The aim of this thesis was to assess the effect of different of intake of polyunsaturated fatty acids on indices of oxidative stress, particularly damage to lipids and DNA and to examine whether this effect could be modified by supplementation with the lipid soluble antioxidant, vitamin E. This was assessed using a split plot/change over dietary study, where half and volunteers consumed a diet containing 5% PUFA (low PUFA) as food energy for 4 weeks and after a washout period of up to 10 weeks, consumed a 15% PUFA (high PUFA) diet for another 4 weeks. The other volunteers completed this protocol in reverse. Total fat, carbohydrates and protein, and vitamins E and C contents of the diets were constant and they were provided either with or without an additional 80mg d-tocopherol acetate/day. The results showed that although plasma total cholesterol showed a small significant increase after the low PUFA diets there was no change after consumption of the high PUFA diets or either of the vitamin E supplemented diets. Indices of oxidative stress (whole blood oxidised glutathione and urinary thiobarbituric acid reactive substances (TBARS)) were increased following consumption of the high PUFA diet. However, there was no change in non-specific plasma indices of lipid peroxidation, conjugated dienes and TBARS, nor in red cell antioxidant enzymes, glutathione reductase, and catalase. These results indicate that increasing dietary levels of PUFA may adversely affect some indices of lipid peroxidation, DNA damage and antioxidant status whilst not appearing to improve lipoprotein profiles. Increasing the dietary intake of vitamin E appears to ameliorate the potentially damaging effects of PUFA. Therefore, care should be taken when providing dietary advice on PUFA intake and an adequate intake of antioxidants to match any increased PUFA may be important for preventing oxidative stress.

Sustaining the integrity of DNA throughout the lifetime is critical for every living organism. Therefore organisms evolved numerous ways to detect and repair different types of DNA damage caused by various endogenous and exogenous factors resulting in replication stress. Defects in these repair mechanisms can lead to severe human diseases such as neurological disorders, familial cancers or developmental syndromes. In presented master thesis, we investigated the function of a yeast protein named Wss1, a metalloprotease that participates in a recently discovered DNA repair pathway that proteolytically removes DNA-protein crosslinks. Wss1 shows strong negative interaction with another DNA repair protease, Ddi1, in which case was discovered, that double-deleted yeast strain lacking WSS1 and DDI1 is hypersensitive to hydroxyurea. Hydroxurea is a ribonucleotide reductase inhibitor that, in the end, arrests cells in the S-phase of cell-cycle. Based on previous studies, we performed rescue experiments with various deletions and single-site mutants of Wss1p to assess the involvement of particular yeast Wss1p domains in the replication stress response to hudroxyurea.

Water quality evaluation is a global concern due to its effect on public health. Different procedures can be implemented to evaluate specific standards of water quality. DNA extraction to characterize the microbial community in the water distribution systems is important. To optimize the DNA extraction process the effect of residual chlorine and water composition was tested. The results exposed the limited effect of the samples dechlorination. Total cell number effect can be varied according to water quality. Also, the study indicated the possible inhibitory effect of the rust on the DNA extraction from drinking water samples.

Fetal alcohol spectrum disorders (FASD) is an umbrella term that describes a range of symptoms associated with prenatal alcohol exposure. Fetal Alcohol Syndrome (FAS) is the most severe disorder in the spectrum and is a major health problem in South Africa, with a prevalence rate of 68.0-89.2 per 1000 children of school-going age. The primary cause of FAS is in utero alcohol exposure. However, secondary factors that contribute to the syndrome include various genetic, epigenetic and additional environmental factors. The proposal that paternal preconception alcohol exposure has adverse effects on offspring development is supported by children born with FASD-like characteristics whose mothers did not drink but whose fathers were alcoholics. Mouse models further support these findings. One of the main epigenetic factors that have been shown to be affected by alcohol is DNA methylation. This chemical modification of DNA is associated with developmentally important genes known as imprinted genes. Imprinted genes are expressed in a parent of origin specific manner. Methylation occurs at specific regions in these genes known as differentially methylated regions (DMRs) or imprinting control regions (ICRs). Alcohol’s ability to alter DNA methylation at imprinted genes raises the possibility that epigenetic disruption could contribute to the clinical features seen in FASD. The main aim of this research was to examine global DNA methylation and locus specific H19 ICR DNA methylation in spermatozoa, related to alcohol exposure. This was done using the luminometric methylation assay (LUMA) and bisulfite based quantitative pyrosequencing, respectively. In this study there was no significant correlation between alcohol exposure and global DNA methylation (p = 0.17), nor was there a significant correlation with drinking frequency (p = 0.31). Although not significant, a slight trend towards decreased global DNA methylation in alcohol-exposed spermatozoa was observed. This suggests that either alcohol does not affect global sperm DNA methylation or that the technique used in this study was not sensitive enough to detect minor changes in global DNA methylation percentage. There was also no significant correlation between alcohol exposure and average H19 ICR DNA methylation (p = 0.051), nor was there a significant correlation with drinking frequency (p = 0.47). There was no significant correlation between alcohol exposure and DNA methylation at individual CpG sites except for CpG 3, where there was a significant increase in DNA methylation in the drinking group (p = 0.03). The findings of this study together with the findings of significant selective demethylation at individual CpG sites within the IG-DMR from another study on the same sperm samples, suggest that alcohol may have the ability to affect DNA methylation levels in spermatozoa at certain loci within the sperm genome. However, these loci-specific effects are not reflected in global DNA methylation levels. These findings do not disprove the hypothesis that there is an epigenetic mechanism responsible for the paternal effects seen in FASD. Instead they suggest that the techniques used in this study were not sensitive enough to detect these changes in DNA methylation or alternatively, alcohol may be exerting its effects through other epigenetic mechanisms.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science / There are a number of recorded difficulties in classing lithop species according to their phenotype alone. DNA profiling, which can provide a quantitative measure of the degree of sequence similarity between any two genomic DNA's, is the method of choice for establishing phylogenetic relationships between species. (Abbreviation abstract} / Andrew Chakane 2018

Thesis advisor: Larry W. McLaughlin / By virtue of encoding and transferring hereditary information, nucleic acids effectively represent the blueprint for life as we know it. Given the biological relevance of this class of polymers, it comes as no surprise that scientists are constantly striving to reach a greater understanding of the innumerable genetic corridors contained within the human genome. This has led to the rational design and synthesis of numerous nucleoside analogues in an attempt to alter and subsequently control native nucleic acid structure and function. The first attempts at harnessing the latent abilities of DNA are described in Chapter 2. Multiple tetrahedral branching "hubs" were designed, synthesized and characterized, at which point single-stranded DNA could be elongated from each of the four points of origin. Ensuing hybridization studies were performed with the goal that the binding traits of these elongated tetrahedral lattices could be monitored, and that fully formed lattices could potentially function as means of drug encapsulation or molecular tethering. Chapter 3 describes direct alteration of the standard DNA backbone. Successive synthetic efforts towards creating a 6'-extended deoxyadenosine molecule are detailed, and its effects on the stability of duplexed DNA (along with sister molecules 6'-deoxythymidine and an elongated 3'-deoxythymidine) are also defined. Upon insertion into DNA, this class of extended nucleosides could ultimately lead to a new duplex structure, as well as novel binding properties. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Larry W. McLaughlin / Thesis advisor: Mary F. Roberts / Nucleic acids are essential biological molecules for life. For example, deoxyribonucleic acid (DNA) is the main genetic information carrier; ribonucleic acid (RNA) plays a critical role in translation and transcription. These characteristics place nucleic acids as the fundamental genetic materials of a living system. Since over a century ago, intensive attempts have been made by researchers to study the nucleic acid properties. For chemists, it is particularly interesting and important to understand the relationship between structures and properties of nucleic acids. For instance chemical modifications can alter stability of nucleic acids, and consequently influence their biochemical behaviors. In this work, we began by investigation of a 5',6-cyclo-modified nucleic acid resembling the product of DNA oxidation, and then developed a library of cyclomodifications. Our research on their structures and properties indicated that by installing cyclo-modifications we might be able to add some properties, that were not observed in nature to nucleic acids. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

PhD - Science / Specialised mechanisms have evolved in pathogenic bacteria to enable adaptation to hostile and fluctuating host environments. Inducible mutagenesis, in particular, has been implicated in the emergence of antibiotic- and stress-resistant mutants. This thesis examined mycobacterial DNA metabolism with specific emphasis on the roles of multiple Y-family polymerases in the evolution of inter-strain variation and drug resistance in M. tuberculosis. The contribution of the nrdZ-encoded class II ribonucleotide reductase (RNR) to the maintenance of dNTP pools for replication and repair under hypoxic conditions was also explored. In addition, the co-factor requirement of NrdZ prompted an investigation into the biosynthesis and transport of adenosylcobalamin (AdoCbl) in M. tuberculosis. The data suggest that the mycobacterial Y-polymerases are tightly regulated and restricted to specialised damage-free repair or replication restart. Disruptions in individual M. smegmatis mc2155 DinB (pol IV) homologues resulted in novel antibiotic-resistance polymorphisms that were suggestive of non-redundant function. In contrast, abrogation of all error-prone polymerase activity failed to impair long-term competitive survival of mc2155 in vitro. Similarly, heterologous overexpression of M. tuberculosis pol IV homologues did not increase spontaneous mutation rates in wild-type mc2155, or complement damage hypersensitivity. However, treatment of M. smegmatis with gyrase inhibitors confirmed the differential induction of pol IV homologues in response to replication stalling and demonstrated elevated rates of spontaneous mutagenesis as a result of GyrB inhibition. The class II RNR does not appear to play a significant role in mycobacterial pathogenesis. Specifically, NrdZ was unable to substitute for the class I RNR under aerobic conditions in vitro, and a M. tuberculosis ÄnrdZ deletion mutant was not impaired in its ability to adapt to hypoxia in vitro. Similarly, infection of immunocompetent mice suggested that nrdZ is not required for the survival or virulence of M. tuberculosis in vivo. Disruptions in genes required for AdoCbl and methionine biosynthesis revealed that complex regulatory functions govern mycobacterial methionine and AdoCbl homeostasis. Loss of early (cobK) or late (cobU) stage AdoCbl biosynthetic enzymes had no effect on the growth of M. tuberculosis H37Rv in vitro. In contrast, deletion of the B12-independent methionine synthase (metE) resulted in impaired growth on solid media that could be rescued by vitamin B12 but not Lmethionine supplementation, simultaneously demonstrating the ability of M. tuberculosis to transport exogenous vitamin B12. Significantly, double ÄcobU/ÄmetE and ÄcobK/ÄmetE deletion mutants in which all predicted methionine synthase activity was eliminated, were not impaired for growth in liquid minimal media, suggesting that M. tuberculosis H37Rv possesses alternative mechanisms for methionine generation. Finally, the attenuated virulence of the ÄcobU and ÄmetE deletion mutants in vivo in immunocompetent mice indicated the relevance of AdoCbl biosynthesis to mycobacterial pathogenesis.

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 thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Doctor of Philosophy February 2013 / Mycobacterium tuberculosis is presented with environmental host assaults that damage its DNA during infection. Tubercle bacilli possess mechanisms to protect against moststresses imposed by the host, including genotoxic stress. However, tolerance of DNA lesions that have escaped the normal repair processes requires the function of specialist DNA polymerases that can introduce mutations during translesion synthesis (replication by-pass), thus leading to damage-induced mutagenesis. Mycobacteria employ a novel DNA polymerase, DnaE2, for DNA damage tolerance and induced mutagenesis. DnaE2 belongs to the C-family of DNA polymerases, which are known to replicate DNA with high fidelity, and has been implicated in virulence and the emergence of rifampicin resistance of M. tuberculosis in vivo. In this study, DnaE2 was shown to function in the same pathway as two accessory proteins, ImuB and ImuA’, for damage tolerance and induced mutagenesis in mycobacteria. In this system, DnaE2 performs the polymerase function in translesion synthesis whereas ImuB is a cryptic Y-family DNA polymerase that lacks critical active site residues. It contains a β-clamp binding motif that allows interaction with the β-clamp and presumably enables DnaE2 and ImuA’ to access the replication fork. ImuB has a C-terminal region extending from the β-clamp binding motif which contains disordered regions that allow the interaction with other proteins and is important for function. ImuA’ is also essential for damage tolerance and induced mutagenesis but its function remains unknown. This protein is structurally similar to Escherichia coli RecA protein in the N-terminus and the middle domain, but it has a distinct C-terminus that was shown to be important for the interaction with ImuB. The essential replicative, C-family polymerase, DnaE1, was shown to be upregulated in response to DNA damage and was also shown to interact with ImuB. To explore the possibility that other proteins are involved in this pathway, ImuB was Cterminally tagged for use as bait in pull-down experiments in M. smegmatis. However, introduction of the tag disrupted ImuB function, further reinforcing the importance of the Cterminal region of ImuB for the function of this protein, presumably via protein-protein interactions. In contrast, a variant of ImuA’ which was N-terminally tagged was shown to retain functionality; however, experiments using this protein as a bait for pull-down proved to be unsuccessful. Proteomic analysis of wild type M. smegmatis, a dnaE2 deletion mutant and complemented derivative was carried out on cells exposed to the same conditions as used in the pull-down assay. Base excision repair (BER) components were identified in this analysis, but did not detect ImuB and ImuA’, suggesting that the levels of expression of these proteins were comparatively lower under the conditions tested resulting in failure of the pull-down experiment. Finally, numerous attempts were made to express and purify recombinant forms of ImuB and ImuA’ in E. coli for use in structural studies. Both proteins were expressed in the soluble and insoluble fractions; however the levels of soluble protein were low, and as a result, purified protein preparations could not be obtained.