Theses and Dissertations from UMD
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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
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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 COMPARATIVE STUDY OF LIPOPROTEIN METABOLISM IN MAREK'S DISEASE SUSCEPTIBLE AND RESISTANT LINES(2010) Yuan, Ping; Song, Jiuzhou; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Marek's disease virus (MDV) infection causes atherosclerosis, and prior vaccination prevented the development of this disease. Two main strategies to resist Marek's disease (MD) have been demonstrated: vaccination and genetic resistance. However, little is known about the role of genetic resistance in the progression of MDV induced atherosclerosis. Atherosclerosis is primarily associated with lipoprotein metabolism. The purpose of this study was to investigate whether lipoprotein metabolisms are different in distinct MD susceptible and resistant chicken lines. Here, we studied different backgrounds of lipoprotein metabolism in the two lines and the changes of lipoprotein levels in response to MDV infection. The results showed that during chicken growth, the increase in total cholesterol was mostly due to the increasing (LDL+VLDL) in MD susceptible line, whereas it was mainly due to the elevating HDL in MD resistant line. These results suggested that different lipoprotein metabolisms exist in MD susceptible and resistant lines.Item GENETIC AND MOLECULAR ANALYSIS OF GERMLINE SEX DETERMINATION IN CAENORHABDITIS BRIGGSAE, A MODEL FOR THE CONVERGENT EVOLUTION OF HERMAPHRODITISM(2010) Doty, Alana V.; Haag, Eric S.; Behavior, Ecology, Evolution and Systematics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Though sex determination and differentiation are critical biological processes, genetic mechanisms that specify sex have undergone profound and rapid evolutionary change across taxa. We may be able to infer processes that generate sex determination diversity by examining closely related species. Within the nematode genus Caenorhabditis, two species, C. elegans and C. briggsae, are androdioecious, producing self-fertile hermaphrodites and males; other Caenorhabditis species generate males/females. Interestingly, phylogenies reveal that C. elegans and C. briggsae independently acquired hermaphroditism, a relatively rare adaptation among animals. In this work, I describe differences in germline sex determination between C. elegans and C. briggsae that may help reveal the molecular basis of their convergent evolution of hermaphroditism. I first describe mutations in the pleiotropic, STAR family RNA-binding protein Cbr-GLD-1 that affect germline sex in C. briggsae. I find that C. briggsae gld-1 mutant hermaphrodites have a sex determination phenotype opposite to that of C. elegans: masculinized versus feminized germlines. I demonstrate that Cbr-GLD-1 coding-plus-regulatory sequences can rescue Ce-gld-1 null animals, arguing that this change in sex determination is not due to changes in GLD-1 function or expression. I further show that gld-1's role in regulating oogenesis is conserved across the Elegans group of Caenorhabditis, demonstrating that the oogenesis function of gld-1 is likely ancient, whereas its sperm-repressing role in C. briggsae has evolved recently. To identify mRNA targets of Cbr-GLD-1 that might be responsible for its sex determination function in C. briggsae, I use an in vivo genome-wide approach to isolate mRNAs associated with Cbr-GLD-1, including potential sex determination targets. I identify 800 putative mRNA targets and confirm specificity of this gene set via qRT-PCR and RNAi. Next, to reveal the roles of GLD-1 in evolutionary context, I create a phylogeny of STAR proteins across metazoans. Finally, I characterize a single feminizing allele recovered through forward screens in C. briggsae for germline sex determination mutants. This work thus begins to dissect the molecular and genetic basis of hermaphroditism in C. briggsae and contributes to a growing body of research on the evolution of germline sex determination differences between C. elegans and C. briggsae.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 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 Genetics of Avian Paramyxovirus serotype 2(2010) Subbiah, Madhuri; Samal, Siba K.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Avian Paramyxovirus (APMV) serotype 2 is one of the nine serotypes of APMV that infect a variety of bird species around the world. In chickens and turkeys, APMV-2 causes respiratory illness and drop in egg production. To understand the molecular characteristics of APMV-2, the complete genome sequences of prototype strain Yucaipa and strains Bangor, England and Kenya were determined. The genome lengths of APMV-2 strains Yucaipa, Bangor, England and Kenya are 14904, 15024, 14904, 14916 nucleotides (nt), respectively. Each genome consists of six non-overlapping genes in the order 3'N-P/V/W-M-F-HN-L5' similar to most of APMVs. Sequence comparison of APMV-2 strains England and Kenya with prototype strain Yucaipa show 94-98% nt and 90-100% aggregate amino acid (aa) identities. However, strain Bangor shares low level of nt and predicted aa sequence identities with the other three strains. The phylogenetic and serological analyses of all four strains indicated the existence of two subgroups: strains Yucaipa, England and Kenya represented one subgroup and strain Bangor represented the other subgroup. All four strains were found to be avirulent for chickens by mean death time and intracerebral pathogenicity test. To further study the molecular biology and pathogenicity of APMV-2, a reverse genetics system for strain Yucaipa was established in which infectious recombinant APMV-2 was recovered from a cloned APMV-2 antigenomic cDNA. The recovered recombinant virus showed in vitro growth characteristics and in vivo pathogenicity similar to wild type virus. Recombinant APMV-2 expressing enhanced green fluorescent protein was also recovered, suggesting its potential use as a vaccine vector. Furthermore, generation and characterization of mutant viruses by replacing the fusion protein (F) cleavage site of APMV-2 with those of APMV serotypes 1 to 9 demonstrated that the amino acid composition at F protein cleavage site does not affect the pathogenicity of APMV-2. Overall, the study conducted here has several downstream applications. The complete genome sequence of APMV-2 is useful in designing diagnostic reagents and in epidemiological studies. The reverse genetics system for APMV-2 would be of considerable utility for introducing defined mutations into the genome of this virus and develop vaccine vector for animal and human pathogens.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 Transcript assembly and abundance estimation with high-throughput RNA sequencing(2010) Trapnell, Bruce Colston; Salzberg, Steven L; Computer Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)We present algorithms and statistical methods for the reconstruction and abundance estimation of transcript sequences from high throughput RNA sequencing ("RNA-Seq"). We evaluate these approaches through large-scale experiments of a well studied model of muscle development. We begin with an overview of sequencing assays and outline why the short read alignment problem is fundamental to the analysis of these assays. We then describe two approaches to the contiguous alignment problem, one of which uses massively parallel graphics hardware to accelerate alignment, and one of which exploits an indexing scheme based on the Burrows-Wheeler transform. We then turn to the spliced alignment problem, which is fundamental to RNA-Seq, and present an algorithm, TopHat. TopHat is the first algorithm that can align the reads from an entire RNA-Seq experiment to a large genome without the aid of reference gene models. In the second part of the thesis, we present the first comparative RNA-Seq as- sembly algorithm, Cufflinks, which is adapted from a constructive proof of Dilworth's Theorem, a classic result in combinatorics. We evaluate Cufflinks by assembling the transcriptome from a time course RNA-Seq experiment of developing skeletal muscle cells. The assembly contains 13,689 known transcripts and 3,724 novel ones. Of the novel transcripts, 62% were strongly supported by earlier sequencing experiments or by homologous transcripts in other organisms. We further validated interesting genes with isoform-specific RT-PCR. We then present a statistical model for RNA-Seq included in Cufflinks and with which we estimate abundances of transcripts from RNA-seq data. Simulation studies demonstrate that the model is highly accurate. We apply this model to the muscle data, and track the abundances of individual isoforms over development. Finally, we present significance tests for changes in relative and absolute abundances between time points, which we employ to uncover differential expression and differential regulation. By testing for relative abundance changes within and between transcripts sharing a transcription start site, we find significant shifts in the rates of alternative splicing and promoter preference in hundreds of genes, including those believed to regulate muscle development.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 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.)