This thesis addresses the following questions: What are the best methods to test the effects of tolaasin, cephalexin and laser on the biofilms of S. aureus and S. epidermidis? Does tolaasin prevent or disrupt biofilm formation in S. aureus and S. epidermidis? Does tolaasin work synergistically with cephalexin to prevent biofilm growth and maturation in S. aureus and S. epidermidis? And, what effects does laser treatment have on E. faecalis biofilms? In order to answer these questions, tolaasin was isolated from P. tolaasii, and biofilms were pre-treated with tolaasin. Trials were performed with tolaasin, cephalexin, or a combination of both. The effectiveness of each treatment was determined by observing the biofilm growth. The protocols were then optimized and trials were repeated. Additionally, E. faecalis biofilms were exposed to laser treatment. Using confocal microscopy, the biofilms were observed and quantitative results were used to determine the effectiveness of the treatment. Overall, the results indicated that tolaasin has little effect on biofilm growth. However, further investigation is necessary to confirm these results due to some inconsistent data obtained over the course of the trials. Variations and improvements to the protocol are necessary to accurately determine tolaasin’s potential role in healthcare. Finally, the results of the laser trials suggest that EDTA in conjunction with laser treatment could be useful in cleaning root canals and eliminating post-procedural biofilms—thereby preventing infections.
X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research.
Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.
The objective of this investigation is to evaluate how environmental changes that are related to the growth environment in the CF lung alters rhamnolipid production. Thirty-five P. aeruginosa isolates from Dartmouth College and Seattle Children’s Hospital were selected to observe the impact of temperature, presence of Staphylococcus aureus metabolites, and oxygen availability on rhamnolipid production. It was found that the rhamnolipid production significantly decreased for 30C versus 37C, but not at 40C. The addition of S. aureus spent media, in any of the tested conditions, did not influence rhamnolipid production. Finally, the change in oxygen concentration from normoxia to hypoxia significantly reduced rhamnolipid production. These results were compared to swarming assay data to understand how changes in rhamnolipid production impact surface-mediated motility.