O desenvolvimento de imunidade naturalmente adquirida na malária é lento e depende de fatores como o número de malárias prévias, intervalo entre cada malária, exposição a variantes antigênicas múltiplas e idade do indivíduo. Mecanismos imunes efetores dependentes de anticorpos são importantes no desenvolvimento dessa imunidade. Vários estudos têm mostrado que as subclasses IgG1 e IgG3 anti-P. falciparum, conhecidas por sua ação citofílica, são anticorpos protetores, enquanto anticorpos não citofílicos como IgG4 reconhecendo os mesmos epítopos seriam bloqueadores dos mecanismos protetores. Dados recentes sugerem que sob determinadas condições, IgG2 também pode ter ação citoíflica e participar da proteção na malária. Neste trabalho, nós estudamos pela primeira vez, a evolução das subclasses de IgG contra formas eritrocitárias de P. falciparum de pacientes com malária falciparum não complicada internados em hospital por até 42 dias, sob tratamento com mefloquina. As subclasses de IgG foram avaliadas por ELISA em 48 pacientes (7 amostras de soro de cada um colhidas nos tempos 0h, 48h, 7, 14, 28, 35 e 42 dias), quanto à quantidade (concentração, ug/ml; índices de reatividade, IR; ou em freqüência, %) e quanto à avidez dos anticorpos (índice de avidez, IA). Amostras de soro de 14 pacientes (tempos: 0, 48h, 7, 21 e 28 dias) também foram avaliadas quanto à especificidade e a avidez de reconhecimento das diferentes bandas protéicas do antígeno de P. falciparum por Immunoblotting. As subclasses IgG1, IgG2, IgG3 e IgG4 anti-P. falciparum, na maioria de alta avidez, estavam presentes no início do tratamento, respectivamente, em 100%, 39,5%, 80,6% e 28,4% dos pacientes e com concentrações médias de 20, 2; 3,8; 1,5 e 0,05 ?g/mL. As concentrações máximas das subclasses de IgG foram alcançadas no 7o dia, e os IAs máximos de IgG1 e IgG3 foram alcançados no 7o dia, e os de IgG2 no 14o dia e os de IgG4 no 2o dia. A concentração inicial dos anticorpos IgG3 anti-P. falciparum apresentou correlação negativa com o tempo de clareamento parasitário (TCP) e a relação das somas dos anticorpos IgG1, IgG2 e IgG3 pelos níveis de IgG4 se correlacionaram negativamente com a parasitemia inicial. No Immunoblotting, foram identificadas frações protéicas que podem estar relacionadas com o reconhecimento imune protetor, por serem reconhecidas pelas subclasses IgG1, IgG2 e IgG3 e não reconhecidas ou reconhecidas tardiamente por IgG4: 125, 96, 86, 75, 55 e 47 kDa. A resposta predominante das subclasses IgG1, IgG2 e IgG3 observada nestes pacientes, todos com malária não complicada, pode indicar que esses anticorpos estão cooperando para o controle de formas graves da doença e refletirem um certo grau de desenvolvimento de imunidade adquirida / The development of naturally acquired immunity to malaria is slow and depends of several factors as number of previous malaria, interval between each malaria attack, exposure to parasite multiple antigen variant and ageassociated maturation of the immune system. Antibody-dependent effector immune mechanisms are believed to be important to the protective immunity. A number of studies have showed that anti-P. falciparum IgG1 and IG3, named cytophilic antibodies, are protective, whereas the noncytophilic, IgG4, that recognize the same epitopes may block the protective mechanisms. Recent data have suggested that in certain situations, IgG2 can also act as cytophilic and to cooperate in protection. In this work, we have studied, for the first time, the evolution of the IgG subclasses against P. falciparum blood stages in uncomplicated falciparum malaria patients taken into hospital upon mefloquine treatment and followed up 42 days. These antibodies were determined by ELISA in 48 patients (7 serum samples from each patient collected in different times: 0h, 48h, 7, 14, 28, 35 and 42 days). The results were expressed in concentration (ug/ml), index of reactivity (IR) or frequency (%) and the avidity were expressed as index of avidity (IA). Serum samples 14 patients (time of collection: 0, 48h, 7, 21 and 42 days) were also evaluated by Immunoblotting as their specificity and avidity against different proteins of the P. falciparum blood stages The subclasses Anti-P. falciparum IgG1, IgG2, IgG3 and IgG4, high avidity predominantly, were present since the beginning of the treatment, respectively, in 100%, 39,5%, 80,6% and 28,4% of the patients with the following concentrations: 20, 2; 3,8; 1,5 and 0,05 ?g/mL. The highest concentrations were reached at day 7, and IgG1 and the highest IgG3 IAs were reached at day 7, and the highest IgG2 IAs at day 14 and the highest IgG4 IAs at day 2. The initial concentration of anti-P. falciparum IgG3 showed a negative correlation with the parasitemia clearance time (PCT) and the ratio between the sum of IgG1, IgG2 and IgG3 levels to IgG4 levels was negatively correlated with the initial parasitemia. Six protein fractions were identified by the Immunoblotting that can be related to protective immune recognition, because they were recognized by IgG1, IgG2 and IgG3 antibodies and not or only later recognized by IgG4 antibodies: 125, 96, 86, 75, 55 and 47 kDa. The predominant IgG1, IgG2 and IgG3 responses observed in these uncomplicated malaria patients may suggest that these antibodies are cooperating to the control of severe disease and reflecting a certain development of protective immunity

Casos de malária no Brasil atingiu seu nível mais baixo em 35 anos e o Plasmodium vivax é responsável por 85% dos casos em todo o país. A vigilância epidemilógica da transmissão residual da malária persistente na Amazônia é o próximo grande desafio para os esforços vigentes de eliminação. Esta situação nos levou a avaliar uma estratégia para detecção de portadores do parasito (sintomáticos ou não) em áreas que se aproximam a eliminação da malária. Nossa hipótese é que o monitoramento sistemático de moradores de domicílios com um ou mais casos de malária vivax clínica confirmados pela vigilância passiva de rotina (referido como casos índices) e seus vizinhos tem um melhor custo-benecífio na identificação de novas infecções em relação aos inquéritos em massa da população em geral. Para testar essa hipótese, foram recrutados 41 casos índices (24 indivíduos classificados como autóctones, 11 como possíveis recaídas e, seis como casos importados), 163 moradores dos domicílios índices (indivíduos morando na mesma residência do caso índice), 878 moradores vizinhos (moradores de domicílios a um raio de < 3 km) e 841 controles (moradores da mesma localidade, porém a > 5 km de distância do domicílio índice) entre os meses de fevereiro a julho de 2013. Participantes residiam em comunidades rurais pertencentes a Acrelândia, onde o P. vivax é a única espécie implicada na transmissão da malária humana. Todos os participantes foram convidados a fornecerem amostra de sangue capilar para realização do diagnóstico para malária no momento de identificação do caso índice (dia 0), 30, 60 e 180 dias após a primeira visita. Em geral, 6028 análises por microscopia revelaram um aumento na prevalência de malária nos domicílios índices (6,1%; odds ratio [OR] = 36,3, P < 0,001) e vizinhos (2,6%; OR = 13,6, P < 0,001) comparados aos controles (0,1%). Não houve casos positivos para P. falciparum. Moradores dos domicílios índices e vizinhos foram associados com uma maior probabilidade de infecção pelo P. vivax em comparação com os indivíduos controles, após análise ajustada por potenciais confundidores (modelos de regressão logística para efeitos mistos), além desses participantes albergarem > 90% da biomassa parasitária circulante. Nos quatro seguimentos da RCD a microscopia identificou somente 49,5% das infecções diagnosticadas por qPCR, porém 76,8% do total da carga parasitária circulante nas proximidades do domicílio índice. Embora, moradores dos domicílios controles foram responsáveis por 27,6% das amostras positivas por qPCR, 92,6% desses indivíduos eram portadores assintomáticos da infecção, que provavelmente não seriam alvos da RCD. Tipagem molecular dos parasitos utilizando três marcadores polimórficos - msp1F3, MS16 e pv3.27 revelaram alta diversidade de P. vivax, consistente com a complexidade das vias de transmissão e múltiplas fontes de infecção dentro dos aglomerados, potenciais complicações para os programas de eliminação de malária. / Malaria burden in Brazil has reached its lowest levels in 35 years and Plasmodium vivax now accounts for 85% of cases countrywide. The epidemiological surveillance residual malaria transmission entrenched in the Amazon is the next major challenge for ongoing elimination efforts. This situation prompted us to evaluate a strategy for targeted detection of parasite carriers (either symptomatic or asymptomatic) in areas approaching malaria elimination. We hypothesize that repeated screening of households (HHs) with one or more slide-confirmed clinical vivax malaria cases diagnosed by routine passive surveillance (referred to as index cases) and their neighbors is more cost-effective for finding new malaria infections than population-wide mass blood surveys. To test this hypothesis, we recruited 41 index cases (24 subjects classified with indigenous, 11 possible relapsing and 6 cases imported) 163 index household members (subjects sharing the household with index cases), 878 neighbors (subjects living within a radius of up to 3 km from index cases) and 841 controls (subjects living in the same locality, but > 5 km from the index case) between January and July 2013. Study participants lived in rural communities surrounding Acrelândia town, where P. vivax is the only malaria parasite diagnosed in humans. They were invited to contribute finger-prick blood samples, for laboratory diagnosis of malaria, at the time of index case diagnosis (day 0) and 30, 60, and 180 days later. Overall, 6,028 microscopical analysis revealed an increased prevalence of infection in index households (6.1%; odds ratio [OR] = 36.3, P < 0.001) and neighbors (2.6%; OR = 13.6, P < 0.001) compared to controls (0.1%). There were no positive cases for P. falciparum. Subjects in index and neighbor households were significantly more likely to be parasitemic than control households members, after adjusting for potential confounders (mixed-effects logistic regression models), and together harbored > 90% of the P. vivax biomass in study subjects. Four rounds of microscopy-based RCD would identify only 49.5% of the infections diagnosed by qPCR, but 76.8% of the total parasite biomass circulating in the proximity of index HHs. However, control HHs accounted for 27.6% of qPCR-positive samples, 92.6% of them from asymptomatic carriers who were beyond the reach of RCD. Molecular genotyping analysis of parasites with three polymorphic molecular markers (msp1F3, MS16, and Pv3.27) revealed high P. vivax diversity, consistent with complex transmission networks and multiple sources of infection within clusters, potentially complicating malaria elimination efforts.

Introduction Nigeria has the highest malaria burden worldwide. The 2010 and 2015 Nigeria Malaria Indicator Surveys (NMIS) suggest an improvement in malaria indicators, with the North West zone lagging behind. This study aimed to identify the individual and household-level malaria determinants in north-west and southern Nigeria, using Rapid Diagnostic Testing (RDT) and microscopy for malaria diagnosis. Methods Data on 3,358 children aged 6-59 months from north-west and southern Nigeria from the 2015 NMIS was used. The two populations were compared using chi-square tests, and logistic regression analysis was done for determinants of malaria infection, based on RDT and microscopic malaria test results. Results Malaria prevalence by RDT in the north-west and south was 55.8% and 29.2%, respectively (37.0% and 14.9%, respectively by microscopy). In both populations, a higher age, positive RDT in an additional household member and rural residence increased the odds of malaria infection; while higher education of the head of household and greater household wealth lowered the odds of malaria infection. Household clustering of RDT-positive cases appeared to be stronger in the south compared to the north-west. There were no statistically significant differences between the results using RDT or microscopy. Conclusion Irrespective of the diagnostic tool used, malaria determinants were similar in north-west and southern Nigeria. However, poorer social circumstances were observed in the north-west, and may account for the delayed progress in malaria control in the region. There may be a need to intensify malaria control efforts, particularly in the north-west, while awaiting socio-economic development.

Central to this research, 40 kDa Heat shock proteins (Hsp40s) are known to partner (or cochaperone) 70 kDa Heat shock proteins (Hsp70s), facilitating the selection and transfer of protein substrate to Hsp70 and the stimulation of the protein folding ability of Hsp70. Members of the diverse Hsp70-Hsp40 protein complement of Plasmodium falciparum have been implicated in the cytoprotection of this malaria parasite, and are thought to facilitate the protein folding, assembly and translocation tasks required by the parasite to commandeer the infected human erythrocyte subsequent to invasion. In particular, the parasite has evolved an expanded and specialised 43- member suite of Hsp40 proteins, 19 of which bear an identifiable export motif for secretion into the infected erythrocyte cytoplasm where they potentially interact with human Hsp70. Although type I Hsp40 proteins are representative of typical regulators of Hsp70 activity, only two of these proteins are apparent in the parasite’s Hsp40 complement. These include a characteristic type I Hsp40 termed PfHsp40, and a larger, atypical type I Hsp40 termed Pfj1. Both Hsp40 proteins are predicted to be parasite-resident and are most likely to facilitate the co-chaperone regulation of the highly abundant and stress-inducible Hsp70 homolog, PfHsp70-I. In this work, the co-chaperone functionality of PfHsp40 and Pfj1 was elucidated using in vivo and in vitro assays. Purified recombinant PfHsp40 was shown to stimulate the ATPase activity of PfHsp70-I in in vitro single turnover and steady state ATPase assays, and co-operate with PfHsp70-I in in vitro aggregation suppression assays. In these in vitro assays, heterologous partnerships could be demonstrated between PfHsp70-I and the human Hsp40, Hsj1a, and human Hsp70 and PfHsp40, suggesting a common mode of Hsp70-Hsp40 interaction in the parasite and host organism. The functionality of the signature Hsp40 domain, the Jdomain, of Pfj1 was demonstrated by its ability to replace the equivalent domain of the A. tumefaciens Hsp40, Agt DnaJ, in interactions with the prokaryotic Hsp70, DnaK, in the thermosensitive dnaJ cbpA E. coli OD259 deletion strain. An H33Q mutation introduced into the invariant and crucial HPD tripeptide motif abrogated the functionality of the J-domain in the in vivo complementation system. These findings provide the first evidence for the conservation of the prototypical mode of J-domain based interaction of Hsp40 with Hsp70 in P. falciparum. Immunofluorescence staining revealed the localisation of PfHsp40 to the parasite cytoplasm, and Pfj1 to the parasite cytoplasm and nucleus in cultured intraerythrocytic stage P. falciparum parasites. PfHsp70-I was also shown to localise to the parasite cytoplasm and nucleus in these stages, consistent with the literature. Overall we propose that PfHsp40 and Pfj1 co-localise with and regulate the chaperone activity of PfHsp70-I in P. falciparum. This is the first study to identify and provide evidence for a functional Hsp70-Hsp40 partnership in P. falciparum, and provides a platform for future studies to elucidate the importance of these chaperone partnerships in the establishment and survival of the parasite in the intraerythrocytic-stages of development.

Chapter 1 revisits the relationship between fiscal decentralization and economic growth by addressing the endogeneity. We use an instrumental variable approach based on two geography variables, namely a Geographic Fragmentation Index (GFI) and country size. We find that both instruments are strong and valid in the first stage regression and that a ten percent increase in decentralization measured by the expenditure or revenue share of subnational government in total government expenditures or revenues increases GDP per capita growth by approximately 0.4 percentage points. Moreover, we find that the results are more pronounced in the case of developed countries with a higher magnitude of the impact of revenue decentralization and lower impact of expenditure decentralization, while for a sample of developing countries both decentralization measures are insignificant. Chapter 2 explores the role of the tax structure and its key elements on the volatility of output growth. We account for both embedded automatic stabilizers measured by progressivity of the tax system and discretionary policy by accounting for the actual levels of revenue and its composition measured by tax mix ratio or the ratio of direct taxes to indirect taxes. We find that higher reliance on direct taxes versus indirect taxes is a significant stabilizing factor for output volatility for the whole sample of all countries and the subsample of lower income countries. For the subsample of high-income countries, we find a significant stabilizing impact of progressivity in the income tax structure, especially when there is higher reliance on personal income tax revenue. Chapter 3 reexamines the causal link between institutional quality and economic development using "Malaria Endemicity" as an instrument for institutions. This instrument is superior to the previously used instruments in the literature which suffered from measurement error. Because the Malaria Endemicity measure captures the malaria environment before the discovery that mosquitoes transmit the disease and before the successful eradication efforts that followed, it is exogenous to both institutional quality and economic development. We find Malaria Endemicity a valid strong instrument which yields larger significant effects of institutions on economic development than those obtained in the previous literature.

Contexte : La transmission du paludisme à Plasmodium falciparum repose sur le développement intra-érythrocytaire de parasites sexués (les gamétocytes) et leur ingestion par un moustique vecteur. Par filtration au travers d’une matrice de microsphères (microsphiltration), nous avons montré que les gamétocytes matures -normalement présents dans la circulation des sujets transmetteurs- sont déformables. Leur capacité à se déformer pour traverser la rate est essentielle à leur présence en circulation. Objectifs : Ce projet vise à découvrir des composés rigidifiant les érythrocytes contenant des gamétocytes pour bloquer la transmission du paludisme. En quelques heures, les gamétocytes rigidifiés seront exclus de la circulation sanguine -donc du cycle de transmission- par la rate. Méthodes et résultats: La microsphiltration a été miniaturisée au format microplaque et couplée à la microscopie à haut débit pour quantifier le nombre de gamétocytes retenus par les filtres. En utilisant la calyculine comme contrôle, l’activité rigidifiante d’antipaludiques de référence a été évaluée par microsphiltration. Les gamétocytes rigidifiés par la calyculine ont également été piégés dans des puces microfluidiques spléno-mimétiques. Leur clairance mécanique splénique a été confirmée dans un modèle murin adapté à la circulation transitoire des globules rouges humains parasités ou non. Conclusions: Un criblage par microsphiltration permet de sélectionner des molécules induisant la rétention mécanique splénique des gamétocytes pour bloquer la transmission du paludisme. Un post-criblage in vitro-in vivo permet de valider l’activité rigidifiante des actifs découverts par microsphiltration. / Background: Human-to-human transmission of Plasmodium falciparum malaria requires the development, within red blood cells (RBC), of sexual parasites termed gametocytes and their ingestion by Anopheles mosquito vector during a blood meal. Using filtration of RBC through microsphere layers (microsphiltration), we had shown that mature gametocytes present in the circulation of infective individuals are deformable. This deformability is a prerequisite for gametocytes circulation as they have (as any other uninfected RBC) to repeatedly cross narrow interendothelial slits in the human spleen. Objectives : This project aims at discovering compounds stiffening RBC harboring mature gametocytes, inducing their mechanical retention into the spleen, thereby removing them from the human bloodstream and interrupting malaria transmission. Methods & Results: Microsphiltration has been miniaturized to the microplate format, then coupled to high content imaging to quantify gametocyte retention in microsphere filters. Using calyculin as positive control, the gametocyte-stiffening activity of a panel of reference antimalarials was evaluated with the microsphiltration assay. Calyculin-stiffened mature gametocytes were held into spleno-mimetic microfluidic chips and were cleared from the circulation of macrophage depleted mice as rapidly as heat-stiffened control RBC, validating the outcomes of the microsphiltration assay. Conclusions: We have developped a microsphiltration assay compatible with screening. The screening/post-screening cascade has the potential to yield potent pharmacological agents blocking malaria transmission based on gametocytes deformability.

Master of Public Health - MPH / Background: Malaria was a major cause of mortality and morbidity in Namibia from 1999 to 2001. Malaria epidemics were recorded in 1990, 1996, 2000 and in 2001. In 2001 alone 733, 509 malaria morbidity cases and 1,728 mortality cases were documented. In recent years, however, malaria morbidity and mortality in the country have drastically declined by over 90%. This has influenced the Ministry of Health and Social Services (MoHSS) in Namibia to adopt a malaria elimination approach as opposed to the malaria case management approach. A malaria programme, known as the National Vector Disease Control Program (NVDCP) was instituted and mandated to coordinate malaria case management (diagnosis and treatment) as well as the current malaria elimination focus (elimination of transmission foci). This is all aimed at effectively addressing the current malaria epidemiology and sustain the decline observed over the last decade. Aim and Methodology: The study’s purpose was to analyse how the Namibia Malaria Strategic Plan (MSP) for 2010-2016 was developed and its relationship to health promotion. It employed an exploratory design which included stakeholders involved in malaria programmes and activities in the country. Data collection methods were of a qualitative nature through in-depth interviews and documentary review. Seven people were interviewed representing stakeholders from the public and private sectors. Those interviewed from the public sector were the Ministry of Environment and Tourism (MET), MoHSS’s division for Malaria, Policy Planning and Information Education and Communication (IEC). Others interviewed included national and international non-governmental organizations’ representatives from the World Health Organisation (WHO) and Society for Family Health (SFH). Documents reviewed include MSP 2010-2016, MSP 2003-2007, Malaria Policy of 2005, National Health Promotion policy 2012 and the Namibian Constitution. Data was analysed using content and thematic processes. Respondents were assured of confidentiality and anonymity. Key findings: The study found that information with regard to the formulation process of the MSP 2010-2016 was limited to a few superficially described events. These events involved, amongst others, a review of the previous (2003-2007) MSP, a pre-assessment questionnaire and two to three workshops. The events were not explicitly described or documented. The researcher concluded that the evidence to better understand the development processes of the MSP 2010-2016 was deficient. Consequently, the study concluded that the policy formulation aspects of the MSP were inadequate. The literature indicates that policy formulation and analysis is a complex undertaking and the MSP process did not meet these criteria. Amongst the limitations were limited stakeholder engagement and incomplete descriptions of the processes undertaken. In relation to the MSP’s focus on health promotion, the study found varied understanding of health promotion among the stakeholders. Some respondents were not aware of their organisation’s health promotion interventions while others believed that health promotion was limited to the health sector only. However, while national documents, such as the Namibian Constitution, advocate for ‘health investment as a just cause’ the MSPs limited inclusion of relevant stakeholders, such as the Ministry of Environment (MET) and the Information, Education and Communication (IEC) unit, restricted the ability of the MSP to offer malaria expansive programmes – that is, those beyond the health realm. Moreover, the implementation of malaria activities through the involvement of a limited range of actors in the malaria programmes will continue to perpetuate the existing narrow focus of health promotion by these stakeholders, as opposed to a more broad-base understanding. As a result malaria prevention will continue to be delivered as silo events or programmes. This poses serious implications in working towards the MSP goal of malaria elimination. Recommendations: The Ottawa Charter advocates for ‘Building healthy public policies’. This specifically refers to multi-disciplinary programmes. This study therefore recommends that the NVDCP follow existing international and national guidelines which systematically guide the development of MSPs and official health documents. Doing so would enable a more streamlined policy development process which would describe and contextualize the dimension of policy formulation, namely context, content, process and actors. It also recommends that the MSP is developed through a broad-based collaborative stakeholder engagement process which would facilitate an appropriately integrated inter-sectoral approach to malaria in the country.

Malaria continues to be a major cause of mortality and morbidity especially in Sub-Saharan Africa. The emergence and spread of drug resistant parasites has highlighted the need for new chemically diverse, effective drugs. Historically, one of the major sources of antimalarial agents and novel template compounds has been higher order plants. The widespread use of medicinal plants for the treatment of malaria in South Africa represents a diverse resource of potential antimalarial drugs. Two South African plants, Vernonia staehelinoides and Oncosiphon piluliferum, were identified as potential sources of new antimalarial drugs through a national multidisciplinary-consortium project aimed at scientifically validating South African medicinal plants for the treatment of malaria. The in vitro antiplasmodial activity of extracts of these plants warranted further investigation to identify the biologically active components. Bio-assay guided fractionation based on in vitro antiplasmodial activity against the D10 P. falciparum strain was used to identify the compounds responsible for the observed activity. Compounds were purified using silica gel column chromatography. The structures of the isolated compounds were elucidated using spectroscopic techniques. Bioassay-guided fractionation of the organic extracts of V. staehelinoides leaves identified a pair of structurally-related hirsutinolides with significant in vitro antiplasmodial activity. The compounds were found to be cytotoxic at similar concentrations but proved to be interesting scaffolds for potential structure-activity relationship studies. Three germacranolides and two eudesmanolides were identified through bioassay-guided fractionation of the organic O. piluliferum extract. Selected derivatizations were conducted in order to fully characterize the compounds. The absolute configuration of the major active germacranolide was determined using Mosher's method. The effect of the reduction of the <font face="symbol"> a</font>-methylene group of the major active germacranolide on antiplasmodial activity and cytotoxicity was also investigated. The 5 compounds and the reduction product were found to possess varying degrees of in vitro antiplasmodial activity and cytotoxicity. None was sufficiently active or selective to be a viable drug candidate but the potential for further structure-activity relationship studies exists. / Dissertation (MSc (Chemistry))--University of Pretoria, 2007. / Chemistry / unrestricted

Plasmodium falciparum causes the most severe form of human malaria, and the continual development of resistance of this parasite to current anti-malarial drugs underpins a pressing need for the discovery of novel chemotherapeutic approaches. Polyamines and their biosynthetic enzymes are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of the malaria parasite’s polyamine biosynthesis pathway causes cytostatic arrest in the trophozoite stage, but does not cure infections in vivo. This may be due to the salvage of exogenous polyamines from the host, replenishing the intracellular polyamine pool; however the mechanism(s) of polyamine uptake by the intraerythrocytic parasite are not well understood. In this study the uptake of the polyamines putrescine and spermidine into P. falciparum-infected erythrocytes (iRBC) well as into P. falciparum parasites functionally isolated from their host cell by saponin-permeabilisation of the erythrocyte membrane was investigated using radioisotope flux techniques. While the characteristics of transport of putrescine into infected erythrocytes were similar to those of transport into uninfected erythrocytes, spermidine entered iRBC in part via the ‘new permeation pathways’ induced by the parasite in the erythrocyte membrane. Both putrescine and spermidine were taken up across the plasma membrane of isolated parasites via a saturable, temperature-dependent process that showed competition between different polyamines as well as the polyamine precursor ornithine and basic amino acids. Inhibition of polyamine biosynthesis led to increased total uptake of both putrescine and spermidine. The influx of putrescine and spermidine into isolated parasites was independent of Na+ but increased with increasing pH and showed a marked dependence on the membrane potential, decreasing with membrane depolarisation and increasing with membrane hyperpolarisation. Both anthracene and polyamine derivatives have been shown to have anti-malarial activity. Anthracene-polyamine conjugates have been developed with the aim of utilising the polyamine uptake mechanisms of cancer cells to deliver the cytotoxic anthracene moieties to these cells. Here, several anthracene-polyamine conjugates showed promising anti-malarial activity. These compounds inhibited parasite proliferation with IC50 values in the nM range, and caused an arrest in the cell cycle, as well as a decrease in the mitochondrial membrane potential. Cytotoxicity could not be reversed by the addition of exogenous polyamines, nor did the conjugates have an effect on intracellular polyamine levels. This doctoral study showed that P. falciparum parasites not only synthesise polyamines, but can also acquire putrescine and spermidine from the extracellular environment and paves the way for interfering with polyamine metabolism as an anti-parasitic strategy. / Thesis (PhD)--University of Pretoria, 2012. / Biochemistry / unrestricted

Plasmodium is a genus of parasites causing malaria, a virulent protozoan infection in humans resulting in over a million deaths annually. Treatment of malaria is increasingly limited by parasite resistance to available drugs. Hence, there is a need to identify new drug targets and authenticate antimalarial compounds that act on these targets. A relatively new therapeutic approach targets proteolytic enzymes responsible for parasite‟s invasion, rupture and hemoglobin degradation at the erythrocytic stage of infection. Cysteine proteases (CPs) are essential for these crucial roles in the intraerythrocytic parasite. CPs are a diverse group of enzymes subdivided into clans and further subdivided into families. Our interest is in Clan CA, papain family C1 proteases, whose members play numerous roles in human and parasitic metabolism. These proteases are produced as zymogens having an N-terminal extension known as the prodomain which regulates the protease activity by selectively inhibiting its active site, preventing substrate access. A Clan CA protease Falcipain-2 (FP-2) of Plasmodium falciparum is a validated drug target but little is known of its orthologs in other malarial Plasmodium species. This study uses various structural bioinformatics approaches to characterise the prodomain‟s regulatory effect in FP-2 and its orthologs in Plasmodium species (P. vivax, P. berghei, P. knowlesi, P. ovale, P. chabaudi and P. yoelii). This was in an effort to discover short peptides with essential residues to mimic the prodomain‟s inhibition of these proteases, as potential peptidomimetic therapeutic agents. Residues in the prodomain region that spans over the active site are most likely to interact with the subsite residues inhibiting the protease. Sequence analysis revealed conservation of residues in this region of Plasmodium proteases that differed significantly in human proteases. Further prediction of the 3D structure of these proteases by homology modelling allowed visualisation of these interactions revealing differences between parasite and human proteases which will lead to significant contribution in structure based malarial inhibitor design.