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>

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

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.

Malaria is a widespread disease caused by parasites of the genus Plasmodium. Each year 500 million clinical cases are reported resulting in over one million casualties. The most lethal species, P. falciparum, 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. In this thesis, the process of computational inhibitor design is illustrated through the development of P. falciparum 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. 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. The influence of the partial charge model on binding free energy calculations with the LIE method was assessed. Two semiempirical and six ab initio 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 ab initio charge models.