Plamodium falciparum heat shock protein (PfHsp70) is believed to be involved in the cytoprotection of the malaria parasite through its action as a molecular chaperone. Bioinformatic analysis reveal that PfHsp70 consists of the three canonical Hsp70 domains; an ATPase domain of 45 kDa, Substrate binding domain of 15 kDa and a C-terminal domain of 10 kDa. At the C-terminus there is a GGMP repeat motif that is commonly found in Hsp70s of parasitic origins. Plasmodium falciparum genome is 80% A-T rich, making it difficult to recombinantly express its proteins in Escherhia coli (E. coli) as a result of rare codon usage. In this study we carried out experiments to improve expression in E. coli by inserting the PfHsp70 coding region into the pQE30 expression vector. However multiple bands were detected by Western analysis, probably due to the presence of rare codons. The RIG plasmid, which encodes tRNAs for rare codons in particular Arg (AGA/AGG), Ile (AUA) and Gly (GGA) was engineered into the E. coli strain resulting in production of full length PfHsp70. Purification was achieved through Ni²⁺ Chelating sepharose under denaturing conditions. PfHsp70 was found to have a very low basal ATPase activity of 0.262 ± 0.05 nmoles/min/mg of protein. In the presence of reduced and carboxymethylated lactalbumin (RCMLA) a 11-fold increase in ATPase activity was noted whereas in the presence of both RCMLA and Trypanosoma cruzi DnaJ (Tcj2) a 16-fold was achieved. For ATP hydrolysis kcat value of 0.003 min⁻¹ was obtained whereas for ADP release a greater kcat value of 0.8 min⁻¹ was obtained. These results indicated that rate of ATP hydrolysis maybe the rate-determining step in the ATPase cycle of PfHsp70.

Plasmodium falciparum is the pathogen that is responsible for the most virulent, severe and dangerous form of human malaria infection, accounting for nearly a million deaths every year. To survive and develop in the unusual environment of the red blood cells, the parasite causes structural remodelling of the host cell and biochemical changes through the export of virulence factors. Among the exportome are the molecular chaperones of the heat shock protein family, of which Hsp40s and Hsp70s are prominent. PF A0660w, a type II P. falciparum Hsp40, has been shown to be exported in complex with PfHsp70-x into the infected erythrocyte, suggesting possible functional interactions. However, the chaperone properties of PF A0660w and its interactions with proteins of parasite and human origin are yet to be investigated. Using a codon optimised coding region, PF A0660w was successfully expressed in E. coli M 15 [pREP4] cells. However, the expressed protein was largely deposited as insoluble pellet, and analysis of the pellets revealed a high percentage of PF A0660w, characteristic of inclusion body formation. PF A0660w was purified from inclusion bodies using additive enhanced solubilisation and refolding buffers followed by nickel affinity chromatography. SDS-PAGE and western analysis revealed that the purified protein was of high purity. Size exclusion chromatography showed that the protein existed as a monomer in solution and the secondary structure analysis using Fourier transformed infrared spectroscopy (FTIR) confirmed the success of the refolding approach. Its monomeric state suggests that PF A0660w may be functionally different from other Hsp40 that form dimers and that for PF A0660w, dimer formation may not be needed to maintain the stability of the protein in solution, but may occur in response to functional necessities during its interaction with partner Hsp70. PFA0660w was able to significantly stimulate the ATPase activity ofPfl-Isp70-x but not Pfl-Isp70-1 or human Hsp70 (HsHsp70), suggesting a specific functional interaction. Also, PF A0660w produced a dose dependent suppression of rhodanese aggregation and cooperated with Pfl-Isp70-1, PfHsp70-x and HsHsp70 to cause enhanced aggregation suppression. Its ability to independently suppress aggregation may help to maintain substrates in an unfolded conformation for eventual transfer to partner Hsp70s during refolding processes. Also, the in vivo characterisation using a PF A0660w peptide specific antibody confirmed that PF A0660w was exported into the cytosol of infected erythrocytes. Its lack of induction upon heat shock suggests that PF A0660w may not be involved in the response of the parasite to heat stress. Overall, this study has provided the first heterologous over-expression, purification and biochemical evidence for the possible functional role of PF A0660w, and has thereby provided the needed background for further exploration of this protein as a potential target for drug discovery.

Please read the abstract in the section 00front of this document / Thesis (DPhil (Medical Immunology))--University of Pretoria, 2007. / Immunology / unrestricted

MSc (Chemistry) / Department of Chemistry / See the attached abstract below

MSc (Biochemistry) / Department of Biochemistry / In 2017 malaria cases were estimated at 219 million and of these 435 000 resulted in death. Malaria is transmitted by female Anopheles mosquitoes which thrive in tropical and sub-tropical areas. Malaria is caused by five species from the genus Plasmodium, namely P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. P. falciparum causes the most severe form of the disease. P. falciparum has a complex life cycle in the human and mosquito hosts exposing the parasite to environmental changes, resulting in upregulation of heat shock proteins (Hsps). These Hsps facilitate protein folding and protein disaggregation. Hsp70 is a molecular chaperone whose function is to facilitate protein folding. P. falciparum Hsp70-x is the only member of this family of proteins that is exported to the erythrocyte cytosol by the parasite. PfHsp70-x has been implicated in the development of malaria pathogenesis. This is largely due to its association with P. falciparum erythrocyte membrane protein 1 (PfEMP1), an important virulent factor that is exposed to the exterior of the infected erythrocyte. In tumour cells, cell surface- bound Hsp70 is known to sensitize the tumour cells to cytolytic attack that is mediated by NK cells. Cell surface bound Hsp70 is thought to recruit NK cells and Granzyme B (GrB) via its 14 amino acid sequence, TKDNNLLGRFELSG, known as the TKD motif. Both PfHsp70-x and human Hsp70 (hHsp70) contain the TKD motif. Thus, this study sought to investigate the role of Hsp70 in facilitating the selective targeting of malaria parasite-infected erythrocytes by GrB. To this end, recombinant hHsp70 and PfHsp70-x were successfully expressed in E. coli and purified. Using slot blot and ELISA, it was observed that both PfHsp70-x and hHsp70 directly interact with GrB. PfHsp70-x showed greater affinity for GrB than hHsp70. In addition, using parasites cultured at the erythrocyte stage it was noted that GrB exhibits potent antiplasmodial activity (IC50 of 0.5μM). In addition, the findings suggest that GrB interacts with both Hsp70s (of parasite and human origin) resident in the infected erythrocyte. This makes GrB a promising antimalarial agent. / NRF

Background: In the Republic of the Congo, malaria is a major cause of death, in particular among children. Therefore, malaria prevention is of high priority. Registered nurses have an important role in illness prevention in sub-Saharan Africa, especially through education of the population. To improve malaria prevention, it is important to understand how registered nurses experience the preventive work. Objective: The objective of this study was to examine the experiences of registered nurses working with malaria prevention in the Republic of the Congo. Method: A qualitative method was used. Semi-structured interviews with six registered nurses in the Republic of the Congo were conducted. Qualitative content analysis was used to analyse the interviews. Findings: Three categories were identified in the analysis: preventive interventions are necessary, possibilities in malaria prevention and difficulties in malaria prevention. The registered nurses experienced that preventive interventions, such as different methods of avoiding mosquito bites, are necessary because malaria affects the entire population in Congo. The registered nurses experienced educating and informing the patients as a possibility in malaria prevention and a lack of resources as a difficulty. Conclusion: The registered nurses experienced education and information as an effective way to communicate preventive interventions against malaria. However, the registered nurses sometimes experienced incomprehension among the patients, which could be improved by adapting the information to the individual. A difficulty that the registered nurses experienced was a lack of resources on many levels. An increased access to resources would lead to improvements in malaria prevention.

Background: Malaria continues to pose a complex public health problem amongst pregnant women in Africa (Schantz-Dunn & Nour 2009; Antwi 2010), accounting for over 90% of the global malaria burden (WHO 2002; Morel et al. 2005; Schantz-Dunn & Nour 2009). However, little is known about the impact of information, education and communication (IEC) strategies in malaria prevention and control among pregnant women in Africa. Aims and Objectives: The study sought to identify: evidence of impact of IEC strategies; feasibility, appropriateness, meaningfulness and effectiveness of the IEC strategies; challenges; best practices and key lessons to inform governments, policymakers, health partners, the academic community, directors, managers of health, frontline health professionals and health educators at institutional and community levels. Moreover, the study aimed to develop a theoretical framework to enhance the understanding of issues related to implementation of IEC strategies. Methods: A mixed method approach was adopted. This consisted of a systematic review of evidence within the African context and an evaluation methodology involving a contextually based survey of Ethiopia, Ghana, Nigeria and Tanzania. The systematic review involved a structured search of relevant databases and websites, and hand search strategies. Three sets of evidence were identified and aggregated using a narrative synthesis approach. A survey questionnaire reflecting the outcomes of the review was sent to health professionals and lay persons in the countries under study. Primary data were analysed using SPSS Version 15.0. Non-parametric tests and sensitivity analyses were conducted to assess the nature of opinions among respondents within and across countries. Findings: 3,440 studies were identified during the systematic review. Out of this number, 57 met the inclusion criteria. Following critical appraisal, 50 studies met the criteria for methodological quality. Ten IEC strategies were identified. These were: Staff training and orientation Advocacy Community mass education campaigns House-to-house sensitisation Health education in health units Visiting places of worship Women’s group meetings Integrated health education campaigns Symbolism versus message delivery Audience segmentation versus information delivery. The survey reveals a high level of awareness of IEC strategies among respondents in the African countries studied. The evidence of impact reported by respondents supported the findings of the systematic review. However, there were some differences, and some concerns still remain regarding the extent of impact. The study suggests that using an IEC strategy implementation equation could enhance the understanding of issues related to implementation of IEC strategies. Implications of the findings are outlined, including implications for professional practice in relation to IEC programme implementation, most especially among nurses and midwives. IEC roles and responsibilities of key actors are also proposed. Conclusions: Despite the challenges of adopting a mixed method approach, the study highlights an important relationship between evidence and practice. This approach also helped to ensure that a comprehensive multiperspective view of IEC strategies was achieved. In designing and implementing IEC programmes, clients must be involved in order to encourage community ownership and programme sustainability. Attitudinal change and commitment is required by all stakeholders in order to achieve and maintain impact on malaria in pregnancy. Finally, while recognising the essence of feasibility, appropriateness and meaningfulness of a given strategy, it is worth noting that the key message from this study is that no one single strategy on its own appears ideal. Therefore, there is the need to pay equal attention to both institutional and community-based strategies. Doing one thing alone will not work; more evidence of impact is required to know what works and in what context.

Heat Shock 40 kDa proteins (Hsp40s) partner with heat shock 70 kDa proteins (Hsp70s) in facilitating, among other chaperone activities; correct protein transport, productive protein folding and assembly within the cells; under both normal and stressful conditions. Hsp40 proteins regulate the ATPase activity of Hsp70 through interaction with the J-domain. Plasmodium falciparum Hsp70s (PfHsp70s) do not contain a Plasmodium export element (PEXEL) sequence although PfHsp70-1 and PfHsp70-3 have been located outside of the parasitophorous vacuole. Studies reveal that a type I P. falciparum (PfHsp40) chaperone (PF14_0359) stimulates the rate of ATP hydrolysis of the cytosolic PfHsp70 (PfHsp70-1) and that of human Hsp70A1A. PFE0055c is a PEXEL-bearing type II Hsp40 that is exported into the cytosol of P. falciparum-infected erythrocytes; where it potentially interacts with human Hsp70. Studies reveal that PFE0055c associates with structures found in the erythrocyte cytosol termed “J-dots” which are believed to be involved in trafficking parasite-encoded proteins through the erythrocyte cytosol. If P. falciparum exports PFE0055c into the host cytosol, it may be proposed that it interacts with human Hsp70, making it a possible drug target. The effect of PFE0055c on the ATPase activity of human Hsp70A1A has not been previously characterised. Central to this study was bioinformatic analysis and biochemical characterisation PFE0055c using an in vitro (ATPase assay) approach. Structural domains that classify PFE0055c as a type II Hsp40 were identified with similarity to two other exported type II PfHsp40s. Plasmids encoding the hexahistidine-tagged versions of PFE0055c and human Hsp70A1A were used for the expression and purification of these proteins from Escherichia coli. Purification was achieved using nickel affinity chromatography. The urea-denaturing method was used to obtain the purified PFE0055c whilst human Hsp70A1A was purified using the native method. PFE0055c could stimulate the ATPase activity of alfalfa Hsp70, although such was not the case for human Hsp70A1A in vitro.

Malaria is one of the three most deadly diseases in Africa. Although there are available treatments, their efficacy has been greatly reduced over the past two decades due to the development of resistance to currently available drugs. This has necessitated the search for new and effective antimalarial agents. This project approached the search for new antimalarial compounds in two ways: (i) by screening natural products isolated from marine algae against the Plasmodium parasite and (ii) by modification of selected isolated active compounds to target 1-deoxY-đ-xylulose 5-phosphate reductoisomerase (DXR), an enzyme found in the nonmevalonate isoprenoid biosynthetic pathway of Plasmodium Jalciparum. It was envisaged that such a compound would exhibit dual action on the Plasmodium parasite. Extracts obtained from 22 marine algae were prefractionated by solvent partitioning and were screened for anti plasmodial activity against the chloroquine sensitive (CQS) P. Jalciparum D 10 strain. Overall, 50% of the algae screened produced at least one crude fraction with activity against P. Jalciparum. Extracts of the algae Sargassum heterophyllum, Plocamium cornutum, Amphiroa ephedrea and Pterosiphonia cloiophylla gave the most promising results. Fractionation of S. heterophyllum afforded three tetraprenyltoluquinols (3.1, 3.2 and 3.5) and an all-trans-fucoxanthin (3.6). Three new compounds (4.5, 4.6 and 4.7) and two known halogenated monoterpenes (4.1 and 4.4) were isolated from P. cornutum. Each of the isolated compounds from both S. heterophyllum and P. cornutum showed antiplasmodial activity with IC₅₀ values ranging from 2.0 - 15.3 μM for S. heterophyllum and 13 - 230 μM for P. cornutum. Attempts to synthetically modify halogenated monoterpene 4.4 by dihydroxylation and phosphorylation in order to inhibit the DXR enzyme was unsuccessful. However, the hemiterpene analogue (5.42) of the halogenated monoterpenes was successfully phosphorylated and dihydroxylated to give compound 5.45 which showed promising activity against DXR. The result obtained indicated that the proposed phosphorylation and dihydroxylation of the halogenated monoterpene 4.4 would result in the synthesis of a potent DXR inhibitor and therefore a potential antimalarial agent with dual mode of action on the Plasmodium parasite.

Background: Insecticide-treated nets (ITNs) are one of the most effective prevention measures against malaria. Malaria is highly endemic in Ghana. The country implemented mass distribution campaigns of ITNs to cover 80% to 95% of the population but the rate of ITNs use among children under 5 years was 52%, which was lower than the universal coverage target of 100%. Objective: The objective of this study was to identify the socio-demographic factors associated with ITNs utilization among children under 5 years in Ghana. Methods: This was a secondary analysis from cross-sectional data of 3,029 children under five years obtained from Ghana Malaria Indicator Survey 2016. Logistic regression analysis was done to identify the determinants of ITNs utilization among children under 5 years in Ghana. Results: Size of the household, number of children ≤5 years old in the household, household wealth index, education level of mother, knowledge of mother on the protection of mosquito nets, place of residence, and region of residence were found to be significantly associated with ITNs utilization in children under 5 years. Conclusion: More interventions are needed to promote the use of ITNs to protect children against malaria. Interventions should focus on households with more than 7 members, households with more than 3 childrens ≤5 years, and on promoting girl’s education.