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 DELINEATING THE ROLES OF C. ELEGANS HEME RESPONSIVE GENES HRG-2 AND HRG-3 IN HEME HOMEOSTASIS(2009) Chen, Caiyong; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme is an essential cofactor for diverse biological processes such as oxygen transport, xenobiotic detoxification, and circadian clock control. Since free heme is hydrophobic and cytotoxic, we hypothesize that within eukaryotic cells, specific trafficking pathways exist for the delivery of heme to different subcellular destinations where hemoproteins reside. To identify molecules that may be involved in heme homeostasis, we conducted a C. elegans microarray experiment on RNA extracted from worms grown at different concentrations of heme in axenic liquid medium. Analysis of the microarrays revealed that the mRNA levels of heme-responsive gene-2 (hrg-2) and hrg-3 increased more than 70 fold when worms were grown at 4 µM compared to 20 µM heme. hrg-2 is expressed in hypodermal tissues in the worm, and the protein localizes to the endoplasmic reticulum and the apical plasma membrane. In vitro hemin agarose pull-down experiments indicate that HRG-2 binds heme. Deletion of hrg-2 in C. elegans leads to reduced growth rate at low heme. Moreover, expression of HRG-2 in hem1δ, a heme-deficient yeast strain, results in growth rescue at submicromolar concentrations of exogenous heme. These results indicate that HRG-2 may either directly participate in heme uptake or facilitate heme delivery to another protein. Unlike hrg-2, hrg-3 is exclusively expressed in the worm intestine under heme deficiency. Following its synthesis, HRG-3 is secreted into the body cavity pseudocoelom. Deletion of hrg-3 results in increased heme levels in the worm intestine, suggesting that HRG-3 may function in intercellular heme transport in C. elegans. To identify the functional network or pathways for HRG-2 and HRG-3, we performed a genome-wide microarray analysis using RNA samples prepared from the worms grown at different concentrations of heme and oxygen. The results showed that a total of 446 genes were transcriptionally altered by heme and/or oxygen. Among them, 41 and 29 genes exhibited similar expression profiles to hrg-2 and hrg-3, respectively. We postulate that these genes may function in conjunction with hrg-2 and hrg-3. Taken together, we have identified two novel heme-responsive genes in metazoa that may play critical roles in modulating organismal heme homeostasis in C. elegans.Item Metarhizium anisopliae's persistence as a saprophyte, genetic basis of adaptation and role as a plant symbiont(2008-11-17) O'Brien, Tammatha Rose; St Leger, Raymond; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Metarhizium anisopliae is a model for elucidating the basis of entomopathogenicity. However, many aspects of the saprophytic life of M. anisopliae remain unclear, including: 1) how the rhizosphere maintains populations of M. anisopliae; 2) the potential for microevolution; 3) the genetic factors that allow M. anisopliae to adapt to a saprophytic life and 4) the extent to which plant ecology is impacted by the rhizospheric competence of M. anisopliae. To extend our knowledge of plant-insect-fungal interactions, a field trial was conducted with M. anisopliae applied to turf. The specific objectives were to: 1) investigate the genetic basis of adaptation and strain stability of M. anisopliae; 2) monitor long-term survival of M. anisopliae, 3) compare the winter survival of M. anisopliae ΔMcl1 (disrupted in an immune evasion gene and nonpathogenic) with M. anisopliae ΔMad2 (adhesin-deficient and unable to adhere to plant epidermis) and 4) investigate root colonization and its impact on plant growth of Triticum aestivum seeds coated with conidia. Results showed that M. anisopliae ΔMad2 had a linear decrease in population and did not colonize roots, while M. anisopliae and M. anisopliae ΔMcl1 cycled with seasons in the soil and colonized roots. Microarray analyses were used to assay for any mutations affecting gene expression during survival in the field. After 3.5 years, field recovered Metarhizium isolates differed in gene expression by an average of 0.26 % for the 1,749 expressed sequence tags. Mutations disproportionately affected cell wall and stress responses, while genes coding for pathogenicity determinants such as adhesins and toxins were highly conserved. Triticum seeds inoculated with M. anisopliae ΔMcl1 and M. anisopliae ΔMad2 prior to planting in the field produced a 14.92%, 4% and 0% increase in seed weight respectively. M. anisopliae increased growth rates of winter wheat in microcosms and may act as a biofertilizer by making insoluble phosphate available to plants. This study showed Metarhizium benefits plants by protecting them from insect pests and by potentially solubilizing otherwise unavailable nutrients. Metarhizium, therefore, may be implemented in an integrated pest management (IPM) approach to reduce the use of chemical insecticides and fertilizers.Item Eye Growth and Differentiation in the Blind Cavefish Astyanax mexicanus: A Study in the Evolution of Development(2006-08-03) Strickler, Allen Gordon; Jeffery, William R; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Modern biological studies often employ a synthesis of multiple fields to accomplish a unified research goal. For instance, evolution of development (evo-devo) answers questions concerning the emergence of unique organismal phenotypes resulting from changes in evolutionary and developmental forces. I am interested in studying these forces on a microevolutionary scale. To accomplish this, I use the teleost fish Astyanax mexicanus. This species, indigenous to Mexico, is comprised of two forms: a surface stream dwelling form (surface fish) and a cave dwelling form (cavefish). Cavefish, which are the evolutionary descendent of surface fish, have evolved a number of constructive and regressive features as a result of being exposed to the subterranean environment, including loss of functional eyes and melanin pigment. Thus, Astyanax is ideal for comparative studies on a microevolutionary scale. I am interested in studying changes in eye development between surface fish and cavefish, and how this may relate to the evolution of the two forms. I initially utilized a comparative approach, using candidate gene, cell proliferation, and cell death studies. I extended these studies to include differential gene expression analyses as a means to better understand differences between surface fish and cavefish development. To further this understanding, I ultimately performed surface fish to cavefish lens transplants and surface fish lens deletions to study the effect of the lens on eye development. Finally, I integrate these data into a theory concerning eye development in Astyanax and put these developmental phenomena in the context of evolution.Item Identification of a single nucleotide polymorphism associated with adiposity following transcriptional profiling of gene expression in the anterior pituitary gland(2006-07-23) muchow, michael; Porter, Tom E; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Although the anterior pituitary secretes three hormones that affect metabolism and body fat stores, a comprehensive analysis of pituitary gene expression associated with body fat has not been performed. This research used cDNA microarrays to investigate pituitary gene expression in two chicken lines that were selected for low and high body fat (Lean and Fat). RNA was extracted from pituitaries at 1, 3, 5, and 7 weeks of age. 386 genes that showed significant differences in expression levels by line or in the line-by-age interaction were analyzed further. Differentially expressed genes between lines are potential candidates as genetic markers for high and low potential for body fat accumulation. One such candidate, the lysophosphatidic acid (LPA) receptor-1 (LPAR1), was identified as a potential marker, being differentially expressed between the 2 lines at the early ages. Genomic DNA from the Fat and Lean F0 generation was sequenced upstream of the LPAR1 coding region. A SNP consisting of a T to C transversion that introduces a GATA-1 transcription factor binding site was identified in the Lean line (Fisher's Exact Test, p ≤ 0.001). The fattest and leanest animals of both sexes in the back-crossed F2 generation (n=48 each) were genotyped by allele-specific PCR, and an association was present between the genotype and phenotype (generalized linear model, p ≤ 0.05). Expression of GATA transcription factors in mice inhibits differentiation of preadipocytes into mature adipocytes. LPAR1 also inhibits differentiation of preadipocytes in mice, and LPAR1 knock-out mice become significantly fatter than wild-type mice. A SNP that introduces a GATA site in the promoter of LPAR1 could up-regulate its expression in the Lean line, and increased LPA signaling could then inhibit preadipocyte differentiation. Conversely, loss of the GATA binding site could explain decreased levels of LPAR1 expression and attenuated inhibition of adipocyte maturation in the Fat line.Item A COMBINATORIAL DESIGN OF A PROTEIN-BINDING DNA MICROARRAY(2004-11-29) Qureshi, Aaron M.; Hunt, Brian R.; Mathematics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The biological process of transcription creates from a template DNA strand (i.e., the gene) copies of short-lived mRNA. The amount of mRNA produced determines the gene's expression in the cell, which affects the activity of the gene at a given time. Transcription factors are proteins which bind to the DNA in the neighborhood of the gene in order to regulate the location and rate of transcription. An important biological question is therefore to find binding locations and binding strengths for transcription factors. This has traditionally been a laborious experimental process, but a new technology called a protein-binding microarray allows us to assay the binding affinities of a given transcription factor for many different DNA sequences in parallel. This thesis addresses a suitable combinatorial design for these microarrays that is both effective and economical.