Bibliography: leaves 203-245. / Delta-aminolaevulinic acid synthase (ALAS) is the first enzyme in the haem biosynthetic pathway, and in mammals is present in two isoforms - differing in regulation and encoded for on different chromosomes. The hepatic form is up-regulated in states of haem deficiency and thus implicated in the acute attack of porphyria. Given the large number of patients who suffer from an acute form porphyria in South Africa the regulation of the human hepatic form of the enzyme is of considerable interest.

This systematic review and meta-analysis forms the mini-dissertation part of the primarily coursework degree of Master of Public Health at the University of Cape Town. The review is divided into three separate parts. The first part constitutes the research protocol which outlines the proposed methods and scope of the review. The second part is a structured literature review further elaborating on the research topic and current knowledge of it. The final third part constitutes the manuscript-ready part of the review. The aim of the review is to systematically explore the current scientific literature regarding the association between narcolepsy and the Pandemrix pandemic influenza vaccine. This particular association has been one of the most studied presumed vaccine adverse effects of the last decade and it has greatly affected the discussion on vaccine safety and vaccine hesitancy still to this date. Despite several studies having been published investigating the association, no systematic review of these studies had been performed prior to the beginning of this this review.

Background Gestational diabetes mellitus (GDM) is one of the most common obstetric complications, affecting 18.4 million live births globally. In South Africa, the high prevalence of GDM estimated to be up to 25.8%, is driven by the high rates of obesity. Women with a history of GDM have a seven-fold increased lifetime risk of developing type 2 diabetes. In addition, children born of women with GDM are susceptible to impaired glucose tolerance and obesity in adulthood. Prioritising GDM as a public health issue is critical for improving maternal and child health care services and non-communicable disease prevention efforts. There is consistent evidence demonstrating that, in at-risk populations, lifestyle change can reduce the risk of developing type 2 diabetes and among women with GDM, continuation of lifestyle changes after a GDM pregnancy can prevent progression to type 2 diabetes. Understanding women's lived experiences and views around GDM is critical for the development of behaviour change interventions. Further, insights from women's experiences of the healthcare system for GDM care are important for informing health policy and improving the quality of care. At present, little is known about the policies and practices relating to the management of GDM in low- and middle- income countries and health systems interventions that support and facilitate continuity of care are lacking. This thesis constitutes the formative research for a complex intervention study aimed at developing and evaluating a novel health system intervention to reduce the subsequent risk of developing type 2 diabetes among women with GDM. The findings will inform the planning, development and testing of an appropriate and innovative intervention for women with GDM, to be integrated into existing health services in South Africa. Methods and Results The first study used document reviews of policy documents and clinical practice guidelines for the management of GDM in South Africa and 11 key informant interviews with policy makers, health service managers from the Department of Health and clinicians working in the public health services to explore the existing policies and reported clinical practices relating to antenatal and postnatal care for women with GDM in the public health sector in South Africa and identified important factors in delivering an integrated mother - baby postpartum health service. We found that the management of GDM in South Africa is aligned with international standards, in addition to locally developed guidelines and protocols for clinical practice. Our findings also confirmed that postpartum follow-up for women with GDM is a significant problem in South Africa as a result of fragmentation of care and the absence of standardised postnatal care services. Key informants also raised patient – related challenges including lack of perceived future risk of developing type 2 diabetes and non-attendance for postpartum follow up, as barriers to postnatal care for women with GDM. The second study was a descriptive qualitative study which explored the lived experiences of 35 women with prior GDM using content analysis and the COM-B model to identify factors influencing lifestyle change during and beyond the GDM pregnancy. The results suggest that the COM-B model's concepts of capability (knowledge and skills for behaviour change), opportunity (resources for dietary change and physical activity) and motivation (perception of future diabetes risk) are relevant to lifestyle change among women with GDM in South Africa. The third study was an in- depth exploration of women's lived experiences of GDM, their context and perceived needs. Women discussed the emotional and psychological burden of having GDM, highlighting (i) their initial emotional reactions to receiving a GDM diagnosis, (ii) their experience of adjusting to the constraints of living with GDM (iii) their feelings of apprehension about childbirth and their maternal role and (iv) their feelings of abandonment in the post-partum period once the intensive support from both health system and family ends. The fourth study further explored women's perspectives of and satisfaction with GDM care in the public health sector as well as their views on the feasibility and acceptability of the proposed intervention using the Donabedian's quality of care framework. Key sub-themes relating to ‘structure' of care were the organisation of GDM health services, efficiency of the referral, efficiency of management practices, quality of hospital facilities and services and the availability of adequate healthcare resources. Overall satisfaction with GDM care processes was influenced by women's unmet need for education and behaviour change counselling, interpersonal support from health care providers and peers and the health services' capacity to enable them to actively participate in their care. Conclusion The management of GDM is currently foetal - centric and over-medicalised, focusing on clinical care processes to achieve glycaemic control and safeguard foetal health. There is a critical need for woman-centred approaches to be incorporated in the management of GDM in order to improve women's overall experiences of care. Behaviour change interventions for women with GDM should target factors within the physical and social environments, that influence women's capacity for lifestyle change. In addition, we recommend routine mental health and psychosocial vulnerability screening and monitoring for women diagnosed with GDM throughout pregnancy and postpartum to improve prognoses. Finally, holistic interventions and health policies that directly support continuity of care are urgently needed if high rates of progression to type 2 diabetes in this population are to be avoided.

Background: The quality of INR control determines the effectiveness and safety of Warfarin. Data on INR control in non-metropolitan settings of South Africa (SA) is sparse. Objectives: To examine the Time in Therapeutic Range (TTR) and its potential predictors in a sample of Garden Route District Municipality INR clinics Methods: INR records from eight Primary Healthcare Clinics (PHCs) were reviewed. The TTR and percentage of patients with a TTR greater than 65% were determined. A host of variables were analysed for association with TTR. Results: The median age of the cohort of 191 was 56 years (IQR 44-69). The median TTR was 37.17% (IQR 20.21-58.78); only 17.8% of patients had a TTR > 65%. Compared to patients older than 50, those under 50 had worse INR control (TTR 26.63%, IQR 16.05-52.98 vs. 43.5%, IQR 23.52- 60.08, p=0.01). Patients hospitalised for any reason during the study period had worse INR control than patients not hospitalised (TTR 26.23%, IQR 16.24-50.16 vs. 42.89, IQR 23.5-61.95, p=0.02). On multivariable regression analysis, participants on warfarin for atrial fibrillation/flutter had better INR control (OR 2.21, CI 1.02-4.77, p=0.04) but the control was still very poor. Conclusion: INR control as determined by TTR and proportion of TTR >65% in these non-metropolitan clinics was poor. Age and markers of illness predicted poor control. There was a difference in control between groups depending on the indication for warfarin. Evidence-based measures to improve the quality of INR control amongst patients on warfarin therapy need to be instituted as a matter of urgency.

Background: It is known that medical male circumcision (MMC) decreases HIV acquisition by up to 60%. One hypothesis is that MMC removes a foreskin (FS) that harbors different immune cells that are HIV target cells such as CD4+ macrophages, T, Langerhans (LCs), and dendritic cells (DCs). However, there have been different reports on whether the inner FS or outer FS has more HIV target cells. While LCs have been implicated in HIV transmission, their role remains controversial. Studies have shown that LCs can transmit the virus to T cells, which increases infection. On the contrary, others have reported that LCs prevent infection by degrading the virus through a langerin-dependent pathway. One of the factors that plays a major role in HIV transmission is their state of maturity and activation, which can be influenced by co-infection and other immunological processes. The aim of this study was to isolate, quantify and characterize Langerhans cells in the inner FS and outer FS from men undergoing MMC and to evaluate the phenotype of matured and activated LCS. Differences in the proteome of the inner FS and outer FS tissues were further investigated. Methodology: FS were obtained from men undergoing voluntary MMC from clinics and hospitals in the Western Cape (Age 18 years or older). Epidermal FS cells were extracted using crawl (migratory) assay and liberase enzyme digestion. Langerhans cells were isolated by density gradient centrifugation, sorted, quantified and immune-profiled by flow cytometry. CD1a and CD207 were used to identify Langerhans cells while HLA-DR, CD80, CD86 and CD40 were used as markers of maturity and activation. The gene expression profile of sorted LCs was also examined by single-cell sequencing with seq-well. Lastly, the differences in the proteome of the inner FS and the outer FS migrated epidermal cells were assessed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Results: Langerhans cells were an average of 85% pure post-sorting. The numbers of Langerhans cells between the inner FS vs. outer FS were not statistically different (mean: 0.56% vs. 0.68% (SD=0.37) from migratory cells and 0.28% vs. 0.45% (SD=0.18) from enzyme digest, p-value >0.05, n=9). Sequencing showed that the sorted cells pooled from 5 participants (inner and outer FS) had different gene expression profiles. Furthermore, two groups of cells were identified from the sorted LCs based on their gene expression profile. The identified cells were monocyte-like and melanocyte-like cells. The monocyte-like cells were identified as LCs based on their gene expression profile while the melanocyte-like cells were identified as the contaminating cells as the cell purity was not 100%. Upon activation with tumor necrosis factor alpha (TNF-α), activated LCs isolated by the migration assay had similar proportions of cells expressing surface maturity and activation markers (HLA-DR, CD40 and CD80/86) when compared to the unstimulated controls (inactivated) (mean: 73.53% vs. 75.66%, n=9, p-value >0.05, SD=4.4) However LCs that were isolated by the migration assay expressed markers of activation at a higher level compared to LCs isolated by Liberase enzyme digestion (mean: 79.4% vs. 40%, p-value < 0.05 n=9, SD=23). Proteomics showed that the inner FS had an over-abundance of proteins involved in the interleukin 7 response and mRNA catabolic processes, while the outer FS had more spindle zones and cornified envelope proteins that were over-abundant when comparing inner and outer FS from 5 participants. Discussion and Conclusion: The study successfully extracted, sorted and immunoprofiled Langerhans cells using different methods and from different FS compartments (inner FS versus outer FS). When LCs were spontaneously migrated and isolated using the “crawl method”, they showed a more mature and activated phenotype compared to non-migrating “skin resident” immune cells. No differences were found between cells that were stimulated with inflammatory cytokines relative to unstimulated migratory cells in proportion of cells expressing activation markers. However, it was observed that cells isolated by liberase enzyme digestion showed significantly lower proportions of activation markers relative to migratory cells. Using LC-MS/MS-based proteomics; the inner FS exhibited high expression of proteins involved in the interleukin 7 response while the outer FS exhibited high expression of structural proteins, which suggests that the inner FS might be more involved in immunity as interleukins can stimulate immune response while the outer FS has a more structural role than the inner FS.

Background: Weight loss often results in various physiological and behavioural compensatory changes which increase susceptibility to weight regain, resulting in unsuccessful weight-loss maintenance in the long term. Evidence suggests that working memory (WM) plays a key role in self-regulation and executive control, which is vital in overcoming these compensatory responses to weight loss. The current study therefore aimed (1) to identify the compensatory responses that occur with weight loss, (2) to investigate whether WM training (WMT) can improve executive control in reduced-obese women, and (3) compare subjective and objective WM methodologies. Methods: Reduced-obese (n=23) and stable-weight (n=6) women were recruited in this study. All 29 women were characterised at baseline testing for various physiological, behavioural and cognitive outcomes. After baseline testing, a subset of 19 reduced-obese women underwent 6-weeks of WMT, after which they completed post-intervention testing and a 6-month follow up. The series of assessments at baseline and post intervention included: anthropometric measurements; eating and behaviour questionnaires; executive function and working memory tests; metabolic rate; blood profile (HbA1c; glucose; insulin); and appetite measures. Results: Reduced-obese women showed signs of greater eating disordered behaviour and greater post-prandial energy efficiency compared to stable-weight women, although there appeared to be no difference in their executive function. The WMT appeared to result in improved WM capacity in reduced-obese women, which was retained in the long term, and there was some evidence of transfer to behaviour, with an observed reduction in eating-disordered behaviour. When comparing WM methodologies there appeared to be a contradictory relationship between subjective and objective WM measures. Conclusions: The results from this study support the evidence that physiological and behavioural changes occur in those that have lost a significant amount of weight, stressing the importance of finding weight-loss therapies that target these compensatory responses to weight loss. WMT was found to improve WM capacity in reduced-obese women, however, study limitations mean that the effects the training may have had on behaviour and weight maintenance could not be accurately determined. Furthermore, the contradictory relationship found between subjective and objective measures of WM add to the uncertainty that they measure the same underlying construct, which highlights the importance of utilising various types of measures in the analysis of WM and executive function.

This thesis records the results of a series of experiments that were designed to examine the biology of human malignant melanoma cells cultured in vitro. The studies were so planned as to document phenotypic differences that exist between melanomas, to define respects in which melanoma cell differentiation could be modulated and to correlate biochemical variability with in vivo behaviour as measured in the nude mouse. Melanoma cell lines were established from biopsy material obtained from 7 patients at Groote Schuur Hospital. Two of these lines synthesized tyrosinase and melanin at a rate that was directly related to cell density. The five remaining lines did not pigment. ii All of the lines showed aneuploidy; 5 of the 7 showed anchorageindependent growth; and 6 of the 7 grew as lethal tumours in nude mice. As has been found with all other melanomas studied, these cells released a plasminogen activator that was chemically and immunologically identical to tissue activator. One of the lines proved to be an exception to this general rule in that it synthesized urokinase-type enzyme. Unlike most other human cells cultured in vitro, melanoma cells proved to be relatively refractory to hormonal stimuli. Addition of estrogen, progesterone, testosterone, dexamethasone or melanocyte-stimulating hormone to the culture medium had very little effect on cellular release of plasminogen activator, upon cell growth, or upon cellular morphology. Although remarkably resistant to hormonal influences, cellular release of plasminogen activator did appear to be inhibited to a striking degree by cocultivation with normal skin fibroblasts. This observation led to the discovery of a phenomenon in which fibroblasts of many types bound tissue-type plasminogen activator and so removed it from the medium. This was accompanied by an apparent change in molecular weight of the melanoma cell enzyme from 72K daltons to approximately 115K daltons, suggesting the presence of a 40-SOK binding molecule. iii In an attempt to influence in vitro differentiation, the tumour promoter tetradecanoylphorbol acetate, and the differentiation-inducing retinoid, retinoic acid, were added to the two pigmenting cell lines. The effects of these compounds on induction of tyrosinase activity, morphological change or plasminogen activator release differed. In the one cell line, tetradecanoylphorbol acetate caused morphological maturation with a decrease in the rate of plasminogen activator release and no obvious effect upon pigmentation. This line was relatively resistant to the action of retinoids. The other pigmenting line responded hardly at all to the tumour promoter. Retinoic acid, on the other hand, inhibited the induction of tyrosinase activity, yet caused an inhibition of growth and plasminogen activator release. A number of interesting observations could be made in experiments in which melanoma cells were inoculated into nude mice. Firstly, the growth rate of the tumours ~n vivo correlated poorly with the doubling times of the corresponding cells cultured in vitro. Secondly, despite a marked inhibitory effect of fibroblasts on plasminogen activator in vitro, coinjection of fibroblasts and melanoma cells in vivo greatly enhanced tumour growth when small tumour cell inocula were used and shortened the latent period for tumour appearance with larger inocula. Thirdly, melanomas growing in nude mice differed strikingly in their ability to elicit a desmoplastic response. Tumours in which large amounts of host connective tissue were deposited tended to be heavily contaminated with murine fibroblasts when re-established in vitro. This contamination was not seen with tumours that contained very little connective tissue. These results point to the existence of a melanoma-associated fibrogenic factor. Finally, by excision of the primary tumour, it was possible to avoid death of the animal from local complications and so allow time for metastases to develop. In three mice, metastatic melanoma deposits could be detected by this device, so establishing a protocol for the use of nude mice as valid models for the experimental study of metastatic spread of human tumours.

Introduction: Traumatic brain injury (TBI) contributes to worldwide death and disability more than any other traumatic event, but it is of particular concern in children in developing resource-scarce countries. Cerebral autoregulation (CA) is a homeostatic mechanism that aims to maintain constant cerebral blood flow within a range of systemic blood pressures, and the loss of this mechanism has been associated with mortality and worse outcomes in adult TBI. Paediatric studies of CA disturbance are few and consist of small cohorts. Given the differences between adult and paediatric TBI pathophysiology, CA needs examination in a larger cohort of paediatric TBI. This study aimed to describe the profile of PRx, a mathematical indicator of cerebrovascular pressure reactivity, in a large cohort of children with severe TBI. The specific aims were to 1) describe the characteristics of PRx; 2) examine associations between PRx, clinical and physiological variables, and 3) examine associations between PRx and mortality at 6 months, and PRx and dichotomized outcome (as well as survivors only) at ≥ 6 months post-injury. Methods: Patient demographics, clinical and monitoring data were recorded, and the temporal profile of median PRx was plotted by outcome groups. The associations between PRx, Glasgow Coma Score (GCS), intracranial pressure (ICP), and cerebral perfusion pressure (CPP) were examined with both summary measures and correlation analysis using high frequency data. Associations between PRx and mortality/outcome were examined with multiple regression analysis, and the prognostic ability of PRx, ICP and CPP was investigated with receiver operating curve analysis. Results: We examined 196 children with severe TBI. Mortality rate was 10.7%, and 70.4% of the cohort had unfavourable outcome. PRx was consistently higher in patients with poor outcome when examined by various summary statistics and over time. Hourly analysis showed that PRx had a moderate positive correlation with ICP (r = 0.35; p < 0.001) and a weak negative correlation with MAP (r = -0.10; p < 0.001) and CPP (r = -0.27; p < 0.001). PRx had a strong and independent association with mortality. Conclusion: This study calculated, described, and analysed PRx in the largest known cohort of children with severe TBI. PRx had a strong association with outcome (particularly mortality) that was independent of ICP, CPP and GCS. However, a combination of several PRx and ICP-related variables will likely be important for overall prognostication in paediatric severe TBI. Whether CA should be incorporated into clinical care, and if so, how, requires separate investigation.

POIKTMP, a multi-systemic fibrosing disease, results from mutations in the human FAM111B gene. Studies have also suggested high expression of this gene in cancers. Despite rising interest in the pathological effects of FAM111B mutations and overexpression of FAM111B, knowledge of the physiological role of this gene remains limited. Therefore, this study sought out to provide insights into the cellular function of FAM111B and to investigate the pathological effect of the FAM111B Y621D mutation. First, bioinformatics studies coupled with quantitative PCR and Western blots analysis were employed to assess FAM111B gene and protein expression in cancerous and non-cancerous cell lines. Subsequently, FAM111B gene expression was downregulated and upregulated in the human fibrosarcoma (HT1080) cell line by RNA-interference mediated gene silencing and recombinant gene expression technologies. The effect of these FAM111B dysregulations was studied using cellbased functional assays: proliferation, apoptosis, migration, and invasion assays. Furthermore, the functional pathways and interacting proteins of the FAM111B protein was determined using mass spectroscopy proteomics. Finally, preliminary studies in a POIKTMP patient-derived fibroblasts were attempted to recapitulate the results obtained using the HT1080 cell line. The results from this study indicated that FAM111B gene and protein overexpression occurs in cancer cells. Second, the depletion of FAM111B suggests a decelerated rate of cell proliferation and migration (14%), and increased apoptosis (1.4-fold). Conversely, overexpression of FAM111B resulted in a marked reduction in apoptosis (3-fold) and increased cell migration by 27 %, howbeit, no evidence of increased proliferation. Furthermore, Y621D FAM111B mutant cells showed reduced expression of FAM111B, decreased apoptosis (1.1-fold), cellular invasion (24%), and indicates an increase in cell proliferation and migration (18 %). The proteomics data suggested wild-type FAM111B interacts with HSP7C, a molecular chaperone, which alongside BAG3 and BCL2 to minimise apoptosis. Similarly, Y621D's interaction with G3V3W4, a component of the 20S proteasome complex involved in the proteolytic degradation of damaged proteins, may suggest the rapid clearance of this mutant protein.

Antibiotic-resistant strains of Mycobacterium tuberculosis (Mtb) are threatening global efforts to eradicate tuberculosis (TB). One attractive approach for target-based drug design proposes to curb the evolution of Mtb during both immune and drug assault. The potential target: mycobacterial DNA metabolism. For this, an in-depth understanding of the mechanisms of DNA repair and mutagenesis in mycobacteria is required. RecA and ImuAʹ are DNA damageinducible proteins implicated in DNA damage repair and tolerance in Mtb. RecA is a key regulatory protein of the SOS response and ImuAʹ is a component of the mycobacterial mutasome, effecting DNA damage tolerance and mutagenesis. In this study, a comprehensive panel of M. smegmatis (Msm) RecA and ImuAʹ reporter strains was generated to explore the dynamics of their expression and subcellular localization within live Msm cells. To this end, fluorescently tagged versions of ImuAʹ and RecA were constructed and shown to retain functional activity in UV-induced mutagenesis but not survival of mitomycin C (MMC) treatment. The discrepant complementation phenotypes observed in UV and MMC assays was unexpected and suggested disruption of a critical protein-protein interaction(s) owing to the presence of the fluorophore. Using fluorescence microscopy, RecA and ImuAʹ expression were monitored in Msm exposed to different types of genotoxic stresses conditions. When mScarletImuAʹ was introduced into wild-type (WT) and ∆imuAʹ backgrounds, diffuse bright red fluorescence was observed in cells treated with MMC and UV; in contrast, no fluorescence expression was observed in untreated cells, confirming the DNA damage-dependent induction of imuAʹ. Following the introduction of RecA-msfGFP into WT and ∆recA backgrounds, discrete green, fluorescent foci were observed in treated and untreated cells in both backgrounds, consistent with the role of RecA in DNA replication in the absence of external DNA damage, and elevated expression under genotoxic conditions. Taken together, these observations support the utility of the fluorescently tagged translational fusions as bioreporters to elucidate the function and regulation of ImuA' and RecA in mycobacteria.