UMD Theses and Dissertations
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Item A NEW APPROACH FOR FINE MAPPING AND CLONING GENES FROM THE TERTIARY GENE POOL MEMBERS OF WHEAT: EXAMPLE OF LR57 FROM AEGILOPS GENICULATA(2020) Steadham, James; Rawat, Nidhi; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Wheat is a staple food for 35% of the world’s population, providing more calories and protein to the world’s diet than any other crop. Wheat rust diseases cause yield losses worth billions of dollars annually. Two rust resistance genes, Lr57 and Yr40, conferring leaf rust and stripe rust resistance, respectively, were previously identified in a wild wheat relative, Aegilops geniculata. Mapping these genes within a wheat background is typically impossible, due to the absence of genetic recombination between wheat and wild species chromatin. We devised a novel technique to overcome this barrier, developing a mapping population from a cross between a resistant Ae. geniculata-wheat introgression line and a susceptible Ae. geniculata disomic addition line. Genotyping and phenotyping 162 individuals from this population, we found 11 recombinants within a 6 Mb interval and fine mapped Lr57, demonstrating the high-resolution power of this strategy for mapping genes from wild relatives of wheat.Item MOLECULAR ISOLATION OF THE MOV-1 LOCUS IN HEXAPLOID BREAD WHEAT(2020) Mahlandt, Alexander Robert; Tiwari, Vijay K; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Wheat provides a substantial portion of globally consumed calories, yet faces growing challenges to meet yield needs. Traits affecting floral architecture and grain number hold immense promise for increasing yield potential. Multi-ovary wheat is a unique mutant line that holds potential in improving yields, characterized by the formation of 2-3 grains per floret in a given wheat spike. Described is a multi-approach mapping effort that refines the chromosomal position of the MOV1 locus at high resolution. A radiation hybrid map, linkage map, and refined physical map are reported. Further mutation analysis recovered missense mutations within a candidate gene, displaying a quantitatively reduced phenotype. Expression profiling of the candidate gene reveals up-regulation at crucial developmental stages. Structural investigation of the gene and surrounding locus identified two insertion/deletion events that suggest a pathway for transcriptional regulation. The results presented implicate the candidate gene in the manifestation of the multi-ovary phenotype.Item LONG-RANGE SIGNALING AT THE INTESTINAL-NEURAL AXIS PROMOTES ORGANISMAL HEME HOMEOSTASIS IN C. ELEGANS(2014) Sinclair, Jason Wallace; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Metazoans synthesize and regulate intracellular heme in a cell autonomous manner although genetic evidence in vertebrates suggests that cell non-autonomous mechanisms may exist at the organismal level. In C. elegans, a heme auxotroph, extraintestinal tissues are intrinsically dependent on the intestine, which acquires dietary heme for sustenance, supporting the concept that intestinal heme status must be coordinated at the systemic level to regulate whole-organism heme homeostasis. Here we show, by conducting a functional genome-wide RNAi screen in an intestinal-restricted heme sensor worm, that an interorgan heme signaling pathway exists and that >30% of the genes identified from the RNAi screen altered heme homeostasis in the intestine even though these genes are not expressed in the intestine. The biological basis for this signaling is underscored by HRG-7, a cathepsin protease-like protein secreted by the intestine and internalized by distally-located neurons. HRG-7 is specifically secreted from the intestine during heme limitation and hrg-7 depletion causes embryonic lethality concomitant with a heme deficiency response. Reciprocally, neuron-to-intestine heme signaling is mediated by the bone morphogenic protein homolog DBL-1, which recapitulates hrg-7 deficiency when depleted. Remarkably, depletion of both genes simultaneously results in markedly enhanced growth and heme deficiency phenotypes, suggesting that bidirectional signaling between the intestine and neurons mediates systemic heme homeostasis. Our results have uncovered an unexpected role for a protease family member in long-range communication between organs at the intestinal-neural axis to regulate systemic heme homeostasis in metazoa. As humans have over thirty cathepsin and cathepsin-like proteases, several of which are secreted, we anticipate that these proteins may play analogous roles in mammalian biology.Item Investigating genetic and health factors related to AA amyloidosis prevalence in captive cheetahs (Acinonyx jubatus): implications for population management(2014) Franklin, Ashley Danielle; Porter, Tom E; Crosier, Adrienne E; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Systemic amyloid A (AA) amyloidosis is an increasingly important cause of morbidity and mortality among captive cheetahs, yet wild cheetahs are virtually unaffected, suggesting the phenomenon is a result of the captive condition. The self-aggregating AA protein responsible for this disease, is a byproduct of serum amyloid A (SAA) protein degradation, an acute phase protein highly upregulated during inflammation. The objective of this study was to identify the relationship between genetics, stress, and inflammation with serum concentrations of the SAA protein and the incidence of AA amyloidosis in captive cheetahs. Fecal and serum samples collected from cheetahs held at the Smithsonian (NZP-SCBI) and Cheetah Conservation Fund (CCF) facilities, as well as wild, free-ranging cheetahs, were examined. Enzyme-linked immunosorbent assays were used to measure SAA protein and proinflammatory cytokine concentrations in serum samples and cortisol concentrations in feces. Additionally, cheetahs were genotyped for the SAA1A-97delG single nucleotide polymorphism (SNP) in the promoter region of the SAA1 gene. This study was the first to demonstrate that serum concentrations of the SAA protein in cheetahs are affected by the SAA1A-97delG SNP (P=0.0453). However, the high prevalence of AA amyloidosis observed among captive cheetahs is not attributable to genetic differences at this locus, but rather appears to be related to stress and/or inflammation, as captive cheetahs at NZP-SCBI have significantly higher SAA protein concentrations in serum compared to captive cheetahs at CCF, regardless of genotype (P=0.0003). Captive cheetahs at NZP-SCBI show levels of stress (fecal cortisol concentrations) greater than their captive counterparts at CCF in Namibia. Interestingly, wild cheetahs and captive cheetahs at CCF in Namibia had significantly higher proinflammatory cytokine concentrations (TNF-α and IL-1β) in serum compared to cheetahs at NZP-SCBI (P<0.0001). It is possible that chronic stress may be suppressing the production of proinflammatory cytokines in the NZP-SCBI cheetah population. Controlling the currently high SAA protein concentrations associated with AA amyloidosis is the best strategy to decreasing the diseases prevalence among captive cheetahs. Promoting management practices that reduce stress could help re-establish proper immune system homeostasis and mitigate the overproduction of SAA protein, decreasing the probability of developing AA amyloidosis.Item EVOLUTION, DEVELOPMENT, AND GENETICS OF OPSIN GENE EXPRESSION IN AFRICAN CICHLID FISHES(2011) O'Quin, Kelly E; Carleton, Karen L; Behavior, Ecology, Evolution and Systematics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The molecular genetic mechanisms that underlie phenotypic evolution include mutations within protein-coding, cis-regulatory, and trans-regulatory factors. Although many studies have examined how these mutations individually contribute to phenotypic divergence and the formation of new species, none have examined how they may do so collectively. In this study, I examine how these molecular genetic mutations collectively contribute to the evolution of color vision among African cichlid fishes. I show that phenotypic divergence in cichlid color vision is achieved by mutations affecting the coding sequence and expression of seven opsin genes. After contrasting the roles of these two mechanisms, I use bioinformatic-, association-, and experimental genetic analyses to determine what role mutations in cis- and trans-regulatory DNA play in the evolution of cichlid opsin expression. Specifically, I demonstrate that: (1) Protein-coding mutations primarily affect cichlid opsins sensitive to the ends of the visible light spectrum (SWS1 [ultraviolet-sensitive] and LWS [red-sensitive]). (2) Changes in opsin gene expression contribute to large differences in color vision among closely related species. These analyses also reveal that the expression of the SWS1 and SWS2B opsins have diverged among closely related cichlids in association with foraging preferences and ambient light intensity, suggesting that their expression has evolved due to natural selection. Ancestral state reconstructions reveal that changes in opsin expression have evolved repeatedly among cichlids in Lakes Tanganyika and Malawi; further, I find that this repeated evolution has likely been achieved by repeated changes to cichlid development. (3) Bioinformatic analyses suggest that cichlids have diverged in multiple cis-regulatory sequences surrounding the opsin genes, and association mapping identified three putative single nucleotide polymorphisms upstream of the SWS2A (blue), RH2B (blue-green), and LWS (red) opsins that may contribute to cichlid opsin expression differences in cis. (4) Genetic mapping in experimental crosses suggests that divergence in multiple trans-regulatory factors also contribute to the evolution of SWS2B (violet), RH2A (green), and LWS (red) opsin expression. The contribution of these trans-regulatory factors to the evolution of cichlid opsin expression may outweigh those in cis. These results reveal that multiple molecular genetic mechanisms can contribute to phenotypic evolution among closely related species.Item Life's Rich Pattern: The Role of Statistics and Probability in Nineteenth Century Argumentation for Theories of Evolution, Variation, and Heredity(2006-04-26) Wynn, James; Fahnestock, Jeanne; English Language and Literature; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Though modern philosophers of science recognize the inappropriateness of the reduction of all scientific investigations to mathematics, mathematics and science share a long history with one another during which mathematics has been employed as a major component of scientific argumentation. Over the last twenty years, rhetoricians have done substantial work studying the role of argumentation in science (Bazerman 1988; Gross 1990, 2002; Myers 1990; Fahnestock 1999); however, despite the importance of mathematics in making scientific arguments, little effort has been made to understand the role mathematics has played in making these arguments. This dissertation represents a move to resolve this shortcoming by investigating the role of mathematics in arguments in evolutionary biology from the middle of the nineteenth to the beginning of the twentieth century. In the first part of the nineteenth century, the mass collection and mathematical assessment of data for scientific purposes provides the context for understanding some of the rhetorical choices of an important group of natural philosophers and biologists who developed arguments in the second half of the century about the nature of variation, evolution, and heredity. In the works of Charles Darwin, Gregor Mendel, Francis Galton, and Karl Pearson, arguments from probability and statistics play important roles as support for their arguments and as a source of invention for their claims. This investigation of the rhetorical situations of these four biologists, their arguments, and the role of the principles, operations, and formulae of probability and statistics supports the position that mathematization had a major impact on the nature of scientific evidence in the nineteenth century. What it also suggests is that, though mathematized arguments may have had a great deal of credibility within the scientific community in general, factors such as the stature of the rhetor and of their biological theory within their specific discourse communities played an equally important role in the persuasiveness of their arguments.Item A DNA REPAIR/PHASE VARIATION REPORTER SYSTEM USING A POLY-GUANINE TRACT IN A NEISSERIA GONORRHOEAE NITROREDUCTASE GENE(2005-11-10) Rodgers, Mark Andrew; Stein, Daniel C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neisseria gonorrhoeae undergoes phase variation to adapt to new environments, increase pathogenesis, and evade the host immune system. This may be due to defects in DNA repair. A reporter system was created to detect phase variation by phenotypic switching from a nitrofuran-sensitive phenotype to a nitrofuran-resistant (NitR) phenotype. Strains were created with poly-guanine tracts from 5 to 12 guanines in the coding region of a nitroreductase gene (nfsB) that would be susceptible to frame-shifting mutations during DNA replication. The minimum number of consecutive guanines needed to observe increased mutation was 5. A strain expressing 7 guanines nfsB possessed nitroreductase activity similar to wild-type and a spontaneous mutation frequency that was increased ~104 fold relative to wild-type. Frame-shifting mutations of strain expressing 8 guanines in nfsB were observed using denaturing gradient gel electrophoresis (DGGE). Future work with the reporter system could lead to new understanding of phase variation and DNA repair.Item Insulin-like Growth Factor 1 Genotype Influences Muscle Strength Response to Sterngth Training in Older Adults(2004-11-03) kostek, matt; Hurley, Ben F; Kinesiology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Strength training (ST) is considered an intervention of choice for the prevention and treatment of sarcopenia. The insulin-like growth factor 1 protein (IGF-I) plays a major role in ST-induced skeletal muscle hypertrophy and strength improvements. A microsatellite repeat in the promoter region of the IGF1 gene has been associated with IGF-I blood levels and phenotypes related to IGF-I in adult men and women. To examine the influence of this polymorphism on muscle hypertrophic and strength responses to strength training (ST), we studied 67 Caucasian men and women before and after a 10-week single leg knee extension ST program. One repetition maximum (1RM) strength, muscle volume (MV) via computed tomography (CT), and muscle quality (MQ) were assessed at baseline and after 10 weeks of training. The IGF1 repeat promoter polymorphism and three single nucleotide polymorphisms (SNP) were genotyped. For the promoter polymorphism, subjects were grouped as homozygous for the 192 allele, heterozygous, or non-carriers of the 192 allele. After 10 weeks of training, 1RM, MV, and MQ increased significantly for all groups combined (P < 0.001). However, carriers of the 192 allele gained significantly more strength with ST than non-carriers of the 192 allele (P = 0.02). There was also a non-significant trend for a greater increase in MV in 192 carriers than non-carriers (P = 0.08). No significant associations were observed for the other polymorphisms studied. Thus, these data suggest that the IGF1 promoter polymorphism may influence the strength response to ST. Larger sample sizes should be used in future studies to verify these results