Master of Science / Department of Biochemistry and Molecular Biophysics / Michal Zolkiewski / ClpB is a molecular chaperone that is essential for infectivity and pathogen survival in a host. It belongs to the AAA+ protein family, which cooperates with the DnaK chaperone system to reactivate aggregated proteins. In this study, we purified and then studied the biochemical properties of the apicoplast targeted ClpB isoform from the malaria parasite Plasmodium falciparum: PfClpB1. Plasmodium falciparum is the parasite responsible for the most severe form of malaria. In contrast to the parasitophorous vacuole targeted PfClpB2 from Plasmodium falciparum which contains all characteristic AAA+ sequence motifs, PfClpB1 also includes a 52-residue long non-conserved insert in the middle domain. The ATPase activity study shows that PfClpB1 hydrolyzes ATP in presence of Poly-lysine and α-casein. Similar to most AAA+ ATPases, addition of ATP induces hexamer formation in PfClpB1. Lastly, PfClpB1 reactivates aggregated firefly luciferase. However, PfClpB1 is unable to efficiently reactivated luciferase in the presence of the E. coli DnaK chaperone system or human Hsp70 and Hsp40 (Hdj1). This can be explained by the extra middle domain sequence of PfClpB1. Our data may suggest that PfClpB1 activity is essential for Plasmodium falciparum survival by preserving the activity of apicoplast proteins.

The Clp chaperones and proteases play a pivotal role in maintaining cellular homeostasis. They are highly conserved across prokaryotes and can also be found in the mitochondria of eukaryotes and chloroplast of plants. For my thesis, I provide an analysis of the Clp chaperones and protease in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which I term PfClpB1, PfClpB2, PfClpC, and PfClpM. One PfClpP, the proteolytic protomer, and one PfClpR, an inactive isoform, were also identified. All proteins, with the exception of PfClpB2, were found to be localized to the apicoplast, a non-photosynthetic relic plastid in P. falciparum. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by various techniques. Through X-ray crystallography, PfClpP assumed a compacted tetradecamer structure similar to that observed for other ClpPs. My data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

The Clp chaperones and proteases play a pivotal role in maintaining cellular homeostasis. They are highly conserved across prokaryotes and can also be found in the mitochondria of eukaryotes and chloroplast of plants. For my thesis, I provide an analysis of the Clp chaperones and protease in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which I term PfClpB1, PfClpB2, PfClpC, and PfClpM. One PfClpP, the proteolytic protomer, and one PfClpR, an inactive isoform, were also identified. All proteins, with the exception of PfClpB2, were found to be localized to the apicoplast, a non-photosynthetic relic plastid in P. falciparum. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by various techniques. Through X-ray crystallography, PfClpP assumed a compacted tetradecamer structure similar to that observed for other ClpPs. My data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

Os membros da sub-família Toxoplasmatinae conhecidos são Hammondia hammondi, Toxoplasma gondii, Neospora hughesi, Neospora caninum, Hammondia heydorni e Besnoitia spp. Os cães (e provavelmente outras espécies de canídeos) são hospedeiros definitivos de N. caninum e H. heydorni. Os oocistos destas espécies de coccídios são morfologicamente indistinguíveis de forma que o diagnóstico coprológico diferencial entre os dois agentes é virtualmente impossível, se utilizadas metodologias convencionais de diagnóstico. Situação análoga é verificada com os gatos (e outras espécies de felídeos) com relação à infecção por T. gondii e H. hammondi. O objetivo deste trabalho foi propor a reconstrução filogenética de protozoários pertencentes à sub-família Toxoplasmatinae pela análise de seqüências de nucleotídeos de genes mitocondriais e de apicoplasto. Foram empregadas seqüências gênicas de CytB mitocondrial e de dois genes de apicoplasto, o gene codificador da subunidade beta de RNA polimerase DNA dependente (RpoB) e o gene codificador de proteína caseinolitica (ClpC). Pelas análises filogenéticas e de variabilidade nucleotídica e de aminoácidos, verifica-se que a espécie H. heydorni é eqüidistante de todas as outras espécies de toxoplasmatineos. Os posicionamentos relativos dos gêneros Toxoplasma, Neospora e Hammondia nas árvores filogenéticas não foram congruentes em todas as reconstruções, pois dependendo dos táxons que são empregados como grupos externos, as topologias das reconstruções variam e os clados formados são estatisticamente pouco suportados. Assim, a reconstrução de topologias produzindo com ramos curtos que derivam nós de baixo suporte estatístico, somado à eqüidistância evolutiva entre os táxons avaliados (Neospora spp., H. heydorny e T. gondii) permite supor que uma politomia consistente explicaria a evolução para estes organismos, ou seja, a resolução para o posicionamento relativo entre estes táxons poderia ser resultado de evolução radiada. Os genes de organelas mostraram-se mais conservados em relação aos genes nucleares. Embora os genes de apicoplasto possam ser mais conservados que genes nucleares, eles parecem ter relações entre substituições não sinônimas e substituições sinônimas consideravelmente superiores àquelas de genes nucleares e mitocondriais, o que pode indicar que os produtos gênicos estejam sendo submetidos a pressão seletiva positiva. No caso dos genes mitocondriais e nucleares, é possível supor que os mesmos estejam submetidos à pressão seletiva negativa, indicando que as substituições tendem a ser deletérias aos organismos e por isso as mudanças nos produtos gênicos devam ser menos freqüentemente registradas. Ainda, a variabilidade em sítios não sinônimos é consideravelmente superior para seqüências de apicoplasto em relação às demais, particularmente no caso das seqüências RpoB. Também em termos de variabilidade em sítios não sinônimos, percebe-se que as seqüências de genes de apicoplasto de H. heydorni são tão distintas das de T. gondii quanto de N. caninum. Nas análises realizadas com genes de apicoplasto, é marcante a divergência entre as duas linhagens de H. heydorni. Vale ressaltar que as diferenças genotípicas entre as duas linhagens de H. heydorni são maiores que as diferenças entre as duas espécies reconhecidas de Neospora, indicando que as duas linhagens de H. heydorni poderiam ser classificadas como duas espécies distintas, se apenas critérios de evolução molecular fossem considerados. / The known members of the sub-family Toxoplasmatinae are Hammondia hammondi, Toxoplasma gondii, Neospora hughesi, Neospora caninum, Hammondia heydorni and Besnoitia spp. Dogs (and probably other species of dogs) are definitive hosts of N. caninum and H. heydorni. The oocysts of coccidia of these species are morphologically indistinguishable and the coprological differential diagnosis between the two agents is virtually impossible if used conventional methods of diagnosis. Similar situation is observed with the cats (and other species of felids) with respect to T. gondii and H. hammondi. The objective of this study was to propose a phylogenetic reconstruction of protozoa belonging to the subfamily Toxoplasmatinae by analyzing nucleotide sequences of mitochondrial genes and apicoplast. We used gene sequences of cytochrome b and two apicoplast genes, the gene encoding the beta subunit of DNA dependent RNA polymerase (RpoB) and the gene encoding caseinolitic protein (ClpC). From the phylogenetic analysis and the analysis of nucleotide and amino acids variability, was shown that the species H. heydorni is equidistant from all other species of toxoplasmatineos. The relative positions of the genera Toxoplasma, Neospora and Hammondia in the phylogenetic trees were not congruent in all reconstructions, because the topologies of the reconstructions varies according to the taxons that are used as outgroups and clades are poorly supported statistically. Thus, reconstructions of topologies with short branches that derive to poorly statistical supported nodes, coupled with the evolutionary equidistance between taxa the assessed (Neospora spp. H. heydorni. and T. gondii) suggests that a consistent polytomous evolution would explain the evolution within this group of organisms, namely the the relative placement of these taxa could be the result of a radiated evolution. The genes of organelles were more conserved than nuclear genes. Although the apicoplast genes may be more conserved than nuclear genes, they have the ratio between non-synonymous substitutions and synonymous substitutions considerably higher than those of nuclear and mitochondrial genes, which may indicate that the gene products are being subjected to positive selective pressure. In the case of nuclear and mitochondrial genes, it is possible to assume that they are subject to negative selective pressure, indicating that the substitutions are likely to be harmful to organisms and therefore changes in gene products to be less frequently recorded. Still, the variability in non-synonymous sites is considerably higher for sequences of apicoplast in relation to others loci, particularly in the case of RpoB sequences. Also in terms of variability in non-synonymous sites, it is observed that the sequences of apicoplast genes of H. heydorni are as different from those of T. gondii as the N. caninum. The analyzes of apicoplast genes revealed a striking divergence between the two strains of H. heydorni. It is noteworthy that the genotypic differences between the two strains of H. heydorni are greater than the differences between the two species of Neospora, indicating that the two strains of H. heydorni could be classified as two distinct species; if solely criteria of molecular evolution were considered.

Os membros da sub-família Toxoplasmatinae conhecidos são Hammondia hammondi, Toxoplasma gondii, Neospora hughesi, Neospora caninum, Hammondia heydorni e Besnoitia spp. Os cães (e provavelmente outras espécies de canídeos) são hospedeiros definitivos de N. caninum e H. heydorni. Os oocistos destas espécies de coccídios são morfologicamente indistinguíveis de forma que o diagnóstico coprológico diferencial entre os dois agentes é virtualmente impossível, se utilizadas metodologias convencionais de diagnóstico. Situação análoga é verificada com os gatos (e outras espécies de felídeos) com relação à infecção por T. gondii e H. hammondi. O objetivo deste trabalho foi propor a reconstrução filogenética de protozoários pertencentes à sub-família Toxoplasmatinae pela análise de seqüências de nucleotídeos de genes mitocondriais e de apicoplasto. Foram empregadas seqüências gênicas de CytB mitocondrial e de dois genes de apicoplasto, o gene codificador da subunidade beta de RNA polimerase DNA dependente (RpoB) e o gene codificador de proteína caseinolitica (ClpC). Pelas análises filogenéticas e de variabilidade nucleotídica e de aminoácidos, verifica-se que a espécie H. heydorni é eqüidistante de todas as outras espécies de toxoplasmatineos. Os posicionamentos relativos dos gêneros Toxoplasma, Neospora e Hammondia nas árvores filogenéticas não foram congruentes em todas as reconstruções, pois dependendo dos táxons que são empregados como grupos externos, as topologias das reconstruções variam e os clados formados são estatisticamente pouco suportados. Assim, a reconstrução de topologias produzindo com ramos curtos que derivam nós de baixo suporte estatístico, somado à eqüidistância evolutiva entre os táxons avaliados (Neospora spp., H. heydorny e T. gondii) permite supor que uma politomia consistente explicaria a evolução para estes organismos, ou seja, a resolução para o posicionamento relativo entre estes táxons poderia ser resultado de evolução radiada. Os genes de organelas mostraram-se mais conservados em relação aos genes nucleares. Embora os genes de apicoplasto possam ser mais conservados que genes nucleares, eles parecem ter relações entre substituições não sinônimas e substituições sinônimas consideravelmente superiores àquelas de genes nucleares e mitocondriais, o que pode indicar que os produtos gênicos estejam sendo submetidos a pressão seletiva positiva. No caso dos genes mitocondriais e nucleares, é possível supor que os mesmos estejam submetidos à pressão seletiva negativa, indicando que as substituições tendem a ser deletérias aos organismos e por isso as mudanças nos produtos gênicos devam ser menos freqüentemente registradas. Ainda, a variabilidade em sítios não sinônimos é consideravelmente superior para seqüências de apicoplasto em relação às demais, particularmente no caso das seqüências RpoB. Também em termos de variabilidade em sítios não sinônimos, percebe-se que as seqüências de genes de apicoplasto de H. heydorni são tão distintas das de T. gondii quanto de N. caninum. Nas análises realizadas com genes de apicoplasto, é marcante a divergência entre as duas linhagens de H. heydorni. Vale ressaltar que as diferenças genotípicas entre as duas linhagens de H. heydorni são maiores que as diferenças entre as duas espécies reconhecidas de Neospora, indicando que as duas linhagens de H. heydorni poderiam ser classificadas como duas espécies distintas, se apenas critérios de evolução molecular fossem considerados. / The known members of the sub-family Toxoplasmatinae are Hammondia hammondi, Toxoplasma gondii, Neospora hughesi, Neospora caninum, Hammondia heydorni and Besnoitia spp. Dogs (and probably other species of dogs) are definitive hosts of N. caninum and H. heydorni. The oocysts of coccidia of these species are morphologically indistinguishable and the coprological differential diagnosis between the two agents is virtually impossible if used conventional methods of diagnosis. Similar situation is observed with the cats (and other species of felids) with respect to T. gondii and H. hammondi. The objective of this study was to propose a phylogenetic reconstruction of protozoa belonging to the subfamily Toxoplasmatinae by analyzing nucleotide sequences of mitochondrial genes and apicoplast. We used gene sequences of cytochrome b and two apicoplast genes, the gene encoding the beta subunit of DNA dependent RNA polymerase (RpoB) and the gene encoding caseinolitic protein (ClpC). From the phylogenetic analysis and the analysis of nucleotide and amino acids variability, was shown that the species H. heydorni is equidistant from all other species of toxoplasmatineos. The relative positions of the genera Toxoplasma, Neospora and Hammondia in the phylogenetic trees were not congruent in all reconstructions, because the topologies of the reconstructions varies according to the taxons that are used as outgroups and clades are poorly supported statistically. Thus, reconstructions of topologies with short branches that derive to poorly statistical supported nodes, coupled with the evolutionary equidistance between taxa the assessed (Neospora spp. H. heydorni. and T. gondii) suggests that a consistent polytomous evolution would explain the evolution within this group of organisms, namely the the relative placement of these taxa could be the result of a radiated evolution. The genes of organelles were more conserved than nuclear genes. Although the apicoplast genes may be more conserved than nuclear genes, they have the ratio between non-synonymous substitutions and synonymous substitutions considerably higher than those of nuclear and mitochondrial genes, which may indicate that the gene products are being subjected to positive selective pressure. In the case of nuclear and mitochondrial genes, it is possible to assume that they are subject to negative selective pressure, indicating that the substitutions are likely to be harmful to organisms and therefore changes in gene products to be less frequently recorded. Still, the variability in non-synonymous sites is considerably higher for sequences of apicoplast in relation to others loci, particularly in the case of RpoB sequences. Also in terms of variability in non-synonymous sites, it is observed that the sequences of apicoplast genes of H. heydorni are as different from those of T. gondii as the N. caninum. The analyzes of apicoplast genes revealed a striking divergence between the two strains of H. heydorni. It is noteworthy that the genotypic differences between the two strains of H. heydorni are greater than the differences between the two species of Neospora, indicating that the two strains of H. heydorni could be classified as two distinct species; if solely criteria of molecular evolution were considered.

Malaria continues to be a global health burden that affects millions of people worldwide each year. Increasing demand for malaria control and eradication has led research to focus on sexual development of the malaria parasite. Sexual development is initiated when pre-destined intraerythrocytic ring stage parasites leave asexual reproduction and develop into gametocytes. A mosquito vector will ingest mature gametocytes during a blood meal. Sexual reproduction will occur in the midgut, leading to the production of sporozoites that will migrate to the salivary gland. The sporozoites will be injected to another human host during the next blood meal consequently, transmitting malaria. Due to decreased drug susceptibility of mature gametocytes, more investigation of the biology and metabolic requirements of malaria parasites during gametocytogenesis, as well as during the mosquito stages, are urgently needed to reveal novel targets for development of transmission-blocking agents. Furthermore, increasing drug resistance of the parasites to current antimalarials, including slowed clearance rates to artemisinin, requires the discovery of innovative drugs against asexual intraerythrocytic stages with novel mechanisms of action. Here, we have investigated the role of the apicoplast during Plasmodium falciparum gametocytogenesis. In addition, we describe drug-screening studies that have elucidated a novel mode of action of one compound from the Malaria Box, as well as identified new natural product compounds that may be serve as starting molecules for antimalarial development. / Ph. D.

Les Apicomplexa sont des parasites intracellulaires obligatoires. Ils peuvent être responsables d’importantes maladies infectieuses. Toxoplasma gondii par exemple, se développe au sein de la cellule hôte, dans une niche protectrice « la vacuole parasitophore » jusqu’à épuisement des ressources de la cellule hôte ou il provoque sa sortie pour ré envahir à nouveau, c’est la phase aigüe de la toxoplasmose. Afin de répondre à leur besoins nutritifs nécessaires à cette expansion rapide, le parasite combine de manière intéressante et très complexe les voies de synthèse de novo et d’import des nutriments depuis la cellule hôte. Dans le cas des lipides, le parasite en a besoin en une importante quantité pour assurer la ségrégation des organites, la formation des nouvelles membranes filles et l’expansion de la membrane de la vacuole parasitophore pendant la division. La synthèse de novo des lipides a été reportée essentielles pour le parasite tout comme la synthèse de novo des acides gras via la voie procaryote de synthèse des acides gras FASII dans l’apicoplaste.Dans cette étude nous apportant des éléments intéressants qui relient la voie de synthèse FASII et la synthèse des lipides. Nous avons pu démontrer que l’apicoplaste possède une voie de synthèse des précurseurs important voire essentielles à la synthèse de tous les lipides membranaires, qui est principalement le LPA dans le cas de T. gondii. Les enzymes acyltransférase impliquées dans la synthèse de ces précurseurs sont TgATS1 et TgATS2 pour former le LPA et le PA respectivement. Elles sont ortologues aux enzymes précédemment caractérisées chez les bactéries et le chloroplaste des plantes et algues. Les modifications de ces enzymes et les analyses de lipidique et de spectrométrie de masse, ont révélé le rôle l’implication de ces enzymes dans la synthèse des phospholipides membranaires à partir des acides gras néo synthétisés de novo (le C14:0). Cette étude présente aussi des résultats préliminaires sur une voie de synthèse du PA dans le réticulum endoplasmique. La être de TgATS2 n’est pas létale et elle est compensée par augmentation de l’abondance des acides gras C16 :0 et C18 :0 dans la fraction des phospholipides extraits. Ces informations suggèrent une importante collaboration entre l’apicoplaste et le réticulum endoplasmique pour la synthèse des lipides nécessaires pour le développement intracellulaire du parasite. / Apicomplexa phylum includes a large number of obligate intracellular parasites responsible for important human and animal diseases, especially malaria and toxoplasmosis. There is current no efficient vaccine against these parasites. Severe toxoplasmosis caused by Toxoplasma gondii, occurs in immunocompromised individuals and during congenital infection. T. gondii is dependent on large amounts of lipids for its intracellular development within the host cell. These lipids are acquired by a combination of host lipid scavenging and de novo biosynthetic pathways. T. gondii is able to de novo synthesis of fatty acid via a prokaryotic FASII pathway in the apicoplast, a relict non-photosynthetic plastid. Genome mining suggests that the apicoplast can generate phosphatidic acid, central phospholipid precursor. Our recent work confirmed that the apicoplast harbors the first step of PA synthesis via a glycerol-3-phosphate acyltransferase enzyme called ATS1 by homology to chloroplast enzyme, which generates lysophosphatidic acid (LPA). This essential LPA can be exported from the apicoplast for the de novo bulk synthesis of phospholipids sustaining parasite membrane biogenesis (Amiar et al. Plos Path. 2016). T. gondii genome encodes for two other acyltransferases named sn-acylglycerol 3-Phosphate acyltransferases (AGPAT). AGPATs ensure the second step of PA synthesis using LPA. In this work we showed that these enzymes are localized in the Endoplasmic Reticulum and the apicoplast (named AGPAT and ATS2, respectively). A genetic disruption of ATS2 using CRISPR-Cas9 strategy affects parasite growth and normal cytokinesis. Lipidomic analysis using mass spectrometry combined to stable isotope labelling of ATS2-KO reveals an important reduction of lipids containing apicoplast-generated fatty acid C14:0. However, an increase of lipids containing C16 and C18 fatty acids was observed, suggesting a compensation of ATS2 loss by AGPAT activity in ER. These data indicated an important collaboration between apicoplast and ER for lipid synthesis that involves massive lipid trafficking between the two organelles.

L'autophagie est un processus d'auto-dégradation conservé chez la plupart des eucaryotes. Généralement induit par un stress nutritif, il requiert la formation d'un compartiment à double membrane appelé l’autophagosome qui séquestre et transporte des composants intracellulaires dégradés et recyclés dans le lysosome. La protéine ATG8, qui occupe une position centrale dans ce processus, est recrutée aux membranes de l’autophagosome par un système de conjugaison très régulé. Toxoplasma gondii est un protozoaire parasite appartenant au phylum des Apicomplexes, qui contient une machinerie d'autophagie réduite. Suite à un stress nutritif, ce parasite intracellulaire obligatoire est néanmoins capable de générer des autophagosomes décorés par TgATG8. De façon surprenante, en condition normale de croissance intracellulaire, cette protéine se localise principalement à l’apicoplaste, un plaste non photosynthétique acquis par endosymbiose secondaire qui contient des voies métaboliques essentielles à la survie du parasite. Le but de ma thèse a été d’élucider les fonctions canoniques et non canoniques d‘ATG8 chez Toxoplasma. La première partie de cette étude porte sur la caractérisation fonctionnelle et spatio-temporelle de l'association de TgATG8 avec l’apicoplaste. Nous avons montré que TgATG8 est recrutée aux extrémités de l’apicoplaste en élongation, ce qui permet le maintien de l’organelle à travers les générations en le connectant aux centrosomes pour une répartition dans les deux cellules filles. La deuxième partie de ce travail vise à isoler et identifier par spectrométrie de masse des partenaires putatifs de TgATG8 qui seraient impliqués dans l’autophagie ou dans le rôle non-canonique à l’apicoplaste. Nous avons analysé la localisation subcellulaire de neuf candidats et des caractérisations fonctionnelles ont été entreprises pour trois protéines. Bien que nous n’ayons pas pu confirmer leurs interactions avec TgATG8, cela a permis l'identification de nouvelles protéines parasitaires: une phospholipase à l’apicoplaste essentielle à la survie du parasite, un régulateur potentiel du cycle cellulaire et un composant du cytosquelette du parasite. / Autophagy is a self-degradative process evolutionary conserved among eukaryotes. Typically induced by starvation, it involves the formation of a double membrane compartment called the autophagosome to sequester and deliver intracellular components for lysosomal degradation and recycling. The protein ATG8 occupies a central position in this process and is recruited to autophagosomal membranes by a highly regulated conjugation system. Toxoplasma gondii is a parasitic protist belonging to the Apicomplexa phylum, which possesses a reduced autophagy machinery. This obligate intracellular parasite is nevertheless able to generate TgATG8-decorated autophagosomes upon nutrient stress. Surprisingly, during normal intracellular parasite growth, TgATG8 mainly localizes to the apicoplast, a non-photosynthetic plastid acquired by secondary endosymbiosis which hosts essential metabolic pathways. My thesis aimed to elucidate the canonical and non-canonical roles of ATG8 in Toxoplasma. The first part of this study is the functional and spatio-temporal characterization of TgATG8 association with the apicoplast. We showed TgATG8 is recruited to both ends of the elongating plastid during parasite division, and allows the maintenance of the organelle across generations by permitting its centrosome-driven distribution into the two daughter cells. The second part of this work is the isolation and mass spectrometry-based identification of putative TgATG8-interacting proteins that would be involved in autophagy-related or non-canonical functions. We analyzed the subcellular localization of nine candidates and functional studies were conducted for three proteins. Although we were unable to confirm their interactions with TgATG8, this approach allowed the identification of novel and important parasite proteins: an essential apicoplast phospholipase, a potential regulator of the cell cycle, and a component of the parasite cytoskeleton.

No description available.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-05-18T10:58:40Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Tese - Lourival de Almeida Silva - 2015.pdf: 2573495 bytes, checksum: 0eee1e3d91463a3f757ecdc4fe7126ce (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-05-18T11:02:27Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Tese - Lourival de Almeida Silva - 2015.pdf: 2573495 bytes, checksum: 0eee1e3d91463a3f757ecdc4fe7126ce (MD5) / Made available in DSpace on 2015-05-18T11:02:27Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Tese - Lourival de Almeida Silva - 2015.pdf: 2573495 bytes, checksum: 0eee1e3d91463a3f757ecdc4fe7126ce (MD5) Previous issue date: 2015-02-27 / Leishmaniasis is a neglected tropical disease responsible for physical, economic and social damages. Even though malaria is not classified as a neglected tropical disease, is responsible for high morbidity and mortality, especially in African countries. Current treatments for both diseases face several drawbacks, including the evolution of drugresistant parasites, the high cost of major drugs and the high toxicity of others. For these reasons, there is an urgent need to develop new drugs that minimize these downsides and, consequently, help eradicate these diseases. To overcome these difficulties, both academics and pharmaceutical companies are increasingly employing the so-called “drug repositioning strategy”. Drug repositioning aims to find new applications for drugs approved for other indications, and has proven valuable for decreasing research costs as well as to decrease the time required to market the "new" drug. In the present study, we used bioinformatics to identify and analyze molecular targets of the energy metabolism of Leishmania spp and of the P. falciparum apicoplast. The energy metabolism of Leishmania and the apicoplast metabolism have various enzymes that can be targeted by specific drugs, leading to lower toxicity and more promising therapies for humans. Using the TDR Targets database, we were able to identify 94 genes and 93 Leishmania energy metabolism targets. We identified 44 positive targets in these databases, and for 11 of these targets we found drugs already approved for use in humans. We used a similar strategy to identify antimalarial drugs that acted specifically against the apicoplast metabolism. The GeneDB database of the P. falciparum genome was used to compile a list of 600 proteins with apicoplast signal peptides. Each of these proteins was treated as a potential drug target and its predicted sequence was used to interrogate three different open access databases (DB TTD, DrugBank and STITCH ). We identified many drugs with the potential to interact with 47 peptides allegedly involved in apicoplast biology in P. falciparum. Fifteen of these hypothetical targets are predicted to interact with drugs are already approved for clinical use, but were never evaluated against malaria parasites. Our results suggest that the drugs identified here show potential activity against leishmania parasite and malaria, but need experimental validation to confirm their effectiveness. / As leishmanioses são parasitoses negligenciadas responsáveis por prejuízos físicos, econômicos e sociais. A malária, embora não seja classificada como negligenciada, é responsável por altos índices de morbidade e mortalidade, principalmente nos países africanos. Ao longo dos anos, o tratamento dos infectados tem sido a forma mais eficaz de controle dessas endemias. Entretanto, os efeitos tóxicos, o alto custo dos fármacos e a resistência dos parasitos têm sido os maiores desafios enfrentados pela terapêutica das leishmanioses e da malária. Sendo assim, é urgente a necessidade de desenvolver novos fármacos que minimizem esses transtornos e contribuam para erradicação dessas parasitoses. Diante disso, laboratórios acadêmicos, governos e organizações não governamentais têm apoiado projetos voltados para o reposicionamento de fármacos aprovados com vistas a reduzir custos e tempo de produção de novos antiparasitários. No presente trabalho, utilizamos a bioinformática para identificar, buscar e analisar alvos moleculares do metabolismo energético de Leishmania spp e do apicoplasto de P. falciparum, visando o reposicionamento in silico de fármacos. Utilizando a base de dados TDR Targets, identificamos 94 genes e 93 alvos do metabolismo energético de Leishmania. Em seguida, utilizando a sequência peptídica de cada alvo, interrogamos as bases de dados Drug Bank e TTD na busca de fármacos. Nossa busca resultou em 44 alvos positivos, dos quais 11 interagiam com 15 fármacos aprovados para uso em humanos. Utilizamos estratégia semelhante para identificar fármacos antimaláricos que atuassem especificamente contra o metabolismo do apicoplasto. A base de dados GeneDB do genoma de P. falciparum foi usada para compilar uma lista de cerca de 600 proteínas com peptídeos sinais do apicoplasto. Cada uma dessas proteínas foi tratada como potencial alvo de fármaco e sua sequência prevista foi usada para interrogar três diferentes bases de dados de acesso livre (TT DB, DrugBank e STITCH ) Identificamos fármacos com potencial de interagir com 47 peptídeos supostamente envolvidos na biologia do apicoplasto do P. falciparum. Quinze desses alvos hipotéticos são previstos interagir com fármacos já aprovados para uso clínico, mas que nunca foram avaliados contra os parasitos da malária. Os nossos resultados sugerem que os fármacos aqui identificados apresentam potencial para tratamentos das leishmanioses e da malária, mas que necessitam de validação experimental para confirmar sua eficácia.