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.

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2012 - 20142

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Subject: search.filters.subject.Stem Cells

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    Pericellular matrix mechanotransduction events in differentiating human mesenchymal stem cells: Modulating the pericellular matrix through silencing type VI collagen and decorin
    (2014) Twomey, Julianne Doreen; Hsieh, Adam H; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Stem cell therapies are currently being explored for their potential in the regeneration of load bearing tissues, such as cartilage. Current therapies lack the ability to intrinsically overcome a mechanically adverse environment at implantation. To advance the implementation of human mesenchymal stem cells (hMSCs) for cartilage repair, the mechanisms by which cells “feel“ and interact with their micromechanical environment need to be understood. Chondrogenic hMSCs develop a thin pericellular matrix (PCM), consisting of type VI collagen (ColVI) and proteoglycans such as decorin (DCN). The PCM is believed to control mechanotransduction events, acting as both a biomechanical and biochemical buffer. This thesis studies the functional role of ColVI and DCN through targeted gene knockdown using shRNA lentiviral vectors complimentary to col6a1 or dcn. In the first part of the work, the biophysical role of the PCM was determined through comparisons of cellular deformability under uniaxial strain with or without ColVI and DCN knockdown. HMSCs were cultured in alginate scaffolds and were stimulated with transforming growth factor β for 1 to 2 weeks. We found that the PCM with ColVI knockdown lacked the ability to withstand applied compression and with DCN knockdown deformed in a strain– dependent manner. Next we analyzed the mechanosignaling initiation caused by a transient sinusoidal compressive load through studying cytoskeletal kinetics and gene expression. Altering the PCM through ColVI and DCN knockdown caused an increase in actin and vimentin cytoskeletal protein concentration that lacked a dynamic response to load. This lead to a stronger fibroblast growth factor gene expression in ColVI knockdown. DCN also demonstrated direct control over cartilage oligomeric matrix protein gene expression, through a loss of TGF– β regulation. These results were further demonstrated during long term compressive culture. Unconfined sinusoidal compressive culture revealed the highest improvement in material properties in knockdown samples at day 14. Through these studies, we demonstrated that ColVI and DCN are integral proteins in maintaining the structural microenvironment through protecting the cell from injurious deformation, maintaining cytoskeletal dynamics in response to load, and regulating the differentiation rate through TGF– β signaling. Finally, we demonstrated the ability to manipulate chondrogenic mechanotransduction events using genetic engineering.
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    On Chip Isolation and Enrichment of Tumor Initiating Cells
    (2012) Saadin, Katayoon; White, Ian M; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    We report for the first time a microdevice that enables the selective enrichment and culture of breast cancer stem cells using the principles of mammosphere culture. For nearly a decade, researchers have identified breast cancer stem cells within heterogeneous populations of cells by utilizing low-attachment serum-free culture conditions, which lead to the formation of spheroidal colonies (mammospheres) that are enriched for cancer stem cells. While this assay has proven to be useful for identifying cancer stem cells from a bulk population, ultimately its utility is limited by difficulties in combining the mammosphere technique with other useful cellular and molecular analyses. However, integrating the mammosphere technique into a microsystem can enable it to be combined directly with a number of functions, including cell sorting and analysis, as well as popular molecular assays. In this work, we demonstrate mammosphere culture within a polydimethylsiloxane (PDMS) microsystem. We first prove that hydrophobic PDMS surfaces are as effective as commercial low-attachment plates at selectively promoting the formation of mammospheres. We then demonstrate the culture of mammospheres as large as 0.25 mm within a PDMS microsystem. Finally, we verify that reagents can be delivered to the cell culture wells exclusively by diffusion-based transport, which is necessary because the cells are unattached. This microsystem component can be integrated with other microfluidic functions, such as cell separation, sorting, and recovery, as well as molecular assays, to enable new discoveries in the biology of cancer stem cells that are not possible today.