Malaria, a mosquito-borne infectious disease, caused by the protozoan Plasmodium genus, is the greatest health challenges worldwide. The plasmodial vitamin B1 biosynthetic enzyme PfThzK diverges significantly, both structurally and functionally from its counterpart in higher eukaryotes, thereby making it particularly attractive as a biomedical target. In the present study, PfThzK was recombinantly produced as 6×His fusion protein in E. coli BL21, purified using nickel affinity chromatography and size exclusion chromatography resulting in 1.03% yield and specific activity 0.28 U/mg. The enzyme was found to be a monomer with a molecular mass of 34 kDa. Characterization of the PfThzK showed an optimum temperature and pH of 37°C and 7.5 respectively, and it is relatively stable (t₁/₂=2.66 h). Ag nanoparticles were synthesized by NaBH₄/tannic acid, and characterized by UV-vis spectroscopy and transmission electron microscopy. The morphologies of these Ag nanoparticles (in terms of size) synthesized by tannic acid appeared to be more controlled with the size of 7.06±2.41 nm, compared with those synthesized by NaBH₄, with the sized of 12.9±4.21 nm. The purified PfThzK was challenged with Ag NPs synthesized by tannic acid, and the results suggested that they competitively inhibited PfThzK (89 %) at low concentrations (5-10 μM) with a Ki = 6.45 μM.

Malaria, a disease transmitted by female mosquitoes, has plagued the world for many centuries. The disease is associated with high mortality rates, severe poverty, and economic burden. These are factors which hamper effective eradication of the disease. Drug resistant forms of the parasite have caused increasing concerns and questioned the longevity of current effective antimalarials. Efforts are therefore aimed at the identification and exploitation of essential parasite proteins as potential drug targets. The polyamine pathway of Plasmodium falciparum is an exploitable pathway which contains two distinct, chemically validated drug targets; a bifunctional PfAdoMetDC-ODC protein and PfSpdSyn. These enzymes ensure intricate regulation of polyamine production and the pathway contains various distinctive features which could be selectively targetable from the mammalian counterpart pathways. However, inhibition of polyamine production through the use of specific enzyme inhibitors has revealed various compensatory responses that negate the efficacy of these inhibitors. An account is given of the metabolomic fluctuations in the parasite during inhibition of polyamine biosynthesis. From co-inhibited P. falciparum extracts, it could be demonstrated that the characteristic growth-arrest coincided with the depletion in spermidine, the metabolic product of PfSpdSyn. The co-inhibition strategy therefore emphasised the importance of spermidine biosynthesis by PfSpdSyn. Moreover, adenosyl-related metabolite levels were not disrupted during polyamine depletion, supporting the notion that these metabolites are intricately recycled within the parasites. The identified metabolic compensatory mechanisms have further potential for exploitation, and can strategically be combined with polyamine biosynthesis inhibition to ensure parasitic attenuation. In addition, several novel inhibitors were previously computationally identified, based on a dynamic receptor-based pharmacophore model of PfSpdSyn. The in vitro inhibiting activity of these compounds was determined against PfSpdSyn. Results from the in vitro experiments supported the in silico predictions, and emphasized the supportive role of pharmacophore modelling has for the identification of novel inhibitors. The research contributed in understanding parasitic polyamine metabolite regulation, and will aid in the future optimization of therapeutic strategies, aimed at exploitation of the polyamine pathway as a potential antimalarial drug target. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Biochemistry / unrestricted

Malaria is a devastating disease which affects almost half of the world’s population. Since the description of the malaria genome sequence, various aspects of the parasite have been studied, including drug resistance mechanisms, epidemiology and surveillance systems. Alarmingly, very little is known about the basic biological processes such as the regulation of the expression of parasite genes. The parasite, Plasmodium falciparum, has developed highly specialized methods of regulating the transcription of genes, starting at the regulation of genes controlling basic cellular processes such as protein synthesis and erythrocyte invasion, followed by the transcriptional regulation of more specialized genes, such as those aiding in immune evasion and pathogenesis. The description of the P. falciparum transcriptome by Bozdech et al. in 2003 revealed a complex, just-in-time and tightly regulated transcription profile of P. falciparum genes. This suggests that the most probable Achilles heel for Plasmodium may be its unique mechanisms of regulating gene expression. Various cis- and trans-regulatory sequences have been identified in P. falciparum, along with possible DNA (and RNA) binding proteins. The first part of this research focussed on transcriptional regulatory mechanisms in which an in silico search identified cis-regulatory sequences in the 5’ untranslated region of the antigenically variant var gene family. Electrophoretic mobility shift assays (EMSA) were used to identify protein binding partners of these sequences, which could ultimately act as transcription factors in regulating the expression of this essential gene family. The second part of the research investigated the involvement of post-transcriptional regulatory mechanisms in the polyamine biosynthetic pathway of P. falciparum. Polyamines have been proven to be crucial for the parasite’s development and therefore, an RNA interference knock-down strategy was used to verify the importance of the polyamine biosynthetic enzymes S-Adenosylmethionine decarboxylase (AdoMetDC), Ornithine decarboxylase (ODC) and Spermidine synthase. It is clear that various mechanisms for gene regulation are used by the parasite and that this is critical for the survival of this organism. The results of this study suggest the potential presence of both double-stranded and single-stranded DNA regulatory proteins within P. falciparum nuclear extract. As controversial as RNA interference remains in P. falciparum, this technique was used as a plausible knock-down strategy of parasite specific genes and certain trends, regarding the visible decreases in gene transcript level after double-stranded RNA treatment, were observed. However, final conclusions as to the feasibility of using RNA interference in P. falciparum remain to be elucidated. This study therefore ultimately lends insight into the transcriptional and post-transcriptional levels of P. falciparum gene regulation. / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted

Of the considerable challenges researchers face in the control and elimination of malaria, the development of antimalarial drug resistance in parasite populations remains a significant hurdle to progress worldwide. Atovaquone is used in combination with proguanil (Malarone) as an antimalarial treatment in uncomplicated malaria, but is rendered ineffective by the rapid development of atovaquone resistance during treatment. Previous studies have established that de novo mutant parasites confer resistance to atovaquone with a substitution in amino acid 268 in the cytochrome b gene encoded by the parasite mitochondrial genome, yet much is still unknown about how this resistance develops, and whether parasites are inherently predisposed to resistance development. Here we report phenotypic characterization of isolates from patients that failed treatment in the original atovaquone Phase II studies in Thailand by using a diverse series of chemotypes that target mitochondrial functions. We defined their structure-activity relationships and observed broad resistance (5-30,000 fold in atovaquone), suggesting that cytochrome b mutations alone are not sufficient to explain this spectrum of resistance. We also report the first known in vitro selection that recapitulates the clinical Y268S mutation using the TM90-C2A genetic background, the pre-treatment parent for TM90-C2B. Selection of the Y268S mutation in TM90-C2A and others indicates that the parasite genetic background is critical in the selection of clinical atovaquone resistance, since selection attempts in multiple other genetic backgrounds results in mutations at positions other than amino acid 268. We implicate mitochondrial heteroplasmy in the development of sporadic, rapid resistance to atovaquone, where pre-existing low-level mutations in the multi-copy mitochondrial DNA can be quickly selected for in parasite populations. High-coverage mitochondrial deep-sequencing data showed that low-level Y268S mutants were present in admission parasites from the atovaquone Phase II clinical trials in Thailand, and recrudescent parasites either maintained high level Y268S mutation frequencies or gradually returned to cryptic Y268S levels. The phenomenon of gradual heteroplasmic conversion back to wild-type was noted in some ex vivo patient isolated parasites as well as some in vitro selected lines, which suggests that other factors are at play that influence heteroplasmy stability. In addition to mitochondrial heteroplasmy, the total mtDNA copy number is likely influencing phenotypes in a gene dose-dependent fashion. Further, pressure on the DHODH enzyme that results in DHODH copy number amplifications/mutations has been shown to influence mitochondrial heteroplasmy directly. Last, mitochondrial diversity was shown to be vastly underestimated without heteroplasmic loci being taken into account, as seen in the re-analysis of the Pf3K MalariaGEN genome dataset we performed. The complex interactions between these drug resistance mechanisms reveal the phenotypic and genotypic plasticity that the Plasmodium falciparum parasite utilizes are a clear fitness advantage in the face of mitochondrial stress, and further studies are required to determine the impact of this wide-ranging phenotype on the development of new mitochondria-targeted drugs.

Vector-borne diseases, such as those transmitted by mosquitoes, pose a significant public health concern in many countries worldwide. In this thesis, I explored the role of a number of risk factors defined at multiple scales on vector-borne disease prevalence, focusing on malaria and arboviral infections in several regions of North-Eastern Tanzania, with the principal aim of improving the overall diagnosis of febrile illness in this region. First, I investigated the influence of household-wealth on prevalence of malaria and arboviral infections using principal component analysis (PCA), and then described the methodological challenges associated with this statistical technique when used to compute indices from smaller datasets. I then employed a multilevel modelling approach to simultaneously incorporate household-level anthropogenic factors and village-level environmental characteristics to investigate key determinants of Anopheles malaria vector density among rural households. These analyses provided methodologically rigorous approaches to studying vector-borne diseases at a very fine-scale and also have significant public health relevance as the research findings can assist in guiding policy decisions regarding surveillance efforts as well as inform where and when to prioritize interventions.

This thesis combines four published research papers and a book chapter investigating aspects of central metabolism in the human malaria parasite Plasmodium falciparum. The publications are preceded by a statement which explores features of the research not fully described in the published texts, incorporates a review of the development over time and the present state of relevant scientific knowledge, and discusses the place of the individual papers and book chapter within malaria research. An assessment of the impact of each publication on its field of study is also included. A general discussion of the combination of papers as representative of the progress of research into the metabolism of malaria parasites concludes the statement section. The first publication is a chapter from a book, which describes detailed methods for the in vitro cultivation of P. falciparum. Such methodology, both robust and reliable, is a prerequisite for any investigation of parasite metabolism. The following publications are all primary research papers. The second publication describes the isolation and characterisation of the gene encoding the glycolytic pathway enzyme enolase from P. falciparum. The inferred amino acid sequence included peptide insertions found only in the enolases of higher plants and other photosynthetic organisms. This raised implications concerning the deep evolutionary history of the malaria parasite and related species. The third is concerned with the elucidation of the molecular basis of resistance to the antimalarial drug sulfadoxine. Resistance was found to result from point mutations within the dihydropteroate synthetase domain of the bifunctional protein hydroxymethylpterin pyrophosphokinase-dihydroptero¬ate synthetase, an enzyme of the parasite folate pathway. Additionally, it was discovered that the presence of exogenous folate has an antagonistic effect on sulfadoxine in some parasites of a defined genotype. This highlighted the importance of folate salvage in parasite metabolism. Fourth is a paper representing the discovery of a novel metabolism in both P. falciparum and the related apicomplexan parasite Toxoplasma gondii. The use of parasite genes in rescuing an Escherichia coli tyrosine auxotroph resulted in a proof of function of the products of these genes as pterin-4a-carbinolaminedehydratases. Pterin recycling, hitherto undetected in apicomplexans, was therefore added to the known metabolic processes of these organisms. The final paper describes an investigation into the subcellular distribution of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) within P. falciparum erythrocytic stage parasites. The use of confocal laser scanning microscopy and immunofluorescent techniques showed that SHMTc, the sole enzymatically active parasite SHMT protein, was found in the cytoplasm but also showed a stage-specific localisation to both the mitochondrion and apicoplast organelles. The otherwise enigmatic, enzymatically inert, SHMTm paralogue revealed a possible function, when in complex, in allowing targeted localisation of SHMTc to the mitochondrion. The spatial distribution of SHMTm also suggested a possible role in the morphogenesis of elongating apicoplasts during schizogony.

Enzymes are immensely powerful and efficient heterogenous catalysts which are essential for life. As essential to life as enzymes are, it is still not well understood exactly how they enhance the rate of their catalyzed reactions up to 19 orders of magnitude over their solution phase counterpart reactions. Recent research has focused on sub--picosecond motions coupled to the reaction coordinate, called rate--promoting vibrations, which are important components of several well--known enzymatic mechanisms and build upon previous models of enzyme activity. Herein I present two studies which are expressly focused on providing tools and knowledge to understand how dynamics affects enzymatic reactions. First, I present a method for the calculation of kinetic isotope effects from first principles, using transition path sampling and centroid molecular dynamics. This method allows for the calculation of kinetic isotope effects without the assumptions necessitated by transition state theory or free energy perturbation methods. It was found that this method could calculate the primary H/D kinetic isotope effect of the conversion of benzyl alcohol to benzaldehyde in yeast alcohol dehydrogenase to within the margin of error of experimentally measured kinetic isotope effects of the same reaction. Second, I examined the role that evolution plays in the preservation of these rate--promoting vibrations, by performing a transition path sampling study of two lactate dehydrogenases, those of Plasmodium falciparum and Cryptosporidium parvum, which evolved through separate gene duplication events from a common malate dehydrogenase ancestor. It was found that though both lactate dehydrogenases share the same rate--promoting vibration, and indeed share the rate--promoting vibration found in other lactate dehydrogenases, the sequence variations in lactate dehydrogenase from P. falciparum causes a diminished contribution of the motions to the reaction coordinate. The studies presented in this dissertation contribute to the our understanding of enzymes on an atomistic level, as well as providing tools necessary for designing novel de novo enzymes and targeted drugs for enzymes of disease--causing organisms.

La pompe ATPase Ca2+ du réticulum sarco-endoplasmique Plasmodium falciparum (PfATP6) est une protéine de dix transmembranes, impliqué dans la régulation de l'homéostasie du calcium dans le parasite. L'importance d'étudier cette protéine repose sur l'hypothèse d'être engagé dans le mécanisme d'action et de résistance des artémisinines. Des travaux précédents, fondé sur l'expression hétérologue dans des ovocytes de Xenopus laevis et Saccharomyces cerevisiae, ont montré des résultats opposés, générant de nombreux corollaires vérifiables. Par conséquent, des travaux supplémentaires sont nécessaires pour mieux comprendre la nature des interactions entre les artémisinines et transporteurs de type SERCA.Afin d'évaluer le caractère essentiel du gène de Plasmodium spp., une approche de génétique inverse a été utilisée. Knockout du gène, soit P. falciparum et berghei, ne pouvant pas être obtenu. La complémentation de sauvetage épisomique a été jugée impossible. Marquage à la fin 3' de PfATP6 et PbATP6 a été, également, tenté pour étudier la localisation et l'expression de la protéine chez les parasites. La manipulation des gènes à cette place n'a pas permis la survie du parasite. Nos résultats, pris ensemble, montrent que ATP6 est essentiel dans Plasmodium spp..Au cours de nos études génétiques, un phénotype stable et particulier de parasites du genre Plasmodium falciparum 3D7 a été distingué. Les étranges parasites “monstres" contiennent une vacuole digestive inhabituelle gonflées à travers toutes les étapes du développement du parasite. Caractérisation de l'insolite Plasmodium a été réalisée, montrant une sensibilité accrue à la chloroquine, mais pas à l'artémisinine ou de la méfloquine. Tenant compte de la similitude du PfATP6 avec la pompe SERCA orthologue mammifère, de nouvelles molécules, connu et synthétisé pour cibler spécifiquement la protéine chez les mammifères, ont été testés sur P. falciparum. Quatre classes différentes de composés (sHA 14-1, BHQ, chalcone et des analogues de l'ACP) a montré le blocage de la croissance in vitro du P. falciparum 3D7 et Dd2 à des concentrations inférieure au range micromolaire. En outre, une nouvelle classe de molécules (thaperoxides), conçu comme un hybride entre l'artémisinine et thapsigargine, a été testé contre le type sauvage 7G8 et la ligne muté L263E. Ce dernier porte une mutation ponctuelle unique de nucléotides dans PfATP6, déjà connu d'être impliqué dans la résistance du l'artémisinine.Compte tenu de la difficulté à manipuler les gènes du parasite, et afin de mieux caractériser PfATP6, un gène synthétique a été optimisé pour l'expression hétérologue chez S. cerevisiae. De cette façon, la complémentation d'une ligne de levure mutée (K616) sans les pompes endogènes Ca2+ de type P a été permis avec succès, montrant le sauvetage de la croissance de la levure en présence de forte concentration de calcium libre. Différents inhibiteurs de SERCA, comme la thapsigargine et l'acide cyclopiazonique, ont été testés sur la levure complémenté K616 PfATP6, afin de vérifier l'inhibition de la croissance. Tous les composés ont bloqué la croissance de levure sélectivement ciblant le PfATP6. En outre, le test a été développé comme un criblage de haute performance, afin de tester de nouvelles molécules pour leur activité. La méthode s'est révélée être un outil rapide et très fiable et reproductible pour l'identification de nouveaux composés actifs. / The Plasmodium falciparum sarco-endoplasmic reticulum ATPase Ca2+ pump (PfATP6) is a ten transmembrane protein involved in the regulation of the calcium homeostasis in the parasite. The importance of studying this protein relies on the fact that it has been hypothesized to be involved in the mechanism of action and resistance of artemisinins. Previous works, based on heterologous expression in Xenopus laevis oocytes and Saccharomyces cerevisiae, have shown contrasting results, generating many testable corollaries. Therefore, further work is needed to better understand the nature of interactions between artemisinins and SERCA-type transporters.In order to assess the essentiality of the gene in Plasmodium spp., a reverse genetics approach has been used. Knockout of the gene, in either P. falciparum and berghei, could not be achieved. Complementation for episomal rescue was found to be not possible. Tagging at the 3' end of PfATP6 and PbATP6 has been, also, attempted to study localization and expression of the protein in parasites. Manipulation of the gene at this position did not permit parasite survival. Our results, taken together, show that ATP6 is essential in Plasmodium spp..During our genetic studies, a stable and peculiar phenotype of Plasmodium falciparum 3D7 parasites has been noticed. The odd “monster” parasites contain an unusual swollen food vacuole throughout all stages of parasite development. Characterization of the unusual Plasmodium has been carried out, showing an increased sensitivity to chloroquine, but not to artemisinin or mefloquine. Taking into account the similarity of PfATP6 with the mammalian orthologue SERCA pump, new molecules, designed and synthesized to specifically target the mammalian protein, were tested on P. falciparum parasites. Four different classes of compounds (sHA 14-1, BHQ, chalcone and CPA analogues) showed to inhibit P. falciparum 3D7 and Dd2 growth in vitro at concentrations in the lower micromolar range. In addition, a novel class of molecules (thaperoxides), designed as an hybrid between artemisinin and thapsigargin, has been tested against 7G8 wild type and mutated L263E line. The latter carries a single nucleotide point mutation in PfATP6 that has been previously shown to be involved in artemisinin resistance. Considering the difficulty in manipulating the gene in the parasite and in order to better characterize PfATP6, a synthetic gene was optimized for heterologous expression in S. cerevisiae. This enabled successful complementation of a mutated yeast line (K616) lacking the endogenous P-type Ca2+ pumps, showing rescue of the yeast growth in presence of high concentration of free calcium. Different SERCA inhibitors, such as thapsigargin and cyclopiazonic acid, have been tested on K616 PfATP6 complemented yeast, in order to check for growth inhibition. All compounds showed to inhibit yeast growth selectively targeting PfATP6. In addition, the assay has been developed as a high throughput screening, in order to test new molecules for their activity. The method has proved to be a fast, highly reliable and reproducible tool for identification of new active compounds.

Titre de la thèse : Développement de thérapies adjuvantes associées à l'anti-cytoadhérence. Résumé : L'antigène de surface PfEMP-1 (P. falciparum Erythrocyte Membrane Protein-1) encodé par 60 gènes de la famille var du parasite, est un facteur de virulence du paludisme touchant l'Homme. Les différents variants de PfEMP-1 sont impliqués dans la cytoadhérence des érythrocytes infectés par P. falciparum (iRBCs) avec plusieurs récepteurs de l'endothélium vasculaire de l'hôte. Parmi eux, l'interaction avec ICAM-1 semble être liée à des manifestations sévères de la maladie telles que le paludisme cérébral. La majorité de la mortalité due à un paludisme sévère est observée dans les 24 heures après admission à l'hôpital, malgré l'utilisation d'antipaludiques efficaces, soulignant ainsi un besoin urgent en thérapies adjuvantes ciblant spécifiquement la cytoadhérence.Le site d'interaction d'ICAM-1 avec les érythrocytes infectés a été identifié au niveau du « BED side » de son domaine N-terminal, similaire à celui des immunoglobulines. Les variants antigéniques de P. falciparum capables de se lier à ICAM-1 présentent des différences subtiles au niveau des résidus responsables de cette interaction. La boucle DE semble être l'élément commun des sites de fixation à ICAM-1 pour trois variants de P. falciparum (ITO4-A4, ITO4-C24 and ItG-ICAM) et fut ainsi choisie par le groupe de Matthias Dormeyer comme cible pour un criblage in silico. Une librairie de structures de petites molécules fut criblée en utilisant une technique d'alignement moléculaire fournie par le programme 4Scan. Grâce à cette étude, la molécule (+)-epigalloyl-cathechin-gallate (EGCG) fut identifiée comme un inhibiteur d'interaction très spécifique et dose-dépendant pour deux des trois variants.Notre projet fut de développer des composés anti-adhésifs capables d'inhiber ou de supprimer l'interaction des hématies infectées avec les récepteurs endothéliaux. Tout d'abord, nous avons réalisé une série de substitues tetrahydroisoquinolines, analogues au composé naturel EGCG précédemment découvert. Ensuite, nous avons basé notre recherche sur la synthèse de composés peptidiques simulant des régions spécifiques d'ICAM-1 qui peuvent être impliquées dans l'interaction avec les variants de PfEMP1. Le design de ces peptides a été dirigé par le groupe du Prof. Tramontano sur la base d'analyses in silico de l'interaction moléculaire entre ICAM-1 (dont la structure cristallographique est connue) et un modèle de PfEMP1. Nous avons étudié les propriétés d'inhibition de la cytoadhérence de plusieurs de ces peptides afin de créer une base pour le design de molécules peptidomimétiques dotées de meilleures propriétés médicamenteuses.Finalement, afin d'élargir notre étude, EGCG a été criblé contre un panel de nouveaux isolats de P. falciparum provenant de patients et capables d'interagir avec ICAM-1. Ses propriétés anti-adhésives ont été étudiées afin de connaitre l'impact des variations de ces souches sur l'interaction et pour guider le design d'un inhibiteur de cytoadhérence à large spectre.INTITULÉ ET ADRESSE DE L'U.F.R. OU DU LABORATOIRE : - Prof. Giuseppe Campiani - Università degli Studi di Siena - Dipt. Farmaco-Chimico Tecnologico. Via Aldo Moro, 2, 53100, Siena (Italy) - Prof. Alister Craig – Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3, 5QA, UK. / Thesis title-: Development of anti-cytoadherence adjunct therapies.Thesis abstract-Parasite derived surface antigen PfEMP-1 (P. falciparum erythrocyte membrane protein-1) encoded by 60 var genes, is a virulence factor of the human malaria parasite. PfEMP-1 variants have been implicated in the cytoadherence of P. falciparum infected erythrocytes (iRBCs) to several binding receptors on host vascular endothelium. Among them, binding to ICAM-1 seems to be related to severe manifestations of the disease such as cerebral malaria. The majority of the mortality with severe malaria is seen within 24 hours of hospital admission despite the use of effective anti-parasite drugs, therefore the development of adjunctive therapies specifically targeting cytoadherence is urgently needed.The binding site for iRBC has been mapped to the BED side of the N-terminal immunoglobulin-like domain of ICAM-1, and shows subtle differences in the contact residues used by ICAM-1 binding P. falciparum antigenic variants. The DE loop appears to be a common feature of the ICAM-1 binding sites for three P. falciparum variants (ITO4-A4, ITO4-C24 and ItG-ICAM) analyzed and was selected by Matthias Dormeyer group for in silico screening of a small-molecule structures library using a molecular-alignment technique based on the program package 4Scan. From this study, (+)-epigalloyl-cathechin-gallate (EGCG) was found to inhibit binding of two variant ICAM-1 binding parasites in a highly specific, dose-dependent manner.Our approach to this need has been the development of anti-adhesive compounds to inhibit and reverse the binding of iRBCs to endothelial receptors. Firstly, we developed a series of substituted tetrahydroisoquinolines as analogues of the natural compound EGCG previously identified. Secondly, we based our research on the synthesis of peptidic compounds mimicking specific ICAM-1 regions hypothesized to be involved in the binding with disease-relevant PfEMP1 variants. The design of the peptides has been conducted by the group of Prof. Tramontano on the basis of an in silico analysis of the molecular interaction between ICAM-1 (whose crystal structure is known) and a homology model of PfEMP1. We have evaluated several peptides for their cytoadherence blocking properties and the results of this studies could form the basis for the design of peptidomimetics endowed with better drug-like properties. Finally, to extend previous studies, EGCG has been screened against a panel of new ICAM-1 binding patient isolates of P. falciparum for its anti-adhesive properties to investigate the impact of iRBC strain variation and to guide the design of a broad-spectrum cytoadherence inhibitor.TITLE AND ADDRESS OF LABORATOIRE : Prof. Giuseppe Campiani - Università degli Studi di Siena - Dipt. Farmaco-Chimico Tecnologico. Via Aldo Moro, 2, 53100, Siena, Italy. Prof. Alister Craig – Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3, 5QA, UK.

The insecticide dichloro-diphenyl-trichloroethane (DDT) is widely used in indoor residual spraying (IRS) for malaria control owing to its longer residual efficacy compared to other World Health Organization (WHO) alternatives. It was envisaged that by investigating mechanisms of degradation of these alternative insecticides, a better understanding would be obtained on strategies of stabilising them and rendering their efficacy comparable with or better than DDT, hence providing less controversial and more acceptable and effective alternative insecticide formulations to DDT. This study sought to investigate the reasons behind the reported longer lasting behaviour of DDT by exposing all the WHO approved insecticides to high temperature, high humidity and ultra-violet light. Interactions between the insecticides and some mineral powders in the presence of an aqueous medium were also tested. Simple insecticidal paints were made using slurries of these mineral powders whilst some insecticides were dispersed into a conventional acrylic paint binder. These formulations were then spray painted on neat and manure coated mud plaques, representative of the material typically used in rural mud houses, at twice the upper limit of the WHO recommended dosage range. DDT was applied directly onto mud plaques at four times the WHO recommended concentration and on manure plaques at twice WHO recommended concentration. All plaques were subjected to accelerated ageing conditions of 40 °C and a relative humidity of 90%. In the accelerated ageing tests, Fourier transform infra-red (FTIR) interferograms showed that pyrethroids were the most stable insecticides compared to carbamates and DDT. High temperature oxidation, ultra violet light and humidity were ruled out as the cause of failure of the alternative insecticides. Gas chromatography (GC) interferograms showed that phosphogypsum stabilised the insecticides the most against alkaline degradation. Bioassay testing showed that the period of efficacy of these formulations was comparable to that of DDT sprayed at 4 times the upper limit of the WHO recommended dosage range. Bioassay testing also showed that these insecticidal “paint” formulations stabilised the insecticides on cattle manure coated surfaces as compared to DDT sprayed directly on similar surfaces. Bioassay experiments indicated that incorporating insecticides into a conventional paint binder or adsorbing them onto phosphogypsum provided effective life spans, under accelerated ageing conditions, comparable to or exceeding that of DDT directly applied to typical soil substrates. Best results were obtained with propoxur in standard acrylic emulsion paint. Similarly, insecticides adsorbed on phosphogypsum and sprayed on cattle manure coated surfaces provided superior lifespans compared with DDT sprayed directly on a similar surface. Copyright / Dissertation (MEng)--University of Pretoria, 2012. / Chemical Engineering / unrestricted