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- All Subjects: Cystic Fibrosis
- All Subjects: Biochemistry
- Resource Type: Text
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
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
Mycobacterium abscessus (Mabs) is a multidrug-resistant nontuberculous mycobacterium capable of causing persistent pulmonary infection. It most prominently threatens those with cystic fibrosis (CF), a progressive and genetic disorder characterized by an immunocompromised respiratory tract. Current treatments fail to eradicate Mabs, meaning novel alternatives are greatly needed. Antimicrobial peptides (AMPs) are short sequences of amino acids that display broad-spectrum antimicrobial activity and play an important role in innate immunity. To maximize their therapeutic potential, key AMP features can be rationally combined through an iterative engineering process to create synthetic, designed AMPs (dAMPs). In this investigation, two dAMPs, RP554 and RP557, reduced Mabs ATCC 19977 viability by 99.99% and were subjected to further testing. In antimicrobial susceptibility testing with Mabs ATCC 19977, RP554 and RP557 demonstrated bactericidal activity at concentrations 16-32 μM. Complete killing of Mabs ATCC 19977 by RP554 and RP557 occurred rapidly in <24 h. RP554 and RP557 also inhibited 20 Mabs clinical isolates obtained from CF patients. Furthermore, RP554 and RP557 retained anti-Mabs activity after pre-exposure to human serum, indicating potential stability in blood. Conversely, the tested dAMPs did not kill Mabs during in vitro experiments in an artificial sputum medium. Novel antimicrobials, such as the RP554 and RP557 dAMPs, offer therapeutic potential for otherwise resistant bacterial pathogens, including Mabs, that afflict both CF and non-CF patients.
ContributorsBrandt, McKenzie (Author) / Haydel, Shelley (Thesis director) / Bean, Heather (Committee member) / Dermody, Roslyn (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2022-05