Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on October 18, 2007) Vita. Includes bibliographical references.

Thesis (M.S. )--Marshall University, 2009. / Title from document title page. Includes abstract. Document formatted into pages: contains 155 p. Includes bibliographical references p. 151-152.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains ix, 132 p.; also includes graphics (some col.). Includes bibliographical references (p. 112-132). Available online via OhioLINK's ETD Center

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (D.P.H.)--University of Michigan.

Dissertation (D.P.H.)--University of Michigan.

P. aerguinosa is a serious human bacterial pathogen. This thesis describes attempts to use structural biology to identify new starting points for drugs against P. aerguinosa .A number of fragment-based screening techniques were used in order to identify potential inhibitors to P. aerguinosa RmlA protein, the first enzyme in the L-Rhamnose pathway. A 500 “Rule of 3” Fragment Library (Maybridge) was investigated. The first approach was the application of Differential Scanning Fluorimetry (DSF) approach to detect ligands that bind and stabilize RmlA protein. The stabilisation of RmlA was determined by thermal unfolding in the presence of each of the 500 compounds. 21 of those compounds were found to increase the protein stability. The library was then screened by NMR spectroscopy for binding to RmlA. Two techniques were evaluated STD and WaterLOGSY. 106 compounds gave positive results in both NMR experiments. These hits were then tested by a simple STD competition binding with dTTP, a natural RmlA substrate, in order to identify those binding at the active or allosteric site. 21 out of the 106 compounds were observed to compete with dTTP. The results were compared to the results of the DSF screening. Compounds that tested positive in the dTTP competition binding STD experiment and in the DSF screening were tested for their ability to inhibit RmlA in a biological assay. A coupled enzyme assay was used to monitor RmlA activity. Only one compound, 3-pyridin-3-ylaniline, showed significant inhibition of the enzyme activity. The PA1645 protein from P. aerguinosa has been identified as essential. The protein was overexpressed, purified and crystallised. Data were collected at Diamond on beamline IO3 and phases were determined by S-SAD at a wavelength of 1.6Å. Final coordinates have been deposited in the protein data bank under entry code 2XU8. The structure has 3 molecules in the asymmetric unit. There is some ambiguity as to the validity of the proposed trimeric arrangement, with results from solution and crystal disagreeing. Fragment-based screening approach has been applied to RmlA protein, using the DSF technique, a number of ligand-based NMR experiments and a coupled enzyme biological assay. 3-pyridin-3-ylaniline was the only compound that showed significant inhibition of the enzyme activity. The structure of PA1645 from P. aerguinosa has been solved. This work will help to design new drugs to combat multi-drug resistant P. aerguinosa and MTB.

The Gram-negative bacterium Pseudomonas aeruginosa is a notorious opportunistic pathogen of individuals with the genetic disease cystic fibrosis (CF). Pseudomonas aeruginosa establishes a chronic infection within the CF lung, where the sputum accumulation characteristic of CF provides a complex and copious growth substrate. P. aeruginosa can grow to high densities in vivo (>10⁹ cells/ml lung sputum), and exacerbations associated with P. aeruginosa high density in vivo growth are primary contributors to CF morbidity and mortality. Surprisingly little is known about the catabolic processes that underlie P. aeruginosa in vivo growth. Unfortunately, nutritional modeling of the CF lung environment in animal models is difficult, as current animal models fail to mimic the sputum accumulation characteristic of CF. In this dissertation, I describe the use of expectorated CF sputum as a P. aeruginosa in vitro growth medium. Using global expression analysis, I show that P. aeruginosa up-regulates genes important for amino acid and lactate metabolism during growth in CF sputum as compared to a laboratory medium. P. aeruginosa also demonstrates enhanced production of the cell-cell communication signal 2-heptyl-3-hydroxy-4-quinolone (the Pseudomonas quinolone signal, PQS), a critical regulator of virulence factor production, during growth in CF sputum. Further, I use chemical analyses of CF sputum samples to develop a defined, synthetic medium that can be used to nutritionally model in vivo conditions. Using this medium, I show that PQS biosynthesis and aromatic amino acid metabolism are intimately linked and that cell-cell communication mediated by PQS is strikingly dependent upon the growth environment of P. aeruginosa. In addition, I demonstrate that P. aeruginosa preferentially consumes specific carbon sources present in the CF sputum milieu during rapid growth. I also describe the use of in vivo-relevant nutrient concentrations to evaluate the potential for P. aeruginosa anaerobic growth in CF sputum. Finally, I describe the purification and characterization of the aromatic amino acid-responsive transcriptional regulator PhhR and discuss its potential role in regulation of P. aeruginosa in vivo carbon substrate preference. / text

M.Cur / According to data from the Centers for Disease Control and Prevention’s(CDC) National Nosocomial Infections Surveillance System of 1996, Pseudomonas aeruginosa(P. aeruginosa) can be rated as the number two cause of nosocomial pneumonia(Chen & Rudoy,2006). Nosocomial pneumonia increases hospital cost and morbidity and mortality in patients. Most of the patients in the critical care unit are immune compromised because of underlying illnesses. Antibiotics eliminates the patient’s normal flora which causes opportunity for pathogens to colonise. Indwelling procedures like endotracheal intubation cause a point of entrance for pathogens like P.aeruginosa. The endotracheal tube bypasses the normal physiological processes and inhibits the cough reflex. It is the nurse’s responsibility to remove secretion through endotracheal suctioning. During the past ten years the closed suction method was increasingly implemented to remove secretions because studies showed closed suction caused less infection than open suction. In a spesific critical care unit in a private hospital in Pretoria the nurses are of the opinion that closed suctioning does not effectively remove secretion. Patients are therefore suctioned open which can cause air contamination because the colonised ventilator circuit is opened. The following question can be asked in view of the above arguments and problem statement: Is there a difference in aircontamintion between open and closed suctioning? The aim of the study is to determine whether any difference in air contamination exists between open and closed suctioning in a spesific critical care unit in Pretoria. v A comparitive contextual design with crossover methods was used. Patients are allocated to group 1 or group 2 through random sampling. An air exstractor is used to take airsamples before, during and after suctioning. There was no significant difference in terms of air contamination for open and closed suction. This is probably because of too small a sample. The null hypothesis is accepted and that is there is no significant difference in air contamination between open and closed suction.