The causative agent of the most deadly form of malaria, P. falciparum, was identified over 130 years ago, yet this disease still causes 430,000 deaths each year. Although naturally-acquired immunity exists, it requires a heavy and sustained exposure to the parasite, with most succumbing as young children, before this immunity has fully developed. Effective treatments exist but with small-molecule drug resistance on the rise and little in the way of affordable alternatives, the need for an efficacious malaria vaccine is as great as ever. A successful malaria vaccine is likely to necessitate targeting each stage of the parasite's lifecycle. Immunity directed to the blood-stage, the stage which causes all the symptoms of malaria, is unique in that it would allow for a concomitant development of naturally-acquired immunity along with a reduction in morbidity and mortality. To date, antibody-mediated immunity to the blood stage requires intractably high levels of antibody and this problem is compounded by a paucity of viable candidates with which to effectively target different strains. Other fields of vaccinology, over the past decade, have been employing various structure-based strategies to increase the specific activity of the immune response thus lowering the antibody levels required for protection. However, very few detailed investigations of this kind have been conducted on a P. falciparum vaccine candidate, and certainly none as promising as PfRH5. In a world's first, fully-human antibodies raised in response to PfRH5 vaccination were isolated and extensively characterised, both functionally and structurally with the intention of elucidating the important features necessary to inform the design of an improved PfRH5-based vaccine. Synergistic and antagonistic effects of antibody combinations were noted and highlight new complexities of the immune response to PfRH5, opening the door to unanticipated potential for rational vaccine design.

Mosquito-borne diseases account for hundreds of thousands of deaths each year, highlighting the need for successful intervention methods, which can be targeted at either the pathogen, mosquito vector, or human host. This thesis aims to contribute to better intervention methods focused against malaria and dengue by either (i) improving available research tools, (ii) enhancing the understanding of a promising intervention method or (iii) designing new intervention candidates. Firstly, a superior method for studying in vitro malaria infection of the liver is shown, with implications for vaccine and drug interventions. Secondly, the biology of Wolbachia infection in Anopheles gambiae mosquitoes in the context of the target of rapamycin signalling cascade is investigated in an attempt to improve our understanding of its malaria inhibitory phenotype and inability to stably infect An. gambiae mosquitoes. Finally, an algorithm is developed for the design of a hypothesis driven conservation-based vaccine against viral mosquito diseases with a particular focus on dengue.

Plasmodium falciparum, the most pestilential of the malaria parasite species, is responsible for ~450,000 direct deaths annually. Clinical disease is a consequence of the blood stage of the parasite’s lifecycle involving a plethora of host-parasite interactions. Key to these interactions are the P. falciparum reticulocyte binding-like homologue (PfRh) proteins responsible for binding erythrocyte receptors and gaining entry to host cells. For example, PfRh4 binds to human complement receptor-1 (CR1) on erythrocytes for sialic-acid-independent invasion. Another protein important for invasion is the PfRh5-interacting protein (PfRipr), an essential member of the PfRh5-associated invasion complex (PAIN-complex) along with CyRPA, the cysteine-rich protective antigen. Loss of function of PfRipr in P. falciparum parasites prevents erythrocyte entry and ablates Ca2+-influx into the erythrocyte; essential events during invasion. This study aimed to biochemically and structurally investigate truncated recombinant versions of PfRh4 and PfRipr. Homology modelling suggested that PfRh4 is rich in alpha-helical secondary structure. The sequence of PfRipr suggested the presence of ten epidermal growth factor-like (EGF) modules, two towards the N-terminus and eight in the C-terminal domain. In this project, monoclonal antibodies made against recombinant PfRh4 were shown, via indirect immunofluorescent assays, to localize to the apical tip of merozoites. Monoclonal antibody 5H12, raised against PfRh4, reduces parasite invasion of erythrocytes by ~75% in growth-inhibition assays with neuraminidase pre-treated erythrocytes. Attempts to produce a stable truncated recombinant PfRh4 protein for structural studies were unsuccessful. An ELISA-based assay using ten alanine-scan mutants suggested the CR1-binding site lies outside of amino acids 283 – 341 of PfRh4. PfRipr truncations, defined by the boundaries of EGF-like repeats predicted based on sequence homology, were produced recombinantly in Escherichia coli and Pichia pastoris. These proteins had a circular dichroism signature suggestive of β-strand-containing proteins with disordered regions. EGF-containing PfRipr truncations did not bind recombinant PfRh5 according to ELISA and size-exclusion chromatography assays. EGFs 1-2, 5-7 and 7-10 of PfRipr did not bind CyRPA via size-exclusion chromatography or NMR. Crystallisation trials performed on EGF modules failed to yield crystals suitable for data collection. A 15N isotopically-labelled sample of EGF5-7 gave good quality HSQC NMR spectra. A suite of three-dimensional NMR spectra collected on a 13C,15N-EGF5-7 sample, at three different temperatures, allowed for >86% of backbone assignments. T1/T2 relaxation analysis and heteronuclear NOE data were suggestive of an elongated, rigid protein undergoing intermolecular self-association. Further evidence for EGF5-7 being an elongated protein was provided via SAXS analysis. Chemical shifts facilitated prediction of secondary structure in EGF 5-7 consistent with an EGF-like fold. Melting studies performed on EGF5-7 showed no evidence of denaturation over the temperature range 20 °C - 95 °C indicating a thermally-stable protein. The addition of Ca2+ to the 15N-EGF5-7 sample caused chemical shift perturbations consistent with high-affinity binding. The discovery of inhibitory monoclonal antibodies recognising a conformational epitope on EGF7 provided evidence of the functional importance of this region within PfRipr. The work described in this thesis provides methods for the industrially-scalable production and biophysical investigations of P. pastoris or E. coli-produced disulfide-rich P. falciparum antigens of interest to vaccinologists.

Plasmodium vivax is the most geographically widespread of the human malarias and is capable of causing severe debilitating disease. The parasite’s unique biology poses challenges to control of the disease and the understanding of its epidemiology. It is less researched and well understood than the more deadly P. falciparum. In this thesis, spatial relapse patterns and models of endemicity and clinical disease were applied to generate robust estimates of the P. vivax burden to address a key knowledge gap in malaria epidemiology. First, a review of the distribution of the parasite, its vectors and populations at risk found nearly one third of the global population living at risk, and more potential vectors than P. falciparum. In spite of low observed endemicity, the public health impact of P. vivax is likely to have been seriously underestimated in the past. To accurately define the burden of P. vivax it was necessary to improve understanding of one of the parasite’s most unique and challenging aspects, its ability to relapse. A meta-analysis of individual records of relapse showed that relapse periodicity varied systematically by geographic region and could be categorized by nine global regions. The nine regions were applied to a model to quantify the relationship between prevalence of infection and incidence of clinical disease. As relapse would have an influence on both measures, separate relationships were drawn for each relapse zone. The prevalence-incidence model was used to translate maps of predicted endemicity into measures of clinical burden. The evidence-base of P. vivax prevalence was poor in some regions and therefore a burden estimate based on surveillance reports was also derived. Reported cases must be adjusted for parameters such as under-reporting and treatment-seeking behaviours. A model used to fill gaps in treatment-seeking data available from national household surveys was developed to allow burden to be estimated using both cartographic modelling and surveillance reporting methods. To improve fidelity, the results of the two approaches were combined to enumerate P. vivax burden globally. The results and conclusions of these studies are discussed with recommendations for how these findings influence our understanding of P. vivax epidemiology and implications for future control and elimination efforts.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Filosofia e Ciências Humanas, Programa de Pós-Graduação em História, Florianópolis, 2015. / Made available in DSpace on 2015-11-24T03:07:15Z (GMT). No. of bitstreams: 1 336126.pdf: 6279737 bytes, checksum: 096432d82db261daf54cf3c88692d4bf (MD5) Previous issue date: 2015 / No final do século XIX e início do XX, a malária atingiu vários municípios do estado de Santa Catarina, em especial a região litorânea e o Vale do Itajaí. Nesse contexto, em 1943, o Serviço Nacional de Malária iniciou uma série de estudos nos quais foram comprovados que a proliferação do mosquito transmissor da malária, no estado catarinense, se dava nas águas armazenadas das bromélias epífitas ou terrestres. As descobertas científicas redesenharam os rumos e os métodos de profilaxia que deveriam ser seguidos para um eficiente combate à malária. A partir da descoberta do chamado complexo malária-bromélia, é possível constatar um novo direcionamento na concepção das autoridades públicas e sanitárias sobre as práticas desenvolvidas para combater a doença, tais como desbromelização, desmatamentos e dedetização intradomiciliar, que certamente provocaram impactos ambientais. Essa tarefa cabia aos ?guardas da malária?. Seus depoimentos, aliado à análise e interpretação de revistas, periódicos e documentos oficiais, livros, artigos e revistas que se debruçam sobre essa temática, evidenciaram questões tanto ambientais e políticas, como também sociais.<br> / Abstract : In the late nineteenth and early twentieth century, malaria affected several municipalities in the state of Santa Catarina, in particular the coastal region and the Valley of Itajaí. In this context, in 1943, the National Malaria Service, initiated a series of studies which considered that the proliferation of the mosquito that transmits malaria, in Santa Catarina state, was given in the waters in the epiphytic or terrestrial bromeliads. Scientific discoveries will redesign the course and prophylaxis methods that should be followed for an efficient struggle against malaria. After the discovery of the bromeliad-malaria complex, public and health authorities start to show new ways for fighting the disease, such as the removal of the bromeliads, deforestation and indoor pest control. All of them have caused several environmental impacts. This task was left to the ?guards? of malaria. Social, environmental and political issues are the main focus of this analysis, which is based on the interpretation of testimonials as well as magazines, newspapers, official documents, books, magazines and articles that focused on the subject, showed both ambiental and political, and social.

Made available in DSpace on 2014-06-11T19:32:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-01-28Bitstream added on 2014-06-13T20:23:14Z : No. of bitstreams: 1 martins_afa_dr_botib.pdf: 1302290 bytes, checksum: f4a20eda14cf780d998f0e02bf28f074 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A malária é uma das principais parasitoses humanas do mundo, causando mais de um milhão de mortes, e quase 500 milhões de casos agudos da doença por ano. No Brasil, esta doença continua sendo uma das mais importantes do país, tendo sido registrados no ano de 2009 mais de 300 mil casos. O mosquito Anopheles darlingi é o principal vetor desta doença no Brasil e outros países da América do Sul. Devido à sua importância como vetor da malária humana, estudos sobre a estrutura populacional de An. darlingi tem sido objeto de vários estudos. A sua distribuição na Região Amazônica é ampla e dados recentes mostram alto grau de heterogeneidade, tanto genética como de comportamento. Grande parte desta variabilidade observada em An. darlingi pode estar relacionada com estratégias adaptativas para explorar nichos ecológicos distintos enquanto que a estrutura populacional e a diferenciação pode ser explicada por diferenças no tamanho efetivo da população, padrões de fluxo gênico e acontecimentos históricos e de colonização recente. De igual modo, alterações ambientais efetuadas pelo homem podem ter um impacto significativo na dinâmica de populações de vetores e, consequentemente, na transmissão da malária. Este projeto utilizou a genotipagem de 10 microssatélites para o estudo populacional de Anopheles darlingi coletados em sete localidades ao longo da extensão das Hidrelétricas de Jirau e Santo Antônio, às margens do Rio Madeira, em Porto Velho – RO. Estes métodos foram aplicados na caracterização de amostras coletadas nestas regiões no 1º e 2° semestres de 2007. O objetivo do trabalho foi analisar a estrutura populacional de An. darlingi ao longo do Rio Madeira, na área de influência das Hidrelétricas de Jirau e Santo Antônio. Os resultados mostraram alto fluxo gênico entre as populações, mesmo distando de 70 km. Foram encontradas diferenças... / Malaria is the major human parasitic diseases in the world, causing more than a million deaths and almost 500 million acute cases of disease per year. In Brazil, this disease remains one of the most important, having been recorded in the year of 2009 more than 300.000 cases. The mosquito Anopheles darlingi is the principal malaria vector in Brazil and other countries in South America. Due to its importance as a vector of human malaria, population structure of Anopheles darlingi has been the subject of several studies. Its distribution in the Amazon region is large and recent data show a high degree of heterogeneity, regarding genetic and behavioral aspects. Much of this observed variability may be related to adaptive strategies to exploit different ecological niches, while the population structure and differentiation can be explained by differences in effective population size, patterns of gene flow and historical events and recent colonization. Similarly, environmental changes made by man can take a significant impact on population dynamics of vectors and hence the transmission of malaria. This project genotyping 10 microsatellites for population-based study of Anopheles darlingi collected at seven locations along Rio Madeira at Porto Velho - RO. These methods were applied in the characterization of samples collected in these regions in the 1st and 2nd semesters of 2007. The objective was to analyze the population structure of Anopheles darlingi along the Madeira River, the area of influence of Hydroelectric of Jirau and San Antonio. The results showed high gene flow among populations, even at 70 km apart. We found significant genetic differences among populations when samples were compared seasonally. The samples collected in the first half of the year showed effective population size 10x greater than those collected in the second half. These differences may represent differences... (Complete abstract click electronic access below)

Orientador: Alda Regina Monteiro Souza Brito / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-07-24T23:17:33Z (GMT). No. of bitstreams: 1 Bighetti_ElieteJanainaBueno_M.pdf: 2479902 bytes, checksum: 810af4e4fa4b9faf8a8e72ffae874611 (MD5) Previous issue date: 1999 / Resumo: A malária, doença parasitária prevalente de países tropicais, atinge aproximadamente 300 milhões de indivíduos/ano no mundo e tem se agravado pela resistência do Plasmodium às drogas. A busca de novos anti-maláricos para substituir ou complementar a terapêutica atual é uma das prioridades da Organização Mundial da Saúde. A espécie Croton cajucara ou "sacaca", uma planta tipicamente brasileira, é indicada popularmente no auxílio do tratamento de malária, inflamações no figado e icterícia, entre outros. Os efeitos da Croton cajucara sobre os aspectos associados aos sinais e sintomas relacionados à infecção malárica, além do efeito sobre o parasita em si foram estudados devido aos danos hepáticos produzidos pela malária, assim como às indicações populares da espécie. Estudamos inicialmente os efeitos farmacológicos do infuso, óleo essencial e desidrocrotonina, obtidos das cascas.de sacaca, em modelos experimentais de inflamação e hepatoproteção. O óleo essencial exerceu efeito antiinflamatório, dose-d.ependente, em modelos de inflamação aguda (edema de pata induzido por carragenina), e de inflamação crônica (granuloma cotton pellet); contudo, não foi capaz de inibir a migração de neutrófílos induzida por lipopolissacarídeo, para a cavidade peritoneal de camundongos. A desidrocrotonina foi capaz de inibir o processo inflamatório somente quando os animais foram tratados cronicamente, enquanto que o infuso não demonstrou efeito antiinflamatório em nenhum dos modelos estudados. Em ensaios de citotoxicidade, realizados em fibroblastos de pulmão de hamster chinês da linhagem V79 e em culturas primárias de hepatócitos de ratos, verificamos que a toxicidade das substâncias ~studadas foi dependente da dose. Esses resultados possibilitaram a comparação da citotoxicidade entre células metabolizantes (hepatócitos) e não metabolizantes (fibroblastos). O óleo essencial apresentou menor citotoxicidade nas células hepáticas do que em fibroblastos, diferente das demais drogas, onde ocorreu o inverso. Quando se testou o óleo essencial no modelo de hepatoproteção in vitro, utilizando como droga hepatotóxica a cumarina, o óleo essencial não foi capaz de proteger o figado desse agente lesivo. Nos testes de hepatoproteção in vivo todas as drogas foram ineficazes em proteger o figado; ao contrário, as substâncias vegetais potencializaram o efeito hepatotóxico do agente estudado, a galactosamina. Estudos preliminares do efeito das drogas na malária demonstraram que o infuso (com maior eficácia), o óleo essencial e a DHC foram capazes de inibir a parasitemia em animais (aves e camundongos) infectados principalmente por P. berghei e gallinaceum. Determinamos também a composição do infuso onde 13% de sua composição é de desidrocrotonina, além de óleo essencial e açúcares em menor quantidade. Nossos estudos demonstraram portanto que, apesar da citotoxicidade apresentada pelo infuso, o chá das cascas de sacaca usado popularmente no auxílio do tratamento da malária está, ainda que, preliminarment~, perfeitamente justificado / Abstract: Malaria a parasitic disease prevalent in tropical countries strikes approximately 300 million individuals/year in the world and has recently become more serious due to Plasmodium resistance to drugs. The search for new antimalarial drugs to replace or complement the current therapeutic arsenal is one of the priorities of the World Health Organization. The species Croton cajucara or "sacaca", a typically Brazilian plant, is popularly indicate as an aid for the treatment of malaria, inflammation of the liver and jaundice, among other conditions. The effects of Croton cajucara on the aspects associated with the signs and symptoms of malarial infection were studied in addition to the effect on the parasite itself due to the tiver damage produced by malaria and in view of the popular indications of this species. We initially studied the pharmacological effect of an infusion, of the essential oil and of dehydrocrotonin obtained from "sacaca" bark on experimental models of inflammation and of liver protection. The essential oil had a dose dependent antiinflammatory effect on models of acute inflammation (paw edema induced by carrageenin) and of chronic inflammation (corton pellet granuloma) but did not inhibit lipopolysaccharide-induced neutrophil migratfon to the peritoneal cavity of mice. Dehydrocrotonin inhibited the inflammatory process only when the animaIs were treated chronically, while the infusion had no anti-inflammatory effect on any of the models studied. In citotoxicity assays carried out on lung fibroblasts from Chinese hamsters of the V79 strain and on primary rat hepatocyte cultures, we noted that the toxicity of the substances under study was dose dependent. These results permitted us to compare citotoxicity on metabolizing cells (hepatocytes) and non-metabolizing cells (fibroblasts). The essential oil showed lower citotoxicity on hepatic cells than on fibroblasts, whereas the opposite occurred with the other products. Wnen the essential oil was tested on the model of in vitro liver protection using coumarin as a hepatotoxic drug, the essential oil was unable to protect the liver against this damaging agent. In the in vivo liver protection assays all products were ineffective in protecting the liver and indeed potentiated the hepatotoxic agent studied, galactosamine. Preliminary studies ofthe effects ofthese products on malaria demonstrated that the infusion (with higher potency), the essential oil and dehydrocrotonin inhibited parasitemia in animaIs (birds in mice) mainly infected with P. berghei and P. gallinaceum. We also determined the composition of the infusion, which consists of 13% dehydrocrotonin plus essential oils and sugars in lower amounts. Thus, the present studies demonstrated that, despite the citotoxicity of the infusion the popular use of tea of "sacaca" bark for the treatment of malaria is perfectly justified, although in a preliminary manner / Mestrado / Farmacologia / Mestre em Farmacologia

Magister Public Health - MPH / The study aims to assess the characteristics of donor funding for national tuberculosis, malaria and HIV programmes to Zambia over an 8 year period in order to inform it more effective and efficient utilization. / South Africa

Malaria is one of the most deadly parasitic diseases known to man. Although the number of malaria cases reported each year is decreasing, this disease continues to pose health and economic problems mainly in developing countries. Significant progress has been made in the fight against this disease. This includes the discovery and development of potent antimalarial agents. However, the development of resistance to most of these potent antimalarials has made the development of new antiplasmodial agents of paramount importance. Several promising antiplasmodial agents have been found from the marine environment. Amongst these are the tetraprenylated toluquinols from the brown alga: Sargassum heterophyllum. These metabolites have been reported to exhibit a range of antiplasmodial activity; however, the mechanisms by which these compounds bring about their antiplasmodial activity and the pharmacophoric groups responsible for such activity are unknown. Two species of Sargassum algae were encountered during the course of this project. From the investigation of the geographical and seasonal variation of metabolites of S. heterophyllum and S. elegans we established that there were no significant intra and inter site variations amongst metabolite profiles of both species both within and between the sampled seasons. These results enabled us to establish that the collection of both species from three different sites on the eastern coast of South Africa namely; Kenton on Sea, Port Alfred and Noordhoek in autumn, winter or spring would qualitatively yield the same metabolites. A comparison of metabolite profiles of both species also revealed no qualitative differences between metabolites of S. heterophyllum and S. elegans. The quantities of selected prenylated metabolites extracted from S. heterophyllum using four different extraction techniques was also assessed using qNMR as the method of quantification. This led to the identification of optimal extraction techniques and conditions for the extraction of sargahydroquinoic acid (1.38), sargaquinoic aid (1.39) and sargachromenol (2.10) from S. heterophyllum. From this study, the extraction of algae by soxhlet extraction using EtOH as the extraction solvent led to the extraction of the highest quantities of sargahydroquinoic acid. The potential of other extraction techniques such as microwave assisted extraction, to yield high quantities of the selected metabolites were also identified. With gram quantities of sargahydroquinoic acid (1.38) in hand, this compound was modified by oxidation, reduction, acetylation, methylation and cyclization reactions to yield nine derivatives. The derivatives and four naturally occurring prenylated toluquinols were assessed for antiplasmodial and cytotoxic activity against the FCR-3 Gambian Chloroquine resistant strain of P. falciparum and the MDA-MB-231 breast carcinoma cell line respectively. Comparison of antiplasmodial data for all twelve compounds showed that the hydroquinone moeity of sargahydroquinoic acid (1.38) is important for antiplasmodial activity while esterification of the carboxylic acid group in 1.38 resulted in more potent antiplasmodial compounds. Of all twelve compounds, compound 5.2, the hydroquinone methyl ester of 1.38 was found to be the most potent antiplasmodial compound with an IC₅₀ value of 1.94 μM and a selectivity index of 22.68.

Please read the abstract in the section 00front of this document / Dissertation (MSc(Biochemistry))--University of Pretoria, 2004. / Biochemistry / unrestricted