Until now, emerging viral diseases have been posing ongoing threats to the global public health. Among the notorious viruses, the HIV that causes the AIDS has been spreading continuously since it was first identified in 1981 and is the most quickly spreading disease of the century. Although considerable advance has been made by drug discovery groups, the therapeutic management is still challenged by the rapid mutations of the virus to yield resistant strains, so as the emergence of side effects. Therefore, the development of novel potent anti-HIV agents is urgently sought. Owing to the chemical diversity, we believe that natural products may serve as potential "lead" compounds for discovery of anti-HIV drugs.;In order to search for novel naturally occurring compounds with potent inhibitory effects against HIV, we began with isolation of natural products from two medicinal plants of Justicia by means of silica gel column chromatography, and preparative HPLC, namely, J. gendarussa that displayed potent anti-HIV activity in our initial screening, and J. procumbens, and their chemical structures and determinated by spectroscopic and chemical methods such as IR, UV, HRESIMS, 1H NMR and 13C NMR spectrometry (including DEPT, 1H-1H COSY, and HMBC techniques). Upon the complete identification of compounds, we focused on the synthesis of one potential lead compound isolated from J. gendarussa, patentiflorin A (3). Nevertheless, we evaluated all the isolated natural compounds and synthetic 3 via bioactivity screening for anti-HIV activity.;In the phytochemical investigation of J. gendarussa, a rare, shade-loving, quick-growing, evergreen scented shrub collected in Vietnam, the bioassay-directed fractionation of the methanol extract of the roots and stems of the plant led to the isolation two new arylnaphthalide lignan glycosides, named justiprocumins A and B (1--2), together with a known one, patentiflorin A (3). On the other hand, the phytochemical investigation of the methanol extract of the aerial parts of J. procumbens resulted in the isolation of four novel arylnaphthalide lignans, procumbenosides G (4), H (5), I (6) and J (7), along with 23 known compounds, namely, tuberculatin (8), procumbenoside B (9), procumbenoside E (10), ciliatoside B (11), ciliatoside A (12), 5-methoxy-4, 42-di-O-methylsecolariciresinol (13), secoisolariciresinol dimethyl ether (14), 2, 3-bis(3, 4-dimethoxybenzyl)-4-hydroxybutyl acetate (15), secoisolariciresinol (16), hemiariensin (17), ariensin (18), secoisolariciresinol dimethyl ether diacetate (19), hinokinin (20), justicidin E (21), justicidin D (22), justicidin C (23), cilinaphthalide A (24), 5'-methoxy-4'-O-methyllariciresinol (25), 3, 5, 7, 32, 42, 52-hexamethoxyflavone (26), 3, 5, 7, 8, 32, 42-heptamethoxyflavone (27), 3, 5, 7, 8, 32, 42, 52-heptamethoxyflavone (28), methyl ferulate (29) and loliolide (30). In addition, the compound 3 was totally synthesized with a yield of 68.3%;In the anti-HIV evaluation for all the isolated compounds using the defective HIV-based pseudotyped assay, patentiflorin A (3) was found to have anti-HIV activity with an IC50 value of 26.9 nM, while justicin E (21) showed 65.4 % inhibitory effect against HIV replication at 2.5 μg/mL. In the evaluation for the broadness of the spectrum of anti-HIV activity using a standardized human PBMC assay, 2 gave IC50 values of 14-21, and 3 gave IC50 values 24-37 nM in inhibiting the particle production of all the four HIV-1 isolates [BAL and SF162 (both are M-tropic), LAV0.04 (T-tropic), and 89.6 (dual tropic)], while the synthetic 3 showed quite similar activity as that of natural 3. In the test of cytotoxicity, natural 3 exhibited no apparent cytotoxicity at 19.0 mM in A549 and Hela cells, and the synthetic 3 displayed much lower cytotoxicity (CC50: 75.5 mM) than that of the natural 3 (CC50: 18.4 mM) in PBMC cells. That means 2 and 3 have great potentials as anti-HIV lead compounds for further drug development.;In conclusion, natural compounds isolated from medicinal plants serves as one of the most important sources of potentially anti-HIV compounds, which can be employed as "lead" compounds to develop novel therapeutic drugs against HIV.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Genetics. Johannesburg, South Africa 2017 / Background: Stavudine (d4T) use is associated with the development of sensory neuropathy (SN), several mechanisms may underlie d4T-induced toxicity, including: (1) Inter-patient genetic variability in the genes modulating the deoxynucleotide triphosphate (dNTP) pool sizes. (2) Variation in intracellular ARV drug concentrations due to genetic variation in drug transporters. In our study we examined the genetic variation in four stavudine transporter genes and seven genes regulating the deoxythymidine triphosphate (dTTP) synthesis and their associations with d4T-induced SN or CD4+ T cell count or mtDNA copy number. Methods: We examined a cohort of HIV-positive South African (SA) adults exposed to d4T, including 143 cases with SN and 120 controls without SN. 26 single nucleotide polymorphisms (SNPs) from the literature were chosen, prioritised on being tagSNPs with minor allele frequency >5% in Kenyan Luhya (a proxy population for the SA Black population); SNP functional effects and suitability for multiplex analysis on the genotyping platform. Genotyping was performed using Sequenom mass spectrometry. A qPCR assay was used to measure the mtDNA copy number. Association of sensory neuropathy, CD4+ T cell count and mtDNA copy number with genetic variants was evaluated using PLINK. Results: All 26 SNPs were in Hardy-Weinberg equilibrium (HWE) in both the cases and controls. SNP rs8187758 of the SLC28A1 transporter gene and a 3-SNP haplotype ABCG2 were significantly associated with CD4+ T cell count after correction for multiple testing (p = 0.043 and p=0.042 respectively), but were not significant in multivariate testing. No SNP remained significantly associated with SN or mtDNA copy number, after correction for multiple testing. Conclusion: Variation in genes encoding molecular transporters of d4T may influence CD4+ T cell counts after ART. This study presents a positive step towards achieving personalized medicine in SA. / MT 2018

Translocation of viral integrase into nucleus is a critical precondition of integration during the life cycle of HIV, a causative agent of Acquired Immunodeficiency Syndromes (AIDS). It has been considered as an important target for the drug development to treat AIDS. In order to understand the detailed mechanisms of integrase-host cell protein complex interactions, we cloned HIV-1 integrase-EGFP into pTRE2hyg as visible tag to monitor the translocation process. When transiently transfected this vector into Tet-off ready HeLa cells, the EGFP: integrase is mainly localized in the nucleus. It has been hypothesized that any drugs that can inhibit the translocation process are novel class of drugs for AIDS treatment. More than 30000 synthetic compounds and 80000 natural products were screened by virtual screening. A total of 34 compounds were obtained and screened for their ability to block the nuclear entry of HIV-1 integrase by monitoring the EGFP fluorescence in the cells by high-throughput live cell imaging. Eight synthetic compounds (DW-IN4, DW-IN5, DW-IN6, DW-IN9, DW-IN15, DW-IN16, DW-IN17, DW-IN21) and one natural product (DW-IN719) were found to block integrase translocation significantly. According to our screening result, six compounds (INNB-1, INNB-2, INNB-3, INNB-4, INNB-5, INNB-6) were designed and synthesized. INNB-1 and INNB-2 had significant inhibition on integrase nuclear translocation. DW-IN6, DWIN719, INNB-1, INNB-2, INNB-3 and INNB-4, showed significant inhibition on P24 production in live virus assay. DW-IN6, INNB-1, INNB-2, INNB-3 and INNB-4 showed significant syncitia formation inhibition in live virus assay. Six compounds (KM7, KM8, KM14, KM30, KM37, KM79) from Kunming were screened as integrase nuclear translocation inhibitors. Using similar cell imaging techniques, we have cloned the GFP-tagged chemokine receptor CXCR4 using the lentivirus transfection system. CXCR4 receptor is a critical co-receptor in CD4 positive lymphocytes mediating the fusion of HIV into the CD4 positive cells. CXCR4-GFP was over-expressed in 293T cells and the results showed that GFP:CXCR4 receptor is expressed at the plasma membrane of the cells. These cells have been used to monitor the blockage of CXCR4 receptor internalization for drug development. Four compounds (KX128, KX166, KX171, KX180) from Kunming showed CXCR4 internalization blockage in imaging assay. The interaction of these compounds with CXCR4 was predicted by molecular docking. KX128 showed significant HIV inhibition in live virus assays. / Gu, Wangang. / Advisers: Pang Chui Shaw; David Chi Cheong Wan. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 165-179). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Non-nucleoside reverse transcriptase inhibitor (NNRTI) is one of the key components of antiretroviral drug regimen against human immunodeficiency virus type-1 (HIV-1) replication. However, the low genetic barriers to drug-resistance or cross-resistance, side effects, as well as the unaffordable cost of NNRTIs compromise their clinical usage. Therefore, to develop novel NNRTIs with potent antiviral activity against HIV-1 becomes a major concern in the treatment and prevention of HIV/AIDS. (+)-Calanolide A, which is a natural product initially extracted from the tropical rainforest tree Calophyllum lanigerum, was identified as an attractive NNRTI against HIV-1 despite virus strains containing drug-resistant K103N/Y181C mutations. In this study, a chemical library was constructed based on the three chiral carbon centers of (+)-Calanolide A. After screening the activity against HIVNL4-3 wild-type and several NNRTI-resistant pseudoviruses, a small molecule 10-chloromethyl-11- demethyl-12-oxo-calanolide A (F18) was identified as novel NNRTI with promising anti-HIV efficacy. Further studies were performed to investigate the antiviral breadth, drug resistance profile and underlying mechanism of the action of F18. F18 consistently displayed a potent activity against primary HIV-1 isolates including various subtypes of M group, CRF01_AE, and laboratory-adapted drug-resistant viruses in PBMC based assay. Moreover, F18 displayed distinct profiles against 17 NNRTI-resistant pseudoviruses, with an excellent potency especially against one of the most prevalent strains with the Y181C mutation (EC50=1.0nM) in cell line based assay, which was in stark contrast from the extensively used NNRTIs nevirapine and efavirenz. F18-resistant viruses were induced by in vitro serial passages, and mutation L100I was appeared to be the dominant contributor to F18-resistance, further suggesting a binding motif different from nevirapine and efavirenz. The efficacy of F18 was non-antagonistic when used in combination with other antiretrovirals against both wild-type and drug-resistant viruses in infected PBMCs. Interestingly, F18 displayed a highly synergistic antiviral effect with nevirapine against nevirapine-resistant virus (Y181C). Furthermore, in silico docking analysis suggested that F18 may bind to the HIV-1 reverse transcriptase in a way different to other NNRTIs. For the potential as an anti-HIV-1 microbicide, F18 also showed the stable and rapid release, as well as the sustained antiviral activity against HIV-1 wild-type virus in a formulation temperature-sensitive acidic gel. In summary, this study presents F18 as a new potential drug for clinical use and also underlies new mechanism-based design for future NNRTI. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

HIV/AIDS remains one of the most pressing challenges facing South African society. South Africa has the highest number of people living with HIV as well as the highest number of people on HIV treatment globally, yet only 37% of persons eligible for treatment have access to treatment. The advent of HAART ushered in a new era in the treatment of HIV infection. HIV infection was no longer a life threatening terminal illness, HIV/AIDS became a chronic manageable disease. The full clinical benefit of HAART can only be achieved with near perfect adherence i.e. > 95%. This means taking the medication exactly as prescribed; on time, no missed doses, every day, lifelong. No other chronic medication requires such stringent adherence rates for optimal therapeutic benefit, which may mean the choice between life and death. Achieving near perfect adherence poses a serious challenge to health service providers and persons on treatment as typical adherence rates for medication prescribed over long periods are in the 50 – 75 % range. Persons on HAART live with the additional burden of drug resistance and limited treatment options if near perfect adherence rates are not achieved. The purpose of this qualitative study was to explore the factors that influence adherence to HAART. These factors may be related to the person, the health care team and system, the treatment regimen, the social and economic environment or to the effects of HIV disease. Factors may either negatively or positively influence a person’s ability to adhere optimally to their prescribed treatment. A small sample of thirteen participants were purposefully selected for this study. Data was collected using in-depth interviews which were tape recorded and transcribed for thematic analysis. The value of this study is that it may assist health care providers, persons on treatment and the health care system to better comprehend the challenges of lifelong optimal adherence to HAART. / Thesis (M.A.)-University of KwaZulu-Natal, Durban, 2011.

This project has focused on exploring the application of Baylis-Hillman (BH) {a.k.a. Morita-Baylis-Hillman (MBH)} scaffolds in the construction of various compounds with medicinal potential. A series of 2-nitrobenzaldehydes has been treated under BH conditions, with two different activated alkenes, viz., (MVK) and methyl acrylate, using (DABCO) or (3-HQ) as catalyst. While most of the BH reactions were carried out at room temperature, some reactions were conducted using microwave irradiation. The resulting BH adducts have been subjected to dehydration, conjugate addition and allylic substitution to obtain appropriate intermediates, which have been used in turn, to synthesize possible lead compounds, viz., cinnamate esters as HIV-1 integrase inhibitors, 3-(aminomethyl)quinolines and quinolones as anti-malarials and cinnamate ester-AZT conjugates as dual-action HIV-1 integrase-reverse transcriptase (IN-RT) inhibitors. Conjugate addition reactions of methyl acrylate-derived BH β-hydroxy esters with the amines, piperidine, propargylamine and 2-amino-5-(diethylamino)pentane, has afforded a range of products as diastereomeric mixtures in moderate to excellent yields. Catalytic hydrogenation of the aminomethy β-hydroxy esters derivatives, using a palladium-oncarbon (Pd-C) catalyst, has afforded the corresponding, novel 3-aminomethyl-2- quinolone derivatives in moderate yields. Effective allylic substitution reactions of the MVK-derived BH β-hydroxy ketones (via a conjugate addition-elimination pathway) using in situ-generated HCl has afforded the corresponding α-chloromethyl derivatives, which have been reacted with various amines, including piperidine, piperazine, propargylamine and 2-amino-5-(diethylamino)pentane, to yield α-aminomethyl derivatives. Catalytic hydrogenation of selected α-aminomethyl derivatives, using a Pd-C catalyst, has afforded the corresponding, novel 3- (aminomethyl)-2-methylquinoline derivatives in low to moderate yields. A bioassay, conducted on a 6-hydroxy-2-methyl-3-[(piperidin-1-yl)methyl]quinoline isolated early in the study indicated anti-malarial activity and prompted further efforts in the synthesis of analogous compounds. Reaction of the methyl acrylate-derived BH adducts with POCl3 has provided access to α-(chloromethyl)cinnamate ester derivatives, which have been aminated to afford α- (aminomethyl)cinnamate ester derivatives as potential HIV-1 integrase inhibitors. The α- (propargylaminomethyl)cinnamates were used, in turn, as substrates for the “click chemistry” reaction with 3'-azido-3'-deoxythymidine (AZT– an azide and an established reverse transcriptase HIV-1 inhibitor) to afford cinnamate ester-AZT conjugates as potential dual-action HIV-1 integrase-reverse transcriptase (IN-RT) inhibitors. Computer modelling and docking studies of a cinnamate ester-AZT conjugate into the HIV-1 integrase and reverse transcriptase active-sites revealed potential hydrogen-bonding interactions with amino acid residues within the receptor cavities. The isolated products have been appropriately characterized using IR, 1- and 2-D NMR and HRMS techniques, while elucidation of the stereochemistry of the double bond in the BH-derived halomethyl derivatives has been assigned on the basis of NOE, computer modelling and X-ray crystallographic data.

Thesis (MFamMed)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction This cross-sectional study assessed factors affecting access to antiretroviral therapy (ART) among HIV patients. The aim of this study was to explore factors that delay patients from accessing antiretroviral treatment at an East London Hospital Complex clinic and to determine the pathway that people from communities in the surrounding area take in order to access antiretroviral treatment at the referral hospital ART clinic Methods The study design was a descriptive cross-sectional survey using both open and closed questions to generate qualitative and quantitative data. The survey used a questionnaire administered via a face to face interview. 200 Adult patients (>18years old) from the local population with a CD4 count of ≤100/mm3 referred to the ART clinic at East London Hospital Complex for the first time during May to October 2011 were interviewed. Results With the health system, some issues were structural (staffing, availability of CD4 counts) and most were process related and reflected a poor patient experience and lack of trust in the quality of care. Contextual related issues were mainly geographic accessibility (cost and lack of transport, distance to health care facility), stigma and discrimination about HIV. Patients related factors included misperceptions and false beliefs about HIV, low level of education, socioeconomic factors, lack of family and social support such as unavailability of treatment supporter and status of patient’s general health discouraged people from seeking ART. Coping strategies such as denial of results and reluctant to disclose results to other people came out as a distrust of patients in community. Conclusions This study gives evidence that people living with HIV experience health system, patient and contextual related barriers to access HIV treatment. The majority accessed care via their local primary care clinic and traditional or alternative practitioners did not appear to play a major role. The distance from and cost of transport to the referral hospital ART service was a major issue compounded by the difficulty of travelling when acutely sick. The expectation of long waiting times and sometimes negative staff attitudes reduced motivation. A number of other factors related to the patient, the health service and the community context were also identified. Many of these factors that reduce access to ART are amenable to change.

Thesis (Ph.D. (Physiology and Environmental Health)) -- University of Limpopo, 2021 / Background: Human immunodeficiency virus (HIV)-infection remains a major public health burden where approximately 38 million people are affected globally. Human immunodeficiency virus infection is associated with chronic inflammation which can lead to endothelial dysfunction and thrombosis, which are precursor events for cardiometabolic abnormalities such as dysglycaemia and dyslipidaemia. The degree of chronic inflammation, endothelial dysfunction, and hypercoagulation among HIVpositive adults on highly active antiretroviral therapy are not well understood in Sub- Saharan Africa. The objective of this study was to determine the effect of highly active antiretroviral therapy (HAART) on chronic inflammation, endothelial dysfunction, and hypercoagulation among HAART-exposed adult South African participants in a rural setting. Aim: The study aimed to determine the effects of HAART on early biomarkers of cardiovascular disease in the HIV-positive subjects. Methods: The study was cross-sectional, descriptive, and quantitative in design. The research population consisted of 158 participants of males and females within the age range of 18 – 81 years from Mankweng Hospital and surrounding clinics. The study population comprised of three groups, HIV-negative (control group), HIV-positive treatment naïve (HAART-naïve group), and HIV-positive participants on HAART (HAART-exposed group). Weight and height were measured using Omron BF 400 and a portable stadiometer respectively, to calculate the body mass index. Glucose and lipid levels were determined on Cobas® Integra 400 plus auto-analyser. The CD4+ T cell count was determined on the Cytomics FC500 Flow Cytometer Multi-Platform loader. The concentration of fibrinogen, c-reactive protein (CRP), L-selectin, D-dimers, P-selectin, von Willebrand factor (VWF), soluble intercellular adhesion molecule (sICAM-1), and soluble vascular cell adhesion molecule (sVCAM-1) in serum samples were determined on the Luminex 200TM. Data were analysed using SPSS version 25.0. Descriptive statistics were performed on all variables and analysis of covariance was used to determine differences across all groups. Correlation coefficients and multiple regression analyses were used to determine associations. Results: Body mass index (BMI) and glucose metabolism were not significantly affected by HAART exposure. However, the HAART-exposed group had significantly increased LDL-C (F (2, 154) = 7.501, p = 0.001) and TC (F (2, 154) = 9.174, 0.0002) levels. The prevalence of high LDL-C levels was significantly elevated in the HAART-exposed group (29.6%) (p = 0.041). The prevalence of pre-diabetes (11.3%) was the highest among the HAART-exposed group (non-significant), although, no significant difference was observed. While P-selectin was significantly reduced in the HAART-exposed group (F (2, 154 = 7.253, p = 0.001). On the other hand, the HAARTexposed group also significantly increased VWF (F (2, 154 = 4.556, p = 0.011). The HAART-exposed group showed no significant effect on L-selectin, sICAM-1, sVCAM- 1, CRP, fibrinogen and D-dimer levels. However, D-dimer was negatively associated with HAART (r = -0.249, p = 0.011). There were significant independent association between the combined HAART regimens and P-selectin (Std β = 0.219, p = 0.032), first-line regimen with both P-selectin (Std β = 0.434, p = 0.004) and sVCAM-1 Std β = 0.328, p = 0.031), second-line regimens with L-selectin (Std β = 1.032, p = 0.005) and, a positive independent association between first-line regimen and D-dimer (β = 0.741, p = 0.0001). Although BMI and glucose metabolism were not significantly affected in both the HAART-exposed and HAART-naïve groups, dyslipidaemia was present across the three groups (HAART-exposed, HAART-naïve and control). HAART-exposure showed a protective effect by reducing endothelial dysfunction (ED) and hypercoagulation. Yet, ED was still present among this rural South African HAART-exposed population. The HAART-exposed group may be at increased risk for CVD. Therefore, CVD should be regularly monitored in the HAART-exposed population. / National Research Fund, the Health and Welfare Sector Education and Training Authority, and the University of Limpopo (UL)

引發獲得性免疫缺陷綜合癥（AIDS）的人類免疫缺陷病毒（HIV）是一種逆轉錄病毒。過去的十餘年間，高效抗逆轉錄病毒治療療法（HARRT），在抗病毒感染方面取得了很大的成功。高效抗逆轉錄病毒治療療法是一種將多種抗逆轉錄病毒藥物複合的藥物聯用療法。然而，因為病毒的逆轉錄過程極易突變，導致HIV已經可以對大多數使用的抑製藥物產生抗藥性。因此，有越來越多的需要去尋找新型的抗病毒複製機理，例如將人體細胞蛋白作為載體，來達到克服病毒抗藥性的目的。 / HIV-1的複製離不開宿主細胞的剪接因子，例如SR蛋白。選擇性剪接因子ASF/SF2，一個典型的調控pre-RNA剪接的SR蛋白，在HIV-1的pre-mRNA剪接和複製中起到了很重要的調控作用。ASF/SF2和其他SR蛋白一樣，都被丝氨酸/苏氨酸蛋白激酶（SRPK）磷酸化，磷酸化位點位於C端的丝氨酸/苏氨酸結構域（RS domain）。SRPK通過磷酸化來調節ASF/SF2在細胞中的分佈。對於SRPK 和ASF/SF2複合物的結構學和功能學研究指出，ASF/SF2的docking motif和SRPK1的遠離活性位點的docking groove存在很強的相互作用。而這種相互作用是調節磷酸化過程關鍵。所以，在我們的研究過程中，我們希望通過阻斷2個蛋白的相互作用來干擾ASF/SF2的磷酸化，進而抑制其在HIV-1 pre-mRNA剪接過程中的活性。 / 我們採用以結構為基礎的藥物模擬篩選，來選擇潛在的抑制物，達到通過抑制物與docking groove的相互作用來阻斷ASF/SF2和SRPK1的相互作用，以達到抑制磷酸化的目的。我們使用的數據庫來自于ZINC數據庫（UCSF），包括天然產物數據庫和SPECS。我們採用AutoDock Vina 和AutoDock 4.2 二個模擬軟件來栓選數據庫中351473个化合物。并從中選出50個潛在的化合物用作之後的化學生物學測試。體外的激酶活性試驗顯示，6個化合物對ASF/SF2的磷酸化有抑製作用。 / 體外的HIV-1 pre-mRNA剪接實驗顯示，5個化合物在逆轉錄PCR（RT-PCR）中有一定得抑制效果。和DMSO對照組相比，在抑製劑作用下剪接產物的生成被抑制。HIV-1病毒合胞體感染實驗顯示，有一個化合物對病毒的感染起到了一定的抑制作用。 / 其他的測試實驗還在進行中，包括對SRPK1和抑制物複合物的結構研究，從而更好的研究抑制物的作用機理。以及，採用表面等離子共振波譜來進行動力學研究和其他關於化合物在病毒複製過程中的實驗測試。 / Human immunodeficient virus (HIV) is a retrovirus that cause acquired immunodeficiency syndrome (AIDS). Highly active antiretroviral therapy (HAART) is a treatment of HIV infection that uses combinations of antiretroviral drugs and has achieved great success in the past two decades. However, since the reverse transcription process of viral RNA is notoriously prone to error, HIV-1 can acquire resistance to nearly all known inhibitors and has started to develop resistance to HAART. Therefore, there is an ongoing search for new drugs with novel inhibitory mechanism such as targeting cellular proteins essential for HIV-1 replication to overcome drug resistance of the virus. / HIV-1 mRNA undergoes complex splicing and the expression of the integrated HIV-1 provirus is largely dependent on the host’s splicing machinery which assembly requires splicing factors such as serine-arginine rich proteins (SR proteins). Alternative splicing factor/splicing factor 2 (ASF/SF2), a prototypic SR protein that is essential for pre-mRNA splicing, has been shown to play critical roles during HIV-1 pre-mRNA splicing and replication. ASF/SF2, like other SR proteins, is phosphorylated by SR protein-specific kinases (SRPKs) at its C-terminal arginine/serine (RS) domain, which governs its localization and metabolism. Structural and functional studies of SRPK1 in complex with ASF/SF2 has revealed that a docking groove on SRPK1 that is distal to the active site interacts strongly with a docking motif and the RS domain of ASF/SF2, leading to high affinity binding as well as regulating the mechanism of phosphorylation. In this study, we propose that by blocking this interaction, we might interfere the phosphorylation of ASF/SF2 and inhibit its activity during splicing of HIV-1 pre-mRNA. / Structure-based in silico screening method is adopted to identify potential inhibitors that bind to the docking groove of SRPK1 to block the binding and phosphorylation of ASF/SF2. The compound libraries being used include the Natual Products Database and SPECS database from ZINC (UCSF). 351,473 compounds have been screened using the program Autodock Vina as well as Autodock 4.0. Until now 50 potential candidates of inhibitor have been selected for biochemical analyses. In vitro kinase assays showed that six compounds exhibit inhibitory activity against the phosphorylation of ASF/SF2. / To test the effect of the selected inhibitors on the splicing of HIV-1 mRNA, ex vivo splicing assay has been performed. Current results showed that the synthesis of splicing products extracted from drug-treated cells was less efficient when compared to untreated cells. Biological assays testing the inhibitory effects of the compounds on viral infection are currently underway. Our preliminary result suggested that one of the compounds could indeed inhibit HIV-1 viral infection. / Other biochemical and biological analyses including structural study of kinase-inhibitor complexes to understand the mode of inhibition; measurement of binding kinetics using surface plasmon resonance spectroscopy (SPR); and biological assays testing the inhibitory effects of the compounds on replication are underway. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yu, Xiyao. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 95-107). / Abstracts also in Chinese. / Abstract --- p.I / 摘要 --- p.III / Acknowledgements --- p.V / TABLE OF CONTENTS --- p.VI / LIST OF FIGURES --- p.IX / LIST OF TABLES --- p.XI / Chapter Chapter I --- : Introduction --- p.1 / Chapter 1.1 --- HIV, HAART and HIV Drug Resistance --- p.2 / Chapter 1.2 --- HIV-1 alternative splicing mechanism --- p.9 / Chapter 1.3 --- SR Protein Family --- p.13 / Chapter 1.4 --- Functional roles of SR protein in HIV pre-mRNA splicing --- p.16 / Chapter 1.5 --- Phosphorylation States of SR Proteins --- p.18 / Chapter 1.6 --- SR protein Kinase --- p.20 / Chapter 1.7 --- Interaction between SRPK1 and ASF/SF2 --- p.23 / Chapter 1.8 --- IDC16 and SPRIN340 --- p.26 / Chapter 1.9 --- Structure-based drug screening --- p.27 / Chapter 1.10 --- AutoDock Suite --- p.29 / Chapter 1.11 --- Kinase-substrate interaction inhibitors --- p.30 / Chapter 1.12 --- Focus of study --- p.34 / Chapter Chapter II --- : Materials and Methods --- p.35 / Chapter 2.1 --- Materials --- p.36 / Chapter 2.1.1 --- Bacterial strain --- p.36 / Chapter 2.1.2 --- Antibodies --- p.36 / Chapter 2.1.3 --- Cell line --- p.36 / Chapter 2.1.4 --- Plasmid --- p.36 / Chapter 2.1.5 --- Reagents --- p.38 / Chapter 2.2 --- Expression and purification of Recombinant protein --- p.38 / Chapter 2.3 --- In silico screening of inhibitors --- p.44 / Chapter 2.4 --- Kinase Glo Assay --- p.45 / Chapter 2.5 --- In vitro kinase assay --- p.45 / Chapter 2.6 --- Cell Culture --- p.46 / Chapter 2.7 --- MTT Assay --- p.46 / Chapter 2.8 --- Immunocytochemistry --- p.47 / Chapter 2.9 --- Ex vivo splicing assay --- p.47 / Chapter 2.10 --- Surface plasmon resonance spectroscope --- p.48 / Chapter Chapter III --- : Results --- p.50 / Chapter 3.1 --- In silico screening of inhibitors --- p.51 / Chapter 3.2 --- Selected Compounds Inhibits SRPK1 in Vitro --- p.60 / Chapter 3.2.1 --- Protein purification --- p.60 / Chapter 3.2.2 --- Inhibits ASF/SF2 Phosphorylation by SRPK --- p.66 / Chapter 3.3 --- Surface Plasmon Resonance Binding Competition Assay --- p.76 / Chapter 3.4 --- Inhibitors Alters HIV-1 Alternative Splicing ex Vivo --- p.79 / Chapter 3.5 --- Cytotoxic effect of candidate compound on HeLa cells --- p.84 / Chapter 3.6 --- Nature compound alters ASF/SF2 localization --- p.86 / Chapter Chapter IV --- : Discussion and Conclusion --- p.89 / References --- p.95

Thesis (MPhil)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Access to antiretroviral therapy has been gradually increasing in resource limited settings, Zimbabwe included. Despite the increasing access to antiretroviral therapy quite a number of patients are still delaying to seek antiretroviral therapy. The purpose of the study was to examine factors associated with delay in seeking antiretroviral therapy. A survey was conducted at Parirenyatwa Hospital Opportunistic Infections/Antiretroviral Therapy Clinic from September and November 2012. A total of 80 participants starting antiretroviral therapy who met the criteria were included in the study. The inclusion criteria included patients 18 years above but less than 65 years, no prior history of antiretroviral therapy and eligibility for antiretroviral therapy based on CD4 count or World Health Organisation clinical staging. An interviewer administered questionnaire containing demographic, socio-economic and health-facility factors were used to collect data. Four weeks was used as a cut off point for delay in seeking antiretroviral therapy. The majority of participants (60%) delayed seeking antiretroviral therapy and the factors which were associated with delay in seeking antiretroviral therapy included female gender; lack of a partner; low level of education; low socio-economic status; treatment of opportunistic infections; extra laboratory tests on top of the CD4 count tests; not being on Cotrimoxazole Prophylaxis; not being referred for antiretroviral therapy by the testing site; stigma and discrimination. However disclosure was not associated with early seeking of antiretroviral therapy. Health system factors such as attitude of health care workers, shortage of staff and long waiting times were also identified as bottlenecks to patients seeking antiretroviral therapy early. Efforts to increase early starting of antiretroviral therapy should focus on addressing the referral system from testing sites to antiretroviral therapy initiating sites, improving efficiency of antiretroviral initiating sites, increasing point of care HIV & AIDS diagnostics tools and addressing patient‟s concerns such as stigma & discrimination. / AFRIKAANSE OPSOMMING: Toegang tot antiretrovirale terapie Geleidelik is steeds in hulpbron beperkte omgewing, Zimbabwe ingesluit. Ten spyte van die toenemende toegang tot antiretrovirale terapie 'n hele aantal van die pasiënte is nog steeds vertraag antiretrovirale terapie te soek. Die doel van die studie was om faktore te ondersoek wat verband hou met vertraging in die soek van antiretrovirale terapie. 'n Opname is by Parirenyatwa-hospitaal opportunistiese infeksies / antiretrovirale terapie Clinic van September en November 2012. 'N totaal van 80 deelnemers begin antiretrovirale terapie wat met die kriteria wat in die studie ingesluit is. Die insluiting kriterium was pasiënte ouer as 18 jaar maar minder as 65 jaar, geen geskiedenis voor antiretrovirale terapie en in aanmerking kom vir antiretrovirale terapie gebaseer op CD4-telling of Kliniese stadiëring Wêreld Gesondheid Organisasie. Was 'n onderhoudvoerder vraelys met demografiese, sosio-ekonomiese faktore en gesondheid-fasiliteit wat gebruik word om data in te samel. 4 weke is gebruik as die afsny punt vir die vertraging in die soeke na antiretrovirale terapie. Die meerderheid van die deelnemers (60%) antiretrovirale terapie en die faktore wat verband hou met die vertraging in die soek na antiretrovirale terapie is vertraag te soek vroulike geslag, gebrek van 'n vennoot, lae vlak van onderwys, 'n lae sosio-ekonomiese status, behandeling van opportunistiese infeksies; Ekstra laboratoriumtoetse op die top van die CD4-telling toetse nie op Cotrimoxazole Profilakse, nie vir antiretrovirale terapie verwys deur die toets site, stigma en diskriminasie. Egter openbaarmaking wat nie verband hou met die vroeë soek van antiretrovirale terapie. Gesondheid stelsel faktore soos houding van gesondheidsorgwerkers, tekort aan personeel en lang wagtye, is ook geïdentifiseer as knelpunte aan pasiënte op soek na vroeë antiretrovirale terapie Pogings om te vroeg begin van antiretrovirale terapie Verhoog Indien Fokus op die verwysingstelsel van die toets sites tot antiretrovirale terapie Inisiëring sites, verbetering van doeltreffendheid van antiretrovirale Inisiëring sites, Verhoog Punt van Care MIV & VIGS diagnose tools en aanspreek van die pasiënt se Kommer Soos stigma en diskriminasie.