Ferroptosis is a non-apoptotic, regulated form of cell death that is characterized by the iron-dependent lethal accumulation of lipid peroxides and lipid peroxide byproducts. Huntington’s disease (HD) is an autosomal neurodegenerative disease with characteristic motor, psychiatric, and cognitive signs and symptoms caused by the expansion of CAG repeats in the Huntingtin gene, resulting in the production of pathogenic protein with an extended polyglutamine tract that is prone to aggregation. Accumulating evidence has identified links between ferroptosis and HD suggesting that ferroptosis inhibition may provide therapeutic benefit. However, the ability to evaluate this potential has been limited by the unavailability of potent, brain-penetrant specific ferroptosis inhibitors. This dissertation evaluates two different types of ferroptosis inhibitors and increases the available molecular tools to investigate the role of ferroptosis in the etiology of HD. In the second and third chapters, two new classes of ferrostatin analogs, termed fourth and fifth generation ferrostatins, are developed and their in vitro and in vivo properties characterized. These efforts identify three, fifth generation analogs that are potent, brain-penetrant, and stable and can be administered chronically to symptomatic HD mice. The fourth chapter provides molecular insights into the mechanism of action of the hypocholesterolemic drug probucol in inhibiting ferroptosis and identifies cellular cholesterol levels and cholesterol import as regulators of ferroptosis. In sum, this work provides new molecular tools and insights that can be utilized to elucidate the contribution of ferroptosis to HD and other disease states.

<p> Photoresponsive polymers can act as controllable drug delivery systems that may revolutionize ophthalmic drug delivery for disease treatment in the posterior segment of the eye. Localized, controlled drug delivery devices have significant therapeutic advantages for treating diseases of back of the eye by increasing patient compliance and maintaining therapeutic levels of drug in the tissue. Sustained-release delivery systems that respond to light/laser stimuli are under development to control the rate of delivery resulting in a tuneable treatment profile ideal for retinal diseases. The use of light as a crosslinking mechanism has the potential to create unique materials with controllable swelling, degradation and diffusion properties. </p> <p> This thesis investigates the synthesis and development of universal, graftable PEG-anthracene molecules and their applications in photosensitive alginate and hyaluronate (HA) "photogels". Anthracene undergoes reversible dimerization with wavelengths above 300 nm and de-dimerization/dissociation below 300 nm; due to its well-understood chemistry and symmetry, it was used as a starting point and proof-ofconcept for the synthesis of reversible dimerizing crosslinkers that may be generically grafted to different polymers to cause crosslinking/decrosslinking. After synthesis, watersoluble PEG-anthracene macromolecules were grafting via carbodiimide chemistry to the carboxyl groups along the polymer backbone of alginate and HA at various densities to create viscous liquids or gels with good handling properties. </p> <p> Light irradiation can be used to control the swelling and effective crosslinking density of the photogels which in tum can control drug delivery from photocrosslinked hydro gels as illustrated through the decrease or increase in the delivery of a variety of low molecular weight (<1000 Da) and high molecular weight (>10,000 Da) model drug compounds from both alginate and HA photogels with various light treatments. Novel loading mechanisms were developed through the loading of compounds into uncrosslinked gels followed by crosslinking 365 nm exposures to "lock" in the model drug compounds. Diffusion coefficients effectively compared the different systems showing increase exposures of 365 nm resulted in greater decrease in release of compounds demonstrating the ability to fine-tune release rates. Different formulations and control gels demonstrate a variety of different release profiles. The photogels were valuable long-term controlled release systems (>80 days) that also demonstrate high cytocompatibility when grown with ophthalmic cell lines. </p> <p> Novel photoresponsive biomaterials for smart delivery of therapeutics which use light-controlled crosslinking and decrosslinking mechanisms have been developed. The PEG-anthracene graftable photocrosslinkers show the ability to introduce photocontrolled crosslinking into hydrogel systems. While anthracene as the photodimerizer and alginate and HA as the bulk materials are used as a proof-of-concept in this work, this grafting system can be further manipulated to include new photosensitive dimerizers and other applicable polymers. The ability to use light stimuli to control release rates in a continual fashion, rather than having delivery that is strictly on or off, is a valuable finding that may lead to the development of drug delivery systems that can be catered towards individuals and the progression of their disease. </p> / Thesis / Doctor of Philosophy (PhD)

AZT (3'-azido-2’, 3’-dideoxythymidine) has been shown to prolong the survival of patients infected with human immunodeficiency virus (HIV) and decrease the severity of opportunistic infections. Such studies have prompted the use of AZT to treat symptomless individuals infected with HIV in the hope of delaying or even preventing the progression to acquired immunodeficiency syndrome (AIDS). However, before chronic use of AZT in symptomless individuals is initiated, it is important to establish whether this anti-viral drug would directly alter the phenotype and functions of the cells involved in the immune system. In the current study, we observed that AZT when administered orally for 7 -14 days into DBA/2 mice at 500 - 1000 mg/kg body weight induced a dose-dependent decrease in cellularity of the thymus. AZT caused significant alterations in the thymus resulting from a significant decrease in the number of double-positive (CD4⁺CD8​​⁺) cells and an increase in the number of double-negative (CD4⁻CD8⁻) cells. Interestingly, after the i.p. administration of interleukin-2 (IL-2) simultaneously with AZT, the total cellularity of the thymus was completely reconstituted. We also observed that AZT effectively suppressed the in vivo T cell response to conaibumin and gp120 of HIV. Furthermore, the addition of AZT to in vitro cultures caused a dose-dependent decrease in T and B cell proliferative responses to mitogens at 50μM or greater concentrations. Also, AZT inhibited the generation of cytotoxic T lymphocytes when added to the culture and this inhibition was reconstituted by the addition of exogenous IL-2. Together, our studies demonstrate that AZT modulates the phenotype and function of cells of the immune system which, in turn, could have marked repercussions on immune responses of the host toward infections and cancers. Also, our data demonstrating that AZT can suppress T cell responsiveness against HIV antigens caution against chronic use of AZT in asymptomatic HIV-infected individuals. / Master of Science

published_or_final_version / Medicine / Master / Master of Philosophy

Thesis (DMed)--Stellenbosch University, 2015 / ENGLISH ABSTRACT: Introduction The increasing intake of traditional medicines among HIV/AIDS patients in sub-Saharan Africa needs urgent consideration by clinicians and other healthcare providers since the safety of such medications are unknown. The pharmacokinetic parameters - Absorption, Distribution, Metabolism and Elimination (ADME) play important role in the safety evaluation of drugs, thus implicating drug metabolizing enzymes and transporters as critical indicators for herb-drug interactions. The objective of this study was to evaluate the risk potential of seven herbal medicines commonly consumed by HIV/AIDS patients for drug interactions applying in vitro models. In this study, inhibition and induction effects of the herbal medicines on cytochrome P450s (CYPs) 1A2, 2C9, 2C19, 2D6 and 3A4 as well as P-glycoprotein (P-gp) were investigated. Methods Herbal medicines – Lessertia frutescens, Hypoxis hemerocallidea, Kalanchoe integra and Taraxacum officinale were sourced from Medico Herbs, South Africa were identified by experts from Compton Herbarium, South African National Biodiversity Institute, Cape Town. Moringa oleifera, Echinacea purpurea and Kalanchoe crenata were obtained from the repository of the National Centre for Natural Product Research (NCNPR), University of Mississippi, USA. Reversible inhibitory effect of aqueous and methanol herbal extracts were evaluated in recombinant CYPs applying the fluorescent metabolites at specified excitation/emission wavelengths; CYP1A2 (3-cyano-7-hydroxycoumarin (CHC); 405/460 nm), CYP2C9, CYP2C19 and CYP3A4 (7-hydroxy-4-(trifluoromethyl)-coumarin (HFC); 405/535 nm) and CYP2D6 (7-hydroxy-4-(aminomethyl)-coumarin (HAMC); 390/460 nm). Comparative studies in human liver microsomes (HLM) and recombinant CYPs were conducted to investigate the inhibitory effect of methanol herbal extracts and fractions on 6β testosterone hydroxylation activity. Time dependent inhibitory (TDI) effect of the herbal extracts were evaluated applying the IC50 shift fold, normalized ratio and the NADPH-, time- and concentration-dependent approaches. Influence of herbal extracts on metabolic clearance of testosterone was assessed in both HLM and human hepatocytes. The effects of each herbal extract on expression of CYP1A2, CYP3A4 and MDR1 genes were evaluated in activated human pregnane X receptor (PXR) co-transfected HepG2 cells. Finally, the inhibitory effect of herbal extracts on P-gp was assessed using the calcein-acetoxymethyl ester (calcein-AM) uptake and the digoxin radiolabelled substrates in MDCKII-MDRI cells. Results The aqueous extracts of Moringa oleifera, Kalanchoe integra, Kalanchoe crenata, Echinacea purpurea and Lessertia frutescens demonstrated high risk of in vivo inhibition on CYPs 3A4 and 1A2 with Cmax/Ki >1.0. Methanol extracts of these herbal medicines also indicated potential risk of reversible drug interaction. The methanol extracts of M. oleifera, K. crenata and L. frutescens showed strong TDI effect on CYP3A4 with IC50 shift fold >1.5 and normalised ratio <0.7. Moringa oleifera intermediately reduced intrinsic clearance of testosterone in human hepatocytes (2 ≤ AUC ratio ≤ 5) when scaled up to humans. Methanol extracts of Echinacea purpurea up-regulated the expression of CYP1A2, CYP3A4 and MDR1 genes in activated PXR. Kalanchoe crenata and Echinacea purpurea indicated strong inhibition on P-gp by reducing transport of digoxin across hMDR1-MDCKII cell monolayer from basolateral to apical with IC50 values of 18.24 ± 2.52 μg/mL and 24.47 ± 4.97 μg/mL, respectively. Conclusion The herbal medicines especially M. oleifera, K. integra and E. purpurea have the potential to cause herb-drug interaction in vivo if sufficient hepatic concentration is achieved in humans. / AFRIKAANSE OPSOMMING: Inleiding Die verhoogde inname van tradisionele medisynes onder MIV/VIGS-pasiënte in sub-Sahara-Afrika verg dringend oorweging deur klinici en ander gesondheidsorgverskaffers, aangesien die veiligheid van sodanige medikasies onbekend is. Die farmakokinetiese parameters – Absorpsie, Distribusie, Metabolisme en Eliminasie (ADME) – speel ’n belangrike rol by die veiligheidsevaluering van geneesmiddels, en impliseer gevolglik geneesmiddel-metaboliserende ensieme en vervoerders as kritiese indikators vir krui-geneesmiddel-interaksies (HDI). Die oogmerk van hierdie studie is om die risikopotensiaal van sewe kruiemedisynes wat algemeen deur MIV/VIGS-pasiënte geneem word, vir geneesmiddel-interaksies te evalueer deur in vitro-modelle te gebruik. In hierdie studie is die inhiberings- en induseringsuitwerkings van die kruiemedisynes op sitochroom P450’s (verkort na CYP’s) 1A2, 2C9, 2C19, 2D6 en 3A4, sowel as P-glikoproteïen (P-gp), ondersoek. Metodes Kruiemedisynes – Lessertia frutescens, Hypoxis hemerocallidea, Kalanchoe integra en Taraxacum officinale – is van Medico Herbs, Suid-Afrika, bekom en deur kundiges van die Compton-herbarium, by die Suid-Afrikaanse Nasionale Biodiversiteitsinstituut, Kaapstad, geïdentifiseer. Moringa oleifera, Echinacea purpurea en Kalanchoe crenata is van die bewaarplek van die Nasionale Sentrum vir Natuurlike Produknavorsing (NCNPR) aan die Universiteit van Mississippi in die VSA verkry. Die omkeerbare inhiberende uitwerking van kruie-ekstrakte in water en metanol is in rekombinante CYP’s geëvalueer deur die gebruik van die fluoresserende metaboliete op gespesifiseerde opwekkings-/emissiegolflengtes; CYP1A2 (3-siaan-7-hidroksikumarien (CHC); 405/460 nm), CYP2C9, CYP2C19 en CYP3A4 (7-hidroksi-4-(trifluoormetiel)-kumarien (HFC); 405/535 nm) en CYP2D6 (7-hidroksi-4-(aminometiel)-kumarien (HAMC); 390/460 nm). Vergelykende studies van menslikelewermikrosome (HLM) en rekombinante CYP’s is uitgevoer om die inhiberende uitwerking van metanolkruie-ekstrakte en -fraksies op 6β-testosteroonhidroksileringsaktiwiteit te ondersoek. Die tydafhanklike inhiberende uitwerking (TDI) van die kruie-ekstrakte is geëvalueer deur gebruikmaking van die IC50-verskuiwingsvou-, die genormaliseerdeverhoudings- en die NADPH-, tyd- en konsentrasieafhanklike benaderings. Die invloed van kruie-ekstrakte op metaboliese testosteroonverheldering is in beide HLM en menslike hepatosiete geëvalueer. Die uitwerkings van elke kruie-ekstrak op die uitdrukking van CYP1A2-, CYP3A4- en MDR1-gene is in geaktiveerde menslike pregnaan-X-reseptor(PXR)-, ko-getransfekteerde HepG2-selle geëvalueer. Laastens is die inhiberende uitwerking van kruie-ekstrakte op P-gp geëvalueer, met gebruikmaking van die kalsien-asetoksimetiel-ester (kalsien-AM)-opname en die digoksien- radiogemerkte substrate in MDCKII-MDRI-selle. Resultate Die ekstrakte in water van M. oleifera, K. integra, K. crenata, E. purpurea en L. frutescens het ’n hoë risiko van in vivo-inhibering op CYP’s 3A4 en 1A2 met Cmaks/Ki >1.0 getoon. Ekstrakte van hierdie kruiemedisynes in metanol het verder potensiële risiko van omkeerbare geneesmiddelinteraksie getoon. Die ekstrakte van M. oleifera, K. crenata en L. frutescens in metanol het sterk TDI-uitwerking op CYP3A4 met IC50-verskuiwingsvou >1.5 en genormaliseerde verhouding <0.7 getoon. M. oleifera het intermediêre vermindering van intrinsieke testosteroonverheldering in menslike hepatosiete (2 ≤ AUC verhouding ≤ 5) tot gevolg wanneer die skaal na mense verhoog word. Ekstrakte van E. purpurea in metanol het die uitdrukking van CYP1A2-, CYP3A4- en MDR1-gene in geaktiveerde PXR opgereguleer. K. crenata en E. purpurea het sterk inhibering van P-gp getoon deur die vervoer van digoksien deur die hMDR1-MDCKII-selmonolaag van basolateraal tot apikaal met IC50-waardes van onderskeidelik 18.24 ± 2.52 μg/mL en 24.47 ± 4.97 μg/mL te verminder. Gevolgtrekking Kruiemedisynes, veral M. oleifera, K. integra en E. purpurea, het die potensiaal om HDI in vivo te veroorsaak indien voldoende hepatiese konsentrasie by mense bereik word.

Thesis (MSc (Mathematical Sciences))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: This thesis takes into account the different levels of infectiousness of the human immunodeficiency virus (HIV) infected individuals throughout their period of infection. Infectiousness depends on the time since infection. It is high shortly after the infection occurs and then much lower for several years, and thereafter a higher plateau is reached before the acquired immunodeficiency syndrome (AIDS) phase sets in. In line with this, we formulated a mathematical model which is structured according to the age of infection. To understand the dynamics of the disease, we first discuss and analyse a simple model in which the age of infection is not considered, but progression of the HIV-AIDS transmission is taken into consideration by introducing three stages of infection. Analysis of these models tells us that the disease can be eradicated from the population only if on average one infected individual infects less than one person in his or her infectious period, otherwise the disease persists. To investigate the reduction of the number of infections caused by a single infectious individual to less than one, we introduce different treatment strategies for a model which depends on the age of infection, and we analyse it numerically. Current strategies amount to introducing treatment only at a late stage of infection when the infected individual has already lived through most of the infectious period. From our numerical results, this strategy does not result in eradication of the disease, even though it does reduce the burden for the individual. To eradicate the disease from the population, everyone would need to be HIV tested regularly and undergo immediate treatment if found positive. / AFRIKAANSE OPSOMMING: Hierdie tesis hou rekening met die verskillende aansteeklikheidsvlakke van die menslike immuniteitsgebreksvirus (MIV) deur besmette individue gedurende hulle aansteeklikheidstydperk. Die graad van aansteeklikheid hang af van die tydperk sedert infeksie. Dit is hoog kort nadat die infeksie plaasvind en daarna heelwat laer vir etlike jare, en dan volg n hoer plato voordat uiteindelik die Verworwe-Immuniteitsgebreksindroom (VIGS) fase intree. In ooreenstemming hiermee, formuleer ons n wiskundige model van MIV-VIGSoordrag met n struktureer waarin die tydperk sedert infeksie bevat is. Om die dinamika van die siekte te verstaan, bespreek en analiseer ons eers n eenvoudige model sonder inagneming van die tydperk sedert infeksie, terwyl die progressie van MIV-VIGS-oordrag egter wel in ag geneem word deur die beskouing van drie stadiums van infeksie. Analise van die modelle wys dat die siekte in die bevolking slegs uitgeroei kan word as elke besmette mens gemiddeld minder as een ander individu aansteek gedurende die tydperk waarin hy of sy self besmet is, anders sal die siekte voortduur. Vir die ondersoek oor hoe om die aantal infeksies per besmette individu tot onder die waarde van een te verlaag, beskou ons verskeie behandelingsstrategiee binne die model, wat afhang van die tydperk sedert infeksie, en ondersoek hulle numeries. Die huidige behandelingstrategiee kom neer op behandeling slegs gedurende die laat sta- dium van infeksie, wanneer die besmette individu reeds die grootste deel van die aansteeklikheidsperiode deurleef het. Ons numeriese resultate toon dat hierdie strategie nie lei tot uitroeiing van die siekte nie, alhoewel dit wel die las van die siekte vir die individu verminder. Om die siekte binne die bevolking uit te roei, sou elkeen gereeld vir MIV getoets moes word en indien positief gevind, dadelik met behandeling moes begin.

Thesis (MBA (Business Management))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: South Africa is on the verge of major changes in the private medical sector. The government’s planned National Health Insurance has far reaching implications for all role players in the industry, as well as for the general public. This paper looks at the changes that have been made since the ANC government came to power in 1994 and then continues to look at possible models for the new National Health Insurance plan. A proposal on practicing cost-effective cardiology within this new system is made. The data of a pilot project between a private service provider and a managed healthcare company is analysed as a basis of this discussion. / AFRIKAANSE OPSOMMING: Suid-Afrika se mediese bedryf staan op die vooraand van groot veranderinge. Die regering se beplande Nasionale Gesondheidsplan het verreikende implikasies vir alle rolspelers in die bedryf, sowel as die algemene man op straat. Die dokument kyk oorsigtelik na die veranderinge wat ondergaan is sedert die ANC regering aan bewind gekom het in 1994 en gaan daarna voort om na moontlike opsies te kyk hoe die nuwe gesondheidsmodel daarna gaan uitsien. Voorstelle word gemaak oor hoe privaat kardiologie in die nuwe sisteem koste-effektief beoefen kan word. ‘n Lootsprojek van ‘n privaat diensverskaffer en ‘n bestuurde gesongheidsorg maatskappy se data word ontleed as basis vir die bespreking.

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

Thesis (MPhil)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: multiple therapeutic categories of medicines have been increasingly targeted for counterfeiting. According to Van Niekerk [Van Niekerk, Anton. (2001). Moral and social complexities of AIDS in Africa. University of Stellenbosch], “it is commonplace to identify and bewail a plethora of problems in the developing world generally, and in Africa in particular. Poverty, illiteracy, famine, political instability, natural disasters, and many more misfortunes dominate the history of this part of the world over the past 50 years. It was therefore adding uncalled (undeserved?) insult to already overwhelming injury when HIV/AIDS visibly struck the world since mid-1980. In spite of all the other calamities that Africa has to deal with, it nevertheless is no exaggeration to claim that HIV/AIDS nowadays constitutes the most serious health and social crisis and challenge that has ever befallen the continent”. Similar patterns involving HIV/AIDS are now emerging on other continents. One objective of this recent research study was to explore possible relationships between the growing scourges of the worldwide counterfeiting of medicines and parallels with the expanding global HIV/AIDS pandemic - as well as to examine potential relationships and risks associated with other diseases that have been observed to have ‘special associations’ with HIV and AIDS [e.g. sexually transmitted infections (STI’s), Tuberculosis (TB) and Malaria] - and possible impact on the “World of Work”. A second and important objective was to develop Recommendations for Workplace Programs. The information gathered has also been used to propose future studies regarding HIV/AIDS and counterfeiting. In the developing world, antibiotics and anti-parasitic medicines are included among the counterfeiters’ favorite targets. Strong parallels exist between locations where counterfeiting of medicines is taking place/product being distributed/sold and where HIV/AIDS is most prevalent and/or where the epidemic is expanding progressively. Counterfeiting of medicines used for treating HIV/AIDS raises the possibility of additional future complications developing in managing other global diseases such as Malaria and Tuberculosis, not to mention exacerbating the potential for developing resistance and encouraging mutation of the HI virus itself. It is also noteworthy that certain medical devices have also been found to be counterfeit. Global demographics and with particular reference to projected growth rates of populations of the developing world are of specific relevance to this subject of anticounterfeiting and medicines used for the treatment of HIV and AIDS. Indeed, next generations of humanity appear to be at unnecessary risk of being caught up in a confluence of forces whereby the practice of the counterfeiting of medicines could result in significant complications and unforeseen consequences regarding management of the global HIV/AIDS crisis. Following the research, recommendations for workplace programs were developed. The research study concludes with a comprehensive set of references. / AFRIKAANSE OPSOMMING: Die problamatiek aangaande die vervalsing (namaak) van medisyne word nou wereldwyd ervaar en het ‘n impak op beide die geindustrialiseerde en die ontwikkelende wereld. Menige medisyne in terapeutiese kategoriee is tot op hede as vervals geidentifeseer, met die direkte resultaat dat hulle ‘n minemale of geen terapeutiese uitwerking het nie. Wat nog erger is, is dat hierdie middels uiters gevaarlik is om te gebruik en selfs lewensgevaarlik kan wees. Dit is van groot betekenis dat ook medisyne wat bestem is om persone met HIV/VIGS te behandel, as vervals aangetoon is – en soedoende tot nog toe onbekende gevolge vir pasiente, die werkomgewing en ongekende risiko’s vir wereldwye gesondheidsorg en internasionale veiligheid en sekuriteit inhou. In hierdie studie word die onderwerp in taamlike besonderhede bestudeer en daar word afgesluit met aanbevelings oor programme in die werkplek wat ontwerp is om sorg en ondersteuning te bied aan werkers met HIV/VIGS. Verdere studie word ook aanbeveel om die tergende probleme wat volg op die vervalsing van medisyne in die behandling van persone met HIV/VIGS, en die implikasies hiervan, die hoof te bide.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2014. / Determining e ective treatment strategies for life-threatening illnesses such as HIV is a signi cant problem in clinical research. Currently, HIV treatment involves using combinations of anti-HIV drugs to inhibit the formation of drug-resistant strains. From a clinician's perspective, this usually requires careful selection of drugs on the basis of an individual's immune responses at a particular time. As the number of drugs available for treatment increases, this task becomes di cult. In a clinical trial setting, the task is even more challenging since experience using new drugs is limited. For these reasons, this research examines whether machine learning techniques, and more speci cally batch reinforcement learning, can be used for the purposes of determining the appropriate treatment for an HIV-infected patient at a particular time. To do so, we consider using tted Q-iteration with extremely randomized trees, neural tted Q-iteration and least squares policy iteration. The use of batch reinforcement learning means that samples of patient data are captured prior to learning to avoid imposing risks on a patient. Because samples are re-used, these methods are data-e cient and particularly suited to situations where large amounts of data are unavailable. We apply each of these learning methods to both numerically generated and real data sets. Results from this research highlight the advantages and disadvantages associated with each learning technique. Real data testing has revealed that these batch reinforcement learning techniques have the ability to suggest treatments that are reasonably consistent with those prescribed by clinicians. The inclusion of additional state variables describing more about an individual's health could further improve this learning process. Ultimately, the use of such reinforcement learning methods could be coupled with a clinician's knowledge for enhanced treatment design.