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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
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
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.
ContributorsLi, Xuanxuan (Author) / Chiu, Chun-Ya (Author) / Wang, Hsiang-Ju (Author) / Kassemeyer, Stephan (Author) / Botha, Sabine (Author) / Shoeman, Robert L. (Author) / Lawrence, Robert (Author) / Kupitz, Christopher (Author) / Kirian, Richard (Author) / James, Daniel (Author) / Wang, Dingjie (Author) / Nelson, Garrett (Author) / Messerschmidt, Marc (Author) / Boutet, Sebastien (Author) / Williams, Garth J. (Author) / Hartman, Elisabeth (Author) / Jafarpour, Aliakbar (Author) / Foucar, Lutz M. (Author) / Barty, Anton (Author) / Chapman, Henry (Author) / Liang, Mengning (Author) / Menzel, Andreas (Author) / Wang, Fenglin (Author) / Basu, Shibom (Author) / Fromme, Raimund (Author) / Doak, R. Bruce (Author) / Fromme, Petra (Author) / Weierstall, Uwe (Author) / Huang, Michael H. (Author) / Spence, John (Author) / Schlichting, Ilme (Author) / Hogue, Brenda (Author) / Liu, Haiguang (Author) / ASU Biodesign Center Immunotherapy, Vaccines and Virotherapy (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Department of Physics (Contributor) / School of Life Sciences (Contributor)
Created2017-04-11
Description
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.
ContributorsDeb, Arpan (Author) / Johnson, William (Author) / Kline, Alexander (Author) / Scott, Boston (Author) / Meador, Lydia (Author) / Srinivas, Dustin (Author) / Martin Garcia, Jose Manuel (Author) / Dorner, Katerina (Author) / Borges, Chad (Author) / Misra, Rajeev (Author) / Hogue, Brenda (Author) / Fromme, Petra (Author) / Mor, Tsafrir (Author) / ASU Biodesign Center Immunotherapy, Vaccines and Virotherapy (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Life Sciences (Contributor) / Biodesign Institute (Contributor) / School of Molecular Sciences (Contributor) / Applied Structural Discovery (Contributor) / Personalized Diagnostics (Contributor)
Created2017-02-22
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
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
Abstract The BIO 189 Life Sciences Career Paths course is a seminar course that is intended to acclimate incoming freshmen into the School of Life Sciences (SOLS). While there are instructors who organize and present in the class, upper division undergraduate students are primarily responsible for facilitating lectures and discussions and mentoring the freshmen. Prior research has demonstrated that the mentor-mentee relationship is a very important predictor of success and retention within all university first-year programs. While past studies focused on the student mentor-mentee relationships, there is limited research that measures student satisfaction within freshmen seminar courses, especially in areas of science, technology, engineering, and mathematics (STEM). The purpose of this project is to survey students about their perception of the BIO 189 course. The effort of the project is on pre-health students, as they initiate their undergraduate careers and attempt to achieve acceptance into professional school four years later. Analysis of Likert scale surveys distributed to 561 freshmen revealed that students with an emphasis on "medicine" in their majors preferred a BIO 189 course geared to pre-health interests whereas students seeking an emphasis on research (ecology and cell biology/genetics) sought a BIO 189 course focused on internship and employment opportunities. Assessment of the mentor-mentee relationship revealed that students (n = 561) preferred one-on-one meetings with mentors outside of class (44%) compared to those who preferred interaction in class (30%). A sizable 61.68% of students (n = 548) were most concerned with attaining favorable GPAs, highlighting strong emphasis on academic performance. Overall, 61% of respondents (n = 561) expressed satisfaction with SOLS resources and involvement opportunities, which was hypothesized. These results give substantial insight into the efficacy of a first-year success seminar-mentoring program for college freshmen in STEM.
ContributorsMaalouf, Nicholas Elie (Author) / Haydel, Shelley (Thesis director) / Harrell, Carita (Committee member) / Capco, David (Committee member) / Department of Psychology (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Pyocyanin is a pigment produced by Pseudomonas aeruginosa that acts as a virulence factor in helping this pathogen to establish chronic infection in the lungs of persons with cystic fibrosis (CF). Then, as lung infections become chronic, P. aeruginosa tends to down-regulate pyocyanin production. The effects of environmental conditions, particularly temperature change, on pyocyanin production in P. aeruginosa has not been widely studied in the past. The goals of this project were twofold: First, we aim to identify how environmental conditions potentially present in the CF lungs affect pyocyanin pigment production in P. aeruginosa. Second, through the examination of effects of environmental changes, we aim to identify methods to modulate phenotypes of P. aeruginosa in order to identify putative biomarkers through metabolic analysis. This paper also identifies a newly derived pyocyanin culturing and extraction procedure that yields increased sensitivity for pyocyanin detection.
Through a liquid-liquid extraction procedure, pyocyanin was quantified in cultures that were incubated at 30°C, 37°C, and 40°C and in the presence of Staphylococcus aureus spent media. In addition, culturing methods for the measurement of pyocyanin under hypoxic conditions were analyzed. I hypothesized that environmental conditions such as temperature, co-infection with S. aureus, and oxygen depletion would influence pyocyanin production. It was found that overall, 30°C incubation produced statistically significant decrease in pyocyanin production compared with incubation at 37°C. These findings will help to determine how phenotypes are affected by conditions in the CF lung. In addition, these conclusions will help direct metabolic analysis and to identify volatile biomarkers of pyocyanin production for future use in breath-based diagnostics of CF lung infections.
Through a liquid-liquid extraction procedure, pyocyanin was quantified in cultures that were incubated at 30°C, 37°C, and 40°C and in the presence of Staphylococcus aureus spent media. In addition, culturing methods for the measurement of pyocyanin under hypoxic conditions were analyzed. I hypothesized that environmental conditions such as temperature, co-infection with S. aureus, and oxygen depletion would influence pyocyanin production. It was found that overall, 30°C incubation produced statistically significant decrease in pyocyanin production compared with incubation at 37°C. These findings will help to determine how phenotypes are affected by conditions in the CF lung. In addition, these conclusions will help direct metabolic analysis and to identify volatile biomarkers of pyocyanin production for future use in breath-based diagnostics of CF lung infections.
ContributorsWitzel, Lea (Co-author) / Bean, Heather D. (Co-author, Thesis director) / Misra, Rajeev (Committee member) / Haydel, Shelley (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
The need for new tuberculocidal drugs is crucial with drug resistance on the rise as the tuberculosis epidemic rages on. One new potential drug target is the PrrAB two component system (TCS) since it does not exist in humans and is essential to viability in Mycobacterium tuberculosis. This project examines Mycobacterium smegmatis, and this nonpathogenic and fast-growing organism possesses two full length PrrAB orthologs, in addition to an orphaned PrrB sensor histidine kinase. While it was determined that PrrAB1 and PrrAB2 are nonessential, the lone PrrB3 is not yet characterized for essentiality. To confirm individual dispensability of PrrAB1 and PrrAB2 and investigate the essentiality of PrrB3 and the full M. smegmatis PrrAB multiplex, we utilized CRISPRi dCas9 to repress the expression (knockdown) of prrAB1 (MSMEG_5662-5663), prrAB2 (MSMEG_0244-0246), and the lone prrB3 (MSMEG_2793) in M. smegmatis independently and simultaneously. Repression of prrAB1 resulted in the greatest growth defect, with a lag of 17 cellular division cycles compared to the control, a strain generated with an empty vector. However, the knockdown of prrAB1 was not lethal to M. smegmatis. The inhibition of all three prrAB orthologs simultaneously, also known as a multiplex knockdown, lagged the control by 13 cellular division cycles. At the 48-hour point, both the single ortholog repression of prrAB1 as well as the whole prrAB system knockdown had a growth defect of 13 replication cycles behind the control. However, the multiplex knockdown stabilized growth at 48 hours, revealing a possible compensatory mechanism in M. smegmatis. Conclusively, we show that the PrrAB TCS is globally inessential for viability in M. smegmatis.
ContributorsHeiligenstein, Piper (Author) / Haydel, Shelley (Thesis director) / Shrivastava, Abhishek (Committee member) / Haller, Yannik (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of Mathematical and Natural Sciences (Contributor)
Created2023-12