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 INVESTIGATION INTO THE ROLE OF UVR8 IN BALANCING GROWTH AND ACCLIMATION TO UV-B RADIATION IN NATURAL AND TRANSGENIC POPULUS VARIANTS(2021) Wong, Tiffany Marie; Eisenstein, Edward; Sullivan, Joseph; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Research on woody plants offers promise for the development of next-generation biofuel feedstocks with reduced lignin recalcitrance and enhanced saccharification for ethanol production. Natural variants of Populus trichocarpa with diverse lignin content and saccharification differences, and transgenic Populus deltoides constructed for reduced lignin levels for improved cellulose extraction, offer clues to enhance biofuel production but with a tradeoff to overall fitness and biomass. One concern of engineering lignin relates to the protection of plants against environmental stress such as UV-B radiation. Secondary metabolite biosynthesis initiated by UV-B, particularly phenylpropanoids (lignin precursors) and flavonoids, plays an important role in managing and protection of UV stress. Genetic modifications affecting the production of these compounds may have significant physiological consequences. Thus, the goal of this research was to develop a model for biosynthetic compensation of low-lignin Populus to UV-B stress. The effect of UV-B on Populus was evaluated by spectroscopic and metabolomic measurements on leaves. UV-B promoted shifts in physiological and metabolomic responses of natural and transgenic Populus with varying levels of lignin were complex, reflecting compensation from variety of biosynthetic alterations. Therefore, the impact of modulating the expression of the photoreceptor, UVR8, in regulating the response of Populus to UV-B was pursued. Modulation of UVR8 expression in Populus hybrid was achieved by constructing transgenic plants using CRISPR and RNAi, in wild-type, and an RNAi-constructed cinnamyl alcohol dehydrogenase knockdown line. UV-B response of UVR8 modulated Populus indicated that flavonoids were upregulated in UVR8 overexpression lines, and that in a CAD knockdown background, these effects were slightly enhanced. Salicylates were upregulated in UVR8 knockout poplars, suggesting metabolic flux in the pathway, but little difference was seen relative to wild-type plants in CAD lines, and UV-B treatment had little effect. An interesting and unexpected finding was that UVR8 modulated Populus exhibited more rapid growth than wild-type plants. The findings underscore the key role of UVR8 in synchronizing protective metabolic responses to UV-B and suggest an additional function of the photoreceptor in regulating growth and development of Populus through shifts in the chemical equilibria of UVR8 monomers and dimers and interactions with other regulatory factors.Item DEVELOPMENT OF SINGLE-CELL MASS SPECTROMETRY TOOLS TO INVESTIGATE METABOLIC REORGANIZATION DURING EARLY EMBRYOGENESIS(2020) Portero, Erika Paola; Nemes, Peter; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Measurement of metabolism in single cells holds the potential to advance our understanding of fundamental biological processes during cell differentiation and development. However, to characterize the metabolic state of single cells, further technological advances are still required. This dissertation discusses the development and application of single-cell mass spectrometry (MS) technologies to investigate metabolism and its role during tissue induction in the early developing vertebrate (frog) embryo. The work presented herein illustrates the strategies devised to advance single-cell analysis using capillary electrophoresis (CE)-MS. Additionally, this work features several contributions to our understanding of cell heterogeneity and the role of small molecules during tissue specification in the vertebrate embryo, providing new information to advance cell and developmental biology.Chapter 1 overviews the current state of metabolomics for cell and developmental biology, as well as the research significance and motivations. Chapter 2 describes the fundamental concepts of CE and the current state of single-cell metabolomics by CE-MS. This chapter also discusses the development of a minimally invasive microprobe sampling technique designed for the Xenopus laevis embryo. Chapter 3 presents the development of a CE-MS approach that enables dual cationic and anionic analysis of metabolites from the same single embryonic cell to deepen the detectable coverage of metabolism. Chapter 4 discusses a stable-isotope labeling strategy and single-cell CE-MS to uncover metabolic pathways involved in cell differentiation. Chapter 5 details the application of our custom-built microprobe sampling technique to investigate spatial cell heterogeneity in the same vertebrate embryo. This chapter examines cell-to-cell communication and small molecule transport between adjacent cells. Moreover, dual-fluorescent cell lineage tracing reveals cell fate changes induced by small molecule transport. Chapter 6 summarizes the results generated from this dissertation work and reflects on technical challenges and potential advancements needed to drive the field of MS-based single-cell metabolomics forward.Item The Effect of Hyper-Osmotic Conditions on the Growth, Metabolism, and Specific Antibody Productivity of a GS-NS0 Cell Line(2009) Brady, Stefanie Ellen; Wang, Nam S; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The effect of cellular growth, metabolism, and monoclonal antibody production on an industrial GS-NS0 cell line to hyper-osmotic medium was studied. The GS-NS0 cell line was found to have an optimum growth rate at a medium osmolality of 350 mosm/kg and an optimum specific productivity at 450 mosm/kg. Medium osmolality was shown to affect cell size as the cell line exhibited a regulatory cell volume increase response after an initial introduction into hyper-osmotic conditions. The response of the cell line to an osmotic shift was also studied. Osmolality of the culture medium was increased, at two different time points, through the addition of NaCl. The shift in osmotic pressure was found to have a positive impact on specific productivity of the monoclonal antibody produced. A finger print of the metabolic response of the GS-NS0 cell line to increased medium osmolality was determined. The application of metabolomics to mammalian cell cultures has not been widely explored. In this study, the cells were quenched and extracted using methods previously developed for microbial and plant cultures. An increase in concentration of internal amino acids, known to be osmolytes, was found under hyper-osmotic conditions.Item TIME SERIES METABOLIC PROFILING ANALYSIS OF THE SHORT TERM Arabidopsis thaliana RESPONSE TO ELEVATED CO2 USING GAS CHROMATOGRAPHY MASS SPECTROMETRY.(2004-08-30) Kanani, Harin Haridas; Klapa, Maria I; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Metabolic profiling has emerged as a high throughput technique for the quantitative analysis of the cellular physiological state at the metabolic level. It allows for the simultaneous relative quantification of hundreds of low molecular weight intra cellular metabolites. In this analysis, the polar metabolic profiles of A. thaliana liquid cultures (grown for 12 days, under light and 23°C) throughout 1-day treatment with 1% CO2, were measured using gas chromatography-mass spectrometry. Despite the advantages of time series analysis, this is the first plant metabolic profiling study of this type reported in the literature. The time series metabolic profiles were analyzed using multivariate statistical techniques. Data analysis revealed repression of photorespiration, repression of nitrogen assimilation and increase in structural carbohydrates. It is for the first time that the latter phenomenon is observed as a result of elevated CO2 in the plant environment.