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Dynamic Sulfur Chemistry : Screening, Evaluation and CatalysisCaraballo, Rémi January 2010 (has links)
This thesis deals with the design, formation and evaluation of dynamic systems constructed by means of sulfur-containing reversible reactions, in organic and aqueous media and under mild conditions. In a first part, the synthesis of thioglycoside derivatives, constituting the biologically relevant starting components of the dynamic systems, is described. In addition, the pD-profile of the mutarotation process in aqueous media for a series of 1-thioaldoses is reported and revealed an astonishing beta-anomeric preference for all the carbohydrate analogs under acidic or neutral conditions. In a second part, the phosphine-catalyzed or -mediated disulfide metathesis for dynamic system generation in organic or aqueous media is presented, respectively. The direct in situ 1H STD-NMR resolution of a dynamic carbohydrate system in the presence of a target protein (Concanavalin A) proved the suitability and compatibility of such disulfide metathesis protocols for the discovery of biologically relevant ligands. In a third part, hemithioacetal formation is demonstrated as a new and efficient reversible reaction for the spontaneous generation of a dynamic system, despite a virtual character of the component associations in basic aqueous media. The direct in situ 1H STD-NMR identification of the best dynamic beta-galactosidase inhibitors from the dynamic HTA system was performed and the results were confirmed by inhibition studies. Thus, the HTA product formed from the reaction between 1-thiogalactopyranose and a pyridine carboxaldehyde derivative provided the best dynamic inhibitor. In a fourth and final part, a dynamic drug design strategy, where the best inhibitors from the aforementioned dynamic HTA system were used as model for the design of non-dynamic (or “static”) beta-galactosidase inhibitors, is depicted. Inhibition studies disclosed potent leads among the set of ligands. / QC 20100621
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Biochemical Characterization of β-galactosidase from Enterobacter sp. YSUMotari, Fred Ondabu 02 May 2023 (has links)
No description available.
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解糖系酵素の構造と機能山根, 隆, 飯島, 信司, 佐藤, 能雅, 田中, 勲, 畑, 安雄, 濱田, 賢作, 原田, 繁春, 樋口, 芳樹, 福山, 恵一, 松浦, 良樹, 松本, 治, 森本, 幸生, 森山, 英明 03 1900 (has links)
科学研究費補助金 研究種目:総合研究(A) 課題番号:03303014 研究代表者:山根 隆 研究期間:1991-1992年度
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Development of a method to detect lysis and investigation if ozone has a lysing effect on Escherichia coli / Utveckling av en metod för att detektera lysering och undersökning om ozon har lyserande effekt på Escherichia coliAndrup, Klara January 2023 (has links)
Detta projekt genomfördes på företaget Sangair som för närvarande utvecklar medicinsk utrustning för att behandla bakteriemi med ozon. Bakteriemi uppstår när bakterier hamnar i blodomloppet, vilket kan trigga sepsis och septisk chock, med potentiellt dödligt utfall om obehandlat. För närvarande används antibiotika för att behandla bakteriemi, men det ökande hotet med antibiotikaresistens världen över innebär att forskare behöver hitta nya vägar för att behandla bakteriemi. Som en del av Sangairs utveckling gjordes en undersökning för att se hur bakterier påverkas av ozon. Projektet syftade till att 1) utveckla en metod för att detektera lysering av bakterier och 2) använda metoden för att se om ozon hade lyserande effekt på model-organismen Escherichia coli. För att testa metoden transformerades stam BL21 av E. coli med en pUC-19 plasmid för att producera beta-galaktosidas. Proteinets aktivitet mättes sedan i sonikerade bakterier, vilket visade att det var en effekitiv metod för att lysera bakterier och sedan mäta aktiviteten. För att undersöka om ozon hade en lyserande effekt, och samtidigt bestämma om man kunde mäta protein fann man att det optimala experimentella upplägget var att använde en ozonkoncentration på 6 g/m3, ett gasflöde på 5 ml/min och ett vätskeflöde på 25 ml/min. Resultatet från studien indikerade att ozon har en lyserande effekt, men fler studier behövs göras för att verifiera resultatet. Man undersökte också om detektionen av Beta-galaktosidas kunde förbättras genom att tillsätta Bovin serumalbumin (BSA) för att inhibera resterande ozon i prover som annars skulle kunna inaktivera Beta-galaktosidas. Resultaten indikerade dock inte någon effekt. Som ett sista experiment mättes även endotoxiner som frigjordes vid behandling, och det visade sig att när bakterier behandlas med ozon frigörs lipopolysackarider (LPS) och peptidoglykaner, vilket också kan tyda på lysis. / This project was conducted at Sangair. The company is currently developing a medical device aimed at treating bacteremia with ozone. Bacteremia is a condition that occurs when bacteria enters the bloodstream, and can trigger sepsis and septic shock, potentially leading to death if untreated. Antibiotics have traditionally been the way to treat bacteremia, but the looming threat of antibiotic resistance worldwide threatens this way of treatment, and research into novel approaches to eradicate the bacteria needs to be done. As part of Sangairs development, an investigation was done to see how ozone interacts with bacteria. The project aimed to 1) develop a method to detect bacterial lysis and 2) use the method to detect if ozone had a lysing effect on the bacterial model organism Escherechia coli. To test the method, the BL21 strain of E. coli was transformed with the pUC-19 plasmid to produce the reporter enzyme Beta-galactosidase. The Beta-galactosidase activity was then measured in a supernatant of sonicated bacteria, which confirmed the suitability of the method to detect bacteria cell lysis. To be able to see if ozone had a lysing effect, while still being able to measure Beta-galactosidase, it was found that the optimal setup for this was using an ozone concentration of 6 g/m3, a gas flow of 5 ml/min and a liquid flow of 25 ml/min. The results acquired with this setup indicated that ozone had a lysing effect on E. coli but more studies are needed to verify this. It was further investigated if Beta-galactosidase detection could be improved by addition of bovine serum albumin (BSA) to quench residual ozone in the samples, but the results showed that it did not have any effect on the Beta-galactosidase enzymatic activity. As a final experiment, endotoxins that were released upon treatment were also measured, and it was found that when bacteria are treated with ozone, lipopolysaccharides (LPS) and peptidoglycans are released, further confirming lysis of the bacterial cells.
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Structural and Functional Characterization of O-Antigen Translocation and Polymerization in Pseudomonas aeruginosa PAO1Islam, Salim Timo 07 June 2013 (has links)
Heteropolymeric O antigen (O-Ag)-capped lipopolysaccharide is the principal constituent of the Gram-negative bacterial cell surface. It is assembled via the integral inner membrane (IM) Wzx/Wzy-dependent pathway. In Pseudomonas aeruginosa, Wzx translocates lipid-linked anionic O-Ag subunits from the cytoplasmic to the periplasmic leaflets of the IM, where Wzy polymerizes the subunits to lengths regulated by Wzz1/2. The Wzx and Wzy IM topologies were mapped using random C-terminal-truncation fusions to PhoALacZα, which displays PhoA/LacZ activity dependent upon its subcellular localization. Twelve transmembrane segments (TMS) containing charged residues were identified for Wzx. Fourteen TMS, two sizeable cytoplasmic loops (CL), and two large periplasmic loops (PL3 and PL5 of comparable size) were characterized for Wzy.
Despite Wzy PL3–PL5 sequence homology, these loops were distinguished by respective cationic and anionic charge properties. Site-directed mutagenesis identified functionally-essential Arg residues in both loops. These results led to the proposition of a “catch-and-release” mechanism for Wzy function. The abovementioned Arg residues and intra-Wzy PL3–PL5 sequence homology were conserved among phylogenetically diverse Wzy homologues, indicating widespread potential for the proposed mechanism. Unexpectedly, Wzy CL6 mutations disrupted Wzz1-mediated regulation of shorter O-Ag chains, providing the first evidence for direct Wzy–Wzz interaction.
Mutagenesis studies identified functionally-important charged and aromatic TMS residues localized to either the interior vestibule or TMS bundles in a 3D homology model constructed for Wzx. Substrate-binding or energy-coupling roles were proposed for these residues, respectively. The Wzx interior was found to be cationic, consistent with translocation of anionic O-Ag subunits. To test these hypotheses, Wzx was overexpressed, purified, and reconstituted in proteoliposomes loaded with I−. Common transport coupling ions were introduced to “open” the protein and allow detection of I− flux via reconstituted Wzx. Extraliposomal changes in H+ induced I− flux, while Na+ addition had no effect, suggesting H+-dependent Wzx gating. Putative energy-coupling residue mutants demonstrated defective H+-dependent halide flux. Wzx also mediated H+ uptake as detected through fluorescence shifts from proteoliposomes loaded with pH-sensitive dye. Consequently, Wzx was proposed to function via H+-coupled antiport. In summary, this research has contributed structural and functional knowledge leading to novel mechanistic understandings for O-Ag biosynthesis in bacteria. / Bookmarks within the document have been provided for ease of access to a particular section in the body of the thesis. Each entry in the Table of Contents, List of Tables, and List of Figures has been "linked" to its respective position and as such can be clicked for direct access to the entry. Similarly, each in-text Figure or Table reference has been "linked" to its respective figure/table for direct access to the entry. / 1.) Canadian Institutes of Health Research (CIHR) Frederick Banting and Charles Best Canada Graduate Scholarship doctoral award, 2.) CIHR Michael Smith Foreign Study Award, 3.) Cystic Fibrosis Canada (CFC) doctoral studentship, 4.) University of Guelph Dean's Tri-Council Scholarship, 5.) Ontario Graduate Scholarship in Science and Technology, 6.) Operating grants to Dr. Joseph S. Lam from CIHR (MOP-14687) and CFC
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