Malaria is a tropical disease affecting millions of people worldwide. Plasmodium falciparum is the causative agent of the most severe form of malaria, and therefore insights into the molecular mechanisms by which it functions are critical. The intraerythrocytic stage of the life cycle is responsible for the clinical manifestations of the disease. Numerous proteins are required for the invasion and remodelling of host erythrocytes, and need to be transported to the highly specialized organelles from which they are secreted (invasion proteins), or to the erythrocyte cytoplasm or membrane (exported proteins). It is postulated that newly synthesized proteins are transported from the Golgi network to their target destinations by specific interactions of target sequences of the proteins with the medium subunit (μ) of an adaptor protein (AP1) complex. Bioinformatic analysis of the putative P. falciparum AP1μ subunit, encoded by Pf13_0062, revealed a cargo-binding domain. Three regions, one of which encompassed the putative binding domain, while the other two interrupted this domain, were cloned into the pGEX-4T-2 expression vector. These recombinant proteins were expressed in E. coli with a GST tag, purified and immobilized on glutathione magnetic beads and used to biopan P. falciparum phage display libraries to identify interacting proteins. No binding was observed with the truncated domains, but several specific interactions were identified with the binding domain. One of these peptides was 13 amino acids long and contained a Yxx motif, indicating that PfAP1, like its homologues in higher eukaryotes, binds specifically to this motif in cargo proteins. Other sequences identified included a RRNIFLFINRKKE peptide; exported protein PHISTa; and conserved protein PFL0675c. In the C-terminal region of PFL0675c an armadillo repeat structure was predicted, just downstream of the binding domain identified by biopanning. This region of PFL0675c was therefore cloned into the pET-15b expression vector and expressed as a recombinant His-tagged protein. Slot overlays and far western blotting confirmed the specificity of the interaction with PfAP1. Since PFL0675c does not display the characteristics typical of AP1 cargo, it is postulated to be an accessory protein to the complex. Localization studies performed by transfection V of P. falciparum parasites with pARL2AP1GFP showed that in vivo, PfAP1 localized to distinct foci around the nucleus. Co-localization studies confirmed that PfAP1 localizes to the cis-Golgi in P. falciparum. PfAP1 may therefore be involved in trafficking proteins from the Golgi network to specific subcellular compartments within the parasite. This is the first study identifying interacting partners of PfAP1, and demonstrating its localization in P. falciparum 3D7 parasites.

A dissertation submitted to the Faculty of Science University of the Witwatersrand Johannesburg, in fulfilment of the requirements of the degree of Master of Science 14 February 2017. / Spirooxindoles are an important class of spirocycles in organic and medicinal chemistry. They are characterised by a spiro-ring fused with the oxindole scaffold and have a wide range of biological activity. We are particularly interested in spirooxindoles because of their antimalarial activity. Malaria is a major health problem in many parts of the world and the burden caused by the disease is still of great concern. In 2015 alone, an estimated 438 000 deaths due to malaria were reported across the world, with 90% of the deaths occurring in Africa. The increase in drug resistance to currently used antimalarial agents has rendered most of them ineffective, thereby contributing to the high mortality rates. As a result, there is a need for the development of new effective antimalarial agents. In the search for a new class of antimalarial chemotypes, cipargamin, introduced as NITD609 by norvatis in 2010 was synthesised. This compound is a novel synthetic antimalarial candidate, with an IC50 of ~1 nM against P. falciparum strains, including multi drug-resistant strains. Previously in our laboratory, several spirooxindole derivatives were synthesised using an imino Diels-Alder reaction, also known as the Povarov reaction. Of all the compounds synthesised, only those derived from a para-substituted aniline displayed activity in the low micromolar range (~5μM) against P. falciparum in vitro. In this project, we aimed to further explore the antimalarial activity of these compounds by designing and synthesising ring-opened analogues. The analogues were successfully synthesised by a Grignard addition reaction using N-Boc protected arylimines as electrophiles. Despite several attempts, we were unable to remove the Boc-protecting group in the final step. The second series of compounds we aimed to synthesise were ring closed analogues lacking one aromatic ring. The compounds were synthesised starting from an imine condensation reaction between benzyl protected isatin with para-substituted 2-allyanilines. The 2-allylanilines were prepared by subjecting N-allylanilines to an aza-Cope rearrangement. The arylimines prepared were then subjected to a nucleophilic Grignard addition reaction with commercially available vinylmagnesium bromide to yield the intermediate necessary for the ring closure step. Unfortunately, the nucleophilic addition reaction was unsuccessful. The ring-closure step is very crucial during the synthetic route as it gives rise to the desired ring closed analogues via ring-closing metathesis. Although we were unable to reach the final step in the synthesis of ring-closed analogues, some progress was made in developing methodology for the synthesis of these analogues. The synthesised ring-opened analogues were screened for antimalarial activity against P. falciparum in vitro. Six hit compounds were identified from the series of compounds tested with tert-butyl 3-(2,4-dichlorophenethyl)-2-oxo-3-(p-tolylamino)indoline-1-carboxylate 60 being the most active compound in the series with an IC50 value of 0.60 nM against the FCR 3 Strain. In general, compounds derived from p-toluidine displayed the most potent activity. / MT2017

A Thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg in the fulfillment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2017. / Malaria continues to be a global health problem. Despite a marked reduction in mortality over the last 15 years, these hard-fought gains are threatened by growing resistance of the Plasmodium falciparum malaria parasite to artemisinin, the frontline drug used in treatment of the disease. Clinical symptoms of malaria are caused by the intra-erythrocytic phase of the parasite life cycle. Entry into the erythrocyte is accomplished by several specialised invasion proteins, which are stored in unique apical secretory organelles known as micronemes and rhoptries. Very little is known about the trafficking signals and transport mechanisms of invasion proteins to these organelles. Three micronemal proteins, Apical Membrane Antigen-1 (PfAMA-1), Subtilisin-like protease 2 (PfSUB2) and Erythrocyte Binding Antigen 181 (PfEBA181) were investigated with the aim of identifying domains responsible for targeting the micronemes. Selected domains were amplified and mini-genes were created by overlap extension PCR. A pARLmCherry plasmid containing a Pfama-1 stage-specific promoter that is only active during the schizont stage of parasite development when micronemes are formed, was used to create mCherry-tagged constructs. P. falciparum parasites were transfected by electroporation of the plasmid constructs. Transgenic parasites were selected by drug pressure and the expression of red fluorescent mCherry-tagged chimaeric proteins was visualised in live parasites. Co-localisation studies were performed with a microneme marker to assess if the transgenic mini-proteins reached their destination. Interestingly, all three proteins required different domains to target the micronemes: PfEBA181 required an extended region of a conserved cysteine-rich domain, PfAMA-1 required the prodomain, and PfSUB2 required the transmembrane domain. Since no common targeting signal was identified, the possibility of a protein escorter was explored. The PfAMA-1 prodomain was expressed as a recombinant histidine-tagged protein and immobilised onto Nickel-coated beads, which were exposed to a P. falciparum phage display library for four rounds of biopanning. Two novel binding partners were identified: a putative Chaperone Binding Protein and a putative Formin 2. The identification of the molecular trafficking determinants of three invasion proteins, as well as a potential protein escorter for microneme targeting, represent novel findings that extend our knowledge of a fundamental biological process in the malaria parasite. This pathway may be exploited for drug development and new malaria treatment strategies. / MT2017

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The way malaria parasites are transmitted in space will have an influence on their genetic relationships. It can be expected that parasites collected within close geographic distances of each other would be more closely related than those across large geographic distances. Further to this, because malaria transmission is focal and heterogeneous in space, then the genetic relatedness between malaria parasites in these foci of malaria transmission would be greater within tightly clustered regions. Thus, using the level of genetic relatedness of these parasites would reveal how they are transmitted not only within these foci but at different geographic settings. This knowledge would offer insight on how malaria control methods can be effectively disseminated. In field settings malaria infections are polyclonal and each of the clones represented within these infections occur at different proportions. With the aid of genetic markers such as single nucleotide polymorphisms (SNPs) or microsatellites, parasite clonal genotypes can be identified. In this study, the genetic markers of choice are SNPs. Using a method that can quantify these SNPs representing the different clones occurring at different proportions in an isolate, then each of the clonal genotypes can be determined. Microsatellites were also used as additional markers in the study. In this thesis, 1.Genetic markers (SNPs) across the P. falciparum genome were identified (Chapter 3); 2. PyrosequencingTM was validated as a technique that would enable the identification of each genetically distinct clone represented in an infection by assigning proportions to the SNPs representing each genetically distinct clone and enabling the identification of parasite clonal genotypes in every isolate analysed. This was validated using laboratory prepared clone mixtures of P. falciparum. In addition; the progeny from a cross derived from genetically characterised 3D7 and HB3 isolates was analysed in preparation for the analysis of the field isolates (Chapter 4). , 4. In Chapter 5, field isolates were tested and clonal genotypes identified using both SNPs and microsatellites. A detailed population genetic analysis was also performed and finally in Chapter 6, evidence for correlation between the genetic relationships of these parasites and geographic distance was investigated. The results from field isolates summarised in this thesis were from analysis of 54 isolates; 7 samples collected from Cameroon, 13 from Kenya and 34 from Mali. The data consists of 13 SNPs analysed by PyrosequencingTM and 8 microsatellites. 84 clonal genotypes were identified by both genetic markers from the analysed isolates. Analysis of both SNPs and microsatellites revealed that microsatellites were more informative than the SNPs based on the observed allelic richness and heterozygosity (genetic diversity) across all loci analysed. The overall FST value was 0.061 using SNPs and 0.043 by microsatellites analysis. These values were low but consistent with what is typically observed in African P falciparum populations. Finally, analyses of the combined data set revealed that no statistically significant levels of spatial autocorrelation existed within the studied parasite populations. However, there was evidence of within host mixed parasite infections exhibiting a high level of genetic relatedness compared to between host infecting clones.

archives@tulane.edu / 1 / Brittany J Dodson

acase@tulane.edu / Since 2005, Senegal has scaled up malaria control interventions nationwide, mainly by an approach that allowed reaching people of highest needs. Activities have included vector control interventions such as Insecticide Treated Nets (ITN) and Indoor Residual Spraying (IRS), prevention of malaria in pregnant women, and diagnosis and treatment with an effective anti-malarial. This study aim to evaluate the impact of malaria interventions on all cause mortality among children under five years following the approach of targeting people of highest needs while scaling up of malaria control in Senegal. A “pre/post” study design following the recommendations of the RBM Monitoring and Evaluation Reference Group (MERG) was used. This assessment of the impact of the scalingup of malaria control interventions is based on a plausibility argument. Given that it is difficult to measure mortality resulting from malaria, the objective of the plausibility argument is to demonstrate the association between the scaling-up of malaria interventions and the reduction of all-cause mortality in children under 5 years of age in Senegal. Efforts in vector control led to an increase in the availability of resources, and substantial improvement in intervention coverage. Use of ITN by children under 5 increased from 7 per cent to 35 percent (p<0.001). The greatest increases were observed among populations most at risk of malaria, namely the poorest two quintiles, southern and central regions. Parasite prevalence decreased significantly from 6 per cent in 2008 to 3 per cent in 2010 (p< 0.001). The greatest reductions in anemia and parasitaemia were observed in populations from rural areas, the poorest populations, and populations from the central and southern epidemiological zones, who also displayed the highest increase in ownership and use of ITNs. All-cause under 5 mortality decreased by 40 per cent. Kaplan-Meier survival analysis showed better child survival over the period 2005–2010 compared to 2000–2005. Except for the region of Dakar, child survival estimates were higher in areas with the lowest prevalence of malaria. In addition, All-cause mortality in children under 5 years was significantly lower during the period after the scaling-up of malaria control interventions (OR: 0 63; 95% CI: 0.46–0.86). Other factors that might affect malaria transmission and child mortality were controlled for in the analysis. Despite increased rainfall malaria morbidity decreased, most strikingly among populations in which access to and use of ITNs increased most. While mortality declined in general during the study period, the greatest decreases in both parasitemia and child mortality were observed among the same populations that had the greatest increase in coverage of malaria control interventions. Similarly, the biggest declines in mortality occurred among the age group most likely to die of malaria, suggesting that malaria control interventions contributed substantially to the decrease in malaria morbidity, and consequently, to all-cause under 5 mortality. Based on the LiST model, the scaling-up of ITNs and IPTp from 2004–2010 averted 5,774 deaths in children under 5. The advent of home-based management to deliver malaria care at home, even in difficult to access rural areas, where the largest number of deaths usually occurs, has greatly contributed to expanding malaria case management across Senegal. All-cause mortality in children under 5 was significantly lower in the period after the scale up of malaria control interventions by targeting people of highest needs. The declines in mortality were greater in the populations and regions where coverage of malaria interventions was highest. The associations held even after taking into account other contextual factors. We drawn the conclusion that malaria control activities reduced malaria related morbidity and mortality, thus contributing to significant declines in all-cause child mortality between 2005- 2010 in Senegal. / 1 / Demba Anta Dione

The primary vector of malaria in the eastern United States, Anopheles quadrimaculatus (Say), was recently discovered to be a complex of five different cryptic species: A - An. quadrimaculatus, B - An. smaragdinus, C1 - An. diluvialis, C2 - An. Inundatus, D - An. maverlius (Reinert et al. 1997). In this research project, the goals were to determine which species were found in Texas, establish overall distribution patterns of those species, and observe the dates in which each specimens were collected so that any seasonal changes in species could be observed. Both An. quadrimaculatus (A) and An. smaragdinus (B) were identified from collections made throughout Texas from September 2002 through January 2005. Anopheles smaragdinus only made up 3% of the total specimens collected and neither An. inundatus nor An. maverlius were collected in Texas, even though they have both been collected in neighboring Parishes in Louisiana. Anopheles. quadrimaculatus' habitat and geographic range was found to be more extensive than An. smaragdinus. While An. smaragdinus was found only in the easternhalf of Texas with no collection south of Fort Bend County, An. quadrimaculatus was found throughout the eastern half of Texas, many of the southern Golf coast counties, and a few counties in far west Texas. The most common land cover where An. quadrimaculatus specimens were collected was on pasture/hay fields. This is very different from An. smaragdinus specimens in that pasture/hay was one of the least common land covers and the dominant land cover was woody wetlands. Overall, An. smaragdinus was usually associated with land covers that could provide shelter, while An. quadrimaculatus could be found among habitat that was more open and urban. There was no observed change in the species composition over time in this study. In fact, when An. smaragdinus was collected, An. quadrimaculatus was usually collected at the same time. Both An. quadrimaculatus and An. smaragdinus were collected throughout late spring, summer and early fall. Of course, the collection times of these species could have been an artifact of when most of the collectors were looking for An. quadrimaculatus (Say) specimens.

<p>Malaria remains the most important parasitic disease worldwide. It is estimated that over 500 million infections and more that 2.7 million deaths arising from malaria occur each year. Most (90%) of the infections occur in Africa with the most affected groups being children of less than five years of age and women. this dire situation is exacerbated by the emrggence of drug resistant strains of Plasmodium falciparum. The work reported in this thesis focuses on improving the purification of PfHPRT by investigating the characteristics of anion exchange DE-52 chromatography (the first stage of purification), developing an HPLC gel filtration method for examining the quaternary structure of the protein and possible end stage purification, and initialcrystalization trials. a homology model of the open, unligaded PfHPRT is constructed using the atoomic structures of human, T.ccruz and STryphimurium HPRT as templates.</p>