MSc (Mathematics) / Department of Mathematics and Applied Mathematics

Thesis (M. Tech.) - Central University of Technology, Free State, 2007 / Malaria and the mosquito that induces the disease in humans have hounded the military for decades. Malaria represents one of the most important infectious disease threats to deployed military forces. Malaria in soldiers has a serious economic impact, both in terms of lost productivity and treatment cost for the state. A contingent of South African National Defence Force members has been deployed in Burundi since November 2001, as part of a peacekeeping mission. At the time of the study no information was available regarding the prevalence of malaria among military personnel during deployments in Burundi and East Africa. In Africa, the saying is that malaria is the disease of poverty and a cause of poverty. To combat malaria, it is of vital importance that the recommended medication be taken exactly as prescribed and that the course is completed. However, one of the greatest challenges facing the African continent in the present fight against malaria is drug resistance. The discovery of Mefloquine and the subsequent development of suitable drugs, have been intimately associated with military imperatives, contingencies and requirements. Since World War II, the development of Chloroquine-resistant falciparum malaria has driven the search for new drugs. Mefloquine, developed by the Walter Reed Army Institute of Research in the United States, was first shown effective as a prophylaxis and treatment of resistant falciparum malaria in the 1970’s. To obtain data, questionnaires were administered to SANDF soldiers deployed in Burundi, East Africa. The total size of the population under investigation was 336 with a final sample size of 111 respondents. The sample was selected by using simple random sampling. The questionnaire aimed to determine the perception of respondents regarding the malaria threat, their compliance with taking the medication, and their experiencing of possible side-effects which might occur due to the chemoprophylaxis and the prophylactic efficacy of Mefliam®. The fact that, of the 111 people who used Mefliam®, only four presented with any malaria symptoms, is an indicator that Mefliam® is an effective option as an antimalarial drug to be used in East Africa and Burundi. The results of this study will be used by the personnel of the South African National Defence Force (SANDF) and other military forces deployed in East Africa. It is envisaged that the results will be used by military policy- and decision-makers as a control programme and by others involved in the control of malaria. The findings and recommendations should also be of interest to anyone visiting the area.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: The African penguin, which occurs along the south-eastern and south-western shores of South-Africa and Namibia, has experienced a severe reduction in population numbers due to guano and egg collection in the first half of the 19th century, and oil pollution in the second half of the 19th century as a result of oil tankers rounding the Cape of Good Hope. The population would have been reduced by a further 19% had it not been for the rehabilitation of penguins at the South African National Council for the Conservation of Coastal Birds (SANCCOB) facility. Although this has been very successful, mortalities as a result of avian malaria infection have considerably reduced the efficiency of rehabilitation. In an effort to assess the role of immunity against malaria in combating the disease, an enzyme-linked immunosorbent assay (ELISA) for the detection of antibody levels to avian malaria was developed. The ELISA was used to detect antibody levels to avian malaria of penguins on entry and during rehabilitation from October 2001 to January 2003. The aim of this study was to continue the determination of antibody levels to avian malaria of penguins entering the SANCCOB facility, in order to allow an evaluation of the antibody levels to avian malaria for two full calendar years. This investigation was combined with a polymerase chain reaction (PCR)-based method, capable of detecting any Plasmodium species in penguin serum. These two methods were also used to investigate avian malaria in several breeding colonies in order to assess the role avian malaria may play in the survival of the African penguin in the wild. Results indicated that the ability of penguins to produce anti-Plasmodium antibodies was not influenced by oiling and that infection with malaria was not due to recrudescence but rather due to infection via mosquitoes. This indicated a possible role of the SANCCOB facility in exposing the penguins to avian malaria. However a large number of penguins arrived at the facility previously infected with malaria, indicating that malaria was present in the breeding colonies. Investigations in the breeding colonies revealed extremely high avian malaria prevalence even though no sick birds or mortalities were observed. This raised the question whether different types of malaria are responsible for infection in the SANCCOB facility and breeding colonies. / AFRIKAANSE OPSOMMING: Die Afrika Pikkewyn kom langs die suid-oostelike en suid-westelike kus van Suid Afrika en Namibië voor. In die afgelope eeu het hierdie spesie ‘n geweldige afname in populasie getalle ondervind. Dit was hoofsaaklik die gevolg van die versameling van guano en pikkewyneiers in die eerste helfte van die 19de eeu en oliebesoedeling in die tweede helfde van die 19de eeu. Die “South African Foundation for Conservation of Coastal Birds” (SANCCOB) is ‘n seevoëlreddings- en rehabilitasiesentrum vir siek, beseerde en ge-oliede pikkewyne. Dit word geskat dat die Afrika Pikkewyn populasie met ‘n verdere 19% sou afgeneem het as dit nie vir die rehabilitasie by die SANCCOB sentrum was nie. Hierdie sentrum het egter aansienlike vrektes in die somer as gevolg van voëlmalaria, wat sodoende die effektiwiteit van die rehabilitasie verlaag. In ‘n poging om die rol van immuniteit teen malaria te bepaal is ‘n “enzyme-linked immunosorbent assay” (ELISA) ontwikkel vir die bepaling van antiliggaam vlakke teen malaria. Hierdie ELISA is gebruik vir die bepaling van die anti-Plasmodium antiliggaam vlakke van die pikkewyne by aankoms en ten tye van rehabilitasie by SANCCOB vanaf Oktober 2001 to Januarie 2003. Die doel van hierdie studie was eerstens om hierdie ELISA bepalings voort te sit om sodoende antiliggaam vlakke teen malaria oor twee kalender jare te kan evalueer. Hierdie ondersoek was gekombineer met ‘n polimerase ketting reaksie (PCR) metode, wat enige Plasmodium spesie in pikkewynserum sou kon opspoor. Hierdie twee metodes is ook gebruik vir ondersoeke in sommige broeikolonies, met die doel om te bepaal watter rol voëlmalaria in die oorlewing van die Afrika pikkewyn in die natuur speel. Resultate het getoon dat olie nie die vermoë van die pikkewyn beïnvloed om anti- Plasmodium antiliggame te vervaardig nie en dat malaria infeksie hoofsaaklik deur muskiete veroosaak word en nie deur heruitbraak van ‘n bestaande infeksie nie. Dit dui egter daarop dat pikkewyne blootgestel word aan voëlmalaria by die SANCCOB sentrum. Daar is ook gevind dat ‘n groot aantal pikkewyne met malaria infeksies by die sentrum opgedaag het wat dui op die voorkoms van malaria in die broeikolonies. Ondersoeke in die broeikolonies het ‘n besonder hoë voorkoms van malaria onthul. Geen vrektes of siek pikkewyne is in die broeikolonies waargeneem nie, wat moontlik kan beteken dat pikkewyne by SANCCOB met ‘n ander tipe malaria geïnfekteer word as in die broeikolonies.

A DISSERTATION SUBMITTED TO THE FACULTY OF MEDICINE, UNIVERSITY OF THE WITWATERSRAND, JOHANNESBURG, FR THE DEGREE OF MASTER OF SCIENCE IN MEDICINE. / The influence of iron on the outcome of various infections have been extensively reviewed. Clinical observations suggests that iron deficiency may be protective against malaria. Various researchers have shown that certain iron chelators blocked the proliferation of plasmodium falciparum in vitro and in vivo. (Abbreviation abstract) / Andrew Chakane 2018

A thesis submitted to the Faculty of Science University of the Witwatersrand Johannesburg in fulfillment for the requirements of the degree of Master of Science. 14 May 2015. / Peptidomimetic compounds have been shown to exhibit both anti-HIV and anti-malarial activity. A multicomponent reaction was used to create a library of peptidomimetic compounds with an α-hydroxy-β-amino acid unit. The Passerini reaction between an aldehyde, carboxylic acid and isocyanide was used to prepare compounds containing both ester and amide functionalities. These compounds were then subjected to a deprotection-acyl migration strategy giving rise to the target compounds. This approach, known as the Passerini Amine Deprotection Acyl Migration (PADAM) sequence was successfully used to create a library of novel peptidomimetic compounds. From this library, 22 compounds were tested for activity against HIV and malaria. The Passerini reaction gives rise to a product containing a new stereogenic centre, and as the starting aldehyde used (N-Boc-phenylalaninal) has a stereogenic centre, the products were isolated as a mixture of diastereomers. Our research was also focused on finding ways of influencing the stereoselectivity of the reaction and the separation of the resulting diastereomers. The diastereomeric ratio of the Passerini products was found to be approximately 2:1 for all the reactions performed. This ratio could be modified slightly when using certain carboxylic acids and isocyanides that were either very bulky or had a stereogenic centre. Attempts to enzymatically resolve the diastereomeric products were not successful after trials using a library of 25 lipase enzymes. However, use of preparative HPLC enabled the successful separation of most of the diastereomeric mixtures, affording compounds with high purity. X-ray crystallography enabled us to identify the major diastereomers as having the R,S configuration, whilst the minor diastereomers had the S,S configuration at the two stereogenic centres. A possible explanation for the observed stereoselectivity is based on the Felkin-Anh chelation control model. It suggests that mono-protected amino aldehydes follow a chelation controlled mechanism in nucleophilic addition reactions. Chelation occurs, albeit in the form of hydrogen bonding, between the NH and carbonyl oxygen. The library of compounds was tested for activity against both HIV-1 and malaria. Only three compounds showed moderate activity against the malaria parasite, inhibiting parasitic growth by 37-42% at 5 μM respectively. Significantly, all of the active compounds contained an adamantyl moiety. Unfortunately no anti-HIV activity was seen for any of the compounds tested in the HIV-assay.

The body of work presented here seeks to illuminate the complex relationship between human society, development, and environment for the case of malaria. While malaria profoundly affects human society and prospects for prosperity, public health measures and anthropogenic environmental change alter the intensity of transmission differentially around the globe. Using global maps of malaria risk, the first chapter finds that the elimination of the disease during the course of the 20th century occurred in places where the strength of transmission was weaker due to suboptimal ecology, and that this result holds even after controlling for income levels. The next chapter employs GIS datasets on population, urbanization, malaria risk, and malaria endemicity to spatially estimate the cost of fully deploying ecology-appropriate anti-malaria interventions in Africa; the cost of curbing malaria is found to be small (around $4 per person at risk per year), especially given its high disease burden and subsequent social and economic costs. I next construct a spatial month-to-month ecological index of malaria transmission strength, and use a climate change model to predict changes in ecological transmission strength of malaria and estimate the implied changes in incidence and mortality given current technology and public health efforts. The final chapter uses the malaria ecology index as an instrumental variable to estimate the effect of child mortality on fertility behavior. The large effect of child mortality indicates that malaria has an indirect effect on society beyond morbidity and mortality: high malaria burdens increase fertility rates, thus slowing the demographic transition. These chapters span the fields of epidemiology, public health systems, climate science, economics and demography in order to holistically model the relationship between malaria and human systems; such understanding of coupled human-natural systems will be vital to policy making for sustainable development.

Tourism is the world's largest earner of foreign currency. It brings an estimated R20 billion a year into the South African economy, second only to the manufacturing and mining industry in its contribution to the Gross Domestic Product (GDP). An estimated 1.7 million overseas and African tourists visited South Africa in 1999. Of the 1.7 million approximately 500 000 or 30% of these tourists visited KwazuluNatal. Forty seven percent of the foreign tourists visited the Zululand and Maputaland area, which falls within a malaria transmission zone. An estimated 8 million domestic tourists from outside or within this province travelled to one or more destinations within KwaZulu-Natal on an annual basis. The Lubombo Spatial Development Initiative is a tri-Iateral initiative between the governments of Swaziland, Mozambique and South Africa to develop the Lubombo region into a globally competitive economic zone. The geographical area targeted by this initiative is broadly defined as eastem Swaziland, southem Mozambique and north-eastem KwaZulu-Natal. Accelerated development with regards to agriculture and tourism is the main objective of the Lubombo Spatial Development Initiative (LSD!). The Lubombo corridor has the potential to develop into an intemational tourist destination but malaria is hampering the growth and development of the region. Perceived malaria risk by tourists is believed to be an important factor that has a negative influence on the tourism industry in the study area. The risk factor, as defined in this study, is the possibility of contracting malaria whilst visiting a tourism facility in the area. It is therefore essential to understand perceptions relating to malaria and malaria risk in the LSDI area. Malaria control plays a pivotal role in the Lubombo Spatial Development Initiative (LSD!). The objective of the malaria control component of the LSDI is to put in place a malaria control programme that will protect the economic interest of the Lubombo Spatial Development Initiative (LSD!) and stimulate development. Malaria control activities have been taking place in the three countries since 1999. Residual house spraying is the method used to control malaria in the Lubombo corridor. Major reductions in both malaria cases and parasite prevalence have been recorded. Swaziland's malaria incidence reduced by 64%, South Africa's malaria incidence plummeted by a staggering 76% and Mozambique saw a parasite prevalence reduction of40% in the first year of residual house spraying in 1999. This study focuses on the scientific study of malaria incidence and distribution as well as on both tourists and tourism operator's perceptions of malaria risk. It considers the factors that drive people's perceptions of risk and investigates how tourists and tourism operators respond to malaria risk. It draws conclusions about how malaria impacts on tourism in the LSDI and recommends how malaria control can play a positive role in tourism development in the area. / Thesis (M.Sc.)-University of Natal, Durban, 2003.

Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is to prevent and reverse protein misfolding. PfHsp70 is a heatinducible cytoplasm/nuclear localised Plasmodium falciparum Hsp70. PfHsp70 is thought to confer chaperone cytoprotection to P. falciparum during the development of malaria fever. The objective of this study was to examine the chaperone properties of PfHsp70 using a bioinformatics approach, coupled to in vivo and in vitro analysis. Structural motifs that qualify PfHsp70 as a typical Hsp70 chaperone were identified. Although PfHsp70 has a higher similarity to human Hsc70 than E. coli DnaK, in vivocomplementation assays showed that PfHsp70 was able to reverse the thermosensitivity of E. coli dnaK756 (a temperature sensitive strain whose DnaK is functionally compromised). Two residues (V401 and Q402) in the linker region of PfHsp70 that are critical for its in vivo function were identified. Constructs were generated that encoded the ATPase domain of PfHsp70 and the peptide binding domain of E. coli DnaK (to generate PfK chimera); and the ATPase domain of E. coli DnaK fused to the peptide binding domain of PfHsp70 (KPf). The two chimeras were tested for their ability to reverse the thermosensitivity of E. coli dnaK756 cells. Whilst KPf was able to reverse the thermosensitivity of the E. coli dnaK756 cells, PfK could not. Previously, PfHsp70 purification involved urea denaturation. Using a detergent, polyethylenimine (PEI), PfHsp70 was natively purified. Natively purified PfHsp70 had a basal ATPase activity approximately two times higher than the previously reported activity for the protein purified through urea denaturation. PfJ4, a type II Hsp40, could not stimulate the ATPase activity of PfHsp70 in vitro. Arch and hydrophobic pocket substitutions (A419Y, Y444A and V451F) were introduced in the PfHsp70 peptide binding domain. Similar substitutions were also introduced in the KPf chimera. PfHsp70-V451F (hydrophobic pocket mutant) had marginally compromised in vivo function. However, a similar mutation (V436F), introduced in KPf abrogated the in vivo function of this chimera. The arch and hydrophobic pocket derivatives of PfHsp70 exhibited marginally compromised in vivo function, whilst equivalent mutations in KPf did not affect its in vivo function. The ability of PfHsp70 and its arch/hydrophobic pocket mutants to suppress the heatinduced aggregation of malate dehydrogenase (MDH) in vitro was investigated. Whilst PfHsp70 arch mutants displayed marginal functional loss in vivo, data from in vitro studies revealed that their functional deficiencies were more severe. This is the first study in which an Hsp70 from a parasitic eukaryote was able to suppress the thermosensitivity of an E. coli DnaK mutant strain. Findings from the in vivo and in vitro assays conducted on PfHsp70 suggest that this protein plays a key role in the life-cycle of P. falciparum. Furthermore, this study raised insights that are pertinent to the current dogma on the Hsp70 mechanism of action.

Ph.D. / Background: Most recent data have shown a slight reduction in the incidence of malaria in Nigeria. However, cases of malaria in children younger than five years of age have continued to escalate amidst ‘simple’ and ‘effective’ treatment options. The realisation of the Millennium Development Goals (MDGs) – to halve the burden of malaria by 2015 – is becoming increasingly unrealistic in Nigeria following the alarming rates of malaria in children. Apart from the ecological and environmental factors, socio-cultural and behavioural factors might be responsible for the staggering cases of malaria in children in local communities in Nigeria. It was against this background that a sociological study of health care service utilisation was conducted among caregivers of children with malaria. The study investigated the perceived threat of malaria; how the local understanding of malaria affects the recognition of signs and symptoms, perceived aetiology, treatment-seeking patterns and the use of insecticide treated nets (ITNs). The socio-generational changes in the healthcare seeking behaviour between young and older mothers as well as differences in the patterns of health care service utilisation between rural and urban subjects were also interrogated. Method: This study adopted a qualitative research design using complementary methods. A total of 40 semi-structured interviews, 20 in-depth interviews and four focus group discussions (FGDs) were conducted with caregivers and health workers. The respondents included young and older parents between the ages of 25 and 80 years whose children or wards below the age of five had manifested malaria symptoms at one time or another. A purposive sampling procedure was used to select sample for the study. The study was conducted in two selected rural areas; Okanle and Fajeromi; and one urban centre; Ilorin, Kwara State of Nigeria. Findings: The research indicated that the perceived aetiology, symptoms and treatment of malaria in children were largely influenced by the socio-cultural patterns of the communities studied. The study found that the first line of treatment for children with malaria in the communities of study was usually home treatment using traditional herbal medicines. The use of modern health care facilities is usually seen as the last resort. The traditional beliefs about causes of malaria, affordability and trust in herbal medicines, on the one hand, were found to be responsible for the widespread use of herbal medicines in the treatment of malaria in children. On the other hand, poor service delivery, lack of money, attitudes of medical personnel, mixed feelings about the efficacy of modern medicines and lack of trust in the community health centres were some factors found to be responsible for delays in seeking modern health care services when children have malaria. More importantly, the decision to seek treatment from either traditional or modern sources was largely influenced by the network of informal social interaction and social support at household and community levels. In addition, the study also found some changes in the patterns of health care seeking behaviour of young and older caregivers but generally found no differences in the patterns of health care seeking behaviour between rural and urban participants. Finally, the study found that the majority of the respondents were not aware of the effectiveness of the ITNs. Consequently, there was a high dependence on the use of traditional preventive measures which included a local leaf known as “ewe-efon” translated as “mosquito leaf”. Apart from the perceived corruption and mismanagement at the level of distribution of the ITNs, lack of appropriate knowledge about the effectiveness of the ITNs was discovered to be responsible for the widespread non-acceptance of the ITN in the prevention of malaria in children.

Malaria poses the greatest threat of all parasites to human life. Current vaccines and efficacious drugs are available however their use is limited due to toxicity, emergence of drug resistance, and cost. The discovery of an alternative pathway of isoprenoid biosynthesis, the non-mevalonate pathway, within the malarial parasite has resulted in development of novel anti-malarial drugs. 1-Deoxy-D-xylulose-5-phosphate (DOXP) reductoisomerase, the second enzyme in this pathway, is responsible for the synthesis of 2-C-methyl-D-erythritol 4-phosphate (MEP) in an intramolecular rearrangement step followed by a reduction process involving NADPH as a hydrogen donor and divalent cations as co-factors. Fosmidomycin and FR900098 have been identified as inhibitors of DOXP reductoisomerase. However, they lack clinical efficacy. In this investigation recombinant DOXP reductoisomerase from Escherichia coli (EcDXR) and Plasmodium falciparum (pfDXR) were biochemically characterized as potential targets for inhibition. (His)6-EcDXR was successfully purified using nickel-chelate affinity chromatography with a specific activity of 1.77 μmoles/min/mg and Km value 282 μM. Utilizing multiple sequence alignment, previous structural data predictions and homology modeling approaches, critical active site amino acid residues were identified and their role in the catalytic activity investigated utilizing site-directed mutagenesis techniques. We have shown evidence that suggests that Trp212 and Met214 interact to maintain the active site architecture and hydrophobic interactions necessary for substrate binding, cofactor binding and enzyme activity. Replacement of Trp212 with Tyr, Phe, and Leu reduced specific activity relative to EcDXR. EcDXR(W212F) and EcDXR(W212Y) had an increased Km relative to EcDXR indicative of loss in affinity toward DOXP, whereas EcDXR(W212L) had a lower Km of ~8 μM indicative of increased affinity for DOXP. The W212L substitution possibly removed contacts necessary for full catalytic activity, but could be considered a non-disruptive substitution in that it maintained active site architecture sufficient for DOXP reductoisomerase activity. EcDXR(M214I) had 36-fold reduced enzyme activity relative to EcDXR, while its Km (~8 μM) was found to be lower than that of EcDXR. This suggested that the M214I substitution had maintained (perhaps improved) substrate and active site architecture, but may have perturbed interactions with NADPH. Rational drug design strategies and docking methods have been utilized in the development of furan derivatives as DOXP reductoisomerase inhibitors, and the synthesis of phosphorylated derivatives (5) and (6) has been achieved. Future inhibitor studies using these novel potential DOXP reductoisomerase inhibitors may lead to the development of effective anti-malarial drug candidates.