Please read the abstract in the section 00front of this document / Dissertation (MSc (Biochemistry))--University of Pretoria, 2005. / Biochemistry / unrestricted

In malaria-endemic regions, Plasmodium falciparum (P. falciparum) infection is characterized by extensive genetic/antigenic diversity. Describing this diversity provides important information about the local molecular epidemiology of infecting P. falciparum parasites. Intriguingly, one of the major obstacles to the development of an effective malaria vaccine has been the genetic polymorphisms exhibited by P. falciparum genes encoding targets of human immune system. This situation has necessitated the development of polyvalent vaccines with wide antigenic coverage that would increase the likelihood of vaccine efficacy that covers wide geographical areas of malaria endemic countries. Limited reports are available on the population genetic diversity and structure of P. falciparum in Malawi, and this is of particular concern as the country has put in place several interventions to combat the disease. The primary aim of the research project was to determine the genetic diversity and population structure of P. falciparum isolates and comparing complexity from four different epidemiological settings in Malawi using msp-2 gene polymorphisms. Samples were collected from four epidemiological locations in the north, centre and southern regions of Malawi. The diversity and genetic differentiation of P. falciparum populations were analyzed based on the highly polymorphic block 3 msp-2 gene. One hundred and twenty patient samples who presented with signs and symptoms of malaria and who had microscopically confirmed P. falciparum infection were enrolled in the study after they had satisfied the inclusion criteria. Parasite DNA was extracted from the blood spot on to filter paper and analyzed by genotyping the msp-2 gene using allele-specific nested PCR. A total of 28 msp-2 block 3 fragments, defined by the size and the allelic types were detected in the 102 patients. The length variants of the PCR product ranged from 240basepairs (bp) to 450bp for the K1/FC and 410bp to 780bp for the 3D7/IC allelic families. Isolates of the 3D7 alleles were predominant in the population (59 percent), compared to isolates of the K1/ FC27 alleles (41 percent) and for 3D7 and K1 most of the isolates were monoclonal infections. In comparisons between the sites, we observed the highest prevalence of mixed infection in Mwanza (46.7 percent) followed by Dwangwa (23.3 percent) compared to Bolero (16.7 percent) and Mitundu (16.7 percent). The difference in prevalence of mixed infections between Mwanza and the other sites was statistically significant (p=0.041). There was also a non-significant trend towards a higher mean genotype number per isolate in the children aged >5 years compared to those below 5 years of age. These data suggest differences in prevalence rates of mixed infections in different geographical/epidemiological settings in Malawi. Further studies are needed to confirm, with larger sample sizes, the observation of a non-significant trend towards higher multiclonality of infection in older children in malaria endemic areas of Malawi.

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Malarial infection in naïve individuals induces a robust innate immune response, but our understanding of the mechanisms by which the innate immune system recognizes malaria and regulates its response remain incomplete. Our group previously showed that stimulation of macrophages with Plasmodium falciparum genomic DNA (gDNA) and AT-rich oligodeoxynucleotides (ODNs) derived from this gDNA induces the production of type I interferons (IFN-I) through a STING/TBK1/IRF3-dependent pathway; however, the identity of the upstream cytosolic DNA receptor remained elusive. Here, we demonstrate that this IFN-I response is dependent on cyclic GMP-AMP synthase (cGAS). cGAS produced the cyclic dinucleotide 2’3’-cGAMP in response to P. falciparum gDNA and AT-rich ODNs, inducing IRF3 phosphorylation and IFNB transcription. In the recently described model of innate immune memory, an initial stimulus primes the innate immune system to either hyperrespond (termed “training”) or hyporespond (“tolerance”) to subsequent immune challenge. Previous work in mice and humans demonstrated that infection with malaria can both serve as a priming stimulus and promote tolerance to subsequent infection. In this study, we demonstrate that initial stimulation with P. falciparum-infected red blood cells (iRBCs) or the malaria crystal hemozoin (Hz) induced human adherent peripheral blood mononuclear cells (PBMCs) to hyperrespond to subsequent Toll-like receptor (TLR) challenge. This hyperresponsiveness correlated with increased H3K4me3 at important immunometabolic promoters, and these epigenetic modifications were also seen in monocytes from Kenyan children naturally infected with malaria. However, the use of epigenetic and metabolic inhibitors indicated that malaria-induced trained immunity may occur via previously unrecognized mechanism(s).

Selon les estimations de l'OMS, le bilan mondial du paludisme a atteint 212 millions de cas et 429 000 décèsen 2015 (OMS, 2016). Cette gravité est principalement due à Plasmodium falciparum. A l’heure actuelle, P. falciparum présente des résistances à tous les antipaludiques donnés en monothérapie.Par conséquent, pour réduire le risque d’échec thérapeutique, l'OMS a recommandé depuis 2001 l’utilisation de bithérapie, notamment d'Artemisinin Combination Therapy (ACT), comme traitement de première intention.Les ACT sont composés essentiellement d’un dérivé d’artéminisine, à demi-vie courte et un autre antipaludique à demi-vie longue, connu en monothérapie.Le parasite a également montré des signes de résistance aux ACT, principalement en Asie du Sud-est, menaçant les programmes d’éradications contre le paludisme.La découverte de nouveaux composés à activité antipaludique ou de nouvelles procédures de traitement sont urgentes.La valorisation d’anciennes molécules est également au cœur des études afin d’améliorer notamment leur biodisponibilité et réverser les mécanismes de résistance du parasite. Ainsi, des études prouvent l’intérêt de l’utilisation de nanotechnologies pour l’amélioration de l’efficacité d’antipaludiques. L’atovaquone en est un exemple, cette modification a notamment permis d’améliorer sa biodisponibilité. Notre étude a porté sur une de ces formulations, de l’atovaquone encapsulée dans une nanoémulsion cationique (NE) appelée ATQ. Une deuxième génération a ensuite été testée par association d’oligonucléotides antisens anti-ARNm de topoisomérase II(AST) de P. falciparum. En effet, des stratégies antisens thérapeutiques font leur preuve en santé humaine et présentent un intérêt croissant en parasitologie. Les NE/AST ont montré une activité anti-palustre spécifique contre P. falciparum in vitro. Leur spécificité a permis d’aboutir à l’arrêt du cycle cellulaire et une forte diminution du taux d’ARNm de la topoisomérase II. Ce phénomène a montré être dépendant de l’action de la RNase H. Un effet synergique de ces NE/AST a également été montré en association avec la chloroquine, l’atovaquone et la dihydroartémisinine sur une souche sensible de P. falciparum et des souches résistantes aux antipaludiques précédemment cités.L’ATQ a également montré une forte efficacité sur une souche résistante à l’atovaquone d’un facteur 5. En présence d’ATQ, la mitochondrie a rapidement été altérée conduisant à une mort précoce du parasite. Un traitement à l’ATQ a abouti à la guérison de souris Swiss infectée par P. berghei après deux injections en i.v. en 5 jours. Enfin, l’ATQ/AST a montré une efficacité in vitro contre P. falciparum et P. berghei in vivo. Un test de cytoadhérance des hématies parasitées a des cellules endothéliales a révélé un fort pourvoir d’inhibition de la cytoadhérance de l’ATQ/AST.Un résultat prometteur dans le cadre de traitement du neuropaludisme. / According to the estimations of theWHO, in 2015, 212million cases ofmalariahave been reported(WHO,2016). These figuresmakemalariathe most deadlyparasitic diseasein the world, with429.000deaths per year. Some treatments against Plasmodium falciparum exist. However, no really good treatment option can be found in monotherapy due to the resistance emergency. Therefore To reduce the risk of resistance, WHO has recommended since 2001 combination therapies, which is basically an Artemisinin Combined Therapy (ACT), as first-line treatment. The main problem of commercialized bi-therapy is that they are composed of two molecules with individual resistance which leaded to the emergence of resistance to the latest ACTs such as a dihydroartemisinin /piperaquine combinationmainly in South-East Asia.Thus the use of new therapeutic combination strategy that can bypass the parasites' mechanisms of resistance is urgent to effectively treat malaria. As the pathway from drug discovery to drug commercialization is both long and very expensive, it is essential to develop ways to improve existing antimalarial treatments. In the first place it’s necessary to find a new antimalarial formulation based on an already commercialized drug to modify its biodisponibility and its mechanism of action in order to revert the resistance. In the second place its necessary to associate this formulation with a novel none commercialized antimalarial strategy such as the antisens oligonucleotides already usedinhumanhealth. In our lab we have developed nanoemulsions containing atovaquone and antisense oligonucleotides anti topoisomerase II against P. falciparum.Nanoemulsionsvectoringantisens oligonucleotidesandused againstP. falciparum topoisomerase II(NE/AST) showed encouraging anti-parasite killing results.Additionalresultshave showna synergistic in vitro effectwithantimalarial drugs(chloroquine, dihydroartemisinin and atovaquone) in sensitive and resistances strains. Moreover NE/ASTrestricted Topoisomerase II gene expression and blocked the cell cycle in G2/M phase leading to parasite’s death by mitophagy.As Drug delivery systemscan improve the efficacy ofcommon antimalarial drugs by delivering the drug to its target, while protecting it from degradation in biological environment and increasing its biodisponibility, our nanoemulsions containing atovaquone (ATQ) leaded to reversion of atovaquone resistance with 5 fold decrease in its IC50. Observations made with confocal microscopy have shown mitochondrial alteration after ATQ treatment.Our novel and original bi-therapy is focused on the association ofATQ with NE/AST (ATQ/AST).We obtained an IC50 8-fold lower than atovaquone’s IC50with total inhibition of parasites’ capacity to reinfect new red blood cells. A cytoadherence test of parasitized erythrocytes to endothelial cells revealed a strong capacity of cytoadherence inhibition of ATQ / AST, a promising result in the treatment of cerebral malaria.

Malaria is caused by the parasite Plasmodium falciparum and still plagues many parts of the world. To date, efforts to control the spread of the parasites have been largely ineffective. Due to development of resistance by the parasites to current therapeutics there is an urgent need for new classes of therapeutics. The vitamin B6 biosynthetic pathway consists of a PLP synthase which produces pyridoxal 5'-phosphate (PLP) within the parasite. The absence of this pathway in humans makes it attractive for selective targeting using small chemical molecules. The PLP synthase condenses D-ribose 5-phosphate (R5P) and DL-glyceraldehyde 3-phosphate (G3P) with ammonia to form PLP. Two proteins make up this PLP synthase – PfPdx1 and PfPdx2. Computational modelling of Pf Pdx1, and mapping of the R5P-binding site pharmacophore facilitated the identification of several ligands with predicted favourable binding interactions. Confirmatory testing of these on the purified Pf Pdx1 in vitro revealed D-erythrose 4-phosphate (E4P) and an analogue 4-phospho-D-erythronhydrazide (4PEHz) were capable of dose-dependently inhibiting the enzyme. The acyclic tetrose scaffold of E4P, with both aldehyde and phosphate group moieties, was thought to affect R5P imine bond formation in Pf Pdx1, possibly allowing the molecule to enter the R5P-binding site of Pf Pdx1. This hypothesis was supported by molecular docking simulations, and suggested that 4PEHz could similarly enter the R5P-binding site. 4PEHz was detrimental to the proliferation of cultured P. falciparum intraerythrocytic parasites and had an inhibitory concentration (IC50) of 10 µM. The selectivity of 4PEHz in targeting Pf Pdx1 was investigated using transgenic cell lines over-expressing Pf Pdx1 and Pf Pdx2, revealing that complementation of PLP biosynthesis rescued the parasites from the detrimental effects of 4PEHz. Functional transcriptomic and proteomic characterisation of 4PEHz-treated parasites revealed that the expression of Pf Pdx2 increased during 4PEHz treatment, moreover showed that other PLP-related processes were affected. These results supported that Pf Pdx1 is targeted by 4PEHz, and affected PLP biosynthesis de novo. Results from this study allude to alternative regulation of de novo PLP biosynthesis within the parasites by E4P. Moreover, contributions from this work showed that the de novo vitamin B6 pathway of P. falciparum is chemically targetable, and a potential strategy for the development of newer antimalarials. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Biochemistry / Unrestricted

Malaria is one of the most serious tropical infectious diseases affecting mankind. The prevention of the disease is hampered by the increasing resistance of the parasite to existing chemotherapy and -prophylaxis drugs. The need for novel therapeutic targets and drugs is therefore enormous and the understanding of the biochemistry of the parasite is imperative. The aim of this study was the identification and molecular characterisation of the eDNA of one such metabolic target protein, ornithine decarboxylase (ODC), in the human malaria parasite P. falciparum. The P. falciparum ODC eDNA was isolated by means of a modified RT-PCR technique, RACE. No sequence data were available and the primers used were based on consensus areas identified in the protein sequences from other related organisms. The isolation and identification of the eDNA with degenerate primers was successful in 3' -RACE, but necessitated the optimisation of the eDNA synthesis protocol and the use of total RNA as starting material. The sequence obtained facilitated the application of 5' -RACE with ODC-specific primers based on the 3' -RACE sequence data. The full-length ODC eDNA sequence was obtained by overlap-alignment of various segments. A novel suppression PCR technology was applied during the 5' -RACE in order to create an uncloned eDNA library of amplified cDNAs representing only the mRNA population. The P. falciparum ODC eDNA contains an open reading frame of ---2847 bp and translates to a large 939 amino acid protein. The protein contained large internal insertions and was extended by '""273 N-terminal residues compared to ODCs from other organisms. Several possible signature motifs were identified for phosphorylation, glycosylation and transamidation. The P. falciparum ODC protein seems to contain more hydrophilic and a-helix forming residues. These characteristics should be further investigated after expression of the recombinant protein. The isolation of the P. falciparum ODC eDNA facilitates the validation of this protein as an antimalarial target. / Dissertation (MSc)--University of Pretoria, 1998. / gm2014 / Biochemistry / unrestricted