Cell Biology & Molecular Genetics Theses and Dissertations

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End date: 2024Subject: search.filters.subject.Biology, Molecular

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    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.
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    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.
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    The Role of Interleukin-19 in Interleukin-10 Production by Regulatory Macrophages
    (2010) Yahil, Ron Jonathan; Mosser, David M.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Interleukin-19 (IL-19) is a recently discovered member of the IL-10 family of Class II cytokines. Although it is known to be secreted by monocytes and has been associated with various models of disease, the biological function of IL-19 remains largely unknown. IL-19 does share many important characteristics with IL-10. Because of this, we hypothesized that IL-19 may be regulated in a manner similar to IL-10, and may provide insight into the molecular mechanism of IL-10 regulation. In addition, IL-19 has been reported to increase IL-10 production in monocytes, and we theorized that it may be able to do the same in macrophages. Like IL-10, IL-19 is expressed in regulatory macrophages. Also, IL-19 is itself able to increase IL-10 production in regulatory macrophages, and the mechanism is independent of ERK phosphorylation. This work suggests that IL-19 can play a central role in the anti-inflammatory processes of IL-10.
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    Role and Regulation of Autophagy During Developmental Cell Death in Drosophila Melanogaster
    (2010) McPhee, Christina Kary; Mount, Stephen M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Two prominent morphological forms of programmed cell death occur during development, apoptosis and autophagic cell death. Improper regulation of cell death can lead to a variety of diseases, including cancer. Autophagy is required for survival in response to starvation, but has also been associated with cell death. It is unclear how autophagy is regulated under specific cell contexts in multi-cellular organisms, and what may distinguish autophagy function during cell survival versus cell death. Autophagic cell death is characterized by cells that die in synchrony, with autophagic vacuoles in the cytoplasm, and phagocytosis of the dying cells is not observed. However, little is known about this form of cell death. Autophagic cell death is observed during mammalian development, during regression of the corpus luteum and involution of the mammary and prostate glands. Autophagic cell death is also observed during development of the fruitfly Drosophila melanogaster, during larval salivary gland cell death. Drosophila is an excellent genetic model system to study developmental cell death in vivo. Cells use two main catabolic processes to degrade and recycle cellular contents, the ubiquitin/proteasome system (UPS) and autophagy. Here I investigate the role of the UPS and autophagy in developmental cell death using Drosophila larval salivary glands as an in vivo model. Proteasome inhibitors are being used in anti-cancer therapies; however the cellular effects of proteasome inhibition have not been studied in vivo. Here I demonstrate that the UPS is impaired during developmental cell death in vivo. Taking a proteomics approach to identify proteins enriched in salivary glands during developmental cell death and in response to proteasome impairment, I identify several novel genes required for salivary gland cell death, including Cop9 signalsome subunit 6 and the engulfment receptor Draper. Here I show that the engulfment receptor Draper is required for salivary gland degradation. This is the first example of an engulfment factor that is autonomously required for self-clearance. Surprisingly, I find that Draper is cell-autonomously required for autophagy during cell death, but not for starvation-induced autophagy. Draper is the first factor to be identified that genetically distinguishes autophagy that is associated with cell death from cell survival.
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    The Role of small RNAs in Regulating APETALA2 and CAULIFLOWER during Flower Development
    (2010) Grigorova, Boyana Zhelyazkova; Liu, Zhongchi; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
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    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.
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    CHARACTERIZATION OF TWO HIGHLY CONSERVED POXVIRUS TRANSMEMBRANE PROTEINS OF UNKNOWN FUNCTION
    (2009) Sood, Cindy Leigh; Moss, Bernard; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The vaccinia virus I5L open reading frame encodes a 79-amino-acid protein, with two predicted transmembrane domains, conserved among all sequenced members of the chordopoxvirus subfamily. No nonpoxvirus homologs or functional motifs have been recognized, and the role of the I5 protein remains unknown. I5 synthesis was dependent on viral DNA replication and occurred exclusively at late times, consistent with a consensus late promoter motif adjacent to the start of the open reading frame. I5 was present in preparations of purified virions and could be extracted with nonionic detergent, suggesting membrane insertion. Transmission electron microscopy of immunogold-labeled thawed cryosections of infected cells revealed the association of an epitope-tagged I5 with the membranes of immature and mature virions. Viable I5L deletion and frameshift mutants were constructed and found to replicate like wild-type virus in a variety of cell lines, indicating that the protein was dispensable for in vitro cultivation. However, mouse intranasal challenge experiments indicated that a mutant virus with a frameshift resulting in a stop codon near the N terminus of I5 was attenuated compared to control virus. The attenuation correlated with clearance of mutant viruses from the respiratory tract and with less progression and earlier resolution of pathological changes. We suggest that I5 is involved in an aspect of host defense that is evolutionarily conserved although a role in cell tropism should also be considered. The vaccinia virus A43R open reading frame encodes a 168-amino acid protein with a predicted N-terminal signal sequence and a C-terminal transmembrane domain. Although A43R is conserved in all sequenced members of the orthopoxvirus genus, no non-orthopoxvirus homolog or functional motif was recognized. Biochemical and confocal microscopic studies indicated that A43 is expressed at late times following viral DNA synthesis and is a type-1 membrane protein with two N-linked oligosaccharide chains. Neither mature nor enveloped virions contained appreciable amounts of A43, which was detected in Golgi and plasma membranes. Loss of A43R expression had no discernible effect on plaque size or virus replication in cell culture and little effect on virulence in a mouse intranasal infection model. Although the A43 mutant produced significantly smaller lesions in the skin of mice than the control, the amounts of virus recovered from the lesions were similar.
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    Resistance to Ionizing Radiation and Oxidative Stress in Halobacterium salinarum NRC-1
    (2009) Robinson, Courtney Kathryn; Dinman, Jonathan; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Oxidative stress results from environmental challenges that cause unchecked production of reactive oxygen species (ROS). We analyzed the cellular damage and stress response of the extremophile Halobacterium salinarum NRC-1 exposed to chemical oxidants and to ionizing radiation (IR). In contrast to IR, cellular damage from H2O2 and superoxide suggested that cell death resulted from interference with major metabolic pathways rather than generalized oxidative lesions. We found that essential ROS scavenging enzymes were not necessary for H. salinarum NRC-1 survival to IR. Protection assays using enzyme-free cellular extracts from H. salinarum NRC-1 demonstrated high level of protection for protein activity but not for DNA integrity against IR. Biochemical analysis of the extracts underlined an essential role in ROS scavenging for specific nucleosides and MnPO4 complexes. These studies contributed novel findings on the critical role played by non-enzymatic systems in IR resistance in H. salinarum NRC-1.
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    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.
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    Investigation of Ethylene Signal Transduction Mechanisms: Characterizing the Novel Gene AWE1 and Testing Hypothesis of Raf-like CTR1's Function In Vivo
    (2009) Kendrick, Mandy Danielle; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Ethylene is a gaseous plant hormone affecting multiple plant processes. Sixteen years ago the first components of the ethylene signaling pathway, the receptor ETR1 and Raf-like kinase CTR1, were identified. Since then many additional components of the pathway have been elucidated through genetic screens. Recent discoveries suggest ethylene signaling, once thought to be a linear pathway from ethylene perception at the endoplasmic reticulum to transcriptional activation at the nucleus, is more complex with multiple auto-feedback loops and potential parallel kinase cascades downstream of the receptors. Although the genetic backbone of the pathway is well established, the signaling mechanisms of the components remain unclear. ETR1 displays histidine kinase activity in vitro and physically interacts with the next-known downstream component of the pathway, CTR1. However the histidine kinase activity of ETR1 is mostly dispensable for signaling to CTR1. How then is CTR1 activated? I proposed that additional proteins, like AWE1, play a role in ETR1 to CTR1 signaling, and that the non-catalytic, amino-terminal region of CTR1 is required both for activation through direct interaction with the ETR1 receptor complex and for auto-inhibition of CTR1 kinase activity. ASSOCIATES-WITH-ETR1 (AWE1) was isolated in a yeast-two-hybrid screen for ETR1-interacting proteins and was of specific interest because the AWE1 clone also interacted with a portion of CTR1. Protein-protein interaction studies and genetic analysis of an awe1 mutant support a role of AWE1 in repressing ethylene responses. However double mutant analysis, over-expression analysis, and protein sub-cellular localization studies suggest that AWE1's function in hypocotyl elongation and cell expansion is more general. AWE1's function may require ETR1 for proper regulation but is likely to lie outside of the direct step from ETR1 to CTR1. To investigate a role of the CTR1 amino-terminal region in CTR1 regulation, I constructed transgenes consisting of truncated ETR1 receptors fused to truncated or full length CTR1 and examined how those transgenes carrying the truncated CTR1 (kinase domain only) affected Arabidopsis seedling growth compared to those transgenes expressing full length CTR1. I concluded that the CTR1 amino-terminal region may have a role in autoregulation, but additional components are required for regulation of CTR1 signaling.