3. Electronic Theses and Dissertations (ETDs) - All submissions

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    Allosteric effects of chicoric acid on human immunodeficiency virus type 1 integrase
    (2018) Fish, Muhammad Qasim
    Human immunodeficiency virus (HIV) integrase (IN) is an essential viral protein involved in the integration of the viral DNA into the host genome. Although having a specific catalytic function, it is apparent from mutagenesis studies that IN is pleiotropic and affects the viral life cycle at multiple points other than integration. Compounds that bind to allosteric sites on IN typically disrupt its ordered multimerization and stalls the viral life cycle at various points. Chicoric acid (CA) is a well-known IN inhibitor, however, its mechanism does not follow a conventional active site inhibition, yet it presents with antiviral activity. We thus, hypothesised that CA has an allosteric inhibitory mechanism. To test the hypothesis we aimed to determine alloteric effects of CA on HIV IN. Site directed mutagenesis was used to develop IN mutants resistant to conventional IN inhibitors and these proteins were purified. These were used in an enzyme-linked immunosorbent assay (ELISA) for comparative resistance profiling of CA and raltegravir (RAL). The ELISA also compared magnesium (Mg2+) and manganese (Mn2+) dependent differences on CA inhibition and to determine the importance of order of addition of assay components. An AlphaScreen assay was developed to test for the disruption of the IN/LEDGF interaction. Crosslinking assays and size exclusion chromatography (SEC) was performed to determine the multimeric state of IN in the presence of CA. Surface Plasmon resonance (SPR) was used to confirm binding of CA to IN and determine the kinetics. In silico docking onto the IN catalytic core (CCD) structures was used to identify a possible binding mode of CA at the allosteric binding site. Resistance profiling depicted a clear distinction between CA and RAL. IN resistance mutants: INQ148H, INN155H, ING140S/Q148H and INE92Q/N155H, showed a fold change in IC50 (FCIC50) of 3.42, 1.09, 1.43 and 2.90 for CA respectively. While the same mutants showed an FCIC50 of 947.99, 392.44, 1262.41 and 583.92 for RAL respectively. Additionally, cooperativity trends between the compound’s inhibition profiles were different. It was concluded that CA and RAL have different resistance profiles. Metal dependent differences show that the IN soluble mutant is resistant to CA inhibition only in the presence of Mg2+. This indicates that metal specific structural differences may play a role in resistance. Order of addition indicated that if DNA was present after CA incubation with IN the inhibition was cooperative, while if DNA was added before incubation with CA the inhibition was non-cooperative. This posits that CA may bind to a single site, possibly only the allosteric site, when DNA is already present in the IN active site. When DNA is absent CA binds to multiple sites, possibly both the active site and allosteric sites. The AlphaScreen indicated that the IN/LEDGF interaction is disrupted by CA with IC50 of 237nM (±27nM). Also the disruption is dependent on the order of addition of assay components. Multimerization of IN was increased in the presence of CA as shown by crosslinking assays as well as SEC. Not only did CA induce multimerization of free IN, a nonreducing gel electrophoresis indicated that virus assembled in the presence of CA had increased multimerization of IN, similar to a control. SPR indicated that CA binds to the full length IN with similar kinetics compared to the catalytic core domain (INCCD) indicating that this domain likely contains the CA binding site. The kinetics indicated a slow kon and koff rate for CA binding. These slow binding kinetics are indicative of a high barrier to resistance. Finally, in sillico molecular docking of CA to the active site as well as the allosteric site indicated that it potentially has a dual binding mode on the IN apo-enzyme. The results show that CA has allosteric effects on IN and may provide a good pharmacophore for further development of allosteric IN inhibitors.
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    Beliefs and perceptions that influence utilization of HIV/AIDS services by newly HIV diagnosed men in rural Mbashe Sub-District in the Eastern Cape Province of South Africa
    (2014) Mubuyayi, Clever
    Introduction: HIV/AIDS services are now given freely at public health facility level. They have been decentralized to the formal primary health facilities in the rural areas. Despite the efforts by the South African government, the utilization of those services remains a challenge. There are gender disparities in utilisation of HIV/AIDS services as females utilize the services in greater numbers compared to their male counterparts. The newly diagnosed seropositive men tend to disappear soon after HIV testing, only to appear in a formal health system when their immune system is seriously suppressed and at a more advanced WHO stage of disease. Therefore, the overall aim of this study was to explore the underlying perceptions and beliefs that influence utilization of HIV/AIDS services by newly diagnosed HIV positive men in Mbashe Sub-District of the Eastern Cape between January 2010 and March 2011 Methods: The study was conducted in the rural Mbashe Sub-District of the Eastern Cape Province and utilized a qualitative methodology. This qualitative approach relied on semi-structured in-depth interviews with newly diagnosed HIV positive men of 18-49 years of age who were either accessing or not accessing the HIV/AIDS services during January 2010 and March 2011.The participants were recruited through purposive sampling and 18 interviews were conducted in 6 different facilities at three different service levels. Interviews were audio-recorded and transcripts were subjected to thematic content analysis based on the Health Belief Model. Results: The results show that both groups of men reacted negatively to HIV positive status. The experiences during HIV Counselling and Testing were not linked to whether men could access services. The barriers to utilizing the available HIV/AIDS services included fear of stigma and discrimination, need for an alternative quick cure which delayed utilization of the services, the clinic as gendered space, compromised Provider-Initiated Counselling and Testing (PICT) model implementation, shortage of food, physical fitness and alcoholism. The facilitators for access included the need for survival, disclosure and social support, and cues to action like witnessing a relative dying due to HIV/AIDS related illness. However, the HBM model could not squarely explain the trends in accessing HIV service since few constructs were found to be relevant and also some issues that are outside the HBM model emerged. Conclusions: The study demonstrates that newly diagnosed men‟s utilization of the subsequent free HIV/AIDS services at the primary health care level is influenced by many factors . There are those factors that trigger men to utilize the services and those that deter them from accessing necessary HIV/AIDS services. The factors that influence their access to services are mainly within the multilevel framework which ranges from individual, family, community and societal factors. Therefore, the targeted interventions to address the issue should focus on addressing stigma and discrimination, policy change on training, recruitment and deployment of male nurses, integration of traditional/spiritual interventions within the mainstream of health services, correct implementation of the PICT model and encouraging couple counselling and testing. The Health Belief Model constructs, especially perceived severity, were not strongly linked to whether men accessed services or not.
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    Attitude and perceived barriers by emergency department staff towards routine HIV testing in the emergency department of three academic centres
    (2014-03-25) Michael, Mojeed Oluwaseyi
    The South African HIV testing guideline, Center for Disease Prevention and Control (CDC), World Health Organisation (WHO), and Joint United Nations Programme on HIV/AIDS (UNSAID) have recommended that routine HIV testing be offered in every healthcare facility. The emergency department(ED) is uniquely placed to be involved in this initiative due the volumes and characteristics of patients seen in the ED. This study seeks to determine the attitude of ED staff and their perceived barriers towards routine testing in the ED. Methods: Paper-based questionnaires were distributed to 170 members of ED staff in 3 academic hospitals. Survey Questionnaires contained 25 questions to reflect staff knowledge of HIV infection, their attitude towards testing, current testing practices and perceived barriers to testing. Chi square test was used to test for associations between various variables and the willingness to test. Result: Response rate was 52% (88/170). Average year of experience in an ED (SD) was 4.4 years. Only 30% of ED staff favoured routine testing in the ED. However, 63% of staff was willing to test if result was available within 20 minutes. Members of ED staff generally prefer that a HIV counsellor disclose the result of a positive test. Members of the white race and those who identified fewer barriers were more likely to test. Important barriers cited include; time constraints (77%), inadequate resources (77%), and lack of support staff (71%). Conclusion: The ED staff generally favoured risk based testing over routine testing. Members of the ED staff are generally willing to offer routine HIV testing, but the presence of barriers may limit the implementation of routine HIV testing in the ED.
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    Parents perceptions of HIV counselling and testing in schools: ethical, legal and social implications
    (2014-02-19) Gwandure, Ruth
    Given the high prevalence of HIV and AIDS in South Africa, particularly among the 12-25 year age group, the study set out to understand the perceptions of parents regarding the proposed school-based HIV Counselling and Testing (HCT) campaign planned by the Department of Health in collaboration with the Department of Education. This campaign is aimed at encouraging teenagers to get tested and to know their HIV status in the hope that such knowledge will reduce the number of new infections. The target market of the HCT campaign includes high schools because they have a significant number of adolescents and young adults who could benefit from HCT campaign (SANAC, 2010:11). The research looked at the ethical, legal and social implications of the HCT campaign in schools as perceived by parents. Semi-structured interviews were conducted with a sample of 20 households. Among the main findings was that parents were generally in favour of the HCT campaign but believed that participation in the programme should be voluntary. They anticipated that the HCT programme could potentially affect children’s emotional well-being, particularly if ethico-legal issues of consent and confidentiality were violated and social issues of stigma and discrimination were not handled sensitively. They emphasised that the campaign needed to consider children’s vulnerability and should seek to protect them in the process. Findings are discussed in terms of their implications for promoting bioethical principles in implementing the HCT campaign.
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