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The characterization of TLR5 /Hayashi, Fumitaka, January 2001 (has links)
Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 108-122).
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Quorum sensing in Yersinia pseudotuberculosisAtkinson, Steven January 1999 (has links)
No description available.
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Rôle des récepteurs Toll-like et de CD14 dans la réponse à Listeria monocytogenes et à la flagelline extraite de Salmonella typhimurium / Implication of the Toll-Like Receptor and CD14 in response to Listeria monocytogenes and flagellin from Salmonella typhimuriumJanot, Laure 18 February 2009 (has links)
L’organisme est exposé à divers agents infectieux et doit mettre en place une réponse immunitaire adéquate pour se protéger. Mes travaux de thèse m’ont permis d’étudier la réponse innée à l’infection par Listeria monocytogenes (L.m) et l’inflammation pulmonaire induite par la flagelline extraite de Salmonella typhimurium. Mes résultats ont mis en évidence l’association du co-récepteur CD14 avec TLR2 (Toll-like Receptor 2) dans la détection de L.m injectée par voie veineuse. En revanche, CD14 ne semble pas être associé au TLR5 dans la reconnaissance de la flagelline. Par ailleurs, l’activation des TLR par leurs ligands permet la synthèse de cytokines intervenant dans l’inflammation. J’ai ainsi pu étudier plus précisément le TNF (Tumor Necrosis Factor). Cette protéine pro-inflammatoire est un des médiateurs principaux de l’immunité et existe sous une forme membranaire qui a été peu étudiée (Mem-TNF) et sous une forme soluble bien connue (sTNF). Mes études ont montré que ce Mem-TNF active la production de cytokines et de médiateurs chimiques de l’inflammation conférant une protection partielle contre Listéria. L’étude de cette cytokine membranaire nous a permis de tester une nouvelle génération de traitements moins agressifs que les anti-TNF contre l’arthrite rhumatoïde ou la maladie de Crohn. / Toll-like receptors (TLRs) recognize a wide range of microbial pathogens and their products modulate the innate immune response that may lead to inflammation. In order to better understand the host-pathogen relationship, we have studied the implication of the co-receptor CD14 in the innate immune response to Listeria monocytogenes and to the bacterial flagellin from Salmonella typhimurium. Our results clearly show that TLR2 requires CD14 to control Listeria infection whereas TLR5 does not. Moreover, TLR activation leads to pro-inflammatory cytokines production such as Tumor Necrosis Factor (TNF). This pleiotropic protein is required for normal development and function of the immune system. TNF can be secreted (sTNF) or associated to the membrane (Mem-TNF). Our results suggest that Mem-TNF can activate the synthesis of cytokines and chemicals mediators of inflammation and partially protect mice from a moderate infection. These experiments open new avenues for the treatment of inflammatory disease like rheumatoid arthritis or Crohn disease.
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Identification and analysis of the flagellin gene and protein from the genus pectinatusChaban, Bonnie 11 December 2003
The use of reduced oxygen-packaging techniques has resulted in anaerobic bacteria emerging as a problem for the brewing industry over the last twenty-five years. The genus Pectinatus, consisting of the species<i>P. cerevisiiphilus</i> and<i> P. frisingensis</i>, is a concern for producers of unpasteurized beer. As a result, there is an ongoing need to both understand this genus and develop rapid detection methodologies to combat its presence in the brewery. The objectives of this study were to sequence and characterize the flagellin genes from both Pectinatus species and evaluate the genes and proteins from a taxonomic and detection-suitability standpoint. <p>A combination of micro-protein sequencing, polymerase chain reaction (PCR) and Bubble-PCR was used to completely sequence one flagellin gene from each Pectinatus species. This knowledge was then utilized to sequence the flagellin gene from four additional Pectinatus isolates, two from each species. To confirm the identity of the flagellin genes, one flagellin gene from each species was cloned, expressed and detected with Pectinatus-specific antibodies. A discrepancy between of the predicted protein size and the actual protein size led to tests for glycosylation, a post-translational modification. Taxonomic analyses, based on the flagellin genes, were conducted at both the superkingdom and genus levels. Finally, genus- and species-specific PCR primer sets were designed and tested for the specific detection of Pectinatus in the brewery. <p>Cloning and expression data confirmed the identity of the sequenced genes as Pectinatus flagellin genes. Glycosylation was positively confirmed to be a post-translational modification for five of the six strains tested. Phylogenetic analysis revealed that both of the Pectinatus species grouped with the phylum Firmicutes (low G+C, Gram-positive bacteria) and that there was more diversity at the species level within the <i>P. frisingensis</i> flagellin gene than the <i>P. cerevisiiphilus</i> flagellin gene. As a final point, the detection of most Pectinatus isolates was achieved with the preliminary PCR primer sets designed, however, some non-Pectinatus beer spoilage organisms, primarily wort spoilage organisms, were also detected. Both the basic science and the applied results generated from this study will aid the brewing industry in its ongoing battle to control Pectinatus contamination.
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Identification and analysis of the flagellin gene and protein from the genus pectinatusChaban, Bonnie 11 December 2003 (has links)
The use of reduced oxygen-packaging techniques has resulted in anaerobic bacteria emerging as a problem for the brewing industry over the last twenty-five years. The genus Pectinatus, consisting of the species<i>P. cerevisiiphilus</i> and<i> P. frisingensis</i>, is a concern for producers of unpasteurized beer. As a result, there is an ongoing need to both understand this genus and develop rapid detection methodologies to combat its presence in the brewery. The objectives of this study were to sequence and characterize the flagellin genes from both Pectinatus species and evaluate the genes and proteins from a taxonomic and detection-suitability standpoint. <p>A combination of micro-protein sequencing, polymerase chain reaction (PCR) and Bubble-PCR was used to completely sequence one flagellin gene from each Pectinatus species. This knowledge was then utilized to sequence the flagellin gene from four additional Pectinatus isolates, two from each species. To confirm the identity of the flagellin genes, one flagellin gene from each species was cloned, expressed and detected with Pectinatus-specific antibodies. A discrepancy between of the predicted protein size and the actual protein size led to tests for glycosylation, a post-translational modification. Taxonomic analyses, based on the flagellin genes, were conducted at both the superkingdom and genus levels. Finally, genus- and species-specific PCR primer sets were designed and tested for the specific detection of Pectinatus in the brewery. <p>Cloning and expression data confirmed the identity of the sequenced genes as Pectinatus flagellin genes. Glycosylation was positively confirmed to be a post-translational modification for five of the six strains tested. Phylogenetic analysis revealed that both of the Pectinatus species grouped with the phylum Firmicutes (low G+C, Gram-positive bacteria) and that there was more diversity at the species level within the <i>P. frisingensis</i> flagellin gene than the <i>P. cerevisiiphilus</i> flagellin gene. As a final point, the detection of most Pectinatus isolates was achieved with the preliminary PCR primer sets designed, however, some non-Pectinatus beer spoilage organisms, primarily wort spoilage organisms, were also detected. Both the basic science and the applied results generated from this study will aid the brewing industry in its ongoing battle to control Pectinatus contamination.
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Molecular basis of NAIP/NLRC4 inflammasome activation by flagellinBittante, Alessandra January 2018 (has links)
The overall aim of this project was to determine the molecular mechanisms by which the flagellin gene from Salmonella enterica serovar Typhimurium (S. Typhimurium) activates the NAIP/NLRC4 inflammasome and its contribution to the host protective immune response against salmonellosis. Inflammasomes are multi-protein complexes formed in response to the activation of pattern recognition receptors (PRRs). The NOD-like receptor (NLR)-family of inflammasome complexes are formed from the cytosolic NLR receptors, ASC adaptor and caspase-1 in response to pathogen- associated molecules or danger-associated signals. The NAIP/NLRC4 inflammasome is activated by the S. enterica flagellar filament protein (FliC), the SPI-1 type III secretion system needle (PrgI) and inner rod proteins (PrgJ). Recognition of these bacterial ligands by the NAIP receptors allows oligomerisation with NLRC4 and subsequent recruitment of caspase-1. Caspase-1 mediates pyroptosis, while recruitment of ASC is also required for cleavage of pro-IL-1β and pro-IL-18 to their active forms by caspase-1. Differential recognition of the flagellar filament proteins (flagellin) by the NAIP/NLRC4 inflammasome forms an important part of my thesis. Here, I have looked at the molecular mechanisms and immunological consequences of the differential recognition of flagellin by the NAIP/NLRC4 inflammasome using S. Typhimurium SL1344 and the non-pathogenic E. coli strain K12-MG1655. An important part of my work was to try and determine which regions of fliC are required for NAIP/NLRC4 inflammasome activation and whether they can be mutated while preserving motility. To do this a panel of ten strains expressing chimeric fliC genes were created and characterised in macrophage infection experiments and bacterial motility assays. My results confirm the C-terminus of FliC is critical for both inflammasome activation and motility in agreement with published reports. To further investigate this differential recognition by the NAIP/NLRC4 inflammasome I modified S. Typhimurium strain of moderate virulence (M525P) to express flagellin from E. coli K12-MG1655. This strain (M525PΔfliC::fliCK12-MG1655CmR) retained motility and both in vitro and in vivo characterisation was carried out in macrophages and using a murine model of sublethal salmonellosis respectively. Activation of the NAIP/NLRC4 inflammasome was impaired in murine macrophages infected with M525PΔfliC::fliCK12-MG1655CmR when compared to those infected with M525P. Mice infected with M525PΔfliC::fliCK12-MG1655CmR had increased liver and spleen bacterial burdens compared to those infected with M525P, indicating that optimal NAIP/NLRC4 inflammasome activation is key for efficient control of microbial spread in vivo. The role of NAIP receptors in inflammasome formation was further investigated with the use of CRISPR/Cas9 to generate mutant murine macrophage cell lines. To investigate the consequence of gene deletions cell lines were designed to lack NAIP 1, 2, 5 and 6, while others were designed to express tagged NAIP proteins to elucidate the cellular localisation of the NAIP proteins during inflammasome formation by microscopy. Characterisation of these cell lines is ongoing, with extensive optimisation of the CRISPR/Cas9 technique undertaken during this study.
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Toll-like receptor 5 recognition fo bacterial flagellin /Andersen-Nissen, Erica, January 2006 (has links)
Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 101-125).
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Diarrheagenic Escherichia coli signaling and interactions with host innate immunity and intestinal microbiotaWang, Gaochan January 1900 (has links)
Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Philip R. Hardwidge / Diarrheagenic Escherichia coli (E. coli) strains are common etiological agents of diarrhea. Diarrheagenic E. coli are classified into enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC or enterohemorrhagic E. coli [EHEC]), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffuse-adherent E. coli (DAEC), and adherent invasive E. coli (AIEC). In addition to encoding toxins that cause diarrhea, diarrheagenic E. coli have evolved numerous strategies to interfere with host defenses.
In the first project, we identified an ETEC-secreted factor (ESF) that blocked TNF-induced NF-[kappa]B activation. One of the consequences of TNF-induced NF-[kappa]B activation is the production of pro-inflammatory cytokines that help to eliminate pathogens. Modulation of NF-[kappa]B signaling may promote ETEC colonization of the host small intestine. In this study, we fractionated ETEC supernatants and identified flagellin as necessary and sufficient for blocking the degradation of the NF-[kappa]B inhibitor I[kappa]B[alpha] in response to TNF[alpha].
In the second project, we attempted to identify an ETEC cAMP importer. ETEC diarrhea leads to cAMP release into the lumen of the small intestine. cAMP is a key secondary messenger that regulates ETEC adhesin expression. We hypothesized that a cAMP importer is present in ETEC, accounting for its hypersensitivity to extracellular cAMP. We used Tn5 transposome-mediated mutagenesis to construct a mutant library and screen for cAMP-hyporesponsive mutants. However, none of the 17,956 mutants we screened were cAMP-hyporesponsive.
In the third project, we focused on gut microbiota and the T3SS effector NleH. We used the mouse-specific pathogen C. rodentium and transplanted performed microbiota between different mouse strains. We evaluated microbiota populations as a function of infection with WT and [Delta]nleH C. rodentium strains before and after microbiota transplantation. Microbiota transfer altered the resistance to WT C. rodentium infection in C57BL/10ScNJ mice and the NleH effector promoted host resistance to C. rodentium.
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Role de la signalisation lipidique chez les plantes en réponse aux contraintes de l'environnement et lors du développement / Deciphering the role of lipid signalling in plant response to environmental stresses and developmental cuesKalachova, Tetiana 09 June 2017 (has links)
La thèse est consacrée à l'étude de la signalisation lipidique comme un mécanisme universel de médiation des réponses cellulaires à phytohormones et élicitors jouant ainsi un rôle clé dans la réorganisation de métabolisme cellulaire pendant l'adaptation de la plante aux changements environnementaux.Phospholipase D (PLD) et son produit acide phosphatidic (PA) ont étés impliqués au cascades de signalisation induites par l’acide salicylique (SA) dans les cellules de garde de Arabidopsis thaliana. On a trouvé une activation de PLD et la production de PA dans les feuilles des plantes après le traitement par SA. En utilisant le marquage radioactif des phospholipides, l'analyse histochimique, les inhibiteurs de la signalisation lipidique et des lignées transgénique des plantes, nous avons montré la participation de la PLD et la NADPH-oxidase RbohD à la formation du superoxyde dans les tissues d’Arabidopsis et à la fermeture des stomates induite par SA.La cooperation entre le SA et l’acide abscisic (ABA) dans la réorganisation de transcriptome induite par ces hormones a été examinée dans la culture de la suspension cellulaire. Tant SA que l'ABA ont inhibé l'activité basale in vivo de phospholipase C dépendante de phosphatidylinositol (PI-PLC), tandis que SA (mais pas ABA) a incité aussi le phosphorylation de phosphatidylinositols. Les transcriptomes de cellules après le traitement par SA ou ABA ont été comparé à ceux obtenus aprés le traitement avec U73122 ou wortmannin. Nous avons trouvé des groupes de gènes, pour qui l'effet d'ABA et des inhibiteurs était semblable; des gènes dependants du SA via l'équilibre des phosphoinositides et des gènes dependants du SA via l’activité de PLD. Basé sur l'analyse bioinformatique de toutes les groupes de gènes choisis, nous proposons le règlement du niveaux des phosphoinositides comme un facteur important dans la regulation du transcriptome basal et également dans les changements du profile transcriptomique induits par l'effet du SA ou d'ABA.L'effet du peptide bactérien flg22 sur l’équilibre des phospholipides a été détecté tant dans des cellules de suspension que dans des plantules. Flg22 a induit l'accumulation de PA par l'activation de PI-PLC couplée a la diacylglycerolkinase 5 (DGK5), et egalement la diminution de niveau de phosphatidylinositol-4,5-biphosphate, qui est un substrat de PI-PLC. L'analyse des effects des inhibiteurs a révélé la participation des DGK et PI-PLC dans la production des espèces d'oxygène réactive (ROS) induite par flg22. La production du PA a été placée dans la cascade de la signalisation en aval de la reconnaissance du flg22 par le complexe de récepteur FLS2-BAK1, mais aprés la formation du ROS par NADPH-oxydase RbohD. Le rôle de DGK5 a été caractérisé dans la regulation du transcriptome; dans l’accumulation du callose induite par flg22 dans l’apoplast et dans la résistance au pathogène biotrophique Pseudomonas syringae pv tomato DC3000. Finalement, nous avons proposé un nouveau modèle de perception du flagellin qui inclut PI-PLC et DGK5.Le rôle de phosphoinositides dans les cascades de la signalisation d’auxin et cytokinin a été révélé dans la morphogenesis racinaire dans le mutant d'Arabidopsis pi4kb1b2 (muté dans deux isoformes de PI4K) et pi4kb1b2sid2 (contient la mutation supplémentaire de l'enzyme de biosynthèse du SA, permettant de séparer les effets du mutation en PI4K qui dependent du SA). Nous avons analysé l'anatomie de la meristem des racines, l'allongement de cellules corticales, la réponse gravitropic, les réponses aux hormones exogènes et nous avons montré la connexion entre l'activité PI4K avec les effets d’auxin et de cytokinin pendant le morphogenesis racinaire et gravitropism. Nos résultats élargissent la connaissance de la nature de la signalisation phytohormonale dans les plantes et peuvent être utilisés comme une base pour augmenter la résistance de céréales agricolement importantes aux contraintes de l’environnement / Thesis is devoted to the investigation of lipid signaling processes as a universal mechanism mediating cellular responses to phytohormones and elicitors thus playing a key role in cell metabolism remodeling during plant adaptation to environmental changes. Phospholipase D (PLD) and its product phosphatidic acid (PA) were found to be involved to the SA-induced signaling cascades in Arabidopsis thaliana guard cells. Using radioactive labeling of phospholipids we found an activation of PLD and production of PA in leaves of 4-week old plants after salicylic acid (SA) treatment. Using histochemical assay, inhibitor assay and transgenic lines knock-out by different isoforms of NADPH-oxidases, we showed the involvement of PLD and NADPH-oxidase RbohD to PA-mediated superoxide formation in Arabidopsis tissues infiltrated by SA and SA-induced stomatal closure. SA- crosstalk with abscisic acid (ABA) in transcriptome remodeling induced by these hormones was investigated in suspension cell culture. Both SA and ABA inhibited basal activity of phosphatidylinositol dependent phospholipase C (PI-PLC) in vivo, while SA (but not ABA) also induced the phosphorylation of phosphatidylinositols. Total transcriptomes of suspension cells after SA or ABA treatment were compared to those obtained from suspension cells treated with U73122 (PI-PLC inhibitor) or wortmannin (inhibitor of phosphatidylinositol-4-kinases (PI4K) that provide the substrate for PI-PLC catalyzed reactions). We found a specific gene clusters, for those the effect of ABA and inhibitors was similar; SA-dependent genes, regulated via the balance of phosphoinositides, and SA-dependent genes, regulated via PLD-mediated pathway. Based on the bioinformatic analysis of the promoters of all selected gene sets, we claim a phosphoinositides level regulation to be an important factor mediating basal cell transcriptome and expression changes induced by SA and ABA.The effect of bacterial peptide flg22 on phospholipid turnover was detected in both suspension cells and seedlings. Flg22 induced accumulation of PA by the activation of PI-PLC coupled with diacylglycerolkinase (DGK) and a corresponding parallel increase of phosphatidylinositol-4,5-biphosphate content, that is a substrate of PI-PLC. Inhibitor analysis revealed the involvement of Ca2+ ions in lipid signaling enzymes reaction to flagellin treatment. We showed the role of DGK and PI-PLC in production of reactive oxygen species (ROS) induced by flg22. PA-production was placed in signaling cascade downstream of flagellin recognition by FLS2-BAK1 receptor complex receptor, but upstream or ROS formation by NADPH-oxidase RbohD. DGK5 was found to be the main source of the detected PA. The role of DGK5 was characterized in basal transcriptome regulation and its flagellin-induced remodeling; in flg22-induced callose accumulation in apoplast and resistance to biotrophic pathogen Pseudomonas syringae pv tomato DC3000. We proposed a new model of flagellin perception that includes PI-PLC and DGK5. Role of phosphoinositides in auxin and cytokinin signaling cascades was revealed studying root morphogenesis in Arabidopsis mutant pi4kb1b2 deficient for two PI4K genes, and pi4kb1b2sid2 that had additional mutation it key enzyme of SA biosynthesis, thus allowing us to separate SA-dependent and independent effects of the PI4K deficiency. pi4kb1b2 mutant plants exhibit the dwarf phenotype both in leaf and root parts, while pi4kb1b2sid2 show the normal rosette growth compared to WT, but still shorter roots. We analyzed root meristem anatomy, cortical cells elongation, gravitropic response, responses to exogenic hormones and firstly showed the connection of PI4K activity with auxin and cytokinin effects during root morphogenesis and gravitropism. Our results broaden the knowledge about the nature of plant phytohormonal signaling and can be used as a basis for increasing the resistance of agriculturally important crop plants to environmental stresses
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Proteomic approaches for the detection of unusual post-translational modifications in simple and complex bacterial protein mixturesSchirm, Michael January 2004 (has links)
Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.
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