Theses and Dissertations from UMD
Permanent URI for this communityhttp://hdl.handle.net/1903/2
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 give thesis/dissertation in DRUM
More information is available at Theses and Dissertations at University of Maryland Libraries.
Browse
57 results
Search Results
Item IDENTIFICATION OF A NON-CLASSICAL GLUCOCORTICOID-RESPONSIVE ELEMENT IN THE 5'-FLANKING REGION OF THE CHICKEN GROWTH HORMONE GENE(2010) Knubel, Kristina Heuck; Porter, Tom E; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Growth hormone (GH) effects growth and contributes to a lean phenotype in broiler chickens. GH secretion by the anterior pituitary begins on embryonic day (e) 14, concomitantly with a rise in adrenal glucocorticoids (GC) or corticosterone (CORT) secretion. CORT treatment of chicken embryonic pituitary (CEP) cells induces GH secretion prematurely. GC induction of the GH gene requires on-going protein synthesis or an intermediary protein, but the gene lacks a classical GC-response element. We hypothesized that a GC-responsive intermediary protein is necessary for the CORT induced increase in GH. Characterization of the upstream region of the gene may help identify such a protein. To this end, a fragment of the GH gene (-1727/+48) was cloned into a luciferase reporter and characterized in e11 CEP cells. CORT treatment increased luciferase activity and mRNA. Inclusion of CHX blocked CORT induction of luciferase mRNA. Through deletion analysis, we found that a GC-responsive region (GCRR) is located at -1045 to -954. By defining the GC-responsive region and cis-acting elements located within, trans-acting proteins involved in GC induction of the GH gene may be identified. The GCRR is CORT-responsive in either orientation, but it is context-dependent. Potential transcription factor motifs in the GCRR include ETS-1 and a degenerate GRE (GREF). Nuclear proteins bound to a GCRR probe in a CORT-regulated manner and unlabeled competitor DNA competed off binding. Mutation of the central portion of the DNA probe resulted in a significant decrease in protein binding. Mutation of the ETS-1 site or GREF site in the -1045/+48 GH construct resulted in ablation of luciferase activity. ETS-1 and GR are associated with the endogenous gene under basal and 1.5 h CORT-treated conditions, while GR recruitment increased after CORT treatment. GC regulation of the GH gene during chicken embryonic development requires cis-acting elements located 1 kb upstream from the transcription start site and includes recruitment of ETS-1 and GR. This is the first study to demonstrate involvement of ETS-1 in GC regulation of the GH gene during embryonic development. Characterization of GC regulation of the GH gene during embryonic development enhances our understanding of growth regulation in vertebrates.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 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.Item 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.Item POLLEN TUBES FAIL TO TARGET OVULE IN THE ABSENCE OF TWO CATION/PROTON EXCHANGERS IN ARABIDOPSIS(2010) Lu, Yongxian; Sze, Heven; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Flowering plant reproduction requires precise delivery of the sperm cells to the ovule by a pollen tube. Guiding signals from female cells are being identified, though how pollen senses and responds to those cues are largely unknown. Here I provide genetic evidence that two predicted cation/proton exchangers expressed in Arabidopsis pollen play essential roles in pollen targeting of ovules. Male fertility was unchanged in single chx21 or chx23 mutant pollen; however, male-specific gene transmission was blocked in chx21chx23 double mutant. Wild-type pistil provided with a limited amount of pollen containing a mixture of single and double mutant produced ~60% less seeds compared to that produced with chx23 single mutant pollen, indicating that chx21chx23 pollen is infertile. The double mutant pollen, visualized by a pollen-specific promoter-driven GUS activity, germinated and extended a tube down the transmitting tract, but the tube failed to turn and target an ovule. Unlike wild-type pollen that targeted isolated ovules in a semi-in vivo assay, tube guidance in chx21chx23 pollen was compromised. As a first step to understand the cellular and molecular bases of tube guidance, membrane localization and activity of CHX23 was determined. GFP-tagged CHX23 was localized to endomembranes, predominantly endoplasmic reticulum (ER), in elongating pollen tubes. Furthermore, expression of CHX23 in E. coli resulted in enhanced K+ accumulation at alkaline pH, suggesting a role for CHX23 in K+ acquisition and pH homeostasis. Based on these studies and observations by others that ER oscillates and enters the apex, a simple model is proposed: Modification of localized pH by CHX21 or CHX23 enables pollen tube to sense female signals and respond by shifting directional growth at the funiculus and micropyle to target pollen tip growth towards the ovule.Item EFFECT OF CHITOSAN ON THE INDUCTION OF DNA DAMAGE RESPONSE BY SELENIUM COMPOUNDS.(2009) Zhang, Shu; Cheng, Wen-Hsing; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Selenium (Se), a nutrient trace mineral, plays important roles in optimizing human health. Chitosan is an effective, natural-oriented material for synthesizing nanopolymers, with preferable properties such as biocompatibility, biodegradation and resistance to certain enzymes. In this study, encapsulated Na2SeO3 and methylseleninic acid (MSeA) with low and medium molecular weight chitosan were used to determine the efficacy of Se in mitigating tumorigenesis. We applied Se compounds, which is from sub-lethal to lethal dose, to colon cancer cell line HCT-116 and normal fibroblasts cell line MRC-5. Analysis of cellular selenium content demonstrated that: 1) Na2SeO3, but not MSeA, treatment resulted in a greater Se retention in HCT-116 than in MRC-5 cells, 2) chitosan encapsulation enhanced Se contents in cells treated with the various Se preparations. Cell survival analysis showed that chitosan encapsulation protected HCT-116 and MRC-5 cells from Na2SeO3 or MSeA induced toxicity. Moreover, this beneficial effect was greater in MRC-5 cells. MSeA encapsulated with chitosan induced phosphorylated ATM Ser-1981 formation in MRC-5 and HCT-116 cells to a less extent as compared to MSeA alone treatment. Taken together, the results suggest that, when encapsulated with chitosan, cells are less susceptible to Se treatment, possibly through a mechanism by which the presence of chitosan attenuates Se-induced activation of ATM and corresponding DNA damage response pathway.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 Myosins IIIa and IIIb regulate stereocilia length by transporting espin 1 to the polymerizing end of actin filaments(2009) Merritt, Raymond Clyde; Kachar, Bechara; Popper, Arthur N; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mutations in the myosin IIIa gene are linked to DFNB30 late onset deafness, in which those affected individuals can hear for the first twenty years of life. Mutations in the espin gene are linked to DFNB36 congenital deafness. Both myosin IIIa and espin 1 are expressed in the inner ear hair cells and colocalize at stereocilia tips, the site of actin polymerization. Overexpression of these proteins in hair cells produce an increase in stereocilia length, and when both are co-expressed together they produce a length increase greater than when each one are overexpressed individually. These results suggest that these proteins interact and influence stereocilia length regulation. We confirmed the interaction of these two proteins in heterologous COS-7 cells. We observed that when co-expressed, these proteins promote elongation of filopodial actin protrusions in a synergistic manner. Mutational analyses show that myosin IIIa3THDI binds to the ankyrin repeats of espin 1. Live and fixed cell imaging shows that myosin IIIa transports espin 1 to the filopodia tips where espin 1 promotes actin polymerizations through its WH2 domain. Because of the late onset of deafness associated with myosin IIIa, it has been speculated that the lack of myosin IIIa function is partially compensated by the paralogous protein, myosin IIIb. Myosin IIIb, encoded by a distinct gene, lacks a C-terminal actin-binding domain shown to be essential for myosin IIIa filopodia tip localization. We observed that myosin IIIb localizes at stereocilia tips and is expressed at an earlier stage than myosin IIIa. We confirmed that myosin IIIb transports espin 1 to stereocilia tips and promotes actin polymerization, consistent with the hypothesis that it partially compensates for myosin IIIa. We found that binding to espin 1 is required for myosin IIIb movement and localization. We observed that myosin IIIb can downregulate the myosin IIIa localization in vestibular, but not in cochlear, hair cells. The interplay of myosins IIIa, IIIb, and espin 1 and their influence on stereocilia length unravels a novel molecular complex at the polymerizing end of F-actin and a framework to understand the cause of DFNB30 and DFNB36 deafnesses.Item Identification and characterization of a heme responsive element in the hrg-1 promoter(2008) Sinclair, Jason; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Despite its biological significance, little is known about how animals sense and respond to heme to maintain homeostasis. C. elegans is a heme auxotroph, which makes it an excellent model to identify and dissect heme homeostasis pathways. Using C. elegans we have identified HRG-1, a vesicular heme transporter that is transcriptionally upregulated when environmental heme is low. The current study seeks to address how hrg-1 is regulated by heme. Here, we show that a putative 23 base pair (bp) heme-responsive element (HRE) and GATA-binding motifs are necessary for heme-dependent regulation of hrg-1. The HRE comprises both enhancer and repressor elements and works in conjunction with ELT-2 to regulate hrg-1 expression. We propose that the HRE could be used as a molecular tool in C. elegans to tightly regulate internal gene expression by modulating environmental heme. Our ultimate goal is to identify the trans-acting factor to eventually create a whole animal sensor for monitoring organismal heme homeostasis.Item THE FUNCTION OF MRN (MRE11-RAD50-NBS1) COMPLEX DURING WRN (WERNER) FACILITATED ATM (ATAXIA-TELANGIECTASIA MUTATED) ACTIVATION(2009) Ma, Junhao; Cheng, Wen-Hsing; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)WRN (Werner) protein is a member of the RecQ family showing helicase and exonuclease activity. WRN protein may lose function upon mutation and causes Werner syndrome (WS) which is an autosomal recessive, cancer-prone and premature aging disease. ATM (Ataxia-Telangiectasia mutated) protein initiates a signaling pathway in response to DNA double strand breaks (DSBs). Genomic disorder ataxia-telangiectasia (A-T) is associated with defective ATM. WRN protein is involved in ATM pathway activation when cells are exposed to DSBs associated with replication fork collapse. Because the Mre11-Rad50-Nbs1 (MRN) complex, a sensor of DSBs, is known to interact with WRN and ATM, we investigated whether the MRN complex mediates the WRN-dependent ATM pathway activation. In this study, we employed short-hairpin RNA to generate WRN- and Nbs1-deficient U-2 OS (osteosarcoma) cells. Cells were treated with clastogens which induce collapsed replication forks, thus provided proof for whether WRN facilitates ATM activation via MRN complex. This study serves as a basis for future investigation on the correlation between ATM, MRN complex and WRN, which will ultimately help understand the mechanism of aging and cancer.