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Controlling and eliminating malaria in Indonesia is a challenging endeavour. Evidence-based strategic plans should be critically formulated to overcome a complex mosaic of infection risk across the 5000-km-long archipelago of thousand islands and distinctive habitats. This project aimed to thoroughly explore the challenges and opportunities for controlling/eliminating malaria and present the application of malaria cartographic tools to allow malaria control agencies and their partners to comprehensively assess the prospects for reaching pre-elimination, monitor and evaluate the effectiveness of future strategies against the baseline generated. First, the historical context of malaria in Indonesia and important methods of control and their impact in the context of the political systems that supported them was comprehensively described. A series of distribution maps of twenty Anopheles malaria vector mosquitoes in Indonesia were also produced, supported by comprehensive reviews of each species’ bionomics and susceptibility to insecticides. Then, the application of malaria cartographic tools for malaria control/elimination in Indonesia was explored. The first high spatial resolution (1 x 1 km) baseline endemicity maps of Plasmodium falciparum and P. vivax malaria were generated, together with corresponding estimates of population at risk and clinical burdens of each species in 2010. Low malaria prevalences of these parasites were predicted in western areas, with high prevalences in eastern Indonesia. Over 132 million people in Indonesia lived at risk of P. falciparum transmission with 70% of them in areas of unstable transmission and 30% in stable transmission. There were an estimated 7.7 million P. falciparum clinical cases across the populations at risk. Meanwhile, nearly 130 million people lived at risk of P. vivax malaria with 79% living in unstable and 21% in stable transmission areas. This infection caused 1.5 million estimated clinical cases in 2010. Both estimates were 30-fold and 6-fold higher than routinely reported numbers, respectively. Finally, this project revealed the substantial multi-faced problems that impede current efforts towards the pre-elimination agenda. High rates of undiagnosed clinical cases, insufficient competence of malaria microscopy, inadequate primaquine dosing against P. vivax malaria infections, insufficient evidence of vector control interventions, wide diversities of vector mosquitoes and their bionomics, mosquito resistance against insecticides and inadequate malaria surveillance systems are challenging the task of controlling and eliminating malaria. A diverse range of strategies enabling locally-specific approaches must be implemented for controlling and eliminating malaria in Indonesia. Strategic recommendations are listed and future research priorities are proposed for further study.

Protozoan parasites of the genus Plasmodium are the causative agent of malaria. The most severe form of human malaria is caused by P. falciparum, responsible for approximately three quarters of a million deaths each year. One major problem in the treatment of malaria is resistance to existing chemotherapies. Consequently, there is an urgent need to identify and validate novel drug targets. A possible recently identified drug target is the PfNitA protein of P. falciparum which contains orthologues in other Plasmodium species but is absent from humans. The gene is annotated as a putative formate-nitrite transporter and orthologues are found in a range of prokaryotes as well as the lower eukaryotes algae and fungi. To determine the biological function of the protein, pfnita was expressed in Escherichia coli strains lacking the endogenous formate and nitrite transporters. In order to analyse the essentiality of the gene a reverse genetics approach was taken and the data discussed. Results indicate that the PfNitA protein is located in the plasma membrane and digestive vacuole of intraerythrocytic parasites suggesting a role in the uptake or excretion of metabolites. A second complexity with regard to treatment is the lack of a vaccine. A problem in crating a vaccine is antigenic variation. The PIR family of proteins contain a so-called hypervariable domain that has led to the suggestion that the family may play a role in antigenic variation. The objective of the work carried out in this thesis was to investigate the topology and structure of the PcCir2 protein of Plasmodium chabaudi, using E. coli as the expression host. The topology of Cir2 has been examined by means of reporter fusions and overexpression/purification studies undertaken towards crystallisation. As the PcCir2 amino acid sequence does not show significant homology to other proteins, structural data may provide insights into potential functional or binding domains.

MSc (Biochemistry) / Department of Biochemistry / Malaria is caused by protozoa of the genus Plasmodium. Malaria accounts for approximately more than half a million deaths yearly. Of the five species of Plasmodium, P. falciparum accounts for the most deadly form of the disease. P. falciparum survives under various physiological conditions during its life cycle. The parasite employs its molecular chaperones machinery particularly heat shock proteins (Hsps) to protect its protein constituents during physiological stress. Hsps are conserved molecules that constitute a major part of the cell’s molecular chaperone system. P. falciparum Hsps play an important cyto-protective role guaranteeing that the malarial parasite survives under the severe conditions that prevail in the host environment. PFF1010c is a type IV P. falciparum heat shock protein 40. PFF1010c is predicted to be expressed only at the gametocyte stage of the malarial parasite’s life cycle. The aim of the current study was to investigate the expression PFF1010c by parasites and the gametocyte stage as well as characterize the structure-function features of the protein. PFF1010c was successfully expressed in E. coli cells. Despite successful expression of the protein, its purification proved problematic. The attempt to purify PFF1010c was carried out under both native and denaturing conditions. Far Western blot analysis to investigate direct interaction between PFF1010c and PfHsp70-1 was conducted and no interaction was observed. Malarial parasites were harvested at different stages and total protein was isolated. The expression of PFF1010c was confirmed to occur at the gametocyte stage of the parasite’s development using Western blot analysis. This study confirmed that PFF1010c is only expressed at the gametocyte stage of the malarial parasite. Furthermore, PFF1010c was not expressed at the asexual stage. Possible interactors of PFF1010c were predicted by STRING, a bioinformatics based tool. The expression of PfHsp90, PfHop and PfHsp70-1 at the gametocyte stage was investigated and confirmed by Western blot analyses.

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

MSc (Chemistry) / Department of Chemistry / This dissertation focuses on the application of 1, 3, 5-triazine in a pharmaceutical point of view since it has wide range of uses as described in chapter 1. Malaria is one of the most prevalent and deadly infectious diseases worldwide though there are already many synthesized anti-malarial drugs which are in use presently, drug resistance seems to be one of the major problem and combination therapy seems to be the only solution for now. Hence in this study we used hybridization as a tool (Figure 9) to synthesize new antimalarial drugs using 1, 3, 5-triazine as an intermediate linker, linking known anti-malarial drug, different amine and chloroquine-like amines together using nucleophilic substitution reaction. As explained in chapter four of this dissertation, triazine is used to synthesize mono-, di- and tri-amino-1, 3, 5-triazine substituted products. Using THF as a solvent and K2CO3 as a base changing in temperatures, from 0 oC 25 oC or reflux. Some products were synthesized using microwave irradiation. The application of triazine as an intermediate linker in the above mentioned condition yielded five mono-amino substituted dichloro-1, 3, 5-triazine (21-25) in an average yield of 82%, three amino substitution chloro-1, 3, 5-triazine (26-28) in an average yield of 87%, two amino substituted-1, 3, 5-triazine (29, 30) in an average of 90%, nine chloroquine-like synthesized compounds (33-41) in 84 % average yields respectively. / NRF

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

Plasmodium vivax malaria threatens nearly half the world’s population. This relapsing disease may be more severe than previously recognised and is proving refractory to current malaria control measures. This thesis aimed to describe the burden of anaemia and mortality attributable to vivax malaria in Southern Papua, Indonesia, an area endemic for multidrug-resistant P. vivax and P. falciparum, and to determine the potential of currently available antimalarial drugs to reduce transmission of P. vivax in co-endemic regions. Approximately 0.5 million uniquely identified clinical records from patients presenting to Mitra Masyarakat Hospital between April 2004 and May 2009 were matched with corresponding laboratory and pharmacy data in order to determine the burden of anaemia in the hospital setting and the effectiveness of primaquine prescription for preventing P. vivax relapses. Clinical information extracted from patient notes was used to clarify the contribution of P. vivax malaria to a series of deaths detected by an active hospital-based surveillance system. Additional secondary sources of data used in this thesis included a large house-to-house survey and multiple clinical trials of antimalarial therapy from both Southern Papua and Northwestern Thailand. In Southern Papua, P. vivax malaria is an important cause of haematological morbidity both in the hospital and community setting. This morbidity is most significant in the first year of life when P. vivax infection accounts for 23% of all severe anaemia (haemoglobin <5g/dL) in the hospital and approximately 28% of all moderate-to-severe anaemia (haemoglobin <7g/dL) in the community. In this region concomitant P. vivax infection accentuates haematological impairment associated with P. falciparum malaria. Plasmodium vivax in Southern Papua rarely causes death directly but rather indirectly contributes to mortality through exacerbation of comorbid conditions. In Northwestern Thailand, 53.8% of patients with falciparum malaria who were treated with a rapidly eliminated drug between 1991 and 2005 had a recurrence of vivax malaria within two months making P. vivax infection the most common cause of parasitological failure in these individuals. Slowly eliminated artemisinin combination therapies (ACT) provided the greatest protection against recurrent P. vivax parasitaemia during 63 days of follow-up. In three randomised controlled trials from Papua and Thailand, P. vivax gametocytaemia was shown to mirror asexual parasitaemia closely and to have the same characteristics in acute and recurrent infections. This emphasises that the most important chemotherapeutic means of blocking P. vivax transmission is prevention of future relapse. Primaquine is recommended for this purpose but analyses in this thesis suggest that in Southern Papua, unsupervised primaquine at a dose of 0.5mg/kg/day for 14 days, does not reduce the risk of subsequent relapse (Adjusted Hazard Ratio = 1.01 [95% confidence interval 0.95-1.07]). Plasmodium vivax malaria should not be neglected. High priority must be given to new hypnozoitocidal drug discovery. In the interim, optimising the safety and effectiveness of primaquine and adoption of a unified ACT-based blood schizontocidal treatment strategy for malaria of any parasitological cause in co-endemic regions will be crucial for controlling P. vivax malaria.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, a common heritable trait whose prevalence mirrors geographic patterns of historic malaria endemicity, is thought to confer a selective advantage owing to partial protection conferred against malaria. Direct evidence supporting this malaria protection hypothesis in the form of clinical association studies remains controversial, however, as conflicting results have been reported with respect to the strength and specificity of a protective effect, if any, conferred to carriers of G6PD deficiency-associated alleles. This thesis examines genetic diversity at the G6PD locus, and then considers how such variation impacts both immediate molecular phenotypes and multifactorial clinical phenotypes. First, Chapter 3 presents a survey of variation at G6PD in several malaria-endemic areas, while Chapter 4 describes a novel technique for polymorphism discovery using pooled massively parallel sequencing. Next, in Chapters 5 and 6, I evaluate the link between genetic variation at the locus and G6PD enzyme activity, identifying major and minor determinants of G6PD deficiency state in an association study conducted in Kenya, and demonstrating a new technique for assaying G6PD deficiency at the level of an individual erythrocyte in a pilot project in Mali. Finally, Chapter 7 addresses the malaria protection hypothesis directly by conducting a fine-mapping case-control association study of severe malaria in the Gambia, where I found that G6PD deficiency alleles exhibited differential direction of association with respect to two important clinical syndromes-- trending towards risk conferred to severe malarial anemia, and protection with respect to cerebral malaria. Overall, these findings suggest that future clinical association studies should consider heterogeneity at the genetic level, as well as at the level of molecular and clinical phenotypes in order to achieve a better mechanistic understanding of the relationship between G6PD deficiency and severe malaria.