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In this study, two plant species, Schefflera umbellifera (Araliaceae) and / Thesis (M.Sc.) - University of KwaZulu-Natal, Pietermaritzburg, 2007.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Having caused the deaths of more than 10 million individuals since 2000 with most of them occurring in Africa, malaria remains a serious disease that requires undivided attention. To this end a detailed kinetic model of Plasmodium falciparum glycolysis was constructed, validated and used to determine potential drug targets for the development of novel, effective antimalarial therapies. The kinetic model described the behaviour of the glycolytic enzymes with a set of ordinary differential equations that was solved to obtain the steady state fluxes and concentrations of internal metabolites. The model included a glycerol branch represented in a single fitted equation. This present study set out to detect, characterise, and incorporate into the model the enzymes that constitute the glycerol branch of P. falciparum glycolysis. The kinetic parameters of glycerol 3-phosphate dehydrogenase (G3PDH), the first enzyme in the branch and catalyst of the dihydroxyacetone phosphosate (DHAP) reducing reaction, was determined and added to the detailed kinetic model. The model was subsequently validated by comparing its prediction of steady state fluxes with experimentally measured fluxes. Once it was evident that the predictions of the unfitted model agreed with experimentally measured fluxes, metabolic control analysis was performed on this branched system to ascertain the distribution of control over the steady state flux through the glycerol branch. The control G3PDH exercised over its own flux was less than expected due to the enzyme’s sensitivity to changes in NADH and thus the redox balance of the cell. Attempts were made to detect the enzymes responsible for the conversion of glycerol 3-phosphate (G3P) to glycerol. Very low levels of glycerol kinase activity was observed. Although G3P-dependent release of inorganic phosphate was detected results were inconclusive as to whether a non-specific phosphatase also mediated the conversion. Overall, the expansion of the model to include G3PDH did not affect the steady state metabolite concentrations and flux adversely. / AFRIKAANSE OPSOMMING: Vanaf die jaar 2000 het malaria die dood van meer as 10 miljoen mense veroorsaak. Die meeste sterftes het in Afrika voorgekom —’n aanduiding van hoe ernstige siekte dit is en een wat onverdeelde aandag moet geniet. Om hierdie rede is ’n gedetaileerde kinetiese model van glikoliese in Plasmodium falciparum gebou, gevalideer en gebruik om potensiële dwelm teikens te identifiseer vir die ontwikkeling van nuwe, meer effektiewe anti-malaria terapieë. Die kinetiese model beskryf die gedrag van die glikolitiese ensieme in terme van gewone differensiële vergelykings wat opgelos is om die bestendige toestand fluksies en interne metaboliet konsentrasies te bepaal. Die model sluit ’n gliserol-tak in wat deur ’n enkele aangepaste vergelyking verteenwoordig word. Hierdie studie het voorgeneem om die ensieme van die gliserol-tak van P. falciparum glikoliese te identifiseer, karakteriseer en in die model te inkorporeer. Ons het die kinetiese parameters van die eerste ensiem in die gliserol-tak, gliserol 3-fosfaat dehidrogenase (G3PDH), die katalis van die dihidroksiasetoon fosfaat(DHAP) reduserende reaksie, bepaal. Die kinetiese parameters is by die gedetaileerde model gevoeg. Validering het plaasgevind deur die model se voorspellings met eksperimenteel bepaalde waardes te vergelyk. Toe dit duidelik geword het dat die voorspellings van die model met die eksperimenteel bepaalde fluks ooreenstem, is metaboliese kontrole analiese op die vertakte sisteem uitgevoer. Dit is gedoen om vas te stel hoe die bestendige toestand fluks deur die gliserol-tak beheer word. G3PDH het nie volle beheer oor sy eie fluks nie, in teenstelling met ons vergewagtinge. Daar is gepoog om vas te stel watter ensieme verantwoordelik is vir die produksie van gliserol vanuit gliserol 3-fosfaat (G3P). ’n Lae gliserolkinase aktiwiteit is waargeneem. Alhoewel G3P afhanklike vrystelling van anorganise fosfaat waargeneem is, is dit nie duidelik vanuit die resultate of die proses deur ’n nie-spesifieke fosfatase uitgevoer word nie. Die uitbreiding van die model om ’n G3PDH vergelyking in te sluit het nie die bestendige toestand metaboliet konsentrasies en fluks negatief geaffekteer nie.

Three protein targets are used in malaria rapid diagnostic tests (RDTs). These are Plasmodium falciparum histidine rich protein 2, Plasmodium lactate dehydrogenase and aldolase. A thrust of research in RDTs is to improve on their specificity and sensitivity. In this study the current diagnostic target, P. falciparum lactate dehydrogenase (PƒLDH) was compared to a new target glyceraldehyde-3-phosphate dehydrogenase (PƒGAPDH) that was identified based on transcriptional data. These proteins are conserved amongst all Plasmodium species, with minor amino acid sequence variations which were evaluated as possible species-specific peptide epitopes for PƒLDH: LISDAELEAIFDRC and PƒGAPDH: CADGFLLIGEKKVSVFA; CAEKDPSQIPWGKCQV, where common peptides were identified as pan-malarial epitopes for pLDH: APGKSDKEWNRDDLC and pGAPDH: CKDDTPIYVMGINH. The chosen peptides were located on the surface of their predicted 3D crystal structure models. Antibodies were raised against these peptides in chickens (IgY) and affinity purified. PƒLDH and PƒGAPDH were recombinantly expressed in E. coli BL21(DE3) cells and their coding inserts confirmed by sequencing. The recombinant proteins were detected in Western blots with specific anti-His₆ tag antibodies at approximately 35 kD (PƒLDH ~ 36 kD and PƒGAPDH ~ 39 kD) which compared with their expected values. Both recombinant proteins were found to form tetramers in solution and were used to raise IgY antibodies for comparison of Pheroids™ and Freund’s adjuvants. Pheroids™, like Freund’s appeared to exhibit a depot effect, however Freund’s adjuvant gave higher affinity purified IgY yields. The anti-recombinant and anti-peptide IgY specifically detected their respective recombinant and native antigens and did not cross-react with other human blood proteins. Immunoprecipitation detected higher levels of PƒGAPDH to PƒLDH in P. falciparum culture lysates. A double antibody sandwich ELISA detected 17.3 ng/ml PƒLDH and 138.5 ng/ml PƒGAPDH at 1% parasitemia in in vitro cultures, however this needs to be further evaluated. These findings suggest PƒGAPDH to be at least as good a protein target as PƒLDH for malaria diagnosis and further trials using it as a target in an RDT format should be considered. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

This dissertation describes the development and application of a new mathematical model for simulating the progression of Plasmodium falciparum infections in individuals with no malarial acquired immunity. The model allows for stochastic simulation of asexual and sexual parasitemias as well as the onset of fever and human to mosquito infectivity on a daily time scale. The model components for the asexual and sexual stages were developed elsewhere but are here extended to allow for simulation of the full range of dynamics observed in a subset of malaria therapy patients. As a first application of the model, I calculate the human component of malarial R0, the basic reproductive number. I then compare this value to those from three other models and describe how this quantity can be used to model malaria transmission. The second application of the model incorporates the effects of drug treatment on progression of infection by utilizing modeled pharmacokinetic and pharmacodynamic properties of a variety of antimalarials. I utilize a stage specific proportional killing model for sexual stages, informed from recent in vitro data. The relationship of effect sizes to treatment coverage and type of treatment in both early and late treatment seeking settings is calculated. In the third chapter, I consider the economic and epidemiological ramifications of antimalarial and rapid diagnostic subsidization for malaria control. For the epidemiological modeling I utilize a semi-mechanistic model of the spread of drug resistance parameterized from historical malaria mortality data; for the economic model I consider the effect of rapid diagnostics on the intensive and extensive margins of antibiotics and antimalarials, as well as the benefits to improved targeting of both. I find that rapid diagnostic testing is justified given our baseline assumptions for areas with low proportions of malarious individuals among all treatment-seekers, but that caution is necessary before deployment worldwide. For antimalarial subsidization, we find that this is a cost-effective method for reducing mortality in developing countries, though efforts to delay the onset and slow the spread of resistance are urgently needed.

Drug resistance is a serious problem in health care in general, and in malaria treatment in particular, rendering many of our previously considered ‘wonder drugs’ useless. Recently, large sums of money have been allocated for the continuous development of new drugs to replace the failing ones. We seem to be one step behind the evolution of antimalarial resistance; is it possible to get one step ahead? Are interventions which slow down the evolution and spread of drug-resistant malaria parasites achievable? In this thesis, I address these issues with experimental data, using the well-established rodent malaria model Plasmodium chabaudi to understand the selective advantages and disadvantages drug-resistant parasites endure within a vertebrate host and the selective pressures various drug treatment regimes exert on these parasites. Competitive interactions between drug-resistant and drug-sensitive parasites were observed within the host, with resistant parasites having a competitive disadvantage in the absence of drug treatment. The frequency of resistant parasites at the start of the infection was an important determinant of the strength of selection: the lower their frequency, the stronger the competitive suppression in non-treated hosts and the greater their competitive release following drug treatment. Genetically similar genotypes, one resistant and one sensitive, showed similar dynamics following drug treatment. Multiplicity of infection did not have an effect on the within-host dynamics: a larger number of co-infecting susceptible genotypes did not lead to greater competitive suppression or release of resistant parasites. Lastly, various drug treatment regimes were compared. Conventional drug treatment resulted in the greatest selective advantage for drug-resistant parasites, while less aggressive treatments were equally as effective, or even better, at improving host health and reducing overall infectiousness. These studies demonstrate that altering the within-host ecology of drug-resistant parasites by administering drugs and hence removing the drug-sensitive competitors has a large influence on the transmission potential of drug-resistant parasites. Furthermore, this thesis provides proof of principle that other drug treatment regimes different from those currently in use could better control drug-resistant parasites, without compromising other treatment goals. In the case of malaria, less drugs may mean extending the useful lifespan of that drug.

Malaria disease poses a serious global health burden as recent reports have indicated that nearly half of the world’s population is at risk (WHO 2008). The World Health Organization (WHO) Expert Consultative Team has reported that 90% of all malaria deaths occur in Sub Saharan Africa. (WHO, 2008). Despite the numerous global efforts to control and manage the disease, through various ways, use of chemotherapeutic agents continues to be the major intervention strategy in the control of malaria. The WHO recommended use of Artermisinin combination therapy (ACT) has been hampered by an imbalance between demand and supply in the poor socioeconomically challenged rural populations of Sub Saharan Africa, the epicenter of malaria infection. Chloroquine (CHQ), therefore, continues to be used in most malaria endemic areas in developing countries despite development of P. falciparum resistance to the drug (WHO, 2006). Oral administration is the major delivery route for CHQ. However, CHQ is a bitter drug, with an inconvenient dosing schedule leading to incomplete courses of therapy by most malaria patients. Oral CHQ administration is also associated with adverse effects in various organ systems resulting from deposition of CHQ in these organs to elicit impairment of glucose homeostasis, renal and cardiovascular function. Alternative methods of CHQ administration such as transdermal delivery have, therefore, been suggested, in an effort not only to avoid the bitter taste, but also to modify the dosing schedule, which may improve patient comfort and compliance. Transdermal delivery of CHQ via an amidated matrix patch, which is envisaged to ensure a slow, controlled and sustained release of therapeutic concentrations of CHQ, may circumvent the previously reported adverse effects of oral CHQ. It is against this background that the current study compared the effects of transdermal CHQ patch and oral chloroquine in the management of malaria as assessed by the ability to clear parasites of P. berghei infected rats. The other aims were to investigate and distinguish between the patho physiological effects of malaria and CHQ treatments on blood glucose and plasma insulin concentration, renal and cardiovascular function in male Sprague-Dawley rats. To investigate and distinguish between the pathophysiological effects malaria infection and CHQ treatments on blood glucose homeostasis, renal and cardiovascular function markers, separate groups of non infected and P. berghei infected male Sprague Dawley rats (90g-150g) were used. The animals were treated twice daily with oral CHQ (60mg/kg) and a once off transdermal delivery of CHQ via topical application of pectin CHQ matrix patch (53mg/kg) in a 21 day study divided into pre treatment, (days 0-7) treatment (days 8-12) and post treatment (days 13-21) periods. The animals were housed individually in metabolic cages for the duration of the study. Treatment was for 5 consecutive days. Measurements of body weight, food and water intake, mean arterial pressure (MAP), blood glucose concentration, % parasitaemia, haematocrit, and 24 hour urine volume, Na+, K+, urea and creatinine outputs were done every day during the treatment period, and every third day during the pre and post treatment periods. Separate groups of non fasted conscious animals (n=6) were sacrificed on days 0, 7, 8, 9, 10, 12, 14 and 21, at 24 hours after the last treatment for oral CHQ administration and after a once off patch application on the first day of treatment. The plasma obtained was assayed for plasma insulin, lipid profile parameters and plasma Na+, K+, urea and creatinine. The harvested liver and gastrocnemius muscle were used for determination of glycogen concentration. The current study has demonstrated the sustained controlled release of CHQ from the pectin matrix patch, demonstrating the therapeutic ability to clear P. berghei malaria parasites from systemic circulation. Malaria infection and oral CHQ treatment exhibited blood glucose lowering effects which were circumvented by topical application of the pectin CHQ matrix patch. Oral CHQ elevated hepatic glycogen concentration through mechanisms that are still to be elucidated. Topical application of CHQ via pectin matrix patch did not alter hepatic and gastrocnemius muscle glycogen concentrations. Malaria infection and oral CHQ delivery reduced food intake, water intake and % body weight changes of the animals as well as inducing natriuresis, reduced urine output and increased urinary creatinine outputs. Malaria infection was also shown to elicit hyperkalaemia and kaliuresis in experimental animals. Hypotensive effects of malaria infection and oral CHQ delivery were also demonstrated in the current study. Malaria infection and oral CHQ delivery elevated plasma total cholesterol and LDL-c as well as reduction in the cardio protective particle, plasma HDL-c, concentrations. Topically delivered CHQ via pectin CHQ matrix patch did not evoke any such alterations, suggestive of its ability to circumvent the observed adverse effects of oral CHQ delivery due to sustained, controlled release of therapeutic concentrations of CHQ from the transdermal formulation. To the best of our knowledge, the results of the present study provides the first evidence of the release of therapeautic CHQ concentrations from pectin CHQ matrix patch that cleared the malaria parasites from systemic circulation as well as demonstrating the ability of the transdermal formulation to circumvent the adverse effects of oral CHQ delivery in glucose homeostasis, renal and cardiovascular function markers. This is clinically relevant as it provides a feasible and novel alternative method of CHQ delivery that could play a major role in the effective management of malaria. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.

Effective case management of malaria is hampered by the spread of parasite resistance to nonartemisinin antimalarials. To counteract the impact of drug resistance, the World Health Organization (WHO) has endorsed artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated Plasmodium falciparum malaria. Currently recommended ACTs are artemether-lumefantrine, artesunate plus amodiaquine, artesunate plus mefloquine, artesunate plus sulfadoxine-pyrimethamine and dihydroartemisinin-piperaquine. This study sought to review evidence of the efficacy and safety of different non-artemisinin antimalarials in combination with artesunate, artemether or dihydroartemisinin for the treatment of uncomplicated P. falciparum malaria in non-pregnant adults and children. The search for randomized controlled trials (RCTs) was conducted in the Cochrane Central Register for Controlled Trials (CENTRAL), MEDLINE, EMBASE and in ClinicalTrials.gov in January 2009. The eligibility and the methodological quality of trials were assessed and data were extracted, using standard forms. Data were captured and analyzed in Review Manager Software, versions 4.2 and 5.0. The outcomes assessed were: treatment failure, fever and parasite clearance time, calculating the relative risk (RR) and a weighted mean difference (WMD) with a 95% confidence interval and p-values, indicating statistical significance at 0.05. Thirty-seven trials with 6862 participants were included. Artesunate combined with amodiaquine had a statistically significant lower risk of treatment failure compared to the combination of artesunate with sulfadoxine-pyrimethamine (RR=0.57, 95% CI [0.33, 0.97], p=0.04, seven trials, N=1341). In addition, treatment with artesunate plus mefloquine was significantly associated with a lower risk of treatment failure compared to artesunate plus azithromycin (RR=0.04, 95% CI [0.00, 0.64], p=0.02, one trial, N=54). There was no significant difference when either mefloquine or atovaquone-proguanil were combination partners with artesunate (RR=2.6, 95% CI [0.93; 7.24], p=0.07, one trial, N=1066). When artesunate was combined with chloroquine, primaquine or azithromycin and compared with artesunate monotherapy, there was no statistically significant difference in the risk of unadjusted treatment failure. Each of these comparisons had one trial each. Artesunate plus chloroquine was quicker at clearing fever compared to artesunate plus sulfadoxinepyrimethamine (WMD= -7.20, 95% CI [-12.53, -1.87], one trial, N=132). Few trials adequately reported adverse events. There was no significant difference observed in the risk of adverse events between artesunate plus amodiaquine compared with artesunate monotherapy, however, adverse events were significantly less in artesunate plus amodiaquine compared to artesunate plus methylene-blue. Artesunate plus amodiaquine on the other hand had significantly more adverse events reported compared to artesunate plus sulfadoxine-pyrimethamine. The findings of this study support the implementation of artemisinin-based combination therapy for the treatment of uncomplicated malaria. Most crucially, this review found a greater advantage of combining amodiaquine with artesunate compared to sulfadoxine-pyrimethamine. The efficacy of artesunate plus mefloquine was superior to that of artesunate plus azithromycin. Furthermore, the combination of artemisinins with chloroquine, primaquine and azithromycin has shown very low efficacy and these combination therapies should not be recommended. The reporting of efficacy was not standardized as many trials did not differentiate between re-infections and recrudescences. Adverse events were also not adequately reported. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Malaria continues to be a major cause of mortality and morbidity especially in Sub-Saharan Africa. The emergence and spread of drug resistant parasites has highlighted the need for new chemically diverse, effective drugs. Historically, one of the major sources of antimalarial agents and novel template compounds has been higher order plants. The widespread use of medicinal plants for the treatment of malaria in South Africa represents a diverse resource of potential antimalarial drugs. Two South African plants, Vernonia staehelinoides and Oncosiphon piluliferum, were identified as potential sources of new antimalarial drugs through a national multidisciplinary-consortium project aimed at scientifically validating South African medicinal plants for the treatment of malaria. The in vitro antiplasmodial activity of extracts of these plants warranted further investigation to identify the biologically active components. Bio-assay guided fractionation based on in vitro antiplasmodial activity against the D10 P. falciparum strain was used to identify the compounds responsible for the observed activity. Compounds were purified using silica gel column chromatography. The structures of the isolated compounds were elucidated using spectroscopic techniques. Bioassay-guided fractionation of the organic extracts of V. staehelinoides leaves identified a pair of structurally-related hirsutinolides with significant in vitro antiplasmodial activity. The compounds were found to be cytotoxic at similar concentrations but proved to be interesting scaffolds for potential structure-activity relationship studies. Three germacranolides and two eudesmanolides were identified through bioassay-guided fractionation of the organic O. piluliferum extract. Selected derivatizations were conducted in order to fully characterize the compounds. The absolute configuration of the major active germacranolide was determined using Mosher's method. The effect of the reduction of the <font face="symbol"> a</font>-methylene group of the major active germacranolide on antiplasmodial activity and cytotoxicity was also investigated. The 5 compounds and the reduction product were found to possess varying degrees of in vitro antiplasmodial activity and cytotoxicity. None was sufficiently active or selective to be a viable drug candidate but the potential for further structure-activity relationship studies exists. / Dissertation (MSc (Chemistry))--University of Pretoria, 2007. / Chemistry / unrestricted

Building supply chain coordination frameworks is a popular practice in the private sector in many developed countries. Despite this fact, in developing countries such as Uganda, the public health sector has hardly adopted this practice. Although the existing frameworks offer a good platform for measuring and improving the understanding of concepts underlying coordination dimensions at the micro-environment, they have limited capacity to analyse coordination interactions within the health sector, especially in developing countries like Uganda. Using the business management environment framework, this study explored the critical supply chain coordination dimensions, logistics activities dimensions and the management environment (market and macro) dimensions affecting the availability of Artemisinin-based Combination Therapies for malaria (ACTs). The overall main research goal of this thesis was to develop a supply chain coordination framework for malaria treatment therapies (ACTs) in general hospitals in Uganda. To understand the coordination dimensions of ACTs, the study adopted an exploratory sequential mixed research design, which involved a mixture of qualitative and quantitative approaches. For the qualitative phase, four focus group discussions were held. From the results, an instrument was developed and later validated using the quantitative approach. Specifically, Exploratory Factor Analysis (EFA) with a maximum likelihood extraction method followed by Confirmatory Factor Analysis (CFA) were used to analyse quantitative data. Considering the volume of the dimensions, Analytical Process Hierarchy (AHP) was carried out to rank the dimensions in order of priority. Analysis of the factor correlation matrix shows no common variance among the components; therefore, the principal components were distinct from one another and there was no discriminant validity. The CFA results showed that the standardised parameter estimates of the initial measurement models were all significant (p<.05). CFA and APH outputs were somehow different simply because each technique has its own purpose and principles. It was indicated that the correlation between critical supply chain coordination dimensions and level of ACTs availability is moderately higher, followed by logistics, macro and market environments. By better understanding the supply coordination dimensions effects on ACTs in Uganda, the research provides important direction to African governments and international donor agencies in their efforts to make malaria treatment therapies available, especially to the rural poor and avert death. The findings serve as a platform to argue for revisiting coordination dimensions in view of conditions that include a resurgent market and macro-environment in developing countries. The insight raises implications for extending coordination frameworks that are geographically focused, and specific to ACTs. It may influence policy direction in this regard and thus contribute to the body of knowledge. / Business Management / D. Phil. (Management Studies)