Includes abstract. Includes bibliographical references (leaves 200-218).

Includes abstract. Includes bibliographical references (leaves 154-171).

Includes bibliographical references. / The clinical success of cisplatin and its derivatives for the treatment of different cancers has had a profound effect on the use of metal-containing agents in medicine. Despite the successes, the drawbacks of platinum-based therapy, such as drug resistance, toxicity and the emergence of unwanted side effects, have bred a need for effective and novel anticancer agents. Hence, the design and study of bioorganometallic complexes as potential therapeutic agents may eventually lead to the identification of new drug candidates. The purpose of this study was to synthesize and characterize a series of polynuclear transition-metal-containing complexes based on a (poly)propyleneimine dendritic scaffold, and investigate the in vitro antiproliferative activity of these complexes.

Includes abstract. / Includes bibliographical references. / The aim of this study was to evaluate the importance of CD28 in initiating protective Th2 immunity against both primary and secondary infections with N. brasiliensis. Our findings demonstrate that CD28 is required for initiation of protective Th2 immunity against primary infection with N. brasiliensis. Furthermore, the absence of CD28 impairs development of memory CD4⁺ T cell responses resulting in failure to clear adult N. brasiliensis worms during secondary infection. Failure to resolve infection was associated with reduced production of Th2 cytokines particularly IL-13 and IL-4, abrogated humoral immunity and failure to expand CXCR5⁺ TFH cells.

A body's axial temperature distribution at death was experimentally demonstrated by the author to predict the postmortem temperature plateau (PMTP), which is known to affect the measured core temperature value and hence death-time estimation. Yet today's methods of death-time estimation apply only a single-point approximation of a body's core temperature in life as well as a single-point measurement of a body's core temperature after death. Four studies were carried out to understand the relationship between a body's axial temperature distribution and the PMTP. The first study numerically approximated antemortem temperature distribution in an MRI-built, high-definition, anatomicallys egmented 3D computational human phantom consisting of several hundred tissues. Metabolic heat generation (QQmm) and blood perfusion (wwbb) parameters were applied to all thermogenic tissue using the Pennes BioHeat Model. The study demonstrated that the antemortem axial temperature distribution was nonlinear, that tissue temperature distribution was inhomogeneous, and that the position and size of the antemortem central isotherm was predicted by the size, shape and location of the most thermogenic internal organ in a given axial plane. Numerical approximation of a body’s antemortem axial temperature distribution using this study’s materials and methods was proposed for death-time estimation. The second study examined postmortem axial heat transfer. The approximated antemortem axial temperature distribution constituted the initial condition. QQmm and wwbb were set to zero to simulate death. Postmortem cooling was simulated in still air, on a cold concrete floor and on a heated floor. The antemortem central isotherm that single-point core thermometry detects was the PMTP. Its size at death, body radius, axial thermometry-depth and length of the postmortem interval (PMI) all predicted PMTP length. The cold concrete floor shifted the central isotherm away from the floor, while the heated floor shifted it towards the floor. Ground temperature and material properties, along with the aforementioned PMTP predictors, result in variation in measured single-point core thermometry values, yet today’s death-time estimation methods do not measure, approximate or standardise them. This is a source of uncertainty. This study demonstrated that a body’s postmortem axial thermal profile was very specific to the PMI at which it exists, including during the PMTP that single-point core thermometry detects. This study proposed a body’s measured postmortem axial thermal profile for death-time estimation to reduce PMTP uncertainties. The study also proposed numerical modelling of the ground, its temperature and material properties. The third study proposed a multipoint axial thermometry (MAT) device to measure a body’s postmortem axial thermal profile. The author designed the device prototype. Its fabrication was outsourced. Empiric and numerical MAT studies were conducted on a cooling dummy and 3D human phantom, respectively. MAT curves indicated a parabolic shape. The fourth study proposed a numerical protocol for death-time estimation that iteratively tested a MAT profile measured at an unknown PMI from a decedent using the proposed MAT device against MAT profiles predicted by numerical simulations of sequentially longer candidate PMIs. A candidate PMI whose MAT profile matched was considered the PMI estimated by the protocol. The proposed protocol applied the exact historical meteorological temperatures that existed during the final estimated PMI. Application of the protocol was demonstrated using a fictitious scenario in which a candidate PMI within 120s of the final estimated PMI was excluded. Potential sources of uncertainty of the proposed protocol were discussed and concluding remarks on future research were made.

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.