Ribonuclease HI (RNase HI), a ubiquitous, non-sequence-specific endonuclease, cleaves the RNA strand in RNA/DNA hybrids. The enzyme has roles in replication, genome maintenance, and is the C-terminal domain of retroviral multi-domain reverse transcriptase (RT) proteins. Murine Leukemia Virus (MLV) and Human Immunodeficiency Virus (HIV) are two such retroviruses and their RNase HI (RNHI) domains are necessary for viral replication, making it an attractive drug target. RNase HI has a “handle region”, an extended loop with a large cluster of positive residues, that is critical for substrate recognition. MLV-RNHI is active in isolation and contains a handle region, but, HIV-RNHI is inactive in isolation and does not contain a handle region. HIV-RT, however, has a region in its polymerase domain (positive charge cluster and aromatic cluster) that makes contact with the RNHI domain that may be serving as a “pseudo” handle region; additionally, insertion of a handle region into isolated HIVRNHI restores its activity. Overall, a breadth of information exists on this region’s dynamics, but important gaps remain unfilled; gaps that may potentially lead to creating effective drugs to treat the above-mentioned viruses. Solution-state nuclear magnetic resonance (NMR) spectroscopy combined with Molecular Dynamic (MD) simulations suggest a model in which the extended handle region domain of the mesophilic Escherichia coli RNHI (EcRNHI) populates "open" (substrate-bindingcompetent) and "closed" (substrate-binding incompetent) states, while the thermophilic Thermus thermophilus RNHI (TtRNHI) mainly populates the closed state at 300 K. In addition, an in silico designed mutant Val98Ala (V98A) EcRNHI was predicted to populate primarily the closed state. Understanding the structural features and internal motions that lead RNase HI to adopt these various conformers is of central importance to better understanding RNase HI’s role in retroviral infection. To formulate a comprehensive model on handle region dynamics, an integrative approach of NMR spectroscopy, X-ray crystallography, and MD simulations is employed. The sensitivity to internal conformational dynamics at multiple time scales of NMR spectroscopy, molecular range and resolution of X-ray crystallography, and structural interpretations of dynamic processes by MD simulations create a synergistic trio capable of tackling this issue. First, the in silico 2-state Kinetic model is validated through NMR observables that correlate with the respective conformers, thus serving as experimental analogs. The NMR parameters also correlate with the Michaelis constants (KM) for RNHI homologs and help to confirm the in silico predictions of V98A EcRNHI. This study shows the important role of the handle region in modulation of substrate recognition. It also illustrates the power of NMR spectroscopy in dissecting the conformational preferences underlying enzyme function. Next, a deeper dive is taken into handle region dynamics, specifically focusing on residue 88 and the impact its identity has on this region. Its sidechain interactions are shown to directly correlate with handle region conformations and helps to amend the originally proposed in silico 2-state Kinetic model. Lastly, looking at RNHI handle region dynamics through an evolutionary lens opens the door to uncovering novel mutations that have been previously overlooked or not identified. Through a phylogenetic analysis, researchers have reconstructed seven ancestral RNHI mutants and three of them have been expressed here. The sequence identity of these three ancestral mutants range from 60-87% to extant homologs and this is reflected by similar peak positions in their 15N HSQC spectra. Requisite experiments to assign the NMR backbone have been completed.

The HIV care continuum captures the proportion of people who engage in various steps of the treatment cascade from the time of HIV diagnosis to the achievement of viral load suppression. Viral load suppression is the ultimate goal of HIV treatment as it is the best way to mitigate the spread of HIV and contain the epidemic. The best pathway to viral load suppression is not always clear. There are several factors that aid or hinder HIV patients from engaging in every step of the care continuum until they achieve and sustain viral load suppression. This dissertation aims to measure the underlying enablers of engagement in HIV care, relate them to potential barriers, and assess the effect of each enabler and barrier on future engagement in care and viral load suppression using data collected from people living with HIV (PLHIV) in Eswatini. Firstly, a systematic review was conducted to summarize the methodologies used to measure and analyze barriers and enablers of engagement in HIV care. A search of all peer-reviewed articles published in English globally since 1996 yielded a final selection of 228 articles. The vast majority of the studies were qualitative and descriptive, and there was a scarcity of quantitative studies utilizing predictive methods that can measure the effect of a barrier or enabler on future engagement in care. Secondly, an empirical analysis was conducted to assess the dimensionality (factor structure) of enablers of engagement in care using a sample largely representative of HIV patients in care in Eswatini. This analysis demonstrated the use of psychometric techniques that can capture underlying latent enablers. These techniques are useful for standardizing the measurements of enablers across studies and programs and can be used to predict future engagement in care. This analysis found financial and access enablers to be the most prominent underlying factors supporting engagement in care in Eswatini, suggesting that these should be an important consideration when designing interventions to retain HIV patients in care in resource-limited settings similar to Eswatini. Thirdly, in an additional empirical analysis, the latent enablers previously identified were used to select potential barriers and assess their effect on linkage to care, retention in care, and viral load suppression. The analysis also involved an assessment of the mediational pathway from the potential barriers to care to viral load suppression that goes through retention in care. Only perceived HIV stigma was related to any step of the care continuum, with low perceived stigma being marginally associated with less viral load suppression. Retention in care did not mediate the relationship between perceived stigma and viral load suppression. More psychometric studies are needed to standardize the measurement of underlying factors affecting engagement in HIV care. This dissertation demonstrated their utility by measuring latent enablers of engagement in care, assessing the downstream effects of the latent enablers and corresponding barriers, and assessing the mechanisms by which the barriers affect viral load suppression.

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Indiana University-Purdue University Indianapolis (IUPUI) / Background: The current treatment for HIV/AIDS is called highly active antiretroviral therapy (HAART) and is a combination of anti-HIV reverse transcriptase inhibitors and protease inhibitors. HAART is capable of suppressing HIV replication and subsequently improving the patients’ survival. However, the issues associated with use of HARRT such as the high cost, severe side-effects, and drug resistance have called for development of alternative anti-HIV therapeutic strategies. In this study, we screened several traditional Chinese medicinal herbal extracts for their anti-HIV activities and determined their anti-HIV mechanisms. Methods: Nine traditional Chinese medicinal (TCM) herbal plants and their respective parts derived from Hainan Island, China were extracted using a series of organic solvents, vacuum dried, and dissolved in dimethyl sulfoxide. Initial anti-HIV activity and cytotoxicity of these extracts were evaluated in HIV-infected human CD4+ T lymphocytes Jurkat. Extracts of higher anti-HIV activities and lower cytotoxicity were selected from the initial screening, and further examined for their effects on HIV-1 entry, post-entry, reverse transcriptase, gene transcription and expression using combined virology, cell biology and biochemistry techniques. Results: Four extracts derived from two different herbal plants completely blocked HIV-1 replication and showed little cytotoxicity at a concentration of 10 g/ml. None of these four extracts had any inhibitory effects on HIV-1 long terminal repeat promoter. Two of them exhibited direct inhibitory activity against HIV-1 reverse transcriptase (RT). All four extracts showed significant blocking of HIV-1 entry into target cells. Conclusions: These results demonstrated that four TCM extracts were capable of preventing HIV-1 infection and replication by blocking viral entry and/or directly inhibiting the RT activity. These results suggest the possibility of developing these extracts as potential anti-HIV therapeutic agents.

Indiana University-Purdue University Indianapolis (IUPUI) / Transcription and alternative splicing play important roles in HIV-1 gene expression and replication and mandate complicated but coordinated interactions between the host and the virus. Studies from our group have shown that a HIV-1 Tat-interacting protein of 110 kDa, Tip110 synergies with Tat in Tat-mediated HIV-1 gene transcription and replication. However, the underlying molecular mechanisms were not fully understood and are the focus of the dissertation research. In the study, we first demonstrated that Tip110 bound to unphosphorylated RNA polymerase II (RNAPII) in a direct and specific manner. We then showed that Tip110 was detected at the HIV-1 long terminal repeat (LTR) promoter and associated with increased phosphorylation of serine 2 within the RNAPII C-terminal domain (CTD) and increased recruitment of positive transcription elongation factor b (P-TEFb) to the LTR promoter. Consistent with these findings, we demonstrated that Tip110 interaction with Tat directly enhanced transcription elongation of the LTR promoter. During these studies, we also found that Tip110 altered HIV-1 mRNA alternative splicing and increased tat mRNA production. Subsequent analysis indicated that Tip110 selectively increased tat exons 1-2 splicing by activating HIV-1 A3 splice site but had no function in tat exons 2-3 splicing. We then showed that the preferential splicing activity of Tip110 resulted from Tip110 complex formation with hnRNP A1 protein, a negative splicing regulator that binds to the ESS2 element within tat exon 2, and as a result, blocked the complex formation of hnRNP A1 with ESS2 and subsequently activated HIV-1 A3 splice site. Taken together, these results show that Tip110 functions to regulate HIV-1 transcription elongation and HIV-1 RNA alternative splicing. These findings not only add to our understanding of Tip110 biology and function but also uncover a new potential target for development of anti-HIV intervention and therapeutic strategies.

Mycobacterium avium and Mycobacterium intracellulare, (M. avium complex, MAC) are human pathogens causing disease in individuals with acquired immunodeficiency syndrome (AIDS) or with thoracic abnormalities. MAC bacteria are difficult to kill because of the resistance of the pathogens to chemotherapeutic agents. One factor affecting treatment of MAC disease is the presence of interconvertible colonial variants. Transparent (T) variants have greater resistance to antibiotics and higher pathogenicity; opaque (O) variants are more susceptible to antibiotics and less pathogenic. The overall goal of this study was to investigate the mechanism for colonial variation. Based on an observation that T variants of M. intracellulare strain Va14 contained a plasmid which was 6 kb smaller than the 68 kb plasmid in O variants, it had been suggested that a transposable element might be responsible for colonial variation. The first objective was to clone the unique DNA fragment present in the 68 kb plasmid but absent from the 62 kb plasmid. The second and third objectives were to determine if the unique fragment contained a transposable element and to analyze the role of that element in the mechanism of colonial variation in M. intracellulare strain Va14. The fourth objective was to determine the distribution of IS1141 in MAC isolates. Fragments containing copies of the putative element were sequenced and a region 1596 basepairs in length with 23 basepair imperfect inverted repeats was designated as insertion sequence IS1141. IS1141 is the first insertion sequence identified in M. intracellulare. Data base searches using open reading frames (ORF) of IS1141, identified ORFb as significantly similar to the transposases of the IS3 family. The presence or absence of IS1141 in strain Va14 plasmids appeared unrelated to colonial variation, but IS1141 was present in another plasmid and the chromosome of the Va14 variants. Hybridization studies with IS1141 identified three chromosomal copies in O variants and two chromosomal copies in T variants. Va14 T variants each had a common IS1141 restriction fragment length polymorphism (RFLP) pattern which was different than the single RFLP pattern found in opaque variants. Based on these differences, it appears that IS1141 may integrate into the gene(s) responsible for the T phenotype preventing their expression. A survey of 64 James River basin non-AIDS, clinical and James River environmental MAC isolates identified 4 of 24 (17%) M. intracellulare isolates as containing IS1141. IS1141 has not been detected in any clinical or environmental M. avium or Mycobacterium species X isolates and may be limited to M. intracellulare. / Ph. D.

Thesis (M. Tech.) - Central University of Technology, Free State, 2009 / The aim of health communication campaigns and visual communication material (VCM) is to positively influence audience health behaviour and attitude. VCM has been used in this respect effectively as a vehicle to convey information about HIV/AIDS over the past three decades. It has been used to promote health knowledge and awareness in order to reduce the transmission of the virus. The aim of this study was to determine the efficacy of graphic imagery in HIV/AIDS VCM. To realise this aim, the researcher set the following objectives: * To review relevant literature in order to isolate key features and process those that communicators must consider/follow when developing HIV/AIDS VCM; * To determine the comprehension of selected outdoor HIV/AIDS messages, the graphics used in these messages, illustration preferences, and an evaluation of the self-efficacy of selected loveLife outdoor visual messages; and * To propose a model that communicators can use as a guideline when developing VCM. The outcome of the review suggests a compilation of features, design guidelines and variables that may contribute to the effectiveness of VCM. The results of the empirical study indicate that suitable graphic imagery fosters message comprehension, while inappropriate imagery inhibits comprehension, and realistic and appropriate imagery is preferred to abstract and representational imagery. Familiar images can contribute towards improved comprehension of HIV/AIDS messages. These findings also gave birth to the proposed ‘O’ communication model, which is a reflection of the results of the empirical study.

Thesis (MA)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The HIV-epidemic that is evident in South Africa today is infecting more women than men. This is mostly due to the vulnerability that women are facing in sexual relationships, where they are not able to negotiate the terms and conditions of their sexual engagement. Patriarchy, the culture of masculinity and a general male dominance influence women’s dependency on their man and agency inside and outside of the home, and contribute to the oppression of women both generally in society and sexually. Women have by this not the control over their own bodies and are for this reason in a high-risk position of contracting HIV. The vulnerability is further linked to the stigmatisation that women experience if they do try to negotiate preventative measures to reduce the risk of transmission. The fear of being stigmatised as ‘loose’ or HIV-positive by both men and women if suggesting condom use, inhibits women to propose the necessary actions for protection. Stigmatising behaviours also impact on a person’s fear of becoming HIV-positive and reduces the likelihood of getting tested, disclose one’s status to sexual partners and receive treatment. This thesis examines cultural and socio-economic issues that contribute to gender inequality in South Africa, and can generate stigma towards women on the basis of HIV and AIDS. This is done by using radical feminism as the theoretical framework for contextualising how women are situated in the South African society, in terms of general and sexual agency. Through the method of content analysis and the findings from the theoretical framework, the thesis further analyses how the three HIVprevention campaigns loveLife, Brothers for Life and TAC manage to address the issues related to stigma based on HIV/AIDS, which are directed towards women. Race, class and gender are all factors that influence the likelihood of becoming HIV-infected and of becoming stigmatised. Women’s low social status situates women in a position where they are more probable to be the object of stigmatisation since they already are considered lower in rank. If the women also are of colour, poor and low educated the chances of becoming stigmatised on the basis of HIV and AIDS are even more likely, the same is the chances of becoming HIV-infected. This indicates that poor, uneducated black women are the group that is most vulnerable towards stigmatisation as well as towards HIV-transmission. Socio-economic and cultural factors have a strong influence on the gender inequality in sexual relationships found in South Africa, which cause HIV to spread and can generate stigmatising behaviours. Stigmatisation on the basis of HIV/AIDS is therefore important to address in order to reduce the number of new HIV-infections. The three campaigns analysed for this thesis did neither directly address stigma on a general level nor directed towards women. The campaigns are therefore considered to be missing an important feature of HIV-prevention in South Africa. / AFRIKAANSE OPSOMMING: Die huidige Suid-Afrikaanse Vigsepidemie infekteer meer vroue as mans. Dit is die geval weens die kwesbaarheid wat vroue ervaar in seksuele verhoudings, waar vroue nie die mag het om die omstandighede van hul seksuele interaksies te onderhandel nie. Patriargie, die kultuur van manlikheid en ‘n algemene manlike dominansie beïnvloed vroue se mag en dra by tot die onderdrukking van vroue, beide in die samelewing in die algemeen en in seksuele verhoudings. Om hierdie rede het vroue nie beheer oor hul eie liggame nie en daarom ervaar hulle ‘n hoë risiko om MIV op te doen. Hierdie kwesbaarheid word ook verbind aan die stigmatisering wat vroue ervaar wanneer hulle probeer om voorkomende aksie te neem ten einde die risiko van Vigsoordrag te verminder. Die vrees om deur mans en ander vroue gestigmatiseer te word as iemand met ‘losse sedes’, of as iemand wat MIV-positief is wanneer hulle kondoomgebruik voorstel, weerhou vroue daarvan om die nodige voorkomende aksie vir selfbeskerming te neem. Stigmatiserende gedrag het ook ‘n impak op ‘n mens se vrees om MIV-positief te word en verminder die waarskynliheid dat jy jouself vir die virus sal laat toets, dat iemand hul status aan seksuele maats sal verklaar, of behandeling sal ontvang. Diegene wat reeds MIV onder lede het is bang om hul status te verklaar weens die gepaardgaande stigma. Hierdie tesis ondersoek kulturele en sosio-ekonomiese kwessies wat bydra tot geslagsongelykheid in Suid-Afrika, en wat stigma kan veroorsaak teenoor vroue met betrekking tot MIV and Vigs. Die studie analiseer dan of Vigsveldtogte hierdie stigma kan aanspreek. Dit word gedoen deur radikale feminisme toe te pas as ‘n teoretiese raamwerk om vroue se plek in die Suid-Afrikaanse samelewing te kontekstualiseer, beide in terme van algemene en seksuele mag. Die metode van inhoudsanalise word toegepas om drie Vigsvoorkomingsveldtogte (loveLife, Brothers for Life en TAC) te analiseer en vas te stel of en hoe hulle kwessies wat betrekking het op stigma teenoor vroue aanspreek. Sosio-ekonomiese en kulturele faktore het ‘n sterk invloed op die geslagsongelykeid in seksuele verhoudings in Suid-Afrika; dit lei daartoe dat MIV versprei word en kan stigmatiserende gedrag vererger. Om hierdie rede is dit belangrik dat MIV/Vigsvoorkomingsveldtogte stigmatisering aanspreek ten einde gedrag te wysig en om die getal nuwe Vigsbesmettings te laat daal. Die drie veldtogte wat in hierdie tesis geanaliseer is het beide nagelaat om stigma direk aan te spreek op ‘n algemene vlak, en was ook nie direk gerig op vroue nie. Die veldtogte kan daarom beskou word as ontoereikend deurdat hulle belangrike komponente van MIV-voorkomig in Suid-Afrika misgekyk het.

Thesis (MScMedSc (Pathology. Medical Virology))--University of Stellenbosch, 2005. / There are currently an estimated 5.3 million people infected with human immunodeficiency virus / acquired immunodeficiency syndrome (HIV/AIDS) in South Africa. HIV-1 group M Subtype C is currently responsible for the majority of HIV infections in sub-Saharan Africa (56% worldwide). The Khayelitsha informal settlement, located 30 km outside Cape Town, has one of the highest HIV prevalence rates in the Western Cape. The objective of this study was to investigate the molecular epidemiology of HIV-1 in Khayelitsha using serotyping and genotyping techniques. Patient samples were received from the Matthew Goniwe general health clinic located at site C in Khayelitsha. Serotyping was performed through a competitive enzymelinked immunosorbent assay (cPEIA). RNA was isolated from patient plasma and a two step RT-PCR amplification of the gag p24, env gp41 IDR, env gp120 V3 and pol genome regions performed. Sequences obtained were used for detailed sequence and phylogenetic analysis. Neighbour-joining and maximum likelihood phylogenetic trees were drawn to assess the relationship between the Khayelitsha sequences obtained and a set of reference sequences obtained from the Los Alamos National Library (LANL) HIV database (http://www.hiv.lanl.gov/). Through serotyping and genotyping the majority of HIV strains were characterised as HIV-1 group M subtype C. One sample (1154) was characterised as a possible C / D recombinant strain. In 9 other samples HIV-1 recombination cannot be excluded, as only one of the gene regions investigated could be amplified and characterised in these samples. The gag p24 genome region was found to be more conserved than the env gp41 IDR, with the env gp41 IDR more conserved than the env gp120 V3. The variability of the env gp120 V3 region indicates that patients might be dually infected with variant HIV-1 subtype C strains or quasispecies. Conserved regions identified in the Khayelitsha sequences can induce CD4+ T-cell responses and are important antibody recognition target sites. These conserved regions can play a key role in the development of an effective HIV-1 immunogen reactive against all HIV-1 subtypes. The majority of subtype C viruses were predicted to use CCR5 as their major chemokine co-receptor. The pol sequences analysed indicate that mutations associated with minor resistance to Protease Inhibitors (PIs) might be present in the Khayelitsha community. The identification of resistant mutations is vital for people receiving antiretroviral treatment (ART). It can influence the success of their treatment and delay the onset of AIDS. Serotyping is a quick characterisation method, but not always accurate. With genotyping detailed molecular analysis can be performed. However, with genotyping the success of amplification often depends on viral load. In Southern Africa a subtype C candidate vaccine appears to be the best option for future vaccine considerations. The sporadic detection of non-subtype C and recombinant subtype C viruses remains a concern and will thus have to be closely monitored. Phylogenetic analysis can help to classify and monitor the spread and evolution of these viruses.