Department of Chemistry / MSc (Chemistry) / Due to the increasing incidence of Plasmodium strains that are resistant to current frontline antimalarial drugs, malaria remains a global public health challenge. In recent years, the emergence of resistance to frontline antimalarial drugs including the more recently discovered artemisinin class drugs has become one of the greatest challenges of controlling malaria incidence and mortality. There is, therefore, an urgent need to develop novel targets and antimalarial drugs that are effective against drug-resistant malarial parasites. Recent studies have demonstrated that calcium dependent protein kinases (CDPKs) regulate a variety of biological processes in the malaria parasite Plasmodium falciparum and that CDPK4 is important for parasite development. The gene disruption of CDPK4 in Plasmodium berghei, which results in major defects in sexual differentiation of the parasite has highlighted the importance of CDPK4 in Plasmodium biology and suggests that it may be used as a target for therapeutic drugs. PfCDPK4 is expressed in the gamete/gametocyte stage, and this could make PfCDPK4 an essential target for malaria drug discovery. The structure of PfCDPK4 was used as a template in the discovery of malaria drug leads and in designing chemical compounds or inhibitors that will show anti-parasitic activity against the target molecule. The model structure of PfCDPK4 was generated through homology modelling, and model structure validation confirmed that the model structure of PfCDPK4 is of stereochemical quality. The molecular modelling approach of in silico screening was utilized in this research, wherein a large library of chemical compounds, some natural chemical compounds, and clinically approved kinase inhibitors were screened against the target molecule PfCDPK4. In silico screening of the Bio-Focus library against PfCDPK4 resulted in twenty-six compounds being identified; in vitro single screening at a concentration of 5 μM confirmed that three compounds exhibit moderate antimalarial activity against the NF54 strain of Plasmodium falciparum, with the percentage inhibition ranging between 42% and 47%.

PhD (Biochemistry) / Department of Biochemistry / Malaria is a parasitic disease that accounts for more than 660 thousand deaths annually, mainly in children. Malaria is caused by five Plasmodium species P. ovale, P. vivax, P. malariae, P. falciparum and P. knowlesi. The most lethal cause of cerebral malaria is P. falciparum. The parasites have been shown to up-regulate some of their heat shock proteins (Hsp) in response to stress. Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is central to protein homeostasis and determines the fate of proteins. Six Hsp70 genes are represented on the genome of P. falciparum. The Hsp70 genes encode for proteins that are localised in different sub-cellular compartments. Of these two occur in the cytosol, PfHsp70-z and PfHsp70-1; two occur in the endoplasmic reticulum, PfHsp70-2 and PfHsp70-y; one in the mitochondria, PfHsp70-3 and one exported to the red blood cell cytosol, PfHsp70-x. PfHsp70-1 is a well characterized canonical Hsp70 involved in prevention of protein aggregation and facilitates protein folding. Little is known about PfHsp70-z. PfHsp70-z was previously shown to be an essential protein implicated in the folding of proteins possessing asparagine rich repeats. However, based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins and is thought to serve as a nucleotide exchange factor (NEF) of PfHsp70-1. The main aim of this study is to elucidate the functional roles of PfHsp70-z as a chaperone and its interaction with PfHsp70-1. In the current study, PfHsp70-z was cloned and expressed in E. coli JM109 cells. This was followed by its purification using nickel chromatography. The expression of PfHsp70-z in parasites cultured in vitro was investigated and its association with PfHsp70-1 was explored using a co-immuno precipitation assay. PfHsp70-z expression in malaria parasites is up regulated by heat stress and the protein is heat stable based on investigations conducted using Circular Dichroism. Furthermore, the direct interaction between recombinant forms of PfHsp70-z and PfHsp70-1 were investigated using slot blot and surface plasmon resonance assays. PfHsp70-z was observed to exhibit ATPase activity. In addition, the direct interaction between PfHsp70-z and PfHsp70-1 is promoted by ATP. Based on limited proteolysis and tryptophan fluorescence analyses, PfHsp70-z binds ATP to assume a unique structural conformation compared to the conformation of the protein bound to ADP or in nucleotide-free state. PfHsp70-z was able to suppress the heat-induced aggregation of malate dehydrogenase and luciferase in vitro. Interestingly, while ATP appears to modulate the conformation of PfHsp70-z, the chaperone function of PfHsp70-z was not influenced by ATP. Altogether, these findings suggest that Characterization of Heat Shock Protein 70-z (PfHsp70-z) from Plasmodium falciparum iii PfHsp70-z serves as an effective peptide substrate holding chaperone. In addition, PfHsp70-z may also serve as the sole nucleotide exchange factor of PfHsp70-1. The broad spectrum of functions of this protein, could explain this PfHsp70-z is an essential protein in malaria parasite survival. This is the first study to show that PfHsp70-z possess independent chaperone activity and that it interacts with its cytosolic counterpart, PfHsp70-1 in a nucleotide dependent fashion. Furthermore, the study shows that PfHsp70-z is a heat stable molecule and that it is capable of forming high order oligomers.

PhD (Biochemistry) / Department of Biochemistry / The main malaria agent, Plasmodium falciparum expresses an Hsp70 (PfHsp70-1) which plays a significant role in parasite survival. PfHsp70-1 is distinct in that it possesses glycine-glycine-methionine-proline (GGMP) tetrapeptide repeats in its C-terminal domain. To date, the GGMP motif of PfHsp70-1 has not been studied. The motif is positioned within the C-terminal lid segment of PfHsp70-1. The motif is also about seven residues upstream the terminal EEVD residues that are responsible for the interaction of PfHsp70-1 with its functional regulators (co-chaperones). P. falciparum Hsp70/Hsp90 organizing protein (PfHop) constitutes one of the functional regulators of PfHsp70-1. PfHop allows PfHsp70-1 and its chaperone partner, PfHsp90 to form a functional partnership. Given the proximity of the GGMP repeats to the C-terminus of PfHsp70-1, it was postulated in this study that the GGMP repeat residues may regulate attachment of PfHop to PfHsp70-1. Hence, this study hypothesized that the GGMP repeat motif is important for the interaction between PfHop and PfHsp70-1 as well as the chaperone activity of PfHsp70-1. Two variants in which the N-terminal and the C-terminal GGMP repeats were conservatively substituted were generated. E. coli Hsp70 (DnaK) lacks a GGMP motif. Thus, the GGMP motif of PfHsp70-1 was introduced into E. coli DnaK in order to generate a third GGMP variant. Recombinant forms of PfHsp70-1, DnaK, and their GGMP variants were heterologously expressed in E. coli XL1 Blue cells. The proteins were purified to homogeneity by using a combination of Ni-NTA affinity chromatography, ion exchange, and size exclusion chromatography. Purified proteins were then biophysically characterized using CD spectroscopy and tryptophan fluorescence. Findings from this study revealed that there were minimal secondary structural differences between PfHsp70-1, DnaK and their GGMP variants. In order to investigate the chaperone function of PfHsp70-1, DnaK and the GGMP variants, a complementation assay in E. coli dnak756 cells whose Hsp70 is functionally compromised was conducted. The PfHsp70-1 GGMP variants were able to suppress the thermosensitivity of the E. coli cells. However, the Investigation of the role of GGMP motif of Plasmodium falciparum Hsp70-1 on the chaperone function of the protein and its interaction with a co-chaperone, PfHop ii DnaK-G variant failed to confer cytoprotection to the E. coli dnak756 cells. To further validate the findings from the complementation assay, the ability of the recombinant proteins to suppress aggregation of heat stressed Malate dehydrogenase (MDH) was elucidated. PfHsp70-1 had better MDH aggregation suppression capabilities than its GGMP variants. Overall, findings from the MDH aggregation suppression assay suggest that the GGMP repeats may contribute towards substrate binding. Substrate binding might be dependent on the specific positioning of a particular repeat in the GGMP motif of PfHsp70-1. Furthermore, the ATPase activity of PfHsp70-G632 and PfHsp70-G648 was significantly reduced compared to PfHsp70-1 (wild type). However, PfHsp70-G632 had the lowest ATPase activity. Interestingly, the ATPase activity of PfHsp70-G632 was enhanced in the presence of synthetic Hsp70 model peptide substrates. Slot blot and ELISA approaches confirmed that the GGMP mutations partially abrogated the interaction of PfHsp70-1 with PfHop. Altogether, the findings suggest that the GGMP motif of PfHsp70-1 has marginal effects on the structure of PfHsp70-1. In conclusion, this study provides the first direct evidence that the GGMP motif is important for the chaperone function of PfHsp70-1 as well as its interaction with PfHop. / NRF

Relief of the global malaria burden relies on the management and application of effective therapies. Unfortunately, the continuous development of resistance to therapies by the deadliest parasite strain, Plasmodium falciparum, has made the treatment and control of malaria much more difficult. Derivatives of the Chinese peroxidic antimalarial drug artemisinin primarily used in first-line combination therapy for treatment of P. falciparum malaria have proved to be highly effective. However, their use also is now compromised by the development of resistance by the parasite to the artemisinin derivative in the drug combination. This event emphasizes the need for ongoing development of new and effective drug combinations. This research aimed to identify efficacious combinations selected from a group of compounds known to induce oxidative stress by redox cycling combined with an artemisinin, which as an oxidant drug also induces oxidative stress but is unable to undergo redox cycling. Combination of the artemisinin with a redox-active compound is expected to both enhance and maintain oxidative stress within the parasite's proliferative environment. These combinations should be used together with a third drug with a completely different mode of action, such as a quinolone. Selected amino artemisinins and redox active phenothiazines, phenoxazines, thiosemicarbazones, and quinolone derivatives were screened for antimalarial activity and mammalian toxicity. These were found to be potently active (11 μM) to Chinese Hamster ovarian (CHO) cells. The compounds are thus highly selective for P. falciparum, as revealed by the selectivity indices (SI) of >270. The in vitro absorption, distribution, metabolism, and elimination (ADME) properties of the compounds were also determined through the application of specific assays. In vivo pharmacokinetic (PK) profiling was also carried out by intravenous and oral administration of the individual compounds to healthy C57BL/6 mice. Biological samples were analysed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical methods, which were validated according to the fit-for purpose recommendations by the FDA. Evaluation of the in vitro and in vivo profiles thereby facilitated the identification of suitable combination candidates. The phenoxazine and phenothiazine derivatives were identified as the best potential redox partners and were each investigated in combination with the amino-artemisinin artemisone through fixed ratio isobole analysis. A substantial synergistic interaction was observed. Overall, the investigation enabled the identification of drug combinations that are potently active in vitro. This synergistic interaction strongly supports the redox cycling rationale for identifying new antimalarial therapies and further suggests that such combinations in chemotherapy may delay the onset of resistance to the new agents. The results strongly encourage further investigation of the in vivo pharmacokinetic and pharmacodynamic (PK/PD) relationships of these combinations in the humanized murine model of P. falciparum

Malaria is an infectious disease, where the parasite, Plasmodium, infects mosquitos, and then infects humans through a mosquito bite. In 2019, there were over 229 million cases of malaria worldwide with children under the age of 5 being the most vulnerable. Risk factors for children under five include not sleeping underneath a mosquito net, having a lower education, and the type of household. Most cases and deaths happened in the sub-Saharan region of Africa, including Sierra Leone. The aim of this project was to study the prevalence and malarial factors that are associated with children under the age of five sleeping underneath mosquito nets at night in Sierra Leone in 2019. Data was extracted from the Demographic and Health Surveys (DHS) Program in the Standard DHS for Children’s recode 2019. SPSS Statistics was used to conduct analysis. This cross-sectional study focused on the prevalence of children under five sleeping underneath a mosquito net, which is the outcome variable. The explanatory variables included type of mosquito net slept under, region, place of residence, and fever in the last two weeks. The first part of the results focused on descriptive tests, finding the frequency and percentage of each variable. Then chi-squared tests were performed between the outcome and explanatory variables, determining the test statistic, p-value, and degrees of freedom. The sample size was 9,899 responses. For the frequency results of the explanatory variables, with missing values excluded, majority of responses lived in the Southern region (n=2,591; 25.5%), lived in a rural area (n=6,873; 69.4%), had children under five sleep under treated nets (n=5,271; 63.4%), and did not have a fever in the last two weeks (n=7,418; 81.8%). The frequency results of the outcome variable were that majority of responses had all their children under five sleep under a mosquito net (n=5,278; 56.6%). There was significant association between children under 5 sleeping underneath a mosquito net with type of net (p<0.001), region (p<0.001), place of residence (p<0.0001), and fever in the last two weeks (p=0.006). One limitation of this study was that it doesn’t ask participants if they had malaria, therefore, variables were chosen based on that they can be used as a surrogate for malaria. Even though, the whole country is endemic to malaria, the Southern region could potentially have the highest cases of malaria, since most responses lived in that region and had many children sleeping under nets. Also, the majority of responses did not have a fever in the last two weeks, and this could be due to having a mosquito net in place to reduce the risk of malaria. In conclusion, studying malaria variables and the possible relationship with children under the age of five on whether they slept under a mosquito net is beneficial to help better understand malaria and the mosquito nets, which is a useful prevention technique.

Despite the availability of effective drugs to prevent malaria during pregnancy using intermittent preventive treatment with Sulfadoxine-Pyrimethamine or Fansidar and insecticide bed net, use of these methods are still little used in Sub-Saharan Africa, including Tanzania. As a result, many pregnant women are at risk of malaria consequences such as maternal anemia and low birth weight babies, which increase the rate of infant mortality. Data from the Demographic Health Survey for Tanzania HIV/AIDs and the Malaria Indicator Survey 2011-2012 were used in a cross-sectional design guided by the health belief model. Logistic regression examined the association between (a) preventive treatment-seeking behavior and (b) SES, malaria media exposure, knowledge of malaria signs and symptoms, perceived seriousness of malaria, and knowledge of malaria preventive measures. After controlling for transportation, family responsibility, and age, significant associations (p < 0.05) were found between SES, malaria media exposure, knowledge of malaria signs and symptom, perceived seriousness of malaria, knowledge of malaria preventive measures, and treatment-seeking behavior. This study contributes to positive social change by helping design and implement policies and programs to improve the knowledge of Tanzanian pregnant women about the risk of malaria infection and the benefits of preventive treatments. Interventions to reduce malaria infection during pregnancy will reduce the associated morbidity and mortality of both mothers and infants; as a result, families and communities will be healthier and prevent unnecessary medical cost of malaria.

ABSTRACT Introduction The use of information systems to understand the dynamics of malaria disease and inform decisions on control proved valuable to a malaria control programme. Development of simple practical and sustainable information system tools has been slow in coming for many resource-poor environments. This thesis addresses many issues relating to the conceptual development and implementation of simple tools and their integration into operational malaria control to support decision making and advocacy. Methods A basic Microsoft Access malaria data collection and repository tool has been in existence since 1997 focussing mainly on case reporting alone. Better utilization of data and further expansion to include outbreak identification and response, cluster detection and intervention monitoring has been the main focus over time. Eight years of retrospective malaria case data from Mpumalanga Province, South Africa were used to explore disease dynamics including spatial as well as temporal variation in malaria epidemiology. The identification of specific risk areas and the confirmation of the unstable nature of malaria occurrence lead to the conceptualization and development of an outbreak model using binomial statistics. The novel three tier outbreak identification and response system was field tested over a two season period to establish acceptance and the ability to direct resources in times of elevated case loads. Comparison against other existing malaria outbreak systems was conducted. SaTScan freely available software was used to detect spatial and spacetime disease clusters within towns in the highest risk area of the province. A malaria case control study was conducted in seven localities/towns/villages to explore risk and protective characteristics of household structure and practices, including the use of impregnated nets. The micro economic status of households as a determinant of malaria risk was also explored. A spray operations component as part of the malaria information system was developed and implemented during the time to allow for routine monitoring and historical exploration of indoor residual spray activities. Results Retrospective malaria case data analysis identified heterogeneity of malaria risk in the Province and spatial analysis identified significant clusters at small geographical area resolution rejecting the hypothesis that malaria is homogeneously distributed over space and time. The importance of intervention monitoring to identify low coverage areas, over or under application of insecticides, and assessment of the productivity of spray operators was identified. The outbreak identification and response system was successfully implemented, integrated and sustained with a set of response activities developed for implementation at defined threshold levels. The outbreak systems can be considered for utilization in other low transmission settings.Results of the case control study indicated that malaria risk was associated with living in traditional housing and the practice of re-opening windows at night when peak biting behaviour of the main mosquito vector, Anopoheles arabiensis is expected. Higher household socio economic status (SES) profile was associated with a lower risk of malaria. Conclusions The conceptualization, development and implementation of operationally feasible malaria information management tools in a rural African environment proved useful for enhancing malaria control. The novel malaria outbreak identification and response, cluster detection as well as the spray monitoring systems were successfully implemented and adopted as an integral part of the routine malaria control programme monitoring and surveillance system. This research has enabled more informed real-time decision-making for effective programme management.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Science in Epidemiology and Biostatistics / Background: Malaria causes an overwhelmingly large number of cases and deaths around the globe every year, with over 90% of deaths occurring in sub-Saharan Africa. Namibia is among the sub Saharan countries that have malaria as a major public health problem, affecting most pregnant women and children in the northern regions. Insecticide treated net (ITN) distribution has been expanded in the northern regions since 2005, yet there is low ITN utilization. The associated factors for low ITN utilization are not well established. Objective: This study aimed to determine factors affecting the utilization of ITN among pregnant women in northern regions of Namibia. Specific objectives were to: (1) describe coverage of ITNs among pregnant women in terms of possession; (2) describe the utilization rate of ITN among pregnant women in northern regions; and (3) determine the association between various factors and utilization of ITN among pregnant women. The first study outcome measure was utilization of ITN, defined as an individual pregnant woman who had used an ITN the night before the survey day. The second outcome measure was coverage of ITNs, defined as possession of at least one ITN in each household, irrespective of whether or not it was being used. Methods: A cross sectional study design was used, using secondary data from a nationally representative survey which collected data on malaria interventions in regions of Namibia. The original survey collected data from a representative sample of 3000 households from 120 primary sampling units (PSUs) in nine regions country wide, using a stratified sampling method of two stages. This study targeted pregnant women in four northern regions, namely; Kavango, Ohangwena, Oshana and Omusati, in both rural and urban areas; who participated in the 2009 Namibia Malaria Indicator Survey (NMIS) from 4 April to 10 June 2009. A total of 83 pregnant women were included in the analysis out of 194 pregnant women who were interviewed during the 2009 survey. In the descriptive analyses, we described the demographic characteristics of pregnant women. In the analytic analyses, univariable and multivariable analysis (logistic regression) were conducted. Logistic regression was used to determine risk factors associated with ITN utilization. Results: The utilization of ITN was high (47%) for young women aged 15-24 years old. Overall, 67% of pregnant women aged 15-44 years old slept under bed nets the night prior the survey day. In the univariable analyses, being 35-44 years of age (OR 0.25; 95% CI: 0.07-0.89, p<0.02) and having information about malaria (OR 0.28, 95% CI: 0.09-0.85, p<0.03), were independently associated with ITN utilization. In the multivariate logistic regression model, none of the explanatory variables were significant at the 5% level. The study showed 98.8% overall coverage of ITNs among pregnant women in terms of possession. Conclusion: These findings have implications for malaria interventions in Namibia. While almost all the pregnant women recruited in the study possessed ITNs, a significant proportion did not utilize them. Older women were more likely to utilize ITNs. Interventions to improve utilization among pregnant women should target younger women below the age of 35. Women that had information on malaria were more likely to utilize ITN. Sensitising women about the epidemiology of malaria across Namibia could lead to improved utilization of ITNs. A national malaria strategic plan needs to incorporate targeted reproductive women’s education for malaria control in Namibia.

Indiana University-Purdue University Indianapolis (IUPUI) / Malaria infects more than 240 million people every year, causing more than 640,000 deaths in 2021 alone. The complex interactions between the Plasmodium parasites that cause malaria and host immune system have made it difficult to identify specific mechanisms of vaccine-induced and naturally acquired immunity. After more than half a century of research into potential immunization methods, reliable immune correlates of malaria protection still have yet to be identified, and questions underlying the reduced protective efficacy of malaria vaccines in field studies of endemic populations relative to non-endemic populations still remain. In this thesis, I use computational methods to identify biological determinants of whole-parasite vaccine-induced immunity and immune correlates of protection from clinical malaria. Our systems analysis of a PfSPZ Vaccine clinical trial revealed that innate signatures were predictive of increased antibody response but also a decrease in the cytotoxic response required for sterilizing immunity. Conversely, these myeloid signatures predicted protection against parasitemia for subjects receiving a saline placebo, suggesting a role for myeloid-lineage cells in clearing pre-erythrocytic parasite stages. Based on these findings, I created a structural equation model to examine the interactions between cellular, humoral, and transcriptomic responses and the effects these have on protection outcome. This revealed a direct positive effect of CD11+ monocyte-derived cells on parasitemia outcome post-vaccination that was mediated by the presence of P. falciparum-specific antibodies at pre-vaccination baseline. Additionally, this model illustrates an indirect role of CD14+ monocyte activation in restricting immune priming by the PfSPZ Vaccine. Together, this data supports our hypothesis that innate immune activation and antigen presentation are uncoupled from cytotoxic cell-dependent immunity from the PfSPZ Vaccine and that this effect may be antibody-dependent.

Raman micro-spectroscopy provides a non-destructive probe with potential applications as a diagnostic tool for cellular disorders. This study presents micro-Raman spectra of live erythrocytes infected with a malaria parasite and investigates the potential of this probe to monitor molecular changes which occur during differentiation of the parasite inside the cell. At an excitation wavelength of 633 nm the spectral bands are dominated by hemoglobin vibrations yielding information the on structure and spin state of the heme moiety. It also demonstrates the novel use of silica capillaries as a viable method for studying the erythrocytes in an environment that is much closer to their native state, thus opening the possibility of maintaining the cell in vivo for long periods to study the dynamics of the parasite's growth.