Depuis sa mise sur le marché à la fin des années 70, le Cisplatin est devenu l’un des pricipaux agents de chimiothérapie anticancéreuse. Actuellement, les composés à base de platine sont présents dans la majorité des coktails de chimiothérapie. Cependant, malgré leur succès clinique, ces composés présentent de nombreux inconvenients comme par exemple de nombreux et graves effets secondaires. Une des stratégies envisagées pour parer à ces défauts a été de remplacer le platine par d’autres métaux de transition. Parmi les différents métaux possibles, l’or est apparu comme particulièrement prometteur. En effet, les propriétés pharmacologiques de l’or sont bien connues depuis des siècles, et aujourd’hui encore certains complexes d’or sont utilisés comme agents anti-arthritiques. Plus récement, il a été démontré que les complexes d’or étaient actifs sur des lignées cellulaires cancéreuses résistantes au Cisplatin. Parmi les différentes classes de composés à base d’or synthétisées et testées, certaines familles de complexes organométalliques tel que les carbènes N-hétérocycliques, les complexes à ligands cyclométallés ou alkynyl ont attiré un intérêt tout particulier à cause de leur grande stabilité en milieu physiologique.Dans le présent manuscrit, nous avons présenté la synthèse de différents types de de complexes à base d’or incluant des carbènes N-hétérocycliques d’or(I), des complexes « bifonctionnels » d’or(I) basés sur le lansoprazole, des complexes (C^N) cyclométalés d’or(III) ainsi que des complexes homo- et hétérobimétalliques présentant un motif or(I)-NHC. Les différents composés ont été testés sur des panels de lignées cellulaires cancéreuses humaines et les résultats ont été comparés à un modèle de cellules rénales humaines saines. Nous avons effectué différentes études mécanistiques pour tenter d’élucider le possible mécanisme d’action de ces composés. Ces investigations ont inclus des études de toxicité sur des tissus sains ex vivo utilisant des tranches de tissus coupés avec précision ainsi que l’identification de possible cibles intracellulaires. Nous avons étudié deux possibles enzymes cibles : la thiorédoxine réductase et la poly(ADP-ribose) polymérase 1 ainsi qu’une structure non-usuelle de l’ADN : la structure G-quadruplexe. L’internalisation cellulaire de certains composés fluorescents a aussi été étudiée par microscopie confocale. Parmi les différents composés testés, certains ont montré une sélectivité pour les cellules cancéreuses qui mérite une étude plus approfondie. / Currently, platinum-based compounds are present in the majority of the chemotherapeutic cocktails. However, despite their clinical success, platinum-based drugs present several limitations including numerous and severe side effects. A strategy envisaged to overcome these limitations is the replacement of platinum by other transition metals. Among the different metals tested over the years, gold compounds have been shown to be promising as they can overcome resistance to cisplatin due to their different modes of action.In the present thesis, we present the synthesis of different types of gold-based complexes including gold(I)-N-heterocyclic carbenes (NHC), (C^N) cyclometalated gold(III) complexes as well as heterobimetallic complexes bearing a gold(I)-NHC moiety. These compounds show higher stability in a physiological environment compared to classical platinum complexes The different compounds have been tested in panels of human cancer cell lines and a model of human healthy kidney cells. Moreover, “bifunctional” lansoprazole-based gold(I) complexes were also evaluated for their biological properties in vitro. On selected compounds we also performed mechanistic studies to try to elucidate their possible mechanisms of action. Specifically, we investigated two possible enzyme targets: thioredoxin reductase and poly(ADP-ribose) polymerase 1, as well as DNA G-quadruplexes. The cellular uptake of some fluorescent compounds was also studied using confocal microscopy techniques. In a few cases, we also assessed their toxicity ex vivo in rat healthy tissues using the precision-cut tissue slices technique. Among the twenty gold-based compounds we synthesized, two of them presented interesting selective toxicity for cancer cells compared to healthy cells and tissues and deserve further investigations as potential anticancer drugs.

Meiosis is a specialized type of cell division where two rounds of chromosome segregation follow a single round of DNA duplication resulting in the formation of four haploid daughter cells. Once the DNA replication is complete, the homologous chromosomes pair and recombine during the meiotic prophase I, giving rise to genetic diversity in the gametes. The process of homology search during meiosis is broadly divided into recombination-dependent (involves the formation of double-strand breaks) and recombination-independent mechanisms. In most eukaryotic organisms, pairing of homologs, recombination and chromosome segregation occurs in the context of a meiosis-specific proteinaceous structure, known as the synaptonemal complex (SC). The electron microscopic visualization of SC has revealed that the structure is tripartite with an electron-dense central element and two lateral elements that run longitudinally along the entire length of paired chromosomes. Transverse filaments are protein structures that connect the central region to the lateral elements. Genetic analyses in budding yeast indicate that mutations in SC components or defects in SC formation are associated with chromosome missegregation, aneuploidy and spore inviability. In humans, defects in SC assembly are linked to miscarriages, birth defects such as Down syndrome and development of certain types of cancer. In Saccharomyces cerevisiae, genetic screens have identified several mutants that exhibit defects in SC formation culminate in a decrease in the frequency of meiotic recombination, spore viability and improper chromosome segregation. Ten meiosis-specific proteins, viz. Hop1, Red1, Mek1, Hop2, Pch2, Zip1, Zip2, Zip3, Zip4 and Rec8, have been shown to be the bona fide components of SC and/or associated with SC function. S. cerevisiae HOP1 (HOmolog Pairing) gene was isolated in a genetic screen for mutants that showed defects in homolog pairing and, consequently, reduced levels of interhomolog recombination (10% of wild-type). Amino acid sequence alignment together with genetic and biochemical analyses revealed that Hop1 is a 70 kDa protein with a centrally embedded essential zinc-finger motif (Cys2/Cys2) and functions in polymeric form. Previous biochemical studies have also shown that Hop1 is a structure-specific DNA binding protein, which exhibits high affinity for the Holliday junction (HJ) suggesting a role of this protein in branch migration of the HJ. Furthermore, Hop1 displays high affinity for G-quadruplex structures (herein after referred to as GQ) and also promotes the formation of GQ from unfolded G-rich oligonucleotides. Strikingly, Hop1 promotes pairing between two double-stranded DNA molecules via G/C-rich sequence as well as intra- and inter-molecular pairing of duplex DNA molecules. Structure-function analysis suggested that Hop1 has a modular organization consisting of a protease-sensitive N-terminal, HORMA domain (characterized in Hop1, Rev7, Mad2 proteins) and protease-resistant C-terminal domain, called Hop1CTD. Advances in the field of DNA quadruplex structures suggest a significant role for these structures in a variety of biological functions such as signal transduction, DNA replication, recombination, gene expression, sister chromatid alignment etc. GQs and i-motif structures that arise within the G/C-rich regions of the genome of different organisms have been extensively characterized using biophysical, biochemical and cell biological approaches. Emerging studies with guanine- and cytosine-rich sequences of several promoters, telomeres and centromeres have revealed the formation of GQs and i-motif, respectively. Although the presence of GQs within cells has been demonstrated using G4-specific antibodies, in general, the in vivo existence of DNA quadruplex structures is the subject of an ongoing debate. However, the identification and isolation of proteins that bind and process these structures support the idea of their in vivo existence. In S. cerevisiae, genome-wide survey to identify conserved GQs has revealed the presence of ~1400 GQ forming sequences. Additionally, these potential GQ forming motifs were found in close proximity to promoters, rDNA and mitosis- and meiosis-specific double-strand break sites (DSBs). Meiotic recombination in S. cerevisiae as well as humans occurs at meiosis-specific double-strand break (DSBs) sites that are embedded within the G/C-rich sequences. However, much less is known about the structural features and functional significance of DNA quadruplex motifs in sister chromatid alignment N during meiosis. Therefore, one of the aims of the studies described in this thesis was to investigate the relationship between the G/C-rich motif at a meiosis-specific DSB site in S. cerevisiae and its ability to form GQ and i-motif structures. To test this hypothesis, we chose a G/C-rich motif at a meiosis-specific DSB site located between co-ordinates 1242526 to 1242550 on chromosome IV of S. cerevisiae. Using multiple techniques such as native gel electrophoresis, circular dichroism spectroscopy, 2D NMR and chemical foot printing, we show that G-rich motif derived from the meiosis-specific DSB folds into an intramolecular GQ and the complementary C-rich sequence folds into an intramolecular i-motif, the latter under acidic conditions. Interestingly, we found that the C-rich strand folds into i-motif at near neutral pH in the presence of cell-mimicking molecular crowding agents. The NMR data, consistent with our biochemical and biophysical analyses, confirmed the formation of a stable i-motif structure. To further elucidate the impact of these quadruplex structures on DNA replication in vitro, we carried out DNA polymerase stop assay with a template DNA containing either the G-rich or the C-rich sequence. Primer extension assays carried out with Taq polymerase and G-rich template blocked the polymerase at a site that corresponded to the formation of an intramolecular GQ. Likewise, primer extension reactions carried out with KOD-Plus DNA polymerase and C-rich template led to the generation of a stop-product at the site of the formation of intramolecular I -motif under acidic conditions (pH 4.5 and pH 5.5). However, polymerase stop assay performed in the presence of single-walled carbon nanotubes (SWNTs) that stabilize I -motif at physiological pH blocked the polymerase at the site of intramolecular I -motif formation, indicating the possible existence of i-motif in the cellular context. Taken together, these results revealed that the G/C-rich motif at the meiosis-specific DSB site folds into GQ and i-motif structures in vitro. Our in vitro analyses were in line with our in vivo analysis that examined the ability of the G/C-rich motif to fold into quadruplex structures in S. cerevisiae cells. Qualitative microscopic analysis and quantitative analysis with plasmid constructs that harbour the GQ or i-motif forming sequence revealed a significant decrease in the GFP expression levels in comparison to the control. More importantly, all the assays performed with the corresponding mutant sequences under identical experimental conditions did not yield any quadruplex structures, suggesting the involvement of contagious guanine and cytosine residues in the structure formation. Prompted by our earlier results that revealed high binding affinity of Hop1 for GQ, we wished to understand the role of the GQ and i-motif structures during meiosis by analysing their interaction with Hop1 and its truncated variants (HORMA and Hop1CTD). In agreement with our previous observations, Hop1 and Hop1CTD associated preferentially with GQ DNA. Interestingly, whereas the full-length Hop1 showed much weaker binding affinity for i-motif DNA, Hop1 C-terminal fragment but not its N-terminal fragment exhibited robust i-motif DNA binding activity. We have previously demonstrated that Hop1 promotes intermolecular synapsis between synthetic duplex DNA molecules containing a G/C-rich sequence. Hence, to understand the functional role of the quadruplex structures formed at the meiosis-specific G/C-rich motif, we examined the ability of Hop1 to promote pairing between linear duplex DNA helices containing the G/C-rich motif. DNA pairing assay indicated that binding of Hop1 to the G/C-rich duplex DNA resulted in the formation of a side-by-side synapsis product. Under similar conditions, Hop1 was unable to pair mutant duplex DNA molecules suggesting the involvement of the G/C-rich motif in the formation of the synapsis product. Our results were substantiated by the observation that yeast Rad17 failed to promote pairing between duplex DNA molecules with a centrally embedded G/C-rich motif. Altogether, these results provide important structural and functional insights into the role of quadruplex structures in meiotic pairing of homologous chromosomes. The second part of the thesis focuses on the biochemical and functional properties of Red1 protein, a component of S. cerevisiae lateral element. RED1 was identified in a screen for meiotic lethal, sporulation proficient mutants. Genetic, biochemical and microscopic analyses have demonstrated the physical interaction between Hop1 and Red1. Given this, hop1 and red1 mutants display similar phenotypes such as chromosome missegregation and spore inviability and thus are placed under the same epistasis group. However, unlike hop1 mutants, red1 mutants show complete absence of SC. RED1 overexpression suppressed certain non-null hop1 phenotypes, indicating that these proteins may have partially overlapping functions. Further, although the functional significance is unknown, chromatin immunoprecipitation studies have revealed the localization of Red1 to the GC-rich regions (R-bands) in the genome, considered to be meiotic recombination hotspots. Although the aforementioned genetic studies suggest an important role for Red1 in meiosis, the exact molecular function of Red1 in meiotic recombination remains to be elucidated. To explore the biochemical properties of Red1, we isolated the S. cerevisiae RED1 gene, cloned, overexpressed, and purified the protein to near homogeneity. Immunoprecipitation assays using meiotic cells extracts suggested that Red1 exists as a Homodimer linked by disulphide-bonds under physiological conditions. We characterized the DNA binding properties of Red1 by analysing its interaction with recombination intermediates that are likely to form during meiotic recombination. Protein-DNA interaction assays revealed that Red1 exhibits binding preference for the Holliday junction over replication fork and other recombination intermediates. Notably, Red1 displayed ~40-fold higher binding affinity for GQ in comparison with HJ. The observation that Red1 binds robustly to GQs prompted us to examine if Red1 could promote pairing between duplex DNA helices with the G/C-rich sequences similar to Hop1. Interestingly, we found that Red1 failed to promote pairing between dsDNA molecules but potentiated Hop1 mediated pairing between duplex DNA molecules. Our AFM studies with linear and circular DNA molecules along with Red1 suggested a possible role of Red1 in DNA condensation, bridging and pairing of double-stranded DNA helices. Bioinformatics analysis of Red1 indicated the lack of sequence or structural similarity to any of the known proteins. To elucidate structure-function relationship of Red1, we generated several N- and C-terminal Red1 truncations and studied their DNA binding properties. Our results indicated that the N-terminal region comprising of 678 amino acid residues constitutes the DNA-binding region of Red1. The N-terminal region, called RNTF-II, displayed similar substrate specificity comparable to that of full-length Red1. Interestingly, site-directed mutagenesis studies with the Red1 C-terminal region revealed the involvement of two cysteine residues at position 704 and 707 in the disulfide bond mediated intermolecular dimer formation. Finally, to understand the functional significance of Red1 truncations we analyzed the subcellular localization of Red1 and its truncations. We made translation fusions of RED1 and its truncations by placing their corresponding nucleotide sequences downstream of GFP coding sequence in yeast expression vector. Confocal microscopy studies with S. cerevisiae cells transformed with the individual plasmid constructs indicated that the N-terminal variants localized to the nucleus, whereas the C-terminal variants did not localize to the nucleus. These results suggest that NLS-like motifs are embedded in the N-terminal region of the protein. Furthermore, other results indicated that the N-terminal region contains functions such as DNA-binding and intermolecular bridging of non-contiguous DNA segments. Altogether, these findings, on the one hand, provide insights into the molecular mechanism underlying the functions of Hop1 and Red1 proteins and, on the other, support a role for DNA quadruplex structures in meiotic chromosome synapsis and recombination.

La maladie d'Alzheimer (MA) est la démence la plus importante dans le monde développé. Cette maladie neurodégénérative rend de plus en plus difficile la capacité d'accomplir les tâches quotidiennes de routine, elle peut également faire oublier les mots aux patients, les désorienter dans le temps et l'espace, et à des stades avancés entraîne une perte de mémoire. Malheureusement, la MA est considérée comme le prochain grand défi pour la santé publique de la plupart des pays, le nombre de cas devant doubler au cours des 20 prochaines années en raison du vieillissement de la population. Cette augmentation du nombre de patients s'accompagne d'une augmentation des besoins de financement et de personnel de santé afin de répondre aux demandes et aux besoins de ces patients. La MA peut être divisée en deux entités distinctes: une maladie héréditaire bien définie et bien comprise qui représente jusqu'à 5% de tous les cas de MA appelés maladie d'Alzheimer familiale, et une maladie moins définie appelée maladie d'Alzheimer sporadique. Le facteur de risque le plus défini pour la MA est l'âge, mais récemment, il a été démontré que le cerveau des patients atteints de MA avait un niveau réduit de BMI1 et que la suppression de BMI1 dans les neurones humains ou chez la souris déclenche les caractéristiques de cette maladie. Alors que BMI1 était connu pour être important dans les stades de développement, nous rapportons ici qu'il est crucial dans les cellules adultes pour maintenir la compaction de la chromatine et l’inhibition de la transcription des séquences répétitives. De plus, ces deux fonctions de BMI1 empêchent l'ADN d'acquérir une conformation G4. Cette conformation peut entraîner une instabilité du génome, une augmentation des dommages à l'ADN et une altération de l'expression des gènes, mais surtout, nous avons montré que dans les neurones corticaux, les structures G4 peuvent influencer l'épissage alternatif de divers gènes, notamment APP. Ces résultats apportent un éclairage nouveau sur l'origine de la maladie et l'importance de BMI1 et de la structure secondaire de l'ADN dans le cadre de la MA. / Alzheimer's disease is the most prominent dementia in the developed world. This neurodegenerative disease renders the ability to do the routine daily tasks more and more difficult; it can also cause patients to forget words, be disoriented in time and space, leading to a memory loss. Unfortunately, AD is considered the next big challenge for most country’s public health, with the number of cases thought to be doubling within the next 20 years due to the aging of the population. This increase in the number of patients comes with an increase in the need for funding and for healthcare personnel to meet the demands and the requirements of these patients. AD is divided into two separate entities: a well-defined and understood hereditary disease that makes up to 5% of all AD cases called familial Alzheimer disease, and a less defined one called sporadic Alzheimer disease. sAD most defined risk factor is age, but recently it was shown that brains of sAD patients had a reduced level of BMI1 and that the knockdown of BMI1 in human neurons or mice triggers the hallmarks of this disease. While BMI1 was known to be important in the developmental stages, we report here that it is crucial in adult cells to maintain the compaction of the chromatin and the silencing of the repetitive sequences. Furthermore, these two functions of BMI1 prevent the DNA from acquiring a G4 conformation. This conformation can lead to genome instability, increased DNA damage, and altered gene expression. However, most importantly, we showed that in cortical neurons, G4 structures could influence the alternative splicing of various genes, notably APP. These results shed new light on the origin of AD, and the importance of BMI1 and the secondary structure of the DNA in its context.

La maturation 3’ des pré-ARNm constitue une étape majeure dans la régulation post-transcriptionnelle de l’expression des gènes, indispensable à la stabilité, l’export vers le cytoplasme et la traduction des ARNm. Elle est composée de deux réactions : un clivage à l’extrémité 3’ suivie de l’addition d’une queue poly(A). Des études ont montré que la maturation en 3’ est inhibée en réponse aux dommages de l’ADN. Cependant, la cellule a mis en place des mécanismes compensatoires qui permettent à certains pré-ARNm d’être correctement maturés assurant ainsi le maintien de son intégrité. Les travaux que nous avons menés ont mis en évidence un mécanisme de résistance à l’inhibition de maturation en 3’ du pré-ARNm codant pour le suppresseur de tumeur p53. Ce mécanisme fait intervenir l’hélicase DHX36 qui déplie une structure secondaire appelée G-quadruplexe située en aval du site de clivage. Par ailleurs dans une deuxième étude, nous avons montré que la maturation en 3’ maintenue du pré-ARNm p53 en réponse aux dommages de l’ADN, est découplée du processus de transcription, contrairement au pré-ARNm TBP dont la maturation 3’ est inhibée en réponse aux dommage de l’ADN. Ce découplage a lieu grâce à un clivage co-transcriptionnelle du pré-ARNm p53 au niveau de la chromatine qui entraîne sa libération dans le nucléoplasme où il subit sa maturation en 3’. Une étude à grande échelle nous a permis de montrer que ce mécanisme de maturation en 3’ survenant dans le nucléoplasme est associé au maintien d'une maturation en 3’ efficace en réponse aux dommages de l’ADN. / The 3’-end processing of pre-mRNA, a key step in the post-transcriptional gene expression regulation, is essential for mRNA stability, export and translation. This process is a two-step reaction composed of a cleavage at the 3’-end followed by the addition of a poly(A) tail. Studies have shown that pre-mRNA 3’-end processing is inhibited in response to DNA damage. However, compensatory mechanisms exist to allow some pre-mRNA to be properly processed at their 3’-end in order to maintain cell integrity. For instance, in response to DNA damage, the 3’-end processing of the pre-mRNA coding for the tumor suppressor p53 is able to escape from its inhibition. In the present work, we have shown that the underlying mechanism involves the DHX36 helicase that unwinds a secondary structure called G-quadruplex located downstream of the cleavage site of the p53 pre-mRNA. Moreover, in a second study, we have shown that the maintained p53 pre-mRNA 3’-end processing in response to DNA damage is uncoupled from the transcription process, unlike the inhibited TBP pre-mRNA 3’-end processing. This uncoupling takes place through a co-transcriptional cleavage of p53 pre-mRNA from the chromatin and its release in the nucleoplasm where it undergoes its 3’-end processing. A genome-wide study allowed us to show that the pre-mRNA 3’-end processing occurring in the nucleoplasm is associated with a maintained 3’end processing in response to DNA damage

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