Biological molecules often undergo large structural changes to perform their function. Computational methods can provide a fine-grained description at the atomistic scale. Without sufficient approximations to accelerate the simulations, however, the time-scale on which functional motions often occur is out of reach for many traditional methods. Natural Move Monte Carlo belongs to a class of methods that were introduced to bridge this gap. I present three novel applications for Natural Move Monte Carlo, two on proteins and one on DNA epigenetics. In the second part of this thesis I introduce a new protocol for the testing of hypotheses regarding the functional motions of biological systems, named customised Natural Move Monte Carlo. Two different case studies are presented aimed at demonstrating the feasibility of customised Natural Move Monte Carlo.

La méthylation de l’ADN est la marque épigénétique la plus connue. Elle consiste en l’ajout d’un groupement méthyle au niveau de la cytosine, produisant la 5-méthyl-cystosine (5mC). Cette réaction chimique est catalysée par des ADN méthyltransférases : DNMT1, DNMT3A et DNMT3B. Peu de choses sont connues concernant les changements de 5mC au cours des lignages cellulaires dans l’embryon et comment cette marque contribue à l’établissement ou au maintien de l’identité cellulaire. Nous avons cherché à mieux comprendre ces mécanismes en étudiant la 5mC dans deux lignages cellulaires : les cellules primordiales germinales (PGCs) et les cellules souches hématopoïétiques (HSCs). Nous avons généré les premiers méthylomes de ces cellules au cours de leur développement chez la souris. Chez les PGCs, nous avons mis en évidence l’existence de deux phases de reprogrammation de la 5mC. Une première phase entre E9,5 et E13,5, où le génome des PGCs se déméthyle et une phase de reméthylation entre E14,5 et E17,5, chez les gamètes mâles uniquement. Néanmoins, certaines régions, dont notamment les éléments transposables sont résistants à la vague de déméthylation. L’utilisation de souris conditionnellement, nous a permis de mettre en évidence l’implication des protéines DNMT1 et UHRF2 dans le maintien de la 5mC au niveau de ces séquences. Concernant les HSCs, nous avons mis en évidence qu’elles acquièrent un profil de 5mC qui leur est propre lors de deux phases. La première a lieu dès l’apparition des HSCs dans l’organisme tandis que l’acquisition de la signature hématopoïétique définitive se déroule à l’âge adulte dans la moelle osseuse. L’utilisation de souris conditionnelles, nous a permis de mettre en exergue l’implication de DNMT3A et DNMT3B dans la mise en place de ces profils, avec un rôle prépondérant de DNMT3B lors de la phase d’acquisition précoce et de DNMT3A lors du verrouillage de leur profil de 5mC. / The methylation of DNA is a well-known epigenetic mark. It consists in adding a methyl group to a cytosine producing the 5-methylcytosine (5mC). This is catalysed by the DNA methyltransferase (DNMT) family: DNMT1, DNMT3A and DNMT3B. Little is known about the changes in DNA methylation that follow lineage decisions in the embryo and how these contribute, establish or maintain cellular identities. We are addressing these questions using as a model the specification of mouse primordial germ cells (PGCs) and mouse hematopoietic stem cells (HSCs) in the mouse embryo. We generate the first genome-wide maps of 5mC during their development. These maps highlight two waves of DNA methylation in PGCs. The first one takes place between E9,5 and E13,5, where the genome demethylates while the second one corresponds to a remethylation phase only in male PGCs between E14,5 and E17,5. Nevertheless, some regions, notably the transposable elements, are resistant to this demethylation wave. We demonstrate the implication of DNMT1 and UHRF2 in maintaining the 5mC on these regions using transgenic mice presenting specific deletion in PGCs. In HSCs, the 5mC maps highlight two wave of DNA methylation. The first one correlates with the first appearance of the HSCs in early embryos while the second one corresponds to their migration to the bone marrow and seems to act as a definitive lock for their hematopoietic identity. Using transgenic mice presenting specific deletions in HSCs, we prove the implication of DNMT3A and DNMT3B in hematopoietic stem cells, with a major role in locking HSC identity of DNMT3B during the first wave and a DNMT3A during the second one respectively.

Les aculéatines et FR235222 sont deux familles de molécules d’origine naturelle qui agissent de façons très efficaces sur les parasites intracellulaires de la famille des apicomplexes, responsables notamment du paludisme ou de la toxoplasmose. Dans la première partie de ces travaux, nous avons développé une nouvelle réaction cascade d’oxydation phénolique en « un pot » utilisant un réactif à base d’iode hypervalent en quantité catalytique. Cette stratégie de synthèse flexible et hautement modulable en peu d’étapes, permettra d’accroître l’accessibilité vers de nouveaux analogues aculéatines bioactives. Dans la deuxième partie, nous nous sommes intéressés à FR235222, un HDACi (inhibiteur des histones désacétylases) qui cible les HDACs de la classe I chez l’homme et l’HDAC3 chez le parasite T. gondii responsable de la toxoplasmose. Les HDACs sont des protéines qui jouent un rôle important dans le contrôle des mécanismes épigénétiques. Dans cette étude, un analogue fluorescent du produit naturel a été synthétisé et a permis de confirmer la cible de FR235222 chez l’homme grâce à des études de localisation cellulaire. Par la synthèse et l’évaluation de l’activité HDACi de nouveaux analogues, associées à des études de modélisation moléculaire, nous avons démontré pour la première fois que la tête chélatante céto-hydroxyle et la flexibilité du linker sont responsable de cette sélectivité sur les HDACs de la classe I humaine. Enfin, grâce à ses résultats et à l’identification de différences structurales entre l’HDAC3 humaine et parasitaire, des études de prédiction (docking) ont permis de dégager des caractéristiques essentielles d’un HDACi potentiellement sélectif sur les parasites apicomplexes. / Aculeatins and FR235222 are two families of natural products that are highly effective against apicomplexan parasites, responsible for malaria and toxoplasmosis. In the first part of this work, we developed a new reaction involving a “one pot” phenolic oxidation cascade sequence using a hypervalent iodine reagent in catalytic condition. This flexible synthetic strategy will increase accessibility to new aculeatin derivatives to achieve bioactive compounds. In the second part, we were interested in FR235222, an HDACi (histone deacetylase inhibitor) which targets the class I human HDAC and parasite T. gondii HDAC3 responsible for toxoplasmosis. HDACs are proteins that play an important role in the control of epigenetic mechanisms. In this study, a FR235222 fluorescent derivative was synthesized to confirm the FR235222 target in human cells through cellular localization studies. The synthesis and assessment of the activity of new HDACi analogues, combined with molecular modelisation studies, allowed to demonstrate for the first time that the keto-hydroxyl zinc binding group and the flexibility of the linker would be responsible for this selectivity on human class I HDACs. Finally, with these results in hand and the identification of structural differences between human and parasitic HDAC3, prediction studies (docking) have revealed structural determinants to design inhibitors selective for apicomplexan parasites HDACi.

Cervical malignancy is the fourth most common cause of cancer-related mortality in women worldwide; infection with high-risk human papillomavirus (HRHPV) is responsible for over 500,000 cases of cervical carcinoma each year, approximately 90% of which are squamous cell carcinomas (SCCs). Over half of all HPV-positive cervical SCCs are caused by the deregulated expression of HPV16 oncogenes E6 and E7 in proliferating basal cells of the cervical squamous epithelium. The major risk factor associated with cervical neoplastic progression is integration of HRHPV into the host genome, which is detected in $~$85% of HPV16-positive cervical carcinomas. The work presented in this doctoral thesis sought to provide insights into our understanding of the process of HPV16 integration as well as to elucidate mechanisms that deregulate both virus and host gene expression following integration. The W12 cell model system used in this project is a polyclonal cervical keratinocyte line generated by explant culture of a low-grade cervical squamous intraepithelial lesion (LSIL) that arose following natural infection with HPV16. The W12 clones were isolated in the absence of selective pressure, and as such represent the range of integration events that occur in a pre-malignant lesion at the early stages of carcinogenesis, prior to integrant selection. Despite identical genetic backgrounds, expression levels of oncogenes E6 and E7 varied up to 16-fold between the W12 clones. Expression of HPV oncogenes is ultimately determined by transcription factor binding to the non-coding long control region (LCR) of the viral genome. The initial result of this study found that genomic mutations affecting transcription factor binding at the LCR of the W12 clones was not a cause of differential viral expression, concluding that epigenetic control may be at play. Higher levels of virus expression per template were associated with increased levels of histone post-translational modification (PTM) hallmarks of transcriptionally active chromatin and reduced levels of repressive hallmarks. There was greater abundance of the active/elongating form of the RNA polymerase-II enzyme (RNAPII-Ser2P), together with CDK9, the component of positive transcription elongation factor-b (P-TEFb) responsible for the Ser2 phosphorylation. The changes observed were functionally significant, as cells with higher HPV16 expression per template showed greater sensitivity to depletion and/or inhibition of histone acetyl transferases and CDK9, as well as reduced sensitivity to histone deacetylase inhibition. Employing next generation sequencing data available for five representative W12 clones, the sites of HPV16 host integration were identified. The three-dimensional (3D) structure of the nucleus and physical interactions between stretches of the genome over long distances (i.e. enhancer and promoters) are known to exert an additional level of gene regulation. Identification of 3D virus-host interactions in the W12 clones employing the newly developed and unique 'Sequence Capture of Regions Interacting with Bait Loci Hi-C' (SCRiBL-Hi-C) protocol showed that both short- ($~$50 kb), and long-range ($~$1 Mb) interactions occur during the early stages of carcinogenesis. Together, the data in this thesis indicate that transcription and subsequent expression of the HPV16 genome is controlled by multiple layers of epigenetic regulation.

Afin d’initier la transcription par l’ARN Polymérase II, la chromatine est modifiée par des coactivateurs, dont certains catalysent des modifications post-traductionnelles des queues des histones. La protéine GCN5 est une enzyme qui possède une activité histone acétyltransférase (HAT). Elle fait partie du complexe coactivateur SAGA, qui acétyle les histones H3. Or, il existe un second complexe HAT contenant GCN5 : le complexe ATAC, mis en évidence chez la drosophile. Chez l’homme en revanche, l’existence d’un tel complexe n’avait pas encore été démontrée au début de ma thèse.L’objectif de ma thèse a consisté tout d’abord en la purification et la caractérisation du complexe HAT ATAC chez l’homme. Puis, j’ai cherché à comprendre le fonctionnement et la spécificité d’action du complexe ATAC, par rapport au complexe SAGA.Dans une première partie, j’ai ainsi pu montrer que GCN5 fait partie d’un second complexe chez l’homme, le complexe ATAC. La composition en sous-unités du complexe ATAC a été déterminée et l’activité de ce dernier sur les histones étudiée. Nous avons pu démontrer que, comme hSAGA, hATAC acétyle les histones in vitro et in vivo, et préférentiellement la lysine 14 de l’histone H3. Chez les vertébrés, un paralogue de GCN5, PCAF peut se substituer à GCN5 dans les complexes ATAC ou SAGA.Par la suite, j’ai poursuivi la caractérisation de ces complexes HAT afin de comprendre le rôle des enzymes au sein des complexes et leurs fonctions. Pour cela, j’ai voulu comprendre le rôle des sous-unités, comment elles influencent l’activité de l’enzyme, et ainsi identifier les protéines qui permettent la spécificité de hATAC par rapport à hSAGA. / In order to initiate the transcription by the RNA polymerase II, chromatin needs to be modified by coactivators. Some of these coactivators are histone post-translational modifying complexes. GCN5 is a histone acetyltransferase enzyme (HAT), which can acetylate the histones. This enzyme is found in a multiproteic complex named SAGA. Recently, a second HAT complex containing GCN5 was discovered: ATAC, in drosophila. At the beginning of my thesis, the existence of such complex in human was not shown.My thesis objectives were then to identify and characterize an ATAC complex in human cells. In a first part, we purified and identified the composition in subunits of human ATAC. Then we studied the activity and specificity of ATAC on histone substrates, compared to SAGA. Next, we were wondering how the subunits of the two HAT complexes could play a role on the regulation of the activity of the enzyme GCN5, in order to understand the histone specificity of ATAC and SAGA.

O desenvolvimento e a progressão do câncer bucal envolvem processos complexos de múltiplas etapas levando a modificações fenotípicas nas células epiteliais, aumento da proliferação e invasão dos tecidos subjacentes. Diversos fatores vem sendo associados à carcinogênese, dentre eles os mecanismos epigenéticos como a acetilação de histonas, que promovem mudanças na expressão de genes independente de mutações. O objetivo do presente estudo observacional transversal foi analisar a relação entre acetilação da histona 3 (acetil Histona H3) com proliferação celular e transição epitélio-mesênquima na mucosa bucal normal (MBN), leucoplasias bucais (LB) e carcinomas espinocelulares (CEC) de boca, bem como correlacioná-los com dados clínico-demográficos, graduação histopatológica e o comportamento das lesões. Foram analisados 10 casos de mucosa bucal normal (MBN), 20 casos de LB e 75 casos de CEC de boca. Todos os casos foram submetidas a análise imunoistoquímica utilizando anticorpos anti-acetil Histona H3, Ki67, vimentina e TGF-β1. A imunomarcação da acetil histona H3 foi significativamente menor nos casos de CEC quando comparados a LB (p=0.03). Não foi encontrado diferença entre os casos de MBN e LB. Paralelamente, foi observado um aumento estatisticamente significativo na proliferação durante o processo de carcinogênese (p<0.00) e o mesmo foi observado quando avaliados os marcadores da transição epitélio-mesênquima, vimentina (p=0.03) e TGF-β1 (p<0.00). A análise da associação dos marcadores com fatores clínicos-demográficos não mostrou diferença significativa. Entretanto, maior média de acetil histona H3 foi associada ao bom prognóstico (p=0.01) e também, foi observado uma tendência de uma melhor taxa de sobrevida (p=0.06). Conclui-se que os CEC de boca são hipoacetilados, exibem maior perfil proliferativo e de transição epitélio-mesênquima. Além disso, a acetil histona H3 pode ser considerada um marcador prognostico nestas lesões. / The development and progression of oral cancer involve multi-step processes leading phenotypic changes in epithelial cells, proliferation increase and invasion of adjacent tissue. Several factors have been associated with carcinogenesis, including epigenetic mechanisms such as histone acetylation, which promote changes in the expression independent of gene mutations. The aim of the present study was to analyze the association of acetylation of histone 3 (acetyl-histone H3) with cell proliferation and epithelial-mesenchymal transition in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) and correlate them with data clinic-demographic, histopathological grading and the behavior of these lesions. We analyzed 10 cases of normal oral mucosa (NOM), 20 cases of OL and 75 cases of OSCC. All samples were submitted to immunohistochemical analysis using anti-acetyl histone H3, Ki67, vimentin and TGF-β1. Acetyl-histone H3 labeling was significantly lower in cases of OSCC compared to LB (p=0.03). It was not found difference between NOM and OL. In parallel, the proliferation analysis revealed a gradual increase on Ki67 labeling (p<0.00) during oral carcinogenesis with highest value detected in OSCC Also, an increase on EMT markers, vimentin (p=0.03) and TGF-β1 (p<0.00) were noted. A higher mean acetyl-histone H3 was associated with good prognosis (p= 0.01) and similarly a tendency to improved survival rate was observed (p=0.06). As conclusion, OSCC are hypoacetylated, exhibit higher proliferative profile and epithelial-mesenchymal transition characteristics. Furthermore, acetyl histone H3 can be considered a prognostic marker in OSCC.

Au cours des dernières décennies, la méthylation de l'ADN en position 5 de la cytosine est apparue comme une importante modification épigénétique qui joue un rôle essentiel dans le contrôle spécifique de l'expression des gènes. Cependant, les mécanismes impliqués dans la régulation de la méthylation de l'ADN restent incompris. Des études récentes ont montré que des protéines de type oxydases, nommées TET, peuvent catalyser l'oxydation de la 5-méthylcytosine (5mC) et générer des dérivés oxydés de celle-ci ce qui soulève la question du rôle biologique des formes oxydées de la 5mC. L'identification et la caractérisation des protéines interagissant avec ces formes oxydées devraient permettre une meilleure compréhension de la fonction de ces modifications de l'ADN et de la régulation de la méthylation de l'ADN. Dans ce projet, nous avons développé des sondes photoactivables basées sur l'ADN pour capturer, isoler et caractériser les protéines associées à ces modifications épigénétiques de l'ADN. Tout d'abord, nous avons conçu et évalué les propriétés de différentes sondes oligonucléotidiques photoactivables. Nous avons ensuite réalisé une étude méthodologique afin de caractériser au niveau moléculaire les photoadduits obtenus par MALDI-TOF. Enfin, nous avons développé une méthode de pull-down couplé à du photomarquage et associée à une analyse protéomique par spectrométrie de masse afin d’identifier les protéines ayant une affinité spécifique pour ces modifications épigénétiques. / Over the past few decades, DNA methylation at the 5-position of cytosine has emerged as an important epigenetic modification that plays essential roles in the specific control of gene expression. However, the mechanisms involved in the regulation of DNA methylation remain unclear. Recent studies have shown that oxidase proteins, called TETs, can catalyze the oxidation of 5-methylcytosine (5 mC) and generate oxidized derivatives thereof, raising the question of the biological role of the oxidized forms of 5mC. The identification and characterization of proteins interacting with these oxidized forms should allow for a better understanding of the function of these DNA modifications and the regulation of DNA methylation.In this project, we develop DNA-based photoactivatable probes to capture, isolate and characterize the proteins associated with these epigenetic DNA modifications. First, we designed and evaluated the properties of different photoactivatable oligonucleotide probes. We then carried out a methodological study in order to characterize at the molecular level the obtained photoadducts by MALDI-TOF. Finally, we developed a pull-down method coupled to photolabelling and associated with proteomic analysis by mass spectrometry to identify proteins with specific affinity for these epigenetic changes.

Introdução: Leiomiomas uterinos ou miomas são tumores benignos que se desenvolvem no miométrio e acometem cerca de 50% da população feminina. Têm como principais sintomas, sangramento excessivo e dor pélvica inespecífica. Convencionalmente o estrogênio é considerado o responsável pelo início da proliferação tumoral, mas recentes evidências clínicas e bioquímicas sugerem que a progesterona apresenta um papel importante no desenvolvimento desses tumores. Além disso, mecanismos epigenéticos parecem estar envolvidos na etiologia dos leiomiomas uterinos, como a metilação de DNA e acetilação de histonas. Somente nos EUA são realizadas 240 mil histerectomias/ano para tratar essa doença sendo considerado um problema de saúde pública. Frente a esses dados é imprescindível o entendimento dos mecanismos moleculares envolvidos no desenvolvimento desses tumores e a busca de tratamentos menos invasivos. A mifepristona (RU 486), um modulador seletivo dos receptores de progesterona e glicocorticoides, é capaz de diminuir o tamanho dos tumores e amenizar os sintomas associados. Objetivos: verificar se (1) nos tecidos de leiomiomas uterinos e miométrio, os mecanismos epigenéticos como a metilação global do DNA e a acetilação de histonas estão alterados (2) se em cultura primária de leiomioma uterino e miométrio o tratamento com estradiol e progesterona é capaz de modular a expressão gênica dos receptores de progesterona total e B, assim como a atividade das enzimas histona acetiltransferase e desacetilase, e (3) se a mifepristona (RU 486) é capaz de modular diretamente a expressão gênica dos receptores de progesterona total e B após tratamento com os hormônios estradiol e progesterona. Métodos: para análise de metilação global do DNA e acetilação de histonas foram utilizadas 25 amostras teciduais para cada grupo de leiomioma uterino e miométrio oriundos de pacientes submetidas à histerectomia no Hospital de Clínicas de Porto Alegre. A metilação global do DNA e a atividade da enzima histona acetiltransferase foram dosadas pelo método de ELISA e a enzima histona desacetilase por detecção fluorimétrica. Para verificar o efeito dos hormônios sexuais ovarianos e o efeito da mifepristona (RU 486) sobre a expressão gênica dos receptores de progesterona total e B foram realizadas 7 culturas primárias de leiomiomas uterinos e miométrio. Resultados: (1) Foram observadas hipermetilação (P = 0,022) e hipoacetilação (P = 0,04) em leiomiomas uterinos quando comparado ao miométrio. Não houve diferença estatística entre estes tecidos em relação à atividade da histona acetiltransferase. (2) Houve aumento da expressão gênica do receptor de progesterona total em cultura primária de leiomioma uterino quando tratado com estradiol (P = 0,028) e o receptor de progesterona B teve sua expressão aumentada quando tratado com estradiol, progesterona e estradiol + progesterona (P = 0,001). (3) O tratamento com mifepristona (RU 486) na dose de 10-6M não foi capaz de diminuir a expressão gênica dos receptores de progesterona total e B em células de leiomiomas uterinos e miométrio. A atividade da enzima histona desacetilase foi maior nas células de leiomioma uterino quando tratadas com estradiol (P = 0,034) e estradiol + progesterona + RU 486 (P = 0,001) quando comparado às células de miométrio, já a atividade da enzima histona acetiltransferase não foi detectada, devido a sua baixa quantidade. Conclusão: Nesse estudo foi observado uma hipermetilação e hipoacetilação nos tecidos de leiomiomas uterinos. Esses resultados sugerem que mecanismos epigenéticos podem estar contribuindo para a diminuição transcricional de genes relacionados ao funcionamento normal do miométrio e, com isso, colaborando para o crescimento desses tumores. Além disso, nossos resultados sugerem que estradiol e progesterona são capazes de modular a expressão gênica dos receptores de progesterona total e B e o medicamento RU 486 na dose de 10-6M não foi capaz de diminuir diretamente a expressão desses receptores. / Introduction: Uterine leiomyoma, also known as fibroids, is a smooth muscle cell tumor, and is clinically apparent in up to 50% of reproductive-age women. Clinical symptoms include abnormal uterine bleeding and pelvic pain. Estrogen has been considered a primary growth promoter of uterine leiomyoma, however clinical and biochemical evidence has suggested that progesterone plays a critical role in the development of these tumors. Furthermore, epigenetic modifications may be involved with etiology of theses tumors, through DNA methylation and histone acetylation. In the United States, 240.000 hysterectomies/year are performed to treat uterine leiomyomas which is being considered to be a public public health problem. The understanding of molecular mechanisms involved in the development of uterine leiomyoma may provide not only opportunities for a diagnostic, but also generation of novel therapeutic approaches. The mifepristone (RU 486), a synthetic steroid that has affinity for progesterone and glucocorticoid receptors has been reported to induce regression of uterine leiomyoma and reduce the symptoms. Objective: verify if (1) DNA global methylation and histone acetylation patterns are altered in uterine leiomioma tissue compared with myometrium; (2) the treatment with estradiol and progesterone are able to modulated de gene expression of total and B progesterone receptors and histone acetylation patterns in primary culture of uterine leiomyoma cells and myometrium as well as (3) the mifepristone (RU 486) are able to modulate the gene expression of total and B progesterone receptors after the treatment with ovarian steroids hormones. Methods: DNA global methylation and histone acetylation patterns were analyzed in 25 tissue samples from uterine leiomioma and myometrium. The DNA global methylation and the activity of histone acetyltransferase were performed using ELISA method. In order to evaluate histone deacetylase activity fluorimetric detection was used. To verify the effect of estradiol and progesterone on total and B progesterone receptors gene expression, as well as the influence of RU486 on this parameters, seven cultured cells from uterine leiomyoma and myometrium cells were performed. Results: (1) Hypermethylation (p = 0.022) and hypoacetylation (p = 0.04) in uterine leiomioma tissues compared with myometrium were observed. There was no statistic difference between these tissues in histone acetyltransferase activity. (2) We observed increased gene expression of total progesterone receptor in culture cells of uterine leiomyoma when treated with estradiol (p = 0.028). There was an increase of B progesterone receptor mRNA when treated with hormones, estradiol and progesterone (p = 0.001). The treatment with RU 486 was not able to modulate progesterone receptors. The histone desacetilase activity was elevated in uterine leiomyoma cells when treated with estradiol (p = 0.034) and estradiol + progesterone + RU 486 (p = 0.001). The histone acetyltransferase activity was barely detectable. Conclusion: In our study we found hypermethylation and hypoacetylation in uterine leiomyoma tissues suggesting that this process may lead to a decreased transcriptional activity of important genes associated with normal myometrium function contributing to the development of these tumors. Furthermore, our results suggest that ovarian steroids hormones increase progesterone receptors expression, being mifepristone (RU 486) at dose of 10-6M unable to decrease total and B progesterone receptors in uterine leiomyoma cells.

Understanding the mechanisms and nature of tumour heterogeneity is a key focus of current cancer research. Tumour heterogeneity can arise from clonal evolution and/or differentiation. This thesis investigated the role of methylation in dynamic regulation of CD24 based heterogeneity in colorectal cancer cell lines. First, E-Cadherin variation between the cell lines LS174T and LS180 was investigated to find out whether E-Cadherin had any causal role in the difference in lumen formation between these two cell lines derived from the same tumour. These studies found no evidence of a causal role of E-Cadherin. However, morphological heterogeneity in LS174T was observed during the E-Cadherin study, suggesting that this cell line might be a mixture of two different clones. Single cell sorting by FACS allowed to isolate and establish these clones which were stable in the culture for long enough (until passage ~30) to allow their characterisations. Between the two clones, the CD24⁺ clone (named the LS174T_Clone 1^CD24+) was found to have shorter doubling time (23 hours) than the CD24- clone (named the LS174T_Clone 2CD24-; 30 hours). When cultured in matrigel, the LS174T_Clone 1^CD24+ showed higher clonogenicity (Chapter 4). Microarray analysis further revealed differences in gene expression including LAPTM4B, CXCR4, TGFIB and IL8 between these clones. Interestingly, when maintained for a long time in culture (around passage 50, which is equivalent to ~7 months), CD24 expression went through a gradual change in these clones, which became more evident from subclones of LS174T_Clone 2^CD24- (Chapter 6) and clones from another CRC cell line SW480 (chapter 5 and 6). To understand the mechanism of this dynamic change in the expression of CD24, it was first shown that no mutation could be responsible for this phenomenon. This suggested that promoter methylation of CD24 might be the mechanism of the observed dynamic changes in CD24 expression. Bisulfite (BS) modification of DNA from the LS174T clones and CD24 differentially expressing CRC cell lines (such as CD24- cell lines: CC20, RKO vs CD24+ cell lines: DLD1, NCIH716) followed by Sanger sequencing showed that direct methylation of seven CpG positions in the CD24 promoter region Chr6:106975560-106975834 is strongly correlated with the expression of CD24. Further subcloning and sequencing revealed that changes in the methylation of only two out of the seven CpG positions might be the main contributor to the CD24 expression differences. This is the first evidence of direct methylation-mediated regulation of CD24, showing, more broadly, how methylation can contribute to and maintain dynamic heterogeneity in cancer cells. Finally, a mixed culture experiment with the CD24+ and CD24- clones was conducted to test a simple mathematical model, which aimed to explain the interaction between the clones that are stably present in the LS174T cell line (Chapter 7). Altogether, these experiments suggest that genes such as REG1A (an inducer of angiogenesis) might be expressed because of synergistic interactions between the clones, whereas CXCR4 and TFF2 might be involved in a receptor-ligand complementary relationship. These findings have set a ground for future studies to confirm such interactions between co-existing subpopulations within a heterogeneous milieu of cancer cells.

Les mécanismes épigénétiques, tels que la méthylation et les modifications d'histones, sont impliqués dans le contrôle à grande échelle de l'expression du génôme et contribuent à la mise en place des profils d'expression des gènes spécifiques de tissus et de types cellulaires. Dans les cellules en cours ou en fin de différenciation, ces mécanismes sont aussi impliqués dans la mise en place et le maintien de la repression d'un grand nombre de gènes. La transformation oncogénique est presque toujours associée à des anomalies de la signalisation épigénétique cellulaire, dont certaines, comme les méthylations aberrantes de gènes suppresseurs de tumeur, sont considérées comme des événements oncogènes. Un aspect beaucoup moins étudié de ces dérégulations épigénétiques est l'activation aberrante de gènes tissu-spécifiques dans des cellules pré-cancéreuses et transformées. De nombreuses études rapportent l'activation « hors contexte » de gènes spécifiques du testicule dans plusieurs cancers somatiques. Ces gènes sont décrits sous le nom de gènes « cancer testis » ou C/T. Il a été suggéré que ces expressions illégitimes pourraient être de bons indicateurs des cancers, et fournir de nouvelles cibles pour une immunothérapie anticancéreuse. Au cours de cette thèse, nous avons développé une approche basée sur ce concept d'activation ectopique de gènes pour identifier les gènes exprimés de manière aberrante dans les lymphoblastes des patients atteints de leucémies lymphoblastiques aigues (LAL). Nous avons ensuite évalué leur intérêt pour une utilisation comme marqueurs pronostics et de prédiction de la réponse au traitement. Nous avons ainsi identifié une signature de huit gènes spécifiques de la lignée germinale / cellules souches embryonnaires, exprimés de manière aberrante dans les LAL pédiatriques et adultes : 4 gènes prédictifs de mauvais pronostic et 4 gènes associés à une issue favorable. Nous avons par la suite montré qu'une combinaison de l'expression de ces 8 gènes peut identifier les formes agressives de LAL chez les enfants ainsi que chez les adultes. Une étude prospective clinique a mis en évidence que notre système de détection des 8 gènes, basé sur un test RT- qPCR, pourrait aider à prédire la réponse précoce à un traitement (induction) dans un groupe de 31 patients adultes nouvellement recrutés, atteints de LAL. Enfin, en exploitant notre méthode de classification, nous avons découvert des traits biologiques communs entre les formes agressives de LAL chez les enfants et chez les adultes. Nos données montrent que les formes les plus agressives de LAL présentent les caractéristiques de cellules souches hématopoïétiques au repos. Cette information pourrait être utilisée pour adapter les approches thérapeutiques. Enfin, en plus de l'amélioration de la détection et du suivi des patients LAL, ce travail a un fort potentiel dans la définition de nouvelles stratégies thérapeutiques ainsi que d'ors et déjà dans les choix thérapeutiques les plus appropriés pour les patients porteurs des formes les plus agressives. / Epigenetic mechanisms such as methylation and histone modifications are involved in large-scale control of the expression of the genome and contribute to the development of specific gene expression profiles of tissues and cell types. In cells, during and after differentiation, these mechanisms are also involved in the establishment and maintenance of the repression of many genes. Oncogenic transformation is almost always associated with abnormalities of cellular epigenetic signalling, some of which, such as aberrant methylation of tumour suppressor genes, are considered as oncogenic events. One much less studied aspect epigenetic deregulations, is the aberrant activation of tissue-specific genes in pre-cancerous and transformed cells. Many studies have reported the “out of context” activation of specific testicular genes in several somatic cancers. These genes are described as the "cancer / testis" genes or C/T. It has been suggested that these illegitimate expressions could be good indicators of cancer and provide new targets for cancer immunotherapy. In this thesis, based on the concept of ectopic activation of genes, we have identified genes aberrantly expressed in lymphoblasts of patients with acute lymphoblastic leukemia (ALL). We have then assessed their potential for a use as markers for prognosis and prediction of treatment response. We have identified a signature of eight genes specific of germline/embryonic stem cells, aberrantly expressed in adult and paediatric ALL. The ectopic activation of four genes was predictive of poor prognosis and the expression of four other genes was associated with a favourable outcome. We have subsequently shown that the combination of the expression of these eight genes can identify aggressive forms of ALL in children and adults. A prospective clinical study showed that a test based on the detection of these 8 genes, by RT- qPCR could help predicting an early response to treatment (induction) in a group of 31 newly recruited ALL adult patients. Finally, using our classification method, we discovered common biological traits between aggressive forms of ALL in children and adults. Our data show that the most aggressive forms of ALL have characteristics of dormant hematopoietic stem cells. This information could be used to refine therapeutic approaches. Finally, in addition to improving the detection and monitoring of ALL patients, this work has great potentials in the definition of new therapeutic strategies as well as in the choice of the most appropriate therapeutic approaches for patients with aggressive forms of ALL.