UMD Theses and Dissertations
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Item Exploring the Function and Regulation of Arabidopsis EIN2 in Ethylene Signaling(2016) Shemansky, Jennifer Marie; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ethylene is an essential plant hormone involved in nearly all stages of plant growth and development. EIN2 (ETHYLENE INSENSITIVE2) is a master positive regulator in the ethylene signaling pathway, consisting of an N-terminal domain and a C-terminal domain. The EIN2 N-terminal domain localizes to the endoplasmic reticulum (ER) membrane and shows sequence similarity to Nramp metal ion transporters. The cytosolic C-terminal domain is unique to plants and signals downstream. There have been several major gaps in our knowledge of EIN2 function. It was unknown how the ethylene signal gets relayed from the known upstream component CTR1 (CONSTITUTIVE RESPONSE1) a Ser/Thr kinase at the ER, to EIN2. How the ethylene signal was transduced from EIN2 to the next downstream component transcription factor EIN3 (ETHYLENE INSENSITIVE3) in the nucleus was also unknown. The N-terminal domain of EIN2 shows homology to Nramp metal ion transporters and whether EIN2 can also function as a metal transporter has been a question plaguing the ethylene field for almost two decades. Here, EIN2 was found to interact with the CTR1 protein kinase, leading to the discovery that CTR1 phosphorylates the C-terminal domain of EIN2 in Arabidopsis thaliana. Using tags at the termini of EIN2, it was deduced that in the presence of ethylene, the EIN2 C-terminal domain is cleaved and translocates into the nucleus, where it could somehow activate downstream ethylene responses. The EIN2 C-terminal domain interacts with nuclear proteins, RTE3 and EER5, which are components of the TREX-2 mRNA export complex, although the role of these interactions remains unclear. The EIN2 N-terminal domain was found to be capable of divalent metal transport when expressed in E. coli and S. cerevisiae leading to the hypothesis that metal transport plays a role in ethylene signaling. This hypothesis was tested using a novel missense allele, ein2 G36E, substituting a highly conserved residue that is required for metal transport in Nramp proteins. This G36E substitution did not disrupt metal ion transport of EIN2, but the ethylene insensitive phenotype of this mutant indicates that the EIN2 N-terminal domain is important for positively regulating the C-terminal domain. The defect of the ein2 G36E mutant does not prevent proper expression or subcellular localization, but might affect protein modifications. The ein2 G36E allele is partially dominant, mostly likely displaying haploinsufficiency. Overexpression of the EIN2 N-terminal domain in the ein2 G36E mutant did not rescue ethylene insensitivity, suggesting the N-terminal domain functions in cis to regulate the C-terminal domain. These findings advance our knowledge of EIN2, which is critical to understanding ethylene signaling.Item DISTINCT MOLECULAR AND MORPHOLOGICAL SUBCIRCUITS OF THE SUBPLATE NEURONS(2014) Viswanathan, Sarada; Kanold, Patrick o; Looger, Loren L; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Subplate neurons (SPNs) are a population of neurons in the mammalian cerebral cortex that exist predominantly in the prenatal and early postnatal period. Loss of SPNs prevents the functional maturation of the cerebral cortex. SPNs receive subcortical input from the thalamus and relay this information to the developing cortical plate and thereby can influence cortical activity in a feed-forward manner. Little is known about potential feedback projections from the cortical plate to SPN. SPNs are also a heterogeneous population in terms of molecular and morphological identity. And the functional role of the different subpopulation of SPN remains poorly defined. This is mainly due to the lack of tools- i.e. transgenic lines and reporters to target and manipulate the SPNs at different stages of development. Hence the functional significance of the molecular diversity remains unexplored. In this study, we used a combination of genetic, molecular, anatomical and physiological approaches to address these questions and also to identify and characterize transgenic `tools' to manipulate the SPN. We identified and characterized a set of reporters and transgenic lines expressing Cre recombinase or green fluorescent protein with different levels of specificity in the subplate (SP). Using these transgenic driver lines and specific antibodies, we find that defined SPNs project to the main thalamo-recipient layers - L4 and L1 - and the spatial pattern of SPN projections to layer 4 is related to the spatial pattern of thalamo-cortical projections. However different subclasses have distinct patterns of projections with respect to the thalamic afferents. While certain subclasses have been shown to project locally, we observe that certain cell types of SPN also extend long-range projections to different thalamic nuclei. Thus molecularly defined SPN cell types are differentially integrated into the thalamo-cortical and intra-cortical connectivity. We also find a laminar difference in intra-cortical connectivity of the SPN. The first class of SPNs receives inputs from only deep cortical layers, while the second class of SPNs receives inputs from deep as well as superficial layers including layer 4 and are located more superficially. These superficial cortical inputs to SPNs emerge in the second postnatal week. Taken together, we demonstrate the presence of distinct laminar and molecular circuits in the developing subplate and characterize yet another level of heterogeneity of this population.Item ALTERING THE AI-2 MEDIATED QUORUM SENSING CIRCUITRY TO QUENCH BACTERIAL COMMUNICATION NETWORKS(2011) Roy, Varnika; Bentley, William E; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The emergence of antibiotic resistant bacteria poses a global threat to human health and has been classified as a clinical super-challenge of the 21st century. This has necessitated research on new antimicrobials that inhibit bacterial virulence by mechanisms other than those that target bacterial growth or viability. Such approaches have been reported to pose less evolutionary pressure on bacteria to evolve and become resistant to antibiotics. Bacterial cell-cell communication, termed quorum sensing (QS), is mediated by signatures of small molecules. QS via these small molecules has been linked to numerous undesirable bacterial phenotypes such as biofilm formation, onset of pathogenicity, triggering of virulence genes etc. The small signaling molecules represent targets for intercepting bacterial communication (and their resultant undesirable phenotypes). We have devised two strategies that interrupt bacterial communication in multispecies bacterial cultures by targeting the interspecies signaling molecule autoinducer-2 (AI-2), which is produced or recognized by over 70 species of bacteria. Our first approach is to bring the native intracellular AI-2 signal processing mechanisms to the extracellular surroundings to quench the QS response of bacteria. Specifically we deliver the Escherichia coli AI-2 kinase, LsrK, to E. coli populations ex vivo and phosphorylate and degrade the extracellular AI-2. This significantly attenuates the native QS response in E. coli. Similar results are obtained in a tri-species synthetic ecosystem comprising E. coli, Salmonella typhimurium and Vibrio harveyi. In our second quenching strategy, we explore a panel of small synthetic molecules that are analogs of AI-2 (C1-alkyl analogs). The analogs are observed to cause species-specific and cross-species quorum quenching in the tri-species synthetic ecosystems of the aforementioned strains. Some of the AI-2 analogs quench pyocyanin (toxin production) in the opportunistic pathogen Pseudomonas aeruginosa. Based on these observations, I used analog cocktails to quench QS en masse in assembled synthetic ecosystems. Finally, I tested the efficiency of the analogs in quenching pathogenic phenotypes such as biofilm formation in E. coli. The analogs inhibit biofilm formation and act in concert with antibiotics to reduce biofilm formation even further. Our results suggest entirely new modalities for interrupting or tailoring the networks of communication among bacteria and identifying drug targets to develop the next generation of antimicrobials based on QS.Item THE ROLE OF EXCITATION-CONTRACTION COUPLING FAILURE IN MUSCLE FATIGUE AND WEAKNESS OF DYSTROPHIC SKELETAL MUSCLE(2011) Mazala, Davi Augusto Garcia; Chin, Eva R; Kinesiology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Alterations in intracellular calcium (Ca2+) are thought to play an important role in skeletal muscle weakness associated with muscular dystrophy due to the activation of Ca2+-regulated proteases (calpains). It was hypothesized that impairments in Ca2+ regulation are exacerbated in dystrophic muscle and that calpain inhibition could attenuate the muscle weakness induced by fatiguing contractions. Single muscle fibres from control and dystrophic mice lacking dystrophin (mdx) and utrophin plus dystrophin (Utr-/-/mdx) were used. Fibres from Utr-/-/mdx mice had similar peak tetanic Ca2+ compared to control and mdx mice, however Utr-/-/mdx mice took longer to clear the released Ca2+. All fibres showed similar time to fatigue but fewer mdx and Utr-/-/mdx fibres remained excitable 1hr after fatiguing contractions. Exposure to a calpain inhibitor improved Ca2+ levels in dystrophic fibres (mdx; trend only in Utr-/-/mdx) after fatigue. Together, these data indicate that calpains play a role in prolonged muscle weakness after fatiguing contractions.Item LOCAL AND GLOBAL GENE REGULATION ANALYSIS OF THE AUTOINDUCER-2 MEDIATED QUORUM SENSING MECHANISM IN ESCHERICHIA COLI(2011) Byrd, Christopher Matthew; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The term `quorum sensing' (QS) is used to define a population density based communication mechanism which uses chemical signal molecules called autoinducers to trigger unique and varied changes in gene expression. Although several communication methods have been identified in bacteria that are unique to a particular species, one type of signal molecule, autoinducer-2 (AI-2) is linked to interspecies communication, indicating its potential as a universal signal for cueing a QS response among multiple bacterial types. In E. coli, AI-2 acts as an effector by binding to the QS repressor LsrR. As a result, LsrR unbinds and relieves repression of the lsr regulon, stimulating a subsequent QS gene expression cascade. In this dissertation, LsrR structure and in vitro binding activity are examined. Genomic binding and DNA microarray analyses are conducted and three novel sites putatively regulated by LsrR, yegE-udk, mppA and yihF, are revealed. Two cAMP receptor protein (CRP) binding locations in intergenic region of the lsr regulon are also confirmed. The role of each CRP site in divergent expression is qualified, indicating the lsr intergenic region to be a class III CRP-dependent promoter. Also, four specific DNA binding sites for LsrR in the lsr intergenic region are proposed, and reliance upon simultaneous binding to these various sites and the resulting effects on LsrR repression is presented. Finally, a complex model for regulation of the lsr regulon is depicted incorporating LsrR, CRP, DNA looping, and a predicted secondary layer of repression by an integration host factor (IHF)-like protein. Further understanding of this QS genetic mechanism may potentially be used for inhibiting bacterial proliferation and infection, modifying the natural genetic system to elicit alternate desired responses, or extracted and applied to a highly customizable and sensitive in vitro biosensor.Item CHARACTERIZATION OF LIVER X RECEPTORS IN PROSTATE CANCER CHOLESTEROL METABOLISM AND PULMONARY IMMUNE RESPONSE(2011) Trasino, Steven E.; Lei, David K; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Liver x receptors (LXRs) are central regulators cholesterol homeostasis and the innate immune response. As modulators of inflammation and cholesterol metabolism LXRs might diminish dyslipidemia and inflammation related pathologies caused by high fat (HF) diets or obesity. There is also data demonstrating that LXRs can protect against progression of prostate cancer (PCa), but little is known about the cholesterol modulating effects of LXRs in transformed cells. The goal of this project is to characterize the cholesterol modulating properties of LXRs in two models of PCa and the anti-inflammatory properties of LXRs in swine bronchial alveolar macrophages (AMs). This project will also examine whether the anti-inflammatory and lipid lowering properties of the dietary probiotic bacteria Lactobacillus casei (L. casei), can interact with the LXR axis in AMs. Studies in two PCa cell lines, LNCaP and PC-3, revealed that LXR ligands regulate the LXR responsive genes, ABCA1 and ABCG1 through the LXR&beta isoform and not LXR&alpha in PC-3 cells, but only ABCG1 in LNCaP. LXR- ABCA1 mediated reverse cholesterol transport (RCT) resulted in a decrease in plasma membrane lipid raft cholesterol domains in PC-3 cells, suggesting a potential anti-cancer axis for LXR activation. Studies in LNCaP and PC-3 cells also demonstrated that soy isoflavones can activate transcriptional activation of ABCA1 and ABCG1 in LNCaP and PC-3 cells through the LXR&beta isoform, but did not lead to an increase in RCT. Metabolic and anti-inflammatory studies of LXR in AM from Ossabaw pigs fed either a control (C) diet, HF, HF plus L casei (HFPB) or L. casei alone (CPB) diet revealed that AM from HF fed pigs had significantly higher concentrations of cholesteryl-esters (CE) compared with AM from control (C) diet fed pigs. Ex-vivo activation of LXR with the LXR ligand T0901317 opposed LPS mediated upregulation of IL-1&beta , IL-6, IL-8 and IL-10 mRNA levels in AM from HF, HFPB and CPB fed pigs. Finally, it was observed that LPS stimulation lead to significant inhibition of LXR transcription of LXR&alpha, ABCA1, ABCG1, cholesterol 25 hydroxylase (CH25H) and PPAR&gamma in AM. This effect was abrogated by L. casei for ABCA1, CH25H and PPAR&gamma mRNA expressionItem Development of Carbon Nanotube Field-Effect Transistor Arrays for Detection of HER2 Overexpression in Breast Cancer(2011) Aschenbach, Konrad Hsu; Gomez, Romel D; Electrical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)We developed a carbon nanotube biosensor platform that was deployed at the National Cancer Institute and successfully detected the HER2 oncogene in real cancer cells at clinically relevant levels. HER2 is a receptor protein that resides on the surface of certain cancer cells and is associated with higher aggressiveness in breast cancers. Overabundance of HER2 at the chromosomal, cell surface, and intermediate gene expression levels can all indicate a dangerous HER2 status. At the present, testing for HER2 status requires labor-intensive laboratory procedures using expensive reagents. Cost remains the major barrier to widespread screening. We propose an integrated electronic testing platform based on direct label-free gene detection. The system would integrate the various labor-intensive processes that are usually performed by skilled laboratory technicians. The heart of the system is an array of carbon nanotube field-effect transistors that can detect unlabelled nucleic acids via their intrinsic electric charges. We developed a scalable fabrication technique for carbon nanotube biosensor arrays, hardware and software for data acquisition and analysis, theoretical models for detection mechanism, and protocols for immobilization of peptide nucleic acid probes and hybridization of nucleic acids extracted from cells. We demonstrated detection of HER2 from real cell lines which express cancer genes, thereby lowering the technological barrier towards commercialization of a low-cost gene expression biosensor. The system is suitable for lab-on-a-chip integration, which could bring rapid, low-cost cancer diagnoses into the clinical setting.Item Ribosomal Protein L11: A Cog in the Nanomachine(2011) Rhodin, Michael Hoover Johannes; Dinman, Jonathan D; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Comprised of two major subunits of both rRNA and proteins, the ribosome is a biological nanomachine, acting as the central player in the process of protein translation. Recent advances in molecular imaging have enabled the visualization of the disparate functional centers within the ribosome, leading to the question of how these critical regions coordinate their actions and communicate with each other. This work examines the essential ribosomal protein L11, located in the central protuberance of the large subunit. L11 maintains connections with the 5S rRNA, H84 of the 25S rRNA, comes in close proximity to the T-loop of the bound peptidyl tRNA, and shares an intersubunit bridge with small subunit protein S18. L11 was found to have a critical dynamic loop which samples the occupancy status of the P-site pocket of the ribosome and communicates this information through H84. L11's intersubunit bridge (the B1b/c bridge) mediates an intersubunit communication network from the decoding center to the peptidyl transferase center of the ribosome. L11 is also involved in proper subunit joining. Mutations in L11 were found to have effects on A- and P-site tRNA binding, translational fidelity, and growth and viability of yeast cells.Item GENETIC POLYMORPHISMS IN DNA EXCISION REPAIR GENES IN RELATION TO CANCER AND CANCER THERAPIES(2011) Gao, Rui; Pick, Leslie; Figg, William Douglas; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)DNA excision repair pathways, including the nucleotide excision repair (NER) and the base excision repair (BER) pathways, play significant roles in maintaining genomic stability. However, these pathways are found to be responsible for therapeutic resistance to cancer therapies causing DNA damage. Platinum-containing drugs are important therapies for various solid tumors. Genetic polymorphisms in NER and BER genes have been identified, and some have been correlated to altered clinical outcome to platinum-based chemotherapies. Here I studied the genetic polymorphisms in the NER genes, ERCC1 and XPD, and the BER genes, XRCC1 and PARP1, and found that the polymorphic variants had significantly higher frequencies in European Americans (EAs) for ERCC1 N118N (p<0.000001), XPD K751Q (p=0.006675), XRCC1 R399Q (p<0.000001) and PARP1 V762A (p=0.000001), compared to those in African Americans (AAs), which may reflect a mild reduction in DNA excision repair function in EA population. However, these polymorphisms were not associated with risk of prostate cancer or the clinical outcome of radiation therapy in prostate cancer in EAs. I also investigated the functional consequences of the most well studied NER polymorphism ERCC1 N118N (500C>T) by introducing the ERCC1 cDNA clones containing either the C or T allele into an ERCC1 deficient cell line UV20. However, neither the ERCC1 expression levels nor the cellular sensitivity to platinum drugs were affected by this silent mutation. These data suggests that the N118N itself does not contribute to the phenotypic differences in ERCC1, but rather this polymorphism may be linked to other causative variants or haplotypes. Therefore, I examined 4 polymorphisms in ERCC1, including rs3212948 (G>C), rs3212950 (C>G) in intron 3, and rs3212929 (T>G) in the 5' UTR, in addition to N118N (500C>T), and found that the haplotypes of these polymorphisms were associated with risk of skin melanoma, indicating the potential functional significance of other ERCC1 polymorphisms. Understanding the functional significance the genetic polymorphisms in DNA excision repair genes may facilitate the administration of personalized medicine.Item CHARACTERIZATION OF THE TWO VACCINIA VIRUS MATURE VIRION-SPECIFIC PROTEINS, A26P AND A25P(2010) Howard, Amanda Rachel; Dinman, Jonathan; Bernard, Moss; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Poxviruses produce two morphologically distinct infectious particles, mature virions (MVs) and extracellular virions (EVs). During replication, some MVs differentiate and become wrapped with cellular membranes, transported to the periphery, and exported as EVs. Some orthopoxviruses, e.g., cowpox virus (CPXV), form large, discrete cytoplasmic inclusions called A-type inclusion bodies (ATIs) within which MVs are embedded by a process called occlusion. ATIs are composed of aggregates of the A-type inclusion protein (ATIp), which is truncated in orthopoxviruses such as vaccinia virus (VACV) that fail to form ATIs. VACV does encode a functional A26p, which along with the ATIp is required for occlusion. A26 lacks a transmembrane domain, and nothing is known regarding how it associates with the MV and regulates occlusion. Additionally, little is known about the formation of ATIs and how MVs become embedded within them. Here, experiments show that A26p is incorporated into MVs by the A27p-A17p complex and interacts with A25p, a truncated form of the CPXV ATIp. Restoration of the full-length ATI gene is sufficient for VACV ATI formation and the occlusion of MVs. A26p directly interacts with ATIp, and this interaction, as well as the A26p-A27p interaction, are required for occlusion. The data demonstrates that ATI mRNAs are transported out of viral factories (VFs) and translated in the cytoplasm. ATIs enlarge both by new protein synthesis and by coalescence, which requires microtubules. ATIs do not nucleate around MVs but rather MVs move along microtubules to embed within ATIs. Taken together, the data suggest a model for occlusion in which MVs move along microtubules to ATIs that are translated from mRNAs in the cytoplasm. At the ATIs, A26p has a bridging role between ATIp and A27p, and A27p provides a link to the MV membrane. Although the specificity of A26p for MVs suggested A26p might regulate wrapping, I did not detect an effect of either the deletion of A26 or occlusion on the production of EVs.