by Cheung, Man Wai William. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 113-123). / ABSTRACT --- p.i / ACKNOWLEDGMENTS --- p.ii / DEDICATION --- p.iii / TABLE OF CONTENTS --- p.iv / LIST OF FIGURES --- p.viii / LIST OF TABLES --- p.x / Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- General Introduction --- p.1 / Chapter 1.2. --- Purpose of Study --- p.3 / Chapter 2. --- Literature Review --- p.7 / Chapter 2.1. --- Central Pathways of Aerobic and Anaerobic Carbon Catabolism --- p.7 / Chapter 2.2. --- Global Regulation of Gene Expression by Oxygen --- p.10 / Chapter 2.2.1. --- Two approaches for the studies --- p.10 / Chapter 2.2.2. --- FNR regulation --- p.12 / Chapter 2.2.3. --- ArcAB regulation --- p.19 / Chapter 2.2.3.1. --- arcA --- p.19 / Chapter 2.2.3.2. --- arcB --- p.20 / Chapter 2.2.3.3. --- A member of the Two- Components regulatory systems --- p.21 / Chapter 2.3. --- Molecular Analysis of Promoters --- p.26 / Chapter 2.3.1. --- S1 mapping --- p.29 / Chapter 2.3.2. --- Primer extension --- p.29 / Chapter 2.3.3. --- DNaseI footprinting --- p.30 / Chapter 2.3.4. --- Mutational analysis of promoters --- p.32 / Chapter 3. --- Materials and Methods --- p.35 / Chapter 3.1. --- Bacterial strains and Plasmids --- p.35 / Chapter 3.2. --- Media --- p.35 / Chapter 3.3. --- Solutions --- p.38 / Chapter 3.4. --- Small Scale Preparation of Plasmid DNA --- p.40 / Chapter 3.5. --- Large Scale Preparation of Plasmid DNA --- p.41 / Chapter 3.5.1. --- Growth of bacterial culture --- p.41 / Chapter 3.5.2. --- Lysis by alkali --- p.43 / Chapter 3.5.3. --- Purification of closed circular DNA by cesium chloride gradient equilibrium centrifugation --- p.44 / Chapter 3.5.4. --- Digestion of DNA with restriction endonucleases --- p.45 / Chapter 3.6. --- Analysis of DNA Samples with Agarose Gel Electrophoresis --- p.45 / Chapter 3.7. --- Cloning of DNA Fragments from Nest-deleted M13mpl8 Clones to pFZYl --- p.47 / Chapter 3.8. --- Introduction of Plasmids into Cells --- p.48 / Chapter 3.8.1. --- Heat shock transformation --- p.48 / Chapter 3.8.1.1. --- Preparation of competent cells (I) --- p.48 / Chapter 3.8.1.2. --- Preparation of competent cells (II) --- p.49 / Chapter 3.8.2. --- High efficiency transformation by electroporation --- p.50 / Chapter 3.8.2.1. --- Preparation of electro- competent cells --- p.50 / Chapter 3.8.2.2. --- Electro-transformation --- p.51 / Chapter 3.9. --- DNA Sequencing by Chain Termination Method --- p.51 / Chapter 3.9.1. --- Preparation of single-stranded M13 templates for sequencing reaction --- p.51 / Chapter 3.9.2. --- Sequencing reactions using single- stranded templates --- p.53 / Chapter 3.9.3. --- Preparation of polyacrylamide gel for sequencing --- p.54 / Chapter 3.9.4. --- Electrophoresis of the DNA samples --- p.55 / Chapter 3.10. --- Construction of Nested Clones by Exonuclease III Unidirectional Deletions --- p.55 / Chapter 3.10.1. --- Unidirectional nested deletion of M13mpl8 clones --- p.55 / Chapter 3.10.2. --- Screening of nested clones by Direct gel electrophoresis --- p.56 / Chapter 3.10.3. --- Screening of nested clones of M13mpl8 and pFZYl by Polymerase Chain Reaction --- p.57 / Chapter 3.11. --- β-galactosidase Assay --- p.59 / Chapter 3.12. --- Primer Extension --- p.60 / Chapter 3.12.1. --- Preparation of total RNA from Gram- negative bacteria --- p.60 / Chapter 3.12.2. --- Labelling the 5' end of the oligonucleotides --- p.61 / Chapter 3.12.3. --- Hybridization and primer extension --- p.62 / Chapter 4. --- Result --- p.63 / Chapter 4.1. --- Subcloning of arcA promoter into M13mpl8/19 --- p.63 / Chapter 4.2. --- Sequencing of p34一18i and p3419i using M13 Sequencing primers (-47) and ArcA-cds Primers --- p.63 / Chapter 4.3. --- Unidirectional Nested Deletion of p3418i using Exonuclease III --- p.65 / Chapter 4.3.1. --- Large scale preparation of p3A18i DNA for Exonuclease III unidirectional nested deletion --- p.65 / Chapter 4.3.2. --- Construction of 3' and 5' overhangs --- p.65 / Chapter 4.3.3. --- Exonuclease III digestion --- p.67 / Chapter 4.3.4. --- Repairing of the 3' and 5' overhangs to generate blunt ends --- p.67 / Chapter 4.3.5. --- Blunt-end ligation of the nested deletion M13mpl8 subclones p3418i --- p.67 / Chapter 4.3.6. --- Transformation --- p.69 / Chapter 4.3.7. --- Screening of nest-deleted p3418i clones by Direct Gel --- p.71 / Chapter 4.3.8. --- Screening of nested deletion p3418i clones by PCR Screening --- p.73 / Chapter 4.3.9. --- Sequencing of the nested deletion p3418i clones --- p.76 / Chapter 4.4. --- Cloning of Nested Deletion DNA Fragments from M13mpl8 into pFZYl --- p.80 / Chapter 4.4.1. --- Screening of pFZYl clones using PCR Screening --- p.80 / Chapter 4.5. --- Expression of Nest-Deleted arcA Promoter Clones in E. coli MC1061-5 --- p.87 / Chapter 4.6. --- Expression of Nest-Deleted arcA Promoter Clones in S. typhimurium JR501 --- p.89 / Chapter 4.7. --- Primer Extension --- p.89 / Chapter 5. --- Discussion --- p.93 / Chapter 5.1. --- Sequencing of arcA Promoter --- p.93 / Chapter 5.2. --- Unidirectional Nested Deletion of p3A18i using Exonuclease III --- p.94 / Chapter 5.3. --- Screening of Nest-deletion p3418i Subclones --- p.95 / Chapter 5.4 --- Cloning of Nest-deleted DNA Fragments from M13mpl8 Subclones into pFZYl --- p.99 / Chapter 5.5. --- Screening of Nest-deleted pFZYl Subclones of p3418i --- p.101 / Chapter 5.6. --- The Effect of 5' Unidirectional Nested Deletion on the Expression of the Cloned arcA promoter in E. coli M1061-5 and S typhimurium JR501 --- p.102 / Chapter 5.7. --- Primer Extension --- p.102 / Chapter 5.8. --- Sequence Analysis of the Cloned arcA Promoter --- p.104 / Chapter 6. --- Conclusion and Further Studies --- p.111 / Chapter 7. --- Reference Cited --- p.113

Thesis (M. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 89-100).

The ventilatory threshold (VT) is measured frequently during graded exercise tests (GXT) for exercise prescription purposes. The computerized V-slope method for VT determination has been reported to be valid yet little data exists concerning the reliability of the measure. The purpose of this study was to determine the reliability of the VT using the computerized V-slope method. Sixteen healthy volunteers, (eight men and eight women) ages 21-27 (23.6 ± 1.7yrs), performed three maximal GXT on an electronically-braked cycle using an individualized ramp protocol. A minimum of 48 hours separated each test; all three tests were completed within two weeks. Breath-by-breath analysis of gas exchange was performed using a SensorMedics 2900 cart. The VT, expressed as L • min1, was selected by a computerized V-slope method. There were no differences in V02 max between the 3 trials, (overall mean 3.18 ± 0.87 L - min-1). No significant differences were observed for V02 at VT (L - min-1) for tests 1, 2, and 3 (1.62 ± 0.44, 1.58 ± 0.49, and 1.49 ± 0.48) respectively. The VT was determined to be 49.7 ± 7.9% of V02 max. Correlations between the VT in trials 1-2, 1-3, and 2-3 were 0.829, 0.791, and 0.925, respectively. The overall coefficient of variation (C.V.) of the VT measures was 10.61 ± 5.1%. The overall (male and female) variability in VT was 1.28 ± 0.63 METS. Due to high variability and a wide range in correlations, the results failed to support the reliability in the computerized V-slope method. / School of Physical Education

Thesis (M.S.)--University of Wisconsin -- La Crosse, 1999. / Digitized and made available by the University of Wisconsin--La Crosse, Murphy Library. Includes bibliographical references. Online version of print edition.

Thesis (M.S.)--University of Wisconsin--La Crosse, 2002. / Includes bibliographical references.

Memoria para optar al Título de Bioquímico / The genus Salmonella belongs to the Enterobacteriaceae family and comprises two species: S. bongori and S. enterica. Both species include more than 2500 serovars. Within S. enterica serovars, the most studied are S. Typhi, S. Typhimurium and S. Enteritidis, the latter being the main causative agent of salmonellosis worldwide. During its infective cycle, Salmonella needs to adapt to variations in different environmental conditions, such as changes in pH, osmolarity and oxygen availability. In addition, these conditions serve as environmental signals that modulate the expression of virulence factors. One of these factors is the lipopolysaccharide (LPS), the main component of the envelope of Gram negative bacteria. The LPS presents three structural domains: the O antigen (AgO), the core region and lipid A, also called endotoxin. The latter anchors the LPS to the outer membrane, and is a known virulence factor responsible for the induction of the immune response. The lipid A domain is capable of undergoing covalent modifications, which modulate its toxicity and helps bacteria to evade antimicrobial agents and the immune system of the host. One of the main environmental cues faced by Salmonella during its infectious cycle is the oxygen availability. During the aerobic-anaerobic transition participates the global regulators ArcA and Fnr. Results from our laboratory show that changes in the polymerization degree of the AgO in response to oxygen availability is modulated by these regulators. This observation led us to examine whether global regulators Fnr and ArcA are involved in the control of probable oxygen-dependent modifications of lipid A in response to oxygen availability. The main goal of this study was to determine the effect of oxygen availability on covalent modifications of lipid A in S. Enteritidis and the participation of transcriptional factors ArcA and Fnr in this process. First, we mounted a method for LPS extraction followed by a hydrolysis step to obtain a highly-purified lipid A that can be used to study the composition of this molecule by MALDI-TOF. Thus, in aerobiosis we observed four major signals corresponding to hexa-acylated and hepta-acylated lipid A molecules and their corresponding hydroxylated species. Under this environmental condition, hydroxylated species were more abundant than non-hydroxylated species. In the case of anaerobic cultures we detected the same four signal, but this relationship is reversed, being more abundant the nonhydroxylated species. To determine the participation of ArcA and Fnr in the modulation of covalent modifications of lipid A, we generated ΔarcA and Δfnr mutant strains and a strain overproducing ArcA (parcA). Our results indicate that both regulators are involved in controlling structural changes of lipid A in response to oxygen availability. Additionally, we observed that these regulators control the incorporation of other covalent modifications into lipid A. LpxO enzyme (encoded by lpxO gene) is responsible of the hydroxylation of lipid A in Salmonella. Therefore, we studied the relative expression (anaerobic/aerobic) of this gene in wild-type S. Enteritidis and their Δfnr and parcA derivatives via qRT-PCR. Our results indicate that the expression of lpxO depends on oxygen availability, an observation that is consistent with changes in the levels of lipid A hydroxylation described herein. Furthermore, ArcA and Fnr participate in the modulation of the expression of this gene. The results of this study reveal a change in the hydroxylation degree of S. Enteritidis lipid A in response to oxygen availability, in which global regulators ArcA and Fnr are involved. These findings contribute to understanding the molecular mechanisms of adaptation to the environment used by Salmonella during infection / Fondecyt

No description available.

Listeria monocytogenes is a Gram-positive facultative anaerobe that is the causative agent of the disease listeriosis and is responsible for nearly 20% of all food-related deaths in the United States. The ability of this bacterium to cause infections is proposed to correlate to its ability to resist the bactericidal properties of bile acids found in bile. Bile resistance mechanisms have exhibited increased activity under anaerobic conditions. Therefore, we hypothesized that limited oxygen could enhance the bile resistance of L. monocytogenes. Upon survival analysis, viability for virulent strains F2365, EGD-e, and 10403S increased upon 10% porcine bile extract under anaerobic conditions. However, avirulent strain HCC23 depicted no difference in bile resistance. The proteomic analysis revealed increased expression of proteins associated with DNA repair and virulence factors under anaerobic conditions in a strain dependent manner. Therefore, oxygen availability may contribute to bile resistance through the regulation of the SOS response.

published_or_final_version / Sports Science / Master / Master of Science in Sports Science

Thesis (M.S.)--Georgia Southern University, 2007. / "A thesis submitted to the Graduate Faculty of Georgia Southern University in partial fulfillment of the requirements for the degree Master of Science." Under the direction of Jim McMillan. ETD. Electronic version approved: May 2007. Includes bibliographical references (p. 32-33) and appendices.