AIDS Dementia Complex (ADC) is a neurodegenerative disorder implicated in HIV-1 infection that is associated with elevated levels of the neurotoxin, quinolinic acid (QA) which causes a cascade of events to occur, leading to the production of reactive oxygen species (ROS), these being ultimately responsible for oxidative neurotoxicity. In clinical studies, Non-nucleoside reverse transcriptase inhibitors (NNRTIs), efavirenz (EFV) and nevirapine (NVP) have been shown to potentially delay the progressive degeneration of neurons, thus reducing the frequency and neurological deficits associated with ADC. Despite these neuroprotective implications, there is still no biochemical data to demonstrate the mechanisms through which these agents offer neuroprotection. The present study aims to elucidate and further characterize the possible antioxidant and neuroprotective mechanisms of NVP and EFV in vitro and in vivo, using QA-induced neurotoxicity as a model. Research has demonstrated that antioxidants and metal chelators have the ability to offer neuroprotection against free radical induced injury and may be beneficial in the prevention or treatment of neurodegeneration. Hence the antioxidant and metal binding properties of these agents were investigated respectively. Inorganic studies, including the 1, 1-diphenyl-2 picrylhydrazyl (DPPH) assay, show that these agents readily scavenge free radicals in vitro, thus postulating the antioxidant property of these agents. The enhancement of superoxide radical generation and iron mediated Fenton reaction by QA is related to lipid peroxidation in biological systems, the extent of which was assayed using the nitroblue tetrazolium and thiobarbituric acid method respectively. Both agents significantly curtail QA-induced lipid peroxidation and potentially scavenge superoxide anions generated by cyanide in vitro. Furthermore, in vivo results demonstrate the ability of NVP and EFV to protect hippocampal neurons against lipid peroxidation induced by QA and superoxide radicals generated as a consequence thereof. The alleviation of QA-induced oxidative stress in vitro possibly occurs through the binding of iron (II) and / or iron (III), and this argument is further strengthened by the ability of EFV and not NVP to reduce iron (II)-induced lipid peroxidation in vitro directly. In addition the ferrozine and electrochemistry assay were used to measure the extent of iron (II) Fe[superscript 2+] and iron (III) Fe[superscript 3+] chelation activity. Both assays demonstrate that these agents bind iron (II) and iron (III), and prevent redox recycling of iron and subsequent complexation of Fe[superscript 2+] with QA which enhances neuronal damage. Both NNRTIs inhibit the endogenous biosynthesis of QA by inhibiting liver tryptophan 2, 3-dioxygenase activity in vivo and subsequently increasing hippocampal serotonin levels. Furthermore, these agents reduce the turnover of hippocampal serotonin to 5-hydroxyindole acetic acid. NVP and not EFV increase 5-hydroxyindole acetic acid and norepinephrine levels in the hippocampus. The results of the pineal indole metabolism study show that NVP increases the synthesis of melatonin, but decreases N-acetylserotonin, 5-hydroxyindole acetic acid and 5-hydroxytryptophol levels. Furthermore, it shows that EFV decreases 5-hydroxyindole acetic acid and melatonin synthesis. Behavioural studies using a Morris water maze show that the post-treatment of rats with NVP and EFV significantly improves QA-induced spatial memory deficits in the hippocampus. This study therefore provides novel information regarding the neuroprotective mechanisms of NVP and EFV. These findings strengthen the argument that these NNRTIs not only have antiviral effects but possess potential neuroprotective properties, which may contribute to the effectiveness of these drugs in the treatment of ADC.

Approximately 33.4 million people are living with HIV/AIDS. Of those, 97% live in low and middle income countries, with 22.4 million in sub-Saharan Africa. Only 42% of the people who require anti-retrovirals (ARVs) in low to middle income countries are receiving anti-retroviral therapy (ART). There is a need to develop novel and cost effective methods for producing antiretroviral drugs. Stavudine and azidothymidine (AZT) were identified as potential targets because they could both be produced through a common intermediate – 5 methyluridine (5-MU). It has been established that the biocatalytic production of 5-methyluridine is possible through a reaction known as transglycosylation, in a process which has not previously been demonstrated as commercially viable. A selection of biocatalysts were expressed either in recombinant E. coli strains or in the wild type organisms, purified and then screened for their ability to produce 5-MU. A combination of Bacillus halodurans purine nucleoside phosphorylase 1 (BHPNP1) and E. coli uridine phosphorylase (EcUP) gave the highest 5-MU yield (80%). This result represents the first combination of free enzymes from different organisms, giving high yields of 5-MU under high substrate conditions. Both enzymes were purified and successfully characterised. The established pH optimum was pH 7.0 for both enzymes. Temperature optima and stability data for BHPNP1 (70 C and t1/2 at 60 C of 20.8 h) indicated that the biocatalytic step was operating within the capabilities of this enzyme and would operate well at elevated temperatures (up to 60 C). Conversely, the temperature optimum and stability data for EcUP (optimum of 40 C and t1/2 at 60 C of 9.9 h) indicated that the enzyme remained active at 40 C for the duration of a 25 h biotransformation, but at 60 C would only be operating at 20% of its optimum activity and would lose activity rapidly. BHPNP1 and EcUP were used in a bench scale (650 ml) transglycosylation for the production of 5-MU. A 5-MU yield of 79.1% was obtained at this scale with a reactor productivity of 1.37 g.l-1.h-1. Iterative saturation mutagenesis was used to rapidly evolve EcUP for improved thermostability. A moderately high throughput colorimetric method was developed for screening the mutants based on the release of p-nitrophenol upon phosphorolysis of a pyrimidine nucleoside analogue. By screening under 20 000 clones the mutant UPL8 was isolated. The mutant enzyme showed an optimum temperature of 60 C and improved stability at 60 C (t1/2 = 17.3 h). The increase in stability of UPL8 is due to only 2 mutations (Lys235Arg, Gln236Ala). These mutations may have caused an increase in stability due to interactions with other structural units in the protein, stabilization of the entrance to the binding pocket, or by decreasing the flexibility of the α-helix at the N-terminus. Transglycosylation experiments showed that the mutant enzyme UPL8 is a superior catalyst for the production of 5-MU. A 300% increase in reactor productivity was noted when free enzyme preparations of UPL8 was combined with BHPNP1 at 1.5% m.m-1 substrate loading. The high yield of 5-MU (75-80% mol.mol-1) was maintained at 9% m.m-1 substrate loading. A commercially viable productivity of 31 g.l-1.h-1 was thus realised. Further optimisation of the process could produce still higher productivities. Future work in directed evolution of nucleoside phosphorylases is envisaged for improved stability and enhanced substrate range for application to other commercially relevant transglycosylation reactions.

The Human Immunodeficiency Virus (HIV) and Acquired Immuno Deficiency Syndrome (AIDS) epidemic has had massive detrimental impacts on rural communities across Africa including in Zimbabwe. In response to the HIV and AIDS epidemic, the government of Zimbabwe has developed and adopted comprehensive programmes to address HIV and AIDS prevention, care and support. One of the critical components of these programmes relates specifically to treatment of the HIV infected given that HIV and AIDS is increasingly seen as a manageable threatening disease. However the success and effectiveness of the treatment regimen (involving antiretroviral drugs or ARVs) is dependent heavily on complete adherence to the rigid and complex regimens. It is against this background that this thesis studies a particular rural community in Zimbabwe called Chivanhu (in Masvingo Province) in terms of the relationship between rural livelihoods and HIV and AIDS (particularly HIV treatment and treatment adherence). Unlike other rural communities (notably in communal areas), Chivanhu is an informal and unstable community with a turbulent history. Most rural studies of HIV and AIDS in Zimbabwe and elsewhere in the region have focused on well-established and stable communities in which agricultural production is still of some significance. In such communities, the impact of HIV and AIDS on livelihoods is severe but, in more informal settlements, the vulnerability of households to the epidemic (and challenges pertaining to treatment adherence) is even more pronounced. Using a rural livelihoods framework, this thesis seeks to identify, understand and analyse the conditions which shape levels of adherence to HIV and AIDS in the informalsettlement of Chivanhu in Zimbabwe.

Homocysteine (Hcy) is a sulfur containing amino acid and is a potent neurotoxin. It has been shown that elevated levels of Hcy, termed hyperhomocysteinemia, plays a role in the pathologies of Alzheimer’s disease (AD) and age-related cognitive decline. Hcy is a glutamate agonist, which causes in increase in Ca[superscript (2+)] influx via the activation of NMDA class of excitatory amino acid receptors, which results in neuronal cell death and apoptosis. Estrogen and progesterone are female hormones that are responsible for reproduction and maternal behaviour. However, in the last decade, it is evident that both female hormones have neuroprotective properties in many animal models of neurodegeneration. Collectively, both estrogen and progesterone reduce the consequences of the oxidative stress by enhancing the antioxidant defence mechanisms, reducing excitotoxicity by altering glutamate receptor activity and reducing the damage caused by lipid peroxidation. However, the mechanisms by which estrogen and progesterone provide such neuroprotection probably depend on the type and concentration of hormone present. Moreover, numerous studies have shown that hormone replacement therapy (HRT, estrogen and progestins) or estrogen-only replacement therapy (ERT) may prevent or delay the onset of AD and improve cognition for women with AD. Clinical trials have also shown that women taking HRT may modify the effects of Hcy levels on cognitive functioning. Oxidative stress increases in the aging brain and thus has a powerful effect on enhanced susceptibility to neurodegenerative disease. The detection and measurement of lipid peroxidation and superoxide anion radicals in the brain tissue supports the involvement of free radical reactions in neurotoxicity and in neurodegenerative disorders. The hippocampus is an important region of the brain responsible for the formation of memory. However, agents that induce stress in this area have harmful effects and could lead to dementia. This study aims to investigate and clarify the neuroprotective effects of estrogen and progesterone, using Hcy-induced neurodegenerative models. The initial studies demonstrate that estrogen and progesterone have the ability to scavenge potent free radicals. Histological studies undertaken reveal that both estrogen and progesterone protect against Hcy-induced neuronal cell death. In addition, immunohistochemical investigations show that Hcy-induced apoptosis in the hippocampus can be inhibited by both estrogen and progesterone. However, estrogen also acts at the NMDA receptor as an agonist, while progesterone blocks at the NMDA receptor. These mechanisms reduce the ability of Hcy to cause damage to neurons, since Hcy-induced neurotoxicity is dependent on the overstimulation of the NMDA receptor. SOD and GPx are important enzymatic antioxidants which can react with ROS and neutralize them before these inflict damage in the brain. Hcy can increase oxidative stress by inhibiting expression and function of these antioxidants. However, it has been shown that the antioxidant abilities of both estrogen and progesterone can up-regulate the activities of SOD and GPx. These results provide further evidence that estrogen and progesterone act as antioxidants and are free radical scavengers. The discovery of neuroprotective agents is becoming important as accumulating evidence indicates the protective role of both estrogen and progesterone in Hcy-induced neurodegeneration. Thus further work in clinical trials is needed to examine whether reducing Hcy levels with HRT can become the treatment of neurodegenerative disorders, such as Alzheimer’s disease.

The human immunodeficiency virus (HIV) is one of the most common and dreaded diseases of the 21th century. Today, the disease is still spreading with increasing incidence. “Since the beginning of the epidemic, almost 75 million people have been infected with the HIV virus and about 36 million people have died of HIV. Globally, 35.3 million [32.2–38.8 million] people were living with HIV at the end of 2012” (http://www.who.int/gho/hiv/en/). Several studies have been conducted on herbs under a multitude of ethnobotanical grounds. The use of medicinal plants for the management of HIV has become a common practice especially, in the Eastern Cape Province of South Africa (Wilfred Otang Mbeng, 2013 PhD thesis, UFH). At the beginning of this programme, an ethnomedicinal survey of plants used for the management of HIV infection was carried out in targeted areas of the Province and information on the names of plants, the parts and the methods of preparation were collected. The survey revealed that 18 species representing 12 families were found to be commonly used for the management of HIV, as well as other opportunistic diseases such as tuberculosis, diabetes mellitus, sores, high blood pressure, etc. Carpobrotus edulis was selected for this research because it was the most frequently used in the Province. The foliar micro morphological contents of the plant, its phytochemical and antioxidant activity, in vitro antimicrobial activity, inhibitory effect against HIV-1 protease and reverse transcriptase, mechanisms of action and cytotoxicity were investigated. In terms of the foliar micro morphological contents in plants, an electron microscopy scanning (SEM) was completed. Investigation revealed that both glandular tricomes and calcium oxalate crystals (CaOX) were observed. Consequently, it is hypothesized that the bioactive therapeutic compounds secreted by C. edulis may be produced in the glandular trichomes. An investigation of phytochemical content of the plant extracts (C. edulis) was completed using four solvent extracts (hexane, acetone, ethanol and water). Results of the phytochemical analysis showed that proanthocyanidins (86.9 ± 0.005%) where highest in the aqueous extract with phenolics at 55.7 ± 0.404% in acetone extract, tannin at 48.9 ± 0.28% in ethanol extract, while the hexane extract had the highest levels of flavonoids (0.12 ± 0.05%) and flavonols (0.12 ± 0.05%). Antioxidant studies of the various extracts revealed that aqueous and ethanol extracts were found to be the best solvents for antioxidant activity in C. edulis leaves. GC-MS analysis of the essential oil from C. edulis leaves revealed that the essential oil contained at least 28 compounds. These included, in order of abundance: Oxygenated monoterpenes (36.61%); fatty acids esters (19.25%); oxygenated diterpenes (19.24%); monoterpenes (10.6%); sesquiterpenes (3.58%); and diterpenes (1.43%). Similarly, a GC-MS analysis of the crude hexane, acetone and ethanol extracts from C. edulis leaves identified a total of 59 compounds. Of the 59 compounds, 12 major phyto-metabolites that are active against infectious diseases were identified. To comfirm the potential use of C. edulis to treat infectious disease, antifungal activity of the crude essential oil extract and the four solvent extracts were tested against Candida albicans, Candida krusei, Candida glabrata, Candida rugosa and Cryptococcus neoformans strains. The essential oil extract was found to be the most active against all the fungal strains tested and performed better than the four extracts used various solvents used to extract (hexane, acetone, ethanol and water) the C. edulis leaves were tested for antibacterial and anti HIV-1 reverse transcriptase (RT) activity. The results indicated that both gram-positive and gram-negative isolates were inhibited by the extracts (hexane, acetone and ethanol) but antimicrobial activity was observed for the water extract. The lowest minimum inhibitory concentration values were obtained for the ethanol extract, followed by acetone and hexane extracts. No inhibition of HIV-1 reverse transcriptase was observed for any of the leaf extracts, even up to concentrations of 16 mg/ml. The potential inhibitory activities of the various solvent extracts against HIV-1 protease were evaluated at four different concentrations (16, 1.6, 0.16 and 0.016 mg/ml). Results indicated that the water extract showed almost 100% inhibition of HIV-1 protease activity, with an IC50 of 0.86 mg/ml leaf extract. Other solvent extracts (hexane, acetone and ethanol) however, did not show any inhibition activity above that observed for the DMSO control. The metabolic components in the water extract were subjected to LC-MS/MS analysis, which identified at least 91 compounds present in the water extract. Further studies involving the molecular modelling need to be carried out to confirm the inhibitory potential of these compounds. The cytotoxicity of the water extract of C. edulis leaves was also screened using human Chang liver cells at concentrations ranging bewteen 0.005 mg/ml and 1 mg/ml. Results indicated that the water extracts were not toxic. In conclusion the results from this study support the use of water extracts of C. edulis leaves by traditional healers to treat HIV infections and have identified possible mechanisms of action of the water extracts of C. edulis.

No description available.

This dissertation is divided into two main sections describing major portions of my Ph.D. research: (1) development of two enzymatic assays for identifying inhibitors of SARS-CoV-2 RNA dependent RNA polymerase (RdRp) and the associated proofreading exonuclease complexes, two key enzymatic activities of SARS-CoV-2, the virus responsible for the COVID-19 pandemic and (2) the design and implementation of four novel single-color fluorescent DNA sequencing by synthesis (SBS) methods, including the synthesis of many of the key nucleotide analogues required for these studies. In response to the COVID-19 pandemic, the first part of my research is focused on the discovery of potential therapeutics for combating coronavirus infections. Chapter 1 describes the identification of several polymerase and exonuclease inhibitors for SARS-CoV-2 using novel mass spectrometry-based molecular assays. SARS-CoV-2 has an exonuclease complex, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RdRp and the exonuclease could overcome this deficiency. Chapter 1 reports the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of identified exonuclease inhibitors, RNAs terminated with the active forms of the prodrugs like Sofosbuvir, Remdesivir and Favipiravir were largely protected from excision by the exonuclease, while in the absence of exonuclease inhibitors, there was rapid excision. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment. Chapters 2-6 describe the single-color DNA SBS studies. Chapter 2 provides essential background on the structure of DNA, the DNA polymerase reaction, and several key DNA sequencing technologies, with an emphasis on the design of nucleotide analogues for the DNA SBS approach. Chapter 3 delineates a one-color fluorescent DNA SBS method based on a set of nucleotide reversible terminators (NRTs) comprising two orthogonal cleavable linkers, one fluorescent dye and one anchor. Chapter 4 describes a one-color hybrid DNA sequencing approach using a set of dideoxynucleotide analogues bearing two orthogonal cleavable linkers, one fluorophore and one anchor as well as a set of unlabeled NRTs. By introducing a pH responsive fluorophore into the design of nucleotide analogues, Chapter 5 demonstrates a novel type of single-color DNA SBS method using a set of NRTs comprising one pH-responsive fluorescent dye or one non-responsive fluorescent dye tethered with one cleavable linker. Chapter 6 presents another option for the single-color DNA sequencing technique using a set of deoxynucleotide analogues comprising the above pH responsive or non-responsive dyes tethered with a cleavable linker, along with a set of unlabeled NRTs. The one-color SBS approaches have the potential for higher sensitivity, miniaturization and cost effectiveness compared with four-color SBS methods. Finally, Chapter 7 summarizes the SARS-CoV-2 antiviral drug discovery and one-color sequencing techniques and discusses potential follow-up research on these projects.

A thesis submitted to the Faculty of Humanities, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Doctor of Philosophy, July, 2017 / This thesis explores the rearticulation of subjectivity in the context of the struggle for antiretroviral therapy in South Africa, and also in the contemporary era of treatment accessibility for HIV and AIDS. Two sub-aims are investigated: the first concerns exploring how, and with what consequences, subjectivity was deployed in the contestations that characterized the South African ‘AIDS war’; the second concerns inquiring into the intelligibility of subjectivity in public and everyday consciousness in the post-AIDS war period. Integrating qualitative analyses with the theoretical lens of an analytics of governmentality, the data set includes policy-related archival materials, a popular HIV advice column and interviews with people living with HIV and on antiretroviral therapy. The thesis brings into sharp focus the adumbration of the right to health with rational decision-making, dignity and autonomy. Much more than a way of organizing interests, advocating for the right to treatment - to prevent the transmission of HIV from mother to child and to slow-down HIV spread - was a strategy of effecting a rationality-cum-affective transfiguration of a widespread helplessness and despair into self-reliance and hope. At the level of public and everyday consciousness, self-government on antiretroviral therapy lies at the intersection of knowledge, self-care and self-management. However, such a subjective positionality is not adopted unproblematically, or even sustained indefinitely, owing to the relative weight of other disparate requirements upon oneself from day to day. What emerged out of the epic battle for antiretroviral therapy, undergirding the prevailing current public and policy orientation to antiretroviral therapy care, was the combination of an optimistic rationality and a hopeful affectivity for the potential of fashioning an HIV-positive subjectivity, contiguously responsibilized and self-responsibilizing. At the experiential level of living on ARVs, where autonomy is synonymous with self-regulation, the thesis demonstrates that self-responsibility is also an unpredictable and fluid undertaking of navigating the affective tumult of hopefulness, uncertainty, sacrifice and tension. / XL2018

Treatment of brain diseases remains extremely challenging partly due to the fact that critical drug delivery is hindered by the blood-brain barrier (BBB), a specialized and highly selective barrier lining the brain vasculature. Focused ultrasound (FUS), combined with systematically administered microbubbles (MBs), has been established as a technique to noninvasively, locally, and transiently open the BBB. The primary mechanism for temporarily opening the BBB using FUS is microbubble cavitation, a phenomenon that occurs when the circulating microbubbles interact with the FUS beam in the brain vasculature. Over the past two decades, many preclinical and clinical applications of FUS-induced BBB opening have been developed, but certain challenges, such as drug delivery route, cavitation control, inflammation onset, and overall accessibility of the technology, have affected its efficient translation to the clinic. This dissertation focuses on optimizing three aspects of FUS-induced BBB opening for therapeutic applications. The first specific aim investigated FUS-induced BBB opening for drug delivery through the intranasal route. Optimal sonication parameters were determined and applied to FUS-enhanced intranasal delivery of neurotrophic factors in a Parkinson’s Disease mouse model. In the second specific aim, cavitation levels affecting the inflammatory response due to BBB opening with FUS were optimized. The relationship between cavitation during FUS-induced BBB opening and the local inflammation was examined, and a cavitation-based controller system was developed to modulate the inflammatory response. In the third specific aim, the devices used for FUS-induced BBB opening were streamlined. A conventional system for FUS-induced BBB opening includes two transducers: one for therapy and another for cavitation monitoring (single element) or imaging (multi-element). In this aim, a single linear array transducer capable of synchronous BBB opening and cavitation imaging was developed, creating a cost-effective and highly accessible “theranostic ultrasound” device. The feasibility of theranostic ultrasound (TUS) was demonstrated in vivo in both mice and non-human primates. In summary, the findings and methodologies in this dissertation optimized FUS-enhanced intranasal delivery across the BBB, developed a cavitation-controlled system to modulate inflammation in the brain, which has been advantageous in reducing pathology and designed a new system for theranostic ultrasound for drug delivery to the brain. Taken altogether, this thesis contributes to the efficient advancement and optimization of FUS-induced BBB opening technology, thus enhancing its clinical adoption in the fight to treat many challenging brain diseases.

Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer’s disease (AD) is a neurological disorder defined by the presence of plaques comprised mostly of amyloid-β (Aβ), and neurofibrillary tangles consisting of hyperphosphorylated microtubule associated protein tau (MAPT). AD is also characterized by widespread synapse loss and degeneration followed by death of neurons in the brain. Inflammatory processes, such as glial activation, are also implicated. In order to study mechanisms of neurodegeneration and evaluate potential therapeutic agents that could slow or reverse this process, a tissue culture system was developed based on primary embryonic cerebrocortical neurons. This culture system was observed to exhibit time-dependent neurodegeneration, glial proliferation, and synaptic marker loss consistent with AD-affected brains. The regulatory promoter regions of several genes implicated in AD, including the Aβ precursor protein (APP), β-amyloid cleaving enzyme (BACE1), and MAPT, were studied in this culture model. The MAPT gene promoter activity followed the pattern of neuronal maturation and degeneration quite closely, increasing in the initial phase of the tissue culture, then reducing markedly during neurodegeneration while APP and BACE1 gene promoters remained active. Deletion series of these promoters were tested to give an initial indication of the active regions of the gene promoter regions. Furthermore, the effects of exogenous Aβ and overexpression of p25, which are two possible pathogenic mechanisms of gene regulation in AD, were studied. Response to Aβ varied between the promoters and by length of the Aβ fragment used. Overexpression of p25 increased MAPT, but not APP or BACE1, promoter activity. This neurodegeneration model was also used to study the putative neuroprotective action of the NMDA receptor antagonist memantine. Treatment with memantine prevented loss of synaptic markers and preserved neuronal morphology, while having no apparent effect on glial activation. The protective action on synaptic markers was also observed with two other structurally distinct NMDA receptor antagonists, suggesting that the effects of memantine are produced by its action on the NMDA receptor. It is concluded that this tissue culture model will be useful for the study of gene regulation and therapeutic agents for neurodegeneration, and that the efficacy of memantine may result from preservation of synaptic connections in the brain.