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Description
This study was conducted as part of an underlying initiative to elucidate the mechanism of action of natural antibacterial clay minerals for application as therapeutic agents for difficult-to-treat infections such as methicillin-resistant Staphylococcus aureus (MRSA)-derived skin lesions and Buruli ulcer. The goal of this investigation was to determine whether exposure to the leachate of an antibacterial clay mineral, designated as CB, produced DNA double-strand breaks (DSBs) in Escherichia coli. A neutral comet assay for bacterial cells was adapted to assess DSB levels upon exposure to soluble antimicrobial compounds. Challenges involved with the adaptation process included comet visualization and data collection. To appropriately account for antimicrobial-mediated strand fragmentation, suitable control reactions comprised of exposures to water, ethanol, kanamycin, and bleomycin were developed and optimized for the assay. Bacterial exposure to CB resulted in significantly longer comet lengths compared to negative control exposures, suggesting that CB killing activity involves the induction of DNA DSBs. The results of this investigation further characterize the antimicrobial mechanisms associated with a particular clay mineral mixture. The adapted comet assay protocol described herein functions as an effective tool to assess double-strand fragmentation resulting from exposure to soluble antimicrobial compounds and to visually compare results from experimental and control reactions.
ContributorsSolanky, Dipesh (Author) / Haydel, Shelley (Thesis director) / Stout, Valerie (Committee member) / Adusumilli, Sarojini (Committee member) / Barrett, The Honors College (Contributor) / College of Liberal Arts and Sciences (Contributor)
Created2012-12
Description
Staphylococcus aureus and Staphylococcus epidermidis are among the most common causes of hospital-acquired infections5, 7, 8. Despite the advancements in modern antimicrobials, infections from these organisms can be very difficult to treat, and equally as difficult to prevent 6,7. These organisms’ abilities to form biofilms are directly related to their abilities to cause infections. In biofilms, the staphylococcal species can survive antibiotics and immune responses much better than planktonic cells7. Tolaasin—a toxin and natural biosurfactant produced by P. tolaasii—has been briefly tested against biofilm formation, and the results suggested that it could have inhibitory effects. In order to further confirm and expand upon this potentially useful data, additional testing was performed to determine the effects of tolaasin on the two organisms. In addition, laser treatment was tested on E. faecalis in order to supplement our current understanding of biofilm behavior, and provide additional data to suggest alternative agents against biofilm growth.
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.
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.
ContributorsChristopher, Zachary Kyle (Author) / Stout, Valerie (Thesis director) / Haydel, Shelley (Committee member) / Muralinath, Maneesha (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
This study examines the effect of the translation of traditional scientific vocabulary into plain English, a process referred to as Anglicization, on student learning in the context of introductory microbiology instruction. Data from Anglicized and Classical-vocabulary lab sections were collected. Data included exam scores as well as pre and post-course surveys on reasoning skills, impressions of biology, science and the course, and microbiology knowledge. Students subjected to Anglicized instruction performed significantly better on exams that assessed their abilities to apply and analyze knowledge from the course, and gained similar amounts of knowledge during the course when compared to peers instructed with standard vocabulary. Their performance in upper-level courses was also better than that of their traditionally educated peers. Hypotheses related to the effect are presented and evaluated; implications for instruction are discussed.
ContributorsRichter, Emily (Author) / Lawson, Anton (Thesis advisor) / Stout, Valerie (Committee member) / Haydel, Shelley (Committee member) / Atkinson, Robert (Committee member) / Arizona State University (Publisher)
Created2011
Description
Pseudomonas aeruginosa is an opportunistic bacterial pathogen commonly associated with increased morbidity and mortality in cystic fibrosis (CF) patients. To adapt to the CF lung environment, P. aeruginosa undergoes multiple genetic changes as it moves from an acute to a chronic infection. The resultant phenotypes have been associated with chronic infection and can provide important information to track the patient’s individualized disease progression. This study examines the link between the accumulation of QS genetic mutations and phenotypic expression in P. aeruginosa laboratory strains and clinical isolates. We utilized several plate-based and colorimetric assays to quantify the production of pyocyanin, rhamnolipids, and protease from paired clinical early- and late-stage chronic infection isolates across 16 patients. Exoproduct production of each isolate was compared to the mean production of pooled isolates to classify high producing (QS-sufficient) and low producing (QS-deficient) isolates. We found that over time P. aeruginosa isolates exhibit a reduction in QS-related phenotypes during chronic infections. Future research of the QS regulatory networks will identify whether reversion of genotype will result in corresponding phenotypic changes in QS-deficient chronic infection isolates.
ContributorsKaranjia, Ava Vispi (Author) / Bean, Heather (Thesis director) / Haydel, Shelley (Committee member) / Davis, Trenton (Committee member) / School of Life Sciences (Contributor) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
AARP estimates that 90% of seniors wish to remain in their homes during retirement. Seniors need assistance as they age, historically they have received assistance from either family members, nursing homes, or Continuing Care Retirement Communities. For seniors not wanting any of these options, there has been very few alternatives. Now, the emergence of the continuing care at home program is providing hope for a different method of elder care moving forward. CCaH programs offer services such as: skilled nursing care, care coordination, emergency response systems, aid with personal and health care, and transportation. Such services allow seniors to continue to live in their own home with assistance as their health deteriorates over time. Currently, only 30 CCaH programs exist. With the growth of the elderly population in the coming years, this model seems poised for growth.
ContributorsSturm, Brendan (Author) / Milovanovic, Jelena (Thesis director) / Hassett, Matthew (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / Economics Program in CLAS (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Catastrophe events occur rather infrequently, but upon their occurrence, can lead to colossal losses for insurance companies. Due to their size and volatility, catastrophe losses are often treated separately from other insurance losses. In fact, many property and casualty insurance companies feature a department or team which focuses solely on modeling catastrophes. Setting reserves for catastrophe losses is difficult due to their unpredictable and often long-tailed nature. Determining loss development factors (LDFs) to estimate the ultimate loss amounts for catastrophe events is one method for setting reserves. In an attempt to aid Company XYZ set more accurate reserves, the research conducted focuses on estimating LDFs for catastrophes which have already occurred and have been settled. Furthermore, the research describes the process used to build a linear model in R to estimate LDFs for Company XYZ's closed catastrophe claims from 2001 \u2014 2016. This linear model was used to predict a catastrophe's LDFs based on the age in weeks of the catastrophe during the first year. Back testing was also performed, as was the comparison between the estimated ultimate losses and actual losses. Future research consideration was proposed.
ContributorsSwoverland, Robert Bo (Author) / Milovanovic, Jelena (Thesis director) / Zicarelli, John (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The current study investigated whether intermittent restraint stress (IRS) would impair fear extinction learning and lead to increased anxiety and depressive- like behaviors and then be attenuated when IRS ends and a post- stress rest period ensues for 6 weeks. Young adult, male Sprague Dawley rats underwent restraint stress using wire mesh (6hr/daily) for five days with two days off before restraint resumed for three weeks for a total of 23 restraint days. The groups consisted of control (CON) with no restraint other than food and water restriction yoked to the restrained groups, stress immediate (STR-IMM), which were restrained then fear conditioned soon after the end of the IRS paradigm, and stress given a rest for 6 weeks before fear conditioning commenced (STR-R6). Rats were fear conditioned by pairing a 20 second tone with a footshock, then given extinction training for two days (15 tone only on each day). On the first day of extinction, all groups discriminated well on the first trial, but then as trials progressed, STR-R6 discriminated between tone and context less than did CON. On the second day of extinction, STR- IMM froze more to context in the earlier trials than compared to STR-R6 and CON. As trials progressed STR-IMM and STR-R6 froze more to context than compared to CON. Together, CON discriminated between tone and context better than did STR-IMM and STR-R6. Sucrose preference, novelty suppressed feeding, and elevated plus maze was performed after fear extinction was completed. No statistical differences were observed among groups for sucrose preference or novelty suppressed feeding. For the elevated plus maze, STR-IMM entered the open arms and the sum of both open and closed arms fewer than did STR- R6 and CON. We interpret the findings to suggest that the stress groups displayed increased hypervigilance and anxiety with STR-R6 exhibiting a unique phenotype than that of STR-IMM and CON.
ContributorsShah, Vrishti Bimal (Author) / Conrad, Cheryl (Thesis director) / Newbern, Jason (Committee member) / Judd, Jessica (Committee member) / School of Life Sciences (Contributor) / Sanford School of Social and Family Dynamics (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Cystic Fibrosis (CF) is a genetic disorder that disrupts the hydration of mucous of the lungs, which promotes opportunistic bacterial infections that begin in the affected person’s childhood, and persist into adulthood. One of the bacteria that infect the CF lung is Pseudomonas aeruginosa. This gram-negative bacterium is acquired from the environment of the CF lung, changing the expression of phenotypes over the course of the infection. As P. aeruginosa infections become chronic, some phenotype changes are known to be linked with negative patient outcomes. An important exoproduct phenotype is rhamnolipid production, which is a glycolipid that P. aeruginosa produces as a surfactant for surface-mediated travel. Over time, the expression of this phenotype decreases in expression in the CF lung.
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.
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.
ContributorsKiermayr, Jonathan Patrick (Author) / Bean, Heather (Thesis director) / Misra, Rajeev (Committee member) / Haydel, Shelley (Committee member) / School of International Letters and Cultures (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
The prrAB two-component system has been shown to be essential for viability in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To study this system, several prrAB mutants of Mycobacterium smegmatis, a close relative of Mtb, were created for study. These mutants included a deletion mutant complemented with prrA from Mtb controlled by Pmyc1_tetO, a deletion mutant, and a deletion mutant complemented with prrAB from M. smegmatis controlled by the native prrAB promoter sequence (~167 bp upstream sequence of prrAB). In a previous study, the prrAB deletion mutant clumped excessively relative to the wild-type strain when cultured in a nitrogen-limited medium. To address this irregularity, the lipid profiles of these mutants were analyzed through several experimental methods. Untargeted lipidomic profiles were analyzed by Electrospray Ionization Mass Spectrometry (ESI-MS). The ESI-MS data suggested the deletion mutant accumulates triacylglycerol species relative to the wild-type strain. This data was verified by thin-layer chromatography (TLC) and densitometry of the TLC images. The mycolic acid profile of each mutant was also analyzed by TLC but no noteworthy differences were found. High-throughput RNA-Seq analysis revealed several genes involved in lipid biosynthetic pathways upregulated in the prrAB deletion mutant, thus corroborating the ESI-MS and TLC data.
ContributorsOlson, Alexandra Nadine (Author) / Haydel, Shelley (Thesis director) / Bean, Heather (Committee member) / Maarsingh, Jason (Committee member) / School of Social Transformation (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
The spread of antibiotic resistant bacteria is currently a pressing global health concern, especially considering the prevalence of multi-drug resistance. Efflux pumps, bacterial machinery involved in various active transport functions, are capable of removing a broad range of antibiotics from the periplasmic space and the outer leaflet of the inner membrane, frequently conferring multi-drug resistance. Many aspects of efflux machinery’s structure, functions, and inter-protein interactions are still not fully understood; further characterization of these components of efflux will provide a strong foundation for combating this resistance mechanism. In this project, I further characterize the channel protein TolC as a part of the AcrAB-TolC efflux pump complex in Escherichia coli by first determining the specificity of compensatory mutations in TolC against defective AcrA and AcrB, and then identifying TolC residues that might influence TolC aperture dynamics or stability when altered. Specificity of compensatory mutations was determined using an array of TolC mutants, previously generated from defective AcrA or AcrB, against a different mutant AcrB protein; these new mutant combinations were then analyzed by real-time efflux and antibiotic susceptibility assays. A vancomycin susceptible TolC mutant—a phenotype that has been associated with constitutively open TolC channels—was then used to generate vancomycin-resistant revertants which were evaluated with DNA sequencing, protein quantification by Western blots, and real-time efflux assays to identify residues important for TolC aperture dynamics and protein stability and complex activity. Mutations identified in revertant strains corresponded to residues located in the lower half of the periplasmic domain of TolC; generally, these revertants had poorer efflux than wild-type TolC in the mutant AcrB background, and all revertants had poorer efflux activity than the parental mutant strain.
ContributorsMcFeely, Megan Elizabeth (Author) / Misra, Rajeev (Thesis director) / Haydel, Shelley (Committee member) / Stout, Valerie (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05