Malária é uma doença causada por protozoários do gênero Plasmodium que causa um milhão de mortes anualmente. Quase 3 bilhões de pessoas vivem em áreas tropicais de risco de infecção com uma das 5 espécies evidenciando um problema mundial carente de solução imediata. Esse parasita apresenta um potencial para o rápido desenvolvimento de resistências contra os fármacos utilizados em seu tratamento, por isso uma das soluções preconizadas pela Organização Mundial de Saúde é o desenvolvimento de vacinas eficazes para seu controle.O objetivo desse trabalho foca-se no aperfeiçoamento de uma metodologia mais efetiva para a formulação de vacinas contra malária. Inicialmente no modelo roedor, foi utilizado o método de carregamento em nanopartículas lipossomais , com proteínas oriundas de merozoítos do gênero Plasmodium. Além da avaliação do potencial vacinal das nanopartículas (sobrevida/morbidade), avaliamos o efeito da vacina contra toxinas (por exemplo, domínios GPI). Concluímos de maneira concisa que proteolipossomos gerados com proteínas GPI ancoradas do gênero Plasmodium possuem efeitos notáveis em relação a controle de crescimento parasitológico, mostrando-se efetivo também em desafios de letalidade in vivo. No que diz respeito a aspectos que afetam humanos, os soros gerados contra proteínas do parasita são capazes de diminuir interleucinas relacionadas com sintomas graves e parecem ter grandes efeitos antiparasitários que se correlacionam diretamente com o perfil genético do hospedeiro imunizado em ensaios in vitro. / Malaria is a tropical disease caused by species of the protozoan Plasmodium and around one million people die of the disease each year, while 3 billion individuals live at risk to acquire infection with one of the five species known to infect humans. Due to the parasite\'s looming resistance against most of the antimalarial compounds used in therapy, the WHO preconizes the development of effective vaccine as an important goal. The purpose of this work was to evaluate the potential of a new method of vaccine formulation against malaria. Initially tested in the rodent model, we loaded liposomal nanostructures with merozoite-derived GPI-anchored proteins. We then monitored parameters such as survival and morbidity after challenge and measured the effect against parasite derived toxines. We observed significant effects in terms of control of parasitemy and in one model complete survival of mice. We also detected the generation of antiGPI antibodies which showed to be functional in decreasing TNF-<font face=\"Symbol\">a production in an in vitro model, however, we detected that this function was dependent on the genetic background of the antibody producing immunized animal.

A malária é uma doença tropical negligenciada causada por protozoários do gênero Plasmodium spp, afeta populações em mais de 100 países ao redor do globo, apresentando 219 milhões de novos casos por ano sendo, portanto, um grave problema de saúde pública. Apresenta um ciclo complexo e digenético, necessitando do mosquito vetor e do hospedeiro vertebrado para se completar - ciclo este que envolve etapas de transformação e adaptação, já que o patógeno passa por 28 formas diferentes ao longo do ciclo, além de enfrentar situações de stress térmico, no momento do contágio e durante os picos febris. Sendo assim, é necessário que o protozoário garanta sua sobrevivência e possibilite a infecção do hospedeiro. Isso é realizado com a assistência de chaperonas moleculares, proteínas estas que são superexpressas no estágio intra-eritrocitário. Uma dessas proteínas é a Hsp90, uma Heat shock protein com diferentes funções, entre elas, maturação de proteínas clientes, encaminhamento de proteínas para translocação por membranas e marcação de proteínas para degradação. Para cumprir adequadamente as diversas funções, as Hsp90 contam com o auxílio de co-chaperonas, como a Hsp70/Hsp90 Organizing Protein (Hop) que modulam sua função. A Hop é uma co-chaperona do sistema foldossoma formado pelas Hsp70 e Hsp90 citoplasmáticas e que atua como proteína adaptadora transferindo proteínas clientes da primeira para a segunda chaperona molecular. A interação da Hop com Hsp70 e Hsp90 ocorre via domínios TPR, que se ligam ao motivo EEVD presente na extremidade C-terminal de ambas as chaperonas citoplasmáticas. É encontrada em diversos organismos, incluindo Plasmodium falciparum, o agente etiológico da malária. Sendo assim, conhecer a Hop de P. falciparum (PfHop), estrutural e funcionalmente, é importante para o entendimento do funcionamento das Hsp90 e Hsp70, proteínas essenciais para a sobrevivência do patógeno e, portanto, possíveis alvos terapêuticos. A PfHop recombinante foi obtida com pureza superior a 95%. A caracterização biofísica da mesma foi feita através de diferentes técnicas. Como outras Hops, a PfHop é majoritariamente constituída por hélices alfa. Os parâmetros hidrodinâmicos determinados sugerem que a PfHop se comporta como um equilíbrio monômero-dímero quando em solução. Dados de espalhamento de raios X a baixo ângulo mostram a PfHop como uma proteína dimérica e alongada. Este trabalho de dissertação de mestrado permitiu alcançar a caracterização estrutural da PfHop e com este conhecimento, espera-se avançar na caracterização funcional da mesma sobre a Hsp70 e Hsp90. / Malaria is a neglected tropical disease caused by protozoa of the genus Plasmodium spp, affects populations in more than 100 countries around the globe, presenting 219 million new cases per year and is therefore a serious public health problem. It presents a complex and digenetic cycle, necessitating the vector mosquito and the vertebrate host to complete - this cycle involves transformation and adaptation stages, since the pathogen goes through 28 different forms along the cycle, besides facing situations of thermal stress , At the time of the contagion and during the feverish peaks. Thus, it is necessary that the protozoan guarantees its survival and makes possible a host infection. This is accomplished with the assistance of molecular chaperones, proteins that are overexpressed in the intra-erythrocyte stage. A life of proteins and Hsp90, a protection of thermal shock with different functions, among them, maturation of client proteins, routing of proteins for membrane translocation and labeling of proteins for degradation. To comply properly, for example, as Hsp90 rely on the help of co-chaperones, such as Hsp70 / Hsp90 Organizing Protein (Hop) that modulate their function. The Hop is a co-chaperone system folded by Hsp70 and Hsp90 cytoplasmic and which acts as an adapter protein transferring client proteins from the first to the second molecular chaperone. The interaction of Hop with Hsp70 and Hsp90 occurs via TPR domains, which bind to the EEVD motif present at the C-terminus of both as cytoplasmic chaperones. It is found in several organisms, including Plasmodium falciparum, the etiologic agent of malaria. Therefore, knowing a Hop of P. falciparum (PfHop), structurally and functionally, is important for the understanding of the functioning of Hsp90 and Hsp70, essential proteins for a pathogen survival and, therefore, in all the therapeutic aspects. A recombinant PfHop was obtained in greater than 95% purity. The biophysical characterization by the same brand made through different techniques. As there is Hops, a PfHop is mostly constituted by alpha helices. The indicated parameters are a PfHop behaves as a monomer-dimer balance when in solution. Higher low-angle X-ray scattering data on PfHop as a dimeric and elongated protein. This work of master\'s dissertation allowed to reach a structural characterization of the PfHop and with this knowledge, it is expected to advance in the functional characterization of the same in Hsp70 and Hsp90.

A malária acompanha os seres humanos há cerca de 50 mil anos. Diferentes fatores genéticos do hospedeiro humano já foram relacionados à suscetibilidade ou resistência a malária: hemoglobinas S, C e E; Duffy; G6PD; TNF; TLRs; entre outros. Poucos estudos genético-epidemiológicos foram, até agora, conduzidos em populações misturadas, como a população brasileira e, em regiões com predominância da infecção por P. vivax. Estudos anteriores mostraram a presença de um fator genético principal, possivelmente localizado no cromossomo 4, relacionado ao número de episódios de malária no município de Monte Negro (RO). O presente trabalho buscou fatores genéticos relacionados à característica em questão em Monte Negro (RO). A análise do polimorfismo I602S do gene TLR1 (4p) pela técnica de RFLP mostrou ausência de associação. O GWAS encontrou sugestões de marcadores, que devem ser replicados em futuros estudos para verificar possíveis associações verdadeiras, sendo os mais relevantes os rs3796504, rs17518475, rs17527389, rs17660753, rs221188, rs7918405, rs7068695. / Malaria is present in humans since 50,000 years ago. Different Human host genetic factors are known to be related to susceptibility or resistance to malaria: S, C and E hemoglobins, Duffy, G6PD, TNF, TLRs, among others. Otherwise, few studies have been conducted in admixed populations, such as Brazilian populations and, in P. vivax predominance regions. Previous studies have shown the presence of a major genetic factor related to the number of malaria episodes of in the municipality of Monte Negro (RO) and, it was also suggested that this genetic factor may be located on chromosome 4. Thus, this study aimed to search genetic factors related to the number of individual malaria episodes in Monte Negro TLR1 candidate gene (4p) using RFLP and by GWAS. The first approach did not show any association with the studied phenotype, whereas GWAS data was able to suggest candidate markers that can be replicated in further studies in order to identify truly associations. The more relevant ones were rs3796504, rs17518475, rs17527389, rs17660753, rs221188, rs7918405, rs7068695.

Plasmodium malariae é um parasita protozoário que causa malária em humanos e é geneticamente indistinguível de P. brasilianum, um parasita que infecta macacos do Novo Mundo nas Américas do Sul e Central. P. malariae possui uma ampla distribuição mundial em regiões tropicais e subtropicais, porém de forma pontual, sendo encontrado na América do Sul, Ásia e África. Entretanto, pouco se sabe sobre a genética destes parasitas e a similaridade entre eles pode devido ao pequeno número de sequências genômicas disponíveis para essas espécies de Plasmodium. Recentemente, seis marcadores microssatélites e a sequencia completa do gene que codifica a proteína de superfície do merozoíta 1 (MSP1) foram descritos para estes parasitas. Neste estudo, o polimorfismo genético de 24 isolados brasileiros de P. malariae e P. brasilianum obtidos de diferentes hospedeiros foi analisado através da utilização desses marcadores microssatélites e da região correspondente ao oitavo bloco da MSP1. Os dados epidemiológicos moleculares foram explorados em relação à origem geográfica e hospedeiros. Para todos os marcadores estudados, as amostras de símios apresentaram um polimorfismo mais elevado que as amostras humanas. Na análise de microssatélites, as amostras humanas foram polimórficas em apenas dois alelos, enquanto as amostras de símios foram polimórficas em cinco alelos. O alelo Pm42- 331 foi monomórfico em todas as amostras analisadas. Na análise do bloco 8 da MSP1, as amostras símias foram altamente polimórficas e as amostras humanas apresentaram quatro tipos alélicos, sendo que dois tipos alélicos (A5 e A7) foram encontrados em alta frequência (90%). Em ambas as análises, a amostra de mosquito foi mais similar a amostras simianas. Nossos dados também mostram que há uma possível ausência de dimorfismo alélico na MSP1 de P. malariae e P. brasilianum, ao contrário de outras espécies de Plasmodium. Pela primeira vez, amostras de humanos, símios e mosquito foram analisadas em conjunto e utilizadas para o primeiro estudo de polimorfismos genéticos de isolados de P. malariae e P. brasilianum do Brasil. / Plasmodium malariae is a protozoan parasite that causes malaria in humans and is genetically indistinguishable from P. brasilianum, a parasite infecting New World monkeys in Central and South America. P. malariae has a wide and patchy global distribution in tropical and subtropical regions, being found in South America, Asia, and Africa. However, little is known regarding the genetics of these parasites and the similarity between them could be because until now there are only a very few genomic sequences available from these Plasmodium species. Recently, six microsatellite markers and the complete sequence of the merozoite surface protein 1 (MSP1) gene have been described for these parasites. In this study, the genetic polymorphism of 24 P. malariae and P. brasilianum isolates obtained from different hosts was analyzed using these microsatellite markers and the corresponding region on the block 8 of MSP1. The molecular epidemiological data were explored in relation to geographical origin and hosts. For all markers studied, the simian samples showed a higher polymorphism than human samples. In microsatellite analysis, the human samples were polymorphic only in two alleles, while simian samples were polymorphic in five alleles. The allele Pm42-331 was monomorphic in all samples analyzed. In the analysis of Block 8 of MSP1, the simian samples were highly polymorphic and human samples showed four allele types with two allelic types (A5 and A7) frequently found (90%). In both analyzes, the mosquito sample was more similar to simian samples. Our data also show that there is a likely absence of allelic dimorphism of MSP1 from P. malariae and P. brasilianum, as opposed to other Plasmodium species. For the first time, samples of humans, monkeys and mosquito were analyzed together and used for the first study of genetic polymorphisms in P. malariae and P. brasilianum isolates from Brazil.

Chan Ping Kei. / Thesis submitted in: December 2001. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 135-143). / Abstracts in English and Chinese. / ACKNOWLEGEMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTECTS --- p.v / LIST OF FIGURE --- p.viii / LIST OF ABBREVIATIONS --- p.xii / CHAPTER / Chapter 1 --- INTRODUCTION / Chapter 1.1 --- Epidemilogy --- p.1 / Chapter 1.2 --- Malaria disease --- p.1 / Chapter 1.3 --- Life cycle of Malaria --- p.1 / Chapter 1.4 --- Current measure to control Malaria --- p.6 / Chapter 1.5 --- Anti-malaria vaccine candidate --- p.7 / Chapter 1.6 --- Anti-erythrocytic malaria vaccine MSP-1 --- p.10 / Chapter 1.7 --- Baculovirus Expression System --- p.20 / Chapter 1.8 --- hexa-histidine tagged fusion protein --- p.25 / Chapter 1.9 --- IMAC --- p.26 / Chapter 1.10 --- Aim of study --- p.26 / Chapter 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.29 / Chapter 2.2 --- Methods --- p.40 / Chapter 3 --- CONSTRUCTION AND CHARACTERIZATION OF RECOMBINANT BmNPV CARRYING PfMSP-l42 / Chapter 3.1 --- Cloning of C-HisPfMSP-l42 into pBM030 --- p.71 / Chapter 3.2 --- Construction of Recombinant BmNPV Carrying PfMSP-l42 --- p.72 / Chapter 3.3 --- Purification of Recombinant BmNPVs --- p.78 / Chapter 3.4 --- In vitro expression of Recombinant --- p.80 / Chapter 3.5 --- In Vivo Expression of Recombinant PfMSP-l42 Protein --- p.80 / Chapter 4 --- PURIFICATION OF BmNPV-EXPRESSED RECOMBINANT C- TERMIAL HEXA-HIS-TAGGED PfMSP-l42 PROTEIN / Chapter 4.1 --- Nickel ion charged Chelating Sepharose Fast Flow (immobilized metal affinity chromatography) --- p.88 / Chapter 4.2 --- POROS HS/M (Strong Cation Exchanger) --- p.105 / Chapter 4.3 --- Combination of chromatographic separations --- p.107 / Chapter 5 --- CHARACTERIZATION OF RECOMBINANT C-HISPfMSP-l42 PROTEIN / Chapter 5.1 --- Proper formation of disulphide bridges in epidermal growth factor (EGF) like domains --- p.115 / Chapter 5.2 --- Characterization of the integrity of hexa-histidines residue on recombinant PfMSP-142 protein --- p.117 / Chapter 5.3 --- Immunogenicity of Recombinant C-HisPfMSP-l42 Protein --- p.117 / Chapter 6 --- DISCUSSION / Chapter 6.1 --- Construction of recombinant BmNPV carrying HisPfMSP-l42 --- p.122 / Chapter 6.2 --- Expression of recombinant HisPfMSP-l42 proteins --- p.123 / Chapter 6.3 --- Purification of recombinant C-HisPfMSP-l42 protein --- p.125 / Chapter 6.4 --- Characterization of recombinant C-HisPfMSP-l42 protein --- p.128 / Chapter 6.5 --- Future prospects --- p.130 / REFERENCE --- p.135 / APPENDICES / Chapter 1. --- Appearance of Mulberry leaves / Chapter 2. --- Biomark 2000 (Beckman) program for sandwich ELISA protocol / Chapter 3. --- Nucleotide Sequence of PfMSP-l42 3D7 Isolate / Chapter 4. --- Nucleotide sequence of PfMSP-l42 FVO isolate / Chapter 5. --- Efficiency of the mAb5.2 immunoaffinity column in purifying the recombinant PfMSP-l42 protein

A highly effective vaccine against malaria is urgently needed, with leading vaccination strategies involving the induction of protective antigen-specific CD8⁺ T-cells via heterologous prime-boost viral vector immunization, targeting primarily the pre- erythrocytic liver stages of the Plasmodium falciparum lifecycle. To date, the greatest immunogenicity has been obtained through a heterologous prime boost regimen, where vaccination with an Adenoviral vector is followed 8 weeks later by a Modified Vaccinia Ankara virus (MVA) boost. Experimental work directed at providing a greater understanding of CD8⁺ T-cell memory responses induced by Ad-MVA vaccinations lead to the development of a novel vaccine strategy aimed at priming CD8⁺ T-cells in the periphery and subsequently targeting them to hepatic tissue with protein loaded poly(lactic- co-glycolic acid) nanoparticles or recombinant viral vectors. Durable Ag-specific CD8+ T- cells exhibiting a phenotype of tissue-resident memory T-cells were generated in the liver, with a ten-fold increase over the conventional heterologous vector regimen. Importantly, in P. berghei sporozoite challenge models of liver-stage malaria, this strategy was found to result in unprecedented levels of sterile protection across multiple clinically relevant antigens and mouse strains. This prime and target immunization strategy for liver-stage malaria may provide a novel general approach for prevention or immunotherapy against other hepato-trophic pathogens.

In Myanmar, Plasmodium falciparum malaria is important because of both the burden of disease and the emergence of parasites resistant to artemisinin-based therapies. In 2012, concomitant with the lifting of international economic sanctions, funding for malaria control and elimination in Myanmar rose significantly. The University of Oxford was asked to support priority setting by assessing the relative cost-effectiveness of insecticide- treated bed nets and community health workers, particularly with respect to planning in the Myanmar Artemisinin Resistance Containment region along the east of the country. In the context of rising artemisinin resistance and, later, the goal of regional malaria elimination by 2030, reduction in malaria transmission was an important consideration in prioritising between interventions. A cost-effectiveness analysis was undertaken using both a static decision tree model and a dynamic disease transmission model. Supporting work towards this analysis included a systematic review of dynamic-transmission economic-evaluations and the creation of a data repository to collate governmental and non-governmental malaria case records. In addition, initially unplanned work on economic evaluation methodology was completed; identifying challenges in the application of cost utility analysis to this decision problem and proposing a framework for budget-based geographic resource allocation as an adaptation of standard methods. The results of this work include a tripling of the number of malaria diagnostic reports available between 2012 and 2014 (71% increase in Plasmodium falciparum cases) with this data showing a decrease in Plasmodium falciparum cases over time, alongside rising testing rates. Cost utility analysis found that, in general, malaria community health workers are more costly yet more effective than insecticide treated bed nets, though in both cases cost effectiveness is very much context dependent. Geographic allocation analyses using both static and dynamic models illustrate the potential for economic evaluation to provide both more detailed and more practical policy recommendations. Parameter uncertainty was explored in both cases. Some township recommendations were robust to both parameter uncertainty and model variation (structural uncertainty). Viewed through the lens of the Reference Case for Economic Evaluation in Low and Middle Income Countries (published during the course of this DPhil), budget-based geographic resource allocation largely adheres to the healthcare economic evaluation principles and offers improvements to dealing with heterogeneity and resource constraints. This DPhil recommends that Myanmar malaria policy is tailored to reflect geographic variation in intervention cost-effectiveness, rather than focusing on universal coverage, and illustrates a framework for economic evaluation to support budget-based geographic allocation.

The human immune-regulatory protein, complement receptor type 1 (CR1, CD35), occurs on erythrocytes where it serves as the immune adherence receptor. It interacts with C3b, C4b, C1q and mannan-binding lectin (MBL). It additionally binds the Plasmodium falciparum protein, Rh4, in the non-sialic acid-dependent erythrocye-invasion pathway, and is also important for rosetting, via an interaction with P. falciparum erythrocyte membrane protein 1 (PfEMP1). A C3b/C4b, and PfEMP1 binding site lies in CCP modules 15-17 (out of 30 in CR1), while polymorphisms that afford advantage to some populations in dealing with severe malaria occur in CCPs 24-25, begging the question central to this thesis – do these polymorphism modulate function, and if so how? We hypothesized that the CR1 architecture apposes CCPs 15-17 and CCPs 24-25 using the exceptionally long linker between CCPs 21 and 22 as a hinge, thus polymorphic variants in CCPs 24-25 modulate functionality in CCPs 15-17. To test this, a panel of recombinant CR1 protein fragments (CCPs 21, 21-22, 20-23, 15-17, 17, 10-11, 17-25, 15-25 and 24-25) were produced in Pichia pastoris along with polymorphic forms of the relevant constructs. After purification, biophysical and biological methods were used to assess whether the linker does indeed act as a hinge, and the comparative abilities of the CCPs 15-25 variants (along with soluble CR1 (sCR1), CCPs 1-3 and the panel of CR1 fragments) to interact with a range of ligands were measured. We found no evidence from NMR for face-to-face contacts between CCPs 21 and 22 that would be consistent with the long linker permitting a 180-degree bend between them. Indeed, based on scattering and analytical ultracentrifugation data, CCPs 20-23 form an extended rather than a bent-back structure. All of the four Knops blood-group variants of the CCPs 15-25 proteins produced similar results according to dynamic light scattering and AUC indicating no structural difference or change in self-association state between variants. In addition, based on the data collected from surface plasmon resonance (SPR), ELISA and fluid-phase cofactor (for factor I) assays, there were no evidence of any difference between the polymorphic forms with respect to their interactions with C3b, C4b, C1q and MBL. Only weak interaction was observed for sCR1, and all CCPs 15-25 variants, with the relevant part of PfEMP1, and there was no measurable difference amongst the variants in disrupting rosettes. The sCR1-Rh4.9 interaction was confirmed by SPR; affinities measured between the binding domain of Rh4 and the panel of CR1 fragments identified CCPs 1-3 (site 1) as the main interaction site. It seemed unlikely therefore that CCPs 24 and 25 could modulate Rh4 binding; indeed none of the four CR1 15-25 variants bound Rh4.9 appreciably. Thus we concluded that allotypic variations in CCPs 24-25 have no measurable effect on the architecture as well as binding of CR1 to its host or parasite ligands The inferred selective pressure acting on these variants likely arise from some other (i.e. besides malaria) geographically localised infectious diseases.

Malaria persists today as a significant burden for a large part of the world. However, over the past few decades, a concerted effort by governments, non-governmental organizations, researchers, and community health workers worldwide has yielded progress in reducing the deadly impact of this disease. Today, some of these gains are threatened by the rise of antimalarial drug resistance, a recurring problem that has impeded global malaria reduction efforts before. Research on Plasmodium falciprum resistance to the numerous antimalarial compounds used today and in the past has made significant progress on determining which specific mutations modulate drug susceptibility and to what degree they do so. To gain a comprehensive understanding of drug resistance, we need to elucidate how and why it arises. Therefore, it is important to elucidate whether some malaria parasites acquire resistance-conferring mutations faster than others and why the native function of the genetic factors involved lend themselves to modulating drug resistance. For instance, resistance to multiple antimalarial therapies has repeatedly emerged in Southeast Asia. We investigated the long-held hypothesis that this was due to the ability of these parasites to mutate significantly faster than non-Southeast Asian strains. Elucidating whether this hypermutability phenotype accurately represents Southeast Asian parasite evolvability is important, as it can inform when resistance would be expected to next arise, particularly in the Greater Mekong Subregion in Southeast Asia. Here, we have adapted a fluctuation assay to Plasmodium falciparum and determined that some contemporaneous Cambodian parasites exhibit a mild mutator, but not a hypermutator, phenotype. We also show that this is likely driven by mutations in DNA repair genes carried predominantly by multidrug resistant Southeast Asian parasites. One of the most common genes in which drug resistance-conferring mutations occurs is the P. falciparum chloroquine resistance transporter (pfcrt). Mutations in pfcrt are associated with parasite susceptibility to many of the antimalarial compounds that have been used in a clinical setting to date. However, beyond its role in drug resistance, little is known about the native function of PfCRT. To facilitate the study of pfcrt, we have designed a zinc-finger nuclease (ZFN)-based gene engineering system that introduces a single double-strand break in intron 1 of pfcrt. Our ZFN strategy enables replacing nearly any endogenous pfcrt locus with a user-defined recombinant pfcrt allele. We show that our method of pfcrt allelic replacement is fast, efficient, and reliable. We used this system to generate a unique mutant parasite encoding a pfcrt-L272F mutation, which enlarges the parasite digestive vacuole, the lysosome-like organelle used to catabolize host-derived hemoglobin for amino acid salvage. Our results provide clear evidence that PfCRT is associated with the terminal steps of hemoglobin degradation, overall parasite fitness, and the balance of osmolytes across the digestive vacuole membrane. Bringing clarity to the native function of PfCRT can reveal how and why this single genetic factor has been and continues to be involved in the resistance to many different antimalarial compounds.

Drug resistance is a serious problem in health care in general, and in malaria treatment in particular, rendering many of our previously considered ‘wonder drugs’ useless. Recently, large sums of money have been allocated for the continuous development of new drugs to replace the failing ones. We seem to be one step behind the evolution of antimalarial resistance; is it possible to get one step ahead? Are interventions which slow down the evolution and spread of drug-resistant malaria parasites achievable? In this thesis, I address these issues with experimental data, using the well-established rodent malaria model Plasmodium chabaudi to understand the selective advantages and disadvantages drug-resistant parasites endure within a vertebrate host and the selective pressures various drug treatment regimes exert on these parasites. Competitive interactions between drug-resistant and drug-sensitive parasites were observed within the host, with resistant parasites having a competitive disadvantage in the absence of drug treatment. The frequency of resistant parasites at the start of the infection was an important determinant of the strength of selection: the lower their frequency, the stronger the competitive suppression in non-treated hosts and the greater their competitive release following drug treatment. Genetically similar genotypes, one resistant and one sensitive, showed similar dynamics following drug treatment. Multiplicity of infection did not have an effect on the within-host dynamics: a larger number of co-infecting susceptible genotypes did not lead to greater competitive suppression or release of resistant parasites. Lastly, various drug treatment regimes were compared. Conventional drug treatment resulted in the greatest selective advantage for drug-resistant parasites, while less aggressive treatments were equally as effective, or even better, at improving host health and reducing overall infectiousness. These studies demonstrate that altering the within-host ecology of drug-resistant parasites by administering drugs and hence removing the drug-sensitive competitors has a large influence on the transmission potential of drug-resistant parasites. Furthermore, this thesis provides proof of principle that other drug treatment regimes different from those currently in use could better control drug-resistant parasites, without compromising other treatment goals. In the case of malaria, less drugs may mean extending the useful lifespan of that drug.