The hypothesis that cell volume and the progression of the cell cycle are interdependent has surfaced off and on in the cell cycle literature for the past 30 years. However, a conclusion as to how cell volume is mechanistically involved in cell division has not been reached in mammalian cells. The aim of this dissertation was to establish how volume changes modulate cell cycle progression. Most of the studies addressing this question have examined mass content yet, more recently, focus has been placed on intracellular water, which is determined by the balance between mechanical and osmotic forces. As a result, ion channels and transporters which regulate intracellular osmotic content are integral to the maintenance of cell volume. In this dissertation, I show that a large, rapid and regulated volume decrease occurs as glioma cells progress through mitosis. I refer to this process as pre-mitotic condensation (PMC). This process is functionally linked to DNA condensation prior to cell division as the two events occur simultaneously, and inhibition of PMC results in a prolongation of DNA condensation. Further, my data demonstrates that glioma cells actively accumulate chloride, which acts as the primary energetic driving for cell volume changes in these cells. During the process of PMC, this gradient drives the efflux of chloride through ClC3 channels, which mediates water loss and the volume decrease. Interestingly, chloride accumulation to similar levels can be observed in immature astrocytes and neurons, suggesting that glioma cells recapitulate the biology of immature proliferating cells in the brain. This also suggests that my findings may have broader applicability to cell division in both neural cells and cancer.

xii, 132 p. : ill., digital, PDF file.

Neurobiology

Joint Health Sciences

Voltage Gated Chloride Channels Pre-miotic Condensation DND Condensation C1C3 Mitosis Volume Regulation

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Aberrant hyaluronan production is frequently associated with tumors where the elevated levels of tumoral hyaluronan are often associated with higher expression levels of cellular hyaluronidases that produce saturated hyaluronan fragments with divergent pro-tumoral activities. In this thesis, we provide evidence that different hyaluronan metabolism of bacteriophage hyaluronidase (HylP) elicits distinct alteration in breast cancer cell behavior. We demonstrate through comparative analysis with bovine testicular hyaluronidase (BTH) that higher enzyme activity, specificity for hyaluronan and production of unsaturated oligosaccharides render HylP to have profound effects on growth, migration and invasion activities of breast carcinoma cells, whereas BTH and its saturated metabolites aggravate cancer cell proliferation. Our experimental data also indicate that HylP reverses hyaluronan-mediated Doxorubicin resistance, which further suggests its therapeutic potential in suppressing hyaluronan-mediated cancer progression. Functional characterization with a truncated form of HylP hyaluronidase suggests that collagen-like region in HylP may have an important role in exerting anti-cancer effects. Hence, the crystal structure of truncated phage enzyme determined as a part of this thesis work will be in use in future investigations to determine the collagenous structure in the native HylP enzyme as well as to identify the active sites of HylP, which would provide better understanding of the distinct catalytic mechanism in metabolizing hyaluronan.

1 online resource (ix, 104 p.) : ill., digital, PDF file.

Physiology and Biophysics

Joint Health Sciences

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Members of the [beta]2-integrin family of adhesion molecules, CD11a, CD11b, and CD11c, have all been shown to play a role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). CD11d had yet to be studied in demyelinating disease and its functions remained unclear. We report here that CD11d is the only member of the [beta]2-integrin family of adhesion molecules that fails to protect against the development of EAE. Surprisingly, the EAE studies suggested that CD11a, CD11b, and CD11c were all contributing to T cell activity during disease development by mechanisms beyond the migration of these cells into the CNS. However, the contributions of individual T cell subsets to the overall phenotypes seen were unclear. Earlier studies show that over the course of EAE a higher proportion of [gamma delta] T cells express the [beta]2-integrins when compared to [alpha beta] T cells. Given this we hypothesized that the [beta]2-integrin family was important to the functions of [gamma delta] T cells that contributed to the development of EAE. However, we show here that even though expression is enriched in this T cell subset the [beta]2-integrins do not seem to be required on [gamma delta] T cells for disease development. The [beta]2-integrin family has also been implicated in regulatory T cell function and homeostasis. Studies using transfer EAE with CD11a-/- mice have suggested these mice may have regulatory defects. Given this we next investigated the role CD11a plays in regulatory T cell biology. We show here that CD11a-/- mice have reduced Treg populations throughout the secondary lymph tissue and that this reduction may be due to a reduced capacity to generate peripheral Tregs. We also found that CD11a is critical to Treg function in vitro, but does not seem to be as important in vivo. Importantly CD11a appears to be mediating its immunosuppressive effects independently of interactions with ICAM-1 on target T cells. Overall, the studies presented here provide further evidence that the ,2-integrin family of adhesion molecules functions in many aspects of T cell biology aside from cellular migration and that these functions differ between T cell subsets.

xi, 100 p. : ill., digital, PDF file

Microbiology

Joint Health Sciences

Integrin EAE Gamma Delta T Cell Regulatory T Cell

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Although bone has a dramatic capacity for regeneration, certain injuries and procedures present defects that are unable to heal properly, requiring surgical intervention to induce and support osteoregeneration. Our research group has hypothesized that the development of a biodegradable material that mimics the natural composition and architecture of bone extracellular matrix has the potential to provide therapeutic benefit to these patients. Utilizing a process known as electrospinning, our lab has developed a bone-mimetic matrix (BMM) consisting of composite nanofibers of the mechanically sta-ble polymer polycaprolactone (PCL), and the natural bone matrix molecules type-I colla-gen and hydroxyapatite nanocrystals (HA). We herein show that BMMs supported great-er adhesion, proliferation, and integrin activation of mesenchymal stem cells (MSCs), the multipotent bone-progenitor cells within bone marrow and the periosteum, in comparison to electrospun PCL alone. These cellular responses, which are essential early steps in the process of bone regeneration, highlight the benefits of presenting cells with natural bone molecules. Subsequently, evaluation of new bone formation in a rat cortical tibia defect showed that BMMs are highly osteoconductive. However, these studies also revealed the inability of endogenous cells to migrate within electrospun matrices due to the inherently small pore sizes. To address this limitation, which will negatively impact the rate of scaf-fold-to-bone turnover and inhibit vascularization, sacrificial fibers were added to the ma-trix. The removal of these fibers after fabrication resulted in BMMs with larger pores, leading to increased infiltration of MSCs and endogenous bone cells. Lastly, we evaluat-ed the potential of our matrices to stimulate the recruitment of MSCs, a vital step in bone healing, through the sustained delivery of platelet derived growth factor-BB (PDGF-BB). BMMs were found to adsorb and subsequently release greater quantities of PDGF-BB, compared to PCL scaffolds, over an 8-week interval. The released PDGF-BB retained its bioactivity, stimulating MSC chemotaxis in two separate assays. Collectively, these re-sults suggest that electrospun matrices incorporating the bone matrix molecules collagen I and HA, with sacrificial fibers, provide a favorable scaffold for MSC survival and infil-tration as well as the ability to sequester PDGF-BB from solution, leading to sustained local delivery and MSC chemotaxis.

PhD

1 online resource (xv, 122 p.) :ill., digital, PDF file.

Joint Health Sciences

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Growth factors are important inducers of vascular cell growth whose regulation is altered during the pathogenesis of atherosclerosis. An increase in growth factor and cytokine production, as well as lipid oxidation is observed in cardiovascular disease (CVD) and contributes to altered vascular cell signaling, exacerbated atherosclerotic lesions and heart failure. A change in cellular bioenergetic status due to mitochondrial dysfunction or damage has also been noted in CVD. In this thesis, we first examine cell signaling pathways in vascular smooth muscle cells (VSMC) which are activated in response to platelet- derived growth factor (PDGF) and regulate the cell cycle protein changes associated with proliferation. Next, we investigate the role of the phosphoinositide-3-kinase in the regulation of glycolysis and examine how glucose utilization and substrate availability for mitochondrial respiration can modulate the proliferative response of VSMC to PDGF. Through this study we highlight a novel link between the regulation of cellular glycolysis products and mitochondrial function. Finally, we examine the regulation of protein Olinked glycosylation in the regulation of mitochondrial bioenergetics and its implications on VSMC proliferation. This study highlights the importance of mitochondrial respiratory function in the observed PDGF-dependent effects. Together, these studies suggest a role for the metabolic changes that alter mitochondrial bioenergetics in the responses of vascular cells to growth stimuli and open avenues for further study for potential therapeutic agents.

1 online resource (xxii, 169 p. : ill., digital, PDF file)

Physiology and Biophysics

Joint Health Sciences;

bioenergetics G1cNAc PDGF P13K proliferation smooth muscle

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