Background: Language discordance, a challenge of miscommunication between health professionals and service users, is a concern for occupational therapy, a profession that foregrounds a client-centred partnership. Occupational therapy literature highlights language discordance as one of the biggest challenges encountered when working in the rural public health sector. Language discordance affects the quality of health services which results in misdiagnosis, informed consent violations, decreased service user satisfaction and safety risks, among others. Occupational therapy is not immune to these negative consequences. In a country as linguistically diverse as South Africa, the need to find effective ways to navigate language discordance in occupational therapy health care, is crucial. However, there is limited literature on language discordance and the strategies used to resolve the issue. Aim: The aim of this study was to describe strategies that occupational therapists working in the public health sector in KwaZulu-Natal use to navigate language discordance and to understand the subsequent role that language discordance has on the quality of occupational therapy care. Methodology: The study adopted a qualitative descriptive design using semi-structured interviews with eight participants recruited using purposive and snowball sampling. Thematic analysis was used to analyse data. Findings: Four themes emerged, namely; using various communication strategies concurrently, language definitely impacts that therapy process, factors perpetuating language discordance and I'm doing everything that I can, what more can I do? Conclusion: The impact of language discordance on the quality of occupational therapy care is undeniable. However, the participants showed agency in navigating language discordance using personal and institutional resources amidst the complexities of applying various strategies concurrently in order to provide the best care that they could.

Comparative genomic analyses have identified an altered capacity for cobalamin biosynthesis as a critical step in the evolution of the pathogenic Mycobacterium tuberculosis Complex strains from a common environmental ancestor. However, resolving the full gene complement involved in the complex, multi-step pathway for de novo cobalamin biosynthesis, assimilation, and salvage in different mycobacterial species is challenging. A genome-scale approach was adopted to yield detailed genetic maps of de novo cobalamin biosynthesis in M. smegmatis, a non-pathogenic saprophyte. To this end, a combination of whole-genome transposon (Tn) mutagenesis and next generation sequencing (TnSeq) was applied in M. smegmatis ΔmetE, a gene-deletion mutant in which the cobalamin-independent methionine synthase is inactivated, rendering the cobalamin-dependent isoform, MetH, essential for viability. Following growth of the metE mutant in rich medium, genomic DNA was extracted, amplified by PCR, and subjected to high-throughput sequencing to quantify all Tn junctions. Thereafter, the library was cultivated in defined minimal medium to enable identification of all conditionally essential genes – including those required for de novo cobalamin biosynthesis. A ∆metE library comprising 400,000 individual Tn insertion mutants (cfu/ml) was generated. Of the predicted 6,716 genes in the M. smegmatis genome, 213 genes were identified as essential for growth on rich agar while 356, 301, and 337 genes were identified as essential in unsupplemented, cyanocobalamin (CNCbl; vitamin B12)-supplemented and cobalt-supplemented Sauton's minimal medium, respectively. A total of 424 genes were identified as essential across all conditions tested with only 10, 13 and 24 genes (ES plus GD) uniquely required for growth in unsupplemented, CNCbl-supplemented and cobalt supplemented Sauton's minimal medium, respectively. On average, predicted cobalamin pathway genes were underrepresented in number of Tn insertions and read counts, indicating the likely essentiality of these genes during growth of the metE mutant in minimal medium. Notably, elucidation of cobalamin biosynthetic and assimilatory genes required the analysis of libraries exposed to CNCbl-unsupplemented minimal media for extended durations, probably reflecting the need to exhaust the organism's capacity for co-factor storage and recycling. Utilizing targeted silencing of individual genes by CRISPR interference, candidate cobalamin biosynthesis genes were validated, providing functional evidence of their essentiality for metE survival in minimal medium, in turn supporting the validity of the cobalamin biosynthetic pathway constructed from the TnSeq results. In addition, the results add further evidence in support of the functionality of the cobalamin riboswitch upstream of metE. This is an important observation as it suggests the potential to apply an analogous approach in M. tuberculosis, a major human pathogen whose ability to synthesize cobalamins remains unresolved. Moreover, elucidating the genetic requirements for optimal growth under specific conditions can inform our basic understanding of mycobacterial physiology and pathogenicity, identifying potential vulnerabilities for novel anti-tuberculosis therapeutics.

Since the advent of antiretroviral therapy (ART) and the resultant suppression of viraemia in the majority of people living with HIV-1 (PLWH) on ART, HIV-1 infection has become manageable and PLWH have similar life expectancies as uninfected persons. However, ART is not curative, is needed lifelong, and its cessation leads to the recrudescence of viraemia. This is due to the formation of a latent reservoir that is long-lived and stable over time, precluding HIV-1 cure. The factors affecting reservoir formation, establishment, and kinetics are not fully understood. Furthermore, differences exist at the population level in disease progression in PLWH depending on ethnicity, biological sex, and infecting viral subtype. Similarly, differences in the latent reservoir of HIV-1 have been described, although less extensively. Understanding what shapes the latent HIV-1 reservoir is critical for developing strategies for cure. Furthermore, it is imperative that cure research is undertaken in diverse populations to ensure coverage of knowledge across different demographics. The latter will ensure that a cure strategy can be developed that will be globally implementable. In the Introductory chapter of this thesis, I provide a detailed review of the current literature and address the need for cure research in low-and middle- income countries. If a global cure is to be achieved, the burden of HIV-1 will need to be addressed in many different populations, most notably African women, as women bear the burden of HIV-1 globally. In South Africa, the country in the sub-Saharan African region with the highest prevalence of HIV-1, women are roughly twice as likely to be living with HIV than men (aged 15 to 49), with a prevalence rate 6% higher than the national average of 19%. Since women are underrepresented in HIV-1 research in general and more specifically in cure studies due to the paucity of research in countries outside of the global North, reservoirs and cure strategies ii need to be characterized in this context. Furthermore, while early treatment is the WHO standard of care for people diagnosed with HIV, a large majority of PLWH only initiated treatment in chronic infection. Since early ART is known to restrict formation of the latent reservoir of HIV-1, research in both early and late ART initiators is necessary. This research focused on characterising the viral reservoir in South African women in a well-established cohort of women who were recruited during acute HIV infection and followed until treatment initiation (which occurred during chronic infection) and beyond. Overall, this thesis focuses on characterising immune activation and inflammation during the course of both untreated and treated HIV-1 infection in a cohort of South African women and subsequently determining whether clinical or immune measures influence characteristics of the latent reservoir of HIV-1. T cell activation and the levels of soluble inflammatory cytokines in plasma were determined in forty-six women in the CAPRISA 002 Acute infection cohort. Chapter 2 describes the cellular immune activation and inflammation profiles of these participants throughout the course of infection at the following timepoints: acute infection, oneyear post-infection, and within a year preceding ART initiation, and two- and four- years postART initiation. T cell activation peaked in chronic infection and reduced dramatically after ART initiation. CD4+ and CD8+ T cell activation reached a post-treatment nadir by two years after ART initiation. Cytokine measures were within the ranges reported in the literature for PLWH. Notably CXCL-10 levels in plasma decreased significantly between two- and four years post-ART, indicating that it may be a sensitive marker of ongoing systemic inflammation in people on ART. In short, the T cell activation and inflammation profiles of the women in this study reflected what has been observed in other cohorts. iii The size of the replication-competent HIV-1 reservoir, measured by quantitative viral outgrowth assay after 5 years of suppressive antiretroviral therapy (ART), was quantified in twenty women of the cohort. In Chapter 3, the clinical and immunological correlates of reservoir size were investigated. Predictive modelling showed that the size of the replicationcompetent reservoir is directly related to viral load and CD4+ T-cell counts over the course of infection, although these measures do not fully predict reservoir size. We found that, in addition to viral load and CD4+ T-cell count, CD8+ T-cell activation within the year preceding ART, nadir CD4+ T-cell count, and baseline as well as on-treatment CD4:CD8 ratio at the time of sizing was associated with replication-competent reservoir size. We provide evidence that the late CD8+ T-cell activation level before treatment, together with viral loads and CD4+ T-cell counts, are directly related to the size of the replication-competent reservoir of HIV-1. Our results are consistent with the hypothesis that the host immune milieu near the time of ART initiation plays an important role in shaping the durable reservoir of HIV infection that persists on ART. Another characteristic of the HIV-1 reservoir is persistence: the presence of all forms of HIV1 within cells and tissues that contribute to pathogenesis, including defective, non-induced, and non-integrated forms of HIV-1. In Chapter 4, total HIV-1 DNA levels were measured as a proxy for viral persistence in thirty-one participants, and the correlates thereof investigated. The HIV-1 DNA levels in this cohort were similar to those reported in the literature for other cohorts where participants initiated therapy in late chronic infection. HIV-1 DNA levels did not differ significantly between two- and four years post-ART, but there was a trend to lower HIV-1 DNA when measuring pol versus gag gene frequencies in peripheral blood mononuclear cells (PBMC). These findings indicate that HIV-1 DNA decay rates may differ depending on the gene being measured, even when using the same assay. A weak significant correlation was iv found between CD4+ T cell counts at ART initiation and the change in HIV-DNA levels between two-and four years on ART. There was a significant correlation between residual CD4+ T cell activation at four years post-ART initiation and gag copies per million PBMC. A trend towards a correlation was found between CD4+ T cell activation and pol copies per million PBMC at the same timepoint. Finally, we found significant correlations between several cytokines at one-year post-infection and within one year pre-ART. These findings further solidify the hypothesis that the immune milieu around the time of ART initiation and after may play a complex role in formation of the viral reservoir of HIV-1. Our studies show a significant link between chronic immune activation and replication competent reservoir size, and also ongoing immune activation and viral persistence on ART. Further studies into whether these immune measures affect the timing of establishment and clonality of the reservoir in this cohort are ongoing and will inform the field about whether differences in cure strategies will need to be explored for those PLWH who had high levels of chronic immune activation before treatment initiation and subsequent shaping for the long-lived viral reservoir.

Includes abstract. / Includes bibliographical references. / Includes bibliographical references (leaves 74-81). / Over-expression of β-arrestin 1 in COS-l cells revealed that the mammalian type GnRH receptor can internalise in a β-arrestin dependent manner whereas the internalisation of the mammalian type I GnRH receptor is β-arrestin independent. investigate which domains on the mammalian type II GnRH receptor are required for β~arrestin dependent internalisation, chimeric receptors were created. / The mammalian type II GnRH receptor possesses an intracellular C-terminal tail that is known to play a role in desensitisation, internalisation and overall signalling in GPCRs. On the other hand, the mammalian type I GnRH receptor, which lacks a C-terminal tail, does not readily desensitise and undergoes slow internalisation compared to the mammalian type II GnRH receptor. Over-expression of ß-arrestin 1 in COS-l cells revealed that the mammalian type GnRH receptor can internalise in a ß-arrestin dependent manner whereas the internalisation of the mammalian type I GnRH receptor is ß-arrestin independent. investigate which domains on the mammalian type II GnRH receptor are required for ß-arrestin dependent internalisation, chimeric receptors were created. Firstly, a chimera in which the full length type II GnRH receptor C-terminal tail was added to the tail-less type I GnRH receptor (TI/T2tail) was created. This chimera internalised in a ß-arrestin and GRK dependent manner, demonstrating that the type II GnRH receptor C-terminal tail confers ß-arrestin JGRK dependent internalisation on the originally ß-arrestin/GRK insensitive GnRH receptor. Mutating the putative GRK and casein kinase phosphorylation sites (serines 338 and 339) on the C-terminal tail of TI/T2tail to alanine residues did not abolish ß-arrestin dependent internalisation but eliminated GRK dependent internalisation, suggesting that other regions on the C-terminal tail are required for ß-arrestin dependent internalisation. A second chimera, in which the whole third intracellular loop of the type II GnRH receptor was replaced with that of the type I GnRH receptor (T2/TIICL3), was created. This chimera could not utilise ß-arrestin in its internalisation, indicating that the third intracellular loop of the type II GnRH receptor is required for ß-arrestin dependent internalisation. An alignment of the amino acid sequences of the two mammalian GnRH receptor third intracellular loops identified a basic residue rich area (R234, R236 and K237) on the type II GnRH receptor that was absent on the type I GnRH receptor. Interestingly, the triple mutant (R234,236,K237 A) still internalised in a ß-arrestin dependent manner, however, truncation of the C-terminal tail of R234,236,K237A abolished the ability of the receptor to internalise in a ß-arrestin dependent manner. This result indicated that the C-terminal tail of the type II GnRH receptor was compensating for the absence of the three basic residues. To summarise, this thesis demonstrates that the C-terminal tail of the type II GnRH receptor can confer ß-arrestin dependent intemalisation on the type I GnRH receptor. Furthermore, the third intracellular loop, and more specifically, basic residues R234, R236 and K237 on the mammalian type II GnRH receptor are required for ß-arrestin dependent intemalisation.

Includes abstract. Includes bibliographical references.

Includes bibliographical references (leaves 108-126). / Infection by the parasitic nematode Nippostrongylus brasiliensis involves migration through the lungs, causing significant damage and generating chronic lung pathology. The resolution of N. brasiliensis infection and also the induction of pulmonary pathology, including goblet cell hyperplasia and acute airway inflammation, depend on IL-4Rá signalling. A key feature of IL-4Rá signalling is the induction of a strong TH2 response which induces the development of alternatively activated macrophages (AAMs). AAMs are associated with tissue remodelling and the control of exacerbated inflammation. In order to investigate potential roles for IL-4Rá in N. brasiliensis' induced lung pathology, we infected mice deficient for IL-4Rá on macrophages and neutrophils (LysMCreIL-4Rá-/lox), IL-4Rá -/- and control mice (IL-4Rá-/lox) with N. brasiliensis and examined lung pathology at days 5, 42 and 180 post infection (p.i.).All three mice strains showed similar emphysemic-like pathology (alveolar dilatation) and airway hyperresponsiveness (AHR) which was well developed by day 42 p.i. and remained chronic. However, LysMCreIL-4Rá-/lox mice consistently demonstrated earlier and increased pulmonary inflammation when compared to IL-4Rá-/lox control mice and IL-4Rá-/- mice. Immunological studies at day 5 p.i. revealed that there were increased CD4+ and CD8+ T-cell numbers and increased CD4+ IL-4 and IL-13 production in the lungs of LysMCreIL-4Rá-/lox mice when compared to control and IL- 4Rá-/- mice. LysMCreIL-4Rá-/lox mice also showed decreased pulmonary arginase activity, indicative of a reduction of AAMs. RNA transcript analysis of isolated alveolar macrophages showed a strong association with promoting inflammation in LysMCreIL-4Rá-/lox mice. Together these data demonstrate that IL-4Rá-responsive macrophages control pulmonary inflammation and play an important protective role in the lung following N. brasiliensis infection.

Includes abstract. Includes bibliographical references (p. 109-125).

Sudden unexpected death in the young (SUDY) is the tragic fatality of seemingly healthy individuals aged between one and 40 years. Little is known about the demographics and risk factors of these cases at Salt River Mortuary (SRM), Cape Town. Therefore, this project aimed to retrospectively investigate the burden and profile of SUDY cases admitted to SRM, between 1 January 2016 and 31 December 2018. Of the total 11 588 cases admitted over this period, 833 (7.2 %) were SUDY cases, wherein males comprised the majority (64.3 %). Individuals were a median age of 31 ± 10.3 years at death, and the main location of death was ‘residential' (43.5 %). There were also significantly more males than females in the age category of 31 - 40 years who were found outdoors compared to all other locations (p < 0.001). Risk factors included physical activity, substance abuse, and co-morbidities with concomitant use of chronic medication. More than a third of individuals experienced breathlessness prior to death (45.0 %). Of cases with a confirmed natural cause of death, the main organ systems involved were pulmonary, cardiovascular, central nervous system and gastrointestinal, which parallels international trends. Akin to local studies, in analogous amounts, TB and pneumonia were the leading causes of death. Additionally, 21.1 % of cases were identified as candidates for genetic testing which may resolve undetermined cases or elucidate underlying predisposing factors to sudden death. Fortunately, 81.8 % had biological samples available for these retrospective analyses. Cases often had missing documentation which advocates for training to ensure compliance to standardised procedures. This study shows that males aged 31 ± 10.3 years with pulmonary and cardiac-related co-morbidities are the most vulnerable for SUDY whilst sleeping. Awareness interventions targeted at this population are thus needed in an attempt to reduce these tragic fatalities.

Background: Pregnancy at advanced maternal age (AMA) is associated with an increased risk of aneuploidy. In the Western Cape's public health sector maternal age alone is widely used to screen women for high risk of pregnancies affected by aneuploidy. The weekly pregnancy counselling clinic (PCC) at Groote Schuur Hospital (GSH) offers genetic counselling (GC) for women who are of AMA to inform them about their age-related aneuploidy risk, offer invasive diagnostic testing (IDT) and discuss the option of voluntary termination of an affected pregnancy. A recent audit at GSH showed that the uptake of IDT was low and other literature reports that South Africans tend to have a conservative view regarding termination of pregnancy (TOP). This study sought to understand what women expect from the GC service at PCC as well as what their experiences are of the service. Methods: This qualitative phenomenological study used a pragmatic approach and participants were recruited through purposive sampling. Semi-structured, in-person interviews were conducted after women had completed their GC sessions at PCC. Thematic analysis was used to analyse the data. Results: The results of this study suggest that participants (n=7) received very little information about their GC appointments at referring clinics, and that they generally did not have prior knowledge about age-related aneuploidy risks. Finding out about the age-related risk of aneuploidy was an emotional experience for the participants, but other factors, including normal ultrasound results, provided relief. The participants' choices regarding IDT and attitudes towards TOP reflected that of available literature as the uptake was low and most participants reported that they would not consider a TOP. The women reported that they would use the knowledge they gained during GC to educate other women in their communities about the pregnancy risks associated with increased maternal age. Generally, the participants believed that GC was useful and appreciated the opportunity. Conclusion: The participants in this study had limited health literacy and knowledge regarding AMA risks and GC. As a result, participants had no expectations of GC. However, the participants felt that GC was useful in helping them prepare for the possibility of a child with DS, and generally viewed the service in a positive light. Additionally, this study's results suggests that there is a need to educate women in local communities regarding AMA pregnancy risks.

We investigated immune responses beyond conventional T cells in the context of BCG vaccination and tuberculosis disease.