Introduction: Cardiovascular disease (CVD) is the leading cause of global mortality and its prevalence is increasing among black South Africans in spite of their favourable lipid profile. Apolipoprotein E (ApoE) is a well-described risk factor for CVD and certain polymorphisms within this gene alter the lipid profile. The author hypothesised that there are population-specific effects within the ApoE gene that are responsible for the favourable lipid profile observed in black South Africans whose effects are being altered by environmental factors. Objectives: The main aim of this study was to investigate the associations between specific ApoE single nucleotide polymorphisms (SNPs) and the lipid profile of a black South African population, taking into account certain environmental and phenotypic factors in order to explore the interaction effects between these variables. Methods: Genotyping within this cross-sectional study (n=1 588), nested within the Prospective Urban and Rural Epidemiology (PURE) study, was achieved using Illumina‘s® GoldenGate Genotyping Assay with VeraCode® technology on the BeadXpress® platform (proprietary multiplex fluorescent hybridisation assays on a bead array substrate) or the Bio-Rad CFX Manager© (version 2.0). The Konelab20i™ auto analyser was used for quantitative determination of serum total cholesterol; high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) concentrations. Low-density lipoprotein cholesterol concentrations were estimated by the Friedewald equation. Results: All SNPs adhered to the assumptions of the Hardy-Weinberg equilibrium, yet the frequency of the SNPs often differed from that reported in other ethnic groups. The well-reported rs429358 and rs7412 SNPs (as the constituent SNPs of the haplotype-genotypes) presented with the strongest associations with various components of the blood lipid profile in the black South African cohort under investigation. Two gene-environment (rs405509 and rs7412) interaction effects on TG remained significant after conducting post hoc tests. Two genotype-phenotype interaction effects between the rs7412 SNP and body mass index and gamma-glutamyl transferase on the HDL-C concentrations remained significant after conducting post hoc tests. Conclusions: The variety of associations between these particular SNPs and the blood lipid profile determined in the present cohort strongly indicates that it is integral to any public health investigation into CVD development that these SNPs be investigated. This study further produced greater insight into the biological mechanisms underlying serum lipid and cholesterol concentrations in a black South African population. Therefore, from these results it is evident that the lipid profile of black South Africans is most definitely influenced by not only genetic variations in the ApoE gene and certain environmental factors, but by the interaction between these factors as well. The present study is the largest study to date to investigate the effect of polymorphisms in the ApoE gene on the lipid profile of black South Africans. / MSc (Nutrition), North-West University, Potchefstroom Campus, 2014

INTRODUCTION AND AIM The prevalence of cardiovascular disease (CVD) has increased significantly in the black South African population in recent years. Early in the development of CVD, atherosclerotic plaques form in the vessel wall. When this plaque becomes unstable and ruptures, the coagulation cascade is activated and a blood clot forms. The function of this clot is to stop bleeding. However, it cannot remain in the vasculature indefinitely and has to be lysed again. The ability of the body to lyse clots can be measured with global fibrinolytic potential (GFP) assays and expressed as lysis time. Increased clot lysis time (CLT) has been shown to be significantly associated with various CVD risk factors and CVD events in Caucasian populations while very little information is available for other ethnicities. In this study we investigated plasma GFP and its relation to CVD risk factors in a large black African population. We also determined the effect of three polymorphisms in the promoter area of the plasminogen activator inhibitor-1 (PAI-1) gene on PAI-1act (activity) levels (a main determinant of CLT) and CLT, together with gene-environment interactions and the effect of urbanisation on these interactions. PARTICIPANTS AND METHODS Apparently healthy men and women between the ages of 35 and 65 years were recruited to take part in the South African arm of the Prospective Urban and Rural Epidemiology (PURE) study. Approximately 1000 rural and 1000 urban black African individuals participated. Data and samples were collected during a 12-week collection period in 2005 for cross-sectional analysis. RESULTS Increased PAI-1act levels, body mass index (BMI), glycosylated haemoglobin (HbA1c), triglycerides, fibrinogen concentration, C-reactive protein, female sex, positive HIV-status and the metabolic syndrome were all associated with prolonged CLTs, while increased habitual alcohol consumption was associated with shorter iv CLTs. Urban-rural differences for CLT existed in women only. This is likely due to the larger extent of rural-urban differences in other CVD risk factors observed in women compared to what was observed in men. Of the CVD risk factors measured, PAI-1 explained the largest proportion of the variance in CLT (27%). Owing to the important role PAI-1act plays in CLT, we investigated three polymorphisms in the PAI-1 gene promoter area (the 4G/5G polymorphism, the novel SNP C428T and SNP G429A (previously identified)), and the influence of these polymorphisms on PAI-1act levels and CLT. The frequency of the 5G allele was high (0.85) in comparison with previously reported literature. PAI-1act increased significantly across genotypes in the urban (5G/5G: 3.84 U/ml; 4G/5G: 4.85 U/ml; 4G/4G: 5.96 U/ml p=0.009) but not the rural subgroup, while CLT did not differ. We found significant interactions between the 4G/5G polymorphism and BMI, waist circumference and triglycerides in determining PAI-1act, and between the 4G/5G polymorphism and fibrinogen and fibrinogen gamma prime in determining CLT. Direct relationships with PAI-1act or CLT were not found for the C428T and G429A polymorphisms; they did, however, influence associations of other environmental factors with PAI-1act and CLT. Several of these interactions differed significantly between rural and urban subgroups, particularly in individuals harbouring the mutant alleles. CONCLUSION CLT associated with many of the same CVD risk factors described in the literature for Caucasian populations, but also with other risk factors. Rural-urban differences in CLT are dependent on the association of CLT with other CVD risk factors in the rural-urban setting. Genetic polymorphisms of the PAI-1 gene did not directly influence CLT, despite influencing PAI-1act. The main contributor to PAI-1act variance, however, was (central) obesity. The effect of the 4G/5G polymorphism on PAI-1act, as well as gene–environment interactions for the C428T and G429A genotypes in determining PAI-1act and CLT, were significantly influenced by urbanisation. / Thesis (PhD (Nutrition))--North-West University, Potchefstroom Campus, 2013.

This work has explored the capability of a mixed film composed of oligonucleotides and oligomers to improve the selectivity for the detection of fully complementary oligonucleotide targets in comparison to partially complementary targets which have one and three base-pair mismatched sites. The intention was to introduce a “matrix isolation” effect on oligonucleotide probe molecules by surrounding the probes with oligomers, thereby reducing oligonucleotide-to-oligonucleotide and/or oligonucleotide-to-surface interactions. This resulted in a more homogeneous environment for probes, thereby minimizing the dispersity of energetics associated with formation of double-stranded hybrids. The mixed film was constructed by immobilizing pre-synthesized oligonucleotides onto a mixed aminosilane layer and then growing the oligomer portion by surface-initiated atom transfer radical polymerization (ATRP) of 2-hydroxy methacrylate (PHEMA). The performance of the mixed film was compared to films composed of only oligonucleotides in a series of hybridization and melt curve experiments. Surface characterization techniques were used to confirm the growth of the oligomer portion as well as the presence of both oligonucleotides and oligomer components. Polyatomic bismuth cluster ions as sources for time-of-flight secondary ion mass spectrometry experiments could detect both components of the mixed film at a high sensitivity even though the oligomer portion was at least 200-fold in excess. At the various ionic strengths investigated, the mixed films were found to increase the selectivity for fully complementary targets over mismatched targets by increasing the sharpness of melt curves and melting temperature differences (delta Tm) by 2- to 3-fold, and by reducing non-specific adsorption. This resulted in improved resolution between the melt curves of fully and partially complementary targets. A fluorescence lifetime investigation of the Cy3 emission demonstrated that Cy3-labeled oligonucleotide probes experienced a more rigid microenvironment in the mixed films. These experiments demonstrated that a mixed film composed of oligonucleotides and PHEMA can be prepared on silica-based substrates, and that they can improve the selectivity for SNP discrimination compared to conventional oligonucleotide films.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 136-152). / Microfiche. / xiv, 152 leaves, bound ill. 29 cm

Elevated total plasma homocysteine (tHcy) is a risk factor for cardiovascular disease and neurocognitive disease such as dementia. The B vitamins folate and B12 are the main de terminants of tHcy. tHcy concentration can also be affected by mutations in genes coding for receptors, enzymes and transporters important in the metabolism of Hcy. This thesis focuses on mutations in the genes for folate receptor-alpha and methylenetetrahydrofolate reductase (MTHFR) and the effect they have on tHcy concentrations. Six novel mutations in the gene for folate receptor-alpha were described in Paper I. Taken together they exist in a population with a prevalence of approximately 1% and thus are not unusual. There may be an association of –69dupA and –18C>T to tHcy but for the 25-bp deletion, –856C>T, –921T>C and –1043G>A there is probably no association to tHcy. Mutation screening was continued and four additional mutations, 1314G>A, 1816delC, 1841G>A and 1928C>T, were described in Paper II. The prevalences for the heterozygotes were between 0.5% and 13% in an elderly population. There was no significant difference in prevalence between the elderly subjects and patients with dementia. The 1816(–)-allele and the 1841A-allele were in complete linkage and the haplotype 1816(–)-1841A may possibly have a tHcy raising effect. The 1314G>A and 1928C>T mutations had no association to tHcy. The genotype prevalences and haplotype frequencies of the MTHFR 677C>T, 1298A>C and 1793G>A polymorphisms were determined in a population sample of Swedish children and adolescents (Paper III). The MTHFR 677T-allele was associated with increased tHcy concentrations in both children and adolescents. A small elevating effect of the 1298C-allele and a small lowering effect of the 1793A-allele could be shown. In an epidemiological sample of adults from the Canary Islands, Spain, data for serum folate and vitamin B12 were used for a broader study of the nutrigenetic impact on tHcy (Paper IV). The 677T-allele had a significant tHcy increasing effect in men but not in women. The 1298C-allele had a minor elevating effect on tHcy in men with the 677CT genotype. It was not possible to document any effect of the 1793A-allele on tHcy due to its low prevalence. A slightly superior explanatory power for the genetic impact was obtained using the MTHFR haplotypes in the analysis compared to the MTHFR 677C>T genotype-based approach in both the Swedish children and adolescents and in the Spanish adults. Therefore MTHFR haplotypes should be considered when analysing the impact of the MTHFR 677C>T, 1298A>C and 1793G>A polymorphisms on tHcy. Notwithstanding the large geographical distance between our study populations the haplotype composition is quite similar. The MTHFR 677T-allele is slightly more prevalent in Spain compared to Sweden but it has only an effect on tHcy in the Spanish men. Age, gender and factors linked to the ethnicity of the studied subjects, seem to be able to override the nutrigenetic impact of tHcy-raising genotypes or haplotypes in particular settings, such as in the Spanish women in our study. Gene-nutrient interactions on plasma tHcy levels thus may or may not exist in a certain population. The transferability of nutrigenetic findings may therefore be limited, and must be re-evaluated for each particular setting of age-gender-ethnicity.

[Formulae and special characters can only be approximated. Please see the pdf version of this abstract for an accurate reproduction.] Tumor necrosis factor (TNF) is a pleiotropic cytokine that mediates a long list of immunological and pathophysiological processes. TNF is produced by a wide variety of cells including immune and non-immune cells, however in most cell types TNF is not expressed prior to stimulation. The function of TNF is mediated via its trimeric domain by binding to TNF receptors that are found on most types of cells, especially of the haematopoietic systems, hence transpiring its effects on a wide variety of cells and organ systems. The cytotoxic (apoptosis) and pro-inflammatory (differentiation, proliferation and activation) functions of TNF are protective but can also result in pathological or deleterious consequences. A biallelic G to A transition polymorphism in the promoter region of TNF at nucleotide position 308 from the transcription start site is suggested to be involved in differential transcriptional regulation of TNF expression. The high TNF producing 308A allele is associated with susceptibility to or worse outcome of many infectious diseases in addition to autoimmune and other pathophysiological conditions. A previous study in our laboratory observed a selective affinity towards the polymorphic 308A allele by an EMSA protein(s) complex, named E. Several other protein complexes were found along with complex E and one of them was identified as Sp1. The identification of complex E was unsuccessful but it was hypothesized to play a major role as transcriptional activator in 308A allele individuals hence transpiring its effect in various pathophysiological states. In this study, the EMSA complexes observed in the TNF promoter region between nucleotides 322 to 283, encompassing the 308 polymorphism, is characterized. EMSA using mutated oligonucleotides mapped the binding sites of complexes B, C, D and E. TRANSFAC database search in addition to previous work revealed the identity of complex C as Sp1 but the rest of complexes remained unknown. Moreover, in contrast to our previous study, the protein(s) in the complex E was found to preferentially bind 308G nucleotide hence posing as a transcriptional repressor, resulting in decreased production state of TNF in 308G allele individuals than 308A allele individuals. In order to characterize putative transcription factors binding to the promoter region, first the biochemical characteristics such as the effects of temperature, salts and cations on DNA binding ability of EMSA complexes were studied. EMSA complexes B, C, DI and E required cations, probably Zn+2, to bind DNA. By optimizing a technique that couples EMSA with SDS-PAGE, the molecular weight of C, DI and E was determined. A novel technique that couples EMSA with IEF determined the pI of complexes B, C, D, DI and E. Although a commonly used technique of identifying unknown DNA-binding protein of interest, Yeast One-Hybrid assay, did not identify complex E, the novel identification method involving chromatography, two-dimensional electrophoresis, EMSA, mass spectrometry and database interrogation successfully identified TNF EMSA complex E as transcription factor Ying Yang 1 (YY1). Supershift EMSA confirmed complex E as YY1. In addition, the supershift assay showed presence of Sp1 and Sp3 in complex C. Similarly, complex DI is identified as Sp3. The novel method in identifying DNA-binding proteins is particularly useful as this technique allows identification of protein seen in EMSA without the need of extensive identification process. YY1 binds to a 6 base pair sequence, 5? TTGAGG 3?, from nt 295 to 290 of TNF promoter. The loss of affinity in 308A allele is caused by transition of underlined G nucleotide to A. The determined and described molecular weight of YY1 in literature is 60 kDa while the theoretical weight is 45 kDa. Both the determined and theoretical pI of YY1 is 5.8. YY1 is a multifunctional transcription factor implicated in both positive and negative regulation of gene expression as well as in initiation of transcription. It is ubiquitously expressed in growing, differentiated, and growth-arrested cells. Although future experiment is yet to establish in vivo presence of YY1 in TNF promoter, our study so far provides convincing evidence that the putative transcription factor that has selective affinity towards 308G allele is indeed YY1.

[Truncated abstract] For the convenience of the reader, detailed figures and tables have been enlarged and compiled in Appendix 2, at the end of this thesis. This thesis is presented as an approach to identify, annotate and detect genomic duplication and polymorphism within large genomic regions. To demonstrate this, I have used as a model, the genomic region known as the Regulators of Complement Activation (RCA). The RCA complex is located on the long arm of chromosome 1 at position 1q32 and is a reservoir of complement regulatory proteins. The genes of the RCA share many similarities implying that all have arisen through multiple complex duplication events. My analysis of this region in the following chapters demonstrates the complexity of this duplication and identifies the many functional units within the RCA. It was my aim at the beginning of these studies to demonstrate an approach that could define the Ancestral Haplotypes (AHs) of the RCA gene cluster. To do this, extensive genomic analysis was required and the ever-increasing availability of genomic sequence has made this thesis possible. Each of the chapters serves to address the following aims set out at the beginning of this thesis: 1. Further characterise the relationship between the genes (Complement Control proteins-CCPs) and domains of the Regulators of Complement Activation (RCA). 2. Identify and examine the duplicated elements within the RCA. - 6 - 3. Examine the effects of retroviruses and other insertions and deletions (indels) in generating the divergence of duplicated genes. 4. Investigate the applicability of the Genomic Matching Technique (GMT) to define AH within the region. 5. Examine association of AHs with CCP implicated diseases. 6. Determine the GMT applicability in non-human species

[Truncated abstract] Tumor Necrosis Factor (TNF) is a potent pro-inflammatory cytokine involved in a range of biological functions including the differentiation, proliferation and survival of many cell types. The TNF gene lies in the class III region of the major histocompatibility complex (MHC), approximately 250 Kbp centromeric of the HLA-B locus and 850 Kbp telomeric of HLA-DR. Due to the genomic location and biological relevance of TNF, it is thought that genetic heterogeneity at this locus may be associated with autoimmune and infectious diseases. A G-to-A single nucleotide polymorphism (SNP) at position -308 (relative to the transcriptional start site) in the TNF promoter has been well described. The less common -308A variant has been shown to be linked with the HLA-A1, B8, DR3 haplotype which in turn has been associated to a high TNF producing phenotype. Determining whether the -308 polymorphism contributes to elevated levels of expression has therefore been a priority for many research groups. Some investigators have shown differences in transcription between the -308G and -308A alleles while others could not. These contradicting results have led to conflicting views regarding the functional relevance of the -308 SNP. In this study, statistical analysis of 18 independent transient transfections of -308 biallelic TNF reporter constructs have provided evidence for a functional consequence of the polymorphism. ... In addition, chromatin accessibility of this region was maximal at greater levels of transcription suggesting a role for both chromatin structure and YY1 binding in -308G regulation. Surprisingly, chromatin structure did not seem to play a role in -308A regulation nor was there any significant binding of YY1, suggesting the -308 region does not affect transcriptional control of TNF. Taken as a whole, the G-to-A SNP relieves YY1 binding and demonstrates an allele-specific regulatory mechanism controlling expression. A growing list of promoter polymorphisms exists in the human genome having associations with certain diseases. Determining the functional consequence of these SNPs has proven difficult and utilized mainly in vitro approaches. In this thesis, a unique approach to investigating the functionality of promoter polymoprhisms has been developed, utilizing in vivo techniques which test their effects in a more natural system. It is hoped that the identification of the allele-specific YY1-mediated control of the -308 region of the TNF promoter may provide insight into overexpression as a consequence of the polymorphism and its role in the genetic susceptibility to MHC-associated autoimmune disease.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 155-182).

Thesis (MSc)--University of Stellenbosch, 2007. / Bibliography. Also available via the Internet.