Neospora caninum é um protozoário Apicomplexa, parasita intracelular obrigatório que possui o cão como hospedeiro definitivo e principalmente os bovinos como hospedeiros intermediários, causando nos primeiros encefalopatia e nos últimos abortos com perda da fertilidade, o que acarreta prejuízos significativos na pecuária mundial. O parasita necessita multiplicar de modo intra-celular, utilizando a descarga de proteínas contidas em organelas filo-específicas, como as micronemas que liberam proteínas relacionadas a adesão e invasão ativa, além das roptrias e os grânulos densos. Este trabalho foi realizado sobre um tipo de proteína micronêmica denominada proteína Anônima Relacionada à Trombospondina (TRAP), relacionada à adesão e ligação com o motor intracelular, responsável pelo processo ativo de invasão. TRAP 1 já havia sido descrita pela literatura e o objetivo deste trabalho foi a caracterização molecular do até então desconhecido homólogo TRAP 2, com expressão de suas formas recombinantes e o uso de seus respectivos anti-soros para ensaios funcionais e localização da proteína nativa. Com base em banco dados de ESTs e genômicos de N. caninum e fazendo uso de RLM-RACE (Reação mediada por RNA ligase- amplificação rápida dos términos de cDNA-RNA,) foi possível a obtenção da sequencia completa do gene NcTrap 2, que possui quatro éxons separados por três íntrons. Sua forma protéica, como seu homológo, é formado por um peptídeo sinal, uma integrina, cinco trombospondinas, uma região transmembrana e a cauda citoplasmática. A integrina e as trombospondinas são motivos relacionados à adesão para início do processo invasivo e foram os alvos deste trabalho. Foram expressos dois recombinantes a partir deste cerne funcional (integrina e trombospondinas), com 52 e 78 kDa, e seus respectivos anti-soros foram obtidos. Estes foram utilizados em western blots 1D e 2D para localização da forma nativa de NcTRAP 2 de aproximadamente 80 kDa no extrato total e de sua forma processada no ESA (extrato secretado, simulando o momento invasivo do taquizoíta) com 70 kDa. O soro contra o recombinante 1 teve a capacidade de inibir o processo invasivo em valores de 53 a 61%, dependendo do método utilizado (contagem manual ou real time PCR). NcTRAP 2 foi encontrada no complexo apical parasitário do taquizoíta por imunofluorêscencia confocal, evidenciando sua provável localização nas micronemas. A nova molécula, NcTRAP 2 possui características que a diferencia de NcTRAP 1 o suficiente para ser considerada homóloga. O soro contra sua forma recombinante detectou as formas nativas integral e secretada e demonstrou que NcTRAP 2 possui capacidade de inibir a invasão dos taquizoítas, o que a torna um interessante alvo vacinal. / Neospora caninum is an Apicomplexan protozoan, obligatory intracellular parasite that has the dog as definitive host and especially cattle as intermediate hosts, causing in the first ones encephalopathy and in the last ones abortion with fertility losses. Such facts lead to significant losses to livestock worldwide. The parasite must invade the cells for its development, using the discharge of proteins contained in phylum-specific organelles, like the micronemes proteins related with adhesion and active invasion, besides the proteins from rhoptries and dense granules. This work was performed on a type of micronemic protein, a thrombospondin-related anonymous protein (TRAP), related with adhesion and connection with the intracellular motor responsible for the active process of invasion. Based on the homologue previously described in the literature, TRAP 1, the aim of this study was the characterization of the undescribed TRAP 2 homologue, the expression of its recombinant forms, the use of its anti-sera for functional assays, and detection of its native form. Based on ESTs and genomic data from N. caninum, and using the RLM-RACE (ligase mediated rapid amplification of cDNA ends) technique, the complete sequence of NcTRAP 2 was obtained, which had four exons separated by three introns. As NcTRAP 1, the protein sequence is composed of a signal peptide, an integrin, five thrombospondin, a transmembrane region and a cytoplasmatic tail. The integrin and thrombospondin motifs are related to adhesion for initiation of the invasive process and were the targets of this work. We expressed two recombinant fragments from the functional core (thrombospondin and integrin), with 52 and 78 kDa, which were utilized for antisera production. The anti-sera localized the native form NcTRAP 2 which had approximately 80 kDa, and its processed form in ESA (Excreted/Secreted Antigen) with 70 kDa, both in western blot 1D and 2D. The serum against recombinant 1 had the ability to inhibit the invasive process from 53 to 61%, depending on the experimental method used (manual counting or real time PCR). NcTRAP 2 was localized at the apical complex of the parasite of the tachyzoites by confocal immunofluorescence, pointing towards its micronemal localization. The new molecule, NcTRAP 2, holds features that differentiates it from NcTRAP 1 in a substantial way to be considered homologue. The serum against the recombinant form detected the native full length and secreted forms of NcTRAP 2 and demonstrated that NcTRAP 2 has the ability to inhibit the invasion of tachyzoites, turning it into an interesting vaccine target to be investigated.

Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2015-07-29T01:17:18Z No. of bitstreams: 1 Disserta??o Final_Sofia Aline Santos.pdf: 1372683 bytes, checksum: 1913347c4bf754b4de49bf2916dbb519 (MD5) / Made available in DSpace on 2015-07-29T01:17:18Z (GMT). No. of bitstreams: 1 Disserta??o Final_Sofia Aline Santos.pdf: 1372683 bytes, checksum: 1913347c4bf754b4de49bf2916dbb519 (MD5) Previous issue date: 2014-03-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The presence of oocysts of pathogenic protozoa in coastal waters resulting from the introduction of humans and animals contaminated feces has been recorded in different regions of the world. Bivalve molluscs such oysters can filter and retain in their tissues encysted protozoa and so act as potential transmitter of parasitic forms capable of causing diseases like toxoplasmosis and cryptosporidiosis. The aim of this study was to detect natural contamination in oysters of the species Crassostrea rhizophorae, grown in areas at the influence of de Todos os Santos Bay, Apicomplexa protozoan (Cryptosporidium and Toxoplasma gondii). A total of 615 oysters were collected from five points in two municipalities in the period from January to April 2013. Gills and digestive glands were dissected and grouped in pools of 3 animals, then resulted in 205 samples of gills and 205 samples of glands. For the molecular detection in tissue samples, DNA was extracted, then a nested-PCR was performed for the detection of genetic material of Cryptosporidium and another for detection of Sarcocystidae in order to amplify fragments of 600 and 290 base pairs, respectively. There was no DNA detection of Cryptosporidium in samples analysed. Genetic material compatible to T. gondii was amplified in 32 (7,8%) oyster tissue samples, and there are significant differences in the presence of positivity at two points of collecting. One of the factors that may be related with this finding is the proximity of these points to a location of soils washing containing feces of animals from farms. With these results we can infer that there is contamination of the aquatic environment and a risk of transmission of oocysts through consumption of oysters produced in these regions, which is an alert to the public health system. / A presen?a de oocistos de protozo?rios patog?nicos nas ?guas costeiras, resultantes da introdu??o de fezes contaminadas de humanos e de animais, tem sido registrada em diferentes regi?es do mundo. Moluscos bivalves como ostras podem filtrar e reter em seus tecidos protozo?rios encistados e assim atuar como potenciais transmissores de formas parasit?rias capazes de provocar doen?as como a criptosporidiose e a toxoplasmose. O objetivo deste trabalho foi detectar contamina??o natural de ostras da esp?cie Crassostrea rhizophorae cultivadas em ?reas de influ?ncia da baia de Todos os Santos por protozo?rios Apicomplexa (Cryptosporidium e Toxoplasma gondii). Um total de 615 ostras foi coletado em cinco pontos de dois munic?pios no per?odo de janeiro a abril de 2013. Br?nquias e gl?ndulas digestivas foram dissecadas e agrupadas em pools de 3 animais, resultando em 205 amostras de br?nquias e 205 amostras de gl?ndulas. Para a detec??o molecular, o DNA das amostras teciduais foi extra?do, em seguida foi realizada uma nested-PCR para a detec??o do material gen?tico do Cryptosporidium e uma outra para detec??o do DNA do Sarcocystidae, afim de amplificar fragmentos de 600 e 290 pares de base, respectivamente. N?o houve detec??o de DNA de Cryptosporidium nas amostras analisadas. Material gen?tico de T. gondii, determinado pela PCR-RFLP, foi amplificado em 32 (7,8%) amostras teciduais de ostras, havendo diferen?a significativa de positividade em dois pontos de coleta. Um dos fatores que pode estar relacionado com esse achado ? a proximidade destes pontos a locais de lichiviamento de solos contendo fezes de animais provenientes de fazendas. Com esses resultados podemos inferir que h? contamina??o do ambiente aqu?tico e um risco de transmiss?o de oocistos atrav?s do consumo das ostras produzidas nestas regi?es, o que ? uma alerta para o sistema de sa?de p?blica.

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.

Neospora caninum é um protozoário Apicomplexa, parasita intracelular obrigatório que possui o cão como hospedeiro definitivo e principalmente os bovinos como hospedeiros intermediários, causando nos primeiros encefalopatia e nos últimos abortos com perda da fertilidade, o que acarreta prejuízos significativos na pecuária mundial. O parasita necessita multiplicar de modo intra-celular, utilizando a descarga de proteínas contidas em organelas filo-específicas, como as micronemas que liberam proteínas relacionadas a adesão e invasão ativa, além das roptrias e os grânulos densos. Este trabalho foi realizado sobre um tipo de proteína micronêmica denominada proteína Anônima Relacionada à Trombospondina (TRAP), relacionada à adesão e ligação com o motor intracelular, responsável pelo processo ativo de invasão. TRAP 1 já havia sido descrita pela literatura e o objetivo deste trabalho foi a caracterização molecular do até então desconhecido homólogo TRAP 2, com expressão de suas formas recombinantes e o uso de seus respectivos anti-soros para ensaios funcionais e localização da proteína nativa. Com base em banco dados de ESTs e genômicos de N. caninum e fazendo uso de RLM-RACE (Reação mediada por RNA ligase- amplificação rápida dos términos de cDNA-RNA,) foi possível a obtenção da sequencia completa do gene NcTrap 2, que possui quatro éxons separados por três íntrons. Sua forma protéica, como seu homológo, é formado por um peptídeo sinal, uma integrina, cinco trombospondinas, uma região transmembrana e a cauda citoplasmática. A integrina e as trombospondinas são motivos relacionados à adesão para início do processo invasivo e foram os alvos deste trabalho. Foram expressos dois recombinantes a partir deste cerne funcional (integrina e trombospondinas), com 52 e 78 kDa, e seus respectivos anti-soros foram obtidos. Estes foram utilizados em western blots 1D e 2D para localização da forma nativa de NcTRAP 2 de aproximadamente 80 kDa no extrato total e de sua forma processada no ESA (extrato secretado, simulando o momento invasivo do taquizoíta) com 70 kDa. O soro contra o recombinante 1 teve a capacidade de inibir o processo invasivo em valores de 53 a 61%, dependendo do método utilizado (contagem manual ou real time PCR). NcTRAP 2 foi encontrada no complexo apical parasitário do taquizoíta por imunofluorêscencia confocal, evidenciando sua provável localização nas micronemas. A nova molécula, NcTRAP 2 possui características que a diferencia de NcTRAP 1 o suficiente para ser considerada homóloga. O soro contra sua forma recombinante detectou as formas nativas integral e secretada e demonstrou que NcTRAP 2 possui capacidade de inibir a invasão dos taquizoítas, o que a torna um interessante alvo vacinal. / Neospora caninum is an Apicomplexan protozoan, obligatory intracellular parasite that has the dog as definitive host and especially cattle as intermediate hosts, causing in the first ones encephalopathy and in the last ones abortion with fertility losses. Such facts lead to significant losses to livestock worldwide. The parasite must invade the cells for its development, using the discharge of proteins contained in phylum-specific organelles, like the micronemes proteins related with adhesion and active invasion, besides the proteins from rhoptries and dense granules. This work was performed on a type of micronemic protein, a thrombospondin-related anonymous protein (TRAP), related with adhesion and connection with the intracellular motor responsible for the active process of invasion. Based on the homologue previously described in the literature, TRAP 1, the aim of this study was the characterization of the undescribed TRAP 2 homologue, the expression of its recombinant forms, the use of its anti-sera for functional assays, and detection of its native form. Based on ESTs and genomic data from N. caninum, and using the RLM-RACE (ligase mediated rapid amplification of cDNA ends) technique, the complete sequence of NcTRAP 2 was obtained, which had four exons separated by three introns. As NcTRAP 1, the protein sequence is composed of a signal peptide, an integrin, five thrombospondin, a transmembrane region and a cytoplasmatic tail. The integrin and thrombospondin motifs are related to adhesion for initiation of the invasive process and were the targets of this work. We expressed two recombinant fragments from the functional core (thrombospondin and integrin), with 52 and 78 kDa, which were utilized for antisera production. The anti-sera localized the native form NcTRAP 2 which had approximately 80 kDa, and its processed form in ESA (Excreted/Secreted Antigen) with 70 kDa, both in western blot 1D and 2D. The serum against recombinant 1 had the ability to inhibit the invasive process from 53 to 61%, depending on the experimental method used (manual counting or real time PCR). NcTRAP 2 was localized at the apical complex of the parasite of the tachyzoites by confocal immunofluorescence, pointing towards its micronemal localization. The new molecule, NcTRAP 2, holds features that differentiates it from NcTRAP 1 in a substantial way to be considered homologue. The serum against the recombinant form detected the native full length and secreted forms of NcTRAP 2 and demonstrated that NcTRAP 2 has the ability to inhibit the invasion of tachyzoites, turning it into an interesting vaccine target to be investigated.

Toxoplasma gondii is an obligate intracellular protozoan parasite of animals and man. The asexual life cycle of Toxoplasma involves three very distinct, but tightly coordinated developmental stages. In nature, the sporozoite (contained within an oocyst) and bradyzoite (contained within a tissue cyst) initiate infection of the intermediate host, followed by rapid differentiation into the actively replicating tachyzoite. When countered by an effective host response, the tachyzoite differentiates back into the latent bradyzoite and this unique ability of Toxoplasma to interconvert between the replicating tachyzoite and the latent bradyzoite within a single host is the cause of life long infection. The transcriptional mechanisms controlling tachyzoite to bradyzoite differentiation and inter-conversion are largely unknown, however, a linkage between the parasite cell cycle and differentiation may underlie these developmental mechanisms. The recent discovery of a family of DNA binding proteins in Apicomplexa (ApiAP2) that are distantly related to the APETALA-2 (AP2) class of plant transcription factors has uncovered an important set of proteins (ApiAP2 factors) that have critical roles in regulating growth and developmental gene expression. Five Toxoplasma ApiAP2s were studied in this thesis project: AP2IX-9, AP2Ib-1, AP2IV-3 (Chapter 2); AP2IV-4 (Chapter 3); and AP2VI-1 (Chapter 4). A major conclusion of this work highlights a novel paradigm in our understanding of the cellular mechanisms regulating stage conversion in Toxoplasma. The study of AP2IX-9 and AP2IV-4 indicate development of the bradyzoite is governed by transcriptional repressors acting at two independent levels, one late in the cell cycle and a second governing the transition from the tachyzoite to the end-stage bradyzoite tissue cyst. The use of repressors to regulate development provides flexibility for the parasite to immediately respond to changing host conditions and modulate the competing needs of expansion and persistence. In addition, the study of AP2VI-1 demonstrates that Toxoplasma employs ApiAP2s that bind chromosome heterochromatin to establish a state of developmental competency AP2IX-9 (Chapter 2) has a unique transient expression profile restricted to early bradyzoite differentiation, and absent form both the tachyzoite and terminal tissue cyst. Disruption of the AP2IX-9 locus resulted in increased tissue cyst formation in vitro while conditional overexpression of AP2IX-9 significantly reduced tissue cyst formation, indicating AP2IX-9 operates as a repressor of bradyzoite development. Consistent with a role as a repressor, AP2IX-9 specifically inhibited the expression of bradyzoite mRNAs including BAG1, B-NTPase and LDH2, common markers for bradyzoite development. Two other ApiAP2s, AP2Ib-1 and AP2IV-3 have similar expression profiles as AP2IX-9 and are candidates for expanding our understanding of this repressor mechanisms regulating development. A number of mRNAs encoding ApiAP2 proteins are dynamically regulated during the tachyzoite cell cycle that also show unique profiles during bradyzoite development. AP2IV-4 (Chapter 3) and AP2VI-1 (Chapter 4) represent two of several cell cycle AP2s whose expression is associated with specific S-phase and mitotic transitions but illustrate divergent roles in regulating growth and development. The expression of AP2IV-4 is exclusive to the tachyzoite stage of development and peak expression coincides with mitosis of the cell cycle. Interestingly, deletion of AP2IV-4 results in the up-regulation of tissue cyst wall and bradyzoite surface antigens in the tachyzoite. The mis-expression of bradyzoite proteins in the tachyzoite indicate AP2IV-4, much like AP2IX-9, is stage specific transcriptional repressor active only late in the tachyzoite cell cycle, likely promoting continued replication of the tachyzoite stage. For AP2VI-1 (Chapter 4), an S phase exclusive factor, we have verified S phase expression using an HA fusion protein at the endogenous locus, determined its DNA binding specificity by EMSA, and developed a genetic model of conditional expression based on the small molecule, Shield-1. Attempts to genetically delete this factor were successful only in laboratory adapted strains of Toxoplasma, indicating AP2VI-1 has an essential function in the bradyzoite developmental pathway. Genome-wide binding (chromatin immunoprecipitation and microarray analysis (ChIP-chip)) regions of AP2VI-1 are indistinguishable from the recently published CenH3 regions (centromere marker) and similarly fall within the H3K9me2 and H3K9me3 methylation patterns (heterochromatin markers) that mark the centromere boundaries. AP2VI-1 was also detected in mature bradyzoites from in vitro or animal tissue cysts. This dual expression profile for AP2VI-1 may suggest this factor has a unique role in chromosome maintenance or stability during developmental transitions.

Apicomplexan parasites are responsible for some of the most devastating human and veterinarian diseases and are parasites of great economic importance. Apicomplexa include Plasmodium, Toxoplasma and Babesia species. The pathogenic mechanisms developed by Apicomplexa parasites, in particular those that reside in a parasitophorous vacuole, involve considerable changes to the host cell, including the expression of variable surface proteins required for immune evasion. In Plasmodium falciparum infections, host cell remodeling is responsible for disease symptomology and severity in the human host. This work represents a multi-faceted study of antigenic secretion and the role of secreted antigens in pathogenesis. We study in detail the mechanisms of antigen secretion in Apicomplexa parasites. By use of comparative genomics, we find Plasmodium Export Element (PEXEL)-like motifs in a subset of Cryptosporidium and Babesia secreted proteins. However, in Babesia the motif functions as a spherical body targeting sequence, suggesting that secretory mechanisms in Apicomplexa are adapted to the parasite's intracellular lifestyle. To elucidate the relationship and function of exported antigens, we first focused on P. falciparum to determine gene co-expression modules. We found that in vivo, export modules are composed of constitutively or variably expressed genes, the latter group associated with patient clinical phenotypes. We then focused on a novel gene family called "phist" and show, using transcriptional expression profiling, its role in P. falciparum cytoadherence. In total, we demonstrate that antigen secretion is an evolutionary mechanism in Apicomplexa parasites and that variant expression of the genes encoding these antigens may allow parasites to adapt to environmental stresses.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 3 uppsatser.

Submitted by Emanoel Martins Filho (emanoelfilho@ufba.br) on 2016-09-13T00:53:42Z No. of bitstreams: 1 Rogerio_Fernando.pdf: 1011706 bytes, checksum: ec29e66873bd428e74520f5f059408bb (MD5) / Approved for entry into archive by Patricia Barroso (pbarroso@ufba.br) on 2016-09-13T20:36:53Z (GMT) No. of bitstreams: 1 Rogerio_Fernando.pdf: 1011706 bytes, checksum: ec29e66873bd428e74520f5f059408bb (MD5) / Made available in DSpace on 2016-09-13T20:36:53Z (GMT). No. of bitstreams: 1 Rogerio_Fernando.pdf: 1011706 bytes, checksum: ec29e66873bd428e74520f5f059408bb (MD5) / CONS NAC DE DESENVOLVIMENTO CIENTIFICO E TECNOLOGICO - CAPES / Toxoplasma gondii é um protozoário coccídeo formador de cistos teciduais, que tem como hospedeiros definitivos os felídeos, e como hospedeiros intermediários mamíferos e aves. É um parasito disseminado em todos os continentes, que infecta aproximadamente um terço da população humana e pode causar encefalite fatal em pacientes imunodeficientes. Nos animais, tem relevância principalmente em pequenos ruminantes, por causar abortos e outras alterações reprodutivas. Quirópteros podem se infectar com T. gondii e atuarem como fonte de infecção para animais silvestres, domésticos e o homem. No Brasil, ocorre uma alta variabilidade genética do parasito, que pode ser explicada pela grande variedade de hospedeiros no ambiente silvestre. Objetivou-se com este estudo determinar a frequência de infecção em quirópteros de vida livre no estado da Bahia por T. gondii e realizar o isolamento in vivo do protozoário a partir desses animais. Foram utilizadas 124 amostras, provenientes de 97 indivíduos de sete espécies de morcegos, capturados entre os anos de 2008 e 2015, sendo encontrados dois indivíduos positivos por meio da PCR de tecidos, correspondendo a 2,06% de positividade. Nenhum isolamento foi realizado uma vez que os tecidos disponíveis para bioensaio apresentaram-se negativos na PCR / Toxoplasma gondii is a cyst-forming protozoan coccidia, which has felids as definitive hosts, and mammals and birds as intermediate hosts. It’s distributed in all continents and infects about a third of the human population. T. gondii can cause fatal encephalitis in immunodeficient patients. In animals, it’s relevant mainly in small ruminants, for causing abortions and other reproductive abnormalities. Bats can become infected with T. gondii and act as a source of infection for wild and domestic animals and man. In Brazil, there is a high genetic variability of the parasite, which can be explained by the great variety of hosts in the wild environment. The objective of this study was to determine the frequency of free-living bats infection in Bahia by T. gondii and perform in vivo isolation of the parasite from these animals. A total of 124 samples were used from 97 individuals of seven species of bats, caught between the years 2008 and 2015. Two animals were positive by tissue PCR, corresponding to 2.06% of positivity. No isolation was achieved once the tissue available for bioassay showed to be negative by PCR.

Five target genes were selected in Toxoplasma gondii tachyzoites for localization studies. These five genes, detected through proteomics studies, included TgME49_227450, TgME49_223080, TgME49_262390, TgME49_230940, and TgME49_269620. Localization of these five target proteins is a first step to confirm their interaction with TgCrk2 and understand their function and role in TgCrk2 regulation of the tachyzoite cell cycle. Gene models for the targets were analyzed using ToxoDB and ApE analysis tools. Endogenous tagging constructs were created for each target. Transgenic parasites were created. Finally, localization analysis of the target proteins in tachyzoites was completed using immunofluorescent microscopy following. One protein was found to be nuclear, two were located in the cytoplasm, and two were unable to be analyzed. Future research should be completed in order to prove these putative interactors are really correlated with TgCrk2 through Co-immunoprecipitation.

Since its description in 2008, Chromera velia has drawn profound interest as the closest free-­-living photosynthetic relative of apicomplexan parasites that are significant pathogens, causing enormous health and economic problems. There-­- fore, this newly described species holds a great potential to understand evolu-­- tionary basis of how photosynthetic algae evolved into the fully pathogenic Apicomplexa and how their common ancestors may have lived before they evolved into obligate parasites. Hence, the aim of this work is to understand how C. velia function and respond to different environmental conditions. This study aims to reveal how C. velia is able to respond to environmental perturbations that are applied individually and simultaneously since, studying stress factors in separation fails to elucidate complex responses to multi stress factors and un-­- derstanding the systemic regulation of involved genes. To extract biologically significant information and to identify genes involved in various physiological processes under variety of environmental conditions (i.e. a combination of vary-­- ing temperatures, iron availability, and salinity in the growth medium) we pre-­- pared strand specific RNA-­-seq libraries for 83 samples in diverse environmental conditions. Here, we report the set of significantly differentially expressed genes as a re-­- sponse to the each condition and their combinations. Several interesting up-­- regulated and down-­-regulated genes were found and their functions and in-­- volved pathways were studied. We showed that the profound regulation of HSP20 proteins is significant under stress conditions and hypothesized that the-­- se proteins might be involved in their movements.