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Bergman, Molly Ann. "Host responses to Salmonella typhimurium infection in vitro and in vivo /". Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/11503.
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Hone, David. "Construction of Salmonella vaccines /". Title page, contents and abstract only, 1988. http://web4.library.adelaide.edu.au/theses/09PH/09phh7721.pdf.
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Salam, Faridah. "Development of immunosensors for Salmonella typhimurium". Thesis, Cranfield University, 2010. http://dspace.lib.cranfield.ac.uk/handle/1826/8193.
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The accidental contamination of Salmonella in raw and processed foods is a major problem for the food and feed industries worldwide. Rapid detection methods for monitoring and identification are required to solve the health and safety problems related to these pathogenic bacteria. Current detection methods require extensive sample preparation and prolonged assay procedures, thus, this research project focused on developing rapid methods which are capable of sensing these microorganisms at a high sensitivity level.
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de, Almeida Pereira Milton César. "Inflammasome signalling during Salmonella Typhimurium infection". Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/283642.
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The innate immune system is the first line of defence against infection. It is comprised of physicochemical barriers and a variety of cell types including macrophages and dendritic cells. Pathogens express specific pathogen associated molecular patterns (PAMP) which are recognised by pattern recognition receptors (PRR) on macrophages to initiate an innate immune response. Gram-negative bacteria such as Salmonella enterica serovar Typhimurium express a range of bacterial PAMPs recognised by Toll-like receptors (TLRs) including lipopolysaccharides (LPS) recognised by TLR-4 and lipoproteins by TLR-2. The activation of TLRs results in activation of nuclear factor κB (NF-κB) to drive transcription of mRNA coding for pro-inflammatory proteins such as tumor necrosis factor α (TNF-α) and pro-interleukin (IL) 1β. Myeloid cells also possess intracellular PRRs including the nucleotide-binding domain and leucine-rich repeat (NLR) family. NLR family CARD domain- containing protein 4 (NLRC4) and NLR family pyrin domain-containing protein 3 (NLRP3) are the main NLRs engaged in recognising S. Typhimurium infection, leading to formation of the inflammasome. The inflammasome is a macromolecular complex assembled in the cytoplasm, and usually contains a NLR, the structural protein apoptosis-associated speck-like protein containing a CARD (ASC) and effector enzymes such as cysteine-dependent aspartate-directed protease (caspase) -1 and caspase-8. This structure is responsible for processing the cytokines pro- IL-1β and pro-IL-18 to their mature form and is involved in triggering a pro-inflammatory process of cell death termed pyroptosis. The formation of the inflammasome therefore results in cell death and secretion of proinflammatory cytokines which play important roles in controlling infections. Inflammasome activity must be tightly coordinated, as its dysregulation is associated with a variety of auto-inflammatory and auto-immune diseases. The signalling events leading to inflammasome assembly are poorly understood and the molecules involved in fine-tuning its activity are only beginning to be discovered. The aim of this thesis was to discover new molecules involved in inflammasome activation and/or in keeping its activity in check. To achieve this goal, I performed S. Typhimurium infection assays in primary bone marrow derived macrophages (BMDM) derived from C57BL/6 mice wild type (WT) and compared the resulting cellular viability, intracellular bacteria counts and IL-1β production to that of BMDMs derived from C57BL/6 mice lacking proteins involved with, or suspected to be involved with, innate immune activity. Amongst the proteins I studied, caspase recruitment domain 9 (CARD9) inhibited inflammasome-mediated IL-1β production. Multiple independent genome-wide association studies link this protein to inflammatory pathologies such as Crohn's disease, but its role in canonical inflammasomes was largely unexplored. To investigate how CARD9 inhibits inflammasome-mediated IL-1β production I have conducted assays in WT and Card9-/- BMDMs, including stimulation of specific NLRs with their purified ligands, infection with bacterial strains deficient in NLRC4 activation, and infection assays in presence of pharmacological inhibitors. By employing these approaches, I observed that CARD9 has a negative role on NLRP3-dependent IL-1β production. Specifically, in response to activation of the NLRP3 by Salmonella infection, CARD9 negatively regulates pro-IL-1β transcription, and decreases IL-1β processing by inhibiting spleen tyrosine kinase (SYK)-mediated NLRP3 activation and represses caspase-8 activity in the inflammasome. CARD9 expression is suppressed in the course of S. Typhimurium infection which may act as a mechanism to increase IL-1β production during the infection. In conclusion, I have established a connection between CARD9 and IL-1β production by the canonical NLRP3 inflammasome and elucidated some of the mechanisms involved in this process. I have also found evidence that other proteins are likely to be involved in inflammasome regulation and the elucidation of their roles will be addressed in future studies.
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Muddiman, Katie. "Functional characterisation of Salmonella Typhimurium CueP". Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/functional-characterisation-of-salmonella-typhimurium-cuep(a9ded192-a63d-48f0-a34e-7d9a1ce0e011).html.
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Metals are used as cofactors for enzymes, but are toxic in excess. In order to avoid the deleterious effects posed by metals, the cell must employ strict metal homeostasis systems. One such system is the Cue copper-resistance system in Salmonella enterica serovar Typhimurium (S. Typhimurium) which includes the periplasmic copper binding protein CueP. Previous studies have shown CueP to be a major periplasmic copper-sequestering protein that has a role in supplying copper to, and thus activating, the periplasmic Cu,Zn-superoxide dismutase enzyme SodCII (Osman et al., 2013). SodCII protects the cell from reactive oxygen species (ROS), due for example to the actions of the respiratory burst oxidase in host macrophages. However, despite its ability to sequester copper and activate SodCII, the precise physiological role of CueP in S. Typhimurium has remained unresolved since cueP mutants of S. Typhimurium strain SL1344 (the wild-type stain used in this study) do not exhibit a phenotype with respect to tolerance to copper or reactive oxygen species. In addition, the copper-binding mechanism of CueP and its interactions with other copper-binding proteins, including SodCII, have not been examined. An aim of this study was to establish a phenotype for a cueP mutant of S. Typhimurium with respect to copper and/or ROS tolerance. It was hypothesised that the possession of KatG (catalase) and multiple superoxide dismutases (SodCI, SodA and SodB), in addition to SodCII, by S. Typhimurium may confer functional redundancy with respect to copper and ROS tolerance. Hence mutants lacking katG (ÎkatG) or the various superoxide dismutase encoding genes (ÎsodA/ÎsodB/ÎsodCI/ÎsodCII) with and without functional cueP were generated. The ÎkatG mutants exhibited reduced catalase activity and reduced tolerance to hydrogen peroxide, consistent with the loss of KatG, however the additional loss of cueP did not reduce tolerance to hydrogen peroxide further. Similarly, tolerance to copper and extracellular superoxide was also unaltered in the ÎkatG/ÎcueP mutant. The tolerance of the various superoxide dismutase mutants to copper and various ROS was also unaffected by the presence or absence of CueP. To examine the role of CueP in SodCII activation in vivo, SodCII was over-expressed in S. Typhimurium (in a ÎsodA/ÎsodB/ÎsodCI/ÎsodCII background) with and without functional cueP and superoxide dismutase activity measured in both whole cells and periplasmic extracts. SodCII-dependent superoxide dismutase activity was successfully identified within the periplasmic extracts. However, surprisingly, the level of activity was unaffected by the presence 16 or absence of CueP and/or the addition of copper. It is possible that SodCII is thus able to scavenge sufficient copper for activity from the reagents used in these assays. Similarly, in an alternative approach to examine the role of CueP in vitro, both SodCII and CueP (WT and potential metal-binding residue mutant forms) were successfully over-expressed in E. coli and methods for their purification optimised (without the use of affinity tags). ICP-MS analysis indicated that a CuePC104S mutant contains > 18-fold less copper than the CueP WT protein. Furthermore, superoxide dismutase activity assays using purified proteins, indicated that the CuePC104S mutant was less able to activate SodCII than the WT CueP. Taken together, these results are consistent with a role for the Cys104 residue in copper-binding by CueP. Bioinformatics results suggest the presence of CueP or homologous genes in the presence of other bacteria, including pathogens such as Klebsiella, Yersinia and Shigella spp. Further understanding of the role of CueP and the systems used by S. Typhimurium to avoid both copper and ROS stress may inform the development of novel treatment strategies for bacterial diseases.
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Damiani, Igor Alexandre Campos 1988. "Estudo do efeito terapêutico de linhagens atenuadas de Salmonella enterica Typhimurium em modelos murinos de câncer". [s.n.], 2011. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317027.
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Orientador: Marcelo BrocchiDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-20T04:43:16Z (GMT). No. of bitstreams: 1 Damiani_IgorAlexandreCampos_M.pdf: 2676549 bytes, checksum: 95c7715f3b322149e80749501a5500a2 (MD5) Previous issue date: 2011
Resumo: Salmonella enterica Typhimurium é uma bactéria anaeróbica facultativa que apresenta tropismo por áreas tumorais. Esta interessante propriedade abre novas perspectivas em relação à pesquisa contra o câncer, pois há muito tempo buscam-se veículos seletivos para a eliminação de neoplasias. A inibição do crescimento tumoral e até mesmo seu total retrocesso foram observados em modelos murinos de câncer tratados com linhagens atenuadas de S. enterica. Além disso, seu potencial como veículo de moléculas antitumorais exógenas (vacina de DNA, RNAi, citocinas e enzimas, por exemplo) também foi descrito. No entanto, as linhagens testadas em humanos até o presente não induziram os mesmos efeitos observados nos modelos animais. Isto indica que estudos adicionais são necessários para otimização desta terapia, incluindo o teste de novas linhagens mutantes atenuadas de S. enterica....Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital
Abstract: Salmonella enterica Typhimurium, a facultative anaerobic bacterium, presents tropism for tumor areas. This interesting property creates new perspectives in cancer research, in which great efforts have been done to seek a drug carrier that could selectively target and destroy malignant cells. Inhibition of tumor growth and even its total elimination were observed in murine cancer models infected by attenuated strains of S. enterica. Besides, its potential as a carrier of exogenous antitumor molecules (DNA vaccine, iRNA, cytokines and enzymes, for example) is also described. Nevertheless, when these strains were tested in humans, they did not induce the same effects observed in murine models. Thus, additional studies are needed to optimize this therapy, including the test of novel S. enterica attenuated strains...Note: The complete abstract is available with the full electronic digital thesis or dissertations
Mestrado
Microbiologia
Mestre em Genética e Biologia Molecular
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Wang, Liying. "Regulation of heme biosynthesis targets the key enzyme HemA by a mechanism of protein stabilization in Salmonella typhimurium". Morgantown, W. Va. : [West Virginia University Libraries], 1999. http://etd.wvu.edu/templates/showETD.cfm?recnum=577.
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Thesis (Ph. D.)--West Virginia University, 1999.Title from document title page. Document formatted into pages; contains xiii, 145 p. : ill. (some col.) Includes abstract. Includes bibliographical references.
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Ohlson, Maikke B. "Characterization of the intracellular activities of SseJ and SifA, two Salmonella enterica serovar typhimurium type III secretion effector proteins /". Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/11485.
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Brahmbhatt, Himanshu N. "Cloning and molecular characterization of the rfb gene cluster from Salmonella typhimurium LT2 /". Title page, contents and abstract only, 1987. http://web4.library.adelaide.edu.au/theses/09PH/09phb813.pdf.
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Tai, Chia-Hui. "Kinetic and Chemical Mechanism of O-Acetylserine Sulfhydrylase-B from Salmonella Typhimurium". Thesis, University of North Texas, 1993. https://digital.library.unt.edu/ark:/67531/metadc279064/.
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Initial velocity studies of O-acetylserine sulfhydrylase-B (OASS-B) from Salmonella typhimurium using both natural and alternative substrates suggest a Bi Bi ping pong kinetic mechanism with double substrate competitive inhibition. The ping pong mechanism is corroborated by a qualitative and quantitative analysis of product and dead-end inhibition. Product inhibition by acetate is S-parabolic noncompetitive, indication of a combination of acetate with E followed by OAS. These data suggest some randomness to the OASS-B kinetic mechanism. The pH dependence of kinetic parameters was determined in order to obtain information on the acid-base chemical mechanism for the OASS-B reaction. A mechanism is proposed in which an enzyme general base accepts a proton from α-amine of O-acetylserine, while a second enzyme general base acts by polarizing the acetyl carbonyl assisting in the β-elimination of the acetyl group of O-acetylserine. The ε-amine of the active site lysine acts as a general base to abstract the α-proton in the β-elimination of acetate. At the end of the first half reaction the ε-amine of the active site lysine that formed the internal Schiff base and the general base are protonated. The resulting α-aminoacrylate intermediate undergoes a Michael addition with HS‾ and the active site lysine donates its proton to the α-carbon to give cysteine and regenerate enzyme to start the second half reaction. In addition, substrate specificity, stereochemistry of the internal Schiff base at C4', and sequence around active site lysine of O-acetylserine sulfhydrylase-A have been determined. The [4'-^3H]pyridoxamine generated by reduction of the internal Schiff base with sodium [^3H]borohydride retained most of its tritium after incubation with apoaspartate aminotransferase. These results agree with the hypothesis put forth by Dunathan (Dunathan, 1971; Dunathan and Voet, 1974) that a single surface (Re face) of the active site PLP is accessible to solvent. The sequence around the active site lysine is AsnProSerPheSerValLysCysArg.
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Howard, Zoe R. "Invasion of avian reproductive tissues by Salmonella typhimurium and Salmonella enteritidis". Texas A&M University, 2003. http://hdl.handle.net/1969/275.
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Olsen, Eric Vincent Petrenko Valery Barbaree James M. "Phage-coupled piezoelectric biodetector for Salmonella typhimurium". Auburn, Ala., 2005. http://repo.lib.auburn.edu/2005%20Summer/doctoral/OLSEN_ERIC_17.pdf.
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Müller, Andreas Johann. "In vivo analysis of Salmonella typhimurium infection /". [S.l.] : [s.n.], 2009. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=18432.
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Osman, Deenah. "Copper Homeostasis in Salmonella Enterica Serovar Typhimurium". Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.509383.
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Okoro, Chinyere Kyna. "Invasive Salmonella typhimurium : linking phenotype to genotype". Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607714.
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McGourty, Kieran D. "Interference with lysosomal biogenesis by Salmonella typhimurium". Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/11096.
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Salmonella enterica is an intracellular bacterial pathogen that replicates within membrane-bound vacuoles by delivering virulence (effector) proteins across the vacuolar membrane by the SPI-2 type III secretion system (T3SS). In this thesis I show that through the action of this T3SS, Salmonella selectively interferes with a subset of endosome-to-trans-Golgi network (TGN) traffic that is involved in mannose-6-phosphate receptor (MPR) recycling. Two pathways of endosome-to-TGN traffic have been described: one involves the Q-SNARE syntaxin 6; the other is defined by the Q-SNARE syntaxin 10 and involves retrograde transport of the MPRs. Salmonella specifically disrupted syntaxin 10-dependent endosome-to- TGN traffic and this was accompanied by redistribution of MPR from the TGN, misrouting of newly synthesized lysosomal enzymes and attenuation of lysosome function. The SPI-2 T3SS effector SifA is an important virulence determinant that is known to interact with the endolysosomal system. SifA was shown to be required and sufficient to mediate the effects on lysosome biogenesis during infection or following transfection of host cells. By contrast, a variant of SifA with a single amino acid substitution that prevents interaction with its host protein target, SKIP, failed to redistribute MPR or to reduce lysosomal function. SKIP was found to contribute to lysosomal biogenesis in uninfected cells. Inhibition of lysosomal enzyme function with a lysosomal protease inhibitor or by depleting cells of syntaxin 10 enhanced intracellular bacterial replication. I conclude that SifA attenuates lysosomal activity through its interaction with SKIP and that this facilitates intracellular bacterial replication.
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Toguchi, Adam James Masao. "Swarming in Salmonella typhimurium and Escherichia coli /". Digital version accessible at:, 1999. http://wwwlib.umi.com/cr/utexas/main.
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Rakeman, Jennifer Leigh. "Transcriptional regulation of Salmonella typhimurium invasion genes /". Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/11531.
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Sigmarsson, Haukur Lindberg. "PATHOGENITÄTSVERGLEICH VON SALMONELLA TYPHIMURIUM DT104 - WILDTYP UND SALMONELLA TYPHIMURIUM - DELETIONSMUTANTEN (sseD::aphT & invC::aphT) IN PERSISTENT INFIZIERTEN SCHWEINEN". Doctoral thesis, Universitätsbibliothek Leipzig, 2012. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-98570.
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ZUSAMMENFASSUNG
Haukur Lindberg Sigmarsson
PATHOGENITÄTSVERGLEICH VON SALMONELLA TYPHIMURIUM DT104 - WILDTYP UND SALMONELLA TYPHIMURIUM - DELETIONSMUTANTEN (sseD::aphT & invC::aphT) IN PERSISTENT INFIZIERTEN SCHWEINEN
Salmonella (S.) Typhimurium DT104 ist ein gram-negatives Bakterium. Es weist keine Wirtsspezifität auf und gilt als Zoonoseerreger. Jährlich erkranken daran allein in Deutschland mehrere Tausend Menschen unter dem Bild einer schwerwiegenden Diarrhö mit zum Teil tödlichem Ausgang. Das Schwein gilt als eines der Reservoire für S. Typhimurium DT104 des Menschen. S. Typhimurium DT104 gelangt über vom Schwein stammende Produkte in den menschlichen Verzehr. Die Kontrolle von S. Typhimurium DT104 einschließlich effektiver Eradikationsmassnahmen in unseren Schweinebeständen ist deshalb von entscheidender Bedeutung, um den Eintrag dieses Bakteriums in die menschliche Nahrungskette wenn möglich zu eliminieren. Dafür ist das Verständnis über S. Typhimurium DT104 einschließlich der Kenntnis seine Pathogenitätseigenschaften notwendig. Ziel dieser Arbeit waren Untersuchungen zur Pathogenität von S. Typhimurium DT104. Dabei wurden der Wildstamm mit zwei seiner Deletionsmutanten (sseD::aphT und invC::aphT) verglichen.
Die Untersuchungen erfolgten im Infektionsversuch an insgesamt 25 sechs Wochen alten männlichen Schweinen, die in einem vollklimatisierten Versuchsstall gehalten wurden. Den Tieren wurde im Anschluss an eine einwöchige Akklimatisierungsphase eines der nachfolgenden Stämme von S. Typhimurium DT104 oral in einer Konzentration von 1 x 1011 KBE verabreicht: Wildtyp (n = 8 Schweine), Deletionsmutante seeD::aphT (n = 8) und Deletionsmutante invC::aphT (n = 9). Bei den Mutanten handelt es sich um Varianten von S. Typhimurium DT104, die an den entsprechenden Abschnitten des Bakteriumgenoms (d.h. sseD-Gen bzw. invC-Gen) deletiert wurden. SseD regelt die Überlebensfähigkeit von S. Typhimurium in Makrophagen, invC dessen Invasionsvermögen. Im Mäusemodel war die Pathogenität beider Mutanten deutlich vermindert. Nach der Infektion schloss sich ein 20 tägiger Beobachtungszeitraum an, während dessen nachfolgend genannte Parameter erfasst bzw. Proben genommen wurden: klinische Symptome (Allgemeinbefinden, Erbrechen, Durchfall, Futteraufnahme, Atmung, Temperatur); Blutentnahme für Erstellung des weißen Blutbildes; Kotentnahme zum Nachweis der Ausscheidung von S. Typhimurium. Einen Tag nach Ende der Beobachtung wurden die Tiere getötet und Proben von insgesamt 15 Organen (unter anderem Tonsille; Colon und Caecum sowie dazugehörige Lymphknoten; Leber; Milz; Muskulatur) genommen. Kot sowie Gewebeproben wurden kulturell und wenn positiv auch mittels PCR untersucht.
Alle mit dem Wildtyp infizierten Schweine wurden mehr oder weniger stark krank. Häufig zeigten erkrankte Schweine zeitgleich mehrere Krankheitssymptome (z. B. Erbrechen und Durchfall). Die Erkrankung hielt über mehrere Tage an. Im Vergleich dazu waren die Krankheitssymptome der Tiere, die mit Mutanten infiziert wurden, mild. Nur wenige Tiere erkrankten und dann auch nur kurzzeitig. Gewöhnlich war nur einer der erfassten Parameter verändert. Typische Veränderungen im weißen Blutbild waren nur bei Wildtyp-infizierten Tieren zu beobachten, während Tiere beider Mutanten kaum auf die Infektion reagierten. Alle 25 infizierten Tiere schieden S. Typhimurium mit dem Kot während der ersten Woche post inocculationem aus. Danach wurden in allen drei Gruppen etwa gleichviel intermittierende Ausscheider beobachtet. Zwischen 65 und 67 % der Gewebeproben der mit dem Wildtyp und mit der sseD::aphT-Mutante infizierten Tiere waren sowohl in der Kultur als auch mittels PCR S. Typhimurium positiv, während dieser Anteil nach Infektion mit invC::aphT nur 49 % betrug. Alle Tiere waren in Mandibularlymphknoten und im Colon positiv, während S. Typhimurium nur selten in Muskulatur und Leber nachzuweisen war.
Die Ergebnisse dieser Arbeit bestätigen, dass Infektionen mit dem Wildtyp von S. Typhimurium zu einer schweren Erkrankung führen können. Gleichzeitig konnte gezeigt werden, dass beide in dieser Arbeit verwendeten Mutanten weniger krankmachend sind. Es muss davon ausgegangen werden, dass die Deletionen in den sseD bzw. invC-Bereichen tatsächlich zu Veränderungen bestimmter Eigenschaften geführt haben, die Teil der Pathogenitätsmechanismen für das Schwein sind. Im Unterschied zur Maus war sseD beim Schwein allerdings invasiv. Es kann vermutet werden, dass die durch sseD kodierten Pathogenitätseigenschaften von S. Typhimurium bei der Maus anders als beim Schwein wirken und somit unterschiedliche Bedeutung haben. Da die invC::aphT-Mutante jedoch und wie erwartet wesentlich schwächer als Wildtyp und sseD::aphT invadierte ist davon auszugehen, dass die Deletion im invC Bereich das Invasionsvermögen der Mutante beim Schwein ähnlich wie bei der Maus verringerte.
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McClure, G. David (George David). "A Study of the Intrinsic Fluorescence of O-Acetyl-L-Serine Sulfhydrylase-A from Salmonella typhimurium". Thesis, University of North Texas, 1993. https://digital.library.unt.edu/ark:/67531/metadc278975/.
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O-Acetyl-L-serine sulfhydrylase-A (OASS-A) forms acetate and L-cysteine from O-acetyl-L-serine (OAS) and sulfide. One molecule of the cofactor pyridoxal 5'- phosphate (PLP) is bound in each holoenzyme protomer.
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Cunning, Christofer Lee. "Regulation of the synthesis and protein stability of the alternative sigma factor RpoS in Salmonella typhimurium". Morgantown, W. Va. : [West Virginia University Libraries], 1999. http://etd.wvu.edu/templates/showETD.cfm?recnum=533.
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Bronstein, Philip Alan. "Identification and characterization of a type III chaperone, InvB /". Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/11524.
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Leiva, Araya Lorenzo Eugenio. "Contribución del sistema de secreción tipo VI codificado en la isla genómica SPI-6 a los mecanismos de virulencia de Salmonella entérica serovar typhimurium". Tesis, Universidad de Chile, 2013. http://www.repositorio.uchile.cl/handle/2250/113484.
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Magíster en Bioquímica área de Especialización en
Bioquímica de Proteínas RecombinantesMemoria de título de bioquímico
Los sistemas de secreción tipo VI (T6SS) corresponden a un mecanismo de interacción célula-célula ampliamente distribuido entre bacterias Gram negativo. Si bien inicialmente al T6SS se le atribuyó un papel en la virulencia de los microorganismos, estudios posteriores dieron cuenta de su versatilidad, indicando que el sistema también toma parte en relaciones mutualistas o comensales entre bacterias y eucariontes, además de relaciones de competencia interbacteriana. Salmonella Typhimurium codifica un T6SS en la isla de patogenicidad SPI-6 (T6SSSPI-6), sin embargo el rol que cumple en la patogénesis de Salmonella aún no ha sido aclarado. Resultados obtenidos en nuestro laboratorio indican que mutantes de este sistema presentan una menor colonización de órganos internos, tanto en ratones BALB/c como en pollos White Leghorn infectados oralmente. Considerando que los componentes celulares del sistema inmune son la principal puerta de entrada de Salmonella para el desarrollo de la infección sistémica, se planteó como hipótesis de este trabajo que “el Sistema de Secreción Tipo VI codificado en la isla genómica SPI-6 de Salmonella enterica serovar Typhimurium se expresa en el interior de macrófagos de origen murino y aviar, favoreciendo la supervivencia bacteriana en estas células”. Para probar esta hipótesis el objetivo fue evidenciar la expresión, funcionalidad y contribución del T6SS durante la interacción de S. Typhimurium con macrófagos murinos y aviares. Para determinar la expresión del T6SS durante la infección de macrófagos, se construyó el vector pLZ01 que permitio la generación de fusiones transcripcionales y traduccionales a la proteína fluorescente verde (GFP) en Salmonella, mediante recombinación homóloga de productos de PCR. De esta manera, se fusionaron componentes estructurales del T6SSSPI-6 (VgrG, Hcp-1, Hcp-2) a GFP y se evaluó su transcripción y traducción en ensayos de infección in vitro mediante microscopía de epifluorescencia. Por otra parte, para determinar si el sistema es translocado al citoplasma de macrófagos durante la infección de S. Typhimurium, se estudió la translocación de una fusión traduccional de VgrG a la β-lactamasa TEM1, construida en el plasmidio pFlagTEM1. La translocación de las fusiones fue determinada mediante un ensayo de pérdida de FRET de la cefalosporina CCF2, observado mediante microscopía de epifluorescencia y cuantificado mediante fluorometría. Finalmente, para determinar la contribución del T6SS en los procesos de internalización y supervivencia en macrófagos se realizaron ensayos de protección con gentamicina. En ellos se comparó la capacidad de la cepa silvestre para invadir y sobrevivir en el interior de macrófagos, versus mutantes que carecen de todo el T6SSSPI-6 o poseen un T6SSSPI-6 no funcional debido a la mutación de clpV, ATPasa esencial para este sistema. Todos los experimentos se realizaron en líneas de macrófagos murinos (RAW264.7) y aviares (HD11), utilizando cepas derivadas de S. Typhimurium 14028s. Los resultados mostraron que ninguno de los componentes estructurales estudiados (VgrG, Hcp-1, Hcp-2) del T6SSSPI-6 de S. Typhimurium se transcribe y traduce en el medio de cultivo celular, sin embargo su transcripción y traducción es gatillada al infectar tanto macrófagos murinos como aviares. A pesar de observar la transcripción y traducción de VgrG, no se detectó su translocación al citoplasma de las células infectadas. Contrariamente a lo esperado, se observó que la presencia del T6SSSPI-6 no contribuye a la supervivencia en el interior de macrófagos murinos o aviares, pero sí tendría una implicancia en la etapa de internalización de Salmonella, puesto que al utilizar mutantes con un T6SSSPI-6 no funcional se observó un fenotipo de mayor internalización en ambos modelos celulares. Estos resultados permiten aceptar una parte de la hipótesis planteada, ya que el Sistema de Secreción Tipo VI codificado en la isla genómica SPI-6 de Salmonella enterica serovar Typhimurium se expresa en el interior de macrófagos de origen murino y aviar, y rechazar una segunda parte de la hipótesis, pues este sistema no tendría un rol en la supervivencia bacteriana en estas células. No obstante, el aumento en la capacidad de internalización de mutantes del T6SS indica que el sistema tendría un rol durante la infección de los macrófagos.
Type VI Secretion Systems (T6SS) correspond to a widely distributed cell-cell interaction mechanism in Gram-negative bacteria. Although initially the T6SS was attributed a role in the virulence of microorganisms, subsequent studies realized its versatility, indicating that this system also takes part in comensal or mutualistic relationships between bacteria and eukaryotes, as well as interbacterial competition. Salmonella Typhimurium encodes a T6SS in the pathogenicity island SPI-6 (T6SSSPI-6), however the role of this island in the pathogenesis of Salmonella has not been clarified. Results obtained in our laboratory indicate that mutants of this system generate a phenotype of reduced colonization of internal organs, both in orally infected BALB/c mice and White Leghorn chicken. Because the initial contact of Salmonella with cellular components of the immune system is the main gateway for the development of systemic infection of Salmonella, the objective of this work was to determine the expression, functionality and contribution of the T6SS during S. Typhimurium interaction with murine and avian macrophages. The vector pLZ01was built to determine the expression of the T6SS during infection of macrophages. This plasmid enables the generation of transcriptional and translational fusions to the green fluorescent protein (GFP) reporter in Salmonella by homologous recombination of PCR products. In this way, structural components of the T6SSSPI-6 (VgrG, Hcp-1, Hcp-2) were merged to GFP and their transcription and translation were assessed by in vitro infection assays and epifluorescence microscopy. On the other hand, to determine whether the system is translocated to the cytoplasm of macrophages during infection of S. Typhimurium, translocation of VgrG was studied using a translational fusion of VgrG to the β-lactamase TEM1, built in the pFlagTEM1 plasmid. The translocation of the β-lactamase fusion was determined by processing of the CCF2/AM fluorescence substrate, detected by epifluorescence microscopy and quantified using fluorometry. Finally, gentamicin protection assays were performed to determine the contribution of the T6SS in the processes of internalization and survival in macrophages. In these experiments, invasion and survive inside macrophages at the wild type strain was compared to a deletion mutant of the T6SS gene cluster and a mutant on the clpV gene, which encodes the ATPase essential for the functioning of the system, All experiments were carried out in murine (RAW264.7) and avian (HD11) macrophage cell-lines, using strains derived from the sequenced wild-type S. Typhimurium 14028s strain. The results showed that none of the studied structural components (VgrG, Hcp-1, Hcp-2) of T6SSSPI-6 of S. Typhimurium are produced in cell culture media, but their transcription and translation are triggered when murine or avian macrophages are infected. Despite observing transcription and translation of VgrG, translocation of this protein into the cytoplasm of infected cells could not be detected. Contrary to expectations, it was observed that the presence of the T6SSSPI-6 did not contribute to Salmonella survival within murine or avian macrophages. However, internalization experiments showed that non-functional T6SSSPI-6 mutants showed a greater uptake into both cellular models. These results indicate that the T6SSSPI-6 of S. Typhimurium is expressed during infection of murine and avian macrophages (the first part of the hypothesis is true), however it did not have an impact on the ability of S. Typhimurium to survive inside murine or avian macrophages (the second part of the hypothesis is false). However, the increase in the internalization of the T6SS mutants suggests a novel role for the T6SS during infection of macrophages.
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Rappl, Catherine. "Charakterisierung eines Typ III-Sekretionssystems für Virulenzproteine aus Salmonella typhimurium". [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=964612909.
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Passerat, Julien. "Exploration du potentiel de virulence de Salmonella typhimurium active mais non cultivable". Montpellier 2, 2005. http://www.theses.fr/2005MON20174.
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Fung, Mei-yuk Ami. "Isolation and characterization of a Salmonella enterica serotype typhi variant". Hong Kong : University of Hong Kong, 2001. http://sunzi.lib.hku.hk/hkuto/record.jsp?B23373106.
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Igue, Patience. "Survival of Salmonella typhimurium in simulated intestinal fluids". Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=31242.
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Salmonella species are among the major foodborne intestinal pathogens that are of public concern with respect to food safety. The ability of intestinal pathogens to resist gastric acidity corresponds to their oral infective dose (ID). The survival and lipopolysaccharide (LPS) profiles of Salmonella typhimurium grown at different pH values and to different phases of growth were examined in simulated gastric fluid (pH 1.5), ileal fluid (pH 7.0), colon fluid (pH 8.0). The survival and growth of S. typhimurium were also examined during sequential passage through all three fluids. Viable cells were rapidly reduced from 106 CFU.ml-1 to <10 CFU.ml-1 within 4 min in gastric fluid. Cells inoculated directly into ilea] and colon fluids survived and multiplied extensively. When low numbers of viable cells of Salmonella in contact with gastric fluid (0.5 min of contact) were transferred sequentially to ileal and colon fluids, only the early and late stationary phase cells were capable of recovery and growth to high numbers. The harsh environment of the gastric fluid did not change the LPS profiles of the inoculated Salmonella cells. Entrapment of S. typhimurium in calcium alginate beads and chocolate increased its survival in gastric fluid. This implies that Salmonella cells are protected from killing when ingested with food. These results may explain why Salmonella species have a very low ID when consumed as part of some contaminated food sources.
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Salcedo, Suzana Pinto. "Analysis of salmonella typhimurium interaction with host cells". Thesis, Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.406437.
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Gunel, Ozcan Aysen. "Cloning and characterization of Salmonella typhimurium dehydroquinate synthase". Thesis, Imperial College London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267843.
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Lai, Jyhmirn. "Epidemiological studies on Salmonella typhimurium DT104 in Scotland". Thesis, University of Glasgow, 2007. http://theses.gla.ac.uk/5439/.
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Salmonella Typhimurium definitive type 104 (ST DT104) isolates resistant to antibiotics have been an issue since the multi-resistant clone was identified in 1985. In order to advance understanding of ST DT104 infections in Scotland, 2,796 human and 2,439 animal isolates with their corresponding antibiotic resistance patterns submitted from 1988 to 2004 were used to conduct descriptive, hierarchical, and geographical cluster studies and to construct a time series model. The analyses showed that using the 13 antibiotics used by the Scottish Salmonella Reference Laboratory, isolates could be allocated into two distinct groups. The first group containing the ApC1SpStSuTe R-type with its associated resistance patterns, mainly the ApCISpStSuTeTm and the ApCISpStSuTeNa R-types, dominated the trend throughout the study period. The second group, mainly composed of fully sensitive isolates, formed a low proportion except during the period from 1988 to 1990. Temporal analyses showed that there was an epidemic from 1993 to 1998 in human ST DT104 and from 1992 to 1999 in animals. Spatial analysis identified the southern part of Scotland as the higher relative risk area for both human and animal infections caused by multi-resistant ST DT104 strains In contrast, the central belt of Scotland was mainly the relative risk lower spatial cluster for the multi-resistant ST DT104 R-types. Of note in the spatio-temporal analysis was the stability and persistence of the chromosomally mediated multiple resistance compared to the more sporadic plasmid mediated resistance types of the second group. Although there were many similarities between the infections in humans and animals, there was no consistent temporal association between the emergence of clones in humans compared with animals suggesting that the ecological and epidemiological direction of the relationships is complex.
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Morgan, Eirwen. "Direct search for Salmonella serotype typhimurium gut invasins". Thesis, University of Birmingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364990.
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Reed, Katharine Anne. "Interactions between Salmonella typhimurium and polarised epithelial cells". Thesis, University of Newcastle Upon Tyne, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242364.
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Komitopoulou, Evangelia. "Environmental sensing and stress resistance in Salmonella typhimurium". Thesis, University of Surrey, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.252342.
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Crago, Aimee Marie. "Virulence-related outer membrane proteins of Salmonella typhimurium". Thesis, University of Cambridge, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624399.
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Kalupahana, Ruwani Sagarika. "Interaction of Salmonella typhimurium with antigen presenting cells". Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620666.
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Avendano, Perez Gaspar. "Interactions between Salmonella typhimurium and human gut bacteria". Thesis, University of East Anglia, 2015. https://ueaeprints.uea.ac.uk/54306/.
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Salmonella enterica subsp. enterica serovar Typhimurium is an important enteropathogen that causes human morbidity and mortality worldwide. It is essential to study the interaction between Salmonella and the gut bacteria to elucidate the elements that influence the ability of the pathogen to overcome the colonisation barrier mediated by the gut microbiota and why Salmonella can persist in ‘healthy’ individuals in a carrier state after infection. In this study the effect of faecal bacteria on the growth and survival of S. Typhimurium was investigated. Initially, experiments involved co-cultures of the pathogen and single strains of intestinal bacteria obtained from culture collections; results showed that when E. coli reached its maximum concentration density, the growth of S. Typhimurium was halted. S. Typhimurium was then inoculated with multi-strain gut bacteria from culture collections and also with faecal samples in batch cultures mimicking the conditions of the human colon. A significant reduction of S.Typhimurium concentration was observed in mixed cultures with faecal samples from different human donors; however, bacteria obtained from culture collection had no effect on S. Typhimurium. Close proximity with faecal bacteria was required as the pathogen was not affected when it was separated from the faecal bacteria by a 0.45 µm pore size membrane. S. Typhimurium was also affected in a continuous culture system. Transcriptomic analysis indicated that some of the functions associated with the genes expressed by S. Typhimurium during Salmonella inactivation were related to stress responses. Molecular profiling of faecal bacteria measured by denaturing gradient gel electrophoresis did not show any change specifically associated to S. Typhimurium inactivation. It was not possible to identify the bacterial strains responsible for the inactivation of S. Typhimurium; however, this effect caused by cell-cell contact with human faecal bacteria is reported for the first time in this study.
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Reyner, Jacqueline Louise. "Characterisation of the CspA paralogues of Salmonella Typhimurium". Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4651.
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In cold temperatures, the survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) requires the action of cold shock protein A (CspA) paralogues. These are thought to melt misfolded ribonucleic acids, facilitating their translation at low temperatures. However, through phenotypic analysis of our SL1344 csp null mutant (lacking all CspA paralogues), it has been shown that CspA paralogues function during other environmental stresses, outwith temperature reduction, and play an essential role in colony formation of an SL1344 rpoS mutant at 37°C. The general stress σ subunit, RpoS, plays an important role in adapting cells to a number of stresses including oxidative stress, temperature changes, low pH and stationary phase. Under such conditions, RpoS acts as an ‘emergency co-ordinator’, subsequently inducing the transcription of necessary stress response genes. In Escherichia coli, RpoS is regulated posttranscriptionally by at least three small RNAs (sRNAs): OxyS, DsrA and RprA; that require interactions with the Sm-like RNA chaperone, Hfq. In S. Typhimurium, the stability of the RpoS protein itself is regulated by ClpXP, an ATP-dependent protease responsible for RpoS degradation, and a specific recognition factor that targets RpoS to this protease, MviA. The present study has shown that the CspA paralogues of S. Typhimurium are involved in the expression of RpoS and aims to elucidate the role of these proteins in RpoS production. Comparative phenotypic tests were carried out in strains carrying mutations in rpoS, hfq and the csp genes to gain insight into the interactions of Hfq and CspA paralogues, with respect to RpoS expression. Both significant phenotypic overlaps, such as peroxide sensitivity, and phenotypes unique to certain mutant strains, such as cold acclimation in the csp null strain, were observed. CspA paralogues and Hfq are functionally distinct, not only in their involvement in RpoS expression, but also in RpoS-independent processes, such as cold acclimation, motility and to some extent, growth at 37°C. The roles of Hfq and the CspA paralogues, in RpoS expression, were also assessed at the molecular level. A combination of qRT-PCR analysis, transcriptional fusions and immunoblotting (with anti-σ antibodies) has shown that DsrA and RprA are not essential for RpoS expression in S. Typhimurium, during stationary phase or exponential cold shock, and do not require Hfq under these conditions. Contrary to reports in E. coli, DsrA is not induced upon cold shock in SL1344. Northern blots have shown that neither Hfq nor the CspA paralogues are involved in regulating rpoS transcription during either stationary phase at 37°C or cold shock in exponential phase. Immunoblotting and translational fusions have identified different pathways for the regulation of RpoS during stationary phase at 37°C and cold shock in exponential phase. Hfq is involved during the former condition only, whilst CspA paralogues are involved in both. Protein stability experiments have shown that the CspA paralogues do not play a major role in stabilising RpoS protein against degradation. Together, these results have pointed to a role for both the CspA paralogues and Hfq in facilitating the efficient translation of rpoS mRNA. An SL1344 csp null rpoS mutant is unable to form colonies on LB agar at 37°C, a phenomenon found when introducing combinations of mutations to SL1344 for phenotypic assessment. A conditional rpoS mutant revealed that the SL1344 csp null rpoS strain is viable but non-culturable. From the csp gene family, only cspA and cspB were able to restore colony forming ability to the rpoS mutant. Further complementation experiments pointed to faulty cell division, due to abnormal RNase E activity, as the cause.
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Bjur, Eva. "Virulence of Salmonella enterica serovar typhimurium and innate antibacterial host responses /". Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-946-7/.
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Valenzuela, Montenegro Camila. "Identificación de genes comunes requeridos para la colonización sistémica de Salmonella enterica serovares Typhi, Typhimurium y Enteritidis mediante un análisis global de mutantes bajo selección negativa in vivo". Tesis, Universidad de Chile, 2013. http://www.repositorio.uchile.cl/handle/2250/113549.
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Magíster en Bioquímica en el área de especialización de
Bioquímica Toxicológica y Diagnóstico MolecularMemoria para optar al Título de Bioquímica
El género Salmonella comprende dos especies, S. enterica y S. bongori, que en conjunto agrupan a más de 2.500 serovares. De éstos, los pertenecientes a S. enterica subespecie enterica son responsables de aproximadamente el 99% de los casos de salmonelosis en animales de sangre caliente. A nivel mundial se producen anualmente millones de casos de salmonelosis en el ser humano y miles de muertes, principalmente en países subdesarrollados. En esta tesis se propuso identificar un conjunto de genes requeridos para la colonización sistémica de un hospedero murino por tres serovares de Salmonella: S. Typhi, S. Typhimurium y S. Enteritidis. Este estudio se realizó mediante un análisis masivo de mutantes bajo selección negativa in vivo. La detección de aquellas mutantes con defectos en la colonización sistémica aguda de ratones BALB/c se realizó mediante hibridaciones comparativas utilizando un microarray genómico de Salmonella. El posterior análisis comparativo de las mutantes bajo selección negativa in vivo en los tres serovares, nos permitió identificar que mutantes en 131 genes serían atenuadas in vivo. Dentro de este grupo identificamos genes codificados en islas de patogenicidad conservadas del género Salmonella, genes necesarios para la biosíntesis de purinas y compuestos aromáticos (aro, pur y gua), genes relacionados con la biosíntesis y modificación del LPS (rfa, rfb) y genes que codifican reguladores globales asociados a patogenicidad (phoP, envZ, rpoN, dam y rsd). Otros genes identificados corresponden a los que codifican el sistema transportador de proteínas Twin-Arginine (tatABC), genes que codifican las diferentes subunidades de una NADH deshidrogenasa (genes nuo); un locus que corresponde a un transportador de péptidos del tipo ABC (sapBF). También pudimos detectar que mutantes en genes involucrados en el transporte de solutos se encuentran bajo selección, como trkH que codifica un transportador de potasio. El sistema de transporte Twin-Arginine corresponde a una de las dos vías de translocación de proteínas hacia el espacio periplasmático en bacterias Gram negativo. La participación de este sistema en la patogenicidad de Salmonella se confirmó mediante ensayos de competencia in vivo entre mutantes definidas del operón y la respectiva cepa silvestre en los tres serovares estudiados. El análisis global de mutantes en tres serovares nos permitió determinar un conjunto de genes comunes necesarios para establecer la colonización sistémica aguda en un hospedero murino. Posteriormente, se confirmó la participación del sistema de transporte de proteínas Tat en la patogenicidad de Salmonella. Los resultados de los ensayos de competencia nos permitieron confirmar la predicción obtenida en el análisis de masivo de mutantes bajo selección negativa in vivo.
The Salmonella genus comprises two species: S. bongori and S. enterica, which can be grouped into more than 2,500 serotypes. Serovars within S. enterica subspecies enterica account for ~99% of all salmonellosis in warm-blooded animals. Worldwide, these organisms are responsible for hundreds of millions of salmonellosis cases and hundreds of thousands of deaths, mainly in underdeveloped countries. In this thesis, we aimed to identify a group of genes required for systemic colonization of a murine host by three Salmonella serotypes: S. Typhi, S. Typhimurium and S. Enteritidis. We used a high-throughput microarray-based screening for mutants with defects in systemic colonization of BALB/c mice. Subsequent comparative analysis of mutants under negative selection in vivo allowed us to identify that mutants in 131 genes are attenuated in the three serotypes under study. Within this group we found genes encoded in some of the pathogenicity islands conserved in the Salmonella genus, genes required for biosynthesis of purines and aromatic compounds (aro, pur and gua), genes related to LPS biosynthesis (rfa and rfb) and genes encoding regulators previously associated with virulence (phoP, envZ, rpoN, dam and rsd). Other genes identified are those encoding the Twin-Arginine transport system (tatABC), genes coding the different subunits of a NADH dehydrogenase (nuo genes) and a locus encoding an ABC peptide transporter (sapBF). We also identified that mutants in genes involved in solute transport (i.e: trkH, that encodes a potassium transporter) are under negative selection in vivo. The Twin-Arginine transport system corresponds to one of the two pathways used by Gram-negative bacteria to translocate proteins to the periplasmatic space. Participation of this system in Salmonella pathogenicity was confirmed in the three serotypes under study by means of in vivo competition assays between targeted mutants of the operon and the corresponding wild-type strains. Overall, the global analysis of mutants under negative selection in vivo in three serotypes of Salmonella allowed us to identify a common group of genes required to establish acute systemic colonization of a murine host. We confirmed the participation of the Tat transport system in the pathogenicity of Salmonella using in vivo competition assays. These results further support the predictions obtained in our global analysis.
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Corrêa, Isadora Mainieri de Oliveira. "Separação imunomagnética associada a bacteriófago para diagnóstico de Salmonella enterica em carne de frango /". Botucatu, 2015. http://hdl.handle.net/11449/138467.
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Orientador: Raphael Lucio Andreatti FilhoBanca: Adriano Sakai Okamoto
Banca: Julio Lopes Sequeira
Banca: Maristela Lovato
Banca: Guilherme Augusto Marietto Gonçalves
Resumo: Utilizou-se o método de Separação Imunomagnética associada a Bacteriófago para detectar os seguintes sorovares: Salmonella Heidelberg, Salmonella Enteritidis e Salmonella Typhimurium, em amostras de sobrecoxas de frango contaminadas artificialmente. Para certificarmo-nos da eficiência do método, comparamos esta técnica com os testes de diagnóstico usuais para este patógeno: a análise bacteriológica padrão, que inclui a etapa de pré-enriquecimento, enriquecimento seletivo, realização de testes bioquímicos de triagem e sorologia, e a reação em cadeia da polimerase (PCR). Na avaliação da capacidade dos testes na detecção de Salmonella em carne de frango, submetemos amostras de sobrecoxas de frango à contaminação artificial com 5, 10 e 100 UFC/25mL de bactéria, para cada um dos sorovares listados anteriormente, totalizando 270 análises, divididas em 90 testes para cada um dos três sorovares, e assim comparamos os resultados obtidos com a análise bacteriológica, PCR e o teste de Separação Imunomagnética associada a Bacteriófago. Ao aferirmos os resultados constatamos que a Separação Imunomagnética associada a Bacteriófago é equiparável ao método bacteriológico recomendado pelo Ministério da Agricultura, Pecuária e Abastecimento (MAPA) e com a técnica de PCR, pois 99,6% das amostras foram positivas ao realizarmos o teste de Separação Imunomagnética associado a Bacteriófago e apenas uma amostra de Salmonella Enteritidis foi negativa neste ensaio, na concentração de 5 UFC/25mL. Já no método bacteriológico verificamos 95,5% de positividade, com nove amostras negativas para S. Heidelberg, duas negativas para S. Enteritidis e duas no ensaio com S. Typhimurium. Na técnica de PCR obtivemos 98,5% de positivos, com uma amostra negativa para S. Enteritidis na concentração de 5 UFC/25mL e três negativas para S. Typhimurium. O tempo despendido para a realização de cada teste foi aferido e constatamos...
Abstract: We used the Immunomagnectic Separarion Assay associated with Bacteriophage to detect the following serovars: Salmonella Enteritidis, Salmonella Heidelberg and Salmonella Typhimurium, in poultry drumstick samples artificially contaminated. We compared the efficiency of this technique to the usual diagnostic tests for Salmonella in food samples: the standard bacteriological analysis, which includes the step of pre-enrichment and selective enrichment, biochemical and serological screening tests, and polymerase chain reaction (PCR). To evaluate the tests capability of Salmonella detection poultry meat samples were submitted to artificial contamination with 5, 10 and 100 CFU/25mL of bacteria, for each of the serovars listed above, totaling 270 analyzes divided into 90 tests for each of the three serovars, and so compare the results obtained with the bacteriological analysis, PCR and Immunomagnetic Separation Bacteriophage assay. We found that the Immunomagnetic Separation Bacteriophage assay was comparable to bacteriological method recommended by the Ministry of Agriculture, Livestock and Supply (MAPA) and the PCR technique, because 99.6% of the samples were positive to accomplish Immunomagnetic Separation Bacteriophage assay and only a sample of Salmonella Enteritidis was negative in this test, in the concentration of 5 CFU/25mL. The bacteriological method check 95.5% positivity, with nine samples negative for S. Heidelberg, two negative for S. Enteritidis and two in the test with S. Typhimurium. In PCR 98.5% positives samples were obtained, with one S. Enteritidis negative sample in the concentration of 5 CFU/25ml and three negative samples for S. Typhimurium. The time for performing each test was measured and the Immunomagnetic Separation Bacteriophage assay was the most rapid test for Salmonella diagnosis, since 20 hours, the conventional bacteriological took 88h and PCR 44h approximately. In this study we confirm the effectiveness of the ...
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Erhardt, Marc. "Genetic Structure and Function Analysis of the Conserved Integral Membrane Components (FliOPQR) of the Flagellar Type III Secretion Apparatus of Salmonella enterica". [S.l. : s.n.], 2006. http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-26404.
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Neves, Meiriele da Silva das 1990. "Caracterização fenotípica e molecular de linhagens atenuadas de Salmonella enterica Typhimurium = Phenotipic and molecular characterization of attenuated strains of Salmonella enterica Typhimurium". [s.n.], 2015. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316402.
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Orientador: Marcelo BrocchiDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O gênero Salmonella pertence à família Enterobacteriaceae que agrupa bacilos Gram-negativos, anaeróbios facultativos, fermentadores e geralmente flagelados. S. enterica é um dos patógenos de origem alimentar mais prevalente, sendo que infecções causadas por essa bactéria podem estar relacionadas a praticamente todos os tipos de alimentos. O trabalho foi proposto com o intuito de realizar a caracterização fenotípica e molecular de linhagens atenuadas de Salmonella enterica Typhimurium para genes codificadores de proteínas associadas ao nucleóide (NAPs Nucleoid associated Proteins). As características fenótipicas dos mutantes nulos de Salmonella enterica para os genes ihfA ou ihfB, codificadores das subunidades A e B de IHF, foram avaliadas através de crescimento in vitro, motilidade, sobrevivência frente ao estresse nutricional (sobrevivência em fase estacionária), sob condições ácidas, na presença de sais biliares e quanto à capacidade de invasão e sobrevivência em macrófagos (linhagem J774A.1). Testes de confirmação da atenuação e avaliação da capacidade de induzir proteção em caso de infecção por S. enterica foram realizados utilizando o modelo murino. Os mutantes não apresentaram diferença no crescimento in vitro e na capacidade de sobreviver na presença de sais biliares em comparação com a linhagem selvagem. As linhagens mutantes para os genes ihfA ou ihf ihf ihfB) apresentaram uma menor capacidade de sobrevivência sob condições ácidas quando comparadas com a linhagem selvagem. A motilidade dos mutantes simples também foi reduzida. Os mutantes simples e duplo apresentaram maior capacidade de sobreviver sob estresse nutricional quando comparados com a linhagem selvagem. O mutante para o gene ihfA e o duplo mutante apresentaram um aumento na capacidade de invadir macrófagos. ihf ihfB mostraram uma capacidade aumentada em sobreviver no interior de macrófagos quando comparadas com a linhagem selvagem. Os mutantes nulos viii de Salmonella enterica para os genes ihfA ou ihfB apresentam atenuação, em diferentes graus, quanto à virulência e apresentaram capacidade de induzir proteção no modelo murino de infecção por S. enterica. Esses resultados demonstram que essa proteína apresenta função relacionada com a virulência bacteriana, sendo um importante alvo de estudo na busca de linhagens atenuadas
Abstract: The genus Salmonella belongs to the Enterobacteriaceae family that comprises Gram-negative bacillus, facultative anaerobe, fermenting and generally flagellate. S. enterica is one of the most prevalent food-borne pathogen, and infections caused by this bacterium can be associated to almost all types of food. The work was proposed with the purpose of performing phenotypic and molecular characterization of attenuated strains of Salmonella enterica Typhimurium for genes encoding proteins associated with the nucleoid (NAPs - Nucleoid associated Proteins). The phenotypic characteristics of the null mutants of Salmonella enterica for genes ihfA or ihfB, encoding the A and B subunits of IHF, were evaluated by in vitro growth, motility, survival under nutritional stress (survival in the stationary phase), under acidic conditions, in the presence of bile salts and for the ability of invasion and survival in macrophages (J774A.1 strain). Attenuation tests and evaluation of the capacity to induce protection in case of infection by S. enterica were performed using the murine model. The mutants showed no difference in the in vitro growth and the ability to survive in the presence of bile salts in comparison with the wild type strain. The single mutant for ihfA or ihf ihf ihfB) showed decreased survival under acidic conditions when compared to the wild type strain. Motility of single mutants was also reduced. Single and double mutants showed higher ability to survive under nutritional stress when compared with the wild type strain. The mutant gene for ihfA and the double mutant showed an increased ability to invade ihf ihfB mutants showed an increased ability to survive within macrophages when compared with the wild type strain. Null mutants of Salmonella enterica for ihfA or ihfB genes exhibited attenuation, to varying degrees, for virulence and showed ability to induce protection in a murine model of infection by S. enterica. x These results demonstrate that this protein has function associated to bacterial virulence and is an important subject of study in search for attenuated strains
Mestrado
Genetica de Microorganismos
Mestra em Genética e Biologia Molecular
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Oliveira, Gláucia Helaine de [UNESP]. "Ensaios imunoenzimáticos (ELISA) para detecção da resposta sorológica contra Salmonella Gallinarum, Salmonella Pullorum, Salmonella Enteritidis e Salmonella Typhimurium em aves". Universidade Estadual Paulista (UNESP), 2004. http://hdl.handle.net/11449/104648.
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Foi desenvolvido um ensaio imunoenzimático do tipo ELISA indireto para a detecção de resposta sorológica de aves para Salmonella sorotipos Gallinarum, Pullorum, Enteritidis e Typhimurium. Utilizou-se antígeno solúvel obtido por meio de sonicação de cultura de Salmonella Gallinarum (AgSG), Salmonella Enteritidis cepa aflagelar (AgSE) e Salmonella Typhimuirum cepa aflagelar (AgTM), os conjugados peroxidase e fosfatase alcalina e amostras de soros positivos e negativos de vários sorotipos de salmonelas. Os resultados demonstraram que o AgSG pode ser utilizado diluído a 1:25.000 (peroxidase e fosfatase alcalina). Observou-se que o ELISA contendo S. Gallinarum como antígeno e fosfatase alcalina como enzima, propicia a separação de reações positivas para Gallinarum e Pullorum de Enteritidis. O AgSE pode ser utilizado diluído a 1:10.000 (peroxidase) ou 1:5.000 (fosfatase alcalina). Nestas condições, o ELISA/AgSE detectou resposta sorológica para os sorotipos Enteritidis, Gallinarum e Pullorum. O ELISA com o AgTM demonstrou que o antígeno pode ser diluído a 1:20.000 para ambos os conjugados. O ELISA/AgTM demonstrou reatividade entre salmonelas dos grupos B e D. Todas as amostras de soros testes devem ser analisadas diluídas a 1:1.000. Concluindo, o ELISA mostrou-se um teste útil para identificar aves com reação sorológica contra S. Gallinarum, S. Pullorum, S. Enteritidis e S. Typhimurium, podendo ainda identificar aves com sorologia positiva para S. Gallinarum, S. Pullorum sem que haja reação cruzada com amostras de soro de aves vacinadas ou infectada por S. Enteritidis.
This study was done to assess the enzyme-linked immunosorbent assays (ELISA) for detection chicken serologic response against Salmonella enterica sorotypes Gallinarum, Pullorum, Enteritidis and Typhimurium. The test was performed using soluble proteins from Salmonella Gallinarum strain 9 (AgSG), from non-flagellate Salmonella Enteritidis strain (AgSE) and from not flagellate Salmonella Typhimurium (AgTM) strain as detecting antigen and peroxidase and alkaline phosphatase enzymes, as conjugate. According to the results, the antigen has to be diluted at 1:25.000 (AgSG, peroxidase and alkaline phosphatase). In addition, using alkaline phosphatase enzyme, the assay was helpful to separate positive serological reaction to serotypes Gallinarum and Pullorum from Enteritidis. To the ELISA/AgSE, the antigen has to be diluted at 1:10.000 for peroxidase assay and at 1:5.000 for alkaline phosphatase assay. In this condition, the ELISA/AgSE can detect serological reaction to S. Enteritidis, S. Gallinarum and S. Pullorum. To the ELISA/AgTM the antigen has to be diluted at 1:20.000 to both enzymes. In this condition the ELISA/AgTM showed sensibility but was no possible to separate positive serological reaction to serotype concerning at the group B and group D. In all test, the sample of serum has to be diluted at 1:1.000. Therefore, the ELISA was able to identity reactors birds to Salmonella antigens and also to detect serological response to S. Gallinarum, S. Pullorum antigen with no cross-reaction with serum samples taken from birds either challenged or vaccinated against S. Enteritidis.
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Oliveira, Gláucia Helaine de. "Ensaios imunoenzimáticos (ELISA) para detecção da resposta sorológica contra Salmonella Gallinarum, Salmonella Pullorum, Salmonella Enteritidis e Salmonella Typhimurium em aves /". Jaboticabal : [s.n.], 2004. http://hdl.handle.net/11449/104648.
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Resumo: Foi desenvolvido um ensaio imunoenzimático do tipo ELISA indireto para a detecção de resposta sorológica de aves para Salmonella sorotipos Gallinarum, Pullorum, Enteritidis e Typhimurium. Utilizou-se antígeno solúvel obtido por meio de sonicação de cultura de Salmonella Gallinarum (AgSG), Salmonella Enteritidis cepa aflagelar (AgSE) e Salmonella Typhimuirum cepa aflagelar (AgTM), os conjugados peroxidase e fosfatase alcalina e amostras de soros positivos e negativos de vários sorotipos de salmonelas. Os resultados demonstraram que o AgSG pode ser utilizado diluído a 1:25.000 (peroxidase e fosfatase alcalina). Observou-se que o ELISA contendo S. Gallinarum como antígeno e fosfatase alcalina como enzima, propicia a separação de reações positivas para Gallinarum e Pullorum de Enteritidis. O AgSE pode ser utilizado diluído a 1:10.000 (peroxidase) ou 1:5.000 (fosfatase alcalina). Nestas condições, o ELISA/AgSE detectou resposta sorológica para os sorotipos Enteritidis, Gallinarum e Pullorum. O ELISA com o AgTM demonstrou que o antígeno pode ser diluído a 1:20.000 para ambos os conjugados. O ELISA/AgTM demonstrou reatividade entre salmonelas dos grupos B e D. Todas as amostras de soros testes devem ser analisadas diluídas a 1:1.000. Concluindo, o ELISA mostrou-se um teste útil para identificar aves com reação sorológica contra S. Gallinarum, S. Pullorum, S. Enteritidis e S. Typhimurium, podendo ainda identificar aves com sorologia positiva para S. Gallinarum, S. Pullorum sem que haja reação cruzada com amostras de soro de aves vacinadas ou infectada por S. Enteritidis.Abstract: This study was done to assess the enzyme-linked immunosorbent assays (ELISA) for detection chicken serologic response against Salmonella enterica sorotypes Gallinarum, Pullorum, Enteritidis and Typhimurium. The test was performed using soluble proteins from Salmonella Gallinarum strain 9 (AgSG), from non-flagellate Salmonella Enteritidis strain (AgSE) and from not flagellate Salmonella Typhimurium (AgTM) strain as detecting antigen and peroxidase and alkaline phosphatase enzymes, as conjugate. According to the results, the antigen has to be diluted at 1:25.000 (AgSG, peroxidase and alkaline phosphatase). In addition, using alkaline phosphatase enzyme, the assay was helpful to separate positive serological reaction to serotypes Gallinarum and Pullorum from Enteritidis. To the ELISA/AgSE, the antigen has to be diluted at 1:10.000 for peroxidase assay and at 1:5.000 for alkaline phosphatase assay. In this condition, the ELISA/AgSE can detect serological reaction to S. Enteritidis, S. Gallinarum and S. Pullorum. To the ELISA/AgTM the antigen has to be diluted at 1:20.000 to both enzymes. In this condition the ELISA/AgTM showed sensibility but was no possible to separate positive serological reaction to serotype concerning at the group B and group D. In all test, the sample of serum has to be diluted at 1:1.000. Therefore, the ELISA was able to identity reactors birds to Salmonella antigens and also to detect serological response to S. Gallinarum, S. Pullorum antigen with no cross-reaction with serum samples taken from birds either challenged or vaccinated against S. Enteritidis.
Orientador: Angelo Berchieri Júnior
Coorientador: Hélio José Montassier
Banca: Raul José Silva Girio
Banca: Fernando Antonio de Ávila
Banca: Paulo Lourenço da Silva
Banca: Ana Maria Iba Kanashiro
Doutor
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馮美玉 i Mei-yuk Ami Fung. "Isolation and characterization of a Salmonella enterica serotype typhivariant". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2001. http://hub.hku.hk/bib/B31970230.
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Hebrard, Magali. "Etude des systèmes antioxydants dans le métabolisme et la virulence de Salmonella typhimurium". Thesis, Aix-Marseille 2, 2010. http://www.theses.fr/2010AIX22022/document.
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Les Formes Actives de l’Oxygène (FAO), molécules dérivées de l’oxygène, sont capables d’oxyder et d’endommager les macromolécules biologiques. Au cours de son cycle de vie, Salmonella typhimurium est exposée à des FAO provenant de deux sources : soit de son métabolisme aérobie, soit du macrophage, sa cellule hôte au cours de l’infection. Parmi les FAO existantes, l’H2O2 est l’une des plus néfastes. Au cours de ma thèse, j’ai étudié la contribution des catalases et des peroxyrédoxines dans le métabolisme et la virulence de S.typhimurium. Cinq enzymes ont ainsi été identifiées pour leur capacité à éliminer l’H2O2 : les catalases KatG, KatE, KatN et les peroxyrédoxines AhpCF et TsaA. Des tests de virulence ont également permis de montrer que ces enzymes participaient à l’établissement de la virulence.A l’aide d’une sonde moléculaire capable de détecter et de signaler l’H2O2, nous avons montré que S. typhimurium percevait cette FAO au cours de l’infection dans des macrophages murins. Ces résultats ont souligné l’importance des catalases et des peroxyrédoxines au cours de la vie intracellulaire de S. typhimurium. L’analyse du mutant DahpCF DtsaA Dtpx a également révélé que les peroxyrédoxines AhpCF, TsaA et Tpx contribuaient à la capacité de prolifération de la bactérie dans le macrophage. Enfin, l’étude des méthionine sulfoxyderéductases a montré que les caractéristiques d’un mutant DmsrA DmsrB étaient proches de celles de la souche sauvage. Les gènes msrA et msrB ont également été inactivés dans une souche dépourvue de katG, katE et ahpCF. Dans cette souche accumulant de l’H2O2endogène, la contribution de MsrA et MsrB devient évidente pour lutter contre les effets liés au stress oxydant. L’ensemble de ces travaux a permis d’identifier et de caractériser l’implication de systèmes antioxydants dans la virulence et le métabolisme de S. typhimuriumReactive Oxygen Species (ROS), produced from molecular oxygen, can oxidize and damagebiological macromolecules. During its lifestyle, Salmonella typhimurium is submitted to ROScoming from two sources: its aerobic metabolism and its host cell upon infection, themacrophage. Among the ROS, H2O2 is one of the most toxic. In this work, the contribution ofcatalases and peroxiredoxins in the metabolism and the virulence of S. typhimurium wasstudied. Five enzymes are implied in H2O2 degradation, the catalases KatG, KatE, KatN andthe peroxiredoxins, AhpCF and TsaA. Virulence tests showed that these enzymes wereinvolved in virulence. Using a molecular probe able to detect and quantify H2O2, we showedthat S. typhimurium sensed H2O2 during infection in murine macrophages. These resultsunderlined the importance of catalases and peroxoxyredoxines for the intracellular life of S.typhimurium. Analysis of the mutant DahpCF DtsaA Dtpx revealed that the peroxiredoxinsAhpCF, TsaA and Tpx contributed to the bacterial proliferation inside macrophage. Finally,the study of the methionine sulfoxyde reductases showed that the phenotype of the mutantDmsrA DmsrB was related to the wild type strain. Then, msrA and msrB were inactivated in astrain deleted of katG, katE and ahpCF. In this strain impaired in H2O2 degradation, thecontribution of MsrA and MsrB to fight against oxidative stress effect is stronger. Altogether,these results allowed the identification and the contribution of antioxidant systems in S.typhimurium virulence and metabolism
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Simmons, James Walter. "O-Acetylserine Sulhydralase-A from Salmonella typhimurium LT-2: Thermodynamic Properties and SPectral Identification of Intermediates". Thesis, University of North Texas, 1993. https://digital.library.unt.edu/ark:/67531/metadc278042/.
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O-Acetylserine Sulfhydrylase (OASS) is a pyridoxal phosphate enzyme that catalyzes the reaction of O-acetyl-Lserine with sulfide to give L-cysteine. OASS is present as two isoforms, designated -A and -B. The kinetic mechanism of OASS-A is well known and there is also much known concerning the acid-base chemistry of the enzyme. However, little is known concerning the location of the rate determining steps, the sequencing of chemical steps that occur at the active site, or the nature of the rate determining transition states. The studies performed to help elucidate these aspects of the OASS-A mechanism included determination of the thermodynamics of both half reactions, along with studies utilizing substrate analogs of OAS halting the reaction at specific points along the reaction pathway allowing the identification of reaction intermediates. The free energy change of the first half reaction was shown to be -5.7 Kcal/mole while the second half reaction was shown to be, for all intents and purposes, irreversible. Intermediates along the reaction pathway that have been previously identified include the internal Schiff base and the a-aminoacrylate. The external Schiff base was identified using the analogs cysteine, alanine, and glycine while the geminal diamine was identified using the analog serine. Formation of the external aldimine was shown to be pH dependent with a pK of 8.1 ± 0.3 most likely representing a general base that accepts a proton from the a-amine of cysteine to facilitate a nucleophilic attack on C4r of the PLP imine. Formation of the geminal diamine was also shown to be pH dependent with two pK values having an average value of 8.1. One of the groups most likely represents the general base which accepts a proton from the a-amine of cysteine while the second group likely interacts with the amino acid side chain to orientate the amino acid into the correct configuration.
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Main-Hester, Kara L. "Counter-silencing of laterally acquired genes, including Salmonella Pathogenicity Island 4, by three DNA binding proteins, HilA, HilD, and SlyA /". Thesis, Connect to this title online; UW restricted, 2008. http://hdl.handle.net/1773/11498.
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Pattni, Krupa. "Subcellular localisation of rat inositol 1,4,5 trisphosphate 3 kinase B and phosphatidylinositol (3) phosphate in living cells". Thesis, University of Bristol, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274843.
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Rosche, Kristin. "Infection with Salmonella typhimurium defaces the splenic tissue architecture and alters the proportion and distribution of cells". OpenSIUC, 2015. https://opensiuc.lib.siu.edu/theses/1686.
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Salmonella enterica serovar Typhimurium (S. typhimurium) is a gram-negative bacteria capable of infecting a variety of warm-blooded vertebrate hosts. In humans, consumption of S. typhimurium through contaminated food or water typically leads to an acute but self-limiting gastroenteritis and oftentimes an enlargement of the spleen (splenomegaly). Splenomegaly has been attributed to the expansion of phagocytes, B and T lymphocytes, and immature CD71+Ter119+ red blood cells (RBCs). The spleen is an important organ with distinct roles in RBC recycling, the capture of blood-borne pathogens, and as a site of initiation of the adaptive immune response. The spleen has a characteristic tissue architecture composed of three compartments. The white pulp (WP), largely populated by B and T lymphocytes is surrounded by the red pulp (RP), which primarily contains F4/80+ macrophages. The border between the WP and RP, the marginal zone (MZ), is populated by MOMA+ and MARCO+ macrophages which are important for the capture of blood-borne pathogens. This precise organization of the spleen allows for efficient antigen capture and activation of adaptive immunity, due to the close proximity of antigen presenting cells and lymphocytes. It is known that Salmonella spp. infections delay the adaptive immune response in comparison to other bacteria, such as Listeria monocytogenes. Therefore, we investigated the effect of an attenuated S. typhimurium strain (÷9088) on in situ splenic organization. We utilized four-color immunofluorescence microscopy (IFM) in combination with flow cytometry to characterize the in situ changes of spleen architecture and cell population profiles during S. typhimurium infection in mice. Within the first week of infection, splenomegaly is evident and after three weeks of infection, the spleen comprises over 5% of the mouse's total body weight. During this time S. typhimurium has not been cleared from the spleen and mice are anemic with decreased pack cell volume. We confirmed previous studies that reported extramedullary erythropoiesis was a major cause of splenomegaly. However, we also show that RP F4/80+ macrophages significantly expand and take over the WP regions of the spleen, increasingly co-localizing with immature (CD71+Ter119+) and mature (CD71-Ter119+) RBC subsets. As a result of these dramatic changes in cell proportions and their in situ distribution, the splenic architecture becomes unrecognizable. The boundary between WP and RP is lost as proportions of MOMA+ macrophages of the MZ are reduced following infection. Likewise, B and T cell zones of the WP are also drastically reduced, most likely due to their increased co-localization with F4/80+ macrophages. As a result of infection, the increased cellularity of splenomegaly results in changing proportions of cell populations, potentially disrupting the link between infection and immune response. Together, these data provide further insight into the disease process of S. typhimurium infection in mice. Understanding how the changes in splenic architecture affect the adaptive immune response has implications for the design of more effective Salmonella-based vaccines and therapies.