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
81 results
Search Results
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 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 The effects of diet and physical activity on telomere length and telomere-related genes in mice bred for high voluntary wheel running(2010) Marshall, Mallory Rachelle; Roth, Stephen M; Kinesiology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The purpose of this study was to determine the effects of diet and physical activity on skeletal muscle telomere length and mRNA levels of components of shelterin, a six-protein complex that protects telomeres, in mice bred over 52 generations for high wheel running activity compared to control mice. Mice were fed either a regular or high-fat diet and were provided wheel access or kept in cages without wheels for 8 weeks. Telomere length was significantly longer in mice fed a high-fat diet compared to those on a regular diet, but no other differences were observed. There were no differences in mRNA levels of the telomere-protecting shelterin components Trf1, Trf2, Pot1a, or Pot1b for diet, wheel access, or selection. High-fat diet may result in telomere dysfunction in these young mice, but we were unable to support our hypothesis that exercise would modify telomere length or shelterin mRNA levels in these mice.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 Genetic Variation in Nitrogen and Phosphorus Levels in Broiler Excreta: Opportunity for Improving both Birds and the Environment(2010) sasikala appukuttan, arun kirshna; Siewerdt, Frank; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The increase in poultry meat consumption has resulted in intensified poultry farming operations with consequent concentration of excreta in major production areas. The nutrient content in the soil surrounding the poultry farms has increased as a result of the high content of nitrogen (N) and phosphorus (P) in the poultry excreta. The current study aimed to propose a strategy to reduce the N and P content in excreta through genetic selection of broilers for efficient nutrient utilization. The traits measured (on a dry matter basis) were the percentage of N in the excreta (PNE) and the percentage of P in the excreta (PPE). Individual 24-hr excreta samples were collected from 6 wk old birds. Excreta samples were collected from a commercial breeding farm at two different time periods from line A and line B birds respectively, and analyzed for PNE and PPE. Analysis of excreta samples collected during the first period (197 bird samples belonging to 15 sire families) and second period (278 birds belonging to 25 sire families) suggested a heritability of 0.08, 0.16 for PNE and 0, 0.20 for PPE, respectively. Phenotypic and genetic correlations between the measured traits from the two lines were very low; however, phenotypic correlation analysis of PNE and PPE with other traits of commercial interest showed some favorable as well as neutral associations. Blood samples collected from the birds were used for an association study of the excreta traits with four candidate genes. The candidate genes were selected based on the results of previous research. Some of the SNPs from the candidate genes were found to have additive and dominance effect on the excreta and production traits and were usually favorably associated with mutations in higher frequency in the populations. The results suggest that genetic selection of birds for PNE and PPE could improve the environment and the market value of the birds.Item ANDROGEN RECEPTOR POLYGLUTAMINE REPEAT LENGTH AFFECTS RECEPTOR ACTIVITY AND C2C12 CELL MYOGENIC POTENTIAL(2010) Sheppard, Ryan Lance; Roth, Stephen M; Kinesiology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Testosterone (T) has a strong anabolic effect on skeletal muscle and is believed to exert its local effects via the androgen receptor (AR). The AR harbors a polymorphic stretch of glutamine repeats demonstrated to inversely affect receptor transcriptional activity in prostate and kidney cells. However, longer AR glutamine repeat lengths are associated with greater lean body mass and higher serum T in humans. The effects of AR glutamine repeat length on skeletal muscle are unknown. Purpose:To determine the effects of AR glutamine repeat length on AR function in skeletal muscle cells. Methods:AR expression vectors carrying 14, 24, and 33 glutamine repeats, respectively, were constructed and AR transcriptional activity was determined in transfected C2C12 myoblasts using an AR sensor plasmid. Each vector was subsequently stably transfected into C2C12 cells to create 3 independent cell lines: C2C12AR14, C2C12AR24, and C2C12AR33. Cellular proliferation and creatine kinase (CK) activity were determined. Gene expression was assessed via RT-PCR. Myosin expression, myotube formation, and myonuclear fusion index were examined immunohistochemically. Results: Transcriptional activity increased with increasing repeat length (3.91±0.26 vs. 25.21±1.72 vs. 36.08±3.22 relative light units in AR14, AR24, and AR33, respectively; p<0.001), in response to T. Ligand activation ratio indicated significant ligand-independent AR transcriptional activity. Significant AR protein expression was only detected in AR14 myoblasts. In contrast, AR mRNA expression was elevated in each stable line in the myoblast stage and throughout differentiation. The proliferation of AR33 cells was significantly decreased vs. AR14 (20512.3±1024.0 vs. 27604.17±1425.3, p<0.001) after 3 days. The CK activity of AR14 cells was decreased in comparison to AR24 and AR33 cells (54.9±2.9 vs. 68.3±2.2 and 70.8±8.1 units/μg protein, respectively; p<0.05) after 5 days of differentiation. The myonuclear fusion index was lower for both AR14 (15.21±3.24%, p<0.001) and AR33 (9.97±3.14%, p<0.001) in comparison to WT C2C12 cells (35.07±5.60%). Both AR14 and AR33 cells displayed atypical myotube morphology. RT-PCR revealed differences in the expression of genes involved in differentiation, cell fusion, and cell cycle progression. Conclusion: AR polyglutamine repeat length affects receptor activity and alters the growth and development of C2C12 cells. This polymorphism may explain some of the heritability of muscle mass in humans.Item Elucidating the Macro- and Micro-evolutionary Relationships of the Federally Listed Endangered Species Agalinis acuta (Orobanchaceae)(2010) Pettengill, James Beaton; Neel, Maile C; Behavior, Ecology, Evolution and Systematics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Agalinis acuta (Orobanchaceae) is a federally listed endangered plant species native to the mid-Atlantic and northeastern coastal plains of the United States. Due to morphological ambiguity and molecular similarity between A. acuta and Agalinis tenella and Agalinis decemloba a conservation priority is to determine whether A. acuta represents an evolutionarily distinct entity worthy of protection under the Endangered Species Act. To resolve this question, a phylogenetic study was first conducted based on seven chloroplast DNA loci and the nuclear DNA locus ITS from 79 individuals representing 29 Agalinis species. A study evaluating the utility of those cpDNA loci and three analytical techniques for the purpose of DNA barcoding was also conducted. The phylogenetic study indicated that A. acuta was perhaps evolutionarily indistinct from A. decemloba and A. tenella. Based on the results of subsequent analyses of 21 microsatellite loci and morphological data evaluated under myriad species concepts, A. acuta, A. decemloba, and A. tenella best represent a single species with two subspecies; the former two putative species would constitute a subspecies called A. decemloba ssp. decemloba and A. tenella would be A. decemloba ssp. tenella. With evolutionary distinct entities described, a phylogeographic study was conducted to determine the extent to which historical processes rather than contemporaneous events can explain extant patterns of genetic and phenotypic diversity within A. decemloba. The dispersal of a few individuals out of southern refugial populations likely represents the process through which northern populations were established; however, recent anthropogenic effects that disproportionately affected northern populations may have also contributed to extant patterns of diversity. Neutral or adaptive explanations for phenotypic variation among populations are also investigated. The conservation implications of population genetic analyses were assessed for members of A. decemloba ssp. decemloba. Despite the evidence that this taxon is self-compatible, the high levels of inbreeding and low levels of heterozygosity are of such a magnitude in certain populations that genetic factors may be negatively impacting fitness. Because of the small effective population sizes and degree of isolation, all populations should be managed to reduce the risk of extinction associated with demographic and environmental stochasticity.Item Genetic Differentiation of Selected Eastern Box Turtle (Terrapene carolina) Populations in Fragmented Habitats, and a Comparison of Road-based Mortality Rates to Population Size(2009) Hagood, Susan; Adams, Lowell; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The decline of eastern box turtle populations is associated with habitat loss and fragmentation, lack of recruitment into breeding populations, removal of individuals from the wild for pets, and road mortality. Box turtle populations in many areas of the eastern United States may effectively be isolated as high traffic volumes on roads adjacent to turtle habitats prevent successful dispersal. If so, populations surrounded by heavily used roads may be less genetically diverse than those in relatively intact habitats. I investigated whether populations in three Montgomery County, Maryland parks that were surrounded by roads were genetically differentiated relative to populations in two larger habitats in Maryland's Prince George's and Anne Arundel counties. Sampling was conducted between 2005 and 2008. I used 10 microsatellite markers to compare these populations, and sampled in an additional five sites (two in Maryland, and one each in Pennsylvania, the District of Columbia, and Florida), to better assess population structure. I found little evidence of genetic differentiation among central Maryland populations regardless of the extent of isolation. I attribute these findings to the slow rate of change in turtle evolution; the observed similarities in genetic diversity may reflect past rather than present gene flow. I found moderate to great differentiation in populations separated by substantial distances. To determine whether road mortality exceeds additive mortality levels believed to be a threat to population persistence, I estimated population size in the three Montgomery County, Maryland, parks using mark-recapture techniques, and compared these estimates to the number of dead, injured, and live turtles in or very near roads observed during walking and driving surveys conducted in 2006. Road-based morality rates fell within the range estimated to be inconsistent with population growth in one of the parks. Road mortality appeared to affect females out of proportion to their abundance in the population. Suggestions for reducing box turtle mortality in areas associated with high mortality rates are included.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 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.