Many severe clinical conditions encountered in Africa involve the effects of iron overload on diseases (AIDS, TB etc) and vital organs (e.g., liver). To intervene successfully in the HIV pandemic, knowledge of AIDS pathogenesis and factors that stimulate or inhibit viral replication are crucial. Iron overload is believed to cause serious damage during HIV infection. Indeed, it is believed that the metal is required by infected cells to synthesize viral particles. The consequences of excess iron in vivo include the stimulation of microorganism growth, an increase in oxidative stress and an impairment of immune system function. Iron chelation have been reported to modulate some of these effects. In a project designed to assess the effect of in vitro iron overload (also synonymously referred to as iron loading) on HIV infection, cells (HIV-infected and controls) were directly loaded with iron and desferrioxamine (DFO, an iron chelator) respectively or combined. We then performed experiments to investigate the effects of these chemicals on host cell defenses and viral replication. Effective iron loading of all cell lines used was confirmed by emission spectroscopy. Through viability assays using tetrazolium salts, flow cytometric analysis of apoptosis and necrosis using Annexin-V, and specialised ELISAS; the effect of iron loading on host cell viability, survival or death and cytokine production was studied. The effect of iron loading on virus infectivity was also investigated by looking at core protein (p24) levels and reverse transcriptase (RT) activity. Prior to the start of the bioassays, several dyes were compared during identical procedures to find an effective and consistently functional dye assay for the assessment of cell growth/viability or proliferation. Viability assays provided a qualitative picture of events while flow cytometric analysis allowed us to compare viability with specific types of cell death (apoptosis/necrosis). Excess iron in the form of 500ƒÝM FeSO4-7H2O in addition to serum iron was found to be non-toxic to cells alone but detrimental to HIV-infected cells. Equimolar amounts of DFO inhibited cell growth, cytokine production and viral replication. Our results indicate that Fe loading stimulates viral replication. Iron chelation on the other hand decreased HIV replication suggesting a possible area for further therapy research using iron chelators in situations of iron loading in the presence of HIV/AIDS. / Dr. Debra Meyer

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2016 / According to UNAIDS, there are ~36.9 million people infected with HIV-1 in the world. Of those, 25.8 million live in sub-Saharan Africa and 6.8 million in South Africa. HIV-1 subtype C accounts for over 95% of HIV infections in South Africa. HIV-1 retrovirus acquires mutations rapidly because of the viral reverse transcriptase. Naturally occurring polymorphisms distinguishing wild type C-SA PR from other proteases make it less susceptible to inhibitors. E35D↑G↑S is a C-SA PR variant with a double insertion in the flap region of the protease. The insertions and background mutations may decrease susceptibility to inhibitors as well as alter kinetic parameters due to increased flap flexibility. This study intended to characterise the effect of the mutations and insertions in E35D↑G↑S on structural, kinetic activity and drug susceptibility. Chemically-synthesised E35D↑G↑S autocatalyses rapidly, impeding further characterisation. There was no detectable overexpression of the E35D↑G↑S protease in Escherichia coli BL21 (DE3)pLysS and Rosetta 2® cells. If the protease is catalytically enhanced, attributed cytotoxicity may prevent overexpression of the protein. Increased autocatalytic activity could also prevent crystallisation. Inactive E35D↑G↑S D25A did not overexpress either, indicating that codon harmonisation with the expression host ought to be performed. C-SA PR was shown to be a predominantly beta-sheeted protein using circular dichroism spectroscopy. The KM of the fluorogenic substrate resembling the capsid/ p2 cleavage site for C-SA PR was 22.02 ±4.09 μM. The specific activity, catalytic turnover and catalytic efficiency of the wild-type C-SA PR protease were found to be 35.68 ±1.06 μmole.min-1.mg-1, 12.79 ±0.38 s-1 and 1.17 ±0.055 s-1.μM-1, respectively. The thermodynamics of binding of atazanavir, ritonavir and darunavir to C-SA PR were determined using isothermal titration calorimetry. The binding of atazanavir and ritonavir to C-SA PR is entropically driven and enthalpically opposed. However, the binding of darunavir to C-SA PR was found to be both entropically and enthalpically favourable. The dissociation constants of the inhibitors in the absence of substrate (Kd) are in the pico-molar range and increased by approximately one order of magnitude when saturating concentrations of substrate were introduced. Atazanavir, ritonavir and darunavir have dissociation constants (Kd) of 160.56 ±54.59 pM, 113.34 ±46.47 pM and 10.24 ±6.02 pM, respectively. Darunavir binds significantly tighter. Keywords: C-SA PR, E35D↑G↑S, insertion mutations, protease, autocatalysis, ITC.

Many researchers have alluded to the inequity in distribution of HIV preventive services in South Africa (SA). Other researchers have demonstrated that socio-demographic factors are main determinants of distribution of preventive services like voluntary counseling and testing (VCT) in SA. VCT is a primary HIV prevention tool through which infected persons enter the treatment, care, and support programs; identifying the impact of socio-demographic determinants (SDDs) on VCT uptake in SA could help direct VCT services to areas and individuals that need them most. The research question in this study examined what and how SDDs impact the uptake of VCT in SA using the integrated theory of health behavior change (ITHBC) as its theoretical framework. A quantitative study with a cross-sectional design using secondary data from a population-based survey by the John Hopkins Education and Health SA (2012) was conducted. In a multivariate logistic regression analysis, SDDs like province, settlement, employment, races, and age were statistically significant while marital status, education, and SES (socio-economic status) did not have statistically significant impact on VCT uptake. This study demonstrated that Black, unemployed men of low to medium SES between the ages of 15-49 years living in peri-urban and urban-informal areas of all provinces but especially Eastern Cape, Northern Cape, and North West provinces of SA had the lowest VCT uptake. This study advocates policies and programs to improve VCT distribution and accessibility in places and individuals with lowest uptake. Improved uptake will help reduce new HIV infection, HIV-associated morbidity, and mortality; as well as ensure equity, equality, and social justice in the distribution of HIV preventive services in SA.

PhD (Health) / Department of Public Health / The Government of South Africa in response to a prevalent human immunodeficiency virus (HIV) has adopted an approach of integrating HIV/AIDS service into primary health care, as a key to achieving universal access to antiretroviral treatment (ART). Despite the government’s efforts of integrating HIV service into Primary Health Care (PHC), insufficient numbers of PHC staff and inadequate infrastructure is challenging when integrating HIV/AIDS service into PHC. This study explored the extent of HIV service integration into PHC and whether the clinic/health centre’s environment is enabling to integrate HIV service into PHC. Barriers to HIV/AIDS services integration as well as attitudes of PHC nurses were assessed. The overall purpose of this study was to develop guidelines to facilitate the integration of HIV/AIDS services into PHC in Vhembe district of Limpopo province, South Africa. An exploratory sequential mixed methods design was used. The qualitative data was collected and analysed before and results for qualitative approach used to build a subsequent quantitative phase. The current study revealed that HIV/AIDS services are integrated into every existing programme at the PHC clinic and health centres; these include: Immunisation programme, Family planning, PMTCT and ANC programmes, STIs, minor ailments and chronic illness and TB. The study further revealed that the environments at PHC clinics and health centres are not enabling the integration of HIV/AIDS services into PHC due to insufficient staff and inadequate infrastructure. Guidelines to facilitate the integration of HIV/AIDS services based on the findings was developed. The study recommendations comprise; increasing knowledge of HIV serostatus, accelerating HIV prevention, accelerating the scale-up of HIV treatment and care, creating of enabling environment for the integration of HIV/AIDS services into PHC, nursing education and training and nursing education and training.