Thesis (Ph. D.)--Brigham Young University. Dept. of Microbiology and Molecular Biology, 2007. / Includes bibliographical references (p. 75-82).

Universiẗat, Diss., 2006--Würzburg.

Made available in DSpace on 2014-06-11T19:32:13Z (GMT). No. of bitstreams: 0 Previous issue date: 1986Bitstream added on 2014-06-13T20:42:59Z : No. of bitstreams: 1 barraviera_b_dr_botfm.pdf: 3578174 bytes, checksum: 1a21940039bc694d01cadb7e41e07ecf (MD5) / Com o objetivo de contribuir para o esclarecimento de alguns pontos obscuros nas relações existentes entre a atividade da via das pentoses e a redução das metahemoglobina pela cistamina, em indivíduos normais e doentes com malária, causada pelo Plasmodium falciparum, bem como a relação entre a atividade da glutationa redutase e o efeito do tratamento pela riboflavina, foi proposto novo método de pesquisa pouco dispendioso, simples e que permitisse aplicação em estudo de campo. Com o método padronizado, foram estudados indivíduos normais de Botucatu, Amazônia e Anhembi e doentes com malária causada pelo Plasmodium falciparum, atendidos em Humaitá... / Click electronic access below.

Orientador: Paulo Eduardo de Abreu Machado / Resumo: Com o objetivo de contribuir para o esclarecimento de alguns pontos obscuros nas relações existentes entre a atividade da via das pentoses e a redução das metahemoglobina pela cistamina, em indivíduos normais e doentes com malária, causada pelo Plasmodium falciparum, bem como a relação entre a atividade da glutationa redutase e o efeito do tratamento pela riboflavina, foi proposto novo método de pesquisa pouco dispendioso, simples e que permitisse aplicação em estudo de campo. Com o método padronizado, foram estudados indivíduos normais de Botucatu, Amazônia e Anhembi e doentes com malária causada pelo Plasmodium falciparum, atendidos em Humaitá... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Click electronic access below. / Doutor

The Plasmodium falciparum parasite is the causative agent of the most severe form of malaria. The increase in resistance against the majority of antimalarial compounds underpins the need for the development of new antimalarial compounds, targeting novel biological activities of the parasite. As the P. falciparum parasite develops through its life cycle stages, the parasite is exposed to different environments, resulting in both strategy-specific differences between the asexual (proliferation) and gametocyte (differentiation) stages, as well as stage-specific (i.e. ring – schizont stages; stage I - V gametocytes) differences within each strategy. These strategy- and stage-specific differences might be supported by the presence of different membrane transport proteins (MTPs) in the asexual and gametocyte stages. P. falciparum-encoded MTPs (permeome) are promising novel drug targets because they are specific to P. falciparum and essential for the survival of the P. falciparum parasite as these proteins mediate the uptake and removal of metabolites and waste products. However, to propose parasite-encoded MTPs as potential novel drug targets in the asexual and gametocyte stages, the presence of these MTPs in these stages should be investigated. The P. falciparum-encoded permeome is well characterised in the asexual stages. However, limited knowledge is available about the permeome in the gametocyte stages. Therefore, to address this knowledge gap, the strategy- and stage-specific expression of the entire complement of parasite-encoded MTPs were investigated in the asexual and gametocyte stages to infer the presence of MTP transcripts in the absence of biochemical uptake data. The transcript expression of the permeome revealed strategy-specific expression, with the entire permeome expressed during asexual stages, as expected, given the metabolic adaptations that support the high proliferation rate. By contrast, the gametocyte stages that are undergoing sexual differentiation towards transmission, as opposed to active proliferation, less than half of the permeome were expressed, indicating a reduced range of MTPs active in the gametocyte stages. Subsequently, stage-specific expression of the permeome was investigated by correlating stage-specific metabolic processes that occur within the asexual and gametocyte stages, to the expression profiles of MTP genes involved in these processes. Most of the MTPs involved in these processes showed stage-specific expression, with a few MTP genes showing no stage-specific expression within the asexual and gametocyte stages, respectively. When comparing the stage-specific expression between the asexual and gametocyte stages, it was observed that during the gametocyte stages, there was an absence of some MTPs (decreased expression) that were expressed during the asexual stages, suggesting that the gametocyte stages require only certain metabolites to maintain the investigated metabolic processes. In conclusion, these expression profiles of the permeome in the asexual and gametocyte stages suggest the differential expression of the permeome in these stages. The data presented in this study provides the first complete evaluation of expression of the permeome across P. falciparum asexual and gametocyte stages and serves as a blueprint for future biochemical investigations of transport in these stages, thereby providing a foundation for identifying novel MTP drug targets in future drug development programmes. / Dissertation (MSc)--University of Pretoria, 2018. / NRF / Biochemistry / MSc / Unrestricted

A hallmark trait of P. falciparum malaria is sequestration, in which parasite infected erythrocytes (IEs) adhere to the vasculature, causing organ failure and death. Current antimalarials only kill the parasites, necessitating development of anti-adhesion drugs. Using our two-step approach, we can efficiently screen for anti-adhesion small molecules. Screenings of 75libraries using Bio-Plex 200 identified the most active TPI libraries, which were deconvoluted to single compounds. Screenings library TPI 1319 yielded 3 inhibiting non-optimized compounds, each of which inhibits binding between two receptors, CSA and ICAM1, and their binding PfEMP1 domains. Two compounds deconvoluted from TPI 2103 prevent binding between PfEMP1 and ICAM1. Cytoadhesion assays with live IEs support the results seen with Bio-Plex, with best hits showing inhibition below 200 nM. Cytotoxicity testing of active compounds showed minimaltoxicity. Identified hits appear to be amenable to Structure Activity Relationship studies to develop powerful anti-adhesion drugs to treat severe malaria. / Includes bibliography. / Thesis (MS)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

Includes bibliographical references / [No subject] Malaria infection caused by the apicomplexa pathogen Plasmodium falciparum has a high rate of resistance to existing anti-malarial drugs. The World Health Organisation recommended interventions are unlikely to eliminate the growth of resistance and it would therefore be prudent to continue the search for new drug targets for the continued combatting of malaria. Plasmodium falciparum is parasitic on the host for its metabolites and therefore inhibiting the transportation of glutamine from the host, has long been considered a potential strategy for combating the spread of infection. The recently sequenced Plasmodium falciparum genome has however shown that pathways for independent survival are also conserved. Therefore, combating the spread of Plasmodium falciparum in the human host, in addition to inhibiting the transportation of glutamine, will also require the inhibition of the de novo expression of essential amino acids within the Plasmodium falciparum cell. This could be achieved by inhibiting the glutamine synthetase gene, which is an essential step in the tri-carboxylic acid cycle.

Introducción: Malaria representa una emergencia médica debido a la posible complicación y muerte del paciente cuando este no fue tratado apropiadamente. Malaria severa y/o complicada (MSC) es causada casi exclusivamente por Plasmodium falciparum. Uno de los factores de riesgo asociado con MSC es el tratamiento inadecuado de los casos de malaria no complicada (MNC). Objetivos: Se genotipificó a los genes dihidrofolato reductasa (Pfdhfr) y dihidropteroato sintasa (Pfdhps) en muestras de 60 pacientes con MSC. La resistencia al tratamiento combinado sulfadoxina-pirimetamina (SP) es causado principalmente por mutaciones puntuales en esos genes Diseño de estudio: Los pacientes con MSC de este estudio fueron enrolados durante el brote de malaria de 1998, cuando SP era la primera línea de tratamiento. Materiales y métodos: Se usó el método de secuenciamiento de Sanger para la identificación de los polimorfismos en el gen Pfdhfr y los métodos PCR-RFLP y PCR alelo-específico para el gen Pfdhps. Resultados: Se encontró que el 84% de las muestras tenían el genotipo del parásito cuádruple mutante N51I/S108N/I164L/inserción repetición Bolivia, y el 16% restante el genotipo mutante simple S108N. Con respecto al gen Pfdhps, encontramos cuatro genotipos, siendo el triple mutante A437G/K540E/A581G el más frecuente (78%). Conclusiones: Observamos que las mutaciones I164L de Pfdhfr y K540E de Pfdhps en los casos de MSC fueron más del doble de frecuente comparado con los reportes publicados en casos de MNC en la misma área y periodo de estudio. / --- Introduction: Malaria represents a medical emergency because it may rapidly progress to complication and death without prompt and appropriate treatment. Severe and/or complicated malaria (SCM) is almost exclusively caused by Plasmodium falciparum. One of the risk factors associated with SCM is an inappropriate treatment of the noncomplicated malaria (NCM). Objectives: We genotyped the dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes from 60 SCM patients. Resistance to SP in P. falciparum is caused mainly by specific mutations at those genes. Study design: SCM patients of this study were enrolled during the malaria outbreak in 1998, when sulfadoxine/pyrimethamine (SP) was the first line of treatment. Material and methods: We used a Sanger sequencing approach for the identification of polymorphisms at Pfdhfr gene codons, and in the case of Pfdhps gene we used a PCRRFLP and PCR allele-specific methodology. Results: We found that 84% of samples harbored a quadruple mutant genotype N51I/S108N/I164L/insertion Bolivia repeat, and the left 16% of the sample contained an infection with a simple mutant genotype (S108N). Regarding the Pfdhps gene, we found four genotypes, the triple mutant genotype A437G/K540E/A581G was the more frequent (78%). Conclusions: We observed that the mutations I164L and K540E, known as highly predictor to SP resistance, in this group of patients with SCM were twice of frequency of the mutations from patients with NCM from published reports, also in the same area and period of study. / Tesis

Malaria is arguably one of the most overwhelming infectious diseases throughout the world's existence. The most virulent parasite, Plasmodium falciparum, has a redundancy of invasion proteins, allowing it to switch between different receptors on the host red blood cell. These invasion proteins are stored in the apical organelles, the rhoptries and micronemes, but very little is known about how newly synthesized proteins are transported to these organelles. The hypothesis in this study was that a common protein is involved in trafficking invasion proteins from the trans-Golgi network and PfSORTILIN was investigated as a potential escorter protein. The CCys domain of PfMAEBL, a rhoptry protein, and the prodomain of PfAMA-l, a microneme protein, have been implicated in trafficking to the apical organelles. These domains and the VPS 10 domain of PfSORTILIN were cloned into expression vectors encoding a GST- or Histag. Recombinant proteins were expressed in E. coli and purified by affinity chromatography on glutathione- or Ni-particles. In vitro binding assays were performed, which showed that PfSORTILIN VPS 10 bound to PfMAEBL ccys but not to the PfAMA-1 prodomain, suggesting that PfSORTILIN is a rhoptry protein escorter and is not involved in microneme trafficking. To identify novel binding partners of PfSORTILIN VPS 10, the protein was biopanned against a P. falciparum phage display library. No binding partners were identified, most likely because the library is not schizont-stage specific, which is when PfSORTILIN and invasion proteins are predominantly expressed. The results from this study were integrated with other studies and a trafficking model for PfMAEBL was proposed. This study enhances our knowledge of trafficking pathways and suggests that PfSORTILIN may serve as a common rhoptry protein escorter. / MT 2019

Plasmodium falciparum est un parasite appartenant au phylum Apicomplexa et est à l’origine de la forme la plus sévère de la malaria. Dans les zones endémiques d'Afrique subsaharienne, la plupart des victimes sont des enfants de moins de cinq ans. L’entrée de P. falciparum dans sa cellule cible, le globule rouge, repose sur la sécrétion de protéines par des organites spécialisés : les micronèmes, les rhoptries et les granules denses. Les mécanismes de biogenèse de ces organites et la coordination de la libération de leur contenu lors de l'invasion sont cependant pour la plupart inconnus. Il a été toutefois été démontré que les protéines destinées à ces organites apicaux se concentrent dans des microdomaines de l’appareil de Golgi, dont la composition en lipides et en protéines détermine leur destination finale. À ce jour, les mécanismes de sélection et de transport des protéines apicales vers les organites d'invasion ainsi que leurs mécanismes de sécrétion durant l’invasion sont pour la plupart inconnus. Nous avons donc posé l’hypothèse que les phosphoinositides (PI) et leurs protéines effectrices sont impliqués dans ces processus chez P. falciparum. Les PI sont sept lipides phosphorylés retrouvés de façon minoritaire dans les différentes membranes cellulaires. Chaque membrane subcellulaire contient une espèce caractéristique de PI qui peut être reconnue et liée spécifiquement par des protéines effectrices. Une large gamme de processus biologiques sont régulés par les PI, tels le trafic vésiculaire, les canaux ioniques, les pompes d’efflux et les transporteurs, ainsi que certains processus endocytiques et exocytaires. Des études antérieures ont été en mesure de détecter seulement cinq des sept espèces de PI chez P. falciparum. Dans le cadre d’un premier projet, nous avons étudié la distribution de six PI, à savoir PI3P, PI4P, PI5P, PI (4,5)P2, PI(3,4)P2 et PI(3,4,5)P3, chez P. falciparum. Pour ce faire, nous avons exprimé chez le parasite des rapporteurs spécifiques correspondant à des domaines humains de liaison aux PI, fusionnés à une protéine fluorescente. Cette méthode nous a permis de confirmer des rapports antérieurs sur la localisation du PI3P dans la membrane de la vacuole alimentaire, dans de petites vésicules près ou sur la membrane plasmique du parasite ainsi qu’à l’apicoplaste. De plus, nous avons révélé pour la première fois la présence de PI5P chez P. falciparum et montré qu’il se localisait à la membrane plasmique, au noyau et potentiellement dans le réticulum endoplasmique de transition. Nous avons aussi montré que le PI4P est localisé dans la membrane plasmique ainsi que dans l’appareil de Golgi et que le PI(4,5)P2 est présent dans la membrane plasmique tout au long du cycle érythrocytaire. Cette carte de la distribution subcellulaire des PI constitue un excellent outil pour mieux déchiffrer les rôles de ces lipides chez le parasite P. falciparum. Dans le cadre d’un second projet, nous avons caractérisé une protéine possédant un domaine conservé chez les Apicomplexa, le domain d’homologie de la Pleckstrine, la protéine PfPH2. En utilisant la stratégie de Knock-sideways pour inactiver conditionnellement la protéine d’intérêt, nous avons montré que PfPH2 est impliquée dans l’attachement initial du mérozoite à la surface du globule rouge. Cet effet est directement lié à un défaut de sécrétion d'une population spécifique de micronèmes en l’absence de la protéine PfPH2. Enfin, nous avons mis en évidence que le domaine PH de PfPH2, lorsque exprimé sous forme de protéine recombinante, se lie aux PI avec une grande spécificité. Pris ensemble, nos résultats démontrent le rôle essentiel des PI dans le processus d’invasion et proposent un modèle mécanistique pour l'exocytose des micronèmes. / Plasmodium falciparum belongs to the phylum of Apicomplexa and causes the most severe form of malaria. In endemic areas of sub-Saharan Africa, most of the victims are among children under the age of five. P. falciparum relies on proteins released from sophisticated invasion organelles called micronemes, rhoptries and dense granules to enter human erythrocytes. The mechanism of biogenesis of invasion organelles and the coordinated release of their contents during invasion are mostly unknown. It has been shown that proteins targeted to the apical organelles accumulate in microdomains of the Golgi apparatus with specific lipid and protein composition that determine the final destination of their cargo. To date, the mechanisms of transport of the cargo molecules to the invasion organelles and their release mechanism are mostly unknown. We proposed that phosphoinositides (PIPs) and their effector proteins could be involved in these processes in P. falciparum. PIPs are seven minor phosphorylated lipids in cellular membranes. Each subcellular membrane contains a characteristic species of PIPs that are specifically bound by PIPinteracting proteins. A wide range of biological processes regulated by PIPs such as vesicular trafficking, ion channels, pumps, and transporters and control both endocytic and exocytic processes. Based on previous reports five out of seven PIP species have been detected in P. falciparum. In my first project, we have studied the distribution of six PIPs namely PI3P, PI4P, PI5P, PI(4,5)P2, PI(3,4)P2 and PI(3,4,5)P3 using expression of specific reporters made up of human PIP-binding domains fused to a fluorescent protein. Here, we have confirmed previous reports on PI3P localization to the food vacuole membrane, small vesicles close/on the parasite plasma membrane and the apicoplast. Also, we have reported for the first time the presence of PI5P in P. falciparum and showed that it localizes to the PM, nucleus and potentially transitional ER. PI4P shows localization to the PM and Golgi and PI(4,5)P2 localizes to the PM all over the erythrocytic cycle. The resulting map of the subcellular distribution of PIPs will now be a great tool to further decipher the roles of these lipids in P. falciparum, In the second project, we have characterized a Pleckstrin Homology domain-containing protein (PfPH2) conserved in all apicomplexan parasites. Using the knock sideways strategy to conditionally inactivate the protein, we show that PfPH2 is involved in an early step of the invasion process, when the merozoites initially attach to red blood cells. We further demonstrate that this is due to the abrogated secretion of a specific population of micronemes. Finally, we reveal that recombinantly expressed PfPH2 binds PIPs with a broad specificity. Taken together, our results present evidence for the role of PI in invasion and propose a mechanistic model for the exocytosis of micronemes.