Resumo: Estudou-se a ocorrência do protozoário Toxoplasma gondii em 70 amostras de linguiças suínas tipo frescal, comercializadas no município de Botucatu-SP, a fim de avaliar a participação deste alimento na cadeia epidemiológica da toxoplasmose. Para tanto, utilizou-se a técnica de isolamento do agente em camundongos e a amplificação do material genético pela reação em cadeia pela polimerase (PCR). Esta última foi realizada com amostras digeridas e não digeridas pela pepsina. Não se isolou o parasita de nenhuma amostra estudada, entretanto, 33 amostras (47,14%) apresentaram resultado positivo à PCR. Para as amostras submetidas à digestão péptica, 21 (30,0%) mostraram-se positivas e para aquelas que não sofreram este tratamento, 25 (35,71%) reagiram positivamente. Não houve diferença significativa entre os dois tratamentos. Os resultados obtidos indicam que a linguiça suína frescal provavelmente tem pouca importância como fonte de infecção para a toxoplasmose humana, na região estudada. No entanto, o elevado índice de amostras positivas na reação de PCR demonstra que o parasita pode estar presente, porém é inviabilizado pela ação do sal adicionado ao tempero das linguiças. Palavras-chave: Toxoplasma, linguiça, PCR, isolamento. / Abstract: Occurrence of the protozoan Toxoplasma gondii was studied in 70 samples of fresh swine sausages commercialized in the city of Botucatu-SP, to evaluate the importance of this kind of food in toxoplasmosis epidemiology. For that, it was proceeded the agent isolation in mice and the polymerase chain reaction (PCR) to amplify the genetic material. The PCR was made with digested samples and no digested by pepsin. The parasite wasn't isolated from any sample, however, 33 (47.14%) samples were positive to the PCR. From digested samples, 21 (30.00%) were positives and from no digested samples 25 (35.71%) showed positives. There wasn't significant difference between the two treatments. Results indicate that swine sausages probably have a little importance as infection source of human toxoplasmosis in the studied region. Nevertheless, the high amount of PCR positives samples show that the parasite may be present, but it is inactivated by the salt added to sausages seasoning. Key words: Toxoplasma, sausage, PCR, isolation. / Orientador: Paulo Francisco Domingues / Coorientador: Helio Langoni / Mestre

Made available in DSpace on 2014-06-11T19:30:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2003Bitstream added on 2014-06-13T18:59:54Z : No. of bitstreams: 1 mendonca_ao_me_botfmvz.pdf: 849559 bytes, checksum: 50a02f9581b73c1f11c3ddadb582e075 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Estudou-se a ocorrência do protozoário Toxoplasma gondii em 70 amostras de linguiças suínas tipo frescal, comercializadas no município de Botucatu-SP, a fim de avaliar a participação deste alimento na cadeia epidemiológica da toxoplasmose. Para tanto, utilizou-se a técnica de isolamento do agente em camundongos e a amplificação do material genético pela reação em cadeia pela polimerase (PCR). Esta última foi realizada com amostras digeridas e não digeridas pela pepsina. Não se isolou o parasita de nenhuma amostra estudada, entretanto, 33 amostras (47,14%) apresentaram resultado positivo à PCR. Para as amostras submetidas à digestão péptica, 21 (30,0%) mostraram-se positivas e para aquelas que não sofreram este tratamento, 25 (35,71%) reagiram positivamente. Não houve diferença significativa entre os dois tratamentos. Os resultados obtidos indicam que a linguiça suína frescal provavelmente tem pouca importância como fonte de infecção para a toxoplasmose humana, na região estudada. No entanto, o elevado índice de amostras positivas na reação de PCR demonstra que o parasita pode estar presente, porém é inviabilizado pela ação do sal adicionado ao tempero das linguiças. Palavras-chave: Toxoplasma, linguiça, PCR, isolamento. / Occurrence of the protozoan Toxoplasma gondii was studied in 70 samples of fresh swine sausages commercialized in the city of Botucatu-SP, to evaluate the importance of this kind of food in toxoplasmosis epidemiology. For that, it was proceeded the agent isolation in mice and the polymerase chain reaction (PCR) to amplify the genetic material. The PCR was made with digested samples and no digested by pepsin. The parasite wasn't isolated from any sample, however, 33 (47.14%) samples were positive to the PCR. From digested samples, 21 (30.00%) were positives and from no digested samples 25 (35.71%) showed positives. There wasn't significant difference between the two treatments. Results indicate that swine sausages probably have a little importance as infection source of human toxoplasmosis in the studied region. Nevertheless, the high amount of PCR positives samples show that the parasite may be present, but it is inactivated by the salt added to sausages seasoning. Key words: Toxoplasma, sausage, PCR, isolation.

Made available in DSpace on 2014-06-12T15:51:38Z (GMT). No. of bitstreams: 2 arquivo4502_1.pdf: 2446634 bytes, checksum: dcb2ca8b11d13cdeb70f90c25cafa768 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2005 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Toxoplasma gondii é um parasita unicelular responsável pela toxoplasmose, doença de alta gravidade em pacientes imunodeprimidos devido ao seu caráter oportunista. Os fármacos empregados no tratamento levam a severos efeitos adversos. Neste cenário, faz-se urgente à descoberta de novas substâncias com potencial atividade anti-T. gondii. A enzima ribonucleotídeo redutase, vem sendo relacionada como possível alvo quimioterapêutico. As tiossemicarbazonas, por sua vez, apresentam comprovada eficácia na inibição desta enzima. Neste sentido, sintetizamos uma série de tiossemicarbazonas e derivados de 4-tiazolidinonas e avaliamos seu potencial contra T. gondii. As tiossemicarbazonas (3a 3j) foram preparadas a partir de nitro-benzaldeídos e tiossemicarbazidas substituídas, em etanol sob refluxo e purificadas por lavagem com solvente apropriado. Na segunda etapa, as tiossemicarbazonas foram ciclizadas utilizando o anidrido maléico para formar as 4-tiazolidinonas (4a 4j). Todas as estruturas foram confirmadas por RMN 1H, 13C e IV. Quanto à atividade biológica, células Vero foram infectadas com taquizoítas de T. gondii por 24 h, e após esse período, incubadas com as substâncias sintetizadas por mais 24 horas. Todas as substâncias reduziram drasticamente a percentagem de células Vero infectadas após o tempo de incubação, bem como o número de parasitas intracelulares, causando graves alterações morfológicas no parasita intracelular sem afetar a célula hospedeira, conforme observado por microscopia ótica e eletrônica. Dentre estas, as substâncias 3a, 3b, 4h e 4i mostraram os melhores resultados

Thesis advisor: Marc-Jan Gubbels / Toxoplasma gondii is an obligate intracellular protozoan parasite capable of invading virtually any nucleated host cell. It is known clinically as the causative agent of toxoplasmosis, a disease that can have disastrous consequences for patients with an impaired immune system or for vertically infected fetuses. Currently available treatments for toxoplasmosis are lacking in long-term efficacy while none target the dormant stage formed by the parasite during infection. Toxoplasmosis results from unchecked completion of the lytic cycle. This cycle is driven by the calcium-dependent processes of host cell invasion and egress. Following a spike in cytoplasmic calcium, apically localized organelles, the micronemes and rhoptries, will secrete their contents and thereby facilitate these events. While much of the signaling pathway leading to microneme secretion is known, there are question marks surrounding microneme trafficking and membrane fusion. Mammalian Ferlins, C2-domain containing proteins, are well-known for facilitating trafficking and membrane fusion events in a calcium-dependent manner. Of the three Ferlin family proteins encoded by T. gondii, FER1 is studied in this thesis. Conditional FER1 overexpression induced premature egress due to an untriggered burst of microneme secretion. Additionally, live imaging micronemes in FER1 overexpressing parasites suggested an additional role for FER1 in trafficking of microneme organelles. Taken together, these data support a role for FER1 in microneme trafficking and the membrane fusion event driving their exocytosis, which are essential for egress, invasion and the successful completion of the lytic cycle. / Thesis (MS) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

No description available.

Toxoplasma gondii, the etiological agent of toxoplasmosis, was first discovered in Ctenodactylus gondi, an small North African rodent, by Nicolle and Manceaux in 1908. Later the same year Splendore observed the organism in a laboratory rabbit. Human infection was first reported by Janku in 1923. Since its discovery, many investigations have shown the organism to be truly unique in the protozoan world. There appears to be only one species and only one serotype. Serologic and microscopic studies reveal Toxoplasma to have a vast host range. At least one representative of virtually every mammalian species tested harbored the parasite or showed the presence of demonstrable antibody. Studies of toxoplasmal infection among mammalian populations show that the prevalence of infection usually increases: 1 ) in warm, moist climates and 2) with age. Sex, however, appears to have little influence on the prevalence of the parasite. One most unusual feature of the organism is its lack of target cell specificity: Toxoplasma is an obligate, intracellular parasite capable of living in any cell except non-nucleated erythrocytes. Infection by Toxoplasma is most often confirmed serologically. Many procedures have been described; however, the Sabin-Feldman dye test, the indirect hemagglutinstion test, and the indirect fluorescent antibody test appear to be superior methods for antibody detection. The indirect fluorescent antibody test was selected for this study because: 1) it does not require the use of live organisms, 2) it is relatively easy to perform, and 3) it correlates well with the standard Sabin-Feldman dye test. This study was undertaken to determine the role of various factors on toxoplasmal prevalence in the Greater Orlando area. Age, sex, and race were considered for each human sample tested. Age, sex, and relative degree of outdoor exposure were considered in the study of each canine and feline sample.

Orientador: Helio Langoni / Resumo: Com relação ao desenvolvimento do quadro clínico e a transmissão transplacentária, a toxoplasmose em humanos e ratos é similar, e a infecção em ratos pode servir como um modelo para a enfermidade humana. Foram inoculados, pela via oral, sete grupos de ratos com 105 (Grupo 1), 104 (Grupo 2) e 103 (Grupo 3) bradizoítos de Toxoplasma gondii cepa BTU-2 (altamente virulenta para camundongos), 105 (Grupo 4), 104 (Grupo 5) e 103 (Grupo 6) bradizoítos de T.gondii cepa ME-49 (pouco virulenta para camundongos) e solução salina estéril (Grupo 7 - Controle). Os animais foram observados durante 84 dias, com colheita semanal de sangue para obtenção de soro e realização das provas sorológicas pelos métodos de aglutinação direta (MAD) e imunofluorescência indireta (RIFI) para anticorpos das classes IgG e IgM. Ao final do período os animais foram sacrificados e fragmentos de tecidos colhidos para reisolamento do parasita em camundongos e para detecção de DNA do T.gondii pela reação em cadeia pela polimerase (PCR), utilizando-se oligonucleotídeos dirigidos para a detecção dos genes SAG- 1, B1, rDNA e para uma seqüência repetida não codificadora (REP). Foi realizada a tipagem da cepa BTU-2 pela avaliação do polimorfismo do gene SAG-2. Todos os ratos desenvolveram títulos de anticorpos detectáveis pelas diferentes técnicas, havendo, independente da cepa inoculada, diferenças mais frequentes entre os grupos que receberam 105 e 103 bradizoítos. Para IgM a partir do 35o DPI e para a RIFI-IgG e o MAD, a cepa BTU-2 induziu títulos mais elevados que a cepa ME-49. A cepa BTU-2 foi isolada de todas as amostras de cérebro e musculatura estudadas, independente da dose utilizada na infecção dos animais, enquanto que para a cepa ME-49, o parasita foi isolado mais frequentemente do cérebro do que da musculatura, com influência da dose infectante... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Referring to the symptoms and transplacentary transmission, toxoplasmosis in humans and rats is similar and rats can be used as a model for the disease in humans. 7 groups of rats were inoculated, by oral administration, using 105 (Group 1), 104 (Group 2) and 103 (Group 3) Toxoplasma gondii bradyzoits from strain BTU-2, which is highly infective to mice and with 105 (Group 4), 104 (group 5) and 103 (Group 6) T.gondii bradyzoits of strain ME-49, which is less infective to mice and, finally, sterile saline solution (Group 7) as a control one. The animals were observed during 84 days, with weekly blood samples, in order to obtain serum and then submitted to serological tests by modified agglutination test (MAT) and imunofluorecent antibody test (IFAT) to antibodies from subtypes IgG and IgM. At the end of time of observation, the animals were killed and samples of their tissues were obtained in order to reisolate the parasite and to detect the T.gondii DNA by polymerase chain reaction (PCR), using specific oligonucleotids to detect the genes SAG-1, B1, rDNA and to no coding repetitive sequence (REP). The BTU-2 strain was classified by the SAG-2 gene polymorphism. All the rats developed detectable antibodies titers, no matter which strain was used between the techniques. The most frequent differences in the serological results were among the groups that received 105 and those who received 103 bradizoits, no matter the strain used. The BTU-2 strain induced higher titles to IgM since 35 DPI and to IFAT-IgG and MAT than the ME-49 strain. The BTU-2 strain also was isolated from all the brain and muscles samples, no matter the dosage used to infect the animals. When the ME- 49 strain was used, the parasite was isolated more frequently... (Complete abstract, click electronic access below) / Doutor

Made available in DSpace on 2014-06-11T19:31:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2003Bitstream added on 2014-06-13T18:42:11Z : No. of bitstreams: 1 silva_av_dr_botfm_prot.pdf: 636648 bytes, checksum: 53bbd69b49e6f85da36a6c425cb8447c (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Com relação ao desenvolvimento do quadro clínico e a transmissão transplacentária, a toxoplasmose em humanos e ratos é similar, e a infecção em ratos pode servir como um modelo para a enfermidade humana. Foram inoculados, pela via oral, sete grupos de ratos com 105 (Grupo 1), 104 (Grupo 2) e 103 (Grupo 3) bradizoítos de Toxoplasma gondii cepa BTU-2 (altamente virulenta para camundongos), 105 (Grupo 4), 104 (Grupo 5) e 103 (Grupo 6) bradizoítos de T.gondii cepa ME-49 (pouco virulenta para camundongos) e solução salina estéril (Grupo 7 – Controle). Os animais foram observados durante 84 dias, com colheita semanal de sangue para obtenção de soro e realização das provas sorológicas pelos métodos de aglutinação direta (MAD) e imunofluorescência indireta (RIFI) para anticorpos das classes IgG e IgM. Ao final do período os animais foram sacrificados e fragmentos de tecidos colhidos para reisolamento do parasita em camundongos e para detecção de DNA do T.gondii pela reação em cadeia pela polimerase (PCR), utilizando-se oligonucleotídeos dirigidos para a detecção dos genes SAG- 1, B1, rDNA e para uma seqüência repetida não codificadora (REP). Foi realizada a tipagem da cepa BTU-2 pela avaliação do polimorfismo do gene SAG-2. Todos os ratos desenvolveram títulos de anticorpos detectáveis pelas diferentes técnicas, havendo, independente da cepa inoculada, diferenças mais frequentes entre os grupos que receberam 105 e 103 bradizoítos. Para IgM a partir do 35o DPI e para a RIFI-IgG e o MAD, a cepa BTU-2 induziu títulos mais elevados que a cepa ME-49. A cepa BTU-2 foi isolada de todas as amostras de cérebro e musculatura estudadas, independente da dose utilizada na infecção dos animais, enquanto que para a cepa ME-49, o parasita foi isolado mais frequentemente do cérebro do que da musculatura, com influência da dose infectante... / Referring to the symptoms and transplacentary transmission, toxoplasmosis in humans and rats is similar and rats can be used as a model for the disease in humans. 7 groups of rats were inoculated, by oral administration, using 105 (Group 1), 104 (Group 2) and 103 (Group 3) Toxoplasma gondii bradyzoits from strain BTU-2, which is highly infective to mice and with 105 (Group 4), 104 (group 5) and 103 (Group 6) T.gondii bradyzoits of strain ME-49, which is less infective to mice and, finally, sterile saline solution (Group 7) as a control one. The animals were observed during 84 days, with weekly blood samples, in order to obtain serum and then submitted to serological tests by modified agglutination test (MAT) and imunofluorecent antibody test (IFAT) to antibodies from subtypes IgG and IgM. At the end of time of observation, the animals were killed and samples of their tissues were obtained in order to reisolate the parasite and to detect the T.gondii DNA by polymerase chain reaction (PCR), using specific oligonucleotids to detect the genes SAG-1, B1, rDNA and to no coding repetitive sequence (REP). The BTU-2 strain was classified by the SAG-2 gene polymorphism. All the rats developed detectable antibodies titers, no matter which strain was used between the techniques. The most frequent differences in the serological results were among the groups that received 105 and those who received 103 bradizoits, no matter the strain used. The BTU-2 strain induced higher titles to IgM since 35 DPI and to IFAT-IgG and MAT than the ME-49 strain. The BTU-2 strain also was isolated from all the brain and muscles samples, no matter the dosage used to infect the animals. When the ME- 49 strain was used, the parasite was isolated more frequently... (Complete abstract, click electronic access below)

Toxoplasma gondii is an obligate intracellular parasite that can cause lifethreatening disease in immunocompromised individuals. Host cell invasion is therefore central to the pathology of the disease and parasite survival. Unlike many intracellular pathogens, T. gondii does not enter cells by manipulating the host’s phagocytic machinery; instead, the parasite enters the cell by a process of active penetration. Gliding motility and active penetration are driven by a complex of proteins termed the glideosome. The glideosome consists of four major proteins: TgMyoA, an unconventional myosin XIV, myosin light chain (TgMLC1) and glideosome-associated proteins 45 and 50 (TgGAP45, TgGAP50). TgMyoA has been shown to be essential for parasite motility, but the role of TgMLC1 in regulating myosin function remains unknown. Our lab has identified an inhibitor of T. gondii motility and invasion that results in a post-translational modification (PTM) to TgMLC1. Using molecular genetic and mass spectrometry methods we have shown cysteine 53 and cysteine 58 of TgMLC1 are essential for the modification to occur. To determine if the TgMLC1 PTM alters TgMyoA activity, glideosomes were isolated from DMSO- and 115556-treated parasites. Using an in vitro motility assay we have shown that the TgMyoA actin filament displacement velocities are decreased after 115556 treatment. This is the first evidence that TgMLC1 plays a role in regulating TgMyoA activity. The TgMLC1 PTM is responsible, at least in part, for the invasion and motility defects seen in the parasite after compound treatment. During the course of our investigations we have shown that TgMLC1 is dimethylated on lysine 95. This is an unusual modification for cytosolic proteins and has not been previously described for MLCs. Experiments using parasites expressing a non-methylatable form of TgMLC1 (TgMLC1-K95A) show that dimethylation is not necessary for TgMLC1 peripheral localization, TgMLC1 protein-protein interactions and is not required for TgMyoA activity in vitro. However, TgMLC1-K95A does not appear to be phosphoryalted indicating that TgMLC1 dimethylation is necessary for efficient phosphorylation of TgMLC1. These experiments will provide new insight into the ways in which TgMLC1 regulates this unconventional myosin motor complex.

Thèse de doctorat : Sciences du Vivant. Aspects moléculaires et cellulaires de la Biologie : Strasbourg 1 : 2007. / Titre provenant de l'écran-titre. Bibliogr. 22 p.