by Chin Miu Ling, Nathalie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 130-143). / ABSTRACT --- p.i / ACKNOWLEDGEMENTS --- p.iii / LIST OF TABLES --- p.vii / LIST OF FIGURES --- p.x / INTRODUCTION --- p.1 / Chapter A --- Aminoglycosides --- p.1 / Chapter 1 --- Structure --- p.1 / Chapter 2 --- Classification --- p.1 / Chapter 3 --- Mode of action --- p.2 / Chapter 4 --- Types --- p.9 / Chapter B --- Mechanisms of aminoglycoside-resistance --- p.13 / Chapter C --- Aminoglycoside-modifying enzymes --- p.14 / Chapter 1 --- Classification --- p.19 / Chapter i --- Phosphotransferases --- p.19 / Chapter ii --- Adenylytransferases --- p.21 / Chapter iii --- Acetyltransferases --- p.22 / Chapter 2 --- Genes encoding AMEs --- p.24 / Chapter 3 --- Applications --- p.27 / Chapter D --- Prevalence of aminoglycoside-resistance --- p.28 / Chapter E --- Methods for the determination of aminoglycoside-modifying enzymes --- p.34 / Chapter 1 --- Examination of resistance phenotype --- p.35 / Chapter 2 --- Phosphocellulose paper binding assay --- p.38 / Chapter 3 --- Hybridization with specific gene probes --- p.39 / Chapter 4 --- Antibiotic inactivation --- p.44 / Chapter 5 --- High performance liquid chromatography (HPLC) --- p.44 / Chapter F --- Prevalence of aminoglycoside-modifying enzymes --- p.45 / Chapter G --- Objectives --- p.52 / MATERIALS AND METHODS --- p.53 / Materials --- p.53 / Chapter A --- Bacterial strains --- p.53 / Chapter 1 --- Standard strains --- p.53 / Chapter 2 --- Clinical isolates --- p.53 / Chapter B --- "Antibiotic, media, chemicals and instruments" --- p.55 / Methods --- p.55 / Chapter A --- Orevalence of aminoglycoside-resistance --- p.55 / Chapter B --- Susceptibility testing --- p.55 / Chapter C --- Characterization of aminoglycoside-modifying enzymes (AMEs) --- p.61 / Chapter 1 --- Extraction of enzymes --- p.61 / Chapter 2 --- Substrate profile analysis by the phosphocellulose paper binding assay --- p.62 / Chapter D --- Localization of resistance genes --- p.64 / Chapter 1 --- Genetic study --- p.64 / Chapter 2 --- Molecular studies --- p.67 / Chapter i --- Preparation of crude plasmid extracts --- p.68 / Chapter ii --- Agarose gel electrophoresis --- p.68 / Chapter E --- Plasmid profile analysis --- p.69 / Chapter F --- Plasmid fingerprinting --- p.69 / Chapter 1 --- Preparation of purified plasmid --- p.69 / Chapter 2 --- Restriction endonuclease digestion of plasmid DNA --- p.70 / Plan to achieve objectives --- p.71 / results --- p.73 / Chapter A --- Prevalence of aminoglycoside-resistant Gram-negative bacteria isolated in the Prince of Wales Hospital from 1989 to1992 --- p.73 / Chapter B --- "Susceptibility to 12 aminoglycosides of aminoglycoside-resistant E. coli, K pneumoniae and Ps. aeruginosa" --- p.78 / Chapter C --- "Aminoglycoside-modifying enzymes (AMEs) produced by E. coli, K pneumoniae and Ps. aeruginosa" --- p.88 / Chapter D --- Plasmid profile analysis --- p.93 / Chapter E --- Localization of aminoglycoside-resistance genes --- p.102 / discussion --- p.114 / Chapter A --- Aminoglycoside-resistance --- p.114 / Chapter B --- Mechanisms of aminoglycoside-resistance --- p.118 / Chapter C --- Genetic location of aminoglycoside-resistance and plasmid profiles --- p.122 / Chapter D --- Characterization of AMEs --- p.126 / Chapter E --- Areas for future research --- p.128 / references --- p.130 / appendix --- p.144

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Phillip E. Klebba / Siderophore uptake systems are one the most prominent methods of Fe³+-iron acquisition in Gram negative bacteria. The catecholate siderophore enterobactin is synthesized and utilized by many members of Enterobacteriaceae as well as several of the ESKAPE pathogens. The outer membrane (OM) transporter of ferric enterobactin (FeEnt), FepA is a ligand-gated porin (LGP) that requires interaction with the inner membrane (IM) protein TonB in order to accomplish active transport. TonB is thought to transduce the electrochemical energy created by the proton gradient across the IM to LGPs like FepA in the OM, to promote siderophore transport through their occluded channels. However, we do not yet have a clear picture of either how TonB transfers energy to FepA, or what kind of conformational changes occur in the occluding domain of FepA to allow ligand passage. The experiments described herein investigate these two questions, building on previously outlined models and observations. Using fluorescence labeling of strategically substituted cysteines in the surface loops of FepA, we unraveled a hierarchy of loop motion during binding of FeEnt to FepA. Additionally, by rendering parts of the FepA protein immobile as a result of engineered disulfide bonds, I identified residues or regions within its occluding domain that may normally unfold to open a size-specific channel for FeEnt. I also elucidated the role of the peptidoglycan polymer beneath the OM a framework for protein-protein interactions between IM and OM proteins. This includes the proposed interaction between a rotating TonB and FepA, or other LGPs, that may transfer kinetic energy to the OM transporter. The role of iron in microbial survival and pathogenesis makes iron-uptake pathways an attractive target for therapeutic intervention. Using the FeEnt-FepA uptake system as a model, we used a fluorescence based high-throughput screening method to identify novel small molecule inhibitors of TonB action in E. coli. The approach used can be potentially adopted to screen bigger chemical libraries as well as used to find inhibitors of ESKAPE pathogens that use FeEnt such as, Acinetobacter baumannii, Klebsiella pneumoniae or Pseudomonas aeruginosa. Finally, we discoverd a TonB-dependent OM transporter of heme/hemoglobin called HutA in the oligotrophic bacterium Caulobacter crescentus.

Chan, Kwok Ho. / Thesis submitted in: November 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 124-129). / Abstracts in English and Chinese. / Statement --- p.I / Acknowledgements --- p.II / Abstract --- p.III / 摘要 --- p.IV / Table of Contents --- p.V / Abbreviations --- p.XIII / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- GTPase in general --- p.1 / Chapter 1.2 --- G proteins and GTP switch --- p.2 / Chapter 1.3 --- Structural similarities in GTPase --- p.3 / Chapter 1.4 --- G proteins in bacteria --- p.3 / Chapter 1.5 --- Background information of the protein family EngB --- p.4 / Chapter 1.6 --- Basic information of EngB in Thermotoga maritima --- p.5 / Chapter 1.7 --- Objectives of this work --- p.6 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Chemical reagents --- p.8 / Chapter 2.1.2 --- Buffers / Chapter 2.1.2.1 --- Preparation of buffers --- p.10 / Chapter 2.1.2.2 --- Buffers for common use --- p.11 / Chapter 2.1.3 --- Expression strains and plasmids --- p.14 / Chapter 2.1.4 --- Primer list --- p.14 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Preparation of competent cells --- p.15 / Chapter 2.2.2 --- Cloning / Chapter 2.2.2.1 --- Cloning of target genes by PCR --- p.15 / Chapter 2.2.2.2 --- Agrose gel electrophoresis --- p.17 / Chapter 2.2.2.3 --- Extraction and purification of DNA from agarose gel --- p.17 / Chapter 2.2.2.4 --- Restriction digestion of DNA --- p.18 / Chapter 2.2.2.5 --- Ligation of digested insert and expression vector --- p.18 / Chapter 2.2.2.6 --- Transformation and plating out transformants for miniprep --- p.19 / Chapter 2.2.2.7 --- Verification of insert by PCR --- p.20 / Chapter 2.2.2.8 --- Mini-preparation of plasmid DNA --- p.21 / Chapter 2.2.2.9 --- Confirmation of miniprep product by restriction enzyme digestion..… --- p.22 / Chapter 2.2.2.10 --- Sequencing of the plasmid DNA --- p.23 / Chapter 2.2.3 --- Expression of the recombinant MBP-TM EngB protein and SBP-CBP EC EngB / Chapter 2.2.3.1 --- Transformation for protein expression --- p.23 / Chapter 2.2.3.2 --- Preparation of starter culture --- p.24 / Chapter 2.2.3.3 --- Expression of recombinant protein --- p.24 / Chapter 2.2.3.4 --- Cell harvesting --- p.24 / Chapter 2.2.3.5 --- Releasing the cell content --- p.25 / Chapter 2.2.3.6 --- Check for protein expression by SDS-PAGE --- p.25 / Chapter 2.2.4 --- Purification of TM EngB / Chapter 2.2.4.1 --- SP ion-exchange chromatography --- p.27 / Chapter 2.2.4.2 --- Thrombin digestion to remove MBP tag --- p.28 / Chapter 2.2.4.3 --- Heparin affinity chromatography --- p.29 / Chapter 2.2.4.4 --- Gel filtration chromatography --- p.29 / Chapter 2.2.5 --- Purification of SBP-CBP EC EngB / Chapter 2.2.5.1 --- SP ion-exchange chromatography --- p.30 / Chapter 2.2.5.2 --- Gel filtration chromatography --- p.31 / Chapter 2.2.6 --- Protein concentration quantitation --- p.32 / Chapter 2.2.7 --- Crystallography of TM EngB / Chapter 2.2.7.1 --- Crystallization preparation --- p.32 / Chapter 2.2.7.2 --- Crystallization screening by sitting drop method --- p.32 / Chapter 2.2.7.3 --- Optimization of crystallization conditions --- p.33 / Chapter 2.2.7.4 --- X-ray diffraction --- p.33 / Chapter 2.2.8 --- Thermodynamics studies of proteins / Chapter 2.2.8.1 --- Preparation of protein sample --- p.34 / Chapter 2.2.8.2 --- Guanidine-induced denaturation experiment --- p.34 / Chapter 2.2.8.3 --- Thermal-induced denaturation experiment --- p.35 / Chapter 2.2.9 --- Binding assay to study affinity for ligands --- p.36 / Chapter 2.2.9.1 --- Using GDP analogue mant-GDP to detect formation of enzyme-ligand complex (TM EngB-mant-GDP) --- p.36 / Chapter 2.2.9.2 --- Basic information of Fluorescence spectroscopy --- p.36 / Chapter 2.2.9.3 --- Determination of λem and λex --- p.37 / Chapter 2.2.9.4 --- Studying ligand affinity by titration with ligand analogue --- p.37 / Chapter 2.2.10 --- Pull down experiment to study interacting partner of E. coli EngB --- p.38 / Chapter 2.2.10.1 --- Preparing protein extracts from E. coli --- p.38 / Chapter 2.2.10.2 --- Preparing streptavidin resin --- p.39 / Chapter 2.2.10.3 --- Binding of dual-tagged E. coli EngB to streptavidin resin --- p.39 / Chapter 2.2.10.4 --- Purifying protein using the prepared streptavidin resin --- p.40 / Chapter 2.2.10.5 --- Preparing calmodulin resin --- p.41 / Chapter 2.2.10.6 --- Binding of dual-tagged E.coli EngB to calmodulin resin --- p.41 / Chapter 2.2.10.7 --- Analysis of dual-tag affinity purified protein --- p.42 / Chapter 2.2.11 --- Silver staining of acrylamide gel / Chapter 2.2.11.1 --- Staining reagents --- p.42 / Chapter 2.2.11.2 --- Staining procedures --- p.43 / Chapter Chapter 3 --- Structure determination of T. maritima EngB by X-ray crystallography / Chapter 3.1 --- Introduction --- p.45 / Chapter 3.2 --- Generation of TM EngB expression construct --- p.45 / Chapter 3.3 --- Expression and purification of TM EngB --- p.46 / Chapter 3.4 --- TM EngB was crystallized with freshly purified TM EngB --- p.47 / Chapter 3.5 --- Data processing of diffraction data and structure refinement of TM EngB …… --- p.48 / Chapter 3.6 --- Apo-form TM EngB was obtained by unfolding and refolding --- p.49 / Chapter 3.7 --- Crystallization of apo-form TM EngB --- p.50 / Chapter 3.8 --- Data processing of diffraction data and structure refinement of apo-form TM EngB --- p.51 / Chapter 3.9 --- Producing EngB-GDP complex crystal from apo-from EngB --- p.52 / Chapter 3.10 --- TM EngB is a monomer in solution --- p.54 / Chapter 3.11 --- Summary of chapter three --- p.55 / Tables and figures of chapter three --- p.57 / Chapter Chapter 4 --- Structural details of TM EngB / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2 --- Overall fold of TM EngB --- p.67 / Chapter 4.3 --- Mode of nucleotide binding of TM EngB --- p.68 / Chapter 4.4 --- Structural differences in switch I region between chain A and chain B in crystal structure of TM EngB/GDP complex --- p.70 / Chapter 4.5 --- Structural difference between TM EngB/GDP complex and apo TM EngB --- p.73 / Chapter 4.6 --- Summary of chapter four --- p.73 / Tables and figures of chapter four --- p.76 / Chapter Chapter 5 --- Purified TM EngB is Active for binding guanine nucleotide but inactive for GTPase hydrolysis activity / Chapter 5.1 --- Introduction --- p.88 / Chapter 5.2 --- Studying ligand affinity by competitive binding experiment --- p.88 / Chapter 5.3 --- GDP binds to TMEngB with higher affinity than GTPyS --- p.91 / Chapter 5.4 --- TM EngB showed very low intrinsic GTPase activity --- p.92 / Chapter 5.5 --- Discussion --- p.93 / Tables and figures of chapter five --- p.95 / Chapter Chapter 6 --- Thermostability of EngB of T. maritima / Chapter 6.1 --- Introduction --- p.98 / Chapter 6.2 --- Guanidine hydrochloride - induced unfolding --- p.98 / Chapter 6.3 --- Thermal-induced unfolding --- p.99 / Chapter 6.4 --- Structural comparison of thermophilic and mesophilic EngB --- p.100 / Chapter 6.5 --- Discussion --- p.102 / Tables and figures of chapter six --- p.105 / Chapter Chapter 7 --- Construction of a dual-tag affinity pull-down system for finding interacting partner of EngB / Chapter 7.1 --- Introduction --- p.112 / Chapter 7.2 --- Preparation of dual-tagged E.coli EngB / Chapter 7.2.1 --- Cloning of SBP-CBP-EC EngB expression construct --- p.113 / Chapter 7.2.2 --- Expression and purification of SBP-CBP-EC EngB --- p.114 / Chapter 7.3 --- Pull down using dual tagged E.coli EngB as bait to isolate potential interacting partners of EngB --- p.114 / Chapter 7.4 --- Discussion --- p.115 / Tables and figures of chapter seven --- p.117 / Chapter Chapter 8 --- Conclusion --- p.122 / References --- p.124

I give an overview of the basic theory of Hilbert spaces necessary to understand the convergence of the Fourier series for square integrable functions. I state the necessary theorems and definitions to understand the formulations of the problem in a Hilbert space framework, and then I give some applications of the theory along the way.

The aim of this study was to examine whether Gram-positive cocci isolated from processed poultry in Western Australia provided a potential risk for the transfer of antimicrobial-resistant organisms to humans via commercially prepared ready-to-eat chicken. Research in this study was conducted in three phases: the characterisation of Gram-positive cocci isolated from poultry, an assessment of the isolates' thermal tolerance and the development of a Hazard Analysis Critical Control Points (HACCP) based food-safety program. In the first phase of the study, three specific objectives were investigated. The first determined the presence of Gram-positive cocci on poultry and on processing equipment from poultry-processing plants. The findings confirm the presence of staphylococci and enterococci on incoming live and slaughtered birds and processed carcasses. The data also indicate that carcasses probably become cross-contaminated during processing, when these bacteria are present on the incoming live birds and equipment. The second objective was to characterise staphylococcal isolates by antimicrobial susceptibility testing, and chromosomal and plasmid DNA analysis. The susceptibility of isolates to antimicrobial agents was tested by the disk diffusion method according to the NCCLS (National Committee for Clinical Laboratory Standards) guidelines. Isolates were typed by contour-clamped homogeneous electric field (CHEF) gel electrophoresis of SmaI digested chromosomal DNA, and plasmids were isolated by the cetyltrimethylammonium bromide (CTAB) method. Approximately 37% of Staphylococcus aureus and 16% of coagulase-negative staphylococcal (CNS) isolates were resistant to six or more of the antimicrobial agents tested. Many isolates exhibited resistance to antibiotics that are commonly used in human medicine and registered for veterinary use in Australia. / Among the S. aureus isolates there were twenty-four epidemiologically unrelated SmaI CHEF groups. All staphylococcal isolates, except three CNS, were found to harbour from one to seven plasmids. Some staphylococcal isolates with epidemiologically related CHEF patterns had similar plasmid profiles and resistance patterns. The third objective was to determine the antimicrobial susceptibility of enterococci isolates to the glycopeptide antibiotics. The isolation of two vancomycin-resistant E. faecalis isolates is the first report of VRE outside the health-care setting in Western Australia. Additionally the detection of the vanA gene in an E. gallinarum isolate, a motile enterococcus, has potentially important implications for infection control practices in hospitals. In the second phase of the study, three specific objectives were established to investigate the practical implications of these findings for the chicken industry. The first objective of this phase of the study was to determine the thermal tolerance (D and Z-values) of antimicrobial-resistant, Gram-positive cocci in ground chicken meat. The results indicate that these isolates do not exhibit enhanced thermal-resistance characteristics compared to antimicrobial-susceptible bacteria. The second objective established the internal time-temperature profiles for cooking commercially prepared chicken and estimated the process lethality (F-values). / From three cooking trials, it was confirmed that the internal temperature of at least 70°C was achieved for at least thirty-eight minutes. The third objective of this phase assessed the effectiveness of the thermal process in reducing the risk of the transfer of antimicrobial-resistant cocci via the food chain. The data confirm that the lethal effect (F-values) of the thermal process destroyed these antimicrobial-resistant cocci in commercially prepared ready-to-eat chicken. In the third phase of the study, the data obtained in the earlier parts of the study was incorporated into a model food-safety program for a fast-food chicken chain. The model was based upon the internationally accepted HACCP system, adopted by the Codex Alimentarius Commission. Mindful that the thermal-process step represents only one critical control point in the safe preparation of chicken, this preventative approach ensures that all hazards are controlled at every other step of the process. The data suggest that antimicrobial-resistant, Gram-positive cocci will be present on some ready-to-cook poultry meat processed in Western Australia. This creates opportunities for the potential spread of resistant strains or resistance genes to humans via the food chain. The information from this study will be useful in providing background data and direction for future planning in preventing antimicrobial-resistant bacteria from poultry meat being transmitted through the food chain. The full implementation of the HACCP program would offer substantial benefits and protection to consumers.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 170-185).

No description available.

Thèse d'exercice : Pharmacie : Nantes : 2003. Mémoire DES : Biologie médicale : Nantes : 2003. / Thèse : 2003NANT026P. Bibliogr. f. 105-115 [129 réf.].

Thèse de doctorat : Sciences de la vie et de la santé : Lille 1 : 2002. / N° d'ordre (Lille) : 3129. Résumé en français et en anglais. Bibliogr. p. 179-200.

Thèse de doctorat : Sciences de la vie et de la santé : Lille 1 : 2000. / Résumé en français et en anglais. Textes en français et en anglais (publications). Bibliogr.