Malaria remains a global health problem, despite over a century of efforts towards control and prevention. It is responsible for over 2 million deaths a year. Plasmodium falciparum, the protozoan parasite that causes malaria, presents quite an unexplored field of study, significant both for the purposes of understanding the complex life cycle of the parasite, and for identifying novel and unique targets for anti-malarial therapy. Cyclin-dependent kinases. (CDK.s) play a number of crucial roles in the progression of the cell cycle such as regulating the onset of DNA replication and entry into mitosis. Plasmodium falciparum protein kinase 5, PfPK5, manifests characteristics of eukaryotic CDKs. It is a serine/threonine kinase, has 60% amino acid identity to eukaryotic cyclin-dependent kinase cdc2, and shares the mechanism of activation with CDKs. To establish if PfPK5 indeed is the major cell cycle regulatory kinase, as well as to expand our knowledge about the signaling networks of the parasite, it is necessary to identify proteins that interact with the kinase, such as its putative substrates. Currently, only one Plasmodium falciparum protein is known to interact with PfPK5 - its cyclin partner, Pfcycl. Identifying substrates of PfPKS is a particularly important research endeavor since it would provide insight into the yet unknown downstream signaling pathways of PfPK5. It is likely that pathways unique to Plasmodium falciparum will be found, which may be specifically targeted for anti-malaria therapy. A potential substrate of Plasmodium falciparum cell cycle regulatory kinase PfPK5 has been identified. The new protein, which we call SPOK, was identified by screening a phage display cDNA library. Since SPOK is a large protein of approximately 140kDa, a domain containing a tandem CDK/cdc2 phosphorylation motif of SPEK (single amino acid code, S/TPXK/R) was expressed in E.coli. Our results show that this domain of SPOK is indeed phosphorylated in vitro by PfPK5. This raises the possibility that SPOK could be an in vivo substrate of PfPK5 and may play a role in regulating the cell cycle of the parasite.

O desenvolvimento de imunidade naturalmente adquirida na malária é lento e depende de fatores como o número de malárias prévias, intervalo entre cada malária, exposição a variantes antigênicas múltiplas e idade do indivíduo. Mecanismos imunes efetores dependentes de anticorpos são importantes no desenvolvimento dessa imunidade. Vários estudos têm mostrado que as subclasses IgG1 e IgG3 anti-P. falciparum, conhecidas por sua ação citofílica, são anticorpos protetores, enquanto anticorpos não citofílicos como IgG4 reconhecendo os mesmos epítopos seriam bloqueadores dos mecanismos protetores. Dados recentes sugerem que sob determinadas condições, IgG2 também pode ter ação citoíflica e participar da proteção na malária. Neste trabalho, nós estudamos pela primeira vez, a evolução das subclasses de IgG contra formas eritrocitárias de P. falciparum de pacientes com malária falciparum não complicada internados em hospital por até 42 dias, sob tratamento com mefloquina. As subclasses de IgG foram avaliadas por ELISA em 48 pacientes (7 amostras de soro de cada um colhidas nos tempos 0h, 48h, 7, 14, 28, 35 e 42 dias), quanto à quantidade (concentração, ug/ml; índices de reatividade, IR; ou em freqüência, %) e quanto à avidez dos anticorpos (índice de avidez, IA). Amostras de soro de 14 pacientes (tempos: 0, 48h, 7, 21 e 28 dias) também foram avaliadas quanto à especificidade e a avidez de reconhecimento das diferentes bandas protéicas do antígeno de P. falciparum por Immunoblotting. As subclasses IgG1, IgG2, IgG3 e IgG4 anti-P. falciparum, na maioria de alta avidez, estavam presentes no início do tratamento, respectivamente, em 100%, 39,5%, 80,6% e 28,4% dos pacientes e com concentrações médias de 20, 2; 3,8; 1,5 e 0,05 ?g/mL. As concentrações máximas das subclasses de IgG foram alcançadas no 7o dia, e os IAs máximos de IgG1 e IgG3 foram alcançados no 7o dia, e os de IgG2 no 14o dia e os de IgG4 no 2o dia. A concentração inicial dos anticorpos IgG3 anti-P. falciparum apresentou correlação negativa com o tempo de clareamento parasitário (TCP) e a relação das somas dos anticorpos IgG1, IgG2 e IgG3 pelos níveis de IgG4 se correlacionaram negativamente com a parasitemia inicial. No Immunoblotting, foram identificadas frações protéicas que podem estar relacionadas com o reconhecimento imune protetor, por serem reconhecidas pelas subclasses IgG1, IgG2 e IgG3 e não reconhecidas ou reconhecidas tardiamente por IgG4: 125, 96, 86, 75, 55 e 47 kDa. A resposta predominante das subclasses IgG1, IgG2 e IgG3 observada nestes pacientes, todos com malária não complicada, pode indicar que esses anticorpos estão cooperando para o controle de formas graves da doença e refletirem um certo grau de desenvolvimento de imunidade adquirida / The development of naturally acquired immunity to malaria is slow and depends of several factors as number of previous malaria, interval between each malaria attack, exposure to parasite multiple antigen variant and ageassociated maturation of the immune system. Antibody-dependent effector immune mechanisms are believed to be important to the protective immunity. A number of studies have showed that anti-P. falciparum IgG1 and IG3, named cytophilic antibodies, are protective, whereas the noncytophilic, IgG4, that recognize the same epitopes may block the protective mechanisms. Recent data have suggested that in certain situations, IgG2 can also act as cytophilic and to cooperate in protection. In this work, we have studied, for the first time, the evolution of the IgG subclasses against P. falciparum blood stages in uncomplicated falciparum malaria patients taken into hospital upon mefloquine treatment and followed up 42 days. These antibodies were determined by ELISA in 48 patients (7 serum samples from each patient collected in different times: 0h, 48h, 7, 14, 28, 35 and 42 days). The results were expressed in concentration (ug/ml), index of reactivity (IR) or frequency (%) and the avidity were expressed as index of avidity (IA). Serum samples 14 patients (time of collection: 0, 48h, 7, 21 and 42 days) were also evaluated by Immunoblotting as their specificity and avidity against different proteins of the P. falciparum blood stages The subclasses Anti-P. falciparum IgG1, IgG2, IgG3 and IgG4, high avidity predominantly, were present since the beginning of the treatment, respectively, in 100%, 39,5%, 80,6% and 28,4% of the patients with the following concentrations: 20, 2; 3,8; 1,5 and 0,05 ?g/mL. The highest concentrations were reached at day 7, and IgG1 and the highest IgG3 IAs were reached at day 7, and the highest IgG2 IAs at day 14 and the highest IgG4 IAs at day 2. The initial concentration of anti-P. falciparum IgG3 showed a negative correlation with the parasitemia clearance time (PCT) and the ratio between the sum of IgG1, IgG2 and IgG3 levels to IgG4 levels was negatively correlated with the initial parasitemia. Six protein fractions were identified by the Immunoblotting that can be related to protective immune recognition, because they were recognized by IgG1, IgG2 and IgG3 antibodies and not or only later recognized by IgG4 antibodies: 125, 96, 86, 75, 55 and 47 kDa. The predominant IgG1, IgG2 and IgG3 responses observed in these uncomplicated malaria patients may suggest that these antibodies are cooperating to the control of severe disease and reflecting a certain development of protective immunity

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 126-137).

Submitted by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2013-08-06T19:23:03Z No. of bitstreams: 1 SARAH_RESENDE_MBCM_Final.pdf: 6553798 bytes, checksum: cb7ea34b3f0a947f3d81c20d5448938f (MD5) / Made available in DSpace on 2013-08-06T19:23:03Z (GMT). No. of bitstreams: 1 SARAH_RESENDE_MBCM_Final.pdf: 6553798 bytes, checksum: cb7ea34b3f0a947f3d81c20d5448938f (MD5) Previous issue date: 2013 / Cepas de Plasmodiumresistentes a diferentes drogas têm sido descritasao redor do mundo. Embora os mecanismos de desenvolvimento de resistência não sejam bem conhecidos, sabe-se que defeitos nos sistemas de reparo do DNA podem estar envolvidos. Esses defeitos estão relacionados principalmente a mutações nas enzimas do sistema de reparo de mal pareamento do DNA ou mismatch repair (MMR) e já foram descritos em populações naturais de diversos organismos. Devido ao conhecimento limitado sobre o sistema MMR de Plasmodium, faz-se necessário um amplo estudo sobre os genes que codificam as proteínas envolvidas nesse sistema. Neste trabalho, foi realizado um estudo sobre as enzimas envolvidas no sistema de reparo do mal pareamento do DNA em Plasmodium: variabilidade intra e interespecífica em Plasmodium, principalmente nos domínios funcionais, e comparação entre níveis de expressão entre cepas/isolados de P. falciparum.Os parasitos foram também avaliados quanto ao número de cópias e expressão dos genes gch-1emdr1. Foram identificadas proteínas pertencentes às classes MSH2, MSH6, MLH1 e PMS1. As sequências de proteínas mostraram-se muito conservadas, tanto entre o gênero Plasmodium, quanto em relação a outros organismos distantes evolutivamente. Foi encontradaum proteína homóloga a MutS que possui os domínios I e V, mas ainda não identificada quanto à sua classificação. O gene codificador desta proteína teve sua expressão confirmada neste e em outros trabalhos. Alguns SNPs foram encontrados em cepas/isolados depositados no PlasmoDB, no entanto, o sequenciamento da região que compreende os principais domínios funcionais apontou apenas 1 SNP na proteína PMS1. Os genes estudados, em sua maioria, apresentaram-se mais expressos entre 10 e 30 horas após a sincronização. W2 e 3D7 apresentam 2 cópias do genes gch-1e mdr1. BHZ apresentou apenas 1 cópia do mdr1. Os resultados da análise de expressão desses genes ligados à resistência concordam com os resultados encontrados para o número de cópias gênicas. Este estudo fornece uma análise ampla das principais enzimas do MMR e será importante para estudos futuros do papel funcional destas enzimas e seu envolvimento no desenvolvimento de resistência às drogas. / Drug-resistant Plasmodiumstrains have been reported world wide. The mechanisms underlying resistance development are not well understood, but failure in DNA repair could be involved in this process. This failure is mainly related to mutations in the enzymes of the DNA mismatch repair (MMR). Because of the limited knowledge about the PlasmodiumMMR system, it is necessary a comprehensive study about the genes encoding proteins involved in this system. In this work, we studied the enzymes involved in the PlasmodiumMMR, considering the intraspecific and interspecific variability in Plasmodium, especially within the functional domains and comparing the expression levels between strains/isolates of P. falciparum.Parasites were also assessed for copy number and expression of the genes pfgch-1and pfmdr1. We identified proteins related to MSH2, MSH6, MLH1 and PMS1. The protein sequences were very conserved among the genus Plasmodium, as well in relation to other evolutionarily unrelated organisms. We found a putative protein homologous to MutS showing the domains I and V, butnot classified yet. The gene encoding this protein has its expression confirmed here and in other previous studies. SNPs were found in some strains/isolates deposited in PlasmoDB, however, the sequencing of the region comprising the main functional domains showed only one SNP in PMS1. The genes studied, mostly, were more expressed between 10 and 30 hours after synchronization. W2 and 3D7 showed 2 copies of the gene gch-1 and mdr1. In BHZ, only one copy of the mdr1 were founded. The results of expression of these genes related to the resistanceagree with the findings for the gene copy number. This study provides a comprehensive analysis of the major enzymes of the MMR and will be important to further functional studies of this enzymes and their role in drug resistance development.

O desenvolvimento de imunidade naturalmente adquirida na malária é lento e depende de fatores como o número de malárias prévias, intervalo entre cada malária, exposição a variantes antigênicas múltiplas e idade do indivíduo. Mecanismos imunes efetores dependentes de anticorpos são importantes no desenvolvimento dessa imunidade. Vários estudos têm mostrado que as subclasses IgG1 e IgG3 anti-P. falciparum, conhecidas por sua ação citofílica, são anticorpos protetores, enquanto anticorpos não citofílicos como IgG4 reconhecendo os mesmos epítopos seriam bloqueadores dos mecanismos protetores. Dados recentes sugerem que sob determinadas condições, IgG2 também pode ter ação citoíflica e participar da proteção na malária. Neste trabalho, nós estudamos pela primeira vez, a evolução das subclasses de IgG contra formas eritrocitárias de P. falciparum de pacientes com malária falciparum não complicada internados em hospital por até 42 dias, sob tratamento com mefloquina. As subclasses de IgG foram avaliadas por ELISA em 48 pacientes (7 amostras de soro de cada um colhidas nos tempos 0h, 48h, 7, 14, 28, 35 e 42 dias), quanto à quantidade (concentração, ug/ml; índices de reatividade, IR; ou em freqüência, %) e quanto à avidez dos anticorpos (índice de avidez, IA). Amostras de soro de 14 pacientes (tempos: 0, 48h, 7, 21 e 28 dias) também foram avaliadas quanto à especificidade e a avidez de reconhecimento das diferentes bandas protéicas do antígeno de P. falciparum por Immunoblotting. As subclasses IgG1, IgG2, IgG3 e IgG4 anti-P. falciparum, na maioria de alta avidez, estavam presentes no início do tratamento, respectivamente, em 100%, 39,5%, 80,6% e 28,4% dos pacientes e com concentrações médias de 20, 2; 3,8; 1,5 e 0,05 ?g/mL. As concentrações máximas das subclasses de IgG foram alcançadas no 7o dia, e os IAs máximos de IgG1 e IgG3 foram alcançados no 7o dia, e os de IgG2 no 14o dia e os de IgG4 no 2o dia. A concentração inicial dos anticorpos IgG3 anti-P. falciparum apresentou correlação negativa com o tempo de clareamento parasitário (TCP) e a relação das somas dos anticorpos IgG1, IgG2 e IgG3 pelos níveis de IgG4 se correlacionaram negativamente com a parasitemia inicial. No Immunoblotting, foram identificadas frações protéicas que podem estar relacionadas com o reconhecimento imune protetor, por serem reconhecidas pelas subclasses IgG1, IgG2 e IgG3 e não reconhecidas ou reconhecidas tardiamente por IgG4: 125, 96, 86, 75, 55 e 47 kDa. A resposta predominante das subclasses IgG1, IgG2 e IgG3 observada nestes pacientes, todos com malária não complicada, pode indicar que esses anticorpos estão cooperando para o controle de formas graves da doença e refletirem um certo grau de desenvolvimento de imunidade adquirida / The development of naturally acquired immunity to malaria is slow and depends of several factors as number of previous malaria, interval between each malaria attack, exposure to parasite multiple antigen variant and ageassociated maturation of the immune system. Antibody-dependent effector immune mechanisms are believed to be important to the protective immunity. A number of studies have showed that anti-P. falciparum IgG1 and IG3, named cytophilic antibodies, are protective, whereas the noncytophilic, IgG4, that recognize the same epitopes may block the protective mechanisms. Recent data have suggested that in certain situations, IgG2 can also act as cytophilic and to cooperate in protection. In this work, we have studied, for the first time, the evolution of the IgG subclasses against P. falciparum blood stages in uncomplicated falciparum malaria patients taken into hospital upon mefloquine treatment and followed up 42 days. These antibodies were determined by ELISA in 48 patients (7 serum samples from each patient collected in different times: 0h, 48h, 7, 14, 28, 35 and 42 days). The results were expressed in concentration (ug/ml), index of reactivity (IR) or frequency (%) and the avidity were expressed as index of avidity (IA). Serum samples 14 patients (time of collection: 0, 48h, 7, 21 and 42 days) were also evaluated by Immunoblotting as their specificity and avidity against different proteins of the P. falciparum blood stages The subclasses Anti-P. falciparum IgG1, IgG2, IgG3 and IgG4, high avidity predominantly, were present since the beginning of the treatment, respectively, in 100%, 39,5%, 80,6% and 28,4% of the patients with the following concentrations: 20, 2; 3,8; 1,5 and 0,05 ?g/mL. The highest concentrations were reached at day 7, and IgG1 and the highest IgG3 IAs were reached at day 7, and the highest IgG2 IAs at day 14 and the highest IgG4 IAs at day 2. The initial concentration of anti-P. falciparum IgG3 showed a negative correlation with the parasitemia clearance time (PCT) and the ratio between the sum of IgG1, IgG2 and IgG3 levels to IgG4 levels was negatively correlated with the initial parasitemia. Six protein fractions were identified by the Immunoblotting that can be related to protective immune recognition, because they were recognized by IgG1, IgG2 and IgG3 antibodies and not or only later recognized by IgG4 antibodies: 125, 96, 86, 75, 55 and 47 kDa. The predominant IgG1, IgG2 and IgG3 responses observed in these uncomplicated malaria patients may suggest that these antibodies are cooperating to the control of severe disease and reflecting a certain development of protective immunity

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.

Malaria is a devastating disease which affects almost half of the world’s population. Since the description of the malaria genome sequence, various aspects of the parasite have been studied, including drug resistance mechanisms, epidemiology and surveillance systems. Alarmingly, very little is known about the basic biological processes such as the regulation of the expression of parasite genes. The parasite, Plasmodium falciparum, has developed highly specialized methods of regulating the transcription of genes, starting at the regulation of genes controlling basic cellular processes such as protein synthesis and erythrocyte invasion, followed by the transcriptional regulation of more specialized genes, such as those aiding in immune evasion and pathogenesis. The description of the P. falciparum transcriptome by Bozdech et al. in 2003 revealed a complex, just-in-time and tightly regulated transcription profile of P. falciparum genes. This suggests that the most probable Achilles heel for Plasmodium may be its unique mechanisms of regulating gene expression. Various cis- and trans-regulatory sequences have been identified in P. falciparum, along with possible DNA (and RNA) binding proteins. The first part of this research focussed on transcriptional regulatory mechanisms in which an in silico search identified cis-regulatory sequences in the 5’ untranslated region of the antigenically variant var gene family. Electrophoretic mobility shift assays (EMSA) were used to identify protein binding partners of these sequences, which could ultimately act as transcription factors in regulating the expression of this essential gene family. The second part of the research investigated the involvement of post-transcriptional regulatory mechanisms in the polyamine biosynthetic pathway of P. falciparum. Polyamines have been proven to be crucial for the parasite’s development and therefore, an RNA interference knock-down strategy was used to verify the importance of the polyamine biosynthetic enzymes S-Adenosylmethionine decarboxylase (AdoMetDC), Ornithine decarboxylase (ODC) and Spermidine synthase. It is clear that various mechanisms for gene regulation are used by the parasite and that this is critical for the survival of this organism. The results of this study suggest the potential presence of both double-stranded and single-stranded DNA regulatory proteins within P. falciparum nuclear extract. As controversial as RNA interference remains in P. falciparum, this technique was used as a plausible knock-down strategy of parasite specific genes and certain trends, regarding the visible decreases in gene transcript level after double-stranded RNA treatment, were observed. However, final conclusions as to the feasibility of using RNA interference in P. falciparum remain to be elucidated. This study therefore ultimately lends insight into the transcriptional and post-transcriptional levels of P. falciparum gene regulation. / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted

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.

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