2AX, RAD51 e topoisomerase III. Positividade para o Bmi-1 foi encontrada em 66 casos (27.6%). A positividade imunoistoquímica do BMI-1 relacionou-se a RE (p=0,004), RP (p<0,001), Ki-67 (p < 0,001), p53 (p=0,003), BRCA-1(p= 0,003), H2AX (p= 0,024) e TopoIII (p < 0,001). Concluindo, nossos resultados mostraram haver relação entre o BMI-1 e genes reguladores da HR, sugerindo que a positividade de BMI-1 pode ser um importante evento na recombinação homóloga em carcinomas ductais invasores da mama. / Bmi-1 is a Polycomb group protein which is able to induce telomerase activity, enabling the immortalization of epithelial cells. Immortalized cells shown more susceptible to double-strand breaks (DSB) and the homologous recombination (HR) are one of DSB repair pathways. Among the regulatory genes in HR, there is BRCA1, involved in the response to DNA damage associated with the RAD51 protein, which accumulates in DNA damage foci after signaling H2AX. H2AX has also been shown to be a good marker of DNA damage. Topoisomerase III (TopoIII) removes HR intermediates before the segregation of chromosomes, preventing damage to the structure of the cellular DNA. The role of proteins involved in HR, in breast carcinomas positive for BMI-1, remains to be investigated. The aim of this study was evaluate the association between BMI-1 and homologous recombination proteins. Using tissue microarrays containing 239 cases of primary breast tumors, the expression of Bmi-1, BRCA-1, H2AX, Rad51, p53, Ki-67, topoisomerase III, RE, RP and HER-2 was analyzed by immunohistochemistry. We observe high expression of Bmi-1 in 66 cases (27.6%). Immunohistochemistry overexpression of BMI-1 was related to RE (p=0,004), RP (p<0,001), Ki-67 (p < 0,001), p53 (p=0,003), BRCA-1(p= 0,003), H2AX (p= 0,024) and TopoIII (p < 0,001). Our results showed a relation between the expression of BMI-1 and HR regulatory genes, suggesting that overexpression of Bmi-1 is an important event in breast cancer homologous recombination.

Os micro-organismos são profícuas fontes de produtos naturais bioativos. Diversos fármacos de importância clínica são de origem microbiana, sendo que a maioria dos antibióticos usados clinicamente é produzida por actinobactérias, principalmente do gênero Streptomyces. A resistência a múltiplas drogas por microorganismos patogênicos e também pelas células tumorais leva à necessidade por novos fármacos antibacterianos e antitumorais. Actinobactérias endofíticas têm demonstrado grande potencial para a busca de produtos naturais bioativos. O presente trabalho relata o estudo químico de duas linhagens de actinobactérias endofíticas, Streptomyces sp. RTd 22 e Streptomyces sp RTd 31, isoladas das raízes de Tithonia diversifolia. As frações ativas nos ensaios biológicos foram fracionadas para a identificação dos compostos bioativos, sendo eles os antibióticos macrolídeos concanamicinas A (S31-1) e B (S31-2), anidro-agliconas das concanamicinas A (S31-3) e B (S31-4), todos produzidos por Streptomyces sp RTd31, e o ionóforo poliéter grisorixina (S22-2), produzido por Streptomyces sp. RTd22. Foi realizado o monitoramento da produção desses compostos bioativos por UPLC-MS através do modo SIM. As concanamicinas A e B tiveram um máximo de produção com 96h, já a grisorixina obteve um máximo com 192h. Outros compostos identificados por desreplicação dos extratos butanólicos de ambas as actinobactérias foram os sideróforos norcardamina (S31-7) e desoxi-nocardamina (S31-8), já o sideróforo desferrioxamina B (S31-9) foi identificado apenas nos extratos butanólicos de Streptomyces sp RTd31. Experimentos de variação do meio de cultivo e co-cultura com bactérias patogênicas foram empregados a fim de estimular a biossíntese de novos compostos, porém nenhum novo metabólito foi identificado. O sequenciamento genético da actinobactéria Streptomyces sp. RTd22 permitiu verificar a presença de vários clusters biossintéticos nesse micro-organismo através da análise feita pelo antiSMASH. Foi possível identificar o cluster da himastatina (S22-4) e da coeliquelina (S22-5), sendo que ambos os compostos não foram biossintetizados nas condições de cultivo utilizadas. O cluster biossintético da grisorixina foi determinado e o experimento de recombinação homóloga para a deleção do gene análogo a flavina mono-oxigenase da nigericina nigC foi realizado. Dois mutantes foram obtidos e um deles foi cultivado para a análise do perfil metabólico por espectrometria de massas. Não houve a produção da grisorixina nem do seu possível precursor pelo mutante, mas outros metabólitos foram produzidos / Microorganisms are prolific sources of bioactive natural products. Several clinically important drugs have microbial origin, and most of the therapeutically used antibiotics are produced by actinobacteria, mainly from the genus Streptomyces. The multidrug resistance observed in pathogenic microorganisms and tumor cells lead to the need for new antibacterial and antitumor drugs . Endophytic actinobacteria have shown great potential in the search for bioactive natural products. This work describes the chemical study of two endophytic actinobacteria strains: Streptomyces sp. RTd 22 and Streptomyces sp RTD 31, isolated from Tithonia diversifolia roots. Active fractions in biological assays were further fractionated for identifying the bioactive compounds, which are: the macrolide antibiotics concanamycins (S31-1) and B (S31-2), anhydrous aglycones of concanamycins A (S31-3) and B (S31-4), all four produced by Streptomyces sp. RTd31, and the ionophore polyether grisorixin (S22-2), produced by Streptomyces sp. RTd22. The production of these bioactive compounds was monitored by UPLC-MS via the SIM mode. Concanamycins A and B had maximum production at 96 h, and grisorixin at 192 h. Other compounds identified by the dereplication of buthanolic extracts of both actinobacteria were the siderophore norcardamine (S31-7) and deoxy-nocardamine (S31-8), the siderophores desferrioxamine B (S31-9) was identified only in buthanolic extracts of Streptomyces sp RTd31. Experiments varying media and co-culture were tested to stimulate the biosynthesis of novel compounds, but nothing new was identified. By genome sequencing of Streptomyces sp RTd22 and antiSMASH analysis it was possible to verify the presence of several biosynthetic clusters in the genome of this strain. It was possible to identify the biosynthetic clusters of himastatin (S22-4) and its analogous compound coelichelin (S22-5); however, these compounds were not biosynthesized in the culture conditions used. The grisorixin biosynthetic cluster was determined, and homologous recombination was performed for deleting the analogue gene of nigericin flavin monooxygenase nigCI. Two mutants were obtained, and one of them was cultured for analyzing its metabolic profile by mass spectrometry. There was no production of grisorixin or its possible precursor by the mutant, but others compounds were produced.

À l'heure actuelle, il y a un regain d'intérêt pour Clostridium acetobutylicum, le biocatalyseur du procédé Weizmann historique, pour produire le n-butanol un produit chimique de commodité et un bio-carburant alternatif et renouvelable . Ce mémoire de thèse décrit un procédé de recombinaison homologue, utilisant plasmide réplicatif, pour la délétion ou l'introdu ction de gènes chez C. acetobutylicum avec une élimination facile des marqueurs utilisés. La souche de C. acetobutylicum cacl502upp et ce système de recombinaison homologue ont été utilisés dans d'autres expériences d'ingénierie pour obtenir une souche produisant du n-butanol avec une sélectivité élevée et en éliminant la plupart des co-produits. Le mutant final, C. acetobutylicum (C. acetobutylicum CAB1060) a été généré avec succès. Cette souche CAB1060 a été utilisée dans un nouveau procédé de fermentation continu qui utilise i) l'extraction in situ des alcools par distillation sous pression réduite et ii) des cultures à haute densité cellulaire (et ne faisant pas intervenir de procédé membranaire) pour atteindre des titre, rendement et productivité en n-butanol qui n'ont jam ais été obtenus chez aucun micro-organisme.Un second procédé de recombinaison homologue utilisant un plasmide non réplicatif pour la modification de gène sans marqueur est également décrit dans le présent mémoire. Cette méthode permet d'inactiver simultaném ent deux gènes. Il a été utilisé avec succès pour la construction d'un mutant incapable de produire de l'hydrogène et utile, comme souche plate-forme, pour l'ingénierie de C. acetobutylicum pour produire en continu des produits chimiques de commodité et des bio­ carburants. / Current ly, there is a resurgence of interest in Clostridium acetobutylicum, the biocatalyst of the historical Weizmann process, to produce n-butanol for use both as a bulk chemical and as a renewablc alternative transportation fuel. This thesis describes a method of homologous recombination by replicative plasmid to delete or introduce genes in C. acetobutylicum . This method was successfull y used to delete genes, includin g CACJ502, CAC3535, CAC2879 (upp), to generate C. acetobutylicum. These strains are readily transformable without any previous plasmid methylation and can serve as hosts for a "marker-less" genetic exchange system. A mutant C. acetobutylicum (C. acetobuty licum CAB 1060) was successfully genera ted. This final mutant produces mainly bu tanol, with ethanol and traces of acetate at a molar rati o of 7:1 :1 . This CAB 1060 strain was subjected to a new continuous fermentation process using i) in situ extraction of alcohols by distillation under low pressure and ii) high cell density cultures to increase the titer, yield and productivity of n-butanol production to levels that have never been previously açhieved in any organism . A second homologous recombination method using non-replicative plasmid for marker less gene modification is also described in this thesis. This method allows the simultaneou s inactivation of two genes. lt has been successfully used to construct a mutant unable to produce hydrogen and useful, as a platform strain, for further engineering of C. acetobutylicum to continuously produce bulk chemicals and fuels.

Bmi-1 é uma proteína do grupo Polycomb capaz de induzir atividade de telomerase, levando à imortalização de células epiteliais. As células, quando imortalizadas, se tronam mais susceptíveis a danos em dupla fita (double-strand breaks (DSB))e a recombinação homóloga é uma das duas vias de reparo dos DSBs. Dentre os genes reguladores da recombinação homóloga temos o BRCA-1, que está envolvido na resposta ao dano associado à proteína RAD51, que por sua vez se acumula rapidamente nos focos de dano ao DNA após a sinalização do H2AX, que têm se mostrado um excelente marcador de dano celular por se acumular rapidamente nos focos de lesão, desencadeando o processo de reparo. Topoisomerase III (TopoIII) remove intermediários da recombinação homóloga antes da segregação de cromossomos, prevenindo danos à estrutura do DNA celular. O papel das proteínas envolvidas na recombinação homóloga, em carcinomas ductais invasores positivos para o BMI-1, necessita ser investigado. Utilizando-se tissue microarrays contendo 239 casos de carcinomas ductais mamários primários, foi analisada a expressão imunoistoquímica de BMI-1, receptor de estrógeno, receptor de progesterona, HER-2, Ki67, p53 e BRCA-1, H2AX, RAD51 e topoisomerase III. Positividade para o Bmi-1 foi encontrada em 66 casos (27.6%). A positividade imunoistoquímica do BMI-1 relacionou-se a RE (p=0,004), RP (p<0,001), Ki-67 (p < 0,001), p53 (p=0,003), BRCA-1(p= 0,003), H2AX (p= 0,024) e TopoIII (p < 0,001). Concluindo, nossos resultados mostraram haver relação entre o BMI-1 e genes reguladores da HR, sugerindo que a positividade de BMI-1 pode ser um importante evento na recombinação homóloga em carcinomas ductais invasores da mama. / Bmi-1 is a Polycomb group protein which is able to induce telomerase activity, enabling the immortalization of epithelial cells. Immortalized cells shown more susceptible to double-strand breaks (DSB) and the homologous recombination (HR) are one of DSB repair pathways. Among the regulatory genes in HR, there is BRCA1, involved in the response to DNA damage associated with the RAD51 protein, which accumulates in DNA damage foci after signaling H2AX. H2AX has also been shown to be a good marker of DNA damage. Topoisomerase III (TopoIII) removes HR intermediates before the segregation of chromosomes, preventing damage to the structure of the cellular DNA. The role of proteins involved in HR, in breast carcinomas positive for BMI-1, remains to be investigated. The aim of this study was evaluate the association between BMI-1 and homologous recombination proteins. Using tissue microarrays containing 239 cases of primary breast tumors, the expression of Bmi-1, BRCA-1, H2AX, Rad51, p53, Ki-67, topoisomerase III, RE, RP and HER-2 was analyzed by immunohistochemistry. We observe high expression of Bmi-1 in 66 cases (27.6%). Immunohistochemistry overexpression of BMI-1 was related to RE (p=0,004), RP (p<0,001), Ki-67 (p < 0,001), p53 (p=0,003), BRCA-1(p= 0,003), H2AX (p= 0,024) and TopoIII (p < 0,001). Our results showed a relation between the expression of BMI-1 and HR regulatory genes, suggesting that overexpression of Bmi-1 is an important event in breast cancer homologous recombination.

DNA double strand breaks (DSBs), which may occur during DNA replication or due to the action of genotoxic agents, are extremely dangerous DNA lesions as they can cause chromosomal rearrangements and cell death. Therefore, accurate DSB repair is vital for genome stability and cell survival. Two main mechanisms serve to repair DNA DSBs: non-homologous end joining, which re-ligates DNA ends together, and homologous recombination (HR), which restores broken DNA using homologous sequence as a template for repair. One-ended DSBs are a subject for the specialised HR-dependent repair pathway known as break-induced replication (BIR). At low frequency, DNA breaks can also be healed by telomerase, which normally extends telomeres at natural chromosome ends, but may also add de novo telomeres to DSBs due to their similarity to chromosome ends. De novo telomere addition is a deleterious event, which is effectively inhibited by the nuclear Pif1 (nPif1) helicase phosphorylated at the TLSSAES motif in response to DNA damage. In this study, it is reported that the same regulatory motif of nPif1 is also required for DSB repair via BIR. The requirement of the nPif1 TLSSAES sequence in BIR is dependent on the functional DNA damage response (DDR). Thus, nPif1 phosphorylation by the DDR machinery might mediate the role of nPif1 in BIR. In contrast, the nPif1 regulatory motif is not essential for BIR at telomeres in cells lacking telomerase. These observations indicate that the mechanism of nPif1 function in DSB repair via BIR and in BIR at telomeres might be different. In this work, a protocol for nPif1 pull-down was optimized to reveal the mechanism of the phosphorylation-dependent nPif1 functions in cells undergoing DNA repair, i. e. the mechanism of nPif1-mediated inhibition of de novo telomere addition and promoting DSB repair via BIR. In future, this protocol can be used to dissect the role of nPif1 in DNA repair through the identification of its potential interacting partners. The Srs2 helicase negatively regulates HR via dismantling Rad51 filaments. According to preliminary data from the laboratory of Sveta Makovets, Srs2 also promotes de novo telomere addition at DSBs in a Rad51-dependent manner. The work presented here establishes that Srs2 is dispensable for telomerase-mediated addition of TG1-3 repeats to DSBs. Instead, Srs2 is required for the reconstitution of the complementary DNA strand after telomerase action, thus ensuring the completion of de novo telomere addition. Overall, this study demonstrates that recombination-dependent DSB repair and de novo telomere addition share common regulatory components, i. e. the nPif1 helicase phosphorylated in response to DNA damage and the Srs2 helicase. Phosphorylated nPif1 promotes DSB repair via BIR in addition to its known role in inhibition of telomerase at DSBs, whereas Srs2 uses its well established ability to remove Rad51 from ssDNA to promote the restoration of dsDNA and thus to complete de novo telomere addition.

Afin de transmettre leurs informations génétiques d'une génération à l'autre, les organismes à reproduction sexuée doivent réduire de moitié leur contenu chromosomique pour former des gamètes haploïdes. Cette réduction se produit lors d'une division cellulaire appelée méiose, durant laquelle une étape de réplication est suivie de deux divisions successives, la méiose I et II. Au cours de la méiose I, les chromosomes homologues se séparent et leur bonne ségrégation dépend de la création entre eux d’un lien physique. En méiose c’est le processus de réparation appelé recombinaison homologue, qui à la suite de l’induction dans le génome de centaine de cassures double brin par la protéine Spo11, permet d’établir ce lien. Spo11 est l'orthologue méiotique de la sous-unité catalytique de la topoisomérase VI, TopoVIA. Comme TopoVI est composée de deux sous-unités, TopoVIA et TopoVIB, l’existence d’un orthologue méiotique de TopoVIB était une question posée depuis l'identification de Spo11. Au cours de ma thèse, j'ai contribué à identifier une nouvelle famille de protéine, que l’on a nommé TopoVIB-like, orthologue à TopoVIB et nécessaire à la formation des cassures double-brin d'ADN méiotiques(Robert et al, 2016). Ces protéines ont des domaines similaires à ceux de TopoVIB, à savoir un GHKL (impliqué dans la liaison et l'hydrolyse de l'ATP), un domaine transducteur et un domaine CTD. Nous avons démontré que chez la souris, SPO11 forme un complexe avec TOPOVIBL. De plus, nous avons démontré que cette protéine est nécessaire à la formation des CDB. Ces résultats suggèrent que chez la souris, les CDB méiotiques sont catalysées par un complexe TopoVI-like. Chez S. cerevisiae, il n'y a pas d'orthologue clair de TopoVIB, mais nous avons trouvé que la protéine Rec102, connue pour être nécessaire à la formation des CDB méiotiques, présente une homologie partielle avec le domaine transducteur des TopoVIB-like. Rec102 forme un complexe avec Rec104, une protéine également requise pour la formation des CDB. Ainsi, nous avons émis l'hypothèse que le complexe Rec102 / Rec104 était l'orthologue méiotique de TopoVIB chez la levure, interagissant avec Spo11 pour former un complexe de type TopoVI-like. Malgré l'importance de Spo11, son mode d'action est mal connu. Cette absence de données biochimiques est due à l’insolubilité de la protéine. Le but de ma thèse était de caractériser le mode d'action et la régulation du complexe TopoVI-like dans la formation des CDB méiotiques. Tout d'abord, biochimiquement, en purifiant in vitro une forme soluble du complexe TopoVI-like de levure composé de Spo11 / Rec102 / Rec104 / Ski8 (un partenaire direct de Spo11) en co-exprimant ces protéines dans deux systèmes d'expression, E. coli et S. cerevisiae. En utilisant E. coli, j'ai réussi à purifier un complexe soluble formé par Spo11 / Rec102 / Rec104 / Ski8 et en utilisant S. cerevisiae, j'ai purifié deux complexes différents, l'un formé par les quatre protéines, et un formé uniquement par Spo11 et Rec102. Néanmoins, les tests d'activité sur différents substrats d'ADN n'ont révélé aucune activité de coupure de l’ADN. Le deuxième objectif de ma thèse était d'étudier comment, chez la souris, TOPOVIBL régule l'activité de SPO11 en interagissant avec d'autres protéines nécessaires à la formation des CDB. En double hybride, j'ai prouvé que, comme chez la levure, l'orthologue méiotique de TopoVIB chez la souris interagissait avec REC114, une autre protéine nécessaire à la formation des CDB. La cartographie de cette interaction à l'échelle de l’acide aminé a conduit à l'identification d'un résidu sur TOPOVIBL essentiel pour l'interaction entre TOPOVIBL et REC114. Afin d'étudier in vivo le rôle de l'interaction entre TOPOVIBL et REC114, une souris mutante pour le résidu identifié de TOPOVIBL a été générée à l'aide de CRISPER-Cas9 et son phénotype a été analysé. / To properly transmit their genetic information from one generation to another, sexually reproductive organisms need to halve their genome to form haploid gametes. This reduction occurs during a special cell division called meiosis, which proceeds through one round of DNA replication followed by two successive divisions called meiosis I and II. During meiosis I homologous chromosomes segregate, and their proper segregation depends on the homologous recombination pathway that establishes a physical link between the homologues. During meiosis, homologous recombination events are triggered by the formation of DNA double strand break (DSB) catalyzed by the evolutionarily conserved Spo11 protein. Spo11 is the meiotic ortholog of the catalytic subunit of the TopoVI topoisomerase, TopoVIA. As TopoVI is composed of two subunits, TopoVIA and TopoVIB, the requirement for meiotic DSB formation of a B subunit was under investigation since the identification of Spo11. During my PhD, I contributed to the identification of a new family of protein, the TopoVIB-like family, ortholog to the Topoisomerase VI B subunit (TopoVIB) and required for meiotic DNA double strand break formation (Robert et al, 2016). These proteins share domains in part similar to the canonical TopoVIB which are a GHKL domain (involved in ATP binding and hydrolysis), a transducer domain and a CTD domain. We demonstrated that in mice, SPO11 forms a complex with TOPOVIBL. Biochemical characterization of this complex showed a structure compatible with an A2B2 organization. Furthermore, we demonstrated that this protein is required for meiotic DSB formation. These results suggest the existence, in mice, of a TopoVI-like complex that catalyzes the formation of meiotic DSB. In S. cerevisiae, there is no clear TopoVIB-like ortholog, but we found that the Rec102 protein, which is known to be required for the formation of meiotic DSB, shows a partial homology with the transducer domain of the TopoVIB-like proteins. Rec102 forms a complex with Rec104, a protein also essential for DSB formation. Thus, we hypothesized that the Rec102/Rec104 complex is the yeast meiotic ortholog of TopoVIB, interacting with Spo11 to form a meiotic TopoVI-like complex. Despite the importance of Spo11 little is known about its mode of action. This absence of biochemical data is due to the lack of solubility of the protein. The aim of my PhD was to characterize the mode of action and regulation of the TopoVI-like complex for meiotic DSB formation. First, biochemically, by purifying in vitro a soluble form of the yeast TopoVI-like complex composed by Spo11/Rec102/Rec104/Ski8. To achieve this objective, I co-expressed these proteins in two different expression systems, E. coli and meiotic culture of S. cerevisiae. Using E. coli I managed to purify a soluble complex formed by Spo11/Rec102/Rec104/Ski8, and using meiotic culture of S. cerevisiae, I purified two different complexes, one formed, by the four proteins, and one formed only by Spo11 and Rec102. Nevertheless, in vitro activity essays on different DNA substrates did not reveal any DNA cleavage activity. The second goal of my PhD was to study how in mouse, the activity of TOPOVIBL / SPO11 may be regulated by other proteins known to be required for DSB formation. Using Y2H experiment I was able to prove that, as in yeast, mouse TOPOVIBL interacts with REC114, a protein required for DSB formation. The mapping of this interaction at the amino-acid scale, leads to the identification of one residue on TOPOVIBL essential for the interaction between TOPOVIBL and REC114. In order to investigate in vivo the role of the interaction between TOPOVIBL and REC114, a mutant mouse carrying a mutation in the identified residue of TOPOVIBL was generated using CRISPER-Cas9, and its phenotype analyzed.

Os micoplasmas são os menores microrganismos capazes de autoreplicação conhecidos na natureza, responsáveis por uma série de doenças no homem e nos animais, infectando ainda plantas e insetos. Constituem um grande grupo de bactérias, ordenadas em diferentes gêneros na classe Mollicutes, cuja principal característica em comum, além do genoma reduzido, é a ausência de parede celular. Mycoplasma mycoïdes subsp. mycoïdes SC, responsável pela Pleuropneumonia Contagiosa Bovina, foi o primeiro microrganismo desta classe de bactérias a ser identificado. Esta é uma doença bastante grave, com altas taxas de morbidade e mortalidade. A variedade Mycoplasma mycoïdes subsp. mycoïdes LC é responsável principalmente por casos de Pleuropneumonia Contagiosa Caprina, mastite no gado bovino, e ainda artrite em ovinos e caprinos em menor extensão. M. capricolum é um patógeno caprino, responsável principalmente por casos de artrite com grande importância econômica na medicina veterinária. M. pulmonis é um patógeno de roedores, considerado como o melhor modelo experimental para o estudo das micoplasmoses respiratórias. M. genitalium, o menor microrganismo conhecido capaz de se autoreplicar, é um patógeno humano responsável por casos de uretrite não gonocócica, cujo seqüenciamento completo do cromossomo tornou-se um marco na era da genômica. O estudo funcional do genoma destes micoplasmas, para a compreensão de sua biologia e patogenicidade, requer o desenvolvimento de ferramentas genéticas eficientes. No presente trabalho, análises in silico das seqüências na região das prováveis origens de replicação cromossômica (oriC) destes micoplasmas demonstraram a existência de possíveis DnaA boxes localizados em torno do gene dnaA. Estas regiões oriC foram caracterizadas funcionalmente após sua clonagem em vetores artificiais e a transformação dos micoplasmas com os plasmídeos recombinantes resultantes. O plasmídeo pMPO1, contendo a região oriC de M. pulmonis, sofreu integração no cromossomo do micoplasma por recombinação homóloga após poucas passagens in vitro. A redução desta oriC para o fragmento contendo somente os DnaA boxes localizados nas estremidades 5´ou 3´do gene dnaA não foi capaz de produzir plasmídeos replicativos em M. pulmonis, exceto quando estes dois fragmentos foram clonados no mesmo vetor, espaçados pelo determinante de resistência à tetraciclina tetM. Um fragmento interno do gene da hemolisina A (hlyA) de M. pulmonis foi clonado nestes plasmídeos oriC, e os vetores resultantes foram utilizados para transformar o micoplasma. A integração destes vetores por um crossing-over com o gene hlyA, causando a sua dirupção, foi documentada. Deste modo, estes plasmídeos oriC podem vir a se tornar ferramentas genéticas valiosas para o estudo do papel de genes específicos, notadamente aqueles potencialmente envolvidos na patogênese. / Mycoplasmas are the smallest microorganisms capable of self replication known to date, responsible for many diseases in man and animals, infecting also plants and insects. They constitute a large group of bacteria, classified in different genera in the class Mollicutes, which main common characteristic, besides the small genome, is the absence of a cell wall. Mycoplasma mycoïdes subsp. mycoïdes SC, responsible for the Bovine Contagious Pleuropneumonia, was the first microorganism of this class of bacteria to be identified. That is a quite severe disease, with high morbidity and mortality rates. Mycoplasma mycoïdes subsp. mycoïdes LC is responsible mainly for cases of Caprine Contagious Pleuropneumonia, mastitis in cattle, and also arthritis in goats and sheep in less extension. M. capricolum is a pathogen of goats, responsible mainly by cases of arthritis with large economic impact in veterinary medicine. M. pulmonis is a rodent pathogen, considered to be the best experimental model for studying respiratory mycoplasmoses. M. genitalium, the smallest microorganism capable of self replication, is an human pathogen responsible for cases of non gonococcal urethritis, which complete chromosome sequencing has become a benchmark in the era of genomics. Functional studies of these mycoplasma genomes, for comprehension of their biology and pathogenicity, requires the development of efficient genetic tools. In the present work, in silico analysis of sequences of the putative origin of chromosome replication (oriC) region of these mycoplasmas demonstrates the existence of putative DnaA boxes located around the dnaA gene. These oriC regions were functionally characterized after cloning into artificial vectors and transformation of mycoplasmas with the resulting recombinant plasmids. The plasmid pMPO1, which contains the M. pulmonis oriC region, has integrated into the mycoplasma chromosome by homologous recombination after a few in vitro passages. Reduction of this oriC to the fragment containing only the DnaA boxes located upstream or downstream the dnaA gene could not produce plasmids able to replicate in M. pulmonis, except when these two fragments were cloned in the same vector, spaced by tetracycline resistance gene tetM. An internal fragment of the M. pulmonis hemolysine A gene (hlyA) was cloned into these oriC plasmids, and the resulting vectors were used to transform the mycoplasma. Integration of these disruption vectors by one crossing-over with the hlyA gene could be documented. Therefore, these oriC plasmids may become valuable genetic tools for studying the role of specific genes of mycoplasmas, specially those potentially involved in pathogenesis.

<p>The aim of this thesis was to investigate the cellular role of the mouse RBBP6 gene using the interference RNA (RNAi) gene targeting technology and also to understand the relevance of two promoters for the RBBP6 gene.</p>