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.

Les DIPG représentent les tumeurs cérébrales pédiatriques les plus sévères. Aucun progrès dans leur prise en charge n’a été accompli au cours des 50 dernières années et la radiothérapie ne demeure que transitoirement efficace. Récemment, une mutation somatique de l’histone H3 (K27M) spécifique des DIPG a été trouvée chez environ 95% des patients. Elle est aujourd’hui considérée comme l'événement oncogénique initiateur de ces tumeurs. Deux sous-groupes majeurs de patients présentant des programmes oncogéniques et une réponse à la radiothérapie distincts peuvent être définis en fonction du gène dans lequel l’altération survient, codant les variantes protéiques H3.1 ou H3.3. Nous avons réalisé deux cribles de létalité synthétique par ARN interférence ciblant le kinome humain afin d'identifier d’une part les gènes nécessaires à la survie des DIPG et d’autre part les gènes dont l’inhibition sensibilise ces tumeurs à la radiothérapie. Le double objectif de ce projet était de mieux comprendre la biologie sous-jacente à l’oncogenèse des DIPG et de découvrir de nouvelles cibles thérapeutiques.Nous avons mis en évidence 41 gènes requis pour la survie des DIPG sans effet délétère majeur sur des cellules contrôles normales. Parmi eux, nous avons identifié VRK3 codant une serine thréonine kinase dont les fonctions restent peu décrites à ce jour et qui n'avait jamais été associée préalablement à l'oncogenèse de DIPG. Nous avons pu confirmer par la suite que son inhibition conduit à un arrêt total de la prolifération des cellules de DIPG associé à d’importants changements morphologiques, plus particulièrement dans les tumeurs mutées pour H3.3-K27M. VRK3 constitue par conséquent une nouvelle cible thérapeutique prometteuse dans cette pathologie à l’issue fatale pour la totalité des patients.En parallèle, un crible de survie similaire a été réalisé en conjonction avec l’irradiation des cellules. Très peu d’ARN interférents ont permis de sensibiliser les cellules H3.3-K27M à la radiothérapie contrairement aux cellules H3.1-K27M. Ce travail nous a permis de mettre en évidence une différence significative de radiosensibilité des modèles vitro de DMG en fonction du sous-groupe de tumeurs considéré, H3.1- ou H3.3-K27M muté, conformément à la survie des patients observée suite à la radiothérapie. Ces résultats inédits laissent entrevoir des perspectives d’amélioration du traitement de référence des patients atteints de DIPG actuellement identique quelle que soit leur génotype. / DIPG is one of the most severe paediatric brain tumours. No progress has been made in their management over the past 50 years and radiotherapy remains only transiently effective. Recently, a specific somatic mutation in the histone H3 (K27M) has been found in approximately 95% of DIPG patients and can be considered as the oncogenic driver of these tumours. Two major subgroup of patients with distinct oncogenic program and response to radiotherapy can be defined according to the gene in which the alteration occurs, encoding the H3.1 or H3.3 protein variants. We performed two synthetic lethality screens by RNA interference targeting the human kinome in order to identify the genes responsible for DIPG cell survival, as well as those sensitizing tumour cells to radiotherapy after inhibition. The dual purpose of this project was to better understand the biology underlying oncogenesis of DIPGs and to discover new therapeutic targets.We identified 41 genes required for DIPG cell survival with no major deleterious effect on normal control cells. Among them, we identified VRK3, a serine threonine kinase never involved in DIPG oncogenesis with functions remaining poorly described to date. We have shown that its inhibition leads to a complete arrest of DIPG cell proliferation and is additionally associated with important morphological changes, more particularly in H3.3-K27M mutated tumours. VRK3 is therefore a promising new therapeutic target for all patients in this fatal pathology.In parallel, a similar survival screen was performed in conjunction to cell radiation and very few interfering RNAs enhance H3.3-K27M cell radiosensitivity, in contrast to H3.1-K27M cells. These data highlighted a significant difference in radiosensitivity of the DMG in vitro models in H3.1- versus H3.3-K27M mutated tumours, in a concordant way with patient survival following radiotherapy. These unprecedented results suggest new opportunities for improving the current treatment of DIPG patients regardless of their genotype.

The N-terminal tails of histone proteins protrude from the nucleosome core and are extensively post-translationally modified. These modifications are proposed to affect many DNA-based processes such as transcription, DNA replication and repair. Post-translational modifications on histone tails do not act independently but are subject to crosstalk. One example of crosstalk is on histone H3 between lysine 14 (H3K14) and trimethylated lysine 4 (H3K4me3), a modification found at the 5’ end of most active or poised genes. In this work, Western blots and chromatin immunoprecipitation (ChIP) experiments show that different amino acid substitutions at histone H3 position 14 cause varying degrees of H3K4me3 loss, indicating that H3K14 is not essential for H3K4me3 but acts as a modulator of H3K4me3 levels. A neighbouring residue, H3P16 is also important for H3K4me3 and may operate in concert with H3K14 to control H3K4me3. These crosstalk pathways have gene-specific effects and the levels of H3K4me3 are influenced to different extents on genes that fall into functionally distinct classes. A model is proposed to explain how H3K14/H3P16 may exert these varying effects on H3K4me3 at individual genes. In addition to its ability to regulate H3K4me3, H3K14 also influences the levels of two modifications on H3K18, acetylation and monomethylation. A ChIP-sequencing experiment has shown that H3K18me1, a previously uncharacterised modification in S. cerevisiae, is widely distributed throughout the genome and correlates strongly with histone H3 levels. The potential for a functional acetyl/methyl switch at H3K18 is explored. Together, these data indicate that, with gene-specific effects, crosstalk between histone modifications may be even more complex than originally thought.

Accelerated Adaptation through Stimulated Copy Number Variation in Saccharomyces cerevisiae Ryan Matthew Hull Repetitive regions of the genome, such as the centromeres, telomeres and ribosomal DNA account for a large proportion of the genetic variation between individuals. Differences in the number of repeat sequences between individuals is termed copy number variation (CNV) and is rife across eukaryotic genomes. CNV is of clinical importance as it has been implicated in many human disorders, in particularly cancers where is has been associated with tumour growth and drug resistance. The copper-resistance gene CUP1 in Saccharomyces cerevisiae is one such CNV gene. CUP1 is transcribed from a copper inducible promoter and encodes a protein involved in copper detoxification. In this work I show that yeast can regulate their repeat levels of the CUP1 gene through a transcriptionally stimulated CNV mechanism, as a direct adaptation response to a hostile environment. I characterise the requirement of the epigenetic mark Histone H3 Lysine 56 acetylation (H3K56ac) for stimulated CNV and its limitation of only working at actively transcribed genes. Based upon my findings, I propose a model for how stimulated CNV is regulated in yeast and show how we can pharmacologically manipulate this mechanism using drugs, like nicotinamide and rapamycin, to stimulate and repress a cell's ability to adapt to its environment. I further show that the model is not limited to high-copy CUP1 repeat arrays, but is also applicable to low-copy systems. Finally, I show that the model extends to other genetic loci in response to different challenging environments, such as formaldehyde stimulation of the formaldehyde-resistance gene SFA1. To the best of our knowledge, this is the first example of any eukaryotic cell undergoing genome optimisation as a novel means to accelerate its adaptation in direct response to its environment. If conserved in higher eukaryotes, such a mechanism could have major implications in how we consider and treat disorders associated with changes in CNV.

This thesis presents a comprehensive phylogenetic and taxonomic study of an acoel subgroup, the Childiidae. Members of this taxon are characterized by well-developed male copulatory organs with conical/cylindrical stylets. The phylogenetic analyses, by means of total evidence approach, based on three molecular markers (18S and 28S rRNA genes and Histone H3) and 50 morphological characters reaffirm the non-monophyly of the Childiidae sensu Dörjes, 1968 (Actinoposthiidae and Childia+Paraphanostoma). The total evidence phylogeny strongly support the Childia+Paraphanostoma clade separate from other former members of the Childiidae, which are now placed in Actinoposthiidae. The monophyly of Childia+Paraphanostoma is well corroborated by several morphological characters. A new taxon Childia is defined, in accordance with the PhyloCode, comprising all former Paraphanostoma species and a member of the monotypic genus C. groenlandica. A new diagnosis for the current members of Childia is provided. Several structures, shown to hold promising phylogenetic signals for unraveling acoel relationships, such as musculature pattern, sperm and male copulatory organs, are investigated, using a combination of traditional and modern techniques (ultrastructure, immunocytochemistry and confocal microscopy), with main focus on Childia and its closest relatives. New characters are described and their phylogenetic significance assessed. Morphological characters relating to body-wall musculature, statocyst muscles, male copulatory organ musculature and ultrastructure, and sperm cytoplasmic granules are shown to carry important phylogenetic signals at lower taxonomic levels, while most of the characters related to sperm ultrastructure are useful at higher taxonomic levels within the Acoela. The data obtained undermine the phylogenetic use of the seminal bursa in Childia. In addition to this, it is shown that most of the classical morphological characters used in acoel taxonomy, obtained using traditional histological methods, may be misleading in identifying monophyletic entities within the Acoela. The most corroborated synapomorphies, identified in this thesis, are used in determining the taxonomic placement of a new viviparous acoel, Childia vivipara, into the taxon Childia.

No description available.

5 Abstract Neuroblastoma is one of the most common cancer diseases diagnosed in children. This rapidly growing solid tumor is usually formed by hypoxic areas which arise as a consequence of inefficient and disorganized neovascularization. The cells stressed by hypoxia triggers transcription of many genes necessary for their survival, and conversely stop the production of proteins which are not necessarily needed for the survival in these severe conditions. The adaptation of cells to hypoxic conditions may appear due to the epigenetic regulation of metabolism associated with chromatin remodeling which involves the DNA methylation and also the posttranslational modifications of histones. Among the most important of these, there is the acetylation of lysine residues of histones associated with the DNA strands loosening, facilitated binding of transcription factors and the activation of gene expression. Thus, the first part of this study is concerned with changes in the acetylation of histones H3 and H4 of human neuroblastoma cell lines UKF-NB-3, UKF-NB-4, SH-SY5Y and SK-N-AS, cultured in parallel under standard culture conditions and in the absence of oxygen (hypoxia, 1% O2) for 24 hours, which are studied by Western blot analysis. Thereupon, the activity of histone deacetylases and histonacetyltransferases,...

A subset of 199 Melissotarsus insularis and 130 Diaspididae specimens were analyzed to 1) determine the species status of M. insularis and 2) to explore the relative intimacy of the relationship between M. insularis and Diaspididae. An analysis of molecular variance and the observed lack of association between clades and distinct habitats on the M. insularis phylogeny suggested that while M. insularis exhibits isolation by distance, it does not apparently diversify by habitat. When cryptic COI pseudogenes were accounted for, the majority of the genetic diversity exhibited by M. insularis was limited to a divergence of 3% or less suggesting that M. insularis represents a single, albeit broadly distributed, species. A cophylogenetic reconstruction of the relationship between M. insularis and Diaspididae yielded 14 “cospeciation” events but was not significant unlike reconstructions of host-parasite relationships. Analyses of reduced datasets suggested that incomplete taxon sampling may significantly affect cophylogenetic reconstruction results. / National Science Foundation (grants No. DEB-0072713, DEB-0344731 to BLF and DEB-0842395 to BLF and MAS), a Natural Sciences and Engineering Research Council of Canada Discovery Grant to MAS and a Leaders Opportunity Fund grant from the Canada Foundation for Innovation to MAS

Due to their phylogenetic position, Psychidae play an important role in the investigation of the W chromosome origin in Lepidoptera. Several species of Psychidae were tested for the presence of sex-chromatin and investigated via comparative genomic hybridization. Furthermore, odd chromosome numbers and a Z univalent were observed in females. Overall, this study brings tangible evidence for the absence of the W chromosome in Psychidae, thus contributes to complex knowledge of the W chromosome evolution. In addition, karyotypes of the given species were analyzed using 18S rDNA and histone H3 probes. The results indicate relative stability of their karyotypes.

Les sirtuines sont une famille conservée de déacétylases NAD+-dépendantes qui sont impliquées dans divers processus. Les humains possèdent 7 sirtuines (SIRT1-7) qui jouent un rôle dans plusieurs voies cellulaires, tandis que la levure Saccharomyces cerevisiae possède 5 membres (Sir2, Hst1-4) qui influencent plusieurs voies comme le cycle cellulaire ou le vieillissement. Une absence d’activité des sirtuines mène toutefois à des défauts de croissance, une thermosensibilité et l’apparition de dommages spontanés à l’ADN par des mécanismes mal élucidés. Pour mieux caractériser ce phénomène, ce mémoire met en lumière certains résultats venant d’un crible chimiogénétique réalisé par traitement au nicotinamide (NAM), un pan-inhibiteur des sirtuines. Nos résultats indiquent que le NAM entraîne chez la levure Saccharomyces cerevisiae une forte activation des voies de réponses aux dommages à l’ADN, et que les défauts de croissance sont principalement dus à l’hyperacétylation de la lysine 56 de l’histone H3 (H3K56), une modification post-traductionnelle qui est renversée par les sirtuines Hst3 et Hst4. Lors d’hyperacétylation de H3K56, la protéine Slx4 et le complexe PP4 sont requis pour la croissance de la levure en modulant les niveaux d’activation de la kinase Rad53 lors de la RDA. Également, certains résultats préliminaires inclus dans ce mémoire mettent en évidence un rôle de l’activité des sirtuines dans la régulation de la recombinaison homologue, l’une des voies de réparation de l’ADN. Ensemble, nos résultats suggèrent que la déacétylation des histones par les sirtuines permet de moduler la réponse aux dommages à l’ADN en présence de stress réplicatif. / Sirtuins are a conserved family of NAD+-dependent deacetylases that are involved in various processes. Humans have seven sirtuins (SIRT1-7) and play a role in several cellular pathways, while the budding yeast Saccharomyces cerevisiae has 5 members (Sir2, Hst1-4) and influence several pathways, such as the cell cycle or aging. Lack of sirtuin activity however leads to growth defects, thermosensitivity and spontaneous DNA damage by poorly understood mechanisms. To further characterize this phenomenon, this thesis highlights results obtained from a chemogenetic screen realized by treatment with nicotinamide (NAM), a pan-inhibitor of all sirtuins. Our results indicate that NAM causes strong activation of DNA damage-induced signaling in budding yeast Saccharomyces cerevisiae, and that growth defects are mainly due to histone H3 lysine 56 (H3K56) hyperacetylation, a post-translational modification reversed by sirtuins Hst3 and Hst4. During H3K56 hyperacetylation, the Slx4 protein and PP4 complex are both required for yeast growth by modulating the activation levels of Rad53 kinase during the DDR. Also, preliminary results included in this thesis highlight that proper regulation of homologous recombination, one of DNA repair pathways, is essential for growth in the presence of NAM-induced sirtuin inhibition. Together, our results suggest that chromosome-wide histone deacetylation by sirtuins can modulate DNA damage response in presence of replicative stress.