Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-11-07T10:55:53Z No. of bitstreams: 2 Dissertação - Mirian de Sylvio - 2009.pdf: 865608 bytes, checksum: bba24a89ef1938c31376520a3eff1e60 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-11-07T11:29:38Z (GMT) No. of bitstreams: 2 Dissertação - Mirian de Sylvio - 2009.pdf: 865608 bytes, checksum: bba24a89ef1938c31376520a3eff1e60 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-11-07T11:29:38Z (GMT). No. of bitstreams: 2 Dissertação - Mirian de Sylvio - 2009.pdf: 865608 bytes, checksum: bba24a89ef1938c31376520a3eff1e60 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2009-12-15 / Infection with Toxoplasma gondii occurs throughout the globe, with a prevalence of up to 90% in the population. The physiological changes caused by this parasite are well studied in immunocompromised individuals and in cases of congenital transmission. In immunocompetent individuals the infection is usually asymptomatic and little explored by researchers. Experimental studies follow the pattern of human studies, and there fow mention about the biochemical changes (liver and kidney metabolisms) in the host infected by T. gondii. This study aimed the quantification of hepatic and kidney alterations caused by acute infections by T. gondii (non cystogenic strain – RH) and by chronic infections (cystogenic strain – ME-49). The control group was formed by mice without infection, only submitted to saline stress. Several enzymes were measured in serum and peritoneal exudate of mice infected and control such as: aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamyltransferase (GGT), alkaline phosphatase (ALP), urea, creatinine and lactate dehydrogenase, using an automated methodology. AST and ALT presented a significative difference in the serum of mice infected with RH strain when compared to controls indicating a destruction of liver cells. The peritoneal exudates did not present significative changes in relation to controls nor did the urea and creatinine levels. The séric lactate dehydrogenase showed gradual changes in all days of the infection in mice peritoneal exudates as early as this change was evident only in the fifth day of infection. All samples of the group infected with ME-49 strain showed changes in serum and peritoneal exudate during all days of analysis. Only ALT peritoneal exudates showed no change during all days of analysis. An increase in urea at all doses was observed, however, creatinine showed a change only within 120 days of infection. The LDH was altered in the serum in all days of analysis. In conclusion, the T. gondii infection may cause hepatic and kidney injuries either when caused by non-cystogenic as by cystogenic strains of the parasite. / A infecção pelo Toxoplasma gondii ocorre em todo o mundo, com prevalência de até 90% na população conforme seus hábitos culturais e condições socioeconômicas. As alterações fisiopatológicas provocadas por este parasito são muito estudadas nos indivíduos imunocomprometidos, nos casos de transmissão congênita, e nos indivíduos imunocompetentes a infecção é, geralmente, assintomática e pouco explorada pelos pesquisadores. Experimentalmente, os estudos seguem o padrão dos estudos humanos, e há pouca referência sobre as alterações bioquímicas (hepáticas e renais) no hospedeiro infectado pelo T. gondii. Este trabalho objetivou avaliar as alterações hepáticas e renais causadas por esse parasito em camundongos na fase aguda, usando a cepa não cistogênica (RH), e na fase crônica, com a cepa cistogênica (ME-49), tendo como controles camundongos sem infecção, somente submetidos ao estresse de inoculação com salina. Foram dosadas no soro e no exsudato peritoneal dos camundongos infectados e controles os níveis das enzimas Aspartato aminotransferase (AST), Alanina aminotransferase (ALT), Gamaglutamiltransferase (GGT), Fosfatase alcalina (FAL), desidrogenase lática (DHL) e dos seguintes compostos: uréia e creatinina, por metodologia automatizada. As enzimas AST e ALT apresentaram diferença significativa no soro de camundongos infectados com cepa RH, demonstraram alterações em relação aos controles indicando uma destruição das células hepáticas. No exsudato peritoneal não foram demonstradas alterações em relação aos controles. A uréia e creatinina dosadas não demonstraram alteração significativa. A enzima lactato desidrogenase sérica apresentou alterações gradativas em todos os dias de infecção do camundongo no soro, já no exsudato peritoneal essa alteração foi evidenciada somente no quinto dia da infecção, mostrando que com o aumento de parasitos e a destruição celular causada por esse, essa enzima presente em várias células é responsável por demonstrar aumentos consideráveis. Todas as amostras de soro analisadas do grupo infectado com a cepa ME-49 demonstraram alterações durante todo período de acompanhamento. Enquanto que no exsudato peritoneal não mostrou nenhuma alteração durante todo período analisado. Houve aumento crescente na uréia em todos os dias de analises, porém, a creatinina não apresentou nenhuma alteração. A LDH mostrou-se alterada no soro em todos os dias de analisado. Conclui-se que a infecção pelo T. gondii pode provocar alterações hepáticas e renais ao longo do curso de infecção, tanto em infecções com cepa cistogênica quanto com cepa não cistogênica.

No / The surface expression and regulated endocytosis of kainate (KA) receptors (KARs) plays a critical role in neuronal function. PKC can modulate KAR trafficking, but the sites of action and molecular consequences have not been fully characterized. Small ubiquitin-like modifier (SUMO) modification of the KAR subunit GluK2 mediates agonist-evoked internalization, but how KAR activation leads to GluK2 SUMOylation is unclear. Here we show that KA stimulation causes rapid phosphorylation of GluK2 by PKC, and that PKC activation increases GluK2 SUMOylation both in vitro and in neurons. The intracellular C-terminal domain of GluK2 contains two predicted PKC phosphorylation sites, S846 and S868, both of which are phosphorylated in response to KA. Phosphomimetic mutagenesis of S868 increased GluK2 SUMOylation, and mutation of S868 to a nonphosphorylatable alanine prevented KA-induced SUMOylation and endocytosis in neurons. Infusion of SUMO-1 dramatically reduced KAR-mediated currents in HEK293 cells expressing WT GluK2 or nonphosphorylatable S846A mutant, but had no effect on currents mediated by the S868A mutant. These data demonstrate that agonist activation of GluK2 promotes PKC-dependent phosphorylation of S846 and S868, but that only S868 phosphorylation is required to enhance GluK2 SUMOylation and promote endocytosis. Thus, direct phosphorylation by PKC and GluK2 SUMOylation are intimately linked in regulating the surface expression and function of GluK2-containing KARs.

This master thesis deals with the development, optimization and validation of an analytical method for determination of neurotoxin -N-methylamino-L-alanine in pond water and cyanobacteria using LC/MS. Firstly, basic parameters of the analytical method developed within authors´s bachelor thesis were verified. Following parameters were selected for verification: selection of suitable MRM transitions, voltage applied to S-lens and F-lens and standardized collision energy. Secondly, the system suitability testing was performed. Thirdly, the analytical method was successfully validated. Then, the testing and optimization of solid phase extraction for analysis of water samples were carried out. The pH of sample solution and composition of elution solution were chosen for the optimization. In addition, the trichloroacetic acid extraction with acid hydrolysis for cyanobacterial samples was carried out too. Finally, both solid phase extraction and trichloroacetic acid extraction were evaluated and applied to the analysis of real samples.

No description available.

Aus den frühen Bindungsstudien des globalen Regulators AbrB mit der ausgedehnten phyC-Promotorregion von Bacillus amyloliquefaciens FZB45 konnte ein mehrstufiger kooperativer Bindungsprozess abgeleitet werden. Dabei verlangt die AbrB-vermittelte Repression von phyC nach Integrität zweier großer Bindungsstellen, ABS1 und ABS2, die 162 bp voneinander entfernt liegen. In der vorliegenden Arbeit wurden die ersten Echtzeitkinetiken zur DNA-AbrB-Interaktion mittels der Oberflächenplasmonresonanz (SPR) gemessen und analysiert. AbrB zeigte hohe Affinitäten zu den 40 bp langen Oligonukleotiden, die den beiden Bindungsstellen entstammen. Dabei verursachten alle Oligonukleotide der ABS2 und nur eine kurze Region innerhalb der ABS1 bei der Bindung von AbrB Konformationsänderungen im Protein und in der DNA (CD - Zirkulardichroismusspektroskopie) und wiesen eine Kooperativität von 2 / In previous binding studies it could be demonstrated that a global regulator AbrB and the extensive phyC promoter region of Bacillus amyloliquefaciens FZB45 interact in a complex manner. AbrB binding is a multistep cooperative process. The integrity of both binding sites, ABS1 and ABS2, which are separated by 162 bp, is crucial for the AbrB-mediated repression of phyC. This work presents the first real-time binding kinetics of the AbrB-DNA interaction using surface plasmon resonance (SPR). AbrB exhibited high affinities to all analyzed 40-bp oligonucleotides that were derived from the ABSs of phyC. All parts of the ABS2, but only a small region within ABS1, were bound cooperatively to AbrB with a stoichiometry of 2 DNA to 1 AbrB tetramer and with 2