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Description
Pathogenic Gram-negative bacteria employ a variety of molecular mechanisms to combat host defenses. Two-component regulatory systems (TCR systems) are the most ubiquitous signal transduction systems which regulate many genes required for virulence and survival of bacteria. In this study, I analyzed different TCR systems in two clinically-relevant Gram-negative bacteria, i.e.,

Pathogenic Gram-negative bacteria employ a variety of molecular mechanisms to combat host defenses. Two-component regulatory systems (TCR systems) are the most ubiquitous signal transduction systems which regulate many genes required for virulence and survival of bacteria. In this study, I analyzed different TCR systems in two clinically-relevant Gram-negative bacteria, i.e., oral pathogen Porphyromonas gingivalis and enterobacterial Escherichia coli. P. gingivalis is a major causative agent of periodontal disease as well as systemic illnesses, like cardiovascular disease. A microarray study found that the putative PorY-PorX TCR system controls the secretion and maturation of virulence factors, as well as loci involved in the PorSS secretion system, which secretes proteinases, i.e., gingipains, responsible for periodontal disease. Proteomic analysis (SILAC) was used to improve the microarray data, reverse-transcription PCR to verify the proteomic data, and primer extension assay to determine the promoter regions of specific PorX regulated loci. I was able to characterize multiple genetic loci regulated by this TCR system, many of which play an essential role in hemagglutination and host-cell adhesion, and likely contribute to virulence in this bacterium. Enteric Gram-negative bacteria must withstand many host defenses such as digestive enzymes, low pH, and antimicrobial peptides (AMPs). The CpxR-CpxA TCR system of E. coli has been extensively characterized and shown to be required for protection against AMPs. Most recently, this TCR system has been shown to up-regulate the rfe-rff operon which encodes genes involved in the production of enterobacterial common antigen (ECA), and confers protection against a variety of AMPs. In this study, I utilized primer extension and DNase I footprinting to determine how CpxR regulates the ECA operon. My findings suggest that CpxR modulates transcription by directly binding to the rfe promoter. Multiple genetic and biochemical approaches were used to demonstrate that specific TCR systems contribute to regulation of virulence factors and resistance to host defenses in P. gingivalis and E. coli, respectively. Understanding these genetic circuits provides insight into strategies for pathogenesis and resistance to host defenses in Gram negative bacterial pathogens. Finally, these data provide compelling potential molecular targets for therapeutics to treat P. gingivalis and E. coli infections.
ContributorsLeonetti, Cori (Author) / Shi, Yixin (Thesis advisor) / Stout, Valerie (Committee member) / Nickerson, Cheryl (Committee member) / Sandrin, Todd (Committee member) / Arizona State University (Publisher)
Created2013
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Description
The ability of microalgae to be mass cultivated and harvested for production of pharmaceuticals, nutraceuticals, and biofuels has made microalgae a focal point of scientific investigation. However, negative impacts on production are essentially inevitable due to the open design of many microalgae mass culture systems. This challenge generates

The ability of microalgae to be mass cultivated and harvested for production of pharmaceuticals, nutraceuticals, and biofuels has made microalgae a focal point of scientific investigation. However, negative impacts on production are essentially inevitable due to the open design of many microalgae mass culture systems. This challenge generates a need for the consistent monitoring of microalgae cultures for health and the presence of contaminants, predators, and competitors. The techniques for monitoring microalgae cultures are generally time-intensive, labor-intensive, and expensive. The scope of this work was to evaluate the use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a viable alternative for the characterization of microalgae cultures. The studies presented here evaluated whether MALDI-TOF MS can be used to: 1) differentiate microalgae at the species and strain levels, 2) characterize simple mixtures of microalgae, 3) detect changes in a single microalgae culture over time, and 4) characterize growth phases of microalgae cultures. This research required the development of a MALDI-TOF MS microalgae analysis protocol for organism characterization. The results yielded in this research showed that MALDI-TOF MS was just as accurate, if not more so, than molecular techniques for the identification of microalgae at the species and strain levels during its logarithmic growth phase. Additionally, results suggest that MALDI-TOF MS is sensitive enough to characterize simple mixtures and detect changes in cultures over time. The data presented here suggests the next logical step is the development of protocols for the near-real time health monitoring of microalgae cultures and detection of contaminants using MALDI-TOF MS.
ContributorsBarbano, Duane (Author) / Sandrin, Todd (Thesis advisor) / Webber, Andrew (Committee member) / Dempster, Thomas (Committee member) / Arizona State University (Publisher)
Created2016