UMD Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/3
New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a given thesis/dissertation in DRUM.
More information is available at Theses and Dissertations at University of Maryland Libraries.
Browse
73 results
Search Results
Item A study of unusual metabolic variants of Aeromonas caviae and Aeromonas hydrophila using a polyphasic taxonomic approach(2010) Chang, Zenas; Joseph, Sam W; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Variation in acid production from carbohydrate metabolism has been identified in Aeromonas as a potential indicator for new subspecies. Therefore, pure cultures of non-lactose fermenting Aeromonas caviae, a cause of waterborne infections in humans and other vertebrates, were studied after noting a mixture of acid producing and non-acid producing colonies after four days of incubation on MacConkey agar at ambient temperature. Unusual arabinose negative strains of A. hydrophila (usually arabinose positive) were added to the project to further study the correlation between carbohydrate fermentation and taxonomy. These metabolic variants of A. caviae and A. hydrophila were studied for phenotypic differences via carbohydrate utilization assays as well as genotypic differences via FAFLP. The results suggest that the A. caviae isolates MB3 and MB7 should be considered novel subspecies, while the arabinose negative strain designated A. hydrophila subsp. dhakensis is correctly identified as a subspecies of A. hydrophila.Item Kinetics of Tetrachloroethene-Respiring Dehalobacter and Dehalococcoides Strains and Their Effects on Competition for Growth Substrates(2010) Lai, Yenjung; Becker, Jennifer G; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The chlorinated solvents tetrachloroethene (PCE) and trichloroethene (TCE) are common groundwater contaminants. Reductive dechlorination of PCE and TCE at contaminated sites is commonly carried out by dehalorespiring bacteria that utilize these compounds as terminal electron acceptors, but often results in the accumulation of cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC), rather than non-toxic ethene. This project focused on evaluating how interactions among dehalorespiring populations that may utilize the same electron acceptors, electron donors and/or carbon source may affect the extent of PCE dechlorination in situ. These interactions may be particularly important if both Dehalococcoides ethenogenes (Dhc. ethenogenes) and Dehalobacter restrictus (Dhb. restrictus) are present because these bacteria utilize the same electron donor (H2) and both respire PCE and TCE. However, unlike Dhc. ethenogenes, Dhb. restrictus cannot dechlorinate PCE beyond cDCE. Therefore, the outcome of the population interactions may determine the extent of detoxification achieved. Monod kinetic parameter estimates that describe chlorinated ethene and electron donor utilization by Dhc. ethenogenes and Dhb. restrictus at non-inhibitory substrate concentrations were obtained in batch assays. Substrate inhibition effects on both populations were also evaluated. Highly chlorinated ethenes negatively impacted dechlorination of the lesser chlorinated ethenes in both populations. In Dhc. ethenogenes, cometabolic transformation of VC was also inhibited by the presence of other chlorinated ethenes. PCE and TCE dechlorination by Dhb. restrictus was strongly inhibited by VC. The microbial interactions between Dhc. ethenogenes and Dhb. restrictus was investigated using reactors and mathematical models under engineered bioremediation and natural attenuation conditions. Under engineered bioremediation conditions, Dhc. ethenogenes became the dominant population, and the modeling predictions suggested that the inhibition of Dhb. restrictus by VC was a key factor in determining this outcome. Dechlorination rates by Dhb. restrictus appeared to be affected very little by low acetate concentrations under natural attenuation conditions, giving it an advantage over Dhc. ethenogenes, which requires relatively high acetate concentrations. This study highlighted that substrate interactions among dehalorespiring bacteria can influence their performance and contaminant fate under common bioremediation scenarios. A better understanding of the factors affecting the outcomes of these microbial interactions was achieved, which should aid in the design of successful bioremediation strategies.Item IDENTIFICATION OF PUTATIVE O-REPEAT BIOSYNTHETIC GENES IN NEISSERIA SICCA 4320(2010) Miller, Clinton; Stein, Daniel C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lipopolysaccharide (LPS) and lipooligosaccharide (LOS) are important virulence determinants found in gram-negative bacteria. LOS differs from LPS in that it lacks the O-repeat characteristic of LPS. While the genetic basis of LOS production in the pathogenic Neisseria has been extensively studied, little research has focused on the genetics underlying LOS production and resulting diversity in commensal Neisseria. A commensal strain that caused a fatal case of bacterial endocarditis, Neisseria sicca 4320, was found to produce a unique polysaccharide similar to the O-repeat of LPS in addition to typical Neisseria LOS. N. sicca 4320 was analyzed by bioinformatic and molecular biological gene-finding screens to identify putative O-repeat biosynthesis genes. Twenty-one open reading frames (ORFs) with similarity to other polysaccharide biosynthesis genes were located in the screens of N. sicca 4320. Two open reading frames with similarity to glycosyltransferases were found to be unique to N. sicca 4320.Item The Role of ErbB Receptors in Neisseria gonorrhoeae Invasion of Genital Epithelial Cells(2010) Swanson, Karen; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, adheres to and invades genital epithelial cells. This study investigates host components that are used by the bacteria for their entry into epithelial cells. I found that the interaction of gonococci with the surface of HEC-1-B, a human endometrial carcinoma, and ME180, a human cervical epidermoid carcinoma, caused redistribution of both epidermal growth factor receptor (EGFR) and ErbB2, a related family member. Both EGFR and ErbB2 were translocated from the basolateral to the apical membrane in polarized HEC-1-B cells and concentrated under the microcolonies. Gonococcal infection increased EGFR and ErbB2 phosphorylation, indicating activation of the receptors. Kinase inhibitors of EGFR and ErbB2 inhibited and enhanced bacterial invasion, respectively, but had no effect on gonococcal adherence or the recruitment of EGFR and ErbB2 to the microcolonies. Gonococcal inoculation upregulated the transcription levels and matrix metalloproteinases (MMP)-mediated surface shedding of ligands of EGFR. Inhibition of the surface shedding of EGFR ligands by an MMP inhibitor and by heparin wash reduced gonococcal invasion without altering their adherence. N. gonorrhoeae induced the activation of the MAP Kinase ERK, PI3K/AKT and PLCγ signaling pathways in an EGFR tyrosine kinase-dependent manner. Blocking Ca2+ flux, the downstream pathway of PLCγ but not ERK and PI3K by inhibitors reduced gonococcal invasion. These data indicate that N. gonorrhoeae utilizes host signaling pathways to drive its invasion. The bacteria modulates host signaling by recruiting and activating EGFR and ErbB2. N. gonorrhoeae induces EGFR activation by increasing the expression and MMP-mediated shedding of EGFR ligands.Item CHARACTERIZATION OF THE ROLE OF MAPKS IN LEISHMANIA INFECTED MACROPHAGES.(2009) Yang, Ziyan; Mosser, David M.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In the current study, we examined the role of the Mitogen Activated Protein Kinases (MAPKs) on the biological responses of macrophages infected with Leishmania. The first section examined the role of MAPK/ERK in IL-10 production by Leishmania-infected macrophages. The macrophage-derived IL-10 has been shown to exacerbate Leishmaniasis. However, the molecular mechanisms whereby Leishmaniasis prompts IL-10 induction are poorly understood. A combination of two signals was necessary for IL-10 induction by the Leishmania amastigotes-infected macrophages. The first signal is mediated by TLR ligation whereas the second signal is mediated by FcgammaR ligation, which yields a population of regulatory macrophages that produce high levels of IL-10. Infection of macrophages with L. amazonensis amastigotes from the lesion sites sparked MAPK/ERK activation, which was required, but not sufficient for IL-10 induction. In combination with an inflammatory stimulus, LMW-HA from the extracellular matrix, these parasites triggered the macrophages to highly produce IL-10. MAPK/ERK activation initiated an epigenetic modification of chromatin at the IL-10 locus, which allowed for transcription factor Sp1 binding to drive IL-10 transcription and subsequent production. U0126, an inhibitor of MAPK/ERK activation, decreased lesion progression in Leishmania infected mice. The second section examined the role of MAPK/p38 in cytokine production and vaccination against Leishmaniasis. TLR agonists activate macrophages to produce pro-inflammatory cytokines and reactive oxygen intermediates. Inhibition of MAPK/p38 reciprocally increased IL-12 but decreased TNFa production from LPS-stimulated macrophages, which also occurred following stimulation by a variety of other TLR agonists, and using different APCs. MAPK/p38 inhibition induced IL-12p40 mRNA accumulation mainly due to enhanced mRNA stability, which was independent of IL-10. Similar results were observed by knocking down MAPK/p38 using specific siRNAs or by targeted deletion of MKK3. IL-12 production following the inhibition of MAPK/p38 skewed antigen-specific T cells to produce more IFN-gamma and less IL-4 in vitro. A MAPK/p38 inhibitor was applied as an adjuvant to vaccine mice against L. major, which resulted in smaller lesions with fewer parasites. Our findings reveal an important role of MAPKs in the Leishmania pathogenesis, and suggest that the manipulation of these kinases may provide novel therapeutics for potential clinical applications.Item Evaluation of the transcription of small RNA SgrS and glucose transporter mRNA ptsG in E. coli B and E. coli K cultures under high glucose conditions(2009) Ng, Weng Ian; Wang, Nam Sun; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Escherichia coli is commonly used as the production system for recombinant proteins. However, acetate accumulation in fermentation affects cell growth and protein yield. Recent studies have showed that the small RNA SgrS regulates the major glucose transporter mRNA ptsG in a post–transcriptional manner when the metabolic intermediate glucose–6–phosphate is accumulated intracellularly in E. coli K. Here, comparative analysis of the transcription of SgrS and ptsG is performed between E. coli B and E. coli K cultures in both shake flasks and bioreactor. Both strains expressed SgrS when grown on the non–metabolizable glucose analog α–methyl–glucoside. However, under high glucose conditions, only E. coli B showed significant expression of SgrS. This behavior is unaffected by oxygen supply and pH control. E. coli B produced less acetate on glucose than E. coli K in the bioreactor settings. This provides evidence of a possible connection between SgrS and acetate production in aerobic fermentation of E. coli.Item Molecular Mechanisms of the Inhibition of Apoptosis by Mycobacterium tuberculosis(2009) Miller, Jessica Lynn; Briken, Volker; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The capacity of infected cells to undergo apoptosis upon insult with a pathogen is an ancient innate immune defense mechanism. Consequently, the ability of persistent intracellular pathogens, such as the human pathogen Mycobacterium tuberculosis (Mtb), to inhibit infection-induced apoptosis of macrophages is important for virulence and to achieve persistence in the host. The nuoG gene of Mtb, which encodes the NuoG subunit of the type I NADH dehydrogenase NDH-1, is important in Mtb-mediated inhibition of host macrophage apoptosis. Here I determine the molecular mechanisms of this host-pathogen interaction. Apoptosis induced by the nuoG deletion mutant (nuoG ) is caspase-8 and TNF-α dependent. This cell death was also reduced in the presence of neutralizers and inhibitors of reactive oxygen species (ROS) and in macrophages derived from NOX2 deficient mice, suggesting that DnuoG induced death is dependent upon NOX2 derived ROS. Correlatively, nuoG infected macrophages also produced more phagosomal ROS than those infected with Mtb, or cells derived from NOX2 deficient mice. NuoG also inhibited apoptosis in human alveolar macrophages in a NOX2 dependant manner. These data suggest that reduction of phagosomal ROS is important for inhibition of apoptosis. Consistent with this hypothesis, Mtb deficient in the ROS neutralizing catalase, KatG, also accumulated ROS in the phagosome and was pro-apoptotic in macrophages. The specific mechanism by which NuoG reduces phagosomal ROS is still unknown. We could not detect secretion of NuoG, so direct neutralization of ROS is unlikely. Interestingly, preliminary data suggests that nuoG may be defective in secretion of SodA and KatG, enzymes known to be important for neutralizing ROS. In conclusion, these studies revealed that Mtb inhibits macrophage apoptosis by neutralizing phagosomal ROS via the NuoG dependent secretion of SodA and KatG. Furthermore, this research suggests a novel function for NOX2 activity in innate immunity, which is the sensing of persisting intracellular pathogens and subsequent induction of host cell apoptosis as a second line of defense for pathogens resistant to the respiratory burst.Item In situ Activity of NAC11-7 roseobacters in Coastal Waters off the Chesapeake Bay based on ftsZ Expression(2009) Yao, Daohong; Suzuki, Marcelino T; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Phylogenetic analysis of sequences of the cell division gene ftsZ retrieved from the Atlantic coast revealed an interesting subgroup NAC11-7, which was targeted by a specifically designed and optimized Taqman assay in diel samples collected in situ and in parallel on-deck incubations. Rapid changes of ftsZ gene copies and the patchy distribution of other phylotypes at different time points suggested that different NAC11-7 populations were sampled. Strong correlations between ftsZ expression and gene abundance (r-squared=0.62), and between ftsZ expression and water temperature (r-squared=0.73) for in situ samples suggested non-synchronous growth of NAC11-7 group. Contrastingly, a sharp 9:00 AM peak of ftsZ expression in the on deck incubation experiment suggested synchronous growth. We propose a possible mixed model in which a certain fraction of the population is synchronously dividing, while a background of asynchronously dividing NAC11-7 cells also exist, some of which are expressing ftsZ at any given time.Item Phylogenetic analysis of swine influenza viruses isolated from humans in Alma-Ata, Kazakhstan(2009) Padmanabhan, Rangarajan; Perez, Daniel; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Continuous surveillance of influenza becomes important considering the economic, epidemic and pandemic implications of influenza infections. This study details phylogenetic & molecular analysis of the genes of four swine influenza viruses isolated from humans in Alma-Ata, Kazakhstan. Phylogenetic analysis placed the eight segments of the four viruses in the classical H1N1 swine clade, along with the isolate A/sw/Jamesburg/1942, except for the HA of A/Alma-Ata/32/98, which was placed in the human H1N1 lineage, along with the isolate A/WS/1933. On amino acid analysis, the viruses displayed mutations on HA and ribonucleoproteins which putatively disrupt antigenic recognition of the virus by the host immune system. The presence of these viruses relatively unchanged for 6 decades after their initial isolation could be speculated to be a combination of laboratory leaks in southern USSR in 1980s, low divergence of classical H1N1 viruses in pigs, and the low population density of Kazakhstan.Item COMPARING LEVELS OF NDH-1 DEHYDROGENASE ACTIVITY IN DIFFERENT MYCOBACTERIAL SPECIES.(2008) Azogue, Sharon; Briken, Volker; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The nuoG gene of Mycobacterium tuberculosis (Mtb) has the ability to inhibit host cell apoptosis. This ability is a virulence factor and does not exist in facultative pathogenic and non-pathogenic mycobacterial species. NuoG is part of the NDH-1 complex, and this study addressed the potential link between the role of NuoG in apoptosis inhibition and the biochemical activity of the NDH-1 complex. Different mycobacterial species were tested for their NDH-1 activities. Among the bacteria tested were bacteria transformed with the Mtb nuoG plasmid, or with the almost entire NDH-1 coding region. Surprisingly, the levels of NDH-1 activity did not correlate with apoptosis levels, suggesting a potential independent, novel mechanism by which NuoG inhibits host cell apoptosis.