Drug resistance is one of the most medically relevant forms of pathogen evolution. To date, vaccines have not failed with the same depressing regularity as drugs. Does that then make vaccines evolution-proof? In the face of vaccination, pathogens are thought to evolve in two ways: by evolving epitope changes at the antigenic target of vaccination (epitope evolution); or by evolving changes at other antigenic loci, some of which may involve virulence (virulence evolution). The fundamental difference between these two forms of evolution is that virulence evolution could lead to disease outcomes in unvaccinated people that are more severe than would have been seen prior to evolution. One of the theoretical assumptions of virulence evolution is that more virulent parasites will have a selective advantage over less virulent parasites in an immunized host, and are thus more likely to be transmitted. The assumption is that more virulent parasites may be competitively more superior in mixed infections, or may be better able to evade/modulate the host immune response. Thus, the aim of this thesis was to experimentally test whether more virulent parasites have a within-host selective advantage in an immunized host or whether vaccine efficacy is more likely to depend on genetic differences at the targeted sites of vaccination. I used clones (genotypes) of the rodent malaria Plasmodium chabaudi originally derived from wild-caught Thicket (Thamnomys rutilans) rats to infect laboratory mice and a rodent analogue of the candidate blood-stage malaria vaccine apical membrane antigen 1 (AMA-1). I found that within-host selection did not depend on parasite virulence, and that protective efficacy depended on genotype-specific differences at the vaccine target. Vaccine-induced protection was not enhanced by including a number of allelic variants. However, such genotype-specific responses were only observed when the vaccine was tested against genetically distinct P. chabaudi parasites. When one P. chabaudi genotype was serially passaged through naïve mice the derived line was more virulent and was subsequently less well controlled by vaccine-induced immunity. In other experiments I found within host competition not to be immune-mediated. Thus my results suggest that vaccination has the potential to select for more virulent parasites but that the selective advantage is likely to be independent of competition. The selective advantage may be attributable to the enhanced immune evasion of more virulent parasites. However, without genetic markers of virulence, the mechanisms that mediate this selection remain unknown. My thesis contributes towards a growing body of evidence that vaccines have the potential to differently alter the within-host parasite dynamics of particular pathogen genotypes and that the selection imposed is likely to be system specific, depending on the fine specificity of the vaccine-induced responses and the identity of infecting parasites. Although vaccine potency may not be enhanced by including more than one allelic variant of an antigen, multi-valent vaccines may be one of the best ways to avoid the inadvertent selection for more virulent malaria parasites.

In recent years there has been growing interest in applying frameworks from evolutionary ecology to understand infectious disease. It is becoming increasingly apparent that the interactions between parasites within the host environment can shape parasite phenotypes underlying infection dynamics and transmission. However, the spread of the disease will crucially depend on both within-host and between-host dynamics. Bridging these scales is challenging and for vector borne parasites, such as malaria and trypanosomes, will involve gaining a much better understanding of infection dynamics both within the host and vector. I apply evolutionary ecology frameworks including social evolution, life history theory, and phenotypic plasticity to investigate how parasite phenotypes are shaped by within-host and within-vector environments and examine the implications for inhost survival and between-host transmission. Specifically, I demonstrate that; 1. Within the host; i. In accordance with theory malaria parasites detect and respond to the presence of competitors by altering reproductive strategies to maximise in-host survival. Furthermore, these strategies are fine tuned in response to variation in the within-host environment, including the availability of resources. ii. The reproductive investment strategies of malaria parasites can be applied to explain the transmission strategies of African trypanosomes. This shows how general evolutionary frameworks can be applied to a novel parasite species and demonstrates the explanatory power of an evolutionary approach. iii. The complexity of the within-host environment poses specific statistical challenges for examining the temporal dynamics of parasite life history traits that are often not adequately dealt with, potentially leading to type 1 errors. Methods to evaluate levels of autocorrelation and how to deal with it are applied to datasets of within-infection dynamics. 2. Within the vector; i. Malaria parasites undergo programmed, apoptotic cell death. The occurrence of, and putative explanation for, apoptosis in protozoan parasites is controversial. I demonstrate the importance of quantitative methods and parasite ecology in testing the evolutionary explanations for parasite apoptosis. ii. The links between within-host dynamics and within-vector dynamics are complex and can lead to counter-intuitive implications for the success of between-host transmission. Density-dependent processes result in diverse fitness costs to parasites of crowding. More broadly, these processes could explain why parasites undergo apoptosis. In general my results demonstrate, across vertebrate hosts and insect vectors, how the interactions between parasites and with their environment shapes traits important for the transmission of infectious disease.

O rompimento eritrocítico característico do ciclo de vida do Plasmodium resulta na liberação de ATP, o qual é reconhecido pelo receptor P2X7. Este estudo da malária experimental causada pelo P. chabaudi em camundongos P2X7-/- mostraram que estes animais são mais suscetíveis à infecção. Tanto in vitro, como ex vivo, as populações celulares fagocíticas mostraram-se sensíveis ao ATP extracelular, de uma forma P2X7 dependente, e a infecção pelo P. chabaudi mostrou ser um fator que aumenta esta sensibilidade. Estudos fenotípicos do baço e do fígado dos animais P2X7-/- indicaram que, com exceção da população CD11b+ Ly6G+, estes animais apresentam número inferior de células fagocíticas quando comparados aos animais C57BL/6. Além disso, há uma leve deficiência na ativação dos fagócitos, na produção de IFN-g e no número de células produtoras de IFN-g, TNF-a e IL-10 no baço dos animais P2X7-/- em relação aos C57BL/6. A maior suscetibilidade dos camundongos P2X7-/- à infecção pelo P. chabaudi deve-se, portanto, a uma resposta imunológica deficiente nestes animais. / The erythrocyte rupture is a step of the life cycle of Plasmodium in which ATP is released and is recognized by the P2X7. This study of experimental malaria caused by P. chabaudi in P2X7-/- mice showed that these mice are more susceptible to the infection. Either in vitro or ex vivo, the phagocyte cell populations were sensible to extracellular ATP in a P2X7-dependent way and the P. chabaudi infection acted as a component that increases this sensitivity. Phenotypic analysis of the spleen and liver from P2X7-/- mice showed that, excepted for the CD11b+ Ly6G+ population, these mice have a lower number of phagocyte cells when compared to C57BL/6 mice. Furthermore, there was a slight deficiency in phagocyte activation, IFN-g production and number of cells that produce important cytokines as IFN-g, IL-10 and TNF-a in P2X7-/- mice in comparison with C57BL/6 mice. The increased susceptibility of P2X7-/- mice to the P. chabaudi infection is due to a deficient immune response.

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 origem geográfica e rotas de dispersão dos dois mais importantes parasitas da malária humana, Plasmodium falciparum e P. vivax, continuam controversos. Para entender a história evolutiva destes parasitos propomos neste projeto inferir as vias e as datas de introdução de P. vivax e P. falciparum nas Américas, com base na análise do genoma mitocondrial completo de parasitos coletados em todas as regiões endêmicas, além de analisar a existência da relação genética entre os isolados de P. vivax e P. simium e inferir a possível transmissão lateral. O alinhamento de 941 sequências de P. vivax e 1795 de P. falciparum permitiram agrupar os isolados em quatro regiões distintas. As rotas migratórias de P. vivax sugere que o continente americano foi colonizado em diferentes momentos e por parasitos de diferentes regiões África, Sul da Ásia e Melanésia, explicando a alta diversidade genética existente neste continente, enquanto que P. falciparum foi introduzido nas Américas por duas regiões distintas, África e Sudeste Ásiático. Já os 10 isolados de P. simium sequenciado neste estudo apresentaram uma menor diversidade genética quando comparado com os isolados de P. vivax, sugerindo que a direção da transmissão lateral foi de humanos para macacos. / The geographical origin and dispersal routes of the two most important human malaria parasites, Plasmodium falciparumand P. vivax, remain controversial. In order to understand the evolutionary history of these parasites this project aims to infer the routes and dates of introduction of P. vivax and P. falciparum in the Americas. Analysis were based on complete mitochondrial genomes of parasites collected in all endemic regions, and we explored the existence of a genetic relationship between P. vivax and P. simium isolates to infer a possible lateral transmission route. The alignment of 941 sequences of P. vivax and 1795 of P. falciparum made it possible to group the isolates into four distinct regions. The migratory routes of P. vivax suggest that the American continent was colonized at different times by parasites from different regions - Africa, South Asia and Melanesia, explaining the high genetic diversity present in this continent, while P. falciparum was introduced in the Americas from two distinct regions, Africa and Southeast Asia. The 10 P. simium isolates sequenced in this study had a lower genetic diversity when compared to P. vivax isolates, suggesting that the direction of lateral transmission was from humans to monkeys.

Student Number : 0110574V - MPh research report - School of Public Health - Faculty of Health Sciences / Pesticide poisoning poses a health risk to individuals throughout the world although the reported global and local risk are not consistent in the literature. Mpumalanga Province has areas of epidemic malaria. Spray teams, applying local insecticides to indoor surfaces operate just prior to the rainy season (October to May) to control malaria. The purpose of this cross sectional study was to compare prescribed safe handling and application practices of Mpumalanga malaria spray operators mixing and applying insecticides versus actual practices in the field. All members of the spray operating teams were included in the study. A tick list and questionnaire was utilized to observe field practices and enquire about reasons for non-compliance. Only 28% of all operators complied with prescribed safety practices and differences in compliance between mixing (38%) and application (36%) were marginal. Gloves, face shields and dust masks were not utilized as recommended and contributed to the highest levels of non-compliance. Compliance was found to be dependent on gender, age, years of experience, education level and employment status. The low compliance rate necessitates further investigation of the malaria programme occupational safety management system. All stakeholders need to be aware of the consequences of pesticide poisoning and collaborate in efforts to work towards prevention rather than cure.

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.

Artemisinin compounds are the most potent anti-malarial drugs available in the market. Today, malaria treatment is largely relies on the artemisinin-based combination therapies. Artesunate (AS) is the most widely used artemisinin derivative. In this thesis, we characterized the population pharmacokinetics of AS and its active metabolite dihydroartemisinin (DHA) following oral administration of AS in different populations. In Chapter II, we developed a population pharmacokinetic model of AS and DHA in healthy subjects. These subjects received either single- or multiple-dosing of oral AS, as a monotherapy regimen or in combination with pyronaridine, with or without food. In Chapter III, we developed a population pharmacokinetic model of AS and DHA in adult and pediatric patients with uncomplicated falciparum and vivax malaria who were administered oral pyronaridine/artesunate combination once daily for 3 days. We modeled the AS and DHA data simultaneously using a parent-metabolite model that assumed complete conversion of AS to DHA. Following oral administration, AS is rapidly absorbed with maximum concentrations reached at about 0.5 hours post-dose. AS is rapidly converted to DHA. DHA then undergoes rapid metabolism, with an elimination half-life of about 0.8 hours in malarial patients. Inter-individual variability for almost all pharmacokinetic parameters and residual variability for both compounds were estimated by the models. Substantial variability was seen in the pharmacokinetic parameters between the subjects. In healthy subjects, intake of food with the dose was found to delay the absorption of AS significantly, but not the extent of absorption. Weight was also included in this model as a determinant of DHA clearance. When modeling the data from patients, we included weight as part of the model a prioria priori using an established allometric function. No other covariates examined in the analysis were statistically significant. The performance of final models was evaluated using non-parametric bootstrap technique and visual predictive check. The models were found to adequately described the data at hand, and robust with sufficient predictive power. The results can be used as the base to develop a population pharmacokinetic-pharmacodynamic model and as prior information in guiding the selection of optimal sampling schedule for future pharmacokinetic studies of AS.

Infection with Plasmodium species leads to nearly 400,000 deaths a year despite widespread use of mosquito bed nets, insecticides, and anti-malarial drugs. To date, there is not a licensed vaccine capable of providing complete protection from Plasmodium infection to vaccinees. Whole parasite vaccination of humans and rodents can achieve complete protection in vaccines, but the dose of sporozoites, number of administrations, and production concerns in generating these types of vaccines will likely prevent these approaches from achieving worldwide use. However, the protective immunological responses against Plasmodium parasites engendered by these vaccination approaches can be studied and aid in the development of advanced subunit vaccines against Plasmodium. Using rodent models of malaria to elucidate the features of protective immunity engendered by whole parasite vaccination, it has been repeatedly shown that CD8 T cell responses directed against liver-stage parasite antigens can provide complete protection with some contribution by CD4 T cells and antibody responses depending on the model system studied. However, the quantatitive and qualitative requirements for CD8 T cell immunity against Plasmodium remains largely undefined. To enhance our understanding of how to generate protective immunity against Plasmodium, I have utilized rodent models of malaria to study the superior protection afforded from single-dose vaccination with virulent sporozoites administered under prophylatic chloroquine-cover, referred to as chemoprophylaxis sporozoites (CPS) vaccination, compared to the well-studied approach of administering radiation-attenuated Plasmodium sporozoites (RAS). RAS vaccination has long been considered the “gold standard” in vaccination due the ability of RAS vaccination to engender complete protection following sporozoite challenge of vaccinated humans and rodents. However, CPS vaccination is arguably a superior vaccination approach since it can achieve protection through less vaccine administrations relative to RAS vaccination, but the immunological basis of this enhanced CPS vaccine-induced immune response was unclear. In my study, I utilized a stringent host/parasite model to find that C57Bl/6 mice administered CPS vaccination with P. yoelii sporozoites elicit substantially higher parasite-specific CD8 T cell responses than RAS vaccination, but CPS-induced CD8 T cells were not necessary for protection following liver-stage sporozoite or blood-stage parasite challenge. CPS vaccination resulted in a low grade, transient parasitemia shortly following cessation of chloroquine treatment, which lead to the generation of potent antibody responses to blood-stage parasites; this blood-stage parasite-specific antibody response correlated with sterilizing protection in sporozoite challenged CPS-vaccinated mice. Therefore, my data provide a mechanistic basis for enhanced protective immunity elicited by single-dose CPS vaccination in a rodent model that is independent of CD8 T cells. The other portion of my work examines how CD8 T cell specificity impacts protective capacity against Plasmodium. I show that robust CD8 T cell responses of similar phenotype are mounted following prime-boost immunization against three novel Plasmodium berghei protein-derived epitopes in addition to a previously described protective, immunodominant epitope. I show that only CD8 T cells specific to sporozoite surface-expressed protein-derived epitopes, but not the intracellular protein-derived epitopes, are efficiently recognized by sporozoite-infected hepatocytes in vitro. These results suggest that antigenic targets must be efficiently presented by infected hepatocytes for CD8 T cells to eliminate liver-stage Plasmodium infection and proteins expressed on the surface of sporozoites may be good target antigens for protective CD8 T cells. Collectively, my work highlights the ability to generate protective CD8 T cell independent and dependent immunity against Plasmodium infections, whether achieved through potent blood-stage-specific antibody responses, or via numerically large monospecific CD8 T cell responses that target parasite antigens that are efficiently presented during liver-stage infection. These studies are relevant in understanding how to efficiency engender protective immunity against Plasmodium, and could aid in the advancement of subunit vaccination approaches that generate immunity through the priming of responses from multiple arms of the immune response, targeting both the liver- and blood-stages of Plasmodium.

Malaria is a mosquito borne infectious disease caused by a unicellular Apicomplexan parasite of the Plasmodia genus. The emergence and subsequent spread of drug resistance in the highly virulent Plasmodium falciparum parasite has been a major setback in eradicating malaria, which affects an estimated 216 million individuals and causes 445,000 deaths annually worldwide. Chloroquine (CQ) was once used as the first-line antimalarial drug treatment, until CQ-resistant parasites emerged in endemic regions including Africa, Southeast Asia, and South America. More recently, parasites have developed resistance to the current first line drug piperaquine (PPQ), used in combination with dihydroartemisinin (DHA) in Southeast Asia. Plasmodium falciparum chloroquine resistance transporter (PfCRT), a member of the drug/metabolite transporter (DMT) superfamily, is a 49-kDa integral transmembrane protein localized in the digestive vacuole (DV) of the pathogenic parasite. Mutations in PfCRT have been identified as the core determinants of Plasmodium falciparum resistance to CQ and PPQ by mediating the efflux of these antimalarial drugs. All CQ resistance-conferring PfCRT isoforms share the K76T mutation, which is widely used as a molecular marker for CQ resistance. Despite the significance in the impact of drug-resistant malaria, a detailed understanding of PfCRT physiological function and the molecular basis of PfCRT-mediated drug resistance have been hampered by a lack of high-resolution structural information. This dissertation describes the first structure of PfCRT and reveals the interaction of drugs with the purified and reconstituted protein. We determined the structure of the 49-kDa PfCRT 7G8, a clinically relevant CQ-resistant isoform found in South America, to 3.2 Å resolution by single-particle cryo-electron microscopy (cryo-EM), in complex with a specific antigen-binding fragment (Fab) to overcome current size limitations in cryo-EM. Our PfCRT structure displays an inward-open conformation, consists of 10 transmembrane (TM) helices with an inverted topology, and has unique elements including two juxtamembrane helices and a highly conserved cysteine-rich loop between TM helix 7 and 8. The architecture of PfCRT is similar to other members of the DMT superfamily. TM helices 1-4 and 6-9 in PfCRT form a central cavity which is a potential binding site for both CQ and PPQ. A striking feature is that virtually all the CQ resistance mutations, identified from decades of investigation into PfCRT variants that have evolved independently across the malaria-endemic world, map around this central, negatively-charged cavity. Distinct mutations that have been proposed to cause high-level PPQ resistance in parasites, which cause a loss of CQ resistance, form a planar ring that also lines this cavity. Functional experiments with various purified PfCRT isoforms or mutants provide evidence that drug resistance is possibly due to pH- and membrane potential-dependent drug transport. We also show that PfCRT CQ-resistant isoforms bind and transport arginine, suggesting that positively charged amino acids may be putative transport substrates for CQ-resistant PfCRT. This work provides a structural and functional framework to understand the mechanism of PfCRT-mediated drug resistance in the malaria parasite.