Mercury occurs naturally in the environment and is released by human activities. Mercury exists in gaseous, liquid, and solid phases, and all phases are of importance when fate s effects of mercury in terrestrial, fresh and marine water, and atmospheric environments. Mercury can be transformed to a highly toxic form of methylmercury. Humans are exposed to the toxicity of methylmercury by eating fish. Methylmercury is bioaccumulated up the food chain by transfer of residues of methylmercury in smaller organisms that are food for larger organisms in the chain. This sequence of process results in higher concentrations in organisms at higher levels in the food chain with human at the top of the food chain. This study is an evaluation of chemical speciation and biological processes that govern mercury distribution and transformation among three environmental media: atmosphere, water, and sediments. Understanding speciation of mercury and biological processes of methylmercury transformation plays an important part in toxicity and exposure of mercury to living organisms. Speciation also influences transport of mercury within and between environmental media while biological processes of methylmercury transformation influence methylmercury production and its transport to the biological communities. Study also covers the demethylation process that can convert methylmercury to inorganic mercury species. Demethylation and methylation processes therefore may occur in parallel further complicating the assessment of mercury fate in the environment. The study will provide integrated fundamental pathways of mercury species transformation through chemical and biological pathways and will contribute to an understanding of fate and transport of mercury species in environmental media. It will also provide a foundation for a state- and region-wide examination of mercury monitoring and control strategies.

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The aims of the work presented in this thesis were: to investigate the role of methylation of 14-3-3σ (a key regulator of p53-mediated G2/M arrest and of translational control during mitosis) in colorectal cancer using colorectal cancer cell lines and fresh colorectal tumours; to investigate any relationship between 14-3-3σ methylation status and gene expression; to determine whether aberrant methylation is associated with cell cycle defects and other factors known to contribute to colorectal carcinogenesis. PCR bisulphite sequencing showed that 78% (7/9) of colorectal cancer cell lines were unmethylated in the 14-3-3σ upstream promoter region (UPR). The unmethylated cell lines expressed high levels of 14-3-3σ, while methylated cell lines expressed negligible levels of 14-3-3σ protein or mRNA. Methylated colorectal cancer cell lines were treated with 5-aza-2’-deoxycytidine and demethylation was confirmed by MSP analysis. However, demethylation did not induce 14-3-3σ re-expression in the methylated cell lines, suggesting that CpG methylation may not be the only mechanism of transcriptional control. In contrast to colorectal cancer cell lines, 90% (89/99) of fresh colorectal tumours were methylated at CpG dinucleotides within the 14-3-3σ UPR. Bisulphite sequencing analysis of individual clones from 14-3-3σ methylated tumours (n =3) demonstrated that the clones displayed methylated CpG levels of approximately 41%. In agreement with previous PCR bisulphite sequencing analysis, there were a low percentage of methylated CpG dinucleotides (~ 15%) in clones from the 14-3-3σ unmethylated tumours (n =3). Unmethylated tumours expressed significantly higher levels of 14-3-3σ in comparison to methylated tumours (p =0.03), indicating that 14-3-3σ methylation may be associated with expression. PCR bisulphite sequencing analysis of matched normal mucosa tissues indicated that the 14-3-3σ UPR was methylated in all samples. Preliminary studies therefore suggest that there is tumour-specific loss of 14-3-3σ methylation in colorectal tumours within the 14-3-3σ UPR and CpG island. There were no apparent clinico-pathological correlations with 14-3-3σ methylation status. Whilst 14-3-3σ methylation was associated with expression in fresh colorectal tumours, there was no significant difference in expression levels between unmethylated colorectal tumours and matched methylated normal tissue. Bisulphite sequencing analysis of individual clones from normal tissues (from patients free of cancer) revealed that the 14-3-3σ UPR and CpG island was methylated at the majority of CpG sites analysed in colonic tissue (422/495, ~85.2%) and approximately half (795/1557, 51.1%) of CpG sites in skin samples (n =3). Furthermore, higher levels of 14-3-3σ protein were observed in skin tissue samples compared to normal colonic tissue, suggesting that 14-3-3σ CpG island methylation may be associated with tissue-specific expression. Experiments to assess the relationship between 14-3-3σ methylation and general methylation defects, suggest that methylation differences in 14-3-3σ were not simply a consequence of more general methylation phenomena well described in colorectal cancer. Nearest Neighbor analysis showed no evidence of generalised hypomethylation. Furthermore, MethyLight analysis of the CpG Island Methylator Phenotype (CIMP) showed no relationship between 14-3-3σ methylation status and CIMP; since, 1/5 (20%) tumours methylated at 14-3-3σ UPR and 1/5 (20%) tumours unmethylated at 14-3-3σ UPR were CIMP positive. In vitro functional assays showed that overexpression of 14-3-3σ in SW480 cells (14-3-3σ methylated) delayed the apoptotic response to UV-C, compared to control SW480 cells. This suggests that 14-3-3σ may protect colorectal cancer cells from apoptosis. MTT assays showed that overexpression of 14-3-3σ in SW480 cells resulted in a trend of increasing proliferation with a significant increase on day 4, compared to controls SW480 cells (p <0.01). Furthermore, FACS-sorted SW480 cells overexpressing 14-3-3σ, showed a significant shift to S-phase from G1 compared to control SW480 cells (p <0.01). Western blot analysis and immunohistochemistry revealed no relationship between p53 status and methylated 14-3-3σ in fresh tumours, while there was no relationship between published p53 status for colorectal cancer cell lines and 14-3-3σ methylation status defined experimentally. I have presented data which shows that methylation status of 14-3-3σ varies between colorectal cancer tissue, colorectal cancer cell lines and normal colonic tissue. Overexpression of 14-3-3σ appears to contribute to colorectal cancer carcinogenesis, raising the hypothesis that 14-3-3σ expression and function may at least in part be dependent on CpG methylation.

E2F1 is a transcription factor which paradoxically has major influence on both apoptosis and cell cycle progression. One of the most important questions in E2F1 biology therefore is the mechanism underlying regulation of these opposing physiological outcomes. Post-translational modifications (PTM) provide proteins with an additional layer of complexity, potentially altering interactions with partner DNA and protein. The importance of arginine methylation has recently been implicated in modulating the activity of the tumour suppression pathway proteins, p53 and E2F1. Previous studies have established that the methyltransferase, PRMT5, is responsible for the symmetrical dimethylation of E2F1, which inhibits its pro-apoptotic activity. In this thesis, E2F1 was found to be a substrate of PRMT1, which catalysed asymmetrical dimethylation of E2F1 at arginine 109. In addition, a positive correlation was found between the percentage of apoptotic cells and levels of PRMT1. Conversely, an increase in cancer cell colony formation was shown when the site of PRMT1 methylation on E2F1 was changed from arginine to lysine at position 109. These findings suggested a growth inhibition effect by PRMT1 methylation on E2F1. At the transcriptional level, depletion of PRMT1 increased E2F1 binding to the promoter region of Cdc6, a cell cycle regulator, and decreased binding to the promoter region of Apaf1, which has a pro- apoptotic role. Genome-wide ChIP-sequencing technology was undertaken and results further clarified that the depletion of PRMT1 preferably enriched E2F1 binding to promoters of positive regulators of cell proliferation and promoters of the cell cycle. Collectively, the findings of this thesis suggested that the opposing roles E2F1 demonstrated in promoting both cell proliferation and apoptosis was due to different types of arginine methylation which trigger E2F1 binding to different promoters. Lastly, arginine methylation was shown to influence protein-protein interactions. PRMT5 induction resulted in the identification by mass spectrometry of &beta;-catenin as an E2F1 interacting partner. As the Wnt/&beta;-catenin signalling pathway is broadly recognised as having pro- cell proliferation activity, this finding is consistent with previous reports that suggest the oncogenic role PRMT5 methylation has on E2F1.

Chez les plantes, les processus de développement global et de plasticité développementale sont contrôlés par des mécanismes épigénétiques. La méthylation de l’ADN peut présenter un polymorphisme (épiallèles) qui est une source possible de biomarqueurs pour la sélection de génotypes d’intérêt agronomique. Pourtant, la recherche de tels biomarqueurs n’a pas encore été initiée. Dans ce contexte, nos objectifs ont concerné l'élaboration d'une stratégie pour la mise en évidence d’un contrôle épigénétique lors d’un processus développemental chez la betterave sucrière (Beta vulgaris altissima), ainsi que la recherche des biomarqueurs épigénétiques associés. Cette stratégie a d’abord été appliquée à la morphogenèse in vitro, sur trois lignées cellulaires de betterave sucrière. Une relation a pu être établie entre le niveau de méthylation de l’ADN et les propriétés morphogénétiques des lignées. Des biomarqueurs de morphogenèse in vitro ont ainsi été identifiés. La même stratégie a ensuite été appliquée in planta à la même espèce. L'existence d'un contrôle épigénétique lors de la vernalisation et de la dévernalisation chez plusieurs hybrides de betterave sucrière, avec des sensibilités à la montaison différentes, a été démontrée. Nous suggérons que l’amplitude et la cinétique des variations épigénétiques contrôlent l’induction de la montaison et sa rapidité, confirmant ainsi le rôle de la méthylation de l’ADN dans ce processus. Les loci cibles de ces remaniements de la méthylation de l'ADN lors de la vernalisation ont été définis. Un criblage a enfin permis d’identifier de potentiels biomarqueurs épigénétiques de la sensibilité à la montaison en vue de la mise au point d’un futur test de sélection agronomique. / In plants, the processes of global development and of developmental plasticity are controlled by epigenetic mechanisms. Polymorphism in DNA methylation (leading to epialleles) is a possible source of biomarkers for the selection of genotypes of agronomic interest. Until now, however, the search for such biomarkers has not been undertaken. Against this background, our objectives were to develop a strategy to investigate the existence of epigenetic control during a developmental process in sugar-beet (Beta vulgaris altissima) and to search for associated epigenetic biomarkers. The strategy was first applied to three sugar-beet cell lines, where we were able to established a relationship between the level of DNA methylation and the morphogenetic statue of the lines, and thus to identified a number of biomarkers for in vitro morphogenesis. We then applied the same strategy in planta in the same species and demonstrated the existence of epigenetic control (DNA methylation) during vernalization and devernalization in several sugar-beet hybrids that differed for bolting susceptibility. We propose that the scale and kinetics of epigenetic modifications control the induction and the rapidity of bolting, confirming the role of DNA methylation in this process. We have identified a number of target loci for these changes in DNA methylation during vernalization, and by screening these have been able to select several potential epigenetic biomarkers for bolting susceptibility, which may prove useful in future beet improvement programmes.

MSc(Med), Human Genetics, Faculty of Health Sciences, University of the Witwatersrand, 2009 / Imprinted loci are critical in foetal development and most are regulated by the methylation-specific CTCF binding protein which binds imprinting control regions (ICRs). The ICR is located between two genes that comprise imprinted loci, which are reciprocally expressed in a parent-of-origin specific manner. Maternally hypomethylated ICRs allow CTCF binding, creating a boundary element which prevents downstream enhancers from acting on the paternally expressed gene upstream of the ICR. Conversely, the hypermethylated (imprinted) paternal ICR prevents CTCF binding, allowing downstream enhancers to act on the gene upstream of the ICR, while suppressing the downstream maternally expressed gene. Alcohol and its metabolites are able to reach the testes via the blood supply and are known to reduce global DNA methylation by disrupting the folate, methyl group and homocysteine pathway. This may therefore affect gene expression at imprinted loci, whose parental alleles are discriminated by the imprinting status at the ICR. The effect of pre-conception paternal alcohol exposure on the DNA methylation of three paternally imprinted ICRs (H19, Rasgrf1, IG-DMR) as well as the maternally imprinted Snrpn ICR was examined in mouse sperm and their offspring. Male mice were gavaged with ethanol or sucrose. DNA was extracted from sperm of treated males and tail biopsies from offspring. Samples were bisulphite modified and the ICRs PCR amplified. DNA methylation patterns of ICRs were analysed by sequencing and quantitatively via pyrosequencing. Sperm samples of ethanol treated males did not show significant demethylation when compared to sucrose treated mice, with the exception of H19 CpG 7, Rasgrf1 CpG 26 and Snrpn CpG 10 (p=0.024, 0.014 and

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree in Master of Science (Medicine) in the Division of Human Genetics / Epigenetic mechanisms regulate gene expression, a particularly important activity during foetal development. DNA methylation contained within promoter and regulatory intergenic regions influence gene activity. In utero alcohol exposure as a result of maternal consumption during pregnancy has been associated with disruption of foetal DNA methylation and gene expression, leading to neurological dysfunction, growth retardation and facial anomalies. While similar phenotypes in offspring have been associated with chronic preconception paternal alcohol exposure, the mechanisms underlying these effects remain largely unexplored. This study aimed to: (1) validate significant changes in sperm DNA methylation in a list of ten candidate genes in male mice chronically exposed for ten weeks to ethanol (n=10) compared to a calorie-equivalent sucrose solution (n=10); (2) validate significant changes in gene expression in candidate genes in the brain, liver and placenta of E16.5 embryos sired by ethanol (n=24) compared to sucrose (n=24) treated male mice; (3) quantify DNA methylation changes in candidate genes in the three embryonic tissues. (4) Lastly, previously generated microarray data were reanalysed using bioinformatics tools to generate a top ranked candidate differentially expressed gene list that was used to identify and analyse biological functions or pathways significantly over represented among these genes using PANTHER and DAVID. This study was unable to provide validation for most of the significant differences observed in the sperm DNA methylome in the original study, most likely because of the low sperm DNA concentration. Significant methylation differences were however observed at individual CpG sites in three candidate genes (Igf1r, Odc1, Depdc1b) in specific tissues of embryos sired by ethanol-exposed males relative to embryos sired by sucrose-treated males. There was concordance in the direction of altered gene expression between the cases and controls using the microarray and real-time PCR approaches for two genes in the brain (Grm7 and Zfp317), three genes in the liver (Igf1r, Vwf and Depdc1b) and one gene in the placenta vii (Vwf). However, none of the candidate genes selected for validation showed statistically significant changes. This may be a result of the modest fold changes observed in the microarray experiment that as shown in many cases, often do not replicate. The remainder of the genes showed no changes in expression in the test embryos relative to the control. The functional enrichment analysis revealed biological processes that were over represented in the brain and liver indicating that they may be more vulnerable to the effects of alcohol, compared to the placenta. Overall, the study could not provide a statistically significant correlation between methylation changes in the sperm that were inherited by the offspring which subsequently dysregulated gene expression in the embryo. However, as trends toward significance and significant DNA methylation changes were observed in the embryonic tissues, this study supports the idea that preconception paternal alcohol exposure can induce epigenetic alterations in a locus and organ specific manner within offspring. / MT2016

One form of protein regulation is accomplished by post-translational modification (PTM). In order to test the importance one type of PTM, methylation, in chromosome segregation, we inhibited protein methylation for brief periods in G2 using the general methylation inhibitor adenosine dialdehyde (AdOx). Inhibiting methylation solely in late G2 leads to mitotic defects. We observed that several methylated histone residues; H3K9me3, H4K20me3 and H4K20me1, are predominantly affected by AdOx in G2. We show both that the kinetochore proteins are not affected and that the mitotic checkpoint is intact. Further, we observed structural defects and chromosome misalignment in mitotic cells. These results indicate that methylation events during late G2 operate to maintain and ensure the structural integrity of pericentromeric heterochromatin prior to mitosis. These results suggest that pericentromeric heterochromatin is required for the proper sensing of kinetochore tension and inactivation of the mitotic checkpoint. / Experimental Oncology

DNA methylation is common in prokaryotes and eukaryotes and has been implicated in various biological roles including gene silencing, X-chromosome inactivation, and genomic imprinting. 5-methylcytosine the "fifth base" of the genetic code comprises 1-3% of the human genome and is primarily found on cytosines within the context of the CpG sequence. Although progress has been made in understanding the biological roles of 5-methylcytosine, we are only beginning to uncover how it changes the local structure and global conformation of DNA. This thesis deals with the local perturbations in structure and hydration and the global conformational changes induced by the presence of 5-methylcytosine in DNA as determined by single crystal x-ray diffraction. 5-methylcytosine induces a novel conformation in the structure of duplex DNA. This conformation has characteristics of both the A-DNA and B-DNA conformations as well as some unique defining characteristics. This distinct duplex provides a structural rationale for the increased rate of deamination in 5-methylcytosine relative to cytosine. In addition to this novel conformation, 5-methylcytosine stabilizes intermediates within the B-DNA to A-DNA transition pathway, thus providing a crystallographic map of the transition from B-DNA to A-DNA. 5-methylcytosine was also used as a tool to probe the stabilizing features of the DNA four-way junction (known as the Holliday junction). The first crystal structures of Holliday junctions were found serendipitously while studying duplex DNA. The DNA four-way junction formation in these crystals was thought to be stabilized by a network of sequence dependent hydrogen bonds at the junction crossover. In this thesis, 5-methylcytosine was used to perturb these hydrogen bonds; however, the junction persisted, suggesting that there is flexibility in the types of sequences that can accommodate junction formation in the crystal, as well as, flexibility in the global structure of the junction. Overall, this work describes the effects of 5-methylcytosine on the local and global structure and hydration of DNA structure, as well as raising some interesting questions regarding the biological impact of methylation induced DNA structure. / Graduation date: 2002