It is estimated that approximately 2 million fires which occur in United States each year result in 1.2 million burn victims. Fibroblasts are responsible for responding to this tissue damage by breaking down the damaged extracellular matrix (ECM) and secreting a new ECM which aids in wound repair and supports the migration of immune cells. Pseudomonas aeruginosa is an opportunistic pathogen commonly associated with health-care infections (HCAIs) due to its ability to take advantage of immunocompromised hosts. However, little research has investigated how wound invading P. aeruginosa interacts with wound repairing fibroblasts. To address this lack of understanding, this thesis focuses on quantifying changes in fibroblast morphology, migratory behavior, and force exertion to investigate this host cell's response to representative cytotoxic (PAO1) and invasive (PA14) strains of P. aeruginosa. These assays study host cell-pathogen interactions on highly aligned nanofibers of varied spacing and diameter, which mimic the fibroblast deposited ECM and dictate fibroblast morphology. We discovered that the cytotoxic strain of P. aeruginosa induced significantly shorter fibroblast death times. Furthermore, two modes of death, sharp and gradual, were identified and found to be dependent on both fiber configuration and strain of P. aeruginosa. In addition, fibroblasts exposed to PAO1 migrating on the parallel formation were found to be significantly slower and less persistent than those exposed to PA14, however, fibroblasts exposed to both strains of bacteria were shown to exert similar forces. Lastly, exposure to PA14 led to the greatest change in actin, evident by increased actin punctae and less prominent actin stress fiber formation. / Master of Science / It is estimated that approximately 2 million fires which occur in United States each year result in 1.2 million burn victims. Fibroblasts respond to burn wounds by breaking down the damaged tissue fibers, termed extracellular matrix (ECM), and secreting a new ECM. Unfortunately, severe thermal injuries place hospitalized burn victims at high risk of infection. Pseudomonas aeruginosa is an opportunistic pathogen commonly associated with health-care infections (HCAIs) due to its ability to take advantage of immunocompromised hosts. However, little research has investigated how wound invading P. aeruginosa interacts with wound healing fibroblasts. To address this knowledge gap, this thesis focuses on quantifying changes in fibroblast shape, migratory behavior, and force exertion to investigate this host cell's response to two strains of P. aeruginosa, which employ different mechanisms of invasion. These interactions are studied on a platform of suspended nanofibers with controlled spacing and diameter, to dictate fibroblast shape and mimic the fibroblast deposited ECM. We discovered that the two strain of P. aeruginosa induced significantly different fibroblast death times. During death, it was observed that fibroblasts either balled up quickly, termed sharp death, or remained spread out, termed gradual death, dependent upon fibroblast shape and strain of P. aeruginosa introduced. In addition, significant differences in migration speed and persistence were found between fibroblasts exposed to the two strains of bacteria, however, both groups were shown to exert similar forces. Lastly, the fibrous proteins which make up the cytoskeleton of the cell, actin stress fibers, were found to vary among the control and bacteria treated cells.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/114117 |
| Date | 22 September 2021 |
| Creators | Berman, Lauren Kathryn |
| Contributors | Mechanical Engineering, Behkam, Bahareh, Kale, Sohan, Nain, Amrinder |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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