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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Subject: search.filters.subject.Metabolic Engineering

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Now showing 1 - 4 of 4
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    ISOTOPE-ASSISTED METABOLIC FLUX ANALYSIS IN THE INVESTIGATION OF PROSTATE CANCER
    (2019) Graham, Trevor; Sriram, Ganesh; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Understanding cancer metabolism is critical to developing treatment strategies which selectively target malignant cells. Toward this objective, we apply isotope-assisted metabolic flux analysis to the investigation of prostate cancer, which kills over 28,000 men every year in the United States alone. We performed metabolic flux analysis (MFA) on immortal prostate cancer cell lines to determine the relative activity of metabolic pathways that constitute central carbon metabolism. We identified multiple deviations of the malignant phenotype from that of benign cells. We found that all cell lines exhibited a preference for the pentose phosphate pathway over glycolysis for glucose catabolism, with an average flux partition of 53% ± 25% in favor of the pentose phosphate pathway. We also identified a drop in TCA cycle flux from 33.5 ± 10.5 for LNCaP to 19.7 ± 7.8 for CSS90 cells, possibly indicating a preference for glutaminolysis and lipogenesis to fuel rapid proliferation.
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    A Systems Engineering Framework for Metabolic Engineering Experiments
    (2011) Johnnie, Joseph George; Austin, Mark; Systems Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Cells of living organisms simultaneously operate hundreds or thousands of interconnected chemical reactions. Metabolic networks include these chemical reactions and compounds participating in them. Metabolic engineering is a science centered on the analysis and purposeful modification of an organism's metabolic network toward a beneficial purpose, such as production of fuel or medicinal compounds in microorganisms. Unfortunately, there are problems with the design and visualization of modified metabolic networks due to lack of standardized and fully developed visual modeling languages. The purposes of this paper are to propose a multilevel framework for the synthesis, analysis and design of metabolic systems, and then explore the extent to which abstractions from systems engineering (e.g., SysML) can complement and add value to the abstractions currently under development within the greater biological community (e.g., SBGN). The computational test-bed that accompanies this work is production of the anti-malarial drug artemisinin in genetically engineered Saccaharomyces cerevisiae (yeast).
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    High-Throughput Time Series Metabolomic Analysis of a Systematically Perturbed Plant System
    (2007-04-27) Kanani, Harin H; Klapa, Maria I; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    In the post-genomic era, availability of high-throughput profiling techniques enabled the measurement of entire cellular molecular fingerprints. Major characteristics of the high-throughput revolution were that (a) studying biological problems did not have to rely on prior hypotheses, while (b) parallel occurring phenomena, previously assumed disconnected, could now be simultaneously observed. Metabolomics is the newest of the "omics" techniques. It enables the quantification of hundreds of free metabolite pools, providing a metabolic fingerprint. Considering the importance of cellular metabolism, which is the net effect of changes at the genomic, transcriptomic and proteomic levels and of the cell with its environment, the metabolomic profile, is a fundamental determinant of cellular physiology. Obtaining accurate and complete metabolomic profiles is thus of great importance. However, being recent technology, metabolomics is currently at its standardization phase. As part of my PhD thesis research, I focused on addressing several current challenges in metabolomics technology development. Specifically a novel data correction, validation and normalization strategy for gas chromatography-mass spectrometry (GC-MS) metabolomic profiling analysis was developed, which dramatically increased the accuracy and reliability of GC-MS metabolomic profiles. The optimized metabolomics protocol was applied to study the short-term dynamic response of systematically perturbed Arabidopsis thaliana liquid culture system to study regulation of its primary metabolism. The biological system was studied under conditions of elevated CO2 stress, salt (NaCl) stress, sugar (trehalose) signal, and hormone (ethylene) signal, applied individually; the latter three stresses also applied in combination with the CO2 stress. Analysis of the obtained results required the appropriate application of multivariate statistical analysis techniques, which are developed mainly in transcriptomic analysis, into metabolomics analysis for the first time. The acquired results identified important new regulatory information about the biological systems resulting in new targets for metabolic engineering of plants. The large number of dynamic perturbation allowed re-construction of metabolic networks to identify possible novel metabolic pathways based on correlations between metabolic profiles. In addition, it demonstrates the advantages of dynamic, multiple-stress "omic" analysis for the elucidation of plant systems function. In this sense, it contributes in further advancing the computational and experimental metabolic engineering and systems biology toolbox.
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    Tissue and Metabolic Engineering of Biohybrid Artificial Organs
    (2005-12-07) Yung, Chong Wing; Barbari, Timothy A; Bentley, William E; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    In an effort to develop a biohybrid artificial organ, mammalian cells were engineered with several properties to make them adept in secreting therapeutic proteins and surviving low oxygen levels in an encapsulated environment. Three cell lines, C2C12, Jurkat, and HEK293, were investigated for their ability to secrete human interleukin 2 (hIL2), which can serve as an anti-cancer agent. An intracellular fluorescent protein marker was independently expressed using an internal ribosome entry site sequence so that hIL2 remained active and free for secretion. DsRed fluorescent protein markers were used since red light is known to be transmissible through mammalian tissue. Transient transfection of all three cell lines proved that internal red fluorescence measurements were indeed linearly correlated with the concentration of hIL2 secreted. To increase the survivability of encapsulated cultures, cells were engineered with an anti-apoptotic gene, bcl-2Delta, placed under the control of a hypoxia sensitive promoter. This protective system was found to lessen both hypoxia induced necrosis and apoptosis. To complete the biohybrid system, a novel hydrogel (mTG-Gel), utilizing microbial transglutaminase (mTG) to enyzmatically crosslink gelatin, was developed as a biocompatible cellular scaffold for encapsulating the engineered cells. These gels were stable at physiological temperature (37 oC) and could be tailored for enzymatic stability. Specifically, HEK293 cells that were metabolically engineered with all the previous characteristics were encapsulated in 4% mTG-Gels. In situ analysis of DsRed fluorescence showed that cells overlayed with mTG-Gel exhibited reductions in fluorescence with increasing height of gel. Human IL2 diffusion through the hydrogel into overlaying media was found to exhibit the expected dependence on the square of the gel thickness. Diffusion cells were used to determine an effective diffusion coefficient for hIL2, which compared well to that obtained from the gel-overlay cell culture experiments.