Scavenger receptors are a large family of structurally diverse molecules that have been implicated in a range of biological functions. In this work, a newly identified multi-domain scavenger receptor-serine protease IrSRP-1 from the tick Ixodes ricinus is characterized. IrSRP-1 is related to the serine protease Sp22D from the mosquito Anopheles gambiae. IrSRP-1 is expressed mainly in the tick gut but also in hemocytes, Malpighian tubules, tracheas and ovaries of fully fed females. This was confirmed with Western blots and immunohistological labeling with antibodies raised against recombinantly expressed IrSRP-1 trypsine-like domain. According to acquired qRT-PCR profiles relative expression of IrSRP-1 is strongly up-regulated during female feeding and remains unchanged in ticks experimentally injected with various microbes. Functional characterization by RNA interference revealed that lowering IrSRP1 expression leads to a higher mortality rate during tick female feeding.

Made available in DSpace on 2014-10-09T12:27:17Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:59:13Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:27:17Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:59:13Z (GMT). No. of bitstreams: 0 / A giroxina é uma enzima serinoprotease do veneno da cascavel sul-americana Crotalus durissus terrificus. É uma toxina apenas parcialmente caracterizada e com múltiplas atividades. Atua na coagulação, na diminuição da pressão arterial e induz um comportamento neurotóxico descrito como rolamento em barril. Os mecanismos envolvidos nestas atividades não são conhecidos. Considerando que a giroxina é uma enzima com alto potencial para ser um novo fármaco com aplicações em clínica médica como a trombina, tripsina, ancrod®, batroxobin® e calicreína, é importante determinar como a giroxina atua. As análises em eletroforese em gel de poliacrilamida e dicroísmo circular confirmaram a pureza e integridade da molécula. A administração intravenosa em camundongos comprovou a neurotoxicidade (rolamento em barril). O estudo in vivo com microscopia intravital comprovou que a giroxina induz vasodilatação com participação dos receptores ativados por proteases (PARs), do óxido nítrico e da Na+K+ATPase. A adesão e rolamento de leucócitos indicaram que não possui atividade pró-inflamatória. A giroxina induziu a agregação plaquetária, que foi bloqueada pelos inibidores dos receptores PAR-1 e PAR-4 (SCH 79797 e tcY-NH2, respectivamente). Finalmente, foi demonstrado que a giroxina alterou temporariamente a permeabilidade da barreira hematoencefálica (BHE). Neste estudo foi comprovado que os receptores ativados por proteases e o óxido nítrico são mediadores envolvidos nas atividades biológicas da giroxina. / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, is one of the most prevalent infectious diseases in our world today. In order to survive within the host the bacteria need to sense and respond to changes in the environment; however, signal transduction in this bacterium is poorly understood. PknB is a serine/threonine kinase essential for the in vitro survival of M. tuberculosis and therefore a potential drug target against the bacteria. It is the goal of the current study to elucidate downstream substrates of PknB. We have found that PknB shares in vitro substrates with another serine/threonine kinase, PknH, implying the potential complexity of the signaling pathways in the bacteria. We have also provided the first description of the coupling between serine/threonine kinases PknB and PknH with a two-component system response regulator DevR, and further proposed Ser/Thr phosphorylation as the negative regulator of DevR transcription activator activity based on LC-MS/MS analysis. Finally, we have identified a previously unknown phosphoprotein glyceraldehyde 3-phosphate dehydrogenase encoded by the ORF Rv1436, which demonstrates autophosphorylation activity and which phosphorylation is independent of PknB. Overall, the current study has contributed to advance our understanding of the signal transduction pathways and phosphoproteome in Mycobacterium tuberculosis. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

No description available.

The amyloid beta peptide (Aβ) plays an important role in Alzheimer’s disease (AD). Increasing evidence suggest that overactivation of extrasynaptic N-methyl-D-aspartate receptors (NMDARs) mediate Aβ-induced excitotoxicity. In serine racemase knockout (SRKO) mice with significantly depleted D-serine levels, Aβ-induced excitotoxicity is attenuated. Using SRKO mice, this thesis attempts to determine the effects of Aβ on synaptic and extrasynaptic NMDAR function, and how D-serine can alter these Aβ- mediated effects. In CA1 pyramidal neurons, Aβ significantly depresses evoked synaptic NMDAR excitatory postsynaptic currents (EPSCs), and this effect is even greater in SRKO mice. The same effect was also observed on isolated evoked extrasynaptic NMDAR currents. During synaptic NMDAR current recordings, Aβ potentiated the holding current in wild type (WT) mice, but not SRKO mice, suggesting an increase in extrasynaptic NMDAR activation in WT, but not in SRKO mice. SRKO mice attenuated Aβ-induced holding current shift and had reduced basal tonic NMDAR activation. These data, along with evidence from previous studies in the literature, suggest that low levels of D-serine can alter NMDAR function in the presence of Aβ. These findings provide insight for future experiments in exploring the importance of D-serine in AD.

The human serine protease inhibitor (serpin) alpha-1-antitrypsin (AAT) was studied for potential interaction with components of the protozoan parasite Cryptosporidium parvum. A homogenate prepared from C. parvum oocysts was incubated with purified human AAT, and complexes formed between the serpin and components of the homogenate were detected using an enzyme-linked immunosorbent assay (ELISA). Serpin:parasite infections were effectively blocked by preincubating AAT with a cognate target enzyme, porcine pancreatic elastase, prior to performing the ELISA on the homogenate. Incubation of a mixture of C. parvum oocysts and sporozoites with AAT demonstrated preferential fluorescence labeling of the sporozoite surface membrane by indirect immunofluorescence assay. Localization of serpin complexes on sporozoites was confirmed by immunogold electron microscopy. AAT was evaluated for in vitro anticryptosporidial activity in a bovine fallopian tube epithelial (BFTE) cell culture system using both oocysts and filter purified sporozoites as inocula. Serial dilutions of AAT were mixed with oocysts (or sporozites) and used to inoculate BFTE cell monolayers. Inoculted cells were maintained at 37ºC/5% CO2 and collected at 24-,48-,72-, and 96-hr post-inoculation intervals. The addition of AAT and other select protease inhibitors (i.e.,antipain, aprotinin, leupeptin, soybean trypsin inhibitor, and phenylmethylsulfonyl floride) significantly inhibited parasite infection (P<0.01) in a concentration- and time-dependent manner when bleach-decontaminated oocysts were used in the inoculum. The anticryptosporidial activity of AAT is postulated to be linked to an antagonistic effect on oocyst excystation and, putatively, the forced expenditure of bioenergetic reserves prior to host cell invasion. This postulate was supported by the observations that serpin activity had no statistically significant effect on reducing established in vitro infections (i.e., 24 hr post-inoculation prior to addition of AAT) and did not inhibit infection of BFTE cells when inoculted with sporozoite preparations. The combined application of AAT and the aminoglycoside paromomycin demonstrated a synergistic anticryptosporidial effect on in vitro infection and suggested the basis for a multi-agent therapeutic protocol in preventing cryptosporidosis. These studies collectively demonstrated an inticryptosporidial potential for serine protease inhibitors, in particular for AAT, and suggest an alternative approach to conventional therapeutic strategies.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23433号 / 農博第2464号 / 新制||農||1086(附属図書館) / 学位論文||R3||N5348(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 谷 史人, 教授 橋本 渉 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20243号 / 医博第4202号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 原田 浩, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.