Matching Items (17)
Description
The Multiple Antibiotic Resistance Regulator Family (MarR) are transcriptional regulators, many of which forms a dimer. Transcriptional regulation provides bacteria a stabilized responding system to ensure the bacteria is able to efficiently adapt to different environmental conditions. The main function of the MarR family is to create multiple antibiotic resistance from a mutated protein; this process occurs when the MarR regulates an operon. We hypothesized that different transcriptional regulator genes have interactions with each other. It is known that Salmonella pagC transcription is activated by three regulators, i.e., SlyA, MprA, and PhoP. Bacterial Adenylate Cyclase-based Two-Hybrid (BACTH) system was used to research the protein-protein interactions in SlyA, MprA, and PhoP as heterodimers and homodimers in vivo. Two fragments, T25 and T18, that lack endogenous adenylate cyclase activity, were used for construction of chimeric proteins and reconstruction of adenylate cyclase activity was tested. The significant adenylate cyclase activities has proved that SlyA is able to form homodimers. However, weak adenylate cyclase activities in this study has proved that MprA and PhoP are not likely to form homodimers, and no protein-protein interactions were detected in between SlyA, MprA and PhoP, which no heterodimers have formed in between three transcriptional regulators.
ContributorsTao, Zenan (Author) / Shi, Yixin (Thesis advisor) / Wang, Xuan (Committee member) / Bean, Heather (Committee member) / Arizona State University (Publisher)
Created2018
Description
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the 10th leading cause of death, worldwide. The prevalence of drug-resistant clinical isolates and the paucity of newly-approved antituberculosis drugs impedes the successful eradication of Mtb. Bacteria commonly use two-component systems (TCS) to sense their environment and genetically modulate adaptive responses. The prrAB TCS is essential in Mtb, thus representing an auspicious drug target; however, the inability to generate an Mtb ΔprrAB mutant complicates investigating how this TCS contributes to pathogenesis. Mycobacterium smegmatis, a commonly used M. tuberculosis genetic surrogate was used here. This work shows that prrAB is not essential in M. smegmatis. During ammonium stress, the ΔprrAB mutant excessively accumulates triacylglycerol lipids, a phenotype associated with M. tuberculosis dormancy and chronic infection. Additionally, triacylglycerol biosynthetic genes were induced in the ΔprrAB mutant relative to the wild-type and complementation strains during ammonium stress. Next, RNA-seq was used to define the M. smegmatis PrrAB regulon. PrrAB regulates genes participating in respiration, metabolism, redox balance, and oxidative phosphorylation. The M. smegmatis ΔprrAB mutant is compromised for growth under hypoxia, is hypersensitive to cyanide, and fails to induce high-affinity respiratory genes during hypoxia. Furthermore, PrrAB positively regulates the hypoxia-responsive dosR TCS response regulator, potentially explaining the hypoxia-mediated growth defects in the ΔprrAB mutant. Despite inducing genes encoding the F1F0 ATP synthase, the ΔprrAB mutant accumulates significantly less ATP during aerobic, exponential growth compared to the wild-type and complementation strains. Finally, the M. smegmatis ΔprrAB mutant exhibited growth impairment in media containing gluconeogenic carbon sources. M. tuberculosis mutants unable to utilize these substrates fail to establish chronic infection, suggesting that PrrAB may regulate Mtb central carbon metabolism in response to chronic infection. In conclusion, 1) prrAB is not universally essential in mycobacteria; 2) M. smegmatis PrrAB regulates genetic responsiveness to nutrient and oxygen stress; and 3) PrrAB may provide feed-forward control of the DosRS TCS and dormancy phenotypes. The data generated in these studies provide insight into the mycobacterial PrrAB TCS transcriptional regulon, PrrAB essentiality in Mtb, and how PrrAB may mediate stresses encountered by Mtb during the transition to chronic infection.
ContributorsMaarsingh, Jason (Author) / Haydel, Shelley E (Thesis advisor) / Roland, Kenneth (Committee member) / Sandrin, Todd (Committee member) / Bean, Heather (Committee member) / Arizona State University (Publisher)
Created2019
Description
One out of ten women has a difficult time getting or staying pregnant in the United States. Recent studies have identified aging as one of the key factors attributed to a decline in female reproductive health. Existing fertility diagnostic methods do not allow for the non-invasive monitoring of hormone levels across time. In recent years, olfactory sensing has emerged as a promising diagnostic tool for its potential for real-time, non-invasive monitoring. This technology has been proven promising in the areas of oncology, diabetes, and neurological disorders. Little work, however, has addressed the use of olfactory sensing with respect to female fertility. In this work, we perform a study on ten healthy female subjects to determine the volatile signature in biological samples across 28 days, correlating to fertility hormones. Volatile organic compounds (VOCs) present in the air above the biological sample, or headspace, were collected by solid phase microextraction (SPME), using a 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) coated fiber. Samples were analyzed, using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS). A regression model was used to identify key analytes, corresponding to the fertility hormones estrogen and progesterone. Results indicate shifts in volatile signatures in biological samples across the 28 days, relevant to hormonal changes. Further work includes evaluating metabolic changes in volatile hormone expression as an early indicator of declining fertility, so women may one day be able to monitor their reproductive health in real-time as they age.
ContributorsOng, Stephanie (Author) / Smith, Barbara (Thesis advisor) / Bean, Heather (Committee member) / Plaisier, Christopher (Committee member) / Arizona State University (Publisher)
Created2018
Description
The purpose of this study was to observe the effectiveness of the phenylalanyl arginine β-naphthylamide dihydrochloride inhibitor and Tween 20 when combined with an antibiotic against Escherichia. coli. As antibiotic resistance becomes more and more prevalent it is necessary to think outside the box and do more than just increase the dosage of currently prescribed antibiotics. This study attempted to combat two forms of antibiotic resistance. The first is the AcrAB efflux pump which is able to pump antibiotics out of the cell. The second is the biofilms that E. coli can form. By using an inhibitor, the pump should be unable to rid itself of an antibiotic. On the other hand, using Tween allows for biofilm formation to either be disrupted or for the biofilm to be dissolved. By combining these two chemicals with an antibiotic that the efflux pump is known to expel, low concentrations of each chemical should result in an equivalent or greater effect on bacteria compared to any one chemical in higher concentrations. To test this hypothesis a 96 well plate BEC screen test was performed. A range of antibiotics were used at various concentrations and with varying concentrations of both Tween and the inhibitor to find a starting point. Following this, Erythromycin and Ciprofloxacin were picked as the best candidates and the optimum range of the antibiotic, Tween, and inhibitor were established. Finally, all three chemicals were combined to observe the effects they had together as opposed to individually or paired together. From the results of this experiment several conclusions were made. First, the inhibitor did in fact increase the effectiveness of the antibiotic as less antibiotic was needed if the inhibitor was present. Second, Tween showed an ability to prevent recovery in the MBEC reading, showing that it has the ability to disrupt or dissolve biofilms. However, Tween also showed a noticeable decrease in effectiveness in the overall treatment. This negative interaction was unable to be compensated for when using the inhibitor and so the hypothesis was proven false as combining the three chemicals led to a less effective treatment method.
ContributorsPetrovich Flynn, Chandler James (Author) / Misra, Rajeev (Thesis director) / Bean, Heather (Committee member) / Perkins, Kim (Committee member) / Mechanical and Aerospace Engineering Program (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
Pseudomonas aeruginosa is a gram-negative bacterium and opportunistic pathogen that is the leading cause of chronic infection in the lungs of adults with cystic fibrosis (CF). During chronic lung infections, P. aeruginosa populations adapt genetically to the CF lung, selecting several important mutations required for long-term persistence. These genetic adaptations lead to phenotypic changes that are associated with the transition from early-stage to late-stage chronic CF infection.
The goal of this project was to develop tools for gene transfer between P. aeruginosa clinical isolates. These tools will allow shuffling of early/late stage of infection genes to restore wild-type phenotypes in late chronic infection isolates and create single-phenotype mutants in the early infection strains. This will allow isolation and investigation of single phenotypes in the clinical isolates to identify metabolic biomarkers specifically for detecting the target phenotypes.
The gene transfer mechanisms of transformation by electroporation, transformation by heat shock, and conjugation were tested using the plasmid pMQ30 with a construct to create an in-frame deletion of the rhlR gene (rhlR) via allelic exchange. The disruption of the P. aeruginosa wild-type rhlR gene leads to rhamnolipids-deficient mutant strains; therefore, rhamnolipids production was assessed to validate successful in-frame deletion of the rhlR gene in the P. aeruginosa clinical isolates and laboratory strains. Based on the efficiencies determined from the gene transfer mechanisms tested, the conjugation mechanism was determined to be the most efficient method for gene transfer in P. aeruginosa laboratory strains, and was used to investigate gene transfer in the P. aeruginosa clinical isolates.
The goal of this project was to develop tools for gene transfer between P. aeruginosa clinical isolates. These tools will allow shuffling of early/late stage of infection genes to restore wild-type phenotypes in late chronic infection isolates and create single-phenotype mutants in the early infection strains. This will allow isolation and investigation of single phenotypes in the clinical isolates to identify metabolic biomarkers specifically for detecting the target phenotypes.
The gene transfer mechanisms of transformation by electroporation, transformation by heat shock, and conjugation were tested using the plasmid pMQ30 with a construct to create an in-frame deletion of the rhlR gene (rhlR) via allelic exchange. The disruption of the P. aeruginosa wild-type rhlR gene leads to rhamnolipids-deficient mutant strains; therefore, rhamnolipids production was assessed to validate successful in-frame deletion of the rhlR gene in the P. aeruginosa clinical isolates and laboratory strains. Based on the efficiencies determined from the gene transfer mechanisms tested, the conjugation mechanism was determined to be the most efficient method for gene transfer in P. aeruginosa laboratory strains, and was used to investigate gene transfer in the P. aeruginosa clinical isolates.
ContributorsBhebhe, Charity Ntando (Author) / Bean, Heather (Thesis director) / Misra, Rajeev (Committee member) / Jenkins, Carrie (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Abstract:
Background: Chronic rhinosinusitis (CRS) is defined as symptomatic inflammation of the nose and paranasal sinuses lasting more than 12 weeks. Persistent inflammation is thought to originate from multiple factors including host physical and innate barrier defects and the exposure of the sinonasal mucosa to exogenous microorganisms. Regional differences in the innate host defense molecules present in nasal and sinus tissue have been recently reported. Thus, a histopathological study was conducted by Lal et al. to compare inflammatory changes in the ethmoid sinus mucosa and nasal turbinate tissue for CRS patients and controls. The objective of this work was to interpret the histopathological data from an immunobiological perspective and describe the significance of the results within the context of current scientific literature.
Methods: Tissue samples were collected from sinonasal surgery patients in three specific regions: ethmoid cells ± uncinate process (EC) in all patients and the inferior (IT) or middle turbinate (MT). EC and IT/MT samples were compared using Cohen’s kappa coefficient to measure agreement based on overall severity of inflammation, eosinophil count per high power field, and the predominant inflammatory cell infiltrate. The results of this study were compared with the current cohort of scientific literature regarding CRS pathogenesis. Both previous and current hypotheses were considered to construct a holistic overview of the development of the current understanding of CRS.
Results: The histopathology study determined that regional differences in degree and type of inflammation may be present in the nose and paranasal cavity. These findings support the current understanding of CRS as an inflammatory disease that is likely mediated by both host and environmental factors.
Conclusions: The histopathology study supports the current cohort of CRS research and provides evidence in support of the involvement of host factors in CRS pathogenesis.
Background: Chronic rhinosinusitis (CRS) is defined as symptomatic inflammation of the nose and paranasal sinuses lasting more than 12 weeks. Persistent inflammation is thought to originate from multiple factors including host physical and innate barrier defects and the exposure of the sinonasal mucosa to exogenous microorganisms. Regional differences in the innate host defense molecules present in nasal and sinus tissue have been recently reported. Thus, a histopathological study was conducted by Lal et al. to compare inflammatory changes in the ethmoid sinus mucosa and nasal turbinate tissue for CRS patients and controls. The objective of this work was to interpret the histopathological data from an immunobiological perspective and describe the significance of the results within the context of current scientific literature.
Methods: Tissue samples were collected from sinonasal surgery patients in three specific regions: ethmoid cells ± uncinate process (EC) in all patients and the inferior (IT) or middle turbinate (MT). EC and IT/MT samples were compared using Cohen’s kappa coefficient to measure agreement based on overall severity of inflammation, eosinophil count per high power field, and the predominant inflammatory cell infiltrate. The results of this study were compared with the current cohort of scientific literature regarding CRS pathogenesis. Both previous and current hypotheses were considered to construct a holistic overview of the development of the current understanding of CRS.
Results: The histopathology study determined that regional differences in degree and type of inflammation may be present in the nose and paranasal cavity. These findings support the current understanding of CRS as an inflammatory disease that is likely mediated by both host and environmental factors.
Conclusions: The histopathology study supports the current cohort of CRS research and provides evidence in support of the involvement of host factors in CRS pathogenesis.
ContributorsElwell, Zachary Andrew (Author) / Blattman, Joseph (Thesis director) / Bean, Heather (Committee member) / Lal, Devyani (Committee member) / School of International Letters and Cultures (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Each year the hospitals in the United States dispose of viable medications worth millions of dollars. These facilities are currently forced to do so not because the medications have expired, or are no longer effective, but rather because to re-use any leftover medications would allow for the possibility of spreading disease. Once a medications sterile seal has been broken, any remaining contents of its container are considered potential pathogenic biohazards, and must be disposed of. The main objective of this thesis was to explore a potential alternative to simply discarding these lifesaving and often expensive leftover medications. The ultimate goal of this work is to establish a process by which excess drugs could be safely and effectively purified for re-use, subsequently cutting costs, and enhancing medication availability. Pseudomonas aeruginosa (P.a.) and Staphylococcus aureus (S.a) were cultured for their commonality in healthcare-associated infections (HAI's), and allowed to contaminate medication-like compounds. These bacterially inoculated solutions were meant to mimic the contaminated medications mentioned above and were then treated with a novel, physical means of pathogen inactivation named SElective PHOtonic DISinfection (SEPHODIS). Pathogen load reduction was determined through plate count assays both before and after exposure to the SEPHODIS system. structural preservation of medication was established through the use of infrared spectroscopy. The results of these experiments furthered the confidence of SEPHODIS as an efficient means of pathogen inactivation, while promoting promise of a real-world application in the form of medication purification.
ContributorsKutemeier, Hayden (Author) / Bean, Heather (Thesis director) / Tsen, Kong-Thon (Committee member) / Barrett, The Honors College (Contributor)
Created2018-05
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
Mycobacterial infections, as represented by leprosy and tuberculosis, have persisted as human pathogens for millennia. Their environmental counterparts, nontuberculous mycobacteria (NTM), are commodious infectious agents endowed with extensive innate and acquired antimicrobial resistance. The current drug development process selects for antibiotics with high specificity for definitive targets within bacterial metabolic and replication pathways. Because these compounds demonstrate limited efficacy against mycobacteria, novel antimycobacterial agents with unconventional mechanisms of action were identified. Two highly resistant NTMs, Mycobacterium abscessus (Mabs) a rapid-growing respiratory, skin, and soft tissue pathogen, and Mycobacterium ulcerans (MU), the causative agent of Buruli ulcer, were selected as targets. Compounds that indicated antimicrobial activity against other highly resistant pathogens were selected for initial screening. Antimicrobial peptides (AMPs) have demonstrated activity against a variety of bacterial pathogens, including mycobacterial species. Designed antimicrobial peptides (dAMPs), rationally-designed and synthetic contingents, combine iterative features of natural AMPs to achieve superior antimicrobial activity in resistant pathogens. Initial screening identified two dAMPs, RP554 and RP557, with bactericidal activity against Mabs. Clay-associated ions have previously demonstrated bactericidal activity against MU. Synthetic and customizable aluminosilicates have also demonstrated adsorption of bacterial cells and toxins. On this basis, two aluminosilicate materials, geopolymers (GP) and ion-exchange nanozeolites (IE-nZeos), were screened for antimicrobial activity against MU and its fast-growing relative, Mycobacterium marinum (Mmar). GPs demonstrated adsorption of MU cells and mycolactone, a secreted, lipophilic toxin, whereas Cu-nZeos and Ag-nZeos demonstrated antibacterial activity against MU and Mmar. Cumulatively, these results indicate that an integrative drug selection process may yield a new generation of antimycobacterial agents.
ContributorsDermody, Roslyn June (Author) / Haydel, Shelley E (Thesis advisor) / Bean, Heather (Committee member) / Nickerson, Cheryl (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2022