The protozoan parasite Plasmodium falciparum is responsible for the deadliest form of malaria, which causes 584,000 fatalities annually and whose complications include coma, anemia, respiratory distress, and renal failure. Although malaria eradication efforts were hindered by the rise of chloroquine (CQ) resistance (CQR), CQ continues to be clinically deployed in resistance-free regions. CQR is primarily mediated by mutations in the P. falciparum chloroquine resistance transporter (pfcrt) gene, which also modulates parasite susceptibility to first-line artemisinin-based combination therapies (ACTs). In certain geographical regions (e.g. Africa), mutant pfcrt alleles display considerable fitness costs and have undergone attrition in the absence of CQ pressure. Surveillance of resistant field isolates presently centers on the PfCRT mutation K76T, ubiquitous among CQ-resistant parasites and always accompanied by ≥3 additional mutations. Despite the global adoption of K76T as a molecular marker of CQR, the contributions of this and other mutations to P. falciparum drug resistance versus fitness had not been previously defined. AIMS: We aimed to address the following: (1) Do PfCRT mutations beyond PfCRT K76T directly contribute to CQR? (2) Do PfCRT mutations contribute to parasite fitness during the pathogenic asexual blood stage? (3) Are there predictable mutational paths in the evolution of pfcrt-mediated drug resistance? (4) How do PfCRT mutations impact current antimalarials, including the first-line ACTs? APPROACH: Using zinc finger nucleases, we generated isogenic, pfcrt-modified blood-stage P. falciparum parasites encoding wild-type (CQ-sensitive) or variant PfCRT haplotypes. Variants included a combinatorial library of alleles harboring 1-4 mutations comprising the simplest CQ-resistant haplotype (Ecu1110). Additional genetic dissections of full-length or partial pfcrt alleles encompassed the most common variants found in Africa and Asia, including a unique fitness-neutral mutant allele (Cam734) that has undergone expansion in Southeast Asia. Parasite antimalarial drug susceptibility was determined using IC50-based (cytostatic) assays or parasite survival-based (cytocidal) assays and was combined with data from flow cytometric parasite growth competition assays to computationally model mutant pfcrt evolution. To further define the biochemical impacts of PfCRT mutations, our studies leveraged metabolomic, heme fractionation, and drug transport studies. RESULTS: Key findings emerging from our studies included the following: (1) PfCRT K76T is insufficient for CQR and an inaccessible first mutational step in pfcrt evolution; (2) Alongside proliferation rates, parasite resistance gains dictate a constrained pfcrt mutational landscape and predict important roles for the active metabolites of CQ and amodiaquine in guiding pfcrt evolution; (3) To various degrees, PfCRT polymorphisms beyond K76T increase the potency of both the artemisinin and partner drug components of first-line ACT regimens; (4) Emerging PfCRT mutations (e.g. A144F) directly contribute to the enhanced fitness of pfcrt alleles and are necessary for multidrug resistance, independent of K76T. CONCLUSIONS: Our studies uncovered multiple pleiotropic contributions of PfCRT mutations to antimalarial drug resistance, countering earlier dogma that non-K76T mutations are merely compensatory. Evolutionary modeling revealed parasites’ ability to navigate constrained mutational landscapes and evolve drug resistance via rare mutational bursts. These results collectively highlight the capacity of PfCRT to acquire novel mutations that successfully balance parasite multidrug resistance with the essential role of PfCRT in maintaining digestive vacuole physiology. Our studies are of direct relevance to the regional recommendations of antimalarials, whose activity is influenced by, and in certain cases enhanced against, pfcrt-mutant parasites.

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No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EstudoFitoquimicoAvaliacao.pdf: 4226562 bytes, checksum: 7edac4fb3f72b9175767c12c0d78fa0a (MD5) Previous issue date: 2013 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / SCTIE - Secretaria de Ciência, Tecnologia e Insumos Estratégicos do Ministério da Saúde do Brasil / DECIT - Departamento de Ciência e Tecnologia da Secretaria de Ciência, Tecnologia e Insumos Estratégicos do Ministério da Saúde do Brasil / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / FAPESPA - Fundação Amazônia de Amparo a Estudos e Pesquisas / Parahancornia fasciculata (Poir.) Benoist (Apocynaceae), também conhecida como Parahancornia amapa (Hub.) Ducke, é uma espécie vegetal empregada popularmente no tratamento da malária, infecções no útero, gastrite, anemia, problemas respiratórios, entre outros. Os objetivos do presente trabalho foram realizar o estudo fitoquímico, avaliar a toxicidade oral aguda e a atividade antimalárica in vitro e in vivo de extratos, frações e substância isolada obtidas a partir de cascas do caule de P. fasciculata. Foram realizados dois tipos de extrações com o pó das cascas de P. fasciculata, por maceração / percolação, com etanol 96°GL e diclorometano, esta última tendo sido realizada a com o pó das cascas alcalinizado com hidróxido de amônio, obtendo-se os extratos secos EEPF e EDAPF, respectivamente. Uma terceira extração foi realizada a partir do EEPF por aquecimento sob refluxo, sucessivamente, com Hex:DCM (1:1), AcOEt:DCM (1:1) e AcOEt. EEPF foi, também, submetido a fracionamento por extrações ácido-base resultando nas frações de neutros (EEPFN) e de alcalóides (EEPFA). A prospecção fitoquímica realizada com o EEPF foi desenvolvida por CCD em cromatoplacas de sílica gel tendo sido detectada a presença de triterpenos, esteróides, heterosídeos flavônicos, saponinas, polifenóis, taninos, heterosídeos antracênicos e heterosídeos cardiotônicos. EDAPF foi submetido à cromatografia em coluna de sílica gel. Foram recolhidas 30 frações sendo que as frações Fr1-3, Fr4, Fr5-7 e Fr11 concentraram a maior parte da massa do extrato cromatografado. Da Fr5-7 foi isolada uma mistura de ésteres do lupeol que representam os componentes majoritários do EDAPF. Esta fração passou por um processo de hidrólise alcalina e o produto obtido (Fr5-7Hid) foi analisado por espectrometrias no IV, RMN de 1H e 13C e foi identificado como o triterpeno lupeol. A fração insolúvel em AcOEt obtida a partir do EEPF, por aquecimento sob refluxo, apresentou resultado positivo para o teste de proantocianidinas e foi submetido a doseamento desta classe de metabólitos. Os resultados foram expressos em porcentagem dos teores para a amostra não diluída (10,46±0,3419%), amostra diluída a 1:10 (9,94± 0,1598%) e amostra diluída a 1:100 (10,55± 0,9299%). A avaliação da atividade antiplasmódica in vitro em culturas de cepas W2 de Plasmodium falciparum foi realizada pelo teste da Proteína II Rica em Histidina (HRP-II) tendo sido testados EEPF, EEPFN, EEPFA, Fr1-3, Fr4, Fr5-7(ésteres do lupeol), Fr11 e o Fr5-7Hid (lupeol). Os melhores resultados obtidos foram para EEPF, EEPFA E EEPFN (CI50= ~ 50 μg/mL) sendo considerados moderadamente ativos. As demais amostras apresentaram CI50 > 50 μg/mL e foram consideradas inativas. Realizou-se também a avaliação da atividade antimalárica in vivo em camundongos fêmeas suíços infectados com cepas ANKA de P. berghei com o EEPF e o EEPF-HEX:DCM (1:1) em concentrações de 500, 250 e 125mg/kg de peso. EEPF foi parcialmente ativo, somente no 8° dia, em todas as concentrações. Já EEPF-HEX:DCM (1:1) foi parcialmente ativo na dose de 500mg/kg de peso e nas demais doses foi inativo. O teste de toxicidade oral aguda foi realizado em camundongos fêmeas suíços, pelo método da dose fixa (5.000mg/kg), com EEPF e não apresentou nenhum sinal de toxicidade evidente, o que foi confirmado pela ausência de alterações nos exames anátomohistopatológicos realizados. / Parahancornia fasciculata (Poir.) Benoist (Apocynaceae), also known as Parahancornia amapa (Hub.) Ducke is a species used in the treatment of malaria, uterus infections, gastritis, anemia, respiratory problems, among other ailments. The objectives of this study were to carry out the phytochemical study of the trunk bark from P. fasciculata, to evaluate the in vitro and in vivo antimalarial activity as well as the acute oral toxicity of extracts and fractions from this plant species. The powder bark of P. fasciculata was submitted to extractions by maceration/percolation with ethanol 96% and with dichloromethane after alkalinization of the bark powder affording the dry extracts EEPF and EDAPF, respectively. EEPF underwent two different re-extractions: 1) acid-base extractions affording the neutral (EEPFN) and alkaloidal fractions (EEPFA) and 2) heating under reflux with different solvents, leading to the fractions EEPF-DCM:HEX (1:1), EEPF-DCM: AcOEt (1:1) and EEPFinsoluble in AcOEt. Phytochemical screening of EEPF by TLC revealed the presence of triterpenes and steroids, flavonoid heterosides, saponins, polyphenols, tannins, anthracene heterosides and cardiotonic heterosides. EDAPF was submitted to chromatography through a silica gel column to give 30 fractions of which Fr1-3, Fr4, Fr5-7 and Fr11 represented most of the extract that was chromatographed. Fr5-7 led to the isolation of a mixture of esters of lupeol which are the major components of this extract. Saponification of this fraction afforded Fr5-7Hid that was analyzed by IV, 1H and 13CNMR and was identified as the triterpene lupeol. The insoluble AcOEt fraction derived from re-extraction of EEPF gave a positive test for proanthocyanidins which were quantitatively determined and the results were expressed in percentage for the content of these metabolites in an undiluted sample (10,46 ± 0,3419 %), a 1:10 diluted sample (9,94 ± 0,1598 %) and a 1:100 diluted sample (10,55 ± 0,9299%). The evaluation of the antiplasmodial activity in vitro was carried out against W2 strains of Plasmodium falciparum by the assay of the Histidine-Rich Protein II (HRPII) with EEPF, EEPFN, EEPFA, Fr1-3, Fr4, Fr5-7 (lupeol esters), Fr11 and Fr5-7Hid (lupeol). The best result was obtained for EEPF, EEPFA, EEPFN (CI50 = ~ 50 μg / mL) that can be considered as moderately active. The remaining samples showed CI50 > 50 μg / mL and were considered inactive. The in vivo antimalarial activity was performed in Swiss female mice infected with ANKA strains of Plasmodium berghei with EEPF and EEPF-DCM:HEX (1:1) at concentrations of 500, 250 and 125mg/kg body weight. EEPF was partially active only on the 8th day in all concentrations tested while EEPF-DCM:HEX (1:1) was partially active at a dosis of 500mg/kg and was inactive in the remaining doses. The acute oral toxicity test was determined for EEPF in Swiss female mice by the method of the fixed dose (5,000mg/kg) when no apparent signs of toxicity were observed what was confirmed by the absence of anatomic and histopathologic changes.

Determina la eficacia de las pruebas DELI y MSF para calcular la IC50 de drogas antimaláricas mefloquina, quinina y cloroquina obtenidas de aislamientos de P. falciparum provenientes de pobladores de la comunidad de Padre Cocha en Iquitos-Perú. Se realizó un estudio cuantitativo descriptivo, prospectivo de corte transversal. La muestra fueron 16 muestras de sangre con diagnóstico de malaria confirmado por gota gruesa. Se realizaron los dos ensayos de sensibilidad in vitro (DELI y MSF) a cada muestra. Se determinaron tres factores de eficacia para el presente estudio; porcentaje de éxito, coeficiente de determinación de curva (R2) y coeficiente de variación (CV). Se hizo un análisis descriptivo y estadístico de los factores de eficacia mediante las pruebas de Wilcoxon y McNemar- Bowker para muestras pareadas con p < 0.05. Las medias aritméticas de los valores de IC50 con el ensayo DELI fueron para cloroquina 231.26 nM, quinina 101.17 nM y mefloquina 16.03 nM. Las medias de los valores de IC50 con el ensayo MSF fueron para cloroquina 227.52 nM, quinina 142.46 nM y mefloquina 35.07 nM. El porcentaje de éxito del cálculo de la IC50 para las tres drogas fueron el 50% (8/16) y 87.5% (14/16) en los ensayos MSF y DELI respectivamente, estas diferencias fueron estadísticamente significativas (p < 0.05). Sin embargo, en el análisis entre los porcentajes de éxito entre drogas, no presentaron diferencias para CQ y QN y si presentaron diferencias para MQ (p < 0.05). No hay diferencias significativas entre los valores de R2 entre las pruebas MSF y DELI. El porcentaje de éxito de CV positivos aumentó de 37.5% con el ensayo MSF a 81.25% con el ensayo DELI, estas diferencias fueron significativas (p < 0.05). Se concluye que el ensayo DELI es más eficaz que el ensayo MSF para calcular la IC50 de las drogas CQ, QN Y MQ. / Tesis

Plasmodium falciparum, one of the parasites that cause clinical malaria, is a continuous public health concern, especially in Asia and Africa. Unfortunately, the parasite has developed resistance to many drugs created to treat and prevent the disease. Artemisinin and its derivatives are the new gold standard for treatment of malaria, yet treatment failures in clinical studies are starting to be reported. Clearly, artemisinin resistance needs to be characterized and dealt with accordingly. In support of the Gates Foundation Artemisinin Consortium, we conducted a blinded study to elucidate the phenotypic response of artemisinin derivatives of parasites derived from patient blood samples from Cambodia and Thailand. Blood samples containing Plasmodium falciparum were cultured and then assayed using SYBR green as an indicator to obtain drug IC50s. The data suggested that many isolates are not demonstrating resistance to artemisinin. However, a select few are showing some resistant characteristics in the form of elevated IC 50s, especially to some of the drugs already identified in previous studies as drugs having resistant characteristics. Compared to studies conducted within the past ten years, no significant changes in parasite susceptibility to the artemisinin drugs have been observed. Additional analysis of clinical outcomes, therapeutic drug levels, and molecular markers needs to be completed before it can be assumed that artemisinin resistance has emerged.

Le paludisme, ou malaria, est une maladie infectieuse qui touche plus de 350 millions d'êtres humains et qui tue chaque année 2,5 millions de personnes à travers le monde. Les parasites responsables de la malaria sont des apicomplexes du genre Plasmodium, essentiellement P. falciparum. Le génome de P. falciparum, est séquencé depuis octobre 2002, et présente un des taux les plus faibles de gènes annotés, avec ~60 % de gènes sans fonction attribuée. Il est difficile, voire impossible, d'identifier dans le génome de P. falciparumi, certains gènes, responsables de fonctions mesurées biochimiquement chez le parasite, par similarité avec des séquences homologues caractérisées dans d'autres organismes. Cette difficulté rencontrée lors des recherches automatiques d'homologie est une limite à tout projet exploratoire du génome malarial fondé sur la phylogénie moléculaire. En particulier, l'inventaire des séquences héritées de l'algue ancestrale, qui a réalisé l'endosymbiose secondaire qui caractérise le phylum des Apicomplexa (sous génome d'origine algale dans lequel il est possible de rechercher des cibles pour des médicaments herbicides), peut être rendu incomplet. Les caractéristiques atypiques du génome et du protéome de Plasmodium, résumées sous le terme de biais compositionnel (en particulier un pourcentage en adénosine+thymidine supérieur à 80%), ont été soupçonnées d'être un cas limite pour les outils d'analyse de séquence existants. L'objet de cette thèse a donc été d'examiner l'influence possible de ce type de biais sur les méthodologies de comparaisons de séquences et de façon plus approfondie sur leurs statistiques.<br />Nous avons proposé des développements théoriques nouveaux, associés à la statistique de la Z-value introduite par Lipman et Pearson pour évaluer la significativité d'un score d'alignement de deux séquences protéiques: (1) le théorème TULIP permettant de déduire un majorant de la probabilité d'un score d'alignement de séquences (i.e. la P-value) par la valeur 1/Z-value2 et (2) la déduction des propriétés remarquables de la distribution des Z-values à partir de quelques hypothèses sur l'évolution des protéines dans le contexte de la théorie de la fiabilité des systèmes. Ces développements théoriques ont permis certaines avancées sur le plan pratique de l'identification de séquences homologues initialement non détectées par le théorème de Karlin-Altschul et d'étayer la relation entre les scores d'alignements et l'information mutuelle, au sens de la théorie de l'information.<br />En construisant un espace de configuration des protéines homologues, permettant une expression du théorème TULIP et ayant une cohérence avec la théorie synthétique de l'évolution, nous avons déduit une méthode de reconstruction de phylogénies de séquences protéiques à l'aide des Z-values. Les phylogénies moléculaires reconstruites par cette méthode sont concordantes avec celles obtenues à partir d'alignements multiples et permettent par ailleurs de résoudre certaines incohérences rapportées avec les méthodes de reconstruction phylogéniques classiques.<br />En prenant en compte le modèle statistique que nous avons élaboré, nous avons entrepris une première analyse de l'évolution du biais en acides aminés chez Plasmodium corrélativement à l'évolution du biais en acides nucléiques dans le génome malarial et en fonction de la divergence évolutive, établie en prenant le génome non biaisé d'Arabidopsis thaliana comme référence. Nous avons observé que le biais des séquences malariales était corrélé au pourcentage de divergence avec leurs homologues végétaux. Nos analyses suggèrent de plus que le biais est vraisemblablement la conséquence d'une évolution au niveau nucléique. Nous avons examiné la possibilité de construire une famille de matrices tenant compte de cette dissymétrie dans le cas de la comparaison de Plasmodium et d'Arabidopsis. Ces matrices appelées DirAtPf, possèdent (1) une sensibilité théorique et (2) une spécificité supérieure aux familles de matrices existantes.<br />Les perspectives des travaux présentés dans ce mémoire incluent une progression de l'annotation automatique de Plasmodium falciparum et la mise en place d'une procédure statistiquement robuste et phylogénétiquement consistante pour caractériser le sous-génome algal du parasite malarial.

Découper, a priori et de façon précise, les séquences en domaines est d'une grande importance dans le champ de la biologie, notamment pour optimiser les études de génomique structurale et de génomique fonctionnelle. Différentes approches basées sur la composition en acides aminés, la complexité de la séquence ou la construction de modèles 3D ab initio, ont été développées par le passé. Nous proposons, dans le cadre de ce travail, une approche nouvelle et originale pour le découpage automatique et sensible des séquences protéiques en domaines structurés distincts par exploitation de leur texture. Cette approche bénéficie de l'information de voisinage 2D apportée par la méthodologie « Hydrophobic Cluster Analysis » (HCA). La distribution des différentes catégories d'amas hydrophobes, tels que définis par l'intermédiaire de HCA, ainsi que l'analyse de leurs caractéristiques en termes de structures secondaires, permettent d'appréhender de façon différenciée les textures des régions globulaires, non globulaires et/ou désordonnées, répétitives, passages membranaires isolés ou multiples.... L'approche développée, DomHCA, permet in fine de segmenter une séquence protéique en une série de régions et sous-régions caractérisées par des textures précises, segmentation qui, appliquée à l'échelle des génomes, autorise une comparaison rapide et originale de l'ensemble des séquences. Une des applications concerne les séquences du génome de Plasmodium falciparum qui, par leurs fortes proportions en acides aminés N et K, rendent les méthodes classiques de détection de similarité peu efficaces.

<p>Protective immunity against <i>Plasmodium falciparum</i> may be obtained after repeated exposure to infection. Several studies indicate that immunity against the blood stages of the <i>P. Falciparum</i> infection is mainly antibody mediated. Protective antibodies may act either on their own, mediate antibody-dependent phagocytosis and/or cell-mediated neutralization of parasites. This thesis describes several aspects of humoral immune responses to <i>P. falciparum</i> infection in individuals of different age groups, different genetic background and with different degrees of malaria exposure.</p><p>Several target antigens for antibody-mediated inhibition of parasite growth or invasion have been identified. One such antigen is Pf332, which appears on the surface of parasitized erythrocytes at late trophozoite and schizont stage. This surface exposure makes the antigen a possible target for opsonizing antibodies. We optimized an <i>in vitro</i> assay for studying cellmediated parasite neutralization in the presence of Pf332-reactive antibodies. Our data demonstrate that, Pf332 specific antibodies are able to inhibit parasite growth on their own and in cooperation with human monocytes.</p><p>The <i>P. falciparum</i> parasites have evolved several mechanisms to evade the host neutralizing immune responses. In this thesis, we show that freshly isolated<i> P. falciparum </i>parasites from children living in a malaria endemic area of Burkina Faso were less sensitive for growth inhibition <i>in vitro</i> by autologous immunoglobulins (Ig) compared with heterologous ones. Analyses of two consecutive isolates taken 14 days apart, with regard to genotypes and sensitivity to growth inhibition <i>in vitro</i>, did not give any clear-cut indications on possible mechanisms leading to a reduced inhibitory activity in autologous parasite/antibody combinations. The frequent presence of persisting parasite clones in asymptomatic children indicates that the parasite possesses as yet undefined mechanisms to evade neutralizing immune responses.</p><p>Transmission reducing measures such insecticide treated nets (ITNs) have been shown to be effective in reducing morbidity and mortality from malaria. However, concerns have been raised that ITNs usage could affect the acquisition of malaria immunity. We studied the effect of the use of insecticide treated curtains (ITC) on anti-malarial immune responses of children living in villages with ITC since birth. The use of ITC did neither affect the levels of parasite neutralizing immune responses nor the multiplicity of infection. These results indicate that the use of ITC does not interfere with the acquisition of anti-malarial immunity in children living in a malaria hyperendemic area.</p><p>There is substantial evidence that the African Fulani tribe is markedly less susceptible to malaria infection compared to other sympatrically living ethnic tribes. We investigated the isotypic humoral responses against<i> P. falciparum</i> asexual blood stages in different ethnic groups living in sympatry in two countries exhibiting different malaria transmission intensities, Burkina Faso and Mali. We observed higher levels of the total malaria-specific-IgG and its cytophilic subclasses in individuals of the Fulani tribe as compared to non-Fulani individuals. Fulani individuals also showed higher levels of antibodies to measles antigen, indicating that the intertribal differences are not specific for malaria and might reflect a generally activated immune system in the Fulani.</p>

<p>Malaria is the most common parasitic disease of humans worldwide. A factor that aggravates the many attempts to control the epidemiologic malaria situation is the spreading of resistance against anti-malarial drugs. In this project the point mutation at position 86 of the <em>Plasmodium. </em><em>falciparum</em><em> </em>multidrug resistance gene (<em>pfmdr1</em>), which is thought to contribute to Chloroquine resistance, was analysed in 188 samples from a low transmission area in eastern Sudan, where malaria endemicity is seasonal. The patient group studied had asymptomatic and sub patent parasitemia that persisted during the transmission-free dry season, after being treated with Chloroquine. To differentiate between wild type and mutant genotypes, nested PCR and restriction fragment length polymorphism with the enzyme Apo1 was used. Out of 188 samples 79 (42%) were successfully analysed. Of those, 72% had parasites with mutant genotypes or where mixed infection. No conclusions on the relevance of the <em>pfmd</em><em>r</em><em>1</em> gene in the studied samples are made due to the many remaining gaps. However, eventual sources of error and previous findings in the study area are discussed.</p>

<p>Malaria is a widespread disease caused by parasites of the genus <i>Plasmodium</i>. Each year 500 million clinical cases are reported resulting in over one million casualties. The most lethal species, <i>P. falciparum</i>, accounts for ~90% of the fatal cases and has developed resistance to chloroquine. The resistant strains are a major problem and calls for novel drugs.</p><p>In this thesis, the process of computational inhibitor design is illustrated through the development of <i>P. falciparum</i> aspartic protease inhibitors. These proteases, called plasmepsins, are part of the hemoglobin degradation chain. The hemoglobin is degraded during the intraerythrocytic cycle and serves as the major food source. By inhibiting plasmepsins the parasites can be killed by starvation.</p><p>Novel inhibitors with very high affinity were found by using a combination of computational and synthetic chemistry. These inhibitors were selective and did not display any activity on human cathepsin D. The linear interaction energy (LIE) method was utilized in combination with molecular dynamics (MD) simulations to estimate free energies of binding. The MD simulations were also used to characterize the enzyme–inhibitor interactions and explain the binding on a molecular level.</p><p>The influence of the partial charge model on binding free energy calculations with the LIE method was assessed. Two semiempirical and six <i>ab initio</i> quantum chemical charge derivation schemes were evaluated. It was found that the fast semiempirical charge models are equally useful in free energy calculations with the LIE method as the rigorous <i>ab initio</i> charge models.</p>

Protective immunity against Plasmodium falciparum may be obtained after repeated exposure to infection. Several studies indicate that immunity against the blood stages of the P. Falciparum infection is mainly antibody mediated. Protective antibodies may act either on their own, mediate antibody-dependent phagocytosis and/or cell-mediated neutralization of parasites. This thesis describes several aspects of humoral immune responses to P. falciparum infection in individuals of different age groups, different genetic background and with different degrees of malaria exposure. Several target antigens for antibody-mediated inhibition of parasite growth or invasion have been identified. One such antigen is Pf332, which appears on the surface of parasitized erythrocytes at late trophozoite and schizont stage. This surface exposure makes the antigen a possible target for opsonizing antibodies. We optimized an in vitro assay for studying cellmediated parasite neutralization in the presence of Pf332-reactive antibodies. Our data demonstrate that, Pf332 specific antibodies are able to inhibit parasite growth on their own and in cooperation with human monocytes. The P. falciparum parasites have evolved several mechanisms to evade the host neutralizing immune responses. In this thesis, we show that freshly isolated P. falciparum parasites from children living in a malaria endemic area of Burkina Faso were less sensitive for growth inhibition in vitro by autologous immunoglobulins (Ig) compared with heterologous ones. Analyses of two consecutive isolates taken 14 days apart, with regard to genotypes and sensitivity to growth inhibition in vitro, did not give any clear-cut indications on possible mechanisms leading to a reduced inhibitory activity in autologous parasite/antibody combinations. The frequent presence of persisting parasite clones in asymptomatic children indicates that the parasite possesses as yet undefined mechanisms to evade neutralizing immune responses. Transmission reducing measures such insecticide treated nets (ITNs) have been shown to be effective in reducing morbidity and mortality from malaria. However, concerns have been raised that ITNs usage could affect the acquisition of malaria immunity. We studied the effect of the use of insecticide treated curtains (ITC) on anti-malarial immune responses of children living in villages with ITC since birth. The use of ITC did neither affect the levels of parasite neutralizing immune responses nor the multiplicity of infection. These results indicate that the use of ITC does not interfere with the acquisition of anti-malarial immunity in children living in a malaria hyperendemic area. There is substantial evidence that the African Fulani tribe is markedly less susceptible to malaria infection compared to other sympatrically living ethnic tribes. We investigated the isotypic humoral responses against P. falciparum asexual blood stages in different ethnic groups living in sympatry in two countries exhibiting different malaria transmission intensities, Burkina Faso and Mali. We observed higher levels of the total malaria-specific-IgG and its cytophilic subclasses in individuals of the Fulani tribe as compared to non-Fulani individuals. Fulani individuals also showed higher levels of antibodies to measles antigen, indicating that the intertribal differences are not specific for malaria and might reflect a generally activated immune system in the Fulani.