MEd (Educational Management) / Department of Educational Management / See the attached abstract below

PhD (Microbiology) / Department of Microbiology / Introduction Human Immunodeficiency Virus (HIV-1) continues to be a global public health concern, even though Antiretroviral drugs (ARV), especially Highly Active Antiretroviral Therapy (HAART) has significantly reduced morbidity and mortality due to AIDS globally in developed and developing countries. However, there is still a great need to explore every avenue for new therapeutic interventions due to the limitations and side effects of HAART. Potential major breakthroughs for future therapeutic development were the discoveries more than 10 years ago of the role of HIV-1 co-receptors and anti-viral activities of host restriction factors such as APOBEC3G protein, which is a member of the DNA cytosine deaminase family. Entry of HIV in to the host cell is through the attachment of the viral envelope glycoprotein to the CD4 receptor, and subsequent interaction, mainly with either CCR5 or CXCR4 co-receptors. Inhibitors, such as Maraviroc, which binds to CCR5 inhibiting entry of CCR5 utilizing viruses (R5 viruses), is currently reserved for salvage therapy in many countries including South Africa. In the course of HIV infection, CXCR4 utilizing viruses (X4 viruses) may emerge and outgrow R5 viruses, and potentially limit the effectiveness of Maraviroc. Several host cell APOBEC3 genes (A3D, A3F, A3G and A3H) have been shown to restrict HIV, and the HIV viral infectivity factor (Vif) protein serves to antagonize the action of APOBEC3 proteins, promoting viral replication. The CCR5 co-receptor and the HIV Env V3 loop have also been documented as playing roles in HIV-1 disease progression. The interplay between host and viral genes still needs widespread attention, given that disease outcomes of HIV depend on many factors, including host cell genetics. Since the discovery of these genes and their role in HIV replication, many studies have been conducted that show their association with viral polymorphism. The polymorphisms found in host cell genes can have significant effects on viral replication, transmission and fitness and can also contribute to the overall diversity in HIV-1 populations. It is hypothesized that there are significant polymorphisms in HIV-1 and cellular genes that may differ among different populations. Population-based studies have tried to establish a relationship between host factors such as APOBEC3 and CCR5 polymorphism and the rate of disease progression, but most studies have focused on Caucasian populations. In vi contrast, little information is available for the effects of variation in these genes in African populations such as South Africa, where the HIV epidemic has expanded at an alarming rate. Although several population studies have focused on African Americans, these do not give us a complete picture of the potential variation in Africans, though the studies can be a good guide on which to base additional studies. A more comprehensive analysis involving different African populations will likely provide a better understanding of the mechanisms underlying host-pathogen interactions, especially in view of the fact that African Americans are primarily infected with HIV subtype B, which is rarely seen in Africa. Methodology This study characterized the genetic variability of the APOBEC3 D, F, G and H genes as well as the HIV-1 vif, in an ethnically diverse HIV-1 infected South African cohort using Next Generation Sequencing (NGS). In addition, polymorphism in CCR5 was analyzed in conjunction with an analysis of the V3 loop sequences in HIV-1 from this cohort. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) of 192 HIV-1 infected drug-experienced individuals who presented for routine care at the HIV/AIDS Prevention Group Wellness Clinic (HAPG) in Bela-Bela, Donald Fraser Hope Clinic (DFHC) in Vhufhuli and in local clinics in the Vhembe district of Limpopo Province, South Africa. Next generation sequencing custom based (Tn5 tagmentation and amplicon based) protocols to prepare libraries for host and HIV-1 genes were developed and validated with commercially available library preparation kits. The Tn5 tagmentation methods were used for longer DNA fragments and the custom amplicon based methods were used mainly for the shorter DNA fragments. To determine the variability of the APOBEC3 and CCR5 host genes, gene-specific primers were designed to amplify complete 12.16 kb A3D, 13.31 kb A3F, 10.74 kb A3G, 6.8 kb A3H and 1.3 kb CCR5 genes targeting the regions containing the exons. Libraries for the resulting amplicons were prepared using Tn5 transposase tagmentation methods and sequenced on an NGS Illumina MiSeq platforms generating millions of reads with good read coverage for variant calling. Single nucleotide polymorphisms (SNPs) and indels were determined, verified in dbSNPs and compared to SNPs in other populations reported in the 1000 Genome Phase III and HapMap. A Chi-square goodness-of-fit was used to verify if whether observed genotype frequencies were in agreement with the Hardy-Weinberg Equilibrium. Haplotypes and Linkage disequilibrium were inferred to determine SNP association. vii The HIV-1 vif and env V3 loop genes were also sequenced to determine their degree of variability of these genes and to infer co-receptor usage in the South African population. Gene-specific primers were designed to amplify the 579 bp Vif region and 440 bp containing the 105 bp V3 loop. Sequencing libraries from the resulting amplicons were prepared using either the Tn5 transposase or custom-based library preparation methods and sequenced on either an Illumina MiSeq or a MiniSeq platform generating millions of reads with good read coverage for variant calling. Phylogenetic analysis was done to determine the relatedness of the sequences. Major and minor variants were determined for HIV-1 and env V3 loop quasispecies was analysed for co-receptor usage; in an effort to draw inferences for the subsequent utility of Maraviroc as salvage therapy in South Africa. Results and Discussion Next generation library preparation; Tn5 tagmentation based and custom amplicon based protocols to sequence host and HIV genes were successfully developed and used to sequence and characterize variability in host cell APOBEC3D, F, G H, CCR5 and the HIV-1 vif gene and the V3 loop region of the env gene. The HIV-1 env V3 loop sequences generated (and quasispecies analyzed) were used to infer co-receptor usage in treatment-experienced individuals; in an effort to draw inferences for the subsequent utility of Maraviroc as salvage therapy in South Africa. Quality V3 loop sequences were obtained from 72 patients, with 5 years (range: 0-16) median duration on treatment. Subtypes A1, B and C viruses were identified at frequencies of 4% (3/72), 4% (3/72) and 92% (66/72) respectively. Fifty four percent (39/72) of patients were predicted to exclusively harbor R5 viral quasispecies; and 21% (15/72) to exclusively harbor X4 viral quasispecies. Twenty five percent of patients (18/72) were predicted to harbor a dual/mixture of R5X4 quasispecies. Of these 18 patients, about 28% (5/18) were predicted to harbor the R5+X4, a mixture with a majority R5 and minority X4 viruses, while about 72% (13/18) were predicted to harbor the R5X4+ a mixture with a majority X4 and minority R5 viruses. The proportion of all patients who harboured X4 viruses either exclusively or dual/mixture was 46% (33/72). Thirty-five percent (23/66) of the patients who were of HIV-1 subtype C were predicted to harbor X4 viruses (χ2=3.58; p=0.058), and 57% of these (13/23) were predicted to harbor X4 viruses exclusively. CD4+ cell count less than 350 cell/μl was associated with the presence of X4 viruses (χ2=4.99; p=0.008). The effectiveness of Maraviroc as a component in salvage therapy may be compromised for a significant number of chronically infected patients harboring CXCR4 utilizing viii viruses in the study cohort. Although from the current study a subset of patients harboring CCR5 utilizing viruses may benefit from Maraviroc, characterizing and identifying if variation in CCR5 are located at Maraviroc binding sites was of importance to investigate. The following variants; P35P, S75S, Y89Y, A335V and Y339F and their varying frequencies were detected in the CCR5 gene. The A335V variant was detected at a higher frequency of 17.4% (29/167). The G265S variant is reported for the first time in this study at 0.6% (1/167) frequency. The SNPs detected were in strong LD (D’= 1, R2 = 0.0) with minor deviation from the Hardy-Weinberg Equilibrium. These variants were not located at the binding motif of Maraviroc. The variants A335V and Y339F were detected at a higher frequency in this study than previously reported in South Africa. Variability in APOBEC3 host cell genes was also characterized in our study cohort. The following APOBEC3 variants compared to the GRCh37 consensus sequence were detected: R97C, R248K and T316T in A3D; R48P, A78V, A108S, S118S, R143R, I87L, Q87L, V231I, E245E, S229S, Y307C and S327S in A3F; S60S, H186R, R256H, Q275E and G363R in A3G and N15Δ, G105R, K140E, K121D, E178D in A3H. Minor allele frequency variants (MAF<5%); L221L, T238I, C224Y and C320Y in A3D; I87L, P97L and S229S in A3F; R256H, A109A, F119F and L371L in A3G, which are frequent in the European population, were also detected. In addition, novel R6K, L221R and T238I variants in A3D and I117I in A3F were detected. Most of the SNPs were in strong LD with minor deviation from the Hardy-Weinberg Equilibrium. Four, six, four, and three haplotypes were identified for A3D, A3F, A3G, and A3H respectively. In general, polymorphism in A3D, 3F, 3G and 3H were higher in our South African cohort than previously reported among other African, European and Asian populations. The APOBEC3 antagonist HIV-1 vif gene was also sequenced to determine the level of diversity in a South African population and also correlated with APOBEC3 variation. Functional Vif without frameshift mutation was observed in all samples except in 4 samples. The functional domain and motifs, such as Zn binding motifs, proline-rich domain, human casein kinase, and the N and C-terminal CBF interaction site were highly conserved. APOBEC binding motifs and the nuclear localization signal were less conserved in the South African HIV-1 Vif. APOBEC3 H variation strongly correlates with Vif variation. All the Vif sequences were subtype C, except one sample, which was identified as an A1/C recombinant. The vif gene in a South African population was under purifying selection, with the dS= 0.2581 and dN= 0.0684 and the dN/dS value = 0.265. There is a high genetic diversity in the South African vif gene, which may ix influence the neutralization and restriction of APOBEC genes. Conclusions In conclusion, the protocols developed in this study can be applied to amplify and sequence any host and HIV-1 genes of interest allowing much deeper and more sensitive profiling of host gene and HIV-1 genetic diversity. Our findings show that a highly significant number of chronically HIV-1 subtype C infected patients in Maraviroc-free treatment harbor CXCR4 utilizing viruses. The data is useful in the consideration of whether to include entry antagonists such as Maraviroc in alternative forms of treatment for patients failing second line treatment regimen in the study setting. The determination of co-receptor usage prior to initiation of therapy consisting of Maraviroc is suggested. Variation in the CCR5 coding region were observed at higher frequencies compare to other studies conducted in South African populations at different locations. This data may suggest that different populations in South Africa have different SNP frequencies. All the polymorphisms identified in the study were not located at the Maraviroc binding motif, therefore the subset of patient infected by R5 viruses may benefit from this drug. We have shown that significant APOBEC3 variation exists among an ethnically diverse population of South Africa by providing extensive data for 4 different A3 genes that are known to restrict HIV infection, but have only been sparsely studied in African populations. This study provides a baseline for future studies that would functionally characterize SNPs identified in this population, in order to understand the role of novel and/or low frequency variants observed. Ex vivo and in vivo studies will increase our understanding of how these variants might have cumulatively impacted the epidemic in Northern South Africa. This study also shows that there is a high level of HIV-1 Vif diversity in the study area. This diversity may impact the expression and packaging of Vif proteins, and the infectivity of HIV. In addition, a significant correlation was observed between HIV-1 Vif variation and APOBEC3 H haplotypes. / NRF

Laser application in the field of biological and medical sciences has significantly grown, thereby strengthening the field of Biophotonics. Research conducted in Biophotonics focuses on the concept of using light especially in the visible and near infrared regions of the electromagnetic radiation for the evaluation of living systems. In this thesis new discoveries are presented about low level laser therapy, optical trapping, transmission spectroscopy, luminescence spectroscopy and structured illumination microscopy (SIM), displaying the impact each technique has on HIV infected cells. The results showed that the irradiation of HIV-1 infected TZM-bl cells with low power red laser reduces HIV-1 infection. The outcomes of this study further proved that when irradiation is used in conjunction with efavirenz, an antiretroviral drug, HIV-1 infection could be reduced to undetectable levels in TZM-bl cells. Through the coupling of transmission spectroscopy with optical trapping, and separately, use of luminescence spectroscopy, label free diagnosis of HIV in infected cell samples was achieved. This finding affirms that HIV-1 infection can be detected in a label free manner when using laser based techniques. Furthermore, the photoluminescence spectrometer system was employed to generate a decay curve, which was necessary so as to have some understanding on lifetime of the luminescent signal in infected TZM-bl cells. Finally, in order to confirm that indeed TZM-bl cells were infected, an established super-resolution microscopy system SIM was used to detect HIV-1 infection in TZM-bl cells. Indeed in the infected cells viral molecules p24 and gp41 were detected through SIM, while they were not detected in uninfected cells. In future studies, super resolution microscopy would be coupled to an optical trapping system in order to confirm that each trapped cells is whether infected or uninfected so as to improve HIV diagnosis. / College of Science, Engineering and Technology / Ph. D. (Science, Engineering and Technology)

PhD (Public Health) / Department of Public Health / Background: South Africa continues to struggle with the high prevalence of Human Immunedeficiency Virus and Acquired Immune deficiency Syndrome. Young people of the university going age are the most affected by this disease. The higher education sector, particularly teachers, are well placed to mitigate this pandemic through teaching and learning. Despite the fact, that a lot has been written on the need to integrate HIV/AIDS content into curricula very few institutions of higher learning are heeding the call mainly due to lack of guidance on how to integrate HIV/AIDS content. Aim:The aim of the study was to develop a framework that facilitate integration of HIV/AIDS content into university curricula. Methods: This cross-sectional study employed quantitative methodology and was conducted in two phases : Data was collected using different methods such as cross sectional surveys, content analysis and systematic reviews. For cross sectional surveys the target population included teachers and students and they were selected using systematic and purposive sampling respectively. The study setting was University of Venda. Data were analysed using SPSS, version 23. Multiple logistic regression and chi-square tests (χ2) were employed to determine the associations. Results: The thesis comprises five interdependent studies. Study one: A systematic review of peer-reviewed journals and grey literature of HIV/AIDS programmes in higher education was conducted. It was found that HIV/AIDS content was integrated mainly into existing , compulsory, undergraduate modules, health sciences disciplines focused on basic facts about HIV/AIDS. The HIV/AIDS content was taught using classroom based teaching strategies. Study two: A quantitative content analysis, to gauge the extent of HIV/AIDS integration into the curricula in various departments at Univen was conducted. The results of this study suggest that HIV/AIDS content was limited as only 68 modules/courses out of 1979 had HIV/AIDS content in different disciplines across all eight schools at the university. Study three: A survey was conducted to assess the knowledge, attitudes and practices of students towards learning about HIV/AIDS content among 340 students . The study found out that majority of the students possessed high knowledge about HIV/AIDS, though they had misconceptions about HIV transmission routes. Further they supported the introduction of formal teaching and learning about HIV/AIDS in their disciplines and very few students were taught about HIV/AIDS in their studies. Study four: A survey was conducted to assess knowledge, attitudes and practices of teachers towards teaching and learning of HIV/AIDS content in the curriculum among 240 teachers . The results showed that the majority of teachers were knowledgeable about HIV/AIDS , had positive attitudes towards the teaching and learning of HIV/AIDS content in the curriculum and very few taught about HIV/AIDS. Study five: Data from the study findings, literature and analysis of the curriculum were integrated within Information ,Motivation and Behaviour Model to develop the proposed framework for integrating HIV/AIDS content. Conclusion: The purpose of the study was to develop a framework that facilitates the integration of HIV/AIDS content into the undergraduate curriculum. The proposed framework in this study may assist HEIs, faculties and teachers to integrate HIV/AIDS content formally into their curriculum and ensure that various academic departments can integrate HIV/AIDS-related issues into the undergraduate curricula. The framework outlines HIV/AIDS competencies for different levels of study in various disciplines and its adoption may assist HEIs in producing graduates who can survive and work in a world ravaged by HIV/AIDS. In order to implement the proposed framework for integration of HIV/AIDS content into undergraduate curricula, recommendations were made. / NRF

Indiana University-Purdue University Indianapolis (IUPUI) / The antiretroviral drug efavirenz remains a cornerstone for treatment-naïve HIV patients. Subsequent to the demonstration that efavirenz is a substrate of cytochrome P450 (CYP) 2B6, a number of clinical studies found that the CYP2B6*6 allele is significantly associated with higher efavirenz exposure and/or adverse reactions. However, the mechanism of reduced efavirenz metabolism by this genetic variant is not fully understood and whether this variant exhibits differential susceptibility to metabolic inhibition is also unknown. Ths use of efavirenz is further complicated by the drug interactions associated with it. Therefore, I hypothezised that 1) the CYP2B6*6 allele reduces efavirenz metabolism by altering catalytic properties of CYP2B6; 2) efavirenz alters the pharmacokinetics of co-administered drugs by inhibiting drug metabolizing enzymes. A series of studies was carried out in hepatic microsomal preparations to determine the functional consequences of the CYP2B6*6 allele and to assess inhibition potency of efavirenz on 8 CYPs. The major findings for these studies include: 1) the CYP2B6*6 allele reduces efavirenz metabolism by decreasing substrate binding and catalytic efficiency; 2) functional consequences of the CYP2B6*6 allele appear to be substrate- and cytochrome b5-dependent; 3) the CYP2B6*6 allele confers increased susceptibility to metabolic inhibition; and 4) efavirenz inhibits the activities of CYP2B6, 2C8, 2C9 and 2C19 at therapeutically relevant concentrations. In addition, I explored the hypothesis that the incorporation of in vitro mechanism by which the CYP2B6*6 allele reduced efavirenz metabolism predicts the genetic effect of this allele on efavirenz clearance after a single oral dose by modeling approach. A pharmacogenetics-based in vitro-in vivo extrapolation (IVIVE) model was developed to predict human efavirenz clearance. Taken together, results from this dissertation provide new mechanistic information on how the CYP2B6*6 allale alters substrate metabolism and drug interactions; demonstrate new mechanisms of efavirenz-mediated inhibition interactions; and demonstrate the utility of a pharmacogenetics-based predictive model that can serve as a basis for future studies with efavirenz and other CYP2B6 substrates. Overall these data provide improved understanding of genetic and non-genetic determinant of efavirenz disposition and drug interactions associated with it.

The aim of this qualitative study was to explore the vulnerability to HIV of rural male youth with regard to their beliefs, sexual attitudes and behaviour. The study took place in the Waterberg, a district of Limpopo in South Africa, and provided insight into and understanding of the youths’ attitudes to and intentions regarding HIV prevention and their perceptions of how they were influenced by the Boys2Men programme. The Theory of Reasoned Action and the Social Constructionist Theory provided a framework for the study. Nine participants between the ages of 19 and 26 were purposefully selected, and data was collected via individual face-to-face and focus group interviews. A number of semi-structured questions were used to guide the study, and data captured from the interviews was analysed by thematic content analysis. / Sociology / M.A. (Social Behaviour Studies in HIV/AIDS)

Cultural factors have been shown to play a role in human decision making and behaviour. The main objective for this research was to identify and evaluate the possible influence of Shangaan cultural beliefs, myths and behaviours, on the spread of HIV within the Mnisi tribe. A qualitative method of investigation was followed; interviews with three participants and observations of the Mnisi culture were used in the construction of the investigation and findings. Through the information obtained two cultures influencing the spread of HIV in the Mnisi tribe were identified, they are (1) The culture of power-rule and fear, and (2) The culture of poverty. / Psychology / M.Sc. (Psychology)

Cultural factors have been shown to play a role in human decision making and behaviour. The main objective for this research was to identify and evaluate the possible influence of Shangaan cultural beliefs, myths and behaviours, on the spread of HIV within the Mnisi tribe. A qualitative method of investigation was followed; interviews with three participants and observations of the Mnisi culture were used in the construction of the investigation and findings. Through the information obtained two cultures influencing the spread of HIV in the Mnisi tribe were identified, they are (1) The culture of power-rule and fear, and (2) The culture of poverty. / Psychology / M.Sc. (Psychology)

Since the earliest reported cases of HIV/AIDS probably in 1959 in Africa, there has been a consistent progression in the new HIV/AIDS infection cases. In South Africa, Gauteng, records one of the highest HIV/AIDS prevalence rates in the country. The Department of Health (DOH) South Africa conducts ongoing studies on HIV/AIDS at provincial levels; these studies monitor the prevalence of HIV/AIDS amongst pregnant women attending antenatal clinics, as a tool for determining and monitoring the prevalence, trends, patterns and spread of the disease in the general population. This study analyses sentinel and spatial data collected from the (DOH) and Statistics South Africa (StatsSA) respectively, and depicts them in the form of spatial maps, and then critically analyses the spatial patterns that occur. The research findings would hopefully contribute to the overall knowledge of HIV/AIDS and provide framework and relevant literature for further investigation. / Geography / M.Sc. (Geography)

Since the earliest reported cases of HIV/AIDS probably in 1959 in Africa, there has been a consistent progression in the new HIV/AIDS infection cases. In South Africa, Gauteng, records one of the highest HIV/AIDS prevalence rates in the country. The Department of Health (DOH) South Africa conducts ongoing studies on HIV/AIDS at provincial levels; these studies monitor the prevalence of HIV/AIDS amongst pregnant women attending antenatal clinics, as a tool for determining and monitoring the prevalence, trends, patterns and spread of the disease in the general population. This study analyses sentinel and spatial data collected from the (DOH) and Statistics South Africa (StatsSA) respectively, and depicts them in the form of spatial maps, and then critically analyses the spatial patterns that occur. The research findings would hopefully contribute to the overall knowledge of HIV/AIDS and provide framework and relevant literature for further investigation. / Geography / M.Sc. (Geography)