Theses and Dissertations at Montana State University (MSU)
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Item Understanding the physiology of Pseudomonas aeruginosa biofilms in an in vitro chronic wound model(Montana State University - Bozeman, College of Letters & Science, 2016) White, Benjamin Michael; Chairperson, Graduate Committee: Michael FranklinPseudomonas aeruginosa is a common colonizer of cutaneous abrasions and burns. These Gram-negative, aerobic bacteria are problematic due to their natural resilience to antibiotics and their metabolic versatility. P. aeruginosa can produce a prodigious extracellular matrix. Within this matrix P. aeruginosa can divide and form a multicellular community called a biofilm. Biofilms have become a health concern worldwide, as these communities are highly resistant to antibiotics. This thesis reports the effort to model the wound environment. A chronic wound exudate medium was designed and P. aeruginosa was grown at 33°C under low flow in a drip-flow biofilm reactor. Bacterial cells were grown planktonically and in biofilms. Biofilms were treated with the fluoroquinone, ciprofloxacin for 24 hours and transcriptomic and metabolomic data were collected from treated and untreated biofilms and planktonic cells. Cells growing in biofilms demonstrated a shift in in the regulation of their tricarboxylic acid cycle, amino acid degradation, and siderophore biosynthesis genes as compared to planktonic cells. Ciprofloxacin treatment altered the transcriptomic landscape within the biofilm. Changes were observed in the transcription of DNA repair, prophage, and phenazine biosynthesis genes. An important virulence factor, the type VI secretion system, was also differently regulated in these samples and is likely important for the persistent infection of wounds. From the information collected, target genes have been identified for future gene-knockout and ciprofloxacin susceptibility assays. A reduction in fitness may indicate genes that are relevant drug targets to enhance antibiotic treatment of these resilient communities.Item The human intestinal organoid as a model system for enteropathogenic Escherichia coli pathogenesis(Montana State University - Bozeman, College of Letters & Science, 2015) Olshefsky, Stephen Christopher (Skip); Chairperson, Graduate Committee: Seth WalkEscherichia coli are Gram-negative, facultative anaerobic bacteria commonly found in the intestine of humans and warm-blooded animals. E. coli can be mutualistic or pathogenic that cause diarrheal disease (diarrheagenic E. coli, DEC). The colonization of E. coli begins with the successful adherence to intestinal epithelial cell (IECs); which is mediated by a variety of colonization factors on the bacterial cell surface. This is the first and most crucial step for E. coli colonization. Therefore, valuable model systems to study E. coli should recapitulate this adherence. Several model systems have been developed and successful reproduce adherence and other important aspects of EPEC pathogenesis, but have significant limitations. However, human intestinal organoids (HIOs) are a 3-dimensional tissue culture composed of a single layer of mature, differentiated, columnar epithelial cells that surround a lumen. When compared to traditional cell cultures, animal models and ligated intestines, HIOs have the potential to be more representative human physiology. Here, we begin to demonstrate the use of HIOs as an in vitro model to study E. coli. We hypothesized that HIOs could be used to study epithelial colonization dynamics of E. coli. In this study, we established working protocols for a novel experimental approach for investigating attachment factors involved in E. coli attachment. HIOs are generated from embryonic or pluripotent stem cells into definitive endoderm that gives rise to 3-dimensional structures. These structures were routinely cultured to a diameter of approximately 3.0 mm and embedding in matrigel. An overnight culture of the prototypic EPEC strain, e2348/69 (O127:H6) was transfected with a plasmid carrying a green fluorescent protein that provided visualization by fluorescent microscopy. Approximately, 2.4 x 10 6 CFU was injected into individual HIOs using a microinjector and incubated for 12 hours at 35°C and 5% CO 2. HIOs were then fixed and either stained for fluorescence imaging or processed in 1% osmium tetroxide for examination by field emission scanning electron microscopy (FE SEM). The observations that EPEC was intimately associated with the intestinal epithelium, supports the use HIO for E. coli colonization investigations.Item Fluid dynamic study of confluent and stratified flow in vascular replicas(Montana State University - Bozeman, College of Engineering, 1978) Valach, Dale AllenItem An analytical model of the patello-femoral joint(Montana State University - Bozeman, College of Engineering, 1978) Hagelin, Jack StephenItem Frequency response analysis of the in-vivo human spine(Montana State University - Bozeman, College of Engineering, 1978) Radons, Stephen WalterItem Determination of macroscopic biological material properties by dynamic, 'in-vivo' testing(Montana State University - Bozeman, College of Engineering, 1973) Garner, Edward Roger