Doctor of Philosophy, The Ohio State University, 2017, Biomedical Engineering

Mammalian cell bioprocessing is a critical part of both diagnostic and therapeutic applications. In terms of diagnostics, blood based biomarkers can be isolated from the peripheral blood and analyzed to help clinicians make informed decisions regarding treatment. Three blood based biomarkers that have gained a lot of attention recently are circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and extracellular vesicles (EVs). Viable CTCs are able to provide information about living tumor cells in circulation and how they survive, while ctDNA is able to provide information about tumor cells that are apoptotic, maybe due to treatment. EVs are produced by living cells and found in several types of bodily fluids including urine, making it a promising diagnostic. Refinement of a negative immunomagnetic depletion approach to CTC separation revealed a population of cells known as efferocytes that can masquerade as CTCs in the peripheral blood. That is, they can appear to be negative for CD45, positive for cytokeratins (CK) and positive for vimentin a profile that would be expected of a CTC that has undergone epithelial to mesenchymal transition (EMT). However, flow cytometric analysis revealed that these cells are a combination of neutrophils and monocytes suggesting that CK positivity is not a random artifact, but rather the result of phagocytosis. Efferocytes are responsible for cleaning up apoptotic tumor cells in the peripheral blood and tumor tissue and have clinical significance as they consist of M2 polarized monocytes (macrophages) that are associated with EMT, angiogenesis and tumor progression. EVs show potential as a delivery vehicle for nucleic acids. However, their manufacture and scale-up is not nearly as well established as production of monoclonal antibodies from Chinese Hamster Ovary (CHO) cells. Using similar approaches to CHO, an upstream bioprocess for EV production from HEK293T cells is presented consisting of cell engineering, clone s (open full item for complete abstract)

Committee: Jeffrey Chalmers (Advisor); Derek Hansford (Committee Member); Jessica Winter (Committee Member) Subjects: Biomedical Engineering

Master of Science in Biological Sciences, Youngstown State University, 2000, Department of Biological Sciences and Chemistry

NK cells, a component of our innate immune system, are responsible for the elimination of virus-infected and tumor cells which bypass detection by T-cells. In these studies, we attempted to create a NK cell hybridoma clone, which would provide a testable pool of NK cells. Mouse splenocytes were fused with P3X mouse non-secreting myeloma cells in the presence of polyethylene glycol (PEG). Following HAT selection, the cells were tested for the presence of the mouse NK cell surface antigen DX5.

Committee: Diane Fagan (Advisor) Subjects: Biology, Cell

Doctor of Philosophy, Case Western Reserve University, 2004, Pathology

Identifying which processes and proteins control glucose transport could provide important clues to understanding and treating a number of clinical entities including diabetes and some cancers. Glucose transport across the plasma membrane occurs by either sodium-dependent or independent glucose transporters. In order to study the mechanisms which control acute changes in glucose transport by sodium-independent glucose transporters, we use the non-transformed rat liver – derived Clone 9 cell line. These cells respond to the acute inhibition of oxidative phosphorylation by azide with a 4-6 fold stimulation of glucose transport and a 1.8 fold increase in the amount of glucose transporter 1 (Glut1) in the plasma membrane. In Clone 9 cells under basal conditions, ~38 % of Glut1 in the post-nuclear lysate is localized to the detergent-resistant membrane (DRM) microdomains. Acute exposure to azide decreased this figure by ~40 %. In order to examine the effects of azide on Glut1 localization to the plasma membrane of the Clone 9 cell, we performed subcellular fractionation of the post-nuclear homogenates. Approximately 30 % of the Glut1 in the post-nuclear homogenate was recovered in the plasma membrane (PM) compartment and 50 % of this PM Glut1 localized to the DRM fraction. Acute inhibition of oxidative phosphorylation with azide resulted in a 1.6-fold increase in the total abundance of Glut1 in the PM and was associated with a 2.9 fold increase in the abundance of Glut1 in the non-DRM fraction but no significant change in the content of Glut1 in the DRM fraction. We conclude that in the Clone 9 cell Glut1 localizes to detergent-resistant membrane microdomains in the plasma membrane. Moreover, in these cells the azide – induced increase in glucose transport is associated with an increase of Glut1 abundance in the non – DRM fraction of the plasma membrane and a decrease of Glut1 association with the DRM fraction of a membrane compartment other than the plasma membrane. The (open full item for complete abstract)

Committee: Faramarz Ismail-Beigi (Advisor) Subjects: Biology, Cell