Includes abstract. / Includes bibliographical references (p. 183-214). / Obesity and obesity-related diseases are a large global problem in both developed and developing nations. In South Africa, a country currently undergoing epidemiological transition, the prevalence of obesity is high, particularly in urban black women. Early detection of overweight and obese individuals is essential for the management of obesity and its related co-morbidities; however, there is no ethnic-specific field measure of body fat percent validated for use in black South African women. Further, despite high levels of adiposity, these women have an atypical presentation of cardiovascular disease (CVD) risk factors, presenting with relatively low levels of visceral adipose tissue (VAT) and a favourable lipid profile compared to white women. As a result of this atypical presentation of CVD risk factors, a high prevalence of “healthy obesity” has been reported, although the determinants of this phenotype have not been systematically investigated. In addition, the applicability of commonly used diagnostic criteria for the determination of insulin resistance, which include enlarged waist circumference and dyslipidemia as components, has not been investigated in this population. Therefore, the overall aim of this thesis was to investigate the impact of body fat and its distribution on the presentation and identification of CVD risk factors in relatively young black South African women, prior to the onset of CVD. More specifically, the objectives were; i) to determine if near infrared interactance (NIR) is a valid field measure of body fat percent in South African women; ii) to determine the agreement between International Diabetes Federation (IDF) and National Cholesterol Education Program (Adult treatment panel III) (ATP III) metabolic syndrome criteria and the degree to which these criteria can predict insulin resistance, and explore the extent to which these phenomena can be explained by body fat and its distribution; iii) to identify determinants of the “metabolically healthy obese” (MHO) and “metabolically obese normal weight” (MONW) phenotypes; and iv) to complete a preliminary investigation of the association between polymorphisms within genes that encode for proteins involved in tissue-specific glucocorticoid metabolism and obesity, body fat distribution and CVD risk factors in black South African women. As obesity is associated with increased risk of cardiovascular disease, accurate quantification of body fatness is particularly important in health risk appraisal. However, in developing countries, “gold standard” measures of body fat percent such as underwater weighing and dual energy x-ray absorptiometry (DXA) are not always practical, as access to facilities and resources are limited. Therefore, a valid field measure of body fat percent is needed for the purpose of health risk appraisal. NIR is a potentially useful field measure of body fat percent that is currently used in South Africa for this purpose. However, NIR cannot be used with confidence in South Africa until it has been validated in different ethnic populations. Therefore, the first study in this thesis examined the validity of singlesite NIR (Futrex-6100 A/ZL) as a measure of body fat percent compared to the criterion method of DXA in black and white South African women.

Includes bibliographical references. / The identification of the genetic variants underpinning bipolar disorder (BPD) has been impeded by a complex pattern of inheritance that may include by genetic heterogeneity, genetic epistasis, gene-environment interactions, incomplete penetrance and variable expressivity. In this thesis three strategies were employed to ameliorate these confounding factors. The first strategy was to focus on a theoretically genetically-homogeneous population with BPD. A unique South African sample including 190 individuals of the relativity reproductively-isolated Afrikaner population yielded promising evidence of linkage to chromosome 1 q31-32 and weaker peaks at lOq23 and 13q32, regions previously implicated in the disorder. A family-based analysis suggested that the 3' variable number tandem repeat (VNTR) variant of the dopamine transporter gene (DAT) is associated with bipolar-spectrum illness in the 132-strong sample of British ancestry. The second strategy was to carry out genetic linkage and association analyses using quantitative traits (elldophenotypes) that were closely associated with BPD. As part of this process a variety of personality traits were evaluated in the cohort, and anxiety related, novelty-seeking, hyperthymic, and cyclothymic personality traits were found to aggregate in participants with BPD and to a lesser extent repeated unipolar illness (MDE-R). These traits were therefore used as quantitative markers or endophenotypes of BPD. The quantitative linkage analysis indicated that a variant in the region of 13q32 may influence the development of novelty-seeking-related traits in the largest Afrikaner pedigree, while the personality trait, ""Stability"", was weakly linked to 4p16 in the total sample. The catechol-o-methytransferase (COM1) Va1l58Mct and the Brain Derived Neurotrophic Factor (BDNF) Va1l66Met polymorphisms were associated with mood-labile-cyclothymic and hyperthymic·-novelty-seeking traits, respectively. the DA T VNTR and the Notch4 exonic repeat variants were associated with a broad range of ""pathological"" personality traits in the sa11lples of British and Afrikaner origin, respectively. The sample was also evaluated with a battery of neuropsychological tasks and the BPD 1 and MDE-R groups displayed both verbal and visual memory recall deficits while the BPD 1 sample also suffered from recognition memory deficits. The neurocognitive trait, ""Memory"" was therefore used as a second endophenotype generating potential linkage signals on IOq23 and 22q 11. The exonic 48bp VNTR polymorphism in the dopamine 4 receptor (DRD4) gene was associated with '""Memory"" performance. As a third strategy, a potentially important aetiological factor, childhood trauma, was measured, and used to test for gene-environment interactions between the various candidate genes and bipolar-illness or BPD-related endophenotypes in the cohort. BPD and M DE-R individuals displayed significantly higher levels of emotional and physical abuse, and the former variable was also associated with the development of anxiety-related and unstable personality traits. A functional variant of the COM1 gene was found to interact with abuse to predispose to anxiety-related, unstable cyclothymic and novelty-seeking related personality traits. The combination of childhood abuse and possession of low-activity MAO-A gene variants was also associated the development of more anxious and unstable personality traits. All interaction between sexual abuse and the B])NF gene modulated performance on verbal and visual memory tasks. A similar interaction between the ApoE gene and sexual abuse was observed. Although a number of theoretical obstacles remain to be resolved, the analyses of isolated populations coupled with the use of endophenotypes and the testing or gene environment interactions, holds out great promise for the eventual elucidation of the genetic basis of hi polar affective disorder.

The reliability and validity of a performance test is important in research to detect meaningful performance differences following an intervention. In accordance with this, the aim of the first study of this thesis was to investigate the reliability and validity of a self-paced 10 km treadmill time trial. This performance measure was then used in the main section of this thesis. This comprised a large training intervention study aimed to answer specific questions following three different high intensity interval training programmes. In particular, changes in 10 km running performance were investigated with respect to various physiological parameters, both immediately following the training intervention, as well as during a subsequent three-week taper period. Methods In the first study, a group of well-trained male runners (n = 8) completed four 10 km treadmill time trials and two 10 km track time trials. Comparisons in performance time were made between the 10 km treadmill time trials to determine the typical percent error between these trials. Additionally, comparisons were made between the track and treadmill time trials. In the second study, well-trained male runners(n = 32) were randomly assigned to one of four groups; a control group, a 400 m interval group, a 1600 m interval group and a mixed (400 m and 1600 m) interval group. The intensity of the intervals was based on the participants' current 10 km time trial time. The high intensity training interventions consisted of eight interval sessions (twice per week) over a four-week period followed by a three-week singlestep30% reduction in total training volume (while maintaining training frequency and some intensity) in all groups.

Includes abstract. / Includes bibliographical references (leaves 118-133). / The polyglutamine disorders are a subgroup of inherited neurodegenerative disorders with a common mutation which confers toxicity via a polyglutamine tract in the protein leading ultimately to various forms of neurodegeneration. One of these disorders, spinocerebellar ataxia 7 (SCA7) exists at a higher frequency in South Africa, than elsewhere in the world, and a founder effect has been demonstrated in South Africa, such that every patient tested thus far is linked to a common ancestor. The manipulation of RNA interference (RNAi) has been used with increasing success to selectively knockdown the expression of disease-causing genes at the RNA level. Thus, the possibility of applying this method to SCA7 in South Africa was considered. However, the wild-type allele of ataxin-7 is likely to be necessary for cellular function therefore a form of allele-specific silencing is required, such as a SNP linked to the mutation.

Bibliography: leaves 272-358. / The hypothesis, upon which this thesis is based, is that repeated bouts of damage-inducing, prolonged, endurance training and racing, over a number of years, may exceed the biological limits of the repair and adaptation process, resulting in maladaptation of the skeletal muscle and malfunctioning of the system.

Background: Inherited retinal diseases (IRDs) constitute a group of clinically and genetically heterogeneous conditions which cause degeneration of retinal photoreceptor cells and result in visual impairment. Characterisation of the genetic basis of IRD is not only beneficial for the affected families, but also contributes towards understanding of the disease pathobiology. Investigations into the molecular basis of IRDs have been ongoing in South Africa (SA) for over 30 years, however the evaluation of reported genetic mutations has yielded low returns in certain populations. Indigenous southern Africans comprise a unique population group with distinct genetic diversity, providing a valuable resource for genetic discoveries; nonetheless, this population remains largely underrepresented in genomic studies. The aim of this investigation was to characterise the underlying genetic mutations in a cohort of indigenous African IRD patients. Methods: The IRD registry in the Division of Human Genetics (University of Cape Town) was reviewed for causative mutations. Subsequently, upon identifying a mutation underlying Usher Syndrome in two indigenous African patients, an assay was designed to screen for this mutation in probands with different IRDs (n=170) and controls (n=51), and haplotype analysis was performed on mutation-positive individuals. The registry review additionally served to identify a suitable cohort for the application of next generation sequencing (NGS) technology. Whole exome sequencing (WES) was performed on genomic DNA samples from 56 individuals from 16 families. The WES data analysis strategy involved prioritisation of variants in reported and candidate IRD genes. Rare, co-segregating, pathogenic, exonic or splice variants were validated by Sanger sequencing. Custom TaqMan assays were designed to screen seven mutations, identified by WES, in 193 unrelated indigenous African probands with IRDs. Results: A homozygous founder mutation, c.6377delC in MYO7A, was identified in 43% of the indigenous African patients with Usher syndrome, which is the most common cause of deaf-blindness. Targeted WES data analysis of all known IRD genes resulted in identification of the underlying genetic defects in six distinct genes (RHO, PRPF3, PRPF31, ABCA4, CERKL, and PDE6B) in six families. Taqman screening revealed four additional probands with identical homozygous mutations in CERKL and PDE6B. An X-linked gene (RP2) mutation was subsequently identified in an affected family with semi-dominant retinitis pigmentosa. Supplementary analysis of the X-linked RPGR ORF15 mutation hotspot (not adequately covered by WES) identified two mutations in three families. A novel IRD gene, IDH3A, was found in one family by analysis of 22 putative candidate genes. The large number of variants in the remainder of the indigenous African exomes presented considerable challenges for identification of additional novel genes. Discussion: The results of this project have important implications for IRD molecular diagnostic services in SA. Using WES, a genetic diagnosis was obtained for ±73% of the indigenous African cohort, and ±70% of the causative mutations identified were novel. This outcome emphasises the superiority of NGS-based approaches over genotyping-based microarrays which screen for IRD mutations previously reported in other (mainly European-derived) populations. The unexpected identification of mutations in known X-linked genes in four families highlighted key considerations for IRD WES analysis. Cascade screening of mutations identified in this study, across larger cohorts of unrelated probands, revealed the genetic cause of IRD in additional cases and the number of indigenous African families in the registry with a genetic diagnosis was effectively doubled. Members of these families can now opt for diagnostic, carrier, or predictive testing of familial mutations. Finally, the information obtained from this research contributes towards a better understanding of the genetic architecture of IRDs in SA.

There is a well described association between participation in exercise and sport and the positive effects of physical, social and psychological health. 1,2 Rugby union is a popular team sport across many countries.3 As a team sport, rugby shares these positive benefits. However, the physical demands of the game means that it is also associated with a risk of serious injuries, such as spinal cord injuries (SCIs). 4–6 SCIs have profound long-term effects on every aspect of a person’s life, including their overall quality of life (QoL).7–10 As such, a nationwide injury prevention programme called ‘BokSmart’ was launched in South Africa in 2009, with the aim to reduce and ultimately prevent these injuries.11,12 However, implementing an injury prevention programme in a country with vast socio-economic disparities, such as South Africa, is a difficult task.13 Additionally, optimal acute care after the injury, rehabilitation services and ongoing health maintenance are essential in the management of SCIs and may play a determining role in enhancing and maintaining health and functioning, and therefore QoL.14–16 In South Africa, socio-economic disparities also have a profound effect on healthcare access and the subsequent health of the population. 14 Thus, the additional burden of an injury with permanent consequences may be substantial and is an important issue to investigate. This introductory chapter summarises the literature on the incidence and risk factors for rugby-related SCIs, and the immediate management of these injuries. It also summarises the long-term healthcare issues and overall QoL of players who sustain these injuries and identifies how these problems present both globally and in South Africa. This chapter also provides the overall structure of this PhD-thesis.

Introduction: The mismatch repair system plays an important role in maintaining the genome integrity as it functions to correct mismatches during DNA replication. Heterozygous mutations in one of the mismatch repair (MMR) genes e.g. MLH1, MSH2, MSH6 and PMS2 cause the dominant adult cancer syndrome termed Lynch syndrome (or hereditary non-polyposis colorectal cancer). In our South African cohort, the MLH1 exon 13 c.1528C>T mutation is the most common Lynch syndrome-causing variant in the Mixed Ancestry population. Recently, a patient homozygous for this mutation, diagnosed with Constitutional mismatch repair deficiency (CMMR-D) syndrome was described within this extended cohort. CMMRD syndrome results in an increased predisposition to a range of cancers, most commonly brain and hematological tumours in early childhood. The aims of this thesis were: (i) to determine the rate of extra-colonic cancers in the cohort of Lynch syndrome families in our colorectal cancer registry, (ii) to determine if MLH1 c.1528C>T is a founder mutation, and (iii) to focus on the CMMR-D syndrome as a branch of Lynch syndrome and to potentially use the hypermutability-status in CMMR-D to understand the diverse carcinogenesis in Lynch syndrome. Methods: The registry consisting of Lynch syndrome families was interrogated and analysed to address the aim (i). Haplotype analysis was performed using microsatellite markers around the MHL1 c.1528C>T mutation to determine founder effect for aim (ii). For aim (iii) whole exome sequencing was also performed in a Lynch/ CMMR-D syndrome family in order to investigate the extent of hypermutability in CMMR-D syndrome, and to develop a working hypothesis for carcinogenesis in CMMR-D and Lynch syndromes. Results: From the analysis of the registry it was noted that 396 individuals carried a disease-causing mutation in either MLH1 or MSH2; females have a relatively later age of onset (for cancer) than males and MLH1 mutation carriers develop cancers relatively earlier in life than in individuals with MSH2 mutations. The most common extra-colonic cancers were endometrial and breast in females; in males small bowel cancer was most common, after CRC. The cohort study revealed a large founder effect with the MLH1 c.1528C>T mutation, with the most common inferred (disease-associated) haplotype found in 25 of the 30 subjects tested; the disease-associated haplotype was not present in controls. The mutation aging analysis traced the mutation to be ~225 years old. The WES investigation of the nuclear family within which the CMMR-D patient, including acquired and germline mutations in tissues from the child with CMMR-D, revealed a range of pathways including the extracellular matrix, WNT signaling, TGFβ and p53 as acquiring significant numbers of variants as a result of the MMR deficiency. Discussion and Conclusion: The results which are indicative of the need to improve the Lynch syndrome mutation testing and management for all patients, also suggests the need to develop surveillance programs for extra-colonic cancers, which will improve compliance and disease-free survival. WES investigation of the nuclear family containing a child with CMMR-D point to the potential involvement of a range of pathways associated with cancer development which may be indirectly invoked in the process of tumorigenesis by the wide range of variants acquired as a result of mismatch repair deficiency. It is likely that some of these processes are also involved in the emergence of extracolonic cancers in individuals affected with Lynch syndrome (i.e. heterozygous for mutations in MMR genes).

Includes abstract. / Includes bibliographical references. / T-box genes constitute an ancient family of developmentally important transcription factors that have gained prominence in cancer biology. For example, endogenous TBX2 is overexpressed in a growing list of cancers and when ectopically overexpressed it can bypass senescence and the tetraploidy checkpoints that protect against cancer. When the current study was initiated it was unclear as to whether the endogenous overexpression of TBX2 was a cause or consequence of the oncogenic process and whether it may be a suitable anti-cancer drug target. The objectives of this study were therefore (1) to explore the role of increased TBX2 levels in melanomagenesis by assessing the effect of knocking it down in melanoma cell lines, (2) to determine the mechanism by which TBX2 is upregulated by the UV-induced DNA damage pathway in melanoma cells and (3) to verify whether TBX2 is a suitable drug target by assessing the cellular sensitivity of control and TBX2-depleted cells in response to the chemotherapeutic drug cisplatin.

Includes abstract. / Includes bibliographical references (p. 185-201). / Measurements of muscle activity derived from surface EMG electrodes are variable due to both intrinsic and extrinsic factors. The intrinsic factors are endogenous in nature (features within the body) and include muscle fiber type, muscle fiber diameter and length, the amount of tissue between muscle and electrode, and depth and location of muscle with respect to the placement of electrodes (24). These biological factors vary between subjects and cannot be controlled. The extrinsic factors are experimental variables which are influenced by the researcher and can be controlled to some extent. Examples of extrinsic factors include the location, area, orientation, shape of electrodes and the distance between electrodes (interelectrode distance). In order to measure biological variation in the EMG signal, which is important in studies where surface EMG is used to gain understanding of physiological regulation, it is important to minimise the variation caused by these factors. This is in part achieved through the appropriate method of normalisation. The isometric maximal voluntary contraction (MVC) has been used as a standardmethod of normalisation for both static and dynamic exercises. However, researchers have recently improved the methods of normalisation by developing alternative techniques for the measurement of EMG during dynamic activities. By using the same type of movement for normalisation as during the trial, experimental errors can be reduced. The appropriate method of normalisation is defined as a method that is capable of showing repeatability, reliability (low intra-subject variation) and sensitivity to changes in EMG amplitude that is due to biological change and not the contribution of experimental factors. The aim of this thesis was to critically analyse alternative methods of EMG normalisation during dynamic exercise. The data should provide possible guidelines to researchers who are planning studies involving measurement of EMG activity during cycling, running and in clinical populations. Furthermore, the thesis aimed to illustrate that decisions regarding the most appropriate method of normalisation should be based on the study design, research question (absolute muscle activity or changes in muscle pattern) and the muscles being investigated.