DNA methylation is a common epigenetic mark that affects gene regulation, genomic stability and chromatin structure. In mammals, methylation is mainly found in the CpG dinucleotides. The CpG methylation signals can be recognised by the Methyl-CpG-Binding Protein (MBP) family which includes MeCP2, MBD1, MBD2, MBD3, MBD4 and Kiaso. MeCP2 and MBD1-4 (except mammalian MBD3) recognise methyl-CpG via their MBD domain whereas Kaiso interprets methylation through its Zn finger DNA binding domain. The TRD domains of MeCP2, MBD1 and MBD2 have been reported to recruit transcriptional co-repressors to the methylated DNA. A thymine DNA glycosylase domain is located at the C-terminal region of MBD4. This study concerns the molecular details of the methyl-CpG recognition by the MBD domain of MeCP2. To achieve this, the MeCP2 MBD domain has been expressed, purified and co-crystallised with a 20 bp DNA fragment from the BDNF promoter. The DNA-protein cocrystal diffracted X-rays to a maximum resolution of 2.5Å using synchrotron sources. It belongs to space group C2 with unit cell dimensions: a = 79.71Å, b = 53.60Å, c = 65.73Å, and β = 132.1°. The X-ray structure of the MeCP2 MBD-DNA complex was solved using the SAD method. Structural analyses of the refined X-ray structure reveal that the methyl groups of the DNA make contact with a predominantly hydrophilic surface that includes tightly bound water molecules. From a structure of the MBD domain in MBD1, established by NMR, the binding specificity of the MBD domain had been thought to depend on hydrophobic interactions between the cytosine methyl groups and a hydrophobic patch within the MBD domain. The findings of this study suggest that MeCP2 recognises the hydration pattern of the major groove of methylated DNA rather than cytosine methylation per se. The X-ray structure also identifies a unique role of T158 and R106, the sites of the two most frequent Rett missense mutations. Both residues stabilise the tandem Asx-ST motif at the C-terminal region of MBD domain. Disruption of this tandem motif destabilises the DNA-protein interaction. The BDNF sequence in this study contains an AT run which displays unique properties of AT tract DNA. Previously, mutation of the AT run has been reported to decrease MeCP2 binding specificity. This study however demonstrated that a significant reduction can only be observed when both AT runs close to the methyl-CpG have been mutated. The X-ray structure of the MeCP2 MBD-DNA complex in this study rationalises the effects of the most common Rett mutations and provides a general model for methylated DNA binding that is dependent on structured water molecules.

In mammals, the correct spatio-temporal patterns of gene expression are coordinated by transcription factor networks in combination with epigenetic signalling pathways. CpG methylation is an epigenetic modification of DNA involved in the heritable transmission of gene silencing patterns. Increasing evidence suggest a primary role for CpG methylation in the direct regulation of gene expression, at least for a subset of promoters. An example of this direct regulation is represented by the ectopic expression of genes involved in genome defence pathways upon global loss of methylation. However, the mechanistic relationship between CpG methylation and transcriptional regulation is not well understood. To explore this, we have applied Cap Analysis of Gene Expression (CAGE), to cells deficient in CpG methylation (Mouse Embryonic Fibroblasts with hypomorphic mutation of Dnmt1 ) and matched controls. This provides a quantitative, single nucleotide resolution, genome wide map of methylation responsive transcription initiation. Integrating this with RNA-seq, genome wide measures of CpG methylation and ChIP-seq for histone modifications in the same system, provides a detailed view of how reduced CpG methylation alters the chromatin and transcriptional landscape of the genome. Our results show dramatic shifts in the cellular RNA pool, with the pronounced up-regulation or de-repression of promoters in a specific sub-family of transposable elements. Tens of other genic and non-coding RNA promoters similarly show dramatic inductions. Contrary to a prior hypothesis, we found no evidence for increased rates of transcriptional initiation from anonymous genomic sites not previously implicated in promoter activity. Transcription initiation at CpG island promoters is generally unaffected by hypomethylation, however a set of TSSs located in CpG island shores and a class to transposable element overlapping TSSs do appear to be sensitive to methylation and are significantly up-regulated upon hypomethylation.

Heavy metal contamination in food is a worldwide concern. Man-made ponds are domestic sites in the production of Procambarus clarkii and Procambarus zonangulus, two edible species of crayfish. Ponds may be constructed in former sugar cane or rice fields. Crayfish farming is an ancillary seasonal business within the rice-growing season. The use of products to control insects, pests, and weeds in rice and sugar cane production, may cause an accumulation of heavy metals in the crayfish tail within pond structures. Arsenic, cadmium, copper, and zinc are heavy metals that are absorbed through the roots of and distributed through rice products. Metabolites associated with rice products are absorbed in the human body. Research suggests that metabolites associated with heavy metals cause disease in animals and humans.

Graduation date: 1995

Nos connaissances sur les mécanismes étiopathogéniques des cancers utérins (endomètre et col) sont en constante amélioration. Cependant, le dépistage précoce des lésions précancéreuses a été réalisé, jusqu'à présent, avec plus de succès pour le col que pour le corps utérin. En outre, un diagnostic histopathologique précis est obligatoire pour adopter la stratégie thérapeutique la plus appropriée. L'objectif général de ce travail a été de valider des marqueurs diagnostiques potentiels des cancers utérins en utilisant la technologie des « Tissus Puces» ou «Tissue Microarray» (TMA) qui permet l'examen uniforme et simultané d'un grand nombre d'échantillons déposés sur une même lame. Grâce à cette technologie, nous avons montré que la détection d'une série de marqueurs (ADN de papillomavirus humain, protéine p16, involucrine et antigène Ki67) peut améliorer le diagnostic histopathologique des différentes lésions (pré)néoplasiques cervicales. En outre, ces biomarqueurs pourraient aider à mieux comprendre la biologie des lésions épithéliales cervicales en les reliant à des critères morphologiques précis. Nous avons également évalué l'intérêt de marqueurs diagnostiques basés sur la méthylation de l'ADN dans la carcinogenèse endométriale. Notre démonstration d'une hyperméthylation des promoteurs de deux gènes suppresseurs de tumeur (RASSF1A et RARB2) au cours de la carcinogenèse endométriale et dans les tissus normaux adjacents aux tumeurs suggère un rôle important de la méthylation de l'ADN dans linitiation et la progression des lésions (pré) cancéreuses de lendomètre.

Pure-breeding, early-flowering lines of flax, derived from treatment of germinating seeds with 5-azacytdine in 1990, flower 7-13 days before controls, have fewer leaves, are shorter, and have hypomethylated total DNA, relative to control lines. This thesis examines the changes in DNA methylation levels in the cotyledons and shoot tips of early-flowering Royal flax lines (i.e. RE1 and RE2) and their control (RC) to determine the changes from 24 days to the onset of flowering (approximately 34 days in RE1 and RE2, and 52 days in RC). It also examines the question of whether DNA is methylated in the chloroplast genome of flax. Finally, the thesis looks at the differences in transcript abundance of the flowering genes LEAFY and TERMINAL FLOWER1 in RC and RE2. Methylation levels in RE1 and RE2 were found to be lower than in RC from 24 days of age to the onset of flowering and the levels of all three lines increase with tissue age and/or differentiation. In addition, buds of RE2 were hypomethylated relative to RC. If plants were placed in the dark prior to DNA extraction, hypomethylation was not seen in the total DNA of RE2. The chloroplast DNA of flax was found to be methylated, and RE2 chloroplast DNA was hypomethylated relative to RC. Differences in transcript levels of LFY were seen in RC and RE2 shoot tips, where a higher accumulation of transcript seen in RE2 compared to RC may be related to its earlier flowering time. In leaves, there was no significant difference in the transcript abundance of LFY between RC and RE2. TFL1 was detected in genomic DNA of RC and RE2; it was not detected in the cDNA of the two lines. In summary, compared to RC, hypomethylation was seen in the total DNA of RE2 plants grown under regular light conditions and the methylation levels in the all lines increased with age in shoot tips, cotyledons, and leaves. The chloroplast DNA of RE2 was also hypomethylated relative to that of RC. RE2 accumulated LFY transcript in shoot tips at flowering, which was not the case in RC. Although these ideas cannot be linked at this time, they are all likely related to the early-flowering phenotype.

Pure-breeding, early-flowering lines of flax, derived from treatment of germinating seeds with 5-azacytdine in 1990, flower 7-13 days before controls, have fewer leaves, are shorter, and have hypomethylated total DNA, relative to control lines. This thesis examines the changes in DNA methylation levels in the cotyledons and shoot tips of early-flowering Royal flax lines (i.e. RE1 and RE2) and their control (RC) to determine the changes from 24 days to the onset of flowering (approximately 34 days in RE1 and RE2, and 52 days in RC). It also examines the question of whether DNA is methylated in the chloroplast genome of flax. Finally, the thesis looks at the differences in transcript abundance of the flowering genes LEAFY and TERMINAL FLOWER1 in RC and RE2. Methylation levels in RE1 and RE2 were found to be lower than in RC from 24 days of age to the onset of flowering and the levels of all three lines increase with tissue age and/or differentiation. In addition, buds of RE2 were hypomethylated relative to RC. If plants were placed in the dark prior to DNA extraction, hypomethylation was not seen in the total DNA of RE2. The chloroplast DNA of flax was found to be methylated, and RE2 chloroplast DNA was hypomethylated relative to RC. Differences in transcript levels of LFY were seen in RC and RE2 shoot tips, where a higher accumulation of transcript seen in RE2 compared to RC may be related to its earlier flowering time. In leaves, there was no significant difference in the transcript abundance of LFY between RC and RE2. TFL1 was detected in genomic DNA of RC and RE2; it was not detected in the cDNA of the two lines. In summary, compared to RC, hypomethylation was seen in the total DNA of RE2 plants grown under regular light conditions and the methylation levels in the all lines increased with age in shoot tips, cotyledons, and leaves. The chloroplast DNA of RE2 was also hypomethylated relative to that of RC. RE2 accumulated LFY transcript in shoot tips at flowering, which was not the case in RC. Although these ideas cannot be linked at this time, they are all likely related to the early-flowering phenotype.

No description available.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences