Orientador: Ana Maria Aparecida Guaraldo / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T00:19:39Z (GMT). No. of bitstreams: 1 Machado_DanielaRodriguezdeAssis_M.pdf: 3566658 bytes, checksum: 69880a3922a0bd457f50c7ba71207380 (MD5) Previous issue date: 2008 / Resumo: A malária é uma parasitose que mata mais de um milhão de pessoas por ano. No Brasil, 99% dos casos ocorrem na Amazônia Legal. O Plasmodium chabaudi chabaudi CR, não letal, representa a malária experimental de autocontrole. A hipoglicemia é uma característica comum da malária causada pelo P. falciparum e também está presente na malária murina. O diabetes tipo 1 é uma doença auto-imune órgão-específica que tem como conseqüência a destruição das células ß produtoras de insulina, nas ilhotas de Langerhans. Alguns trabalhos observaram que a infecção de camundongos com o estágio sangüíneo de P. chabaudi induziu uma hipoglicemia em camundongos normais e normalizou a hiperglicemia em camundongos moderadamente diabéticos por indução pela streptozotocina (STZ). No camundongo NOD ("non-obese diabetic"), o diabetes é determinado geneticamente e simula o desenvolvimento do diabetes tipo 1. O presente trabalho se propõe avaliar a influência do diabetes mellitus tipo 1 na infecção experimental por Plasmodium chabaudi chabaudi, linhagem CR, em camundongos fêmeos NOD/Uni, com diferentes graus de diabetes (diabetes moderada e diabetes severa). Camundongos fêmeos livres de patógenos específicos (SPF) da linhagem NOD/Uni com oito a 42 semanas de idade foram infectados via intraperitoneal com 1x107 eritrócitos parasitados pelo Plasmodium chabaudi chabaudi CR. A glicemia foi determinada pela deposição de sangue coletado diretamente da cauda do animal em fita reativa do aparelho Roche Accu-Chek Active® em dias alternados. A parasitemia foi expressa em percentagem de hemácias parasitadas mediante observação de esfregaço sangüíneo corado com May-Grünwald e Giemsa. Os camundongos NOD/Uni foram classificados segundo o grau de diabetes em moderada (300 a 500 mg/dL) e severa (>500 mg/dL). Foi observada redução da glicemia a partir do quinto dia de infecção nos camundongos NOD/Uni, em geral no pico da infecção. Esta redução não ocorreu nos camundongos NOD/Uni sem infecção e nos camundongos da linhagem BALB/c/Uni infectados pelo Plasmodium chabaudi chabaudi CR. As principais lesões renais observadas foram edema, necrose e infiltração glomerular, descamação e dilatação dos túbulos contorcidos e espessamento da membrana basal glomerular. Os camundongos da linhagem NOD/Uni infectados pelo Plasmodium chabaudi chabaudi CR apresentaram maior sobrevida em relação aos camundongos sem infecção. A redução da glicemia parece estar relacionada a diversos fatores dentre eles, a influência das proteínas de membrana GPIs (glycosylphosphatidylinositols) e o equilíbrio entre a resposta dos linfócitos Th1/Th2. / Abstract: Plasmodium chabaudi chabaudi CR in Non-Obese Diabetic (NOD) mice Malaria is a parasitic disease that kills more than one million people worldwide each year. In Brazil, the 99% of the cases occur in the northern States that encompass the Amazon Basin. Plasmodium chabaudi chabaudi is an important experimental model for several aspects of malaria research, including parasite biology and immunology. It is recognized as a developmental analog to human infection by Plasmodium falciparum. The non-lethal Plasmodium chabaudi chabaudi CR strain represents the experimental autocontrol disease. Hypoglycemia is a common symptom of P. falciparum malaria and also occurs in murine malaria. The Type 1 diabetes is an organ-specific autoimmune disease that destroys the insulin-producing ß cells of the Islets of Langerhans. In non-obese diabetic (NOD) mice, diabetes is genetically determined and also develops type 1 diabetes. The present work evaluated type 1 diabetes mellitus during experimental infection by Plasmodium chabaudi chabaudi CR strain in female NOD mice with moderate and severe diabetes. Eight to 42 week-old female specific pathogens free (SPF) NOD/Uni mice were inoculated with red blood cells parasitized sample containing the total of 1x107 Plasmodium chabaudi chabaudi CR by intraperitoneal injection. Routine measurement of blood glucose in NOD/Uni mice was performed every other day for 11 days post-infection. Glycemia was quantified using Roche Accu-Chek Active kits with blood collected from the tail of each animal. The parasitemia was expressed as percent of infected erythrocytes counted by microscopy analysis of tail blood films stained with Giemsa. The animals were classified according to their degree of diabetes into two groups: moderate diabetes (300 to 500 mg/dL) and severe diabetes (>500 mg/dL). The control group was composed of animals of the BALB/c/Uni strain. The natural death of NOD/Uni mice was recorded for both infected and healthy animals. Glycemia decreased at the height of parasitemia around the fifth day of infection. The diabetic infected animals presented higher survival after the onset of diabetes when compared with non-infected control. The main renal lesions found were glomerular edema, glomerular necrosis, cell infiltration, tubular atrophy and expansion of the glomerular basement membrane. The decrease of blood glycemia remains incompletely understood and it seems to be related to GPIs (glycosylphosphatidylinositols) proteins and the balance between Th1/Th2 responses. / Mestrado / Parasitologia / Mestre em Parasitologia

Made available in DSpace on 2015-04-20T12:31:40Z (GMT). No. of bitstreams: 1 Tese Final Sergio Roberto.pdf: 559548 bytes, checksum: 0d333ac1a39ed9593fd449588f71054c (MD5) Previous issue date: 2009-02-27 / Fundação de Amparo à Pesquisa do Estado do Amazonas / A malária é a mais importante doença parasitária do mundo, tendo exercido um forte efeito na evolução humana com um crescente corpo de evidências indicando que tanto o risco de adquirir esta infecção, quanto o de desenvolver severas complicações, tem sido determinado por fatores genéticos do hospedeiro e do parasito infectante. Isolados selvagens de parasitas de malária tem mostrado uma maior variabilidade para invadir hemácias humanas quando comparados com os cultivados em laboratórios, mostrando que em áreas endêmicas, estes podem ter desenvolvido diferentes habilidades para invadir particulares tipos de hemácias. Existem na literatura, resultados controversos quanto a associação entre o sistema ABO, antígenos Rh e a malária vivax, porém já foi bem demonstrada a importância da glicoproteína eritrocitária Duffy na invasão de hemácias humanas por merozoítos de Plasmodium vivax, entretanto ainda sendo pouco conhecido se mutações nesta glicoproteína podem estar associadas à frequência de infecção do P. vivax ou na densidade parasitária da malária vivax. Neste estudo investigamos associações entre o sistema sanguíneo ABO, antígeno RhD, as mutações da proteína Duffy (125 G>A (FYA/FYB) 265 C>T e 298 G>A (antígeno FyX) e do promotor GATA box, -33(T>C),) e a malária vivax, em habitantes do Estado do Amazonas, Brasil. Neste estudo, tanto a identificação do P. vivax, como a verificação das densidades parasitárias na malária vivax, foi determinada por testes microscópicos e leucometria em 497 pacientes infectados, nos quais foram realizadas fenotipagens dos sistemas ABO, Duffy antígeno RhD pela técnica da hemaglutinação, assim como as genotipagens do sistema sanguíneo Duffy pela técnica da PCR/RFLP. As possíveis associações entre as frequências encontradas dos sistemas sanguíneos citados e a malária vivax foram analisadas através do teste qui quadrado, assim como as possíveis associações das genotipagens Duffy com o nível de densidades parasitárias encontradas foram analisadas através do teste de kruskal wallis. Nossos dados mostraram, que a presença do antígeno A, genótipos FYA/FYB-33 e FYB/FYB-33 pode ser uma vantagem seletiva na população, reduzindo a taxa de infecção pelo P. vivax nesta região, porém sem estarem associados ao nível de densidade parasitária das infecções de malária. Não encontramos associação entre o antígeno RhD e a susceptibilidade ao P. vivax, no entanto os genótipos FYA/FYB, FYA/FYA mostraram-se associados ao aumento da frequência de infecção pelo P. vivax na região estudada. Os genótipos FYB/FYX e FYA/ FYX não mostraram-se associados à frequência de infecção pelo P. vivax, mas sim aos baixos níveis de densidades parasitárias encontrada entre os pacientes infectados com este genótipo. Reportamos, ainda, neste estudo, indivíduos com o genótipo FYB-33/FYB-33 com antecedentes de malária vivax. Estes resultados sugerem que, em regiões endêmicas de malária, pode estar havendo adaptações naturais tanto quanto ao surgimento de mecanismos parciais de defesa contra o Plasmodium vivax distintos dos já descritos em descendentes africanos como adaptações que podem estar também levando a um aumento a susceptibilidade a este tipo de malária.

Le paludisme est causé par des parasites du genre Plasmodium et la gamétogenèse est une étape essentielle à sa transmission. Afin d'améliorer les connaissances sur la genèse des gamètes, nous avons mené des études protéomiques sur des gamétocytes de P. berghei sauvages ou mutants avec le gène moteur de la kinésine 8 interrompu. Le mutant est morphologiquement semblable au type sauvage mais incapable de compléter la gamétogenèse et les gamètes mâles d'exflageller. A partir d'échantillons enrichis en gamétocytes, la gamétogenèse a été suivie sur 15 min après induction par l'acide xanthurénique. Les peptides marqués par iTRAQ à partir de triplicatas biologiques ont été analysés par nanoLC/MS-MS et interprétés par les logiciels Patternlab et Blast2GO. Les phosphopeptides ont été enrichis au TiO2. Chez le parasite sauvage, 443 protéines et 206 phosphoprotéines ont été identifiées à partir de 2617 peptides et chez le mutant, 530 protéines et 218 phosphoprotéines à partir de 3198 peptides. L'induction de la gamétogenèse est marquée par une transcription, une biosynthèse de protéines et de l'ADN importantes. Chez le mutant, la formation des fuseaux mitotiques et des axonèmes des flagelles des gamètes mâles est fortement affectée. Des protéines du complexe du protéasome dépendant de l'ubiquitine, de la réponse au stress, du métabolisme énergétique sont également affectées. Cette étude apporte de nouveaux éléments de compréhension sur le rôle joué par la kinésine 8 dans la gamétogenèse de Plasmodium. / Malaria is caused by parasites from Plasmodium genus and its gametogenesis is an essential step to ensure the malaria transmission. In efforts to improve the knowledge on gamete biology we did quantitative proteomic and phosphoproteomic comparative experiments with P. berghei WT and gametocytes disrupted for the motor gene kinesin 8. The mutant is morphologically similar to wild-type parasite but impaired gametogenesis and unable to exflagellate. The Gametocytes-enriched sample from mice at time T0 were xanthurenic acid-induced to exflagellate and harvested at time T7 and T15. The iTRAQ labelled peptides from independent biological triplicate sample were analyzed overtime through nanoLC/MS-MS Orbitrap after TiO2 enrichment, and interpreted by Patternlab and Blast2GO softwares. The wild type had 443 proteins and 206 phosphoproteins identified from 2,617 peptides. The mutant had 530 proteins and 218 phosphoproteins identified from 3,198 peptides. GO biological processes related to RNA translation, DNA and protein biosynthesis were most prominent and phosphorylated proteins are mainly RNA, ATP or protein binding proteins. Within the mutant, the axoneme and mitotic spindle microtubules disorganization were strongly affected. The nucleosome components are key to nuclear division disorganization. The ubiquitin-dependent proteasome complex and stress/folding response, energy metabolism and egress proteins were affected. The Plasmodium proteomic approach brings that insight into kinesin 8 critical importance for male gametogenesis in Plasmodium with effect on protein expression, phosphorylation modulation, flagellar organization and biology.

Le paludisme est une des maladies tropicales les plus dévastatrices au monde causée par des parasites protozoaires intracellulaires du genre Plasmodium. Cinq espèces de plasmodies sont responsables du paludisme chez l'homme et causent 600 000 décès par an principalement chez les enfants de moins de 5 ans et les femmes enceintes vivant dans les régions les plus pauvres du globe. Les parasites ont généré une résistance contre les chimiothérapies existantes et aucun vaccin efficace n'est encore disponible. Il est donc impératif d'identifier et de valider de nouvelles cibles qui peuvent être exploitées pour la découverte de nouveaux médicaments.Cette étude a porté sur la caractérisation d'un enzyme, la phosphoénolpyruvate carboxylase (PEPC), produit d'un gène spécifique au parasite et absent chez l'hôte humain, ce qui constitue l'un des pré-requis d'une cible potentielle pour la découverte de médicaments. Le gène avait été montré comme essentiel pour des parasites seulement en absence de malate ou de fumarate, suggérant un rôle de la protéine dans le métabolisme du carbone intermédiaire des parasites.Mes études de thèse avaient pour but de caractériser le rôle de la PEPC en utilisant la métabolomique. J'ai d'abord établi et normalisé une méthodologie d'analyses métabolomiques des globules rouges infectés par Plasmodium et optimisé l'analyse des métabolites hydrophiles présents dans le parasite intracellulaire et sa cellule hôte. Nous nous sommes concentrés sur les métabolites du métabolisme du carbone intermédiaire, où la PEPC pouvait jouer un rôle déterminant par analogie avec les plantes et les bactéries. Des analyses ciblées utilisant un marquage isotopique du métabolome à partir de 13C-U-glucose, 13C-bicarbonate et 13C, 15N-glutamine ont aussi été réalisées permettant de mieux appréhender les conséquences d'un KO de l'enzyme PEPC sur le métabolisme du parasite.Les données montrent que l'enzyme PEPC permet une fixation du bicarbonate et catalyse une réaction anaplérotique conduisant à du malate qui est introduit dans le cycle de l'acide tricarboxylique mitochondrial, transférant ainsi des équivalents réducteurs du cytoplasme à la mitochondrie et fournissant aussi un point d'entrée du squelette carboné dans le cycle. Les résultats montrent surtout que les parasites possèdent un cycle complet et de type oxydatif de l'acide tricarboxylique mitochondrial. Il parait y avoir trois points d'entrée: 1. l'acétyl CoA résultant du pyruvate généré par la glycolyse et décarboxylé dans la mitochondrie; 2. l'acide alpha-cétoglutarique provenant du glutamate, qui lui-même résulte de la désamination de la glutamine essentiellement fournie par l'environnement externe; 3. le malate, produit en aval de la malate déshydrogénase qui réduit l'oxaloacétate produit par la PEPC. En aval de la PEPC, la biosynthèse des pyrimidines opère grâce à l'activité de l'aspartate aminotransférase agissant sur oxaloacétate.En dehors du malate, le fumarate est le seul autre métabolite qui permet de s'opposer au défaut de croissance des parasites déficients en PEPC, ce qui a conduit à évaluer le rôle de la fumarase. À cette fin, l'étiquetage du gène endogène fumarase avec une étiquette HA, a permis de montrer que la protéine est exprimée dans les stades intra-érythrocytaires de P. falciparum et de montrer que la protéine se trouve à la fois dans la mitochondrie et le cytoplasme. La protéine recombinante a été exprimée avec succès et partiellement caractérisée biochimiquement. De nombreuses tentatives visant à générer des mutants de délétion génétique de P. falciparum n'ont pas abouti, laissant en suspens la question du caractère essentiel du gène pour les parasites. Cependant, il est possible de cibler le locus du gène via un marquage C-terminal. Ceci suggère que l'enzyme peut être essentielle pour la survie du parasite et donc une cible exploitable pour la découverte d'un type nouveau de médicament antipaludique. / Malaria is one of the world's most devastating tropical diseases caused by obligate intracellular protozoan parasites of the genus Plasmodium. Five species of these parasites cause malaria in humans and infection results in ~600,000 deaths annually primarily in children under the age of 5 and pregnant women living in the poorest areas of the globe. The parasites have an outstanding ability to generate resistance against existing chemotherapies and an efficacious vaccine is not available yet. Therefore it is imperative that attempts are being made to identify and validate new targets that can be exploited for future drug discovery.This study focused on the validation and elucidation of a parasite-specific gene product namely phosphoenolpyruvate carboxylase (PEPC), which is not present in the human host and thus has one of the pre-requisites of a potential drug target. The gene had been previously genetically validated and it was demonstrated that mutant parasites lacking pepc were only viable in the presence of malate or fumarate, suggesting a role of the protein in intermediary carbon metabolism of the parasites.My studies had the goal to assess the role of PEPC using a metabolomics approach. Initially the methodologies to perform metabolomics analyses of Plasmodium-infected RBCs were established and standardised and it was assessed how to best analyse the hydrophilic metabolites present in the intracellular parasites and its host cell. We focused on metabolites of intermediary carbon metabolism, as it is likely that PEPC is important for metabolic functions linked to this in the parasites, in analogy to plants and bacteria. While global metabolomics analyses were appealing, it was decided to apply a targeted metabolomics and comparative approach using stable isotope labelling of the parasite metabolomes with 13C-U-glucose, 13C-bicarbonate and 13C-,15N-glutamine to assess the consequences of the pepc knockout on parasite metabolism.The data demonstrated that PEPC has an anaplerotic function fixing bicarbonate and leading to generation of malate that is fed into the mitochondrial tricarboxylic acid cycle and so transfers reducing equivalents from cytoplasm to mitochondrion as well as providing an entry point of carbon skeleton into the cycle. The most important findings with respect to parasite mitochondrial metabolism were that the parasites possess a complete and oxidative tricarboxylic acid cycle, which appears to have three entry points: 1. Acetyl CoA resulting from glycolytically generated pyruvate that is decarboxylated in the mitochondrion; 2. α-ketoglutarate from the reaction of glutamate dehydrogenase and 3. malate, which is a downstream product of malate dehydrogenase that reduces oxaloacetate the reaction product of PEPC. Other downstream reactions supported by PEPC activity are pyrimidine biosynthesis through the activity of aspartate aminotransferase also acting on the PEPC-derived oxaloacetate.Apart from malate, fumarate was the only other metabolite that reversed the growth defect of pepc mutant parasites. Hence the role of fumarase in the parasites was also assessed. To this end the endogenous fumarase gene of P. falciparum was tagged with an HA-tag, which showed that the protein is expressed in the intra-erythrocytic stages of P. falciparum and demonstrated that the protein is located in both mitochondrion and cytoplasm. In addition, the recombinant protein was produced and partially biochemically characterised. Numerous independent attempts to generate genetic deletion mutants of P. falciparum were unsuccessful, leaving the question whether the gene is essential for the parasites unanswered. However, it was possible to manipulate the locus by C-terminal tagging of the fumarase gene suggesting that fumarase might be indeed essential for parasite survival and therefore possibly suitable for future drug design and discovery.

Plasmodium is a genus of parasites causing malaria, a virulent protozoan infection in humans resulting in over a million deaths annually. Treatment of malaria is increasingly limited by parasite resistance to available drugs. Hence, there is a need to identify new drug targets and authenticate antimalarial compounds that act on these targets. A relatively new therapeutic approach targets proteolytic enzymes responsible for parasite‟s invasion, rupture and hemoglobin degradation at the erythrocytic stage of infection. Cysteine proteases (CPs) are essential for these crucial roles in the intraerythrocytic parasite. CPs are a diverse group of enzymes subdivided into clans and further subdivided into families. Our interest is in Clan CA, papain family C1 proteases, whose members play numerous roles in human and parasitic metabolism. These proteases are produced as zymogens having an N-terminal extension known as the prodomain which regulates the protease activity by selectively inhibiting its active site, preventing substrate access. A Clan CA protease Falcipain-2 (FP-2) of Plasmodium falciparum is a validated drug target but little is known of its orthologs in other malarial Plasmodium species. This study uses various structural bioinformatics approaches to characterise the prodomain‟s regulatory effect in FP-2 and its orthologs in Plasmodium species (P. vivax, P. berghei, P. knowlesi, P. ovale, P. chabaudi and P. yoelii). This was in an effort to discover short peptides with essential residues to mimic the prodomain‟s inhibition of these proteases, as potential peptidomimetic therapeutic agents. Residues in the prodomain region that spans over the active site are most likely to interact with the subsite residues inhibiting the protease. Sequence analysis revealed conservation of residues in this region of Plasmodium proteases that differed significantly in human proteases. Further prediction of the 3D structure of these proteases by homology modelling allowed visualisation of these interactions revealing differences between parasite and human proteases which will lead to significant contribution in structure based malarial inhibitor design.

La pompe ATPase Ca2+ du réticulum sarco-endoplasmique Plasmodium falciparum (PfATP6) est une protéine de dix transmembranes, impliqué dans la régulation de l'homéostasie du calcium dans le parasite. L'importance d'étudier cette protéine repose sur l'hypothèse d'être engagé dans le mécanisme d'action et de résistance des artémisinines. Des travaux précédents, fondé sur l'expression hétérologue dans des ovocytes de Xenopus laevis et Saccharomyces cerevisiae, ont montré des résultats opposés, générant de nombreux corollaires vérifiables. Par conséquent, des travaux supplémentaires sont nécessaires pour mieux comprendre la nature des interactions entre les artémisinines et transporteurs de type SERCA.Afin d'évaluer le caractère essentiel du gène de Plasmodium spp., une approche de génétique inverse a été utilisée. Knockout du gène, soit P. falciparum et berghei, ne pouvant pas être obtenu. La complémentation de sauvetage épisomique a été jugée impossible. Marquage à la fin 3' de PfATP6 et PbATP6 a été, également, tenté pour étudier la localisation et l'expression de la protéine chez les parasites. La manipulation des gènes à cette place n'a pas permis la survie du parasite. Nos résultats, pris ensemble, montrent que ATP6 est essentiel dans Plasmodium spp..Au cours de nos études génétiques, un phénotype stable et particulier de parasites du genre Plasmodium falciparum 3D7 a été distingué. Les étranges parasites “monstres" contiennent une vacuole digestive inhabituelle gonflées à travers toutes les étapes du développement du parasite. Caractérisation de l'insolite Plasmodium a été réalisée, montrant une sensibilité accrue à la chloroquine, mais pas à l'artémisinine ou de la méfloquine. Tenant compte de la similitude du PfATP6 avec la pompe SERCA orthologue mammifère, de nouvelles molécules, connu et synthétisé pour cibler spécifiquement la protéine chez les mammifères, ont été testés sur P. falciparum. Quatre classes différentes de composés (sHA 14-1, BHQ, chalcone et des analogues de l'ACP) a montré le blocage de la croissance in vitro du P. falciparum 3D7 et Dd2 à des concentrations inférieure au range micromolaire. En outre, une nouvelle classe de molécules (thaperoxides), conçu comme un hybride entre l'artémisinine et thapsigargine, a été testé contre le type sauvage 7G8 et la ligne muté L263E. Ce dernier porte une mutation ponctuelle unique de nucléotides dans PfATP6, déjà connu d'être impliqué dans la résistance du l'artémisinine.Compte tenu de la difficulté à manipuler les gènes du parasite, et afin de mieux caractériser PfATP6, un gène synthétique a été optimisé pour l'expression hétérologue chez S. cerevisiae. De cette façon, la complémentation d'une ligne de levure mutée (K616) sans les pompes endogènes Ca2+ de type P a été permis avec succès, montrant le sauvetage de la croissance de la levure en présence de forte concentration de calcium libre. Différents inhibiteurs de SERCA, comme la thapsigargine et l'acide cyclopiazonique, ont été testés sur la levure complémenté K616 PfATP6, afin de vérifier l'inhibition de la croissance. Tous les composés ont bloqué la croissance de levure sélectivement ciblant le PfATP6. En outre, le test a été développé comme un criblage de haute performance, afin de tester de nouvelles molécules pour leur activité. La méthode s'est révélée être un outil rapide et très fiable et reproductible pour l'identification de nouveaux composés actifs. / The Plasmodium falciparum sarco-endoplasmic reticulum ATPase Ca2+ pump (PfATP6) is a ten transmembrane protein involved in the regulation of the calcium homeostasis in the parasite. The importance of studying this protein relies on the fact that it has been hypothesized to be involved in the mechanism of action and resistance of artemisinins. Previous works, based on heterologous expression in Xenopus laevis oocytes and Saccharomyces cerevisiae, have shown contrasting results, generating many testable corollaries. Therefore, further work is needed to better understand the nature of interactions between artemisinins and SERCA-type transporters.In order to assess the essentiality of the gene in Plasmodium spp., a reverse genetics approach has been used. Knockout of the gene, in either P. falciparum and berghei, could not be achieved. Complementation for episomal rescue was found to be not possible. Tagging at the 3' end of PfATP6 and PbATP6 has been, also, attempted to study localization and expression of the protein in parasites. Manipulation of the gene at this position did not permit parasite survival. Our results, taken together, show that ATP6 is essential in Plasmodium spp..During our genetic studies, a stable and peculiar phenotype of Plasmodium falciparum 3D7 parasites has been noticed. The odd “monster” parasites contain an unusual swollen food vacuole throughout all stages of parasite development. Characterization of the unusual Plasmodium has been carried out, showing an increased sensitivity to chloroquine, but not to artemisinin or mefloquine. Taking into account the similarity of PfATP6 with the mammalian orthologue SERCA pump, new molecules, designed and synthesized to specifically target the mammalian protein, were tested on P. falciparum parasites. Four different classes of compounds (sHA 14-1, BHQ, chalcone and CPA analogues) showed to inhibit P. falciparum 3D7 and Dd2 growth in vitro at concentrations in the lower micromolar range. In addition, a novel class of molecules (thaperoxides), designed as an hybrid between artemisinin and thapsigargin, has been tested against 7G8 wild type and mutated L263E line. The latter carries a single nucleotide point mutation in PfATP6 that has been previously shown to be involved in artemisinin resistance. Considering the difficulty in manipulating the gene in the parasite and in order to better characterize PfATP6, a synthetic gene was optimized for heterologous expression in S. cerevisiae. This enabled successful complementation of a mutated yeast line (K616) lacking the endogenous P-type Ca2+ pumps, showing rescue of the yeast growth in presence of high concentration of free calcium. Different SERCA inhibitors, such as thapsigargin and cyclopiazonic acid, have been tested on K616 PfATP6 complemented yeast, in order to check for growth inhibition. All compounds showed to inhibit yeast growth selectively targeting PfATP6. In addition, the assay has been developed as a high throughput screening, in order to test new molecules for their activity. The method has proved to be a fast, highly reliable and reproducible tool for identification of new active compounds.

Le développement du parasite Plasmodium falciparum, responsable du paludisme, nécessite la synthèse de phospholipides et plus particulièrement de phosphatidylcholine (PC) et phosphaditylethanolamine (PE) qui représentent environ 85% de la totalité des phospholidipes du parasite. Leur synthèse s'effectue principalement par les voies métaboliques de novo, voies de Kennedy, en trois étapes enzymatiques. Les enzymes CTP: phosphoethanolamine cytidylyltransferase (ECT) et CTP: phosphocholine cytidylyltransferase (CCT) catalysent les étapes limitantes des deux voies de biosynthèse de la PE et de la PC, respectivement. Ces deux enzymes sont essentielles à la survie du parasite murin, P. berghei et représentent ainsi des cibles thérapeutiques potentielles. La PfCCT est constituée de deux domaines cytidylyltranférases (CT) répétés alors que l'enzyme homologue chez l'homme est composée d'un seul domaine. En revanche, pour la ECT, la présence de deux domaines CT est retrouvée chez toutes les espèces mais les analyses de séquences et de structures ont montré que des résidus importants du site catalytique liant le substrat n'étaient pas conservés dans le domaine CT C-terminal de la PfECT. Ce travail a eu pour but de déterminer les propriétés enzymatiques et les caractéristiques cellulaires de la PfECT et de la PfCCT. Les paramètres cinétiques de ces enzymes ont été quantifiés in vitro à l'aide protéines recombinantes ainsi que sur les enzymes endogènes à l'aide d'extraits parasitaires. Grâce à l'utilisation de protéines recombinantes ponctuellement mutées, nous avons montré que seul le domaine CT N-terminal de la PfECT est catalytiquement actif. Chez P. falciparum, la PfECT et la PfCCT sont exprimées tout au long du cycle intra-érythrocytaire du parasite. La PfECT est présente dans la fraction soluble du parasite alors que la PfCCT apparait aussi bien dans la fraction soluble qu'insoluble. Des expériences d'immunofluorescence ont montré que la PfECT est cytosolique. L'ensemble des résultats présentés apportent un éclairage important sur les fonctions et les propriétés de ces deux cibles potentielles et constituent les premières étapes indispensables à l'élaboration d'une approche thérapeutique. / Phospholipids are essential for the growth and development of Plasmodium falciparum malaria parasite. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are its major structural phospholipids. This study focused on CTP: phosphoethanolamine cytidylyltransferase (ECT) and CTP: phosphocholine cytidylyltransferase (CCT) that catalyzes the rate-limiting steps of the de novo Kennedy pathways for PE and PC biosynthesis respectively. Both ECT and CCT are essential in the rodent malaria parasite P. berghei and constitute potential chemotherapeutic targets to fight against malaria. PfCCT consists of two very similar cytidylyltransferase (CT) domains whereas the human enzyme consists of only one CT domain. The presence of two CT domains in ECT seems to be widespread in all the organisms. Sequence and structural analysis showed that the C-terminal CT domain of ECT lacks key residues in the substrate binding motif. This study aimed at unravelling the enzymatic properties and cellular characteristics of PfECT and PfCCT enzymes. In addition, these studies addressed the key question if C-terminal CT domain of PfECT is catalytically active. Kinetic parameters of the enzymes were evaluated in vitro on native proteins as well as on recombinant proteins, the latter being produced in bacterial system. Cellular characterisation studies using polyclonal antisera showed that PfECT and PfCCT are expressed throughout the intra-erythrocytic life cycle of the parasite. PfECT is found mainly in soluble form in the parasite while PfCCT is present in soluble as well as insoluble forms in the parasite. Furthermore, immunofluorescence studies for PfECT revealed that it is mainly cytosolic. To assess the contribution of each CT domain to overall PfECT enzyme activity, recombinant PfECT mutants were generated by site-directed mutagenesis. Kinetic studies on these mutants indicated that the N-terminal CT domain was the only active domain of PfECT. Collectively, these results bring new insights into the kinetic and cellular properties of the enzymes and will pave the way in developing a future pharmacological approach.

O objetivo do estudo foi confirmar se a redução da fecundidade dos mosquitos Aedes aegypti infectados por Plasmodium gallinaceum ocorre por morte das células foliculares dos ovários. Mosquitos infectados produzem menos ovos quando comparado aos mosquitos sadios. Uma explicação é a redução da viabilidade celular que ocorre nos ovários de fêmeas 18, 22 e 24 horas após o repasto sanguíneo infectado (RSI) como foi observado pela técnica MTT. Utilizando o acridine orange, não foi possível observar a morte das células foliculares no intervalo de 18 horas, mas 22 e 24 horas após o RSI essas células estão em morte em relação ao mesmo intervalo do repasto sanguíneo controle (RSC). A análise do DNA fragmentado foi realizada através do TUNEL. Ovários de 22 e 24 horas após RSC e RSI foram negativos nas regiões dos cortes histológicos examinados. Sendo assim, podemos concluir que, utilizando esses ensaios foi possível identificar a morte das células foliculares como uma resposta a redução da fecundidade, porém não foi possível determinar que o tipo de morte é apoptose. / The objective of this study was to confirm the hypothesis that the fertility reduction in Plasmodium gallinaceum-infected Aedes aegypti occurs by follicular cells death. A significant reduction in the number of eggs laid by infected mosquitoes was confirmed. It was observed a reduction of viable cells in 18, 22 and 24 hours PBM infected by MTT assay. It was not possible to observe cell death in ovary tissue 18 hours PBM infected, but the follicular cells showed orange color 22 and 24 hours indicating they are in death in relation to the same interval of PBM control. To determine if these cells exhibit apoptosis, we use the TUNEL which mark the fragmented DNA, a characteristic of the apoptosis process. Ovaries 22 and 24 hours PBM infected and control were negative for TUNEL marker from ovary histological preparations. Thus, we conclude that fecundity reduction occurs as a response to follicular cells death caused by P. gallinaceum infection but it was not possible to affirm if the type of follicular cells death is apoptosis.

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.

Malaria remains one of the most serious tropical infectious diseases affecting mankind. The prevention of the disease is hampered by the increasing resistance of the parasite to existing chemotherapies. The need for novel therapeutic targets and drugs is therefore of the utmost importance and detailed knowledge of the biochemistry of the parasite is imperative. This study was directed at the biochemical characterisation of the polyamine metabolic pathway of P. falciparum in order to elucidate differences between the parasite and its human host that can be exploited in the design of novel antimalarials. The thesis focussed on the two rate-limiting enzymes in polyamine biosynthesis, S¬adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), which occur as a unique bifunctional complex in P. falciparum. The genomic structure of the bifunctional gene indicated a single, monocistronic transcript with large untranslated regions that were predicted to be involved in unique translational regulatory mechanisms. This gives rise to a bifunctional protein containing both decarboxylase activities on a single polypeptide forming a heterotetrameric complex. Activity of the decarboxylases decreases dramatically if these proteins are expressed in their monofunctional forms as homodimeric ODC and heterotetrameric AdoMetDC. The deduced amino acid sequence indicated that all the essential residues for catalysis are conserved and highlighted the presence of three parasite-specific insertions. The parasite-specific inserts were shown to be essential for the catalytic activity of the respective domains and also to influence the activity of the neighbouring domain, indicating that intramolecular communication exists in the heterotetrameric complex. The most structured and smallest insert was also shown to mediate protein-protein interactions between the two domains and to stabilise the complex. Further structure- functional characterisations of specifically the ODC domain were deduced from a comparative homology model. The model predicted an overall structure corresponding to those of other homologous proteins. The validity of the model is supported by mutagenesis results. However, certain parasite-specific properties were identified in the active site pocket and dimerisation interface. The former was exploited in the rational design of novel putative ODC inhibitors directed only against the P. falciparumprotein by in silico screening of chemical structure libraries. This study therefore describes the identification of certain parasite-specific properties in a unique bifunctional protein involved in regulation of polyamine metabolism of P. falciparum. Such discoveries are invaluable in strategies aimed at elucidating biochemical and metabolic differences between the parasite and its human host that could be exploited in the design of alternative, parasite-specific chemotherapies. Moreover, the thesis also contributed new knowledge on certain less well-understood biological phenomena characteristic of P. falciparum, the nature and origin of bifunctional proteins and the functional properties of parasite-specific inserts found in some proteins of the parasite. / Thesis (PhD (Biochemistry))--University of Pretoria, 2002. / Biochemistry / unrestricted