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

Few studies regarding treatment seeking behaviour related to Malaria have been conducted in Uganda, and most of the studies regarding use of mosquito nets that have been conducted are quantitative. There is thus a need for qualitative studies for a deeper understanding of the complex issue of coping with malaria in a resource limited setting. The aim of this study is to understand sociocultural and structural factors influencing the coping with malaria in rural Uganda. Focus is on the local experiences, perceptions and hardships regarding seeking treatment for malaria and preventative measures like the use of mosquito nets. A theoretical framework based on Political ecology of health, with a focus on human agency, is used to analyse these issues. The village Nyakasojo in Kasese district was selected for a field study. The main source of information was open-ended interviews conducted in March 2020. The study showed that the main reason for not using mosquito nets was lack of access to nets and easily torn nets, rather than unwillingness to use mosquito nets. Further, the study showed that the unequal access to healthcare in combination with livelihood vulnerability forces people to wait before seeking treatment and/or taking half doses of antimalarials, probably leading to unnecessary malaria transmission. A more holistic approach to malaria control would be beneficial, including the aspect of treatment in addition to the present narrower focus on prevention.

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.

Malaria is both a preventable and curable disease if treated early and appropriately. However, it is estimated that every 30 seconds a child dies of malaria in sub-Saharan Africa. The use of innovative eHealth/mHealth tools for malaria that seamlessly integrate into the workflow of healthcare workers could potentially ameliorate this problem. Successful design and development of these tools require an overarching understanding of the socio-technical context for the problems and opportunities in the application domain. A series of studies involving malaria management in the Millennium Village Project (MVP) cluster in rural Ghana were performed. A new method introduced by the author was applied to prioritize health information needs of stakeholders that have the potential to have a higher impact in solving health related problems. The result of applying this method was a group of impactful interventions for the MVP malaria program in Ghana. Findings from this study were validated with the MVP Ghana eHealth team, and after mutual agreement, an eHealth/mHealth intervention around malaria supply chain management was selected for further study. User-Centered Design (UCD) methods were adapted for use in a resource poor setting. Functional and non-functional requirements were identified. A low fidelity prototype was created and early usability inputs were collected. A high fidelity prototype was created to provide decision support to health workers through visualizations of stock levels and recommendations of quantities to order. Results from evaluation studies of the high fidelity prototype with end users suggest that they perceive the prototype as both easy to use and useful, with a potential for adoption and with a low risk of implementation. Usability problems found during the course of the study should be addressed to increase the potential of adoption. To obtain a more complete list of usability issues, both users’ and experts’ evaluations are recommended as well as the use of native and foreign test facilitators.

MSc (Biochemistry) / Department of Biochemistry / Malaria is a disease that claims about half a million lives annually, mainly children. There are 5 Plasmodium species that cause malaria; namely, P. falciparum, P. ovale, P. malariae, P. knowlesi and P. vivax. P. falciparum is the most virulent of them all. The parasite upregulates some heat shock proteins (Hsps) in response to stress it encounters during its life cycle. These Hsps play a major role in proteostasis. The drug resistance of P. falciparum to traditionally used remedies has led to a need for the development of novel drugs. Hsps have been implicated as antimalarial drug targets. Hsps act as molecular chaperones and some make complexes, which are important in facilitating protein folding. As an example, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) form a functional complex through an adaptor protein, Hsp70-Hsp90 organizing protein (Hop). P. falciparum expresses six Hsp70s that are localized in different subcellular compartments. Amongst them, P. falciparum Hsp70-x (PfHsp70-x), is exported to the erythrocyte where it is implicated in host cell remodeling. PfHsp70-x possesses an ATPase domain, substrate binding domain and a C-terminal subdomain. PfHsp70-x possesses an EEVN motif on its C-terminus which is implicated in interactions with co-chaperones amongst them, Hop. Although some of the chaperone functions of PfHsp70-x have been reported, its interaction with human chaperones has not been investigated. The availability of PfHsp70-x in the infected erythrocyte cytosol presents a possibility that this protein may functionally cooperate with human Hsp90 via human Hop (human Hop). This hypothesis that PfHsp70-x interacts with human chaperones is strengthened by the absence of Hsp90 and Hop of parasite origin in the infected erythrocytes. The main aim of this study was to explore the chaperone activity of PfHsp70-x and its functional co-operation with human Hop. Recombinant PfHsp70-x (full length and EEVN deletion mutant) proteins were expressed in E. coli XL1 Blue cells and purified using nickel affinity chromatography. PfHsp70-x was found to be structurally comprised of mostly alpha helices and demonstrated heat stability based on circular dichroism (CD) spectrometry studies. It was established that the EEVN motif may be important for the ATPase activity of PfHsp70-x. However, it was established that the EEVN motif was not important in regulating the holdase chaperone (protein aggregation suppression) function of PfHsp70-x. Furthermore, PfHsp70-x and its mutant preferentially bound to asparagine-rich peptides. Parasite proteins have high asparagine repeat regions as compared to human proteins. In addition, preference for asparagine-rich proteins iii could signify that PfHsp70-x is biased towards binding proteins of parasitic origin. Surface plasmon resonance (SPR) analysis suggested that PfHsp70-x interacts with human Hop with relatively higher affinity compared to its EEVN minus derivative. In conclusion, the removal of the EEVN motif of PfHsp70-x does not affect the chaperone function of PfHsp70-x. However, the EEVN motif is essential for the interaction of PfHsp70-x with human Hop.

MSc Applied Mathematics) / Department of Mathematics and Applied Mathematics / See the attached abstract below

MSc (Biochemistry) / Department of Biochemistry / Plasmodium falciparum is a unicellular protozoan parasite that causes malaria in humans. The parasite is passed to humans through mosquito bites and migrates to the liver before it infects host erythrocytes. It is at the erythrocytic stage of development that the parasite causes malaria pathology. Malaria is characterized by the modification of host erythrocytes making them cytoadherent. This is as a result of formation of protein complexes (knobs) on the surface of the erythrocyte. The knobs that develop on the surface of the erythrocyte are constituted by proteins of host origin as well as some proteins that the parasite ‘exports’ to the host cell surface. Nearly 550 parasite proteins are thought to be exported to the infected erythrocyte. Amongst the exported proteins is P. falciparum heat shock protein 70-x (PfHsp70-x). Hsp70 proteins are known to maintain protein homeostasis. Thus, the export of PfHsp70-x may be important for maintaining protein homeostasis in the host cell. PfHsp70-x is not essential for parasite survival although is implicated in the development of parasite virulence. This is possibly through its role in facilitating the trafficking of parasite proteins to the erythrocyte as well as supporting the formation of protein complexes that constitute the knobs that develop on the surface of the infected erythrocyte. The main objective of the current study was to investigate protein interaction partners of PfHsp70-x. It is generally believed that PfHsp70-x interacts with various proteins of human and parasite origin. Potential candidate interactors include its protein substrates, Hsp70 co-chaperones such as Hsp40 members, and human Hsp70-Hsp90 organizing protein (hHop). The establishment of the PfHsp70-x interactome would highlight the possible role of PfHsp70-x in the development of malaria pathogenicity. Based on bioinformatics analysis, PfHsp70-x was predicted to interact with some exported P. falciparum Hsp40s, hHop and human Hsp90 (hHsp90). Recombinant forms of PfHsp70-x (full length and a truncated form that lacks the C-terminal EEVN motif implicated in co-chaperone binding) were expressed in E. coli BL21 Star (DE3) cells. Recombinant hHop and hHsp70 were expressed in E. coli JM109 (DE3) cells. The proteins were successfully purified using nickel affinity chromatography. Co-affinity chromatography using recombinant PfHsp70-x and immuno-affinity chromatography using PfHsp70-x specific antibody did not confirm the direct interaction of PfHsp70-x with human Hop. However, the direct interaction of hHop and PfHsp70-x has previously been validated in vitro and the current bioinformatics data support ii the existence of such a complex. PfHsp70-x was not stable in the cell lysate that was prepared and this could explain why its interaction with hHop could not be ascertained. However, taken together the evidence from a previous independent study, and the predicted interaction of PfHsp70-x with human chaperones suggests cooperation of chaperone systems which possibly facilitates the folding and function of parasite proteins that are exported to the infected erythrocyte. / NRF

MSc (Biochemistry) / Department of Biochemistry / Sustaining proteostasis is essential for the survival of the cell and altered protein regulation leads to many cellular pathologies. Heat shock proteins (Hsps) are involved in the regulation of the protein quality control. Hsps are a group of molecular chaperones that are upregulated in response to cell stress and some are produced constitutively. The Hsp70 family also known as DnaK in Escherichia coli (E. coli) is the most well-known group of molecular chaperones. Structurally, Hsp70s consist of a nucleotide binding domain (NBD) and a substrate binding domain (SBD) conjugated by a linker sub-domain. ATP binding and hydrolysis is central to the Hsp70 functional cycle. Hsp70s play a role in cytoprotection especially during heat stress in E. coli. Hsp70s from different organisms are thought to exhibit specialized cellular functions. As such E. coli Hsp70 (DnaK) is a molecular chaperone that is central to proteostasis in E. coli. On the other hand, Plasmodium falciparum Hsp70s are structurally amenable to facilitate folding of P. falciparum substrates. The heterologous production of P. falciparum proteins in E. coli towards drug discovery has been a challenge. There is need to develop tools that enhance heterologous expression and proper folding of P. falciparum proteins in an E. coli expression system. To this end, a chimeric Hsp70, KPf consisting of E. coli DnaK NBD and P. falciparum Hsp70-1 (PfHsp70-1) SBD was previously designed. KPf was shown to confer cytoprotection to E. coli DnaK deficient cells that were subjected to heat stress. In this study it was proposed that KPf has an advantage over E. coli DnaK and PfHsp70-1 in its function as a protein folding chaperone. Therefore, the main aim of this study was to characterize the chaperone function of KPf relative to the function of wild type E. coli and P. falciparum Hsp70s. The recombinant forms of KPf, DnaK and PfHsp70-1 proteins were successfully expressed and purified using nickel affinity chromatography. Circular Dichroism (CD) structural study demonstrated that KPf and PfHsp70-1 are predominantly α-helical and are also heat stable. Tertiary structure studies of PfHsp70-1 and KPf using tryptophan fluorescence revealed that both confirmations of recombinant proteins are perturbed by the presence of ATP more than ADP. Interestingly, the substrate binding capabilities of these proteins were comparable both in the absence or presence of nucleotides ATP/ADP. KPf is an independent chaperone, that exhibit nucleotide binding and hydrolysis. The current study has established unique structure-function features of KPf that distinguishes it from its “parental” forms, DnaK and PfHsp70-1. / NRF