Matching Items (29)
Description
The study of bacterial resistance to antimicrobial peptides (AMPs) is a significant area of interest as these peptides have the potential to be developed into alternative drug therapies to combat microbial pathogens. AMPs represent a class of host-mediated factors that function to prevent microbial infection of their host and serve as a first line of defense. To date, over 1,000 AMPs of various natures have been predicted or experimentally characterized. Their potent bactericidal activities and broad-based target repertoire make them a promising next-generation pharmaceutical therapy to combat bacterial pathogens. It is important to understand the molecular mechanisms, both genetic and physiological, that bacteria employ to circumvent the bactericidal activities of AMPs. These understandings will allow researchers to overcome challenges posed with the development of new drug therapies; as well as identify, at a fundamental level, how bacteria are able to adapt and survive within varied host environments. Here, results are presented from the first reported large scale, systematic screen in which the Keio collection of ~4,000 Escherichia coli deletion mutants were challenged against physiologically significant AMPs to identify genes required for resistance. Less than 3% of the total number of genes on the E. coli chromosome was determined to contribute to bacterial resistance to at least one AMP analyzed in the screen. Further, the screen implicated a single cellular component (enterobacterial common antigen, ECA) and a single transporter system (twin-arginine transporter, Tat) as being required for resistance to each AMP class. Using antimicrobial resistance as a tool to identify novel genetic mechanisms, subsequent analyses were able to identify a two-component system, CpxR/CpxA, as a global regulator in bacterial resistance to AMPs. Multiple previously characterized CpxR/A members, as well as members found in this study, were identified in the screen. Notably, CpxR/A was found to transcriptionally regulate the gene cluster responsible for the biosynthesis of the ECA. Thus, a novel genetic mechanism was uncovered that directly correlates with a physiologically significant cellular component that appears to globally contribute to bacterial resistance to AMPs.
ContributorsWeatherspoon-Griffin, Natasha (Author) / Shi, Yixin (Thesis advisor) / Clark-Curtiss, Josephine (Committee member) / Misra, Rajeev (Committee member) / Nickerson, Cheryl (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2013
Description
In somatic cells, the mitotic spindle apparatus is centrosomal and several isoforms of Protein Kinase C (PKC) have been associated with the mitotic spindle, but their role in stabilizing the mitotic spindle is unclear. Other protein kinases such as, Glycogen Synthase Kinase 3â (GSK3â) also have been shown to be associated with the mitotic spindle. In the study in chapter 2, we show the enrichment of active (phosphorylated) PKCæ at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases, PKC and GSK3â are associated with the mitotic spindle, first, the co-localization and close molecular proximity of PKC isoforms with GSK3â was studied in metaphase cells. Second, the involvement of inactive GSK3â in maintaining an intact mitotic spindle was shown. Third, this study showed that addition of a phospho-PKCæ specific inhibitor to cells can disrupt the mitotic spindle microtubules. The mitotic spindle at metaphase in mouse fibroblasts appears to be maintained by PKCæ acting through GSK3â. The MAPK pathway has been implicated in various functions related to cell cycle regulation. MAPKK (MEK) is part of this pathway and the extracellular regulated kinase (ERK) is its known downstream target. GSK3â and PKCæ also have been implicated in cell cycle regulation. In the study in chapter 3, we tested the effects of inhibiting MEK on the activities of ERK, GSK3â, PKCæ, and á-tubulin. Results from this study indicate that inhibition of MEK did not inhibit GSK3â and PKCæ enrichment at the centrosomes. However, the mitotic spindle showed a reduction in the pixel intensity of microtubules and also a reduction in the number of cells in each of the M-phase stages. A peptide activation inhibitor of ERK was also used. Our results indicated a decrease in mitotic spindle microtubules and an absence of cells in most of the M-phase stages. GSK3â and PKCæ enrichment were however not inhibited at the centrosomes. Taken together, the kinases GSK3â and PKCæ may not function as a part of the MAPK pathway to regulate the mitotic spindle.
ContributorsChakravadhanula, Madhavi (Author) / Capco, David G. (Thesis advisor) / Chandler, Douglas (Committee member) / Clark-Curtiss, Josephine (Committee member) / Newfeld, Stuart (Committee member) / Arizona State University (Publisher)
Created2012
Description
The Philadelphia chromosome in humans, is on oncogenic translocation between chromosomes 9 and 22 that gives rise to the fusion protein BCR-Abl. This protein is constitutively active resulting in rapid and uncontrolled cell growth in affected cells. The BCR-Abl protein is the hallmark feature of chronic myeloid leukemia (CML) and is seen in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) cases. Currently, the first line of treatment is the Abl specific inhibitor Imatinib. Some patients will, however, develop resistance to Imatinib. Research has shown how transformation of progenitor B cells with v-Abl, an oncogene expressed by the Abelson murine leukemia virus, causes rapid proliferation, prevents further differentiation and produces a potentially malignant transformation. We have used progenitor B cells transformed with a temperature-sensitive form of the v-Abl protein that allows us to inactivate or re-activate v-Abl by shifting the incubation temperature. We are trying to use this line as a model to study both the progression from pre-malignancy to malignancy in CML and Imatinib resistance in Ph+ ALL and CML. These progenitor B cells, once v-Abl is reactivated, in most cases, will not return to their natural cell cycle. In this they resemble Ph+ ALL and CML under Imatinib treatment. With some manipulation these cells can break this prolonged G1 arrested phenotype and become a malignant cell line and resistant to Imatinib treatment. Cellular senescence can be a complicated process requiring inter-play between a variety of players. It serves as an alternate option to apoptosis, in that the cell loses proliferative potential, but does not die. Treatment with some cancer therapeutics will induce senescence in some cancers. Such is the case with Imatinib treatment of CML and Ph+ ALL. By using the S9 cell line we have been able to explore the possible routes for breaking of prolonged G1 arrest in these Ph+ leukemias. We inhibited the DNA damage sensor protein ataxia telangiectasia mutated (ATM) and found that prolonged G1 arrest in our S9 cells was broken. While previous research has suggested that the DNA damage sensor protein ataxia-telangiectasia mutated (ATM) has little impact in CML, our research indicates that ATM may play a role in either senescence induction or release.
ContributorsDixon, Sarah E (Author) / Chang, Yung (Thesis advisor) / Clark-Curtiss, Josephine (Committee member) / Touchman, Jeffrey (Committee member) / Arizona State University (Publisher)
Created2011
Description
Intrinsic antibiotic resistance is of growing concern in modern medical treatment. The primary action of multidrug resistant strains is through over-expression of active transporters which recognize a broad range of antibiotics. In Escherichia coli, the TolC-AcrAB complex has become a model system to understand antibiotic efflux. While the structures of these three proteins (and many of their homologs) are known, the exact mechanisms of interaction are still poorly understood. By mutational analysis of the TolC turn 1 residues, a drug hypersensitive mutant has been identified which is defective in functional interactions with AcrA and AcrB. Antibiotic resistant revertants carry alterations in both TolC and AcrA act by stabilizing functional complex assembly and opening of the TolC aperture, as monitored by stability of a labile TolC mutant and sensitivity to vancomycin, respectively. Alterations in the AcrB periplasmic hairpin loops lead to a similar antibiotic hypersensitivity phenotype and destabilized complex assembly. Likewise, alterations in TolC which constitutively open the aperture suppress this antibiotic sensitivity. Suppressor alterations in AcrA and AcrB partially restore antibiotic resistance by mediating stability of the complex. The AcrA suppressor alterations isolated in these studies map to the three crystallized domains and it is concluded they alter the AcrA conformation such that it is permanently fixed in an active state, which wild type only transiently goes through when activated by AcrB. Through this genetic evidence, a direct interaction between TolC and AcrB which is stabilized by AcrA has been proposed. In addition to stabilizing the interactions between TolC and AcrB, AcrA is also responsible for triggering opening of the TolC aperture by mediating energy flow from AcrB to TolC. By permanently altering the conformation of AcrA, suppressor mutants allow defective TolC or AcrB mutants to regain functional interactions lost by the initial mutations. The data provide the genetic proof for direct interaction between AcrB and that AcrA mediated opening of TolC requires AcrB as a scaffold.
ContributorsWeeks, Jon William (Author) / Misra, Rajeev (Thesis advisor) / Stout, Valerie (Committee member) / Shi, Yixin (Committee member) / Clark-Curtiss, Josephine (Committee member) / Arizona State University (Publisher)
Created2012
Description
First-semester student retention is a constant priority for undergraduate institutions. The transition to the collegiate level, and to a new scholastic program and format, is frequently challenging academically and socially—for this reason, many first-semester course schedules for incoming freshman undergraduates feature an introductory seminar to ease transition to an undergraduate lifestyle. Arizona State University features a required “Careers in the Life Sciences” course for its first-semester School of Life Sciences students, which has had tractable results in first semester student retention and academic success. Here, we evaluate a component of the seminar, the peer-mentorship program, for its efficacy in students’ first semester experience. Analysis of self-reports from 168 first-semester “mentees” and their 25 mentors indicates frequency of mentee-mentor contact was the best indicator of a higher first semester GPA, comfort with academic resources and study habits, and desire to engage in extracurricular activities and internships. These data indicate that access to a mentor who actively engages and verbally connects with their mentees is a valuable component of first-semester student academic integration and retention.
ContributorsMathews, Ian T. (Author) / Capco, David (Thesis director) / Clark-Curtiss, Josephine (Committee member) / Harrell, Carita (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05
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
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