"Host resolution of viral infection is dependent upon components of the innate and acquired immune system. The mammalian protein Apobec3 plays an important role as part of the immune system’s innate defenses through its modification of reverse transcribed viral DNA. Recently, Apobec3 was found to directly inhibit HIV-1 and HBV replication through deaminating newly transcribed deoxycytidine residues to deoxyuridine. The ability of mouse and simian Apobec3 variants to inhibit human retroviruses and vice versa highlights the utility of analyzing cross-species homologues. To better understand this editing enzyme, differentially pathogen-susceptible inbred mice were used as an experimental model. The purpose of this project is to examine the effects of murine Apobec3 (muA3) alternative splicing on its DNA-editing characteristics. Three distinct Apobec3 isoforms were isolated from pathogen-susceptible BALB/cByJ (“C”) inbred mice, and two Apobec3 isoforms came from pathogen-resistant C57BL/6ByJ (“Y”) mice. The five muA3 isoforms were cloned, sequenced, and expressed from a constitutive promoter in a haploid Saccharomyces cerevisia strain. MuA3 DNA-editing activity was measured via the CAN1 forward mutation assay. The five isoforms studied in this project were discovered to be strain-specific. One isoform from each mouse strain mutated the yeast CAN1 locus significantly. Additionally, both muA3 isoform mRNAs derived from the pathogen-resistant Y mice were found to persist at a higher level (2.7 -12.4 fold) than any of the C mouse isoforms. This suggests that the absence of exon 5 or some other signal in the Y mice may influence transcript stability. Evidence also suggests that the murine Apobec3 start codon is actually 33bp upstream of its reference start, with implications for previous research performed using muA3. Sequencing analysis of genomic DNA revealed the presence of a 4bp insertion in a region of BALB/cByJ muA3 which may have disrupted an intronic splicing enhancer signal. Furthermore, a novel BALB/cByJ Apobec3 isoform was characterized. This is the first report of strain-specific processing with regard to muA3."

As access and use of antiretroviral therapy continue to increase, the need to improve antiretroviral toxicity monitoring becomes more critical. This is particularly so in underserved settings, where patterns of antiretroviral toxicities possibly alter the need for and frequency of antiretroviral toxicity monitoring. However, barriers such as few skilled healthcare providers and poor infrastructure make antiretroviral toxicity monitoring in underserved settings difficult. The purpose of this dissertation was to investigate how standard clinical guidelines, knowledge-based clinical decision support, and task delegation could be leveraged to overcome barriers to antiretroviral toxicity monitoring in underserved settings. The strategy adopted in this dissertation was guided by the Design Science Research Methodology that emphasizes the generation of scientific knowledge through building novel artifacts. Two qualitative descriptive studies were conducted to characterize the contextual factors associated with antiretroviral toxicity monitoring in underserved settings. Supported by the findings from these studies, a knowledge-based software application prototype that implements clinical practice guidelines for antiretroviral toxicity monitoring was developed. Next, a quantitative validation study was used to evaluate the structure and behavior of the prototype’s knowledge base. Lastly, a quantitative usability study was conducted to assess lay health worker perceptions of the satisfaction and mental effort associated with the use of checklists generated by the prototype. This dissertation research produced empirical evidence about the broad motives and strategies for promoting medication adherence, safety, and effectiveness in underserved settings. It also identified barriers and facilitators of antiretroviral toxicity monitoring within ambulatory HIV care workflows in underserved settings. Additionally, it provided evidence about the extent to which antiretroviral toxicity domain knowledge could be implemented in a knowledge-based application for supporting point-of-care antiretroviral toxicity monitoring. Lastly, the research provided previously unavailable empirical evidence about the perceptions of lay peer health workers on the use of checklists for the documentation of antiretroviral toxicities.

HIV-associated lipodystrophy (HIVLD) syndrome is a condition comprising abnormalities in distribution of body fat and metabolism of lipids and glucose that arises in HIV-infected patients on long-term antiretroviral therapy. This thesis describes clinical research into aspects of the natural history and treatment of HIVLD, as well as molecular research into its pathogenesis centred on subcutaneous adipose tissue. Results demonstrate HIVLD to be a treatment-induced syndrome characterised by initial gains in body fat followed by selective, progressive loss of limb fat. Exposure to thymidineanalogue nucleoside reverse transcriptase inhibitors (tNRTI) induces lipoatrophy through mitochondrial dysfunction of which inhibition of mitochondrial RNA expression, rather than mitochondrial DNA depletion, is an early feature. Mitochondrial dysfunction is associated with decreases in expression of peroxisome proliferatoractivated receptor gamma (PPAR??), an adipocyte transcription factor, which helps explain how tNRTI exposure leads to the loss of adipocyte function. Once established, lipoatrophy is characterised by mitochondrial DNA depletion, although this depletion occurs throughout the mitochondrial genome, suggesting an underlying cause other than inhibition of DNA polymerase gamma. HIVLD is a difficult syndrome to treat. Lipoatrophy is resistant to treatment with rosiglitazone, an agonist of PPAR??, which is ineffective in the setting of ongoing tNRTI therapy and mitochondrial dysfunction. Dyslipidaemia is also difficult to treat as use of pravastatin in the setting of ongoing exposure to protease inhibitors results in only modest declines in fasting cholesterol concentrations. Gains in central fat, such as that seen in patients with buffalo hump, are associated with insulin resistance and diabetes, but only occur in a relatively small percentage of treated patients, suggesting a role for genetic factors in its development. Use of strategies such as avoidance of tNRTI in firstline ART, genetic screening to identify those at risk of toxicities and targeted selection of interventions in subgroups of affected patients, may help prevent this syndrome occurring and better treat those patients in which it has already occurred.

Amendments appended. / Bibliography: leaves 140-165. / xi, 165 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 2005

Thesis (MCur. (Faculty of Health Sciences))--University of Pretoria, 2006. / Summary in English. Includes bibliographical references.

The overall goals of this project were to develop microstructurally based constitutive models to characterize the mechanical behavior of arteries and to investigate the effects of HIV proteins and antiretroviral drugs on the microstructure and mechanical behavior. To this end we created several constitutive models in aim 1 using a rule of mixtures approach, investigated the role of viral proteins in aim 2 through the use a transgenic mouse model, and studied the effects of the antiretroviral drug AZT administered to mice in aim 3. It is well known that the local mechanical environment which cells experience mediates growth and remodeling and that subsequent growth and remodeling can change that mechanical environment. This remodeling includes changes in the content and organization of the constituents of arteries (collagen, elastin, and smooth muscle cells). The first aim thus created models that incorporated the content and organization of these constituents using a rule-of-mixtures approach. The models we developed were able to capture the mechanical behavior of the arteries as well as previously developed phenomenological models while providing more physical meaning to the parameters, some which can be measured experimentally for incorporation into future models. Aims 2 and 3 investigated the mechanical and microstructural changes to murine arteries in response to HIV proteins or the drug AZT. While the development of antiretroviral therapy has greatly increased the life expectancy of patients with HIV, a number of other complications and co-morbidities including cardiovascular disease have become apparent. While clinical data has implicated both the virus and the antiretroviral drugs as playing roles, this work addressed the need of investigating these effects in a controlled manner. Specifically we used mouse models and focused on the two subclinical markers of increased intima-media thickness and arterial stiffening. Aim 2 used a transgenic mouse that expressed most of the human HIV proteins. We observed both intima-media thickening and arterial stiffening in alignment with clinical data. Other changes that also support a proatherogenic phenotype included decreased elastin content and changes in cathepsin activity. Aim 3 administered the antiretroviral drug AZT to healthy mice and we also observed the same subclinical markers of atherosclerosis including intima-media thickening and arterial stiffening as well as the other proatherogenic changes of decreased elastin and changes in cathepsin activity. Several other parameters including axial behavior, opening angles, collagen content, and collagen fiber angles were also quantified. These were important to fully characterize the vessel and may also be incorporated in the future into the constitutive models developed in aim1. In conclusion, in aim 1 we developed a microstructurally based constitutive model of arteries that effectively captures the mechanical behavior and includes parameters that have more physical meaning and some of which are experimentally tractable. Aims 2 and 3 both observed several subclinical markers of atherosclerosis in mice that express HIV proteins or were given AZT, providing a good model for future work and suggesting that both the HIV virus and antiretroviral drugs may play roles in the development of atherosclerosis in HIV.

published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

A systematic review and meta-analysis was conducted to evaluate the efficacy of different infant antiretroviral (ARV) prophylaxis regimens for prevention of mother to child transmission (MTCT) of human immunodeficiency virus (HIV) infection in breastfeeding infants who were born to HIV positive mothers but were HIV uninfected at birth. The systematic review of the literature published during January 2000 to April 2012 resulted in ten randomized and controlled clinical studies which met the study inclusion criteria. Two datasets were identified from the ten selected clinical trials. One dataset contains six studies evaluating short-course ARV prophylaxis regimens, and the second dataset contains four studies evaluating short-course versus extended ARV prophylaxis regimens. The odds ratio was used as the effect size to measure the efficacy between two comparative infant ARV prophylaxis regimens. Meta-analyses were conducted to assess the overall (pooled) treatment effect of the two comparative infant ARV prophylaxis regimens of the two datasets. The pooled ARV treatment effect was calculated as a weighted average of the effect estimated in the individual studies. If no heterogeneity was identified, a fixed-effect meta-analysis by the Mantel-Haenszel method was used. The random-effects method was used when there was heterogeneity in the meta-analysis. The inverse-variance method was used in the random-effects method of meta-analysis. Heterogeneity in the meta-analysis was accessed by the Chi-squared (χ2) test and I2 test. The combined sample size of all ten clinical trials was a total of 10,316 breastfeeding infants, and the overall postnatal HIV transmission rate regardless of ARV regimens and the timing of HIV infection status was approximately 8.7%. The overall HIV transmission rates of the short-course ARV prophylaxis regimen groups were 10.3% at 4-8 weeks and 9.0% at 6-9 months, respectively. The overall late postnatal HIV transmission rate (at 6-9 months after birth) was 5.5% in the extended ARV prophylaxis regimen group. The first dataset contains six randomized and controlled studies to evaluate the efficacy outcome (defined as the unadjusted HIV infection status at 4-8 weeks after birth) of two short-course infant ARV prophylaxis regimens, the nevirapine (NVP) regimen and the zidovudine (ZDV) with or without combination of lamivudine (3TC) or NVP regimen. Due to the existence of substantial heterogeneity, a random-effects method was used to test for the overall treatment effect. The results show that there was no significant difference between the two short-course infant ARV prophylaxis regimens (odds ratio:1.07; 95% CI: 0.69-1.66; Z=0.31, p=0.76). The results of the meta-analysis of five comparative short-course versus extended infant ARV prophylaxis regimens from four randomized and controlled clinical trials, demonstrate a favorable efficacy outcome (defined as the unadjusted HIV infection status at 6-9 months after birth), of the extended ARV regimens. There was no heterogeneity found in this dataset. There was a highly significant difference in the overall effect between the two ARV prophylaxis regimens by a fixed-effect model (odds ratio: 1.72; 95% CI:1.45-2.04; Z=0.68, p<0.00001). In summary, there was no significant difference in the overall treatment effect in reducing the early postnatal MTCT of HIV infection by infant short-course regimens of ARV prophylaxis, which include NVP, ZDV and their combination regimens. In comparison with the short-course ARV regimens, the extended ARV prophylaxis further reduced the risk of the late postnatal MTCT of HIV infection in breastfeeding infants. / published_or_final_version / Public Health / Master / Master of Public Health

HIV-associated lipodystrophy (HIVLD) syndrome is a condition comprising abnormalities in distribution of body fat and metabolism of lipids and glucose that arises in HIV-infected patients on long-term antiretroviral therapy. This thesis describes clinical research into aspects of the natural history and treatment of HIVLD, as well as molecular research into its pathogenesis centred on subcutaneous adipose tissue. Results demonstrate HIVLD to be a treatment-induced syndrome characterised by initial gains in body fat followed by selective, progressive loss of limb fat. Exposure to thymidineanalogue nucleoside reverse transcriptase inhibitors (tNRTI) induces lipoatrophy through mitochondrial dysfunction of which inhibition of mitochondrial RNA expression, rather than mitochondrial DNA depletion, is an early feature. Mitochondrial dysfunction is associated with decreases in expression of peroxisome proliferatoractivated receptor gamma (PPAR??), an adipocyte transcription factor, which helps explain how tNRTI exposure leads to the loss of adipocyte function. Once established, lipoatrophy is characterised by mitochondrial DNA depletion, although this depletion occurs throughout the mitochondrial genome, suggesting an underlying cause other than inhibition of DNA polymerase gamma. HIVLD is a difficult syndrome to treat. Lipoatrophy is resistant to treatment with rosiglitazone, an agonist of PPAR??, which is ineffective in the setting of ongoing tNRTI therapy and mitochondrial dysfunction. Dyslipidaemia is also difficult to treat as use of pravastatin in the setting of ongoing exposure to protease inhibitors results in only modest declines in fasting cholesterol concentrations. Gains in central fat, such as that seen in patients with buffalo hump, are associated with insulin resistance and diabetes, but only occur in a relatively small percentage of treated patients, suggesting a role for genetic factors in its development. Use of strategies such as avoidance of tNRTI in firstline ART, genetic screening to identify those at risk of toxicities and targeted selection of interventions in subgroups of affected patients, may help prevent this syndrome occurring and better treat those patients in which it has already occurred.

Thesis (MSc)--University of Stellenbosch, 2007. / Bibliography. Also available via the Inernet.