Thèses sur le sujet « R-methylation »

Restriction Modification (R-M) systems prevent the invasion of foreign genetic material into bacterial cells and are therefore important in maintaining the integrity of the host genome. The spread of antibiotic resistance, which is proposed to occur via the transfer of foreign genes to the bacterial genome, makes the subject of R-M systems extremely relevant. R-M systems are currently classified into four types (I to IV) on the basis of differences in composition, target recognition, cofactors and the manner in which they cleave DNA. Kennaway et al (2012) proposed that there is an evolutionary link between Types I and II. Comparing the structures of examples from two of the subfamilies of Type II systems (IIB and IIG) to those of Type I structures, similarities can be observed. Due to the fact that Type II R-M systems cut DNA at fixed positions, they can be used to obtain genetic material selectively. They have therefore proven to be invaluable in molecular biology. One aspect of this project aims to create a novel R-M system, a pseudo-Type II system, by removing the molecular motors from the restriction subunit of a Type I system and fusing the remaining nuclease domain to a known Type I methyltransferase (MTase). This will not only provide evidence to support the theory that evolution has produced a pared down form of the Type I systems in the Type II systems, but it may also become a useful biological tool. This thesis describes the several attempts at doing this and how the subsequent constructs were expressed, purified and assayed to varying degrees of success. An important characteristic of the Type I systems is their ability to methylate DNA, and it is the mechanism via which host DNA is protected from restriction. This is another subject investigated in this project. As with the nuclease activity of the Type I systems, the site at which DNA is methylated is dictated by the HsdS subunit. It is described here how this subunit can be altered to change the sequence of DNA that is recognised by the system. Again, using Type II system subtypes as a reference, various mutations were made to the HsdS subunit of an MTase from Staphylococcus aureus. This is in an effort to bring about a new mode of action, but also to provide further evidence for an evolutionary link between the two system types. The HsdM and HsdS subunits are expressed from two separate genes at the same locus. There is a frameshift between the genes where the start of the hsdS gene occurs a few base pairs upstream from the stop codon of the hsdM gene. This work shows that removing this frameshift creates an MS fusion product, and in vivo studies show that this product has methylase activity and can form an active restriction complex when the HsdR subunit is added. The product can also be over-expressed and purified, and shows in vitro restriction activity on addition of the HsdR subunit protein. The HsdS subunit is composed of two target recognition domains (TRDs), each dictating one part of the bipartite recognition sequence. These TRDs can be altered, bringing about a change in the sequence of DNA recognised by the enzyme. In this thesis, it is shown that the C-terminal TRD can be removed and that the subsequent “Half S” enzyme possesses both methylase and restriction activity in vivo and that its recognition sequence is different from that of the wild-type enzyme. After the successful creation of both “MS fusion” and “Half S” recombinant proteins of the Sau1, Type I system from a CC398 strain of Staphylococcus aureus, a further construct was produced. This possesses both in vivo and in vitro activity. The novel “M Half S Fusion” enzyme not only links the two aspects of this project but also creates a structure similar to some seen in the Type II systems. This shows that the Type I systems can be manipulated to change their mode of action but also supports the idea that Types I and II are evolutionarily linked. By making the alterations in a step-wise fashion identifies that these structural changes can create viable enzymes, and that they could have occurred through the process of evolution.

Various post-translational modifications (PTMs) have been described to regulate RNA-binding protein (RBP) activity, subcellular localization, and interactions with other proteins or RNAs. Proteome-wide experiments recently carried out in our group have shown that RBPs are the most abundant arginine (R)-methylated proteins. Protein Arginine Methyltransferases (PRMTs) are the enzymes responsible for the deposition of methylation on arginine. Recent evidence has indicated that R-hypomethylation could influence RBP phase-separation and consequent formation of Membrane-Less Organelles (MLOs). In my Ph.D. project, we implemented a quantitative proteomic approach to profile global changes of RBP-RNA interactions upon the modulation of R-methylation, both directly by the use of PRMT inhibitor and indirectly through cisplatin (CDDP)-induced PRMT1 re-localization on chromatin. In particular, by coupling the Orthogonal Organic Phase Separation (OOPS) strategy with mass spectrometry (MS) analysis, we profiled RNA-protein interaction in dependence on R-methylation remodeling. Biochemical and immunofluorescence analysis validated the differential association of a set of RBPs with RNA upon PRMT1 inhibition but also that altered modification is linked to MLOs formation. We then applied our strategy in ovarian cancer in the context of CDDP-induced protein R-methylation rewiring to understand if it may affect RBP-RNA interaction. Preliminary analysis of the significantly regulated RBPs from a new OOPS-MS experiment carried out in these conditions revealed a strong modulation of RBP-RNA interaction and suggested new interesting targets. We are currently analyzing the R-methylation state of the most promising RBPs and in parallel the RNAs which interact differentially with the RBPs in the same conditions.

DNA methylation is a postreplicative covalent modification of the DNA which is catalysed by the DNA methyltransferase enzyme. DNA methylation plays an important role in controlling the gene expression profile of mammalian cells. The hypothesis presented in this thesis is that the expression of the DNA methyltransferase gene is upregulated by cellular oncogenic pathways, and that this induction of MeTase activity results in DNA hypermethylation and plays a causal role in cellular transformation. Novel DNA methyltransferase inhibitors may inhibit the excessive activity of DNA methyltransferase in cancer cells and induce the original cellular genetic program. These inhibitors may also be used to turn on alternative gene expression programs. Therefore specific DNA methyltransferase antagonists might provide us with therapeutics directed at a nodal point in the regulation of genetic information.

One of the main questions of modern biology is how our cells interpret our genetic and epigenetic information. DNA methylation is a covalent modification of the genome that is essential for mammalian development and plays an important role in the control of gene expression, genomic imprinting and X-chromosome inactivation (Bird and Wolffe, 1999; Szyf et al., 2000). Furthermore, changes in DNA methylation and DNA methyltransferase 1 (DNMT1) activity have been widely documented in a number of human cancers (Szyf, 1998a; Szyf et al., 2000).
In Escherichia coli, timing and frequency of initiation of DNA replication are controlled by the levels of the bacterial methyltransferase and by the methylation status of its origin of replication (Boye and Lobner-Olesen, 1990; Campbell and Kleckner, 1990). In mammalian cells, however, the importance of methyltransferase activity and of DNA methylation in replication is only now starting to emerge (Araujo et al., 1998; Delgado et al., 1998; DePamphilis, 2000; Knox et al., 2000).
The work described in this thesis focuses mainly on understanding the functional relationship between changes in DNA methylation and DNMT1 activity on mammalian DNA replication. In higher eukaryotes, DNA replication initiates from multiple specific sites throughout the genome (Zannis-Hadjopoulos and Price, 1999). The first part of the thesis describes the identification and characterization of novel in vivo initiation sites of DNA replication within the human dnmt1 locus (Araujo et al., 1999). Subsequently, a study of the temporal relationship between DNA replication and the inheritance of the DNA methylation pattern is presented. We also demonstrate that mammalian origins of replication, similarly to promoters, are differentially methylated (Araujo et al., 1998). We then tested the hypothesis that DNMT1 is a necessary component of the replication machinery. The results presented indicate that inhibition of DNMT1 results in inhibition of DNA replication (Knox et al., 2000). Finally, results are presented, demonstrating that the amino terminal region of DNMT1 is responsible for recognizing hemimethylated CGs, DNMT1's enzymatic target. Taken together, the results presented in this thesis demonstrate that DNMT1 is necessary for proper DNA replication and that DNA methylation may modulate origin function.

Cancer cells have aberrant DNA methylation patterns which are characterized by hypomethylation of a large set of promoters and hypermethylation of tumor suppressor genes. The dynamic nature of the epigenome makes it a valuable target for therapeutic interventions. This thesis focuses on understanding the use of various inhibitors towards DNA methylation-related proteins and their respective anti-cancer activities at both global and gene-specific levels. The widely used demethylating agent 5-azacytidine and 5-aza-2'-deoxycytidine (5-azaCdR) are FDA-approved drugs for the treatment of myelodysplastic syndrome. However, these nucleoside analogs which trap the DNA methyltransferases (DNMTs) are non-specific. Studies have shown that 5-azaCdR induced pro-metastatic genes and caused long distance metastasis. This raises serious safety concerns for their clinical use. On the contrary, targeting the DNMTs individually or in combination did not result in dramatic induction of pro-metastatic genes as with 5-azaCdR treatment. In particular, single DNMT1-specific inhibition resulted in maximum growth suppression when compared to inhibition of all three major DNMTs, while not increasing cell invasiveness. DNMT1 has been shown to be important for cancer growth. Our study supports the idea that DNMT1 has a major role in cancer over the other DNMTs and that DNMT1 inhibitors could be effective anti-cancer drugs. 5-azaCdR has nevertheless been proven to be a potent suppressor of cancer growth. We tested the idea of a combinatorial treatment that may minimize its side-effects on cell invasion while maintaining its growth suppressor effects. The methyl-CpG binding protein 2 (MBD2) protein has been shown to demethylate pro-metastatic genes. Its inhibition in concurrent with 5-azaCdR treatment synergistically suppressed cancer growth, while reversed the 5-azaCdR-induced invasion. In order to have a deeper understanding of the impact of the treatments, microarrays studies on the methylome and transcriptome of the treated cells were carried out. Bioinformatics analysis indicated that the combined treatment suppressed gene networks that were involved in cell mobility, while synergistically enhanced gene networks that were involved in cell death. This data indicate that combining 5-azaCdR treatment with MBD2 inhibition results in more potent anti-cancer effects than either treatment alone. In order to explore the currently available drugs that inhibit MBD2, we tested the combination of S-adenosylmethionine (SAM) with 5-azaCdR on the same cancer cell lines. SAM remethylated gene promoters of pro-metastatic genes and repressed 5-azaCdR-induced invasion similarly to MBD2 inhibition. We then investigated the relationship between SAM and MBD2 downregulation and observed hypermethylation on both CpG and non-CpG sites in the MBD2 promoter upon SAM treatment. Interestingly, inhibition of MBD2 using short interference RNA also resulted in hypermethylation of its own promoter. This observation suggested that SAM treatment could directly downregulate MBD2 expression, which is further downregulated through a feedback loop. These results also suggested that SAM treatment could have a direct effect on MBD2 promoter, which in turn affects multiple MBD2 targets that are involved in invasion. Together, the data from this thesis support the idea that targeting the epigenome could be a highly efficacious anti-cancer therapy and that combining drugs that target DNA methylation could increase the potency over individual treatments.
Les cellules cancéreuses présentent un profil de méthylation caractérisé par l'hypométhylation d'un grand nombre de promoteurs et l'hyperméthylation de gènes suppresseurs de tumeur. La nature dynamique de l'épigénome en fait une cible de choix pour les interventions thérapeutiques. Cette thèse vise à comprendre l'utilisation de divers inhibiteurs visant des protéines liées à la méthylation de l'ADN et leurs activités anticancéreuses à une échelle génomique globale et au niveau de gènes particuliers. Les agents déméthylants 5-azacytidine et 5-aza-2'-deoxycytidine (5-azaCdR) sont des médicaments pour le traitement du syndrome myélodysplasiqueapprouvés par la FDA. Cependant, ces analogues de nucléosides qui piègent les DNA méthyltransférases (DNMTs) ne sont pas spécifiques. Des études ont montrées que la 5-azaCdR induisait l'expression de gènes pro-métastatiques et l'apparition de métastases. Ceci soulève de sérieuses interrogations quant à leur utilisation en clinique. À l'inverse, le ciblage spécifique des DNMTs ne conduit pas à une induction dramatique des gènes pro-métastatiques. Plus particulièrement, l'inhibition spécifique de DNMT1 résulte en une suppression de la croissance maximale des tumeurs, sans effet sur l'invasion cellulaire, lorsque l'on compare à l'inhibition des trois principales DNMTs. Notre étude supporte l'idée que DNMT1 à un rôle majeur dans le cancer et que le développement d'inhibiteurs de DNMT1 pourraient conduire à des médicaments anti-cancéreux efficaces.Il a néanmoins été montré que la 5-azaCdR était un suppresseur potentiel de la croissance cancéreuse. Nous avons testé l'hypothèse qu'un traitement combiné permettrait de minimiser ses effets secondaires sur l'invasion cellulaire tout en maintenant ses effets suppresseurs de croissance. Il a été montré que la protéine methyl-CpG binding protein 2 (MBD2) participait à la déméthylationde gènes pro-métastatiques. Son inhibition simultanée à un traitement 5-azaCdR abolit de façon synergétique la croissance cancéreuse, tout en inhibant l'invasion induite par la 5-azaCdR. Des analyses du méthylome et du transcriptome ont été réalisées par micropuces à partir de cellules traitées avec un siRNA dirigé contre l'ARNm de MBD2 et la 5-azaCdR afin d'avoir une meilleure compréhension de l'impact de la combinaison des traitements. Les analyses bioinformatiques ont indiqué que le traitement combiné réprimait des réseaux de gènes impliqués dans la mobilité cellulaire tandis que les réseaux de gènes activés étaient impliqués dans la mort cellulaire. Ces données indiquent que le traitement à la 5-azaCdR combiné avec l'inhibition de MBD2 résulte en de plus puissants effets anti-cancéreux que l'un ou l'autre des traitements individuels.Nous avons également testé la combinaison de la S-adenosylmethionine (SAM), un médicament actuellement disponible sur le marché et inhibant l'activité de MBD2, avec la 5-azaCdR sur les lignées cellulaires utilisées précédemment. La SAM, de façon similaire à l'inhibition de MBD2 par un siRNA, permet la méthylation des promoteurs de gènes pro-métastatiques et réprime l'invasion induite par la 5-azaCdR. Nous avons ensuite examiné la relation entre la SAM, la diminution de l'expression de MBD2 et l'hyperméthylation observée à la fois aux sites CpG et non-CpG au niveau du promoteur de MBD2 après traitement avec la SAM. De façon intéressante, l'inhibition de MBD2 par des petits ARN interférant résulte également en une hyperméthylation de son propre promoteur. Cette observation suggère que le traitement avec SAM pourrait directement réduire l'expression de MBD2, qui serait réduite encore plus via une boucle de rétrocontrôle. L'ensemble des données de cette thèse supporte l'idée que le ciblage de l'épigénome pourrait être une thérapie anti-cancéreuse hautement efficace et que la combinaison de médicaments qui ciblent la méthylation de l'ADN pourrait augmenter l'efficacité des traitements individuels.

Arginine methylation is a post-translational modification occurring in higher eukaryotes that results in the addition of one or two methyl group on the nitrogen in the side chain of arginines. The enzymes responsible for protein arginine methylation have been classified in three groups. Type I enzymes promote the formation of both NG-monomethylated and asymmetric o-NG,NG-dimethylated arginines (aDMA). Type II enzymes catalyze the formation of monomethylated and symmetrical o-N G,N'G-dimethylated arginines (sDMA). The type III enzyme found in yeast catalyzes the monomethylation of the delta-guanidino nitrogen atom of the arginine residue. Although some abundant proteins have been described as being substrates for arginine methyltransferases for some time, there are still few known proteins to bear this modification. The primary goal of the work presented in this thesis was to identify new arginine methylated proteins and functionally characterize the roles of arginine methylation in new cellular processes. First, we generated four arginine methyl-specific antibodies: ASYM24 and ASYM25 are specific for aDMA whereas SYM10 and SYM11 recognize sDMA. Cell extracts were used to purify the protein complexes recognized by each of the four antibodies and the proteins were identified by microcapillary reverse-phase liquid chromatography coupled on line with electrospray ionization tandem mass spectrometry (LC/MS/MS). The analysis of 2 tandem mass spectra for each methyl-specific antibody resulted in the identification of 247 proteins, of which 197 are putatively arginine methylated.
The DNA repair MRE11/RAD50/NBS1 (MRN) complex was purified using one of the aDMA specific antibody. Since a role of protein arginine methylation in DNA damage checkpoint control and DNA repair had not been previously reported we chose to investigate the consequence of MRE11 methylation in DNA damage. Our results show that the MRE11 checkpoint protein is arginine methylated as determined by mass spectrometry and methylarginine-specific antibodies. The glycine-arginine rich (GAR) domain of MRE11 was specifically methylated by protein arginine methyltransferase 1 (PRMT1). Mutation of the arginines within MRE11 GAR domain severely impaired the exonuclease activity of MRE11. Cells treated with methyltransferase inhibitors displayed a DNA damage-resistant DNA synthesis phenotype and prevented the re-localization of the MRN complex to sites of DNA damage. Downregulation of PRMT1 with small interfering RNAs (siRNA) also yielded a damage-resistant DNA synthesis phenotype that was rescued with the methylated MRE11 complex. Taken together, the work presented in this thesis allowed the identification of many new potentially arginine methylated proteins and demonstrated a novel role for arginine methylation in the regulation of DNA repair enzymes and of the intra-S phase DNA damage checkpoint.

Maintaining appropriate patterns of gene expression in the gametes and during early embryogenesis is essential for normal development. DNA methylation is an epigenetic means of regulating gene expression and is an important molecular mark regulating the sex-specific expression of genes subject to genomic imprinting. Imprinted genes are expressed from only one of two inherited chromosomes and are differentially marked during gametogenesis to allow for their parental allele specific expression. These genes affect embryo growth, placental function, behavior after birth and are implicated in the etiology of a number of human diseases. The primary objective of this thesis was to gain a better understanding of the developmental dynamics and origins of DNA methylation profiles regulating maternally methylated imprinted genes during mouse oocyte development. Studies revealed that maternally methylated imprinted genes acquire methylation within their DMRs during postnatal oocyte growth and that this acquisition occurs in a gene and allele specific manner. It was also observed that maternal methylation imprint acquisition is related to oocyte diameter and that a repetitive parasitic element also acquires methylation during this period. DNA methylation is catalyzed by DNMTs and investigations into the developmental expression profiles of Dnmt3a, Dnmt3b and Dnmt3L indicated that transcript accumulation of these enzymes during oocyte development coincided with the timing of maternal methylation imprint establishment. Moreover, expression analysis in DNMT-depleted oocytes suggested these enzymes to be coordinately regulated. Additional studies aimed at developing another model of oocyte imprinting lead to the identification and characterization of a putative bovine Snrpn DMR. Its DNA methylation profile was found to be conserved with that of mouse and human. Snrpn DNA methylation analysis in bovine IVF and SCNT embryos revealed slight loss of methylatio

One of the elements commonly seen in cancer is the change in methylation status of the genome. These aberrations in methylation appear to be critical for the neoplastic phenotype and manifest as changes to gene expression of oncogenes and tumour suppressors. In addition to epigenetic alterations, the proteins involved in maintaining the plastic methylation status of the genome, DNA methyltransferases and demethylases, also show methylation-independent protein-protein interactions that have effects on cell cycle progression and proliferation. As changes in gene expression and mitotic regulation are seminal elements of cancer, and because several methylated DNA binding proteins show differential expression in a wide variety of cancers, these proteins serve as prime targets for anticancer therapies. This thesis relates to exploring both current and forthcoming possibilities and mechanisms of utilizing the DNA methylation machinery for pharmacological intervention of cancer. Chapter two deals with an antisense drug, currently in clinical trials, targeted to reduction of DNA methyltransferase 1, the maintenance methylation enzyme in mammalian cells. Our data indicate that the existence of a common truncation mutation of the adenomatous polyposis coli gene seen in some forms of sporadic and familial colorectal cancer may lead to downstream upregulation of DNA methyltransferase 1, as reconstitution of the wildtype protein reduces DNA methyltransferase 1 mRNA and protein. Reduction of the transcripts of this methylation enzyme with an antisense oligonucleotide decreases the tumourigenicity of these colorectal cancer cells, and provides a rationale for use of this drug in colorectal cancer patients and prophylactic treatment of adenomatous polyposis coli mutation-bearing individuals. Chapter three describes the rationale, design, and in vitro and in vivo testing of antisense molecules against the methylated DNA binding protein MBD2. These drugs red

Proteins are known to be post-translationally modified. This thesis will discuss arginine methylation, one of the many post-translational modifications that occur within the cell. The enzymes that catalyze this post-translational modification are called arginine methyltransferases. The three main types of methylated arginines include monomethylated arginine (MMA), asymmetric dimethylated arginine (aDMA) and symmetric dimethylated arginines (sDMA). Type I arginine methyltransferases catalyze the formation of MMA and aDMA; Type II enzyme catalyze the formation of MMA and sDMA. Protein arginine methylation has been implicated in the regulation of many different cellular processes, including transcription, cellular localization, protein-protein interaction and signal transduction.
The purpose of this work was to further characterize arginine methylation by identifying new members of the arginine methyltransferase enzyme family in Drosophila melanogaster and to study the effects of protein arginine methylation on novel substrates. I identified and characterized nine homologues of arginine methyltransferases in Drosophila that were named DART1 to DART9, for drosophila arginine methyltransferases 1-9. All nine enzymes are expressed at various developmental stages. I discovered that a substrate of mammalian enzyme protein arginine methyltransferase 1 (PRMT1) can also be methylated by PRMT5. I also identified HIV-1 Tat protein as the first substrate of the novel enzyme PRMT6.

DNA methylation is vital in genome functions as diverse as parental imprinting, X chromosome inactivation, regulation of gene expression and precipitation of inactive chromatin structures. A growing line of evidence suggests that aberrations in proper homeostatic regulation of genomic methylation status are causal in oncogenic processes. Recent evidence has also revealed novel roles of the DNA Methyltransferase (DNMT1), the enzyme believed to be largely responsible for maintenance of the DNA methylation pattern, in replication and in direct regulation of chromatin structure which may help to explain its role in cancer. Despite the importance of DNMT1 in the regulation in many critical genome functions, little has been done to clearly elucidate its regulation of expression. Chapters 1--3 of this thesis describe three intriguing mechanisms employed in the regulation of dnmt1 gene expression which further implicate this protein as an important player in cellular transformation and tumorigenesis. Chapter 1 describes dnmt1 as a novel downstream effector of the commonly studied T Antigen oncogenic signaling pathway and points to a novel mechanism by which DNMT1 inhibitors might target tumorigenesis. dnmt1 has also been previously characterized as an effector of the Ras-jun oncogenic signaling pathway and Chapter 2 describes how the c-jun proto-oncogene can recruit and synergize with the tumor suppressor Rb to transactivate dnmt1. Chapter 3 describes a novel and unique mechanism by which dnmt1 can feedback regulate its own expression via methylation of its own promoter. The discovery of active demethylation activity in transformed cell types has also changed our understanding of genomic methylation as a means of epigenetic control and suggests the possibility that aberrant regulation of a demethylase gene may also contribute to cellular transformation and tumorigenesis. Chapter 4, therefore, describes the upregulation of Mbd2/Demethylase in various human tumors and the

A growing line of evidence indicates that the proper control of DNA methyltransferases (DNMT) and DNA demethylases is critical for maintaining correct gene expression. In addition, misregulation of this machinery likely plays a role in the aberrant DNA methylation patterns and gene expression that is a hallmark of many pathologies such as cancer. A greater comprehension of the mechanisms involved in regulating the expression and activity of these proteins should provide new therapies aimed at restoring gene expression gone wrong.

O6-Methylguanine DNA methyltransferase (MGMT) is an inducible DNA repair protein that acts to repair damage by DNA alkylating agents currently used in chemotherapy, such as temozolomide. MGMT removes the alkyl group placed at the O6-position of guanine by these alkylating agents, decreasing their efficacy. It has been shown that epigenetic methylation of the O6-MGMT DNA promoter region in tumour tissue from glioblastoma multiforme (GBM) is associated with improved survival from patients treated with temozolomide and concomitant radiotherapy. We wanted to assess the levels of MGMT promoter methylation, RNA and protein expression of cell lines to determine if there is a correlation between these three variables. We also hypothesized that MGMT promoter methylation mosaicism exists in glial tumours and would affect response to temozolomide. To assess this mosaicism we sampled multiple regions of each tumour intra-operatively and analyzed them using methylation specific PCR. Blood was drawn from these patients and the aforementioned MGMT assays were assessed in the peripheral blood mononuclear cells (PBMCs) to determine its usefulness as a prognostic tool. Our results show that MGMT promoter methylation is not a binary event, as currently calculated, but that an intermediate levels of promoter methylation percentage can be assessed. Promoter methylation also does not correlate with RNA or protein expression, but they do trend together. MGMT promoter methylation and RNA expression also vary intratumourally. MGMT promoter methylation can also be assayed in the PBMC fraction of blood in certain patients with high grade gliomas. These methylation levels appear to be associated with recurrence of the tumour and were altered after resection. Our study shows that promoter methylation may need to be looked at as a percentage-based variable and not as a binary system. Furthermore, due to intratumoural heterogeneity more areas of a tumour may need to be assessed for promoter me
O6-Methylguanine DNA methyltransferase (MGMT) est une protéine qui répare l'ADN à la suite de dommages génétiques causées par des traitements de chimiothérapiques tel que Temozolomide (TMZ). MGMT corrige l'addition alkyle à la position O6 de guanine et par conséquence, diminue l'efficacité des agents alkylateurs. La méthylation épigénétique de la région promoteure de MGMT dans les tissues de glioblastomes corrèle avec l'augmentation de la survie des patients traités avec le TMZ et la radiothérapie. Le but de notre recherche était d'évaluer le niveau de méthylation du promoteur de MGMT, de l'ARN, et de l'expression de la protéine dans des lignées cellulaires afain de déterminer si une corrélation existe entre ces trois facteurs. De plus, nous avons prédit que le niveau de méthylation du promoteur de MGMT varierait entre chaque échantillon de tumeur cérébrale. Nous avons testé plusieurs régions d'une tumeur par (MSP). Ce teste a été performé sur les cellules périphérales mononucléaires sanguines (CPMS). Nos résultats suggèrent que le niveau de la méthylation varie énormément. De plus le niveau d'expression de l'ARN et de la protéine de MGMT ne corrèle pas avec le niveau de méthylation du promoteur de MGMT. Ces niveaux sont souvent différents chez des échantillons qui proviennent de la même tumeur. Chez certains patients diagnostiqués avec un glioblastome, le niveau de méthylation dans les CPMS semble indiquer la récidive de la tumeur. Nous suggérons que la méthylation du promoteur de MGMT n'est pas binaire, et qu'elle doit être évaluée en terme de spectre variant de zero à cent pour cent. En plus, plusieurs spécimens d'une même tumeur doit être évalué par MSP. Finallement, l'analyse des CPMS peut servir d'outil pour prédire la réponse des patients traités avec des agents alkylateurs tel que TMZ.

Aggressive behaviour is a complex phenomenon that often arises in early childhood and typically decreases with age. Studies have shown that adults with severe aggression often have lower serotonin (5-HT) neurotransmission. The hypothesis of this thesis is that 5-HT alterations associated with childhood aggression are also defined by epigenetic mechanisms through differential methylation of critical genes in the 5-HT pathway that can be detected in peripheral white blood cells. Serotonin transporter (SLC6A4) was chosen in this study based on its importance in 5-HT function and preliminary genomic DNA microarray data. We first determined the state of DNA methylation of SLC6A4 promoter using pyrosequencing in T cells and monocytes isolated from blood of adult males with low or high childhood-limited aggression (N=25) and who have been followed since childhood. We then examined whether the state of DNA methylation of SLC6A4 promoter in the blood is associated with in vivo measures of brain 5-HT synthesis by integrating previously obtained Positron emission tomography (PET) data from these participants. Lastly, a luciferase reporter construct was generated to test whether SLC6A4 promoter methylation plays a functional role in its transcriptional activity. Significantly higher levels of methylation in high childhood-limited physical aggression (C-LHPA) group were observed in both T cells and monocytes at specific CpG sites. In addition, greater mean methylation of significantly altered CpGs was associated with lower 5-HT synthesis in the left and right lateral orbitofrontal cortex (OBFC) in both cells (N = 20). Moreover, in vitro methylation of the SLC6A4 promoter dramatically suppressed gene expression suggesting that methylation plays a functional role in gene regulation. Taken all together, these novel findings imply that SLC6A4 is epigenetically modulated by DNA methylation and that DNA methylation is associated with in aggression level observed during childhood. The association between higher DNA methylation and lower brain 5-HT synthesis supports the relevance of using DNA methylation in peripheral white blood cells as a marker for human brain 5-HT function. If these results can be confirmed in a larger sample, the identification of such 5-HT biomarkers may be beneficial for the prediction, prevention and evaluation of treatment of psychiatric disorders with 5-HT involvement.
Le comportement agressif est un phénomène complexe qui survient souvent lors de la petite enfance et diminue typiquement avec l'âge. Des études ont montré qu'une agressivité sévère chez l'adulte est associée à une plus faible neurotransmission de sérotonine (5-HT). L'hypothèse de cette thèse est que les modifications de neurotransmission de 5-HT associées à l'agressivité infantile sont également dépendantes de mécanismes epigénétiques, notamment du niveau de méthylation des gènes critiques à la régulation de la neurotransmission de la 5-HT qui peut être mesuré dans les leucocytes périphériques. Le transporteur de la sérotonine (SLC6A4) a été choisi dans cette étude dû à son importance dans la régulation du niveau de 5-HT ainsi qu'au vu de données provenant d'analyses préliminaires de micropuces d'ADN génomique. Nous avons premièrement déterminé les niveaux de méthylation d'ADN du promoteur de SLC6A4 en utilisant la méthode de pyroséquençage dans des cellules T et des monocytes isolés à partir de sang prélevé chez des hommes adultes ayant présenté des niveaux d'agressivité faibles ou importants durant l'enfance (N=25). Nous avons ensuite examiné si les niveaux de méthylation d'ADN du promoteur de SLC6A4 mesurés dans les cellules sanguines sont corrélés aux niveaux de synthèse cérébrale de 5-HT mesurés in vivo par tomographie par émission de positrons (TEP) chez les mêmes participants. Enfin, nous avons évalué in vitro grâce à une technique utilisant le gène rapporteur luciférase si le niveau de méthylation du promoteur de SLC6A4 est directement impliqué dans la modulation de son niveau de transcription. Nous avons mesuré des niveaux de méthylation de CpG spécifiques de SLC6A4 significativement plus importants dans les cellules T ainsi que dans les monocytes du groupe d'hommes ayant présentés une forte agressivité durant l'enfance (C-LHPA) par rapport au groupe ayant présenté une faible agressivité durant l'enfance. De plus, nous avons montré que ces niveaux de méthylation importants sont associés à une synthèse réduite de 5-HT au sein des cortex orbitofrontaux latéraux (OBFC) gauche et droit (N = 20). De plus, nous avons mesuré in vitro que la méthylation du promoteur SLC6A4 réprime fortement son expression. Ces données suggèrent que le niveau d'expression de SLC6A4 est modulé épigénétiquement par la méthylation de l'ADN et que les niveaux de méthylation du gène corrèlent avec les niveaux d'agressivité observés durant l'enfance. L'association entre les niveaux de méthylation d'ADN et de synthèse cérébral de 5-HT renforce la pertinence d'utiliser les niveaux de méthylation d'ADN de leucocytes périphériques comme indicateur du niveau de neurotransmission de 5-HT cérébrale. Ces résultats demandent à être confirmés au sein d'une cohorte plus importante. Cependant, l'identification d'un tel marqueur biologique pourrait être utile à la prédiction, la prévention et l'évaluation de traitements de désordres psychiatriques associés à des problèmes de neurotransmission de 5-HT.

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by cystic degeneration of chorionic villi and absence of embryo. It has been correctly proposed that deregulation of imprinted genes, expressed in a parent-of-origin specific pattern, leads to this pathology due to the fact that biparental and androgenetic HMs are indistinguishable at the phenotypic level. To determine the extent of the abnormal DNA methylation in two biparental moles from a family with a mutation in NALP7, we assessed long interspersed nuclear elements (LINEs), inactive X-linked genes and three tumour suppressor genes and demonstrated their normal levels of methylation. Since the identification of the NALP7 as the causative gene of recurrent HMs, the role of inflammation and immunity has come into light as a possible cause of this disease. Due to the known role of NALP7 in cytokine processing, we addressed the ability of the patients' peripheral blood mononuclear cells (PBMCs) to secrete cytokines in response to stimulation with various antigenic molecules. We found a reduced level of IL-1beta and TNF-alpha secretion by the patients' PBMCs suggesting that abnormal processing of several cytokines may underlie this disease.

The development of healthy gametes is paramount to the health of progeny and to the survival of a species. Epigenetic information contained within gametic DNA in the form of DNA methylation is essential for germ cell and embryo development. DNA methylation is a genome-wide phenomenon involved in the control of gene expression and chromosome structure and stability. During germ line development, patterns of DNA methylation are established in a sex- and sequence-specific manner. The primary goal of the work presented in this thesis is to gain an understanding of the nature of the genome-wide pattern of DNA methylation in germ cells and to study its progression during germ cell development. The complexity of male germ cell development has been well studied in mice and thus makes an excellent system in which to study germ cell DNA methylation. Firstly, genome-wide patterns of DNA methylation in adult male germ cells were determined using a variety of techniques. Results from these studies demonstrate that the DNA methylation pattern in male germ cells is highly distinct from that of somatic cells. The reorganization of the germ cell pattern is associated with chromosomal features such as the chromosomal banding pattern and regional GC content. Secondly, by examining purified populations of male germ cells, we have determined that patterns of DNA methylation are being acquired during spermatogenesis. De novo methylation and demethylation events occur in a sequence-specific manner prior to the meiotic phase of germ cell development. Finally, the stability of these patterns was studied by perturbing DNA methyltransferase activity. The study of germ cells lacking a functional Dnmt3L gene demonstrates that the abnormalities displayed in these cells are associated with a failure to acquire normal levels of DNA methylation. In addition, the treatment of mice with the hypomethylating agent, 5-aza-2'-deoxycytidine, results in adverse effects on sperm function and is associated with sequence-specific hypomethylation. Collectively, these studies have uncovered several novel aspects of DNA methylation in male germ cells and contribute to our understanding of the roles) for epigenetic phenomena in the development and maintenance of healthy gametes.

For the past 45 years, QKI has been studied for its role in the processes of development and central nervous system myelination using the qkv mouse. The presence of a single KH domain and the recent identification of a high-affinity binding site in mRNAs, suggests that it can bind to and regulate mRNAs through processes such as stability, splicing and transport. As a member of the STAR RNA binding family of proteins the QKI isoforms may also be involved in cell signaling pathways.
QKI's involvement in all of these processes, lead us to examine both the protein partners and the mRNA targets of the QKI complex in order to identify potentially new pathways regulated by QKI. In doing so, we identified a novel direct protein-protein interaction with PABP and for the first time described the relocalization of QKI to cytoplasmic granules following oxidative stress. In addition, in vivo mRNA interaction studies were performed and allowed the identification of approximately 100 new mRNA targets in human glioblastoma cells. One of the targets identified was VEGF mRNA.
Another QKI target mRNA is MBP, a major protein component of the myelin sheath and the candidate auto-antigen in multiple sclerosis (MS). In vivo MBP is symmetrically dimethylated on a single arginine residue. To further establish the role of the methylation of MBP in myelination, a methyl-specific antibody and an adenovirus expressing a recombinant protein arginine methyltransferase 5 (PRMT5) was generated. We show that methylated MBP is found in areas of mature myelin and that overexpression of the PRTM5 blocked the differentiation of oligodendrocytes.
Taken together these datas implicate QKI for the first time in the process of human cancer angiogenesis and could explain the vascularization defects observed in some of the qkI mutant mice. In addition, arginine methylation of MBP may prove to have an important role in the process of myelination and in the pathogenesis of demyelination and the autoimmune reaction in diseases such as MS.

Suicide is a problem resulting from the interaction between several factors. Among these factors, early-life adversity, characterized by child sexual and physical abuse as well as parental neglect, is one of the strongest risk factors for depression and suicidal behaviors. While it is clear that child abuse increases the risk for depression and suicide, the mechanisms mediating these effects are still unknown. Recent evidences suggest that epigenetic mechanisms may be involved in mediating the effects of early-life adversity on behavior. Early-life stress in animals has been shown to alter DNA methylation in genes regulatory regions what, in turn, has been associated with changes in gene expression and behavioral modifications. Importantly, similar changes have been reported in several genes in the human brain. However, although epigenetic modifications have been found in several genes, the extent of epigenetic changes induced by early-life adversity is still unknown and their impacts on increasing suicide risk are unclear. This thesis aims at expanding our understanding of suicide by investigating one potential mechanism by which early-life adversity may increase vulnerability to mood disorders and suicide, namely epigenetic regulation of gene expression. We first expanded our characterization of DNA methylation alterations induced by early and adult-life stress in the regulatory regions of the glucocorticoid receptor gene which is one of the most consistent alterations associated with stress, depression and suicidal behaviors. We then opted for a genome-wide characterization of DNA methylation alterations found in the brains of suicide completers with and without a history of child abuse using microarray analyses focusing on gene promoters throughout the genome. Our findings revealed several DNA methylation alterations in genes with known roles on behavioral regulation and identified several new genes and networks which may be specifically altered by early-life adversity. We also characterized the functional impact of these alterations on gene expression and promoter transcriptional activity. Overall, this work identifies and characterizes the molecular mechanisms by which environment induces behavioral changes conferring vulnerability toward mood disorders and suicidal behaviors.
Le suicide est un problème qui résulte de l'interaction entre plusieurs facteurs. Un de ces facteurs, l'adversité durant l'enfance se définissant par une histoire d'abus sexuel et/ou physique de même que par la négligence parentale, est un des facteurs de risque les plus importants en ce qui à trait à l'augmentation des risques de développer des comportements dépressifs et suicidaires à l'âge adulte. Or, si une histoire d'abus augmente les risques de dépression et de suicide, les mécanismes responsables de ces effets comportementaux demeurent néanmoins méconnus. De récentes avancées laissent présager que l'impact comportemental d'une histoire d'abus serait transmis par des mécanismes épigénétiques. En effet, de nombreuses études suggèrent que le stress au cours du jeune âge chez les rongeurs interfère avec l'établissement des schémas de méthylation dans les régions régulatrices des gènes ce qui, en retour, modifie les niveaux d'expression des gènes et induit des changements comportementaux semblables aux symptômes de la dépression. Fait important, des changements semblables ont également été rapportés chez l'humain. Malgré tout, l'ampleur des modifications épigénétiques induites par une histoire d'abus durant l'enfance, de même que leurs conséquences comportementales, demeurent toujours inconnues. Cette thèse vise à améliorer notre compréhension du suicide en élucidant un mécanisme potentiel, soit les mécanismes épigénétiques, par lequel le stress au cours du jeune âge augmenterait la vulnérabilité aux troubles de l'humeur et au suicide. Nous avons premièrement poursuivit la caractérisation des changements de methylation de l'ADN induits par une histoire d'abus dans la région régulatrice du gène du récepteur des glucocorticoides, un des gènes les plus fréquemment associés au stress, la dépression et le suicide. Nous avons ensuite opté pour une approche globale visant à identifier l'ensemble des modifications de methylation de l'ADN dans les régions promotrices de l'ensemble du génome dans le cerveau des suicidaires avec ou sans histoire d'abus durant l'enfance. Nos résultats révèlent la présence de nombreux changements de methylation de l'ADN dans plusieurs gènes impliqués dans la régulation comportementale. Nos résultats supportent également l'implication de plusieurs autres gènes affectés spécifiquement par une histoire d'abus durant l'enfance. Les travaux de cette thèse ont également mis l'emphase sur l'impact fonctionnel de ces altérations sur l'expression des gènes et la régulation de l'activité transcriptionnelle des régions promotrices affectées. Dans l'ensemble, ce travail détaille et définit les mécanismes moléculaires par lesquels l'environnement induit des changements comportementaux conférant une vulnérabilité envers les troubles de l'humeur et les comportements suicidaires.

DNA methylation is an epigenetic modification that is essential for germline and embryo development. DNA methylation establishment occurs in the germline in a gender and site specific manner through the action of DNA cytosine-5-methyltransferase (DNMT) enzymes. Spermatozoa have been shown to have a unique configuration of DNA methylation as compared to somatic cells. Germline DNA methylation may also depend on the availability of methyl donors provided by the folate pathway through the action of 5,10-methylenetetrahydrofolate reductase (MTHFR). Absence of MTHFR has been associated with methylation loss in mature sperm as well as decreased male fertility. This thesis presents new data exploring the dynamics of DNA methylation at nonpromoter intergenic sites during the perinatal period in normal conditions and in the setting of perturbed expression of Dnmt3L and Mthfr. The key phase of DNA methylation acquisition in male germ cells was determined to occur during prenatal development of the germline suggesting that DNA methylation may be particularly vulnerable to in utero insults. DNA methylation acquisition was found to be a dynamic process at individual sites including loci that demonstrated incomplete erasure suggesting a potential for transgenerational inheritance of DNA methylation errors. Perturbation of the expression of Dnmt3L, a DNMT with no endogenous methylation activity, revealed that it may be responsible for not only directing DNA methylation to specific loci but also for controlling the timing of DNA methylation acquisition. Spermatogonial stem cell (SSC) cultures were developed as a model for examining DNA methylation in the male germline. While DNA methylation was stable in normal culture conditions, it could be perturbed by haploinsufficiency of either Dnmt3L or Mthfr. Further modification of culture conditions through changes of methionine concentration in Mthfr+/- SSC cultures resulted in increased variability of DNA methylation indicating a generalized effect of MTHFR on DNA methylation and possibly its interaction with other methyl group dependent processes such as histone methylation. This thesis presents a number of novel findings that begin to unravel the mechanisms of DNA methylation and epigenetic modification of the male germline.
La méthylation de l'ADN est une modification épigénétique essentielle au développement germinal et embryonnaire. Elle est établie en une région et un genre spécifiques sous l'action des ADN cytosine-5-méthyltransférases (DNMT). Il a précédemment été démontré que les spermatozoïdes se distinguent des cellules somatiques au niveau de la méthylation de leur ADN. Celle-ci dépend entre autres de la disponibilité des groupes méthyles fournis par l'action de la 5,10-méthylènetétrahydrofolate réductase (MTHFR), une enzyme impliquée dans le cycle des folates. L'absence de cette enzyme a été associée à une perte de la méthylation de l'ADN spermatique ainsi qu'à une baisse de la fertilité masculine. De nouvelles données sur la méthylation de régions intergéniques en période périnatale, impliquant ou non une altération de l'expression des gènes Dnmt3L et Mthfr, sont exposées dans la présente thèse. Une méthode d'analyse chromosomique a d'abord confirmé que l'acquisition de la méthylation sur l'ADN des cellules germinales mâles a lieu durant l'embryogénèse. Cette assimilation de la méthylation s'est avérée être un processus dynamique en lien à des loci démontrant un effacement incomplet de l'empreinte génomique. Ces données suggèrent non seulement une vulnérabilité de la méthylation de l'ADN aux perturbations in utero, mais également un possible héritage transgénérationnel des erreurs de méthylation. L'altération de l'expression de Dnmt3L a révélé que cette enzyme joue probablement un rôle dans l'emplacement et la synchronisation de la méthylation de l'ADN. Afin d'étudier cette modification épigénétique dans l'ADN spermatique, des cellules souches spermatogoniales (CSS) ont été cultivées. Les cellules développées dans des conditions normales ont présenté une régulière méthylation de leur ADN, alors que les cellules haploinsuffisantes des gènes Dnmt3L ou Mthfr ont révélé une méthylation variable. Des modifications apportées aux concentrations de méthionine des cultures de CSS Mthfr+/- ont également exercé un effet perturbateur sur la méthylation. Ces résultats indiquent que MTHFR influence la méthylation de l'ADN et intervient possiblement dans d'autres processus impliquant des groupes méthyles, tels que la méthylation des histones. Les conclusions présentées dans cette thèse permettent une meilleure compréhension des mécanismes de méthylation de l'ADN et de modification épigénétique de la lignée germinale masculine.

It is well known that DNA methylation in gene promoter regions inhibits gene transcription and that tissue-specific gene expression is partially under the control of this transcription regulatory mechanism. In this study, bovine mammary gland tissues were collected from individual animals in lactating and non-lactating stages to investigate the DNA methylation patterns in the kappa-casein gene and alpha-lactalbumin gene core promoter regions using the bisulphite treatment in combination with polymerase chain reaction (PCR) sequencing. Different methylation status of each sample was classified into three categories, namely methylation at known transcription factor binding domains, methylation at core promoter non-binding domains and the absence of cytosine methylation. Real-time quantitative PCR was used to quantify the transcription levels of the kappa-casein and alpha-lactalbumin genes from the collected samples. A comparative method was used and fold-change values were calculated based on the comparison of the normalized threshold values of samples from different physiological stages as well as on various methylation patterns observed in their core promoter regions. Statistical analyses showed that the expressions of the kappa-casein and alpha-lactalbumin genes were significantly different in lactating and non-lactating mammary gland tissues. The methylation observed in the core promoter region of bovine alpha-lactalbumin gene was found to be associated with its gene expression. On the other hand, the methylation found in the core promoter region of bovine kappa-casein gene did not have any effect on its gene transcript levels.

DNA methylation has been shown to be involved in switching a number of genes on or off in particular cells. The relationship between DNA methylation and $ beta$-casein gene expression in the mammary tissue of lactating cows and mammary epithelial cells was examined. A positive correlation existed between hypomethylation of two MspI/HpaII sites in the body and one MspI/HpaII site in the 3$ sp prime$ end of the $ beta$-casein gene and its expression. In addition to these sites, hypomethylation of a distal MspI/HpaII site and HindIII sensitivity at a HindIII site also correlated with gene expression. Five DNase I hypersensitive sites were located within a 8 kb fragment. These sites designated as H1 to H5 were mapped approximately $-5, -1.3, -0.2,$ 1.7 and 2.5 kb with respect to the start site of transcription, respectively. The H2 and H3 sites were within a 1790 bp sequence that has been reported to contain a responsive element for prolactin and extracellular matrix dependent regulation and the binding site for mammary gland specific factor.
To study the dynamic changes in hypomenthylation at the MspI/HpaII sites and HindIII sensitivity, mammary tissues from pregnant heifers were evaluated. Site specific demethylation was observed depending on the stage of gestation. Demethylation of two MspI/HpaII sites (denoted M2 and M4) occurred during the early gestation, progressed slowly until mid-pregnancy, and rapidly during the last part of pregnancy. During the early stages of gestation, changes in the HindIII sensitivity in the coding domain of the $ beta$-casein gene also took place. Despite changes in HindIII sensitivity, the second HindIII site remained resistant to HindIII. By the fifth stage of gestation, the third MspI/HpaII site (M3) became less methylated and during this time the H2 site became more sensitive to HindIII. Northern analysis confirmed that demethylation of the M3 site and the acquisition of HindIII sensitivity at the H2 site was correlated with $ beta$-casein transcription.
Although $ alpha$-lactalbumin and $ beta$-casein genes are structurally and evolutionarily unrelated, they likely share common regulatory features, since both are expressed in the mammary gland during lactation. To investigate this possibility, methylation of the $ alpha$-lactalbumin gene was examined. In vivo studies revealed hypomethylation of the bovine $ alpha$-lactalbumin gene at two MspI sites and a cluster of two HhaI sites during the first and second stage of gestation, respectively. Furthermore, hypomethylation events occured only in the functional gene and not in pseudogenes, and the hypomethylation pattern was established prior to gene expression.
Taken together, the present finding suggest that DNA hypomethylation is necessary for the expression of two mammary-specific milk protein genes, $ beta$-casein and $ alpha$-lactalbumin. Hypermethylation within the body of these genes may silence these genes in non-expressing tissues and in non-epithelial cells within the mammary gland during lactation.

Antisocial personality disorder (ASPD) is a condition characterized by elevated impulsive aggression and increased risk for criminal behaviour and incarceration. Deficient activity of the monoamine oxidase A (MAOA) gene is suggested to contribute to serotonergic system dysregulation strongly associated with impulsive aggression and ASPD. The potential contribution of epigenetic processes, which modulate gene expression without altering the underlying genomic code, towards the dysregulation of MAOA in ASPD is not yet understood.The current study aimed to elucidate the role of epigenetic processes in altered MAOA expression and serotonin regulation in a population of offenders with ASPD compared to healthy controls. Results suggest MAOA promoter hypermethylation contributes to downregulated gene expression and elevated whole-blood serotonin in offenders with ASPD. These results are consistent with prior literature suggesting MAOA and serotonergic dysregulation in antisocial populations. Further, our results offer the first evidence suggesting epigenetic mechanisms may contribute to MAOA dysregulation in antisocial offenders.
Le trouble de personnalité antisociale est une condition qui se caractérise par un niveau élevé d'agression impulsive ainsi qu'un risque accru de comportements criminels et d'incarcération. Il a été suggéré qu'une activation déficitaire du gène monoamine oxydase A (MAOA) contribuerait à une dysrégulation du système sérotogénique, qui est fortement associé à l'agression impulsive et au trouble de personnalité antisociale. La potentielle contribution des processus épigénétiques modulant l'expression génétique sans altérer le code génomique sous jacent dans la dysrégulation du MAOA chez les individus atteint du trouble de personnalité antisociale n'est pas encore comprise. L'étude suivante avait comme objectif d'élucider le rôle des processus épigénétiques dans l'altération de l'expression du MAOA et de la régulation de la sérotonine dans une population incarcérée avec un trouble de personnalité antisociale, lorsque comparés à des contrôles sains. Les résultats suggérent que le promoteur d'hyperméthylation MAOA contribue à une réduction de l'expression génétique et à un niveau élevé de sérotonine sanguin chez les incarcérés avec un trouble de personnalité antisociale. Ces résultats sont cohérents avec la littérature suggérant que la dysrégulation du MAOA et de la sérotonine est présente dans les populations antisociales. De plus, nos résultats représentent la première évidence suggérant que les méchanismes épigénétiques pourraient contribuer à la dysrégulation du MAOA chez les incarcérés avec un trouble de personnalité antisociale.Le trouble de personnalité antisociale est une condition qui se caractérise par un niveau élevé d'agression impulsive ainsi qu'un risque accru de comportements criminels et d'incarcération. Il a été suggéré qu'une activation déficitaire du gène monoamine oxydase A (MAOA) contribuerait à une dysrégulation du système sérotogénique, qui est fortement associé à l'agression impulsive et au trouble de personnalité antisociale. La potentielle contribution des processus épigénétiques modulant l'expression génétique sans altérer le code génomique sous jacent dans la dysrégulation du MAOA chez les individus atteint du trouble de personnalité antisociale n'est pas encore comprise. L'étude suivante avait comme objectif d'élucider le rôle des processus épigénétiques dans l'altération de l'expression du MAOA et de la régulation de la sérotonine dans une population incarcérée avec un trouble de personnalité antisociale, lorsque comparés à des contrôles sains. Les résultats suggérent que le promoteur d'hyperméthylation MAOA contribue à une réduction de l'expression génétique et à un niveau élevé de sérotonine sanguin chez les incarcérés avec un trouble de personnalité antisociale. Ces résultats sont cohérents avec la littérature suggérant que la dysrégulation du MAOA et de la sérotonine est présente dans les populations antisociales. De plus, nos résultats représentent la première évidence suggérant que les méchanismes épigénétiques pourraient contribuer à la dysrégulation du MAOA chez les incarcérés avec un trouble de personnalité antisociale.

DNA replication is cruciaI for the transmission of genetic information. Understanding the enzymology involved in this complex process will allow further insight into its mechanism. Experimental evidence indicates a role for DNA ligase I in DNA replication. Techniques of molecular and cellular biology and immunology were utilized in this study to further investigate DNA ligase I and clarify its involvement and interaction with other proteins in DNA replication. Immunofluorescence studies were performed to examine the intracellular distribution of DNA ligase I. Confocal analysis of indirect immunofluorescence detection of DNA ligase I using affinity purified anti-human DNA ligase I antibodies showed nuclear localization of DNA ligase I in distinct foci resembling those structures seen in detection of centres of DNA replication and other DNA replication proteins. Immunoprecipitation experiments were performed on extracts of MDBK cells to examine possible interactions of DNA ligase I with the DNA replication cofactor, PCNA; and no interactions were detected.

Recent studies indicate that children conceived using assisted reproductive technologies are at increased risk of growth disorders and genomic imprinting diseases. Reports have suggested that these disorders could result from epimutations including abnormal DNA methylation patterns in imprinted genes. DNA methylation, catalyzed by a family of DNA methyltransferases (DNMTs), is both heritable and reversible and is the major epigenetic mark associated with imprinting. We propose that techniques used in ART, such as ovarian stimulation, and factors underlying infertility might interact to disrupt DNA methylation. In this study, we investigated whether superovulation in combination with DNMT1o deficiency could induce more severe perturbations of imprinted methylation in mouse blastocysts, than ovarian stimulation alone. Female mice heterozygous for lack of oocyte-derived DNMT1o and Dnmt1o +/+ controls were subjected to moderate (6.25 IU PMSG/hCG) or high (10 IU PMSG/hCG) hormone doses and then mated with B6 (CAST7) males. At 3.5 days, ovulation sites were counted and blastocysts were collected. Methylation patterns of the imprinted genes Snrpn, H19 and Kcnq1ot1 were assessed by bisulfite sequencing from pools of 5 to 8 blastocysts. The high hormonal dose induced higher ovulation rates but also the lowest yield of blastocysts. The moderate and high doses were selected for the DNA methylation analysis. DNMT1o deficiency did not significantly perturb Snrpn methylation patterns in blastocysts of females injected with the moderate dose of hormones. H19 methylation patterns were more susceptible to perturbation, showing a preliminary indication of a slight gain of methylation on the maternal allele after superovulation at the moderate and high doses. The high dosage regimen showed evidence of perturbed Kcnq1ot1 methylation patterns in one of the pools of blastocysts with the paternal allele showing an unexpected gain of methylation. Follow-up studies will be conducted by transferring blastocysts into pseudopregnant females and examining the effects at later times in development (i.e. mid-gestation). The aim will be to determine whether superovulation and DNMT1o-deficiency are associated with abnormalities in genomic imprinting in postimplantation development.
Des études récentes indiquent que les enfants conçus à l'aide de procréation médicalement assistée (PMA) sont à risque accru de troubles de la croissance et de maladies à empreinte génomique. Des rapports ont suggéré que ces troubles pourraient résulter d'épimutations anormales au niveau de la méthylation de l'ADN dans les gènes imprimés. La méthylation de l'ADN, catalysée par une famille d'ADN méthyltransférases (DNMTs), est la principale marque épigénétique associée à l'empreinte. Nous proposons que les techniques utilisées dans le cadre de la reproduction assistée, telles que la stimulation ovarienne, et l'infertilité pourraient interagir de manière à perturber la méthylation de l'ADN. Dans cette étude, nous avons examiné dans quelle mesure la superovulation jumelée avec un déficit au niveau de la DNMT1o pourrait induire davantage de perturbations graves de la méthylation dans les blastocystes de souris, que la stimulation ovarienne seule. Des souris femelles hétérozygotes pour le manque d'ovocyte dérivé DNMT1o et les contrôles ont été soumis à une stimulation de l'ovaire à dose modérée (6,25 UI de PMSG / HCG) ou élevée (10 UI de PMSG / hCG) d'hormones et puis accouplées avec des mâles B6 (CAST7). À 3,5 jours, les sites d'ovulation ont été comptés et blastocystes recueillis. Les profils de méthylation des gènes à empreinte génétique Snrpn, H19 ainsi que Kcnq1ot1 ont été évalués à l'aide du séquençage au bisulfite à partir d'un pool de 5 à 8 blastocystes. La dose élevée hormonale induit des taux d'ovulation élevés, mais aussi le plus faible rendement de blastocystes. Nous avons sélectionné les doses modérées et élevées pour l'analyse de la méthylation d'ADN. La carence en DNMT1o n'a pas perturbé de manière significative les profils de méthylation dans le gène Snrpn au sein de blastocystes de femelles injectées avec la dose modérée d'hormones. Les profils de méthylation de H19 ont été plus sensibles aux perturbations, révélant une hyperméthylation sur l'allèle maternel après superovulation à la dose modérée et élevée. Le dosage élevé induit une plus grande perturbation des profils de méthylation de Kcnq1ot1 dans l'un des pools: l'allèle paternel a arboré un gain surprenant de méthylation.D'autres études seront menées en effectuant un transfert de blastocystes chez des femelles pseudo-enceintes plus tard dans le développement (c'est-à-dire à mi- gestation), afin de déterminer si la combinaison de la superovulation et de la carence en DNMT1o est associée à des anomalies dans les gènes à empreinte génétique après l'implantation de l'embryon.

DNA methylation is an epigenetic phenomenon catalysed by a family of DNA methyltransferases (DNMTs) and is tightly regulated throughout spermatogenesis. In these studies, I employed pharmacological and genetic means to disrupt DNA methylation in the male germ line. First, to lower levels of DNMT activity, I treated adult wild-type (Dnmt1+/+) and DNMT-deficient (Dnmt1c/+) male mice with the anticancer agent 5-aza-2'-deoxycytidine (5-azaCdR), which incorporates into DNA and inhibits methylation. Dnmt1+/+ males treated with clinical doses of 5-azaCdR for 7 weeks, to expose germ cells throughout their development, exhibit abnormal testicular histology, as well as dose-dependent reductions in testis weight, sperm count, fertility, and sperm motility. In contrast, Dnmt1c /+ males are evidently partially protected from the deleterious effects of 5-azaCdR, likely because of lowered levels of DNMT1. Although 5-azaCdR-treated Dnmt1c/+ males display reduced testis weight, they do not have altered sperm number or testicular histology; these males also have greater changes in sperm DNA methylation relative to treated Dnmt1+/+ males. Interestingly, elevated preimplantation loss is observed in females mated with treated males of either genotype. This preimplantation loss appears to result from a reduction in oocyte fertilisation, perhaps because of altered sperm motility. Moreover, higher levels of blastocyst loss were observed in embryos derived from matings with treated Dnmt1 +/+ males. I suggest that the testicular effects and reduced fertility of treated Dnmt1+/+ males are the result of both the cytotoxic and hypomethylating effects of 5-azaCdR, whereas the changes observed in 5-azaCdR-treated Dntnt1c /+ males may be due more to hypomethylation and its consequences.
Our second genetic model targetted the methyl supply necessary for DNA methylation. Methylenetetrahydrofolate reductase (MTHFR) is integral to the folate pathway and is necessary for methionine and S-adenosylmethionine formation; levels of MTHFR are highest in the testis, which suggests a role for this enzyme in spermatogenesis. Indeed, severe MTHFR deficiency (Mthfr -/-) results in severely abnormal spermatogenesis and infertility. Within my studies, I showed that maternal administration of betaine, an alternative methyl donor, throughout gestation and nursing, results in reduced germ cell apoptosis in male pups soon after birth. When betaine supplementation is maintained post-weaning, spermatogenesis is partially restored and fertility increases significantly. Here, I provide evidence that perturbation of the components of the DNA methylation pathway detrimentally affects male germ cell development and fertility.

Improper acquisition of DNA methylation patterns during the fetal period in germ cells of male mice is associated with impaired meiosis and infertility. MTHFR is a key folate pathway enzyme involved in providing methyl groups for DNA methylation. The goal of these studies was to evaluate DNA methylation patterns in spermatogonia from mice heterozygous for a targeted deletion in Mthfr (Mthfr+/-) as compared to those of their wildtype, Mthfr+/+, littermates. DNA methylation patterns were similar at imprinted genes and intergenic sites across chromosome 9 in neonatal spermatogonia of Mthfr+/+ and Mthfr+/- mice. We then established spermatogonial stem cell (SSC) cultures from Mthfr+/+ and Mthfr+/- mice to examine the stability of DNA methylation patterns over time in culture and determine whether methionine supplementation could restore any DNA methylation defects caused by MTHFR haplosufficiency. There were no differences detected between early and late passages, suggesting DNA methylation patterns in SSC clusters are generally stable in culture. Cultured SSC clusters treated with 20-fold normal medium methionine concentrations showed an overall increase in the levels of DNA methylation across chromosome 9, indicating DNA methylation can be perturbed in culture. Additionally, Mthfr+/- SSC clusters cultured in varying concentrations of methionine demonstrated a significantly increased variance of DNA methylation at multiple loci across chromosome 9 compared to Mthfr+/+ SSC clusters treated the same way. Together our results provide evidence that DNA methylation patterns of SSC clusters are stable in culture but can be perturbed by altered methionine and MTHFR levels.
L'acquisition inexacte de patrons de méthylation d'ADN des cellules germinales pendant la période fœtale chez la souris mâle est associée à des désordres de méiose et d'infertilité. L'enzyme MTHFR joue un rôle clé dans le processus de méthylation d'ADN puisqu'elle est impliquée dans une cascade produisant les groupements méthyles nécessaires à la réaction. L'objectif des travaux présentés dans ce mémoire était d'évaluer les profiles de méthylation d'ADN dans les spermatogonies provenant de souris hétérozygotes pour une suppression ciblée de Mthfr (Mthfr+/-), en comparaison avec celles découlant des compagnons de type sauvage de la même portée (Mthfr +/+). Nous avons déterminé que dans les spermatogonies néonatales de souris Mthfr+/+ et Mthfr+/- les patrons de méthylation sont demeurés similaires au niveau des gènes à empreinte ainsi qu'à divers sites intergéniques retrouvés à travers le chromosome 9. Subséquemment nous avons établi un système de culture de cellules souches spermatogoniales (SSC) à partir de souris Mthfr+/+ et Mthfr+/- afin d'examiner la stabilité des profiles de méthylation en culture et de déterminer si un supplément de méthionine peut restaurer un dérèglement de méthylation d'ADN causé par une haploinsuffisance de MTHFR. La période de culture (nombre de passages faible vs élevé) n'a nullement altérée les patrons de méthylation d'ADN, ce qui suggère que cette modification épigénétique est globalement très stable dans les SSCs en culture. Le traitement de ces même SSCs avec un milieu 20 fois plus élevé en méthionine occasionne par contre une augmentation des niveaux globaux de méthylation d'ADN à travers le chromosome 9, démontrant que cette modification post-traductionnelle peut être perturbée en culture. De plus, la culture des SSCs Mthfr+/- dans diverses concentrations de methionine démontre une augmentation significative de la variance des niveaux de méthylation d'ADN pour une multitude de loci à travers le chromosome 9 comparativement aux SSCs Mthfr+/+ soumis aux mêmes traitements. Ensemble, nos résultats suggèrent que les patrons de méthylation d'ADN des SSCs sont normalement stables en culture mais peuvent cependant être perturbés par les niveaux de méthionine et MTHFR.

Formation of gametes capable of supporting development is dependent on a number of genetic and epigenetic events. DNA methylation is an epigenetic modification catalyzed by enzymes named DNA methyltransferases (DNMTs). In the mouse, methylation of DNA is associated with the control of gene expression and proper embryo development. Methylation patterns are established in a sex- and sequence-specific manner during male and female germ cell development and further modified during early embryonic development. Even though new DNMTs have recently been identified, little information is known on the origin of the methylation marks during male germ cell development (spermatogenesis). The main goal of the work presented in this thesis was to gain a better understanding of the enzymes involved in creating the epigenetic program of the male genome. The first step in doing so involved comparing the temporal expression profiles of DNA methyltransferases in the developing testis and ovary. The expression profiles obtained indicated that in the male, DNMT3a and DNMT3L could be involved in de novo methylation while DNMT3b and DNMT1 could be responsible for maintaining methylation patterns following DNA replication. Next, characterization of Dnmt3a and Dnmt3b expression in isolated postnatal male germ cells revealed how tightly regulated the expression of these genes is during spermatogenesis: specific transcript variants and protein isoforms of each DNMT are differentially expressed during male germ cell development. Finally, assessing the effect of Dnmt3L inactivation on the male germ line exposed the presence of a mitotic defect in germ cells lacking this protein. DNA methylation analyses revealed that many loci throughout the genome are marked for methylation by DNMT3L, indicating a more global role for this enzyme than that previously reported in genomic methylation patterning in the male germ line. As methylation patterns instituted during spermatogenesis have to be properly established for accurate transmission of epigenetic information to the next generation, the studies presented here contribute to our knowledge of the events leading to the creation of the epigenetic program necessary for the formation of healthy gametes.

Folate is a critical determinant in male reproductive health. During spermatogenesis, there are massive alterations to the epigenome associated with tightly regulated gene transcription and chromatin reorganization including a tightly regulated pattern of histone H3 methylation. In vitro experiments were conducted to determine whether folate depletion could alter global histone H3 methylation at lysine 4 and 9 in cultured spermatogonia-like GC-1 cells. Folate depleted media did not alter global levels of histone H3 methylation at lysine 4 and 9 in spermatogonia-like GC-1 cell. In vivo experiments were used to determine the extent to which folate deficiency, from early embryonic development to adulthood, impacts histone methylation and male reproductive health in a mouse model. C57BL/6 females were fed either a folate-sufficient diet or a folate-deficient diet two weeks prior to breeding and through pregnancy and lactation. Male offspring received the same diet as their mother until sacrifice. Testes and epididymides were collected at postnatal day 6 to 18, and at 6 and 13-14 weeks of age. At 8 weeks of age, male pups were assessed in fertility trials. Folate deficiency severely compromised male reproductive health. Folate deficient males had reduced epididymis weight and defects in spermatogenesis. Abnormalities included the presence of multinucleated cells, sloughing of germ cells, and a slight increase in germ cell apoptosis. Alterations in key fertility parameters included an increase in sperm morphological defects and consequently decreased pregnancy rate and litter size. Remarkably, gene activating histone H3 tri-methylation at lysine 4 was significantly reduced i
Le folate joue un rôle déterminant au niveau de la santé reproductrice de l'homme. Au cours de la spermatogenèse, il y a d'importantes modifications à l'épigénome liées à la transcription de gènes et à la réorganisation de la chromatine, incluant une régulation essentielle de la méthylation de l'histone H3. Des expériences in vitro furent effectuées afin de déterminer si une réduction de la concentration de folate dans le milieu de culture pourrait altérer la methylation globale de l'histone H3 aux lysines 4 et 9 de cellules cultivées spermatogonia-like GC-1. La réduction de folate n'a pas affecté la méthylation globale de l'histone H3 aux lysines 4 et 9 de cellules spermatogonia-like GC-1. Des expériences in vivo, chez un modèle murin, furent réalisées afin de déterminer les répercussions d'une déficience en folate, du développement embryonnaire précoce jusqu'à l'âge adulte, sur la méthylation de l'histone et la santé reproductrice de l'homme. La santé reproductrice masculine fut sévèrement compromise par une déficience en folate. Les males déficients en folate démontrèrent une réduction du poids des épididymes et une augmentation des anomalies spermatogéniques incluant des cellules multinuclées, la desquamation de cellules germinales et une légère augmentation de cellules germinales apoptotiques. Les principaux paramètres de fertilité ont démontré une diminution du nombre de spermatozoïde morphologiquement normal et par conséquent une réduction du taux de grossesse et de la taille des portées. Remarquablement, la tri-méthylation de l'histone H3 à la lysine 4, associée à l'activation de l'expression des gènes, a été f

The enzyme DNA methyltransferase 3-like (DNMT3L) is essential for de novo DNA methylation in germ cells, proper germ cell development, and has been reported as the first paternal effect gene in mammals. In mice, although Dnmt3L heterozygous or haploinsufficient (+/-) males are fertile, there are associated chromosomal compaction abnormalities and the males have been shown to sire an increased number of aneuploid offspring even if the offspring do not possess the mutant allele (the paternal effect). Despite this, there have been no reported DNA methylation abnormalities in Dnmt3L haploinsufficient mature male germ cells. This is unexpected as DNMT3L's only known function is its role in DNA methylation and some of the phenotypes seen in Dnmt3L+/- males were observed in sperm. Therefore we assessed DNA methylation defects in Dnmt3L haploinsufficient mature sperm using a genome-wide, bisulfite sequencing based method, reduced representation bisulfite sequencing (RRBS). Our experiments revealed a total of 367 hypomethylated sites and 88 hypermethylated sites found in the sperm of Dnmt3L+/- as compared to wildtype mice. These differentially methylated sites were determined by comparing 100bp genomic tiles that had DNA methylation levels that were at least 20% different between Dnmt3L+/+ (n=4) and +/- (n=5) sperm. The hypomethylated regions were mainly found in intergenic LINE1 sequences, while the hypermethylated regions were found more frequently in regulatory and exonic sequences. The findings confirm that a dosage change, without complete obliteration, of a key epigenetic regulator in the male germline can result in DNA methylation abnormalities. These gene dosage effects associated with Dnmt3L haploinsufficiency also prompted us to test the effects of Dnmt3L overexpression in the murine germline. Therefore we created a transgenic mouse model that overexpresses endogenous DNMT3L at a higher level than in wildtype germ cells. This model was successfully created and initial mating trials of the F0 revealed a trend toward female sub-fertility. Although preliminary, it appears as though the overexpression of Dnmt3L in the mouse using a germ cell specific promoter is of greater consequence to the female germline in comparison to that of the male.
L'enzyme ADN méthyltransférase 3-like (DNMT3L) est essentielle durant l'établissement initial de la méthylation de l'ADN dans les cellules germinales ainsi que dans le développement de ces dernières. Dnmt3L est le premier gène à effet paternel caractérisé chez le mammifère. Bien que les mâles Dnmt3L hétérozygotes (+/-) sont fertiles, ils démontrent des anomalies au niveau de la compaction chromosomique et de l'expression des gènes post-méiotiques. La descendance de ces mâles démontre des taux d'aneuploïdies supérieurs, et ce même si la progéniture ne possède pas d'allèle mutante (effet paternel). Malgré ces faits, aucune perturbation de la méthylation d'ADN n'a été exposée dans les cellules germinales matures de mâles Dnmt3L haploinsuffisants. Cette situation est énigmatique puisque les seules fonctions rattachées à DNMT3L sont un rôle dans la méthylation d'ADN et certains phénotypes observés dans les spermatozoïdes de mâles Dnmt3L+/-. Conséquemment, nous avons examiné les irrégularités de méthylation d'ADN, causé par une haploinsuffisance de Dnmt3L, dans les spermatozoïdes matures et ce à l'échelle du génome entier par technique de représentation réduite de séquençage bisulfite (RRBS). Nos travaux ont révélé qu'un total de 367 régions hypométhylées et 88 régions hyperméthylées dans les spermatozoïdes de Dnmt3l+/- en comparaison avec les souris de type sauvage. Ces loci différentiellement méthylés ont été déterminés en comparant des tuiles génomiques de 100 pb démontrant au moins 20% de différence entre les spermatozoïdes Dnmt3L+/+ (n=4) et Dnmt3+/- (n=5). Les régions hypométhylées sont principalement retrouvées dans les séquences intergéniques LINE1, tandis que les régions hyperméthylées sont plutôt localisées à l'intérieure de séquences régulatrices et exoniques. Nos résultats confirment qu'un simple changement de dosage, sans complètement annihiler l'expression, d'un régulateur épigénétique clé à l'intérieur des cellules germinales mâles engendre la déformation des patrons de méthylation de l'ADN. Les conséquences associées à l'effet-dose, due à l'haploinsuffisance de Dnmt3L, nous ammène à s'interroger sur la répercussion qu'aurait une surexpression Dnmt3L dans la lignée germinale murine. Par conséquent, nous avons créé un modèle de souris transgénique qui surexprime une forme endogène de DNMT3L à un niveau plus élevé que des cellules germinales de type sauvage. Ce modèle a été créé avec succès et les essais d'accouplement initiaux de la F0 révèlent une tendance de sous-fertilité des femelles. Bien que les résultats soient préliminaires, il semble que la surexpression de Dnmt3L chez la souris, en utilisant un promoteur spécifique aux cellules germinales, a des conséquences plus sévères au niveau de la lignée germinale femelle comparativement à celle du mâle.

"Epigenetic" refers to a heritable change in the gene expression pattern that is not mediated by any alterations in the primary nucleotide sequence of a gene in the genome. This change involves methylation of DNA in the gene promoter regions, modification of histone residues and chromatin remodeling. Among them, methylation of DNA promoter region is an essential step in epigenetic gene silencing and is known to be closely related to carcinogenesis and cancer progression.
Our preliminary study on effect of treatments of some potential anti-cancer drug candidates, namely Pheophorbide A (Pa), Pa combining with photodynamic therapy, Polyphyllin D (designated as HK-18), and its derivative designated as HK-27 on human breast cancer cell lines MCF-7 and MDA-MB-231 showed that the promoter methylation of CDH1 was decreased in response to treatments of Pa, HK-18, and HK-27 in MDA-MB-231 cells.
The aim of this study was to explore whether any methylation of DNA promoters mechanism is involved in drug resistance of a doxorubicin-induced human multidrug resistant hepatocellular carcinoma sub-linage R-HepG2 which was established from the doxorubicin sensitive HepG2 cell line in our laboratory. In this project, it was observed that the DNA promoter methylations of ESR1, Rassf2A, CDH1 and MDR1 in R-HepG2 were higher than those in HepG2 cells respectively by methylation specific polymerase chain reaction method. Bisulfite sequencing showed that the total 32 CpGs of CDH1 promoter region in R-HepG2 cells were hypermethylated while they were hypomethylated in HepG2 cells. CDH1 is the encoding gene of E-cadherin. The promoter hypermethylation induced CDH1 silencing in R-HepG2 cells was confirmed by reverse transcription polymerase chain reaction and Western blotting that CDH1 transcription and E-cadherin expression were maintained in HepG2 cells but both were lost in R-HepG2 cells. RT-PCR of 10 multidrug resistant related genes revealed that transcription of MDR1 was obviously increased in R-HepG2 cells, transcription of MRP1 and MRP5 were slightly increased in R-HepG2 cells, transcription of MRP6 and BCRP were slightly decreased in R-HepG2 cells comparing to those in the parental HepG2 cells. This result suggests that up-regulation of P-glycoprotein expression which is the protein product of MDR1 may be one of the major causes of multidrug resistance in R-HepG2 cells. Transient transfection of CDH1 cDNA increased the CDH1 transcription and E-cadherin expression in R-HepG2 cells. I also found that the CDH1 transfected R-HepG2-CDH1 cells showed increased amount of doxorubicin uptake, increased apoptotic population of cells exposed to doxorubicin, suppressed cell migration, and decreased P-glycoprotein expression comparing to those in R-HepG2 cells. It was also found that the transcription levels of SNAI2, TWIST1, ASNA1 and FYN were obviously higher in R-HepG2 cells than those in HepG2 cells. The transcription of FYN and TWIST1 were obviously decreased in CDH1 cDNA transfected R-HepG2-CDH1 cells which displayed a negative correlation with the transcription level of CDH1 and these results imply a suppressive role of CDH1 in regulating these genes which were involved in cancer metastasis and multidrug resistance.
Jiang, Lei.
Adviser: Kwok-Pui, Fang.
Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2010.
Includes bibliographical references (leaves 144-171).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
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Abstract also in Chinese.