UMD Theses and Dissertations

Permanent URI for this collectionhttp://hdl.handle.net/1903/3

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 given thesis/dissertation in DRUM.

More information is available at Theses and Dissertations at University of Maryland Libraries.

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Subject: search.filters.subject.metabolic engineering

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    The Regulation Of Intervertebral Disc Cell Interactions With Their Surrounding Microenvironment
    (2010) Rastogi, Anshu; Hsieh, Adam H; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Intervertebral disc degeneration is the major cause of back pain in the US, which can be both physically debilitating and costly to treat. Current treatments include invasive surgeries, which can be effective in ameliorating pain, but also contain the risk of complications. Additionally, these strategies target clinical manifestations of disc degeneration, rather than examine the cause of degenerative changes. Therefore, current research focuses on finding minimally invasive treatments for disc disease such as gene therapy. Regulating intervertebral disc cell interactions with their immediate environment can be a useful tool in the development of therapeutic strategies. This was explored through environmental changes to assess shifts in cell phenotype as well as genetic modulation to elucidate alterations in cell function. Biochemical, nutritional, and physical factors were examined in immature nucleus pulposus cells to assess changes in gene expression, attachment, and proliferation. It was found that nutritional and physical factors can alter gene expression levels of NP cells, thereby altering cell phenotype. In addition, down-regulation of the proteolytic enzyme MMP-2 was explored through RNAi interference. Five shRNA lentiviral vectors were designed and validated for the sustained gene silencing of MMP-2. Silencing MMP-2 activity resulted in the inability of disc cells to focally degrade gelatin films as well as reduced ability of disc cells to remodel fibers in type I collagen gels, resulting in weakened gel architecture. These functional consequences were further explored in an in vivo study utilizing an annular needle-puncture model of disc degeneration. Injection of the shMMP lentiviral construct lead to decreased expression of MMP-2 in the disc, as well as improved disc height and morphology. Thus, the functional consequences of silencing MMP-2 were examined, elucidating its role in the degradative pathway leading to degenerative disc disease. The results of these studies can lay the foundation for developing therapeutic treatments for intervertebral disc degeneration.
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    RNA Interference Mediated Suppression of Tn-Caspase-1 as a means of investigating apoptosis and improving recombinant protein production in Trichoplusia ni cells
    (2008-11-17) Hebert, Colin G; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The baculovirus expression system has proven to be a robust and versatile system for recombinant protein production in insect cells. A wide range of promoters is available for the facile expression of transgenes, and yields of up to 50% of total protein have been reported. However, in many cases production is decreased as a result of proteases and host cell apoptosis. To combat this problem, RNA interference (RNAi) has been used as a metabolic engineering tool to knockdown host genes responsible for decreasing the yield of recombinant protein. A novel caspase (Tn caspase-1) derived from Trichoplusia ni cells has been identified and characterized. Through modulation of caspase levels via either RNAi or through interaction with baculovirus protein p35, the overall level of apoptosis present in cell culture has been decreased. In addition, the use of in vitro RNAi targeted against Tn caspase-1 has increased the production of recombinant green fluorescent protein. To further study the effect of suppressing Tn caspase-1, a stable cell line producing in vivo RNAi was developed, resulting in a nearly 90% decrease in caspase enzymatic activity. This suppression was able to improve culture viability under adverse conditions and increase recombinant protein production levels up to two-fold that of standard cells.