In eukaryotes, cell proliferation and cell cycle transitions are strictly controlled by the anaphase-promoting complex/cyclosome (APC/C). The APC/C is an E3 ubiquitin ligase that regulates chromatid segregation at the metaphase to anaphase transition, exit from mitosis and the establishment and maintenance of G1. The APC/C’s catalytic activity and substrate specificity are controlled by its interactions with two coactivators, Cdc20 and Cdh1. In contrast to Cdh1, APC/C activation by Cdc20 during mitosis requires hyper-phosphorylation of APC/C subunits by cyclin-dependent kinase (Cdk) and polo kinase. The aim of the first part of this thesis was to understand how mitotic phosphorylation regulates APC/C activity. Using cryo-electron microscopy and biochemical analysis, we found that an auto-inhibitory segment of the Apc1 subunit acts as a molecular switch that in apo unphosphorylated APC/C interacts with a coactivator-binding site (C-box binding site), thereby obstructing engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box binding site to relieve APC/C auto-inhibition. Efficient phosphorylation of the auto-inhibitory segment requires the recruitment of the kinase Cdk-cyclin-Cks to a hyper-phosphorylated loop of Apc3. In addition to regulation of APC/C activity by phosphorylation, ordered cell progression is ensured by the ability of the APC/C to target substrate degradation in a defined order. At mitosis onset, degradation of securin and cyclin B1 is inhibited by the spindle assembly checkpoint, exerted by the mitotic checkpoint complex (MCC), whereas both cyclin A2 and Nek2A are not subject to MCC inhibition. The aim of the second part of the thesis was to elucidate the mechanism of how the APC/C achieves its substrate specificity. Our biochemical analysis showed that the resistance of cyclin A2 to MCC inhibition is due to its ABBA motif and the Cdk-associated Cks2 subunit. Furthermore, we found that it is the Cdc20 molecule of the MCC that binds to the ABBA motif to allow for cyclin A2 ubiquitination. Strikingly, mutating all three known degrons (KEN box, D box and ABBA motif) of cyclin A did not affect its ubiquitination by APC/CCdc20. Deletion of a potential novel degron found within residues 60-80 of cyclin A2 impaired cyclin A2 ubiquitination.

Psidium cattleianum Sabine, also known as strawberry guava, is a Brazilian native species whose fruits are valued for a high level of vitamin C, which are consumed in natura. Besides, it is also a species used for medicinal purposes. The use of plants as a therapeutic resource is a widely spread practice in popular medicine worldwide, although there have been few studies about their chemical composition as well as their potential risks to human health. Besides, the germplasm characterization of a species, as the pollen viability determination for instance, is important in maintaining a database that may be useful for future research. Considering that some plants cause adverse effects, the purpose of the present study was to analyze the antiproliferative and genotoxic effect of strawberry guava juice and strawberry guava leaf aqueous extracts by Allium cepa test system. This study also aimed at analyzing strawberry guava pollen grains through different colorimetric techniques so as to assess their viability. Fruits from three accessions were used for preparing juice at the concentration of 125 g.L-1, and dried leaves from four accessions were used for preparing aqueous extracts at the concentrations of 15 g.L-1 and 75 g.L-1. Distilled water was used as negative control, whereas glyphosate served as positive control. Inhibition of cell division as well as the occurrence of chromosomal abnormalities in the meristematic cells of Allium cepa roots were also analyzed. In order to identify the phenolic compounds, juice and aqueous extract samples were analyzed by high performance liquid chromatography (HPLC). The pollen viability of twenty accessions was determined by two colorimetric methods. The statistical analysis was carried out using Chi-square and Scott-Knott tests (p < 0,05). The analysis of genotoxicity showed that the strawberry guava juice induced cell proliferation and chromosomal abnormalities, besides inhibiting the formation of the mitotic spindle. The extracts prepared with leaves from four strawberry guava accessions caused a decrease in cell proliferation, however, only the extracts prepared with leaves collected in Cerro Largo (CL) induced chromosomal alterations. The following phenolic compounds were identified in the composition of the strawberry guava juice and the leaf aqueous extracts: gallic acid, catechin, chrologenic acid, caffeic acid, ellagic acid, epicatechin, rutin, quercitrin, isoquercitrin, quercetin and kaempferol. The pollen viability determined through 2% acetic orcein was higher than 97,9%, and between 43% and 97% when determined through Alexander s stain. One concluded that the strawberry guava juice has proliferative, genotoxic and anti-mitotic action upon Allium cepa. Moreover, the leaf aqueous extracts produce an antiproliferative effect, whereas the accession collected in Cerro Largo is genotoxic, which may be valid to other types of eukaryotic cells. The most predominant phenolic compounds identified in the strawberry guava juice were epicatechin (two accessions) and isoquercitrin (one accession). Quercitrin was the most predominant compound in the leaf aqueous extracts from one accession, and quercetin was the most predominant one in the remaining accessions. One also concluded that the Alexander s stain was more accurate in regard to the actual pollen viability mainly due to its two stains (green malachite and fuchsine). / Psidium cattleianum Sabine, também conhecido como araçá, é uma espécie nativa do Brasil, valorizada pelo alto teor de vitamina C em seus frutos, que são consumidos in natura. Além disso, também é uma espécie utilizada com fins medicinais. O uso de plantas como recurso terapêutico é uma prática bastante difundida na medicina popular mundial e, apesar dessa generalidade da fitoterapia, são poucos os estudos sobre a composição química das plantas, bem como sobre os seus potenciais riscos à saúde da população. Além disso, a caracterização de germoplasma de uma espécie, a exemplo da determinação da viabilidade polínica, é importante na manutenção de um banco de informações que podem ser úteis em trabalhos futuros. Tendo em vista que algumas plantas apresentam efeitos adversos, o presente trabalho objetivou analisar o efeito antiproliferativo e genotóxico do suco dos frutos de P. cattleianum e do extrato aquoso de suas folhas usando o sistema-teste de Allium cepa L. (cebola). Ademais, o presente também objetivou realizar a análise de seus grãos de pólen e estimar sua viabilidade através de diferentes técnicas de coloração. Frutos de três acessos foram utilizados no preparo dos sucos na concentração de 125 g.L-1, e folhas secas de quatro acessos foram utilizadas na preparação dos extratos aquosos em duas concentrações diferentes: 15 g.L-1 e 75 g.L-1, sendo utilizada a água destilada como controle negativo e o glifosato como controle positivo. Foi analisada a inibição da divisão celular e também a presença de danos cromossômicos nas células meristemáticas de raízes de A. cepa. Para a determinação dos compostos fenólicos, as amostras dos sucos e dos extratos aquosos foram analisadas por cromatografia líquida de alta eficiência (CLAE). A viabilidade polínica de vinte acessos de P. cattleianum foi determinada usando dois métodos de coloração. A análise estatística foi realizada pelos testes Qui-quadrado e Scott-Knott (p < 0,05). A análise da genotoxicidade mostrou que o suco de araçá induziu a proliferação celular e alterações cromossômicas, além de inibir a formação do fuso mitótico em A. cepa. Os extratos preparados com as folhas dos quatro acessos reduziram a proliferação celular, no entanto, apenas o extrato das folhas coletadas no município de Cerro Largo (CL) induziu alterações cromossômicas. Os sucos e os extratos aquosos das folhas apresentaram na sua constituição os compostos fenólicos: ácido gálico, catequina, ácido clorogênico, ácido cafeico, ácido elágico, epicatequina, rutina, quercitrina, isoquercitrina, quercetina e canferol. A viabilidade polínica determinada pela orceína acética 2% foi superior a 97,9%, enquanto que com o reativo de Alexander variou entre 43% e 97%. Conclui-se que o suco de araçá possui efeito proliferativo, genotóxico e antimitótico em A. cepa, enquanto que os extratos aquosos são antiproliferativos e o acesso CL é genotóxico, o que pode ser extrapolado para outros tipos celulares eucarióticos. Os compostos fenólicos majoritários no suco são de epicatequina (dois acessos) e isoquercitrina (um acesso). Quercitrina é o composto fenólico predominante no extrato aquoso das folhas de um acesso e quercitina nos demais. Conclui-se também que o reativo de Alexander foi mais fiel à viabilidade real, principalmente devido à dupla coloração oferecida por esse corante (verde de malaquita e fucsina ácida).

For the first time in human history, more people worldwide suffer from obesity than are undernourished. Numerous health complications are associated with obesity including cardiovascular disease, Type 2 Diabetes, cancers of reproductive tissues, stroke, depression, anxiety disorders, and Alzheimer’s disease. A deeper understanding of the anti-adipogenic effects and mechanism of action of sesquiterpene lactones may have pharmacological import in the continuing search for therapeutic modalities to ameliorate the effects of this global obesity epidemic. Dehydroleucodine (DhL), 11,13-dihydro-dehydroleucodine (DH-DhL), and dehydroparashin-B (DhP), sesquiterpene lactones extracted from or derived from compounds extracted from Artemisia douglasiana, were investigated for their anti-adipogenic effects on 3T1-L1 preadipocytes. Dehydroleucodine inhibited the expression of C/EBPa and PPARg, and also strongly blocked the expression of C/EBPβ, an early stage biomarker of early adipogenesis, in a concentration-dependent manner. Dehydroleucodine arrested the cell cycle at the G0/G1 phase, increased p27 and decreased both cyclins A and D and their partners (e.g., CDK2 and CDK4). Furthermore, DhL downregulated expression of histone demethylase JMJD2 as well as repressed the expression of histone methyltransferase MLL4, which in turn diminished the expression of C/EBPb and PPARg, respectively. 11,13-dihydro-dehydroleucodine blocked the accumulation of lipid droplets and inhibited the expression of PPARγ and C/EBPβ. Collectively, the results indicate that the inhibition of early stage preadipocyte differentiation by DH-DhL may be associated with cell cycle arrest at the G0/G1 phase. Dehydroparashin-B significantly decreased the accumulation of lipid content and downregulated the expression of CEBPβ, PPARγ and CEBPα as well as FAS. Interestingly, the addition of DhP inhibited the number as well as the size of the lipid droplets during the differentiation of 3T3-L1 preadipocytes. Taken together, this data suggests that DhP has an important inhibitory effect on cellular pathways regulating adipocyte differentiation.

Nous étudions une nouvelles familles d'inhibiteurs de kinase: les benzopyridoindole. Ces molécules ont des effets antiprolifératifs sur des lignées cancéreuses et représentent les têtes de série de possibles agents anti-cancéreux. We study on a new family of kinase inhibitors: benzopyridoindole. These molecules have antiproliferative effects on cancer cell lines and represent the lead of potential anti-cancer products. / Benzo[e]pyridoindoles are novel potent inhibitors of aurora kinases. We performed a SAR study to improve their activity and water solubility. Amino-benzo[e]pyridoindolones were found to be potent hydrosoluble anti-proliferative molecules. They induced a massive arrest in mitosis, prevented histone H3 phosphorylation as well as disorganizing the mitotic spindles. Upon a delay, cells underwent binucleated and finally died. Taking into account their interesting preclinal characteristics, their efficiency towards xenografts in nude mice and their apparent safety in animals, these molecules are promising new anti-cancer drugs. They probably target a metabolic signaling pathway, besides aurora B inhibition. In addition to their possible applications, these inhibitors are tools for cell biology studies. C4, a low ATP affinity inhibitor of aurora B kinase, revealed that the basal activity of the kinase is required for histone H3 phosphorylation in prophase and for chromosome compaction in anaphase. These waves of activation/deactivation of the kinase, during mitosis, corresponded to different conformations of the passenger chromosomal complex.

Com o objetivo de se caracterizar o processo de recombinação somática em Trichoderma pseudokoningii foram feitos cruzamentos via anastomose de hifas entre duas linhagens contrastantes para quatro marcadores de auxotrofia, coloração dos conídios e marcadores de RAPD. Foram feitos quatro cruzamentos, sendo analisados um total de 1052 colônias obtidas a partir de suspensões de conídios provenientes das colônias heterocarióticas. Sessenta e oito colônias recombinantes foram analisadas quanto às marcas de auxotrofia em quatro gerações de crescimento, sendo observado que 58 mantiveram o fenótipo recombinante, enquanto que as colônias restantes reverteram para um dos parentais. A maioria das colônias recombinantes se mostrou instável. Entretanto, após 4 gerações de crescimento estas colônias se tornaram estáveis para as marcas de auxotrofia avaliadas. As colônias recombinantes instáveis apresentaram bordas de crescimento irregular, esporulação esparsa e a freqüente formação de setores. Estas colônias recombinantes foram analisadas quanto aos marcadores RAPD, tendo mostrado grande similaridade, em relação ao perfil de bandas apresentado, com a maioria dos primers analisados. Somente com um primer foi possível visualizar a presença de uma banda polimórfica entre os recombinantes e a presença de bandas nos parentais não existentes em alguns recombinantes. Cinco colônias recombinantes foram analisadas quanto ao perfil de bandas cromossomais (PFGE), tendo sido observado que 2 colônias apresentaram padrões cromossomais igual a um dos parentais e 3 colônias apresentaram padrões recombinantes. Nos estudos citológicos verificou-se a formação de conídios uninucleados na conidiogênese, e a presença de conídios verdes maduros multinucleados, devido a prováveis divisões nucleares durante o processo de maturação dos conídios. Observou-se durante a formação dos heterocários a ocorrência de anastomoses e a passagem de núcleos, tendo sido observado a presença de núcleos com várias conformações, sugerindo um movimento ativo dos mesmos. Os resultados acima sugerem a ocorrência de mecanismos de recombinação no heterocário (recombinação somática), diferentes daqueles descritos para o ciclo parassexual ou parameiose, sendo proposto a ocorrência da degradação, no heterocário, dos núcleos de um dos parentais envolvidos nos cruzamentos (parental não prevalente) e a incorporação de segmentos destes em núcleos íntegros do parental prevalente. Se estes eventos realmente estiverem ocorrendo, sugere-se que estes sejam devido a possíveis reações limitadas de incompatibilidade vegetativa, ocasionando processos de lise e morte celular em algumas regiões do micélio heterocariótico. / To understand the somatic recombination process in Trichoderma pseudokoningii, auxotrophic complementary mutant strains were used to produce 4 heterokaryons. These strains were contrasting for four auxotrophic markers, conidia colors and for some RAPD markers. It was analyzed a total of 1052 colonies obtained from conidial suspensions of the heterokaryotic colonies. Stability of auxotrophic markers was evaluated in 68 recombinant colonies after four growing generations. In this analysis, 58 colonies kept the recombinant phenotype, while 10 reverted to one parental strain. Most of the recombinant colonies were initially unstable, but after at least 4 growing generations these recombinants became stable for auxotrophic markers. The unstable recombinant colonies showed irregular growing borders, sparse sporulation and frequent sector formation. The recombinant colonies were analyzed by RAPD technique. These colonies showed high similarity for the most of used primers. However, one primer showed a polymorphic band and some recombinants missing bands observed in parental strains. Chromosomal band profile of 5 recombinants and two parental strains were analyzed by Pulsed Field Gel Electrophoresis technique (PFGE). Two recombinants showed parental profiles and 3 showed recombinant profiles, respectively. In cytological studies of the conidiogenesis was observed the formation of only uninucleated conidia. However, presence of multinucleated mature green conidia was evident, probably due to nuclear divisions in course of maturing process of the conidia. During the process of heterokaryotic mycelium formation was possible to observe the occurrence of anastomosis that showed nuclear transfer. The presence of nuclei in several conformations was observed at the different regions of the heterokaryon, suggesting an active movement. The results presented in this study suggest the occurrence of recombination mechanisms in the heterokayon (somatic recombination), different from those described in classic parasexual cycle or parameiosis. Thus, it was proposed that may occur during this recombinant process the degradation of nuclei from one parental (non-prevalent parental) in the heterokaryon, and that the resulting chromosomal fragments may be incorporated into whole nuclei of the another parental (prevalent parental). If this natural transformation is occurring during this recombination process could be suggested that this event is due to a limited incompatible vegetative reactions, generating cellular lyses and death in some regions of the heterokaryotic mycelium.

La mitose assure la répartition égale du génome. La kinase mitotique Aurora B y joue un rôle majeur en contrôlant la fidélité de la ségrégation des chromosomes de par sa localisation aux centromères et aux microtubules, qui nécessite son ubiquitination par CUL3. Cependant, le mécanisme conduisant la forme ubiquitinée d’Aurora B sur ces structures mitotiques reste à déterminer. Dans ce contexte, j’ai pu identifier la protéine UBASH3B, qui contient un domaine de liaison à l’ubiquitine (UBD) comme un régulateur essentiel de la ségrégation chromosomique, agissant comme un récepteur de l’ubiquitine pour Aurora B. UBASH3B interagit directement avec Aurora B et cette interaction est dépendante de la modification d’Aurora B par l’ubiquitine ainsi que de CUL3. UBASH3B ne régule pas le niveau d’expression d’Aurora B. En revanche, UBASH3B se localise aux fuseaux mitotiques et est à la fois nécessaire et suffisant pour transférer Aurora B aux microtubules. De plus, la redistribution d’Aurora B des centromères vers les microtubules contrôle le déroulement et la fidélité de la ségrégation des chromosomes et donc le contenu correct du matériel génétique des cellules. Ainsi, mes résultats expliquent comment la modification par l’ubiquitine régule la localisation et la fonction d’Aurora B, reliant une voie de signalisation impliquant un récepteur à l’ubiquitine à la mitose. / Mitosis ensures equal segregation of the genome. The major mitotic kinase Aurora B controls fidelity of chromosome segregation by its localization to centromeres and microtubules, which requires CUL3-mediated ubiquitylation. However, it remains unknown how ubiquitylated Aurora B is targeted to mitotic structures. Here, I identify ubiquitin-binding domain (UBD) protein UBASH3B that critically regulates chromosome segregation, acting as ubiquitin receptor for Aurora B. UBASH3B directly binds Aurora B, and this interaction is dependent on CUL3 and on ubiquitin recognition. UBASH3B does not regulate protein levels of Aurora B. Instead, UBASH3B localizes to the mitotic spindle and is both required and sufficient to transfer Aurora B to microtubules. Moreover, redistribution of Aurora B from centromeres to microtubules controls timing and fidelity of chromosome segregation and thereby euploidy of cells. Thus, my findings explain how ubiquitin attachment regulates localization and function of Aurora B, linking receptor-mediated ubiquitin signaling to mitosis.

O principal objetivo deste trabalho é o de estabelecer uma estratégia confiável relacionada a genotoxicidade, proteção genômica e recombinação mitótica em Aspergillus nidulans. A genotoxicidade foi induzida por vapor de benzeno e, para testar a proteção genômica, a soja foi usada devido às suas propriedades anticarcinogênicas muito conhecidas. O principal resultado obtido foi que a soja transgênica mostrou maiores propriedades antigenotóxicas do que a soja tradicional. Isto foi duplamente confirmado, tanto através de estratégias de genética clássica, como molecular. Além disso, cada experimento foi repetido três vezes. Principais conclusões foram as seguintes: a) estabelecimento de um protocolo adequado para atender às complexidades dos eventos epigenéticos e, b) a aplicação desse tal protocolo e experimentos afins para testar outros agentes ambientais suspeitos de apresentarem propriedades antigenotóxicas, ou que precisem de sua identificação como tal. Evidentemente, levando-se em conta a grande preocupação relacionada aos alimentos transgênicos e aos muitos produtos de biotecnologia, que entram no mercado, o elenco de prováveis candidatos aos tipos de testes apresentados, será muito grande. Como os resultados obtidos sugerem íntima relação entre metilação de DNA eucariótico, a recombinação mitótica e outros eventos danosos às células, os eventos envolvidos serão epigeneticamente discutidos. / Main aim is to provide a reliable approach to deal with the aspects related to induced genotoxicity, genomic protection and eukaryotic mitotic recombination in Aspergillus nidulans. Genotoxicity was benzene fumes induced, and to test genomic protection soybean has been used on account of its putative anticarcinogenic properties. Main outcome is that transgenic soybean bears higher antigenotoxic properties than traditional soybean. This has been twofold confirmed through basic genetic approaches and molecular approaches, as well. In addition, each experimental approach has been three times repeated. Additional important outcomes are: a- establishment of a reliable protocol to deal with the complexities of the epigenetic events, and b- likely use of present protocol and experimental set-up to test other environmental agents of which antigenotoxic properties are either suspected, or need precise identification. Evidently, on account of present wide concern the transgenic foodstuffs, and many other biotechnological products, as well, are obvious candidates for similar approaches. Obtained results suggest close relationships among eukaryotic DNA methylation, mitotic recombination, and other cells damaging events reputedly leading to carcinogenesis.

Des stress réplicatifs sont rencontrés à chaque phase S du cycle cellulaire et différents mécanismes permettent leur prise en charge. La recombinaison homologue (RH) tient un rôle important dans le maintien de la stabilité du génome au cours de la réplication. En effet, la RH escorte la progression des fourches et prévient les défauts mitotiques. Toutefois, le lien moléculaire entre le stress réplicatif et les défauts mitotiques n’est pas élucidé. De façon générale, les fourches de réplication bloquées peuvent être sauvées grâce à leur fusion avec la fourche convergente ou au redémarrage de fourche par la RH. Le laboratoire a développé un essai génétique reposant sur l’utilisation d’une barrière de réplication conditionnelle afin d’étudier le mécanisme par lequel la RH contribue au sauvetage des fourches de réplication bloquées. L’équipe a montré que le redémarrage des fourches bloquées par la RH est conditionné par l’exposition d’un ADNsb et non d’une cassure double-brin. Ainsi, les fonctions de la RH au cours de la gestion du stress réplicatif peuvent être adressées indépendamment de sa fonction de réparation des cassures (fonction relativement bien documentée). Les travaux décrits dans ce manuscrit s’inscrivent autour des mécanismes que la RH engage afin d’assurer la stabilité génétique en réponse au stress réplicatif. Plus précisément, je me suis intéressée à l’implication des facteurs de la RH dans la protection des fourches de réplication bloquées au niveau moléculaire et cellulaire. En absence de la recombinase Rad51 ou de son chargeur Rad52, le blocage d’une seule fourche de réplication est suffisant pour induire des défauts mitotiques, incluant des ponts anaphasiques (lien physique entre chromatides sœurs). Il s’avère que les fourches bloquées sont extensivement dégradées par la nucléase Exo1 en absence de Rad51/Rad52. De manière intéressante, l’accumulation excessive d’ADNsb à la fourche est à l’origine de la non-disjonction des chromatides sœurs en mitose et ce malgré l’arrivée de fourches convergente. Ainsi, les fourches de réplication non protégées sont le siège de terminaison pathologique mettant à mal la ségrégation des chromosomes. La RH étant impliquée dans la protection et le redémarrage des fourches de réplication, l’utilisation du mutant de séparation de fonction Rad51-3A a permis de montrer que ces deux fonctions sont génétiquement séparables. Les fourches de réplication protégées et incapables d’être redémarrées par la RH ne présentent pas de symptômes de terminaison pathologique. Ainsi, au-delà de sa capacité à redémarrer les fourches inactivées, les facteurs de la RH assurent la complétion de de la réplication en maintenant les fourches de réplication dans une conformation propice à une fusion avec la fourche convergente. Ces résultats contribuent à une meilleure compréhension des mécanismes moléculaires invoqués par la RH afin de maintenir la stabilité génétique au cours du stress réplicatif. / At each cell cycle, cells undertaking the DNA replication process face several sources of replication stress (RS) compromising the progression of the replicating forks and threatening both chromosome duplication fidelity and their correct segregation during mitosis. Replication stresses emerged as a major source of genetic instability and cancer development. Several mechanisms, among which homologous recombination (HR), operate to buffer the deleterious effects of RS. HR acts as an escort to fork progression and prevents mitotic defects. Nonetheless, the molecular connection between replication stress and mitotic defects remains elusive. A conditional replication fork barrier (RFB) acting in a polar manner was developed in the lab to terminally-arrest fork progression. In this system, HR functions handling replication stress can be assessed independently of its well-known function in double strand break repair. The work described here aims to understanding the mechanism that HR performs to ensure genetic stability in response to replication stress. In general, blocked replication forks can be rescued either by fork convergence or by active HR-mediated fork restart. However, in absence of Rad51 recombinase or it loader Rad52, a single activated RFB is sufficient to induce mitotic abnormalities including anaphase bridges. The involvement of HR factors in fork protection was explored at the molecular and cellular levels. It turns out that terminally-arrested forks are extensively resected by the Exo1 nuclease in the absence of Rad51/Rad52. Interestingly, the excess of ssDNA accumulation at the fork triggers sister chromatid non-disjunction in mitosis despite the arrival of an uncorrupted converging fork to rescue replication. Thus, unprotected replication forks are prone to pathological termination threatening chromosome segregation. HR being involved in fork protection and restart, the use of a Rad51 mutant showed that these two functions are genetically separable. Indeed, protected forks unable to restart by HR do not show any pathological termination. Thus, beyond their ability to restart inactivated forks, HR factors ensure replication completion by maintaining the forks in a suitable conformation for a fusion with the converging fork. Overall, these results shed light on the molecular events engaged by RH to ensure genome stability in response to replication stress.

Le cancer du sein touche une femme sur neuf dans le monde et constitue un problème majeur de santé publique. L’identification de nouveaux biomarqueurs pour un traitement personnalisé pour les tumeurs du sein de plus mauvais pronostic, dites « triple-négatives », est extrêmement urgent. ATIP3, le produit majeur du gène candidat suppresseur de tumeurs MTUS1, a été identifié par l’équipe comme étant un biomarqueur des tumeurs du sein les plus agressives. De plus, ATIP3 inhibe la prolifération et la migration in vitro, ainsi que la progression tumorale et la formation de métastases in vivo et constitue une cible thérapeutique. ATIP3 est une protéine associée aux microtubules (MT) en interphase et au fuseau mitotique durant la mitose. Mon projet de thèse a pour objectif principal d’identifier les partenaires d’interaction d’ATIP3 impliqués dans ses mécanismes d’action antitumoraux. Dans une première partie, j’ai montré qu’ATIP3 interagit avec EB1, une protéine majeure de la dynamique du MT. L’interaction ATIP3-EB1 diminue l’accumulation d’EB1 à l’extrémité croissante du MT. Un nouveau mécanisme a été proposé dans lequel l’interaction ATIP3-EB1 réduit indirectement la vitesse d’échange d’EB1 à son site de liaison au bout plus du MT, ayant pour conséquence une diminution de la dynamique du MT. Dans une deuxième partie, j’ai montré qu’une déplétion d’ATIP3 induit une réduction de la taille du fuseau mitotique. Une analyse protéomique a permis d’identifier la kinésine Kif2A comme partenaire d’interaction d’ATIP3. ATIP3 forme un complexe avec Kif2A et Dda3 qui est dépendant d’une phosphorylation par Aurora kinase A. ATIP3 maintient la taille du fuseau en diminuant le recrutement de Kif2A et Dda3 au pôle de façon dépendante d’AurKA. ATIP3 régule donc négativement ses partenaires d’interaction. Enfin, dans une troisième partie, la relevance clinique du couple ATIP3-EB1 a été évaluée et j’ai montré que l’expression combinée des deux biomarqueurs ATIP3 et EB1 était associée à l’agressivité de la tumeur et à une survie diminuée. Ainsi, l’ensemble de mes travaux a permis de mettre en évidence de nouvelles cibles thérapeutiques afin de mettre en place des traitements personnalisés / Breast cancer is a leading cause of death by malignancy in women worldwide. The identification of new molecular markers for personalized treatment of poor prognosis breast tumors, such as those of the triple negative subtype, is urgently needed. Our team is leader in the study of ATIP3 protein, encoded by candidate tumor suppressor gene MTUS1. ATIP3 is down-regulated in 85% of triple negative breast tumors, and low levels of ATIP3 are associated with poor survival of the patients. We have shown that ATIP3 reduces proliferation and migration in vitro, and tumor growth and metastasis formation in vivo. ATIP3 localizes along the microtubule (MT) in interphase and on the mitotic spindle and spindle poles during mitosis. My PhD project aimed at identifying ATIP3 partners involved in its anti-tumoral effects. In the first part, I will present data showing that ATIP3 interacts with EB1, a major regulator of MT dynamics. ATIP3-EB1 interaction prevents EB1 accumulation at MT growing ends. I proposed a novel mechanism by which ATIP3-EB1 indirectly reduces EB1 turnover at its binding site at MT plus end, which consequently reduces MT dynamics. In the second part of my thesis, I showed that ATIP3 silencing induces reduced spindle length. In parallel, I identified the MT-depolymerizing kinesin Kif2A as an ATIP3 partner by proteomic analysis. ATIP3 forms a complex with Kif2A and Dda3 in an AurKA-dependent manner. I showed that ATIP3 maintains mitotic spindle size by inhibiting Kif2A and Dda3 recruitment at the spindle pole. My study also revealed a recriprocal regulation between ATIP3 and AurKA. Thus, ATIP3 negatively regulates its binding partners. Finally, in a third part, clinical relevance of ATIP3-EB1 in breast cancer has been evaluated and I showed that combinatorial expression of ATIP3 and EB1 is associated with tumor agressiveness and reduced patient survival. Altogether, this work highlighted new therapeutic targets to propose personalized treatments.

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