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Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2013 Evolutionary studies are critical in eliciting the fundamental phylogeny within and among populations of living organisms. Genetic diversity is displayed in human mitochondrial DNA (mtDNA) as haplogroups that consist of shared mutations, which are carried to the following generation through the maternal lineage. The current haplogroup hierarchies commonly used to describe and compare the genetic diversity of global human populations are based on the available mtDNA sequence variation datasets of numerous continent-specific populations. The description of mtDNA variation in human populations is furthermore of importance, as it allows the identification of population-specific genetic variation that has an effect on gene function, as well as on adaptation and susceptibility to disease. Owing to the limited amount of available mtDNA variation data from the numerous African populations currently residing in Africa, a lack of genetic diversity data exists for the determination of a sufficient baseline standard sequence representing the genetic variation present in African populations and thus also for a representative African haplogroup hierarchy. In this study, the mtDNA variation of 50 Tswana-speaking individuals from South Africa was determined and a novel Tswana consensus sequence was constructed to contribute to the urgent need for information of the mtDNA variation present in African populations. The consensus mtDNA sequence variation data obtained through this analysis should be regarded as a baseline for the observed sequence variance and genetic diversity of the maternal ancestral genetic pool of a Bantu-speaking population of South Africa. This study therefore contributes novel information regarding the mitochondrial genetic diversity of a South African Tswana-speaking population to the current body of literature. The results of this study provide strong evidence to support the ancient nature of African haplogroups and also provide evidence in support of the presence of Khoi-San maternal ancestry in the origins of the current Bantu-speaking populations of southern Africa. In addition, the observed sequence variation contributes to the current haplogroup hierarchy of African lineages and provides information in support of the previously reported distinct phylogenetic relationship between individuals of African and non-African origin, thereby explaining the high level of genetic diversity among and between African populations. Doctoral

Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2010. Type 2 diabetes (T2D) is a complex disease that affects 4% of the general population and is expected to increase to 5.4% by the year 2025. A clear understanding of the aetiology of T2D susceptibility and pathogenesis will thus have a noticeable impact on global health. The black South African population is currently under increased risk for developing T2D due to the impact of urbanisation. Since the mechanisms of disease risk in this population differ to that of the so-called developed countries, it is necessary that the exact pathogenesis of this disease be elucidated in order to define suitable screening and therapeutic strategies for the black South African population. The purpose of this study was to initiate this process. Four genotypes were investigated, including alterations in the IRS-1, IRS-2, PPAR?2 and calpain 10 genes. This study was therefore the first to evaluate these specific genotypes in the context of the T2D risk phenotype in the black South African population, aiming towards a novel and population specific contribution towards current T2D research. The results of this study indicated that none of the screened genotypes were significant predictors of impaired glucose in the black South African population. A biphasic glucose curve shape (GCS) was associated with female gender, whereas a monophasic GCS, a high BMI, female gender as well as a high HbA1c level were linked to glucose intolerance. A high HbA1c level proved to be a significant predictor for glucose intolerance, although the four screened loci were not good predictors of the HbA1c level. The study also illustrated that it is not possible to simply adopt T2D screening strategies from those developed in other ethnic groups and that different genetic and environmental risk factors that play a role in the pathophysiology of T2D should be taken into account. The need for optimised and population specific T2D screening strategies is therefore emphasised. By further elucidating the complexities of T2D, a step towards providing more accurate screening strategies to the immediate population will be achieved. This will directly result in a significant decrease in the national burden of care, morbidity and mortality, paving the way to optimal health care strategies for this developing country. Doctoral

Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2010. Since the early 1980s, laboratory managers in the field of forensic science were introduced to the first international standards for testing and calibration laboratories. Now almost three decades later every laboratory should have some level of quality management system implemented to assure the quality of results. With all the quality standards and requirements in the global arena, proper assessment is necessary to ensure that quality standards are harmonised across laboratories. The aim of this study was first to establish the extent of quality standards and recommendations within the forensic drug environment and secondly, the level to which they were implemented in forensic drug laboratories globally. A questionnaire was developed to measure quality variables according to five categories in forensic drug laboratories, namely equipment, personnel, quality assurance and quality control, customer relationship as well as productivity. A total of seventy international drug laboratories participated in the study which consisted of laboratories from the United States of America, Canada, Australia, New Zealand, Belgium, Finland, Netherlands, Switzerland, Taiwan and Israel. To make statistical inferences on the greater population of forensic drug laboratories, all data was converted to proportions. These proportions were compared to international quality standards such as ISO 17025 and ASCLO/LAB. International forensic drug laboratories use similar analytical methodology when analysing drug samples. These techniques comply with ISO standards and can be accepted within any jurisdiction, if operated and maintained correctly. Laboratory managers should however pay more attention to maintenance and procurement plans. All the laboratories appoint qualified scientists and have internal training programs to ensure that technical staff are qualified and competent when performing specialised tasks. More attention should however, be given to mentorship programs to assess and coach new technical staff. The majority of laboratories complied with the technical and managerial ISO requirements on quality control and quality assurance, in spite of a non-standardised sampling scheme. Although laboratories have a good relationship with their customers, staff shortages will lead to extended turn around times which will influence customer satisfaction over time. Furthermore, small drug laboratories, translating to a small staff quotient, were determined to be more productive than larger laboratories. This study ultimately, underscores the fact that an established quality system and an effective laboratory information management system will contribute to higher productivity in a forensic laboratory environment. Masters

Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2010. The revised Cambridge Reference Sequence (rCRS) is used as a standard for the human mitochondrial DNA (mtDNA) sequence in studies of human evolution and the identification of disease-causing mutations. Due to the large number of differences observed between the rCRS and mitochondrial sequences obtained from individuals of African descent, it is frequently difficult to differentiate between alterations that are population-specific or have possible pathological significance. To address this problem, two human consensus sequences consisting of mitochondrial sequences from different continents and different haplogroups have been constructed. However, combining data from different continents and haplogroups led to a loss of variation in the human mitochondrial consensus sequences. This can be countered by using an African reference sequence, of which two sequences are currently available, namely NC_001807 (L3a1) and D38112 (L0c2). However, these sequences are not representative of the most ancient African populations (i.e. hunter-gatherers) or haplogroups (i.e. L0). In the current investigation, the complete mitochondrial genome sequences of 30 Khoi-San individuals from southern Africa were determined. Twenty-two of these Khoi-San sequences, which belong to the L0a and L0b sub-haplogroups, were combined with 13 L0 Khoi-San sequences generated previously to compile a consensus sequence for the Southern African Khoi-San population. This Khoi-San consensus sequence will represent the first example of an African population-specific consensus sequence. The results presented in the current investigation provide support for previous findings regarding the existence of a high level of genetic variation in the mtDNA of the Khoi-San population, as well as the ancient character of the Khoi-San population. In addition, it offers novel insights into the complexity of the L0 haplogroup within the Khoi-San population and the African population as a whole. The high level of genetic variation and increased age of mtDNA lineages in the Southern African Khoi-San population compared to other populations, support the use of the Khoi-San consensus sequence, alone or in conjunction with the rCRS, as a standard in studies of human evolution and mitochondrial disease. Doctoral

Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2008. Malignant hyperthermia (MH) is an autosomal dominant, potentially lethal pharmacogenetic disorder of skeletal muscle, which is elicited by exposure to volatile anaesthetics and depolarising muscle relaxants. Susceptible individuals appear clinically normal, but may present with a hypermetabolic crisis and muscle contracture when exposed to triggering substances that elicit excessive release of calcium ions from the sarcoplasmic reticulum. Diagnosis of MH susceptibility is currently made via the in vitro contracture test. Genetically, in more than 50% of the affected families, MH occurs due to alterations in the skeletal muscle ryanodine receptor gene (RYR1) on chromosome 19q13.1. However, the disorder is genetically heterogeneous, as six other loci have to date been associated with MH susceptibility (MHS). Thus far, molecular tests have focused on three mutation hotspots of the RYR1 gene, which refer to regions that are more frequently mutated. Screening the entire RYR1 has led to a higher detection rate in a variety of populations. In this study the entire coding region of the RYR1 gene was screened via sequencing for novel or reported alterations for the first time in 15 South African probands. Eight different RYR1 alterations were observed in seven MHS South African probands, six of which were previously reported and two of which were novel. Compound heterozygous alterations and alterations outside the mutation hotspots were detected. Screening of the entire coding region of the RYR1 gene is crucial for genetic investigations into MHS. It was postulated that MH in the South African population is due to multifactorial inheritance in which a network of several genetic, environmental and epigenetic factors interact and cumulatively result in the development of the MH phenotype. Data generated in this study highlight the complexity of this disorder, further supporting a novel epigenetic aetiology for MH in the South African population. Doctoral

Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2006. Mitochondrial NADH:ubiquinone oxidoreductase (complex I) carries out a number of well defined functions required for cell physiology. Deficiencies of complex I lead to multi-system disorders that include several well-known phenotypes such as type 2 diabetes mellitus, Alzheimer's disease as well as less known phenotypes such as MELAS, Leigh syndrome and MERRF. It was recently identified that ROS sensitive proteins known as metallothioneins (MTs), are over-expressed in complex I deficient cell lines and that these proteins have a protective effect against ROS related pathologies. It is still not clear if isoform-specific MT expression occurs in this disease and if it plays a significant role in vivo. This study investigated the expression of different MT isoforms in rotenone-treated Sprague Dawley rats, an in vivo model that has been used to study cellular biological responses of mitochondrial complex I deficiency. The hypothesis of this study states that a rotenone-induced complex I deficiency would lead to an increase of MT mRNA expression in vivo. The specific aim was to determine the relative mRNA expression levels of the three main MT isoforms in rotenone-treated rat tissues. Real-time PCR was used to achieve this aim. In this dissertation the differential expression of MT-1, MT-2 and the brain specific isoform, MT-3 in brain, liver, heart- and skeletal muscle tissues of rotenone-treated Sprague Dawley rats is described. The results indicate that MT-1 expression is significantly increased in the liver as well as, but to a lesser extent, in the brain and heart muscle. MT-1 expression in skeletal muscle was not detected. In contrast, significant increases in expression were observed for MT-2 in all the tissue types with an approximate two-fold increase at the highest rotenone dosage in liver, brain and heart muscle. Skeletal muscle had the smallest increase. For MT-3, no detectable levels of expression could be observed in skeletal and heart muscle. Surprisingly, levels of expression occurred in the liver which slightly (43%), but significantly increased at the highest rotenone dose. As expected, much higher relative levels of MT-3 expression were observed in brain tissue with a more pronounced increase (almost two-fold) at the highest rotenone dose. As the hypothesis of this study proposed, the in vivo data generated from this study supports the published in vitro data which showed that a rotenone-induced complex I deficiency results in MT expression. This over expression may contribute to a protected effect on the pathology of this disease although this still needs to be established. Furthermore, the results of this study show that the expression of the various MT isoforms in rotenone-treated rat tissues is not expressed in a similar way to the induced deficiency which may point to a differential regulation and response of the three MT isoforms to such a deficiency. Masters

Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2005. Type 2 diabetes mellitus (T2D) is currently one of the fastest growing non-communicable diseases in the world. It is induced by the pathogenic interaction between insulin resistance and secretion. There are numerous forms of these disorders which are characterised by hyperglycaemia and affect approximately 4% of the general population. This percentage is however rapidly increasing especially in developing regions such as sub-Saharan Africa and Latin America. During this investigation two diabetic cohorts and two control cohorts consisting of adult black Southern African and Cuban individuals respectively, were screened for reported single nucleotide polymorphisms (SNPs) within the adiponectin and calpain 10 genes. Genotyping was achieved via a real time PCR strategy. Frequency differences between the various genetic configurations of the two cohorts were calculated utilising appropriate statistical analyses. With regards to the black Southern African cohort, it was determined that certain factors in the calpain 10 gene, e.g. the wild type homozygote at UCSNP-56, were associated with protection towards T2D. Investigation of the Cuban cohort alternatively resulted in the elucidation that this group presents with a differential risk pattern than that of the reported European populations. Analysis of the adiponectin gene resulted in the determination that within the South African cohort, the G-11391A locus and the 11/12 haplotype combination were associated with protection towards T2D. The variant allele homozygote at the C-11377G locus was associated with increased disease risk within the Cuban cohort investigated. The associations detected in the aforementioned genes were not maintained upon meta-analysis. When compared to various non-African populations, the investigated SNPs have population specific effects in T2D susceptibility depending on the population investigated. This is most likely due to certain epistatic factors, determination of which will be integral to future investigations of T2D. Data from this investigation indicates that the elucidation and implementation of prevention strategies should be population specific. Doctoral

Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2005. Increased reactive oxygen species (ROS) have been reported to be at the centre of various diseases. Although several reports have implicated elevated levels of ROS in the pathogenesis of diabetes mellitus, the early detection of ROS is still not attainable. This limitation causes difficulty in the early diagnosis of ROS related disorders. The presence of high levels of ROS was reported to result in differential expression of antioxidant genes involved in protecting cells from their deleterious effects. Among the antioxidant genes that are expressed, it was postulated that expression of metallothioneins (MTs) are also induced. MTs are low molecular weight, cysteine-rich proteins involved in metal homeostasis and reported to harbour antioxidant function. The aim of this investigation was to explore MTs as biomarkers for elevated levels of ROS in whole blood of type 2 diabetic (T2D) individuals. The level of ROS in diabetic, non-diabetic as well as individuals at risk of developing T2D was determined via the use of biochemical assays. Real-Time PCR was utilised to analyse the expression of MTs and the presence of MT proteins was analysed via the ELISA. In this study it was observed that diabetic individuals had elevated levels of ROS. However, no significant difference in the expression of MTs and the presence of MT proteins between the diabetic and non-diabetic individuals was observed. In vitro experimental conditions indicated that MT expression is induced by elevated levels of ROS. In pathological conditions the ROS-dependent induction of MT expression needs to be elucidated further. It therefore can be suggested that MTs can not yet be utilised as biomarkers for the detection of elevated levels of ROS in pathological conditions with ROS aetiology. This investigation also highlights the fact that blood is not an optimal medium in which this objective can be attained. Doctoral

Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2005. The relevance of haplogroups in mitochondrial disease was investigated in this study. Twenty-seven paediatric patients with clinically suspected mitochondrial disorders from diverse ethnic origins were investigated via an automated sequencing strategy. Previous studies in a similar population identified only the A3243G reported causative mutation in a single patient, in addition to a number of reported and unreported polymorphisms. It suggested that the aetiology of mitochondrial disorders in Africa, specifically South Africa, might be different from that of other continents. Comparison between these sequences and the Revised Cambridge Reference Sequence revealed 37 polymorphic sites (7 novel changes, 30 reported alterations). No previously reported causative mutation was detected. The findings support the hypothesis that the aetiology of mitochondrial disorders in Africa is unique. Sixty-three percent of patients in the current study belonged to haplogroup L3 (48% in L3b), 22% to LO, 11% to L2a, and 4% to M. No patients with an L1 haplogroup were observed in this study. The above-mentioned observations have important implications. There was distinct clustering of affected patients in macrohaplogroup L. In this patient cohort, certain sub-haplogroups may play a susceptibility or protective role with regard to mitochondrial dysfunction. Results generated in this study suggested that differential haplogroup-tissue-specific reliance on mitochondrial ATP may culminate in specific phenotypic consequences. Masters

MSc (Biochemistry) North-West University, Potchefstroom Campus, 2004 Maternal inheritance and the absence of recombination have contributed to mitochondrial deoxyribonucleic acid (mtDNA) being utilised to study human evolution. This, together with an increased mutation rate in mtDNA, provides information about the most recent common ancestor of modern humans. Previous studies suggested that Africa harbours the highest mtDNA diversity, supporting an out-of-Africa hypothesis for modern human evolution. From subsequent studies it was suggested that the Khoi-San population, in particularly the !Kung, cluster at the deepest root of the global phylogenetic tree. The Cambridge reference sequence is used worldwide in mitochondrial studies as a reference. However, recent studies have observed discrepancies from this sequence, which were confirmed by reanalysis. During this investigation the complete mitochondrial sequences of 13 !Kung individuals were determined. From phylogenetic analyses their clustering in the African LO-Iineage was revealed. The evolutionary rate of the derived sequences was investigated through statistical analysis and the hypothesis of neutral evolution was rejected. Pairwise nucleotide distribution suggested that sequences representing haplogroups LO, L 1 and L2 are examples of populations that were of stable population size for a long time. However, L3 was suggested to have been subjected to population expansion, in support of the out-of-Africa theory of evolution. From the comparative analysis of the 13 !Kung sequences with an LO-specific haplogroup tree it was observed that the 13 individuals clustered in two main groups. Ten individuals were added to one branch of the phylogenetic tree, revealing further branching, while three individuals were added to the terminal branches of another tree topology. A consensus sequence was derived from the 13 Khoi-San sequences, which was 99.25% similar to each of the sequences. This sequence could be utilised to investigate evolution of the mitochondrial genome over time as well as to evaluate the pathogenicity of mutations in patients. Masters