Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3298. Adviser: Steven R. Blanke. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007. / Source: Dissertation Abstracts International, Volume: 68-11, Section: B, page: 7105. Adviser: James Imlay. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 70-10, Section: B, page: 5972. Adviser: Joanne N. Engel.

<p> Previous work has identified a protein-lipid complex from human milk (HAMLET) that effectively kills the prominent human respiratory bacterial pathogen <i>Streptococcus pneumoniae</i> (pneumococcus), and was serendipitously found to also kill tumor cells by inducing apoptosis. Two of the most attractive characteristics of HAMLET's bactericidal activity are that it employs a specific mechanism separate from common antibiotics, and pneumococci are unable to develop resistance to it <i>in vitro.</i> The <b>objective</b> of our studies was to develop a greater mechanistic understanding of HAMLET-induced death in the pneumococcus. Based on phenotypic parallels observed in bacteria and tumor cells following exposure to HAMLET, we <b>hypothesized</b> that HAMLET kills <i>S. pneumoniae</i> by binding to the surface and inducing an apoptosis-like pathway of death, including related ion transport events. In <b>Part 1</b>, we sought to elucidate mechanistic events that occur during this pathway, with a focus on the pneumococcal membrane, including membrane polarity and ion transport events. After establishing methods of monitoring these events using fluorescent indicator dyes and radioisotopes, we found that HAMLET dissipates the polarity and integrity of the pneumococcal membrane, and causes a sodium-dependent calcium influx that is required for death, which was effectively inhibited by both calcium and sodium transport inhibitors. The addition of kinase inhibitors also inhibited death, indicating a role for Ser/Thr kinases. Significantly, this mechanistic pathway appears to also be employed during starvation-induced death, as the same transport and kinase inhibitors also disrupted autolysis activation and biofilm formation. In additional studies, we found that pneumococcal death with these same mechanistic features can be induced by a related protein, equine lysozyme, in complex with oleic acid (ELOA). In <b>Part 2</b>, we explored the interaction of HAMLET with the pneumococcal surface and found that choline, a major component of cell surface teichoic acids, is important for HAMLET's bactericidal activity, as choline-free pneumococci were less susceptible to HAMLET-induced death and other mechanistic features including membrane perturbations and ion transport. Further studies showed that addition of exogenous lipoteichoic acid could effectively block HAMLET-induced death, suggesting that this molecule concentrates HAMLET at the surface to initiate its lethal activity. As the incidence of infections caused by antibiotic-resistant strains of <i>S. pneumoniae</i> continues to rise, developing a better understanding of HAMLET's bactericidal activity and the molecular targets and activities involved is important, as it has great potential to uncover new targets for antimicrobial intervention.</p>

<p>Septins are a highly conserved family of eukaryotic proteins having significant homology within and among species. In the budding yeast, Saccharomyces cerevisiae, a septin-based hierarchy of proteins is required to localize chitin in the bud neck prior to septum formation. However, this process has not been clarified in a filamentous, conidiogenous fungus capable of yeast growth, such as Wangiella dermatitidis, a polymorphic agent of human phaeohyphomycosis. Prior studies of this melanized mold showed that some chitin synthase mutants (wdchsa??) have defects in yeast septum formation, suggesting that the septins of W. dermatitidis might functionally associate with some of its chitin synthases (WdChsp). To test this hypothesis, four vegetative septin homologs of S. cerevisiae were cloned from W. dermatitidis and designated WdCDC3, WdCDC10, WdCDC11, and WdCDC12. Of the four, only WdCDC3 functionally complemented completely a strain of S. cerevisiae with a ts mutation in the corresponding gene, although WdCDC12 did so partially. Functional characterizations by mutagenesis of the four W. dermatitidis septin genes revealed that resulting mutants (wdcdca??) each had unique defects in yeast growth and morphology, indicating that each septin carried out a distinct function. Furthermore, when a wdcdc10a?? mutation was introduced into five different wdchsa?? strains, weak genetic interactions were detected between WdCDC10 and WdCHS3 and WdCHS4, and a strong interaction between WdCDC10 and WdCHS5. Cytological studies showed that WdChs5p was mislocalized in some septin mutants, including wdcdc10a??. These results confirmed that in W. dermatitidis septins are important for proper cellular morphogenesis, cytokinesis, and especially septum formation through associations with some chitin synthases.

Thesis (Ph.D.)--University of California, San Francisco, 2005. / Source: Dissertation Abstracts International, Volume: 66-12, Section: B, page: 6408. Adviser: Jeffery S. Cox.

Thesis (Ph.D.)--University of California, San Francisco, 2008. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0877. Adviser: Ronald D. Vale.

<p> In bacteria, stress can produce a variety of distinct phenotypes, including competence, sporulation, persistence, dormancy, and lysis. Yet despite a veritable mountain of genomic data and a growing understanding of the molecular mechanisms, characterizing the rules that dictate the expressed phenotype remains a challenge. This work bridges the gap for three systems at the heart of bacterial stress response: toxin-antitoxin regulation under stress-induced proteolysis, amino acid biosynthesis subject to starvation and the stringent response, and lysogen induction triggered by DNA damage. In each case, a model of the molecular mechanisms is analyzed using novel techniques, and the results quantitatively, rather than qualitatively, describe the kinetic or environmental changes that produce distinct phenotypic behaviors. The results agree with published experiments, answer several open questions, and offer new insights into the links between molecular mechanisms, stress, and cell fate. The core process&mdash;the construction and analysis of the system design space&mdash;is formalized and automated, offering a tantalizing glimpse of the future in which the full phenotypic repertoire of a system can be predicted and explored.</p>

<p> <i>Sinorhizobium meliloti</i> is a Gram-negative alphaproteobacterium and nitrogen-fixing symbiont, which undergoes a novel cell cycle modification during its' host-microbe interaction. I intend to monitor the transcriptional regulation of cell cycle-related genes during free-loving growth, in addition to monitoring their expression during symbiosis. Using genes known to be regulated by CtrA in <i>C. crescentus</i> or predicted to be regulated by CtrA in <i>S. meliloti,</i> I aim to show how certain cell cycle genes are regulated in <i>S. meliloti.</i> In <i>C. crescentus, </i> CtrA acts as a transcription factor that is active when phosphorylated and inactive when not phosphorylated. In <i>S. meliloti,</i> CbrA is a histidine kinase that ultimately inhibits CtrA phosphorylation. Using a &Delta;<i>cbrA</i> null mutant, which leads to increased levels of CtrA in <i>S. meliloti,</i> and the &beta;-glucuronidase (GUS) reporter gene, I can monitor the expression levels of target genes that are potentially regulated by CbrA and CtrA. Promoter regions, transcription start sites, and translation start sites of target genes have been cloned into the plasmid pVO155 upstream of the GUS gene. I measured the GUS enzymatic activity using the 4-methylumelliferyl-beta-D-glucuronide (MUG) substrate. Additionally, after infecting <i>Medicago sativa</i> seedlings with these fusions strains, I used a different GUS substrate to test for the presence of target gene expression in root nodules. Results thus far have shown some target genes with large differences in expression coinciding with the absence of <i> cbrA</i> and increased CtrA levels while some target genes show only slight differences, if any at all. Tracking the expression location and patterns of target genes in root nodules has shown that some genes are expressed ubiquitously throughout the nodule while other genes are expressed in specific locations. These results are significant because no one has looked at genes regulated by CbrA or CtrA in <i>S. meliloti,</i> which is more applicable to host-microbe interactions than <i>C. crescentus,</i> especially since <i>Agrobacterium tumefaciens</i> and <i>Brucella abortus </i> both have a CbrA homologue. Additionally, I will provide critical insight into the molecular biology of the <i>S. meliloti</i> host-microbe interaction.</p>

Thesis (Ph.D.)--University of California, San Francisco, 2006. / Source: Dissertation Abstracts International, Volume: 67-08, Section: B, page: 4235. Adviser: Joseph M. McCune.