Browsing by Subject "Plasmid"

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    Characterization of the effects of regulators PrgY and PrgZ on the inducibility of plasmid pCF10 in Enterococcus faecalis
    (2012-09) Barr, Frank T.
    Enterococcus faecalis is an increasingly significant pathogenic bacterium in humans, as well as a commensal member of the intestinal flora. Pheromone-responsive plasmid systems play an important role in the dissemination of antibiotic resistance among this and other species. pCF10 is a well-characterized member of this class of plasmids, and allows for transmission of tetracycline resistance in E. faecalis. The experiments described in this thesis were designed to assess the effects of three genes thought to be important for the response of plasmid pCF10 to the peptide mating pheromone cCF10 in E. faecalis: 1) the chromosomal locus responsible for pheromone production- ccfA, 2) the plasmid-encoded negative regulator prgY thought to be responsible for sequestration of endogenous pheromone at the cell surface, and 3) the plasmid-encoded positive regulator prgZ, an oligopeptide permease subunit A homolog responsible for specific import of pheromone. In this study transcription emanating from regulatory regions of this plasmid was characterized over time using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results from these assays were confirmed using phenotypic analysis and Northern blotting. Expression of ccfA was shown to be important to moderate the pheromone response. This moderation effect is believed to be the result of basal induction of donor cells, resulting in production of the peptide inhibitor iCF10 from the plasmid. As the first study to probe PrgY function at the level of transcription, RNA quantitation revealed that levels of endogenous pheromone cCF10 resulting from processing of the CcfA lipoprotein precursor are insufficient to fully induce a conjugation response in the absence of negative regulator PrgY, suggesting secondary negative regulation. Finally, PrgZ was shown to be important for regulated shutdown of the conjugation response in addition to initiation. This lends evidence to previous research suggesting specific import of inhibitor iCF10 by PrgZ. Probing the effects of these regulators over time in strains that differ in their ability to secrete pheromone allows us to paint a more complete regulatory picture of this complex and dynamic system.
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    Enhanced Efficacy of Plasmid DNA Vaccines for Cancer Therapy
    (2010-11) Dietz, Wynette
    Since cancer is the second most common cause of death in the United States, it is of great importance to pursue new and improved methods for treating cancer. The goal of cancer immunotherapy is to exploit the specificity and longevity of immune responses through the use of vaccines to treat cancer. DNA vaccines have many advantages over protein and viral vaccine-based strategies including low cost, ease of production, flexibility and low toxicity. Plasmid DNA vaccines encoding tumor antigens can produce powerful anti-tumor immune responses in animal models, but clinical trials have shown only modest responses. This lack of clinical efficacy is thought to reflect the two major limitations of plasmid DNA vaccines: transient protein expression and low transfection efficiency. Transient protein expression is likely the result of gene silencing due to transcriptionally repressive chromatin within the plasmid backbone. To overcome this limitation, we removed the bacterial backbone sequences and produced a minicircle DNA consisting of the gene expression cassette with only a few bases of the bacterial backbone. This resulted in persistent protein expression, increased transfection efficiency and enhanced immunogenicity. In an effort to further enhance the transfection efficiency, we produced cationic carriers that bind plasmid DNA and protect it from degradation. The addition of these cationic carriers significantly increased transfection efficiency in vitro but has yet to show the same effect in vivo. Additionally, we administered these vaccines transdermally using a tattoo device and achieved rapid and potent immune responses. Our results suggest transdermal delivery of a minicircle DNA vaccine elicits potent antigen-specific immune responses, and as such, holds great promise for cancer therapy. Future work will include increasing transfection efficiency in vivo and increasing the immunogenicity of the vaccines through the use of adjuvants with the goal of producing feasible, efficacious DNA vaccines for cancer therapy.