Doctor of Philosophy (PhD), Ohio University, 2016, Chemistry and Biochemistry (Arts and Sciences)

Cell adhesion is an important early step of cancer metastasis, yet the roles of sialylation in regulating integrin-mediated breast cancer cell adhesion in comparison to migration and invasion are not well-understood. The role of sialylation on alpha5 beta1 and alpha2 beta1 integrins in the regulation of adhesion between breast cancer cells and extracellular matrix (ECM) was studied. Our data showed that alpha2, alpha5 and beta1 integrins had considerable alpha 2,6 sialylation on MDA-MB-231 cells, whereas signals from MCF-7 cells were undetectable. Cleavage of alpha 2,6 sialylation increased adhesion of MDA-MB-231 cells to ECM, while adhesion of MCF-7 cells was unaffected, consistent with the latter's lack of endogenous alpha 2,6 sialylated surface integrins. Neither surface expression of alpha5 beta1 and alpha2 beta1 integrins, nor activated beta 1 integrin, changed in MDA-MB-231 cells after sialidase treatment. However, sialidase treatment did not have significant impact on migration or invasion of MDA-MB-231 cells. Integrins not only play an important role in adhesion of cancer cells, but also have a direct connection with ionizing radiation-induced atherosclerosis, which is an adverse effect observed after radiotherapy. However, minimal attention has been given to monocytes/macrophages, which are exposed to the radiation at the same time. Under flow conditions using a parallel plate flow chamber to mimic physiological shear stress, we demonstrate here that the avidity between very late antigen-4 (VLA-4) of RAW264.7 cells and its ligand vascular cell adhesion molecule-1 (VCAM-1), was increased after low dose (0.5 Gy), but was reduced after higher dose (5 Gy) treatment of ionizing radiation. Treating the cells with free radical scavenger N-acetyl-L-cysteine reduced the avidity between RAW264.7 cells and VCAM-1 to a similar level. These results suggest that ionizing radiation regulates adhesive interactions between VLA-4 and VCAM-1, and that reactive oxygen spe (open full item for complete abstract)

Committee: Shiyong Wu (Advisor) Subjects: Biochemistry

PHD, Kent State University, 2016, College of Arts and Sciences / School of Biomedical Sciences

The secretion of soluble pro-angiogenic factors by tumor cells and stromal cells in the perivascular niche promotes the aggressive angiogenesis that is typical of glioblastoma (GBM). Here, we show that angiogenesis also can be promoted by a direct interaction between brain tumor cells, including tumor cells with cancer stem-like properties (CSCs), and endothelial cells (ECs). As shown in vitro, this direct interaction is mediated by binding of integrin αvβ3 expressed on ECs to the RGD-peptide in L1CAM expressed on CSCs. It promotes both EC network formation and enhances directed migration toward basic fibroblast growth factor. Activation of αvβ3 and bone marrow tyrosine kinase on chromosome X (BMX) is required for migration stimulated by direct binding but not for migration stimulated by soluble factors. RGD-peptide treatment of mice with established intracerebral GBM xenografts significantly reduced the percentage of Sox2-positive tumor cells and CSCs in close proximity to ECs, decreased integrin αvβ3 and BMX activation and p130CAS phosphorylation in the ECs, and reduced the vessel surface area. These results reveal a previously unrecognized aspect of the regulation of angiogenesis in GBM that can impact therapeutic anti-angiogenic targeting.

Committee: Candece Gladson M.D. (Advisor); Gail Fraizer Ph.D. (Committee Member); Derek Damron Ph.D. (Committee Member); Matthew Summers Ph.D. (Committee Member); Songping Huang Ph.D. (Committee Member) Subjects: Biomedical Research; Cellular Biology; Molecular Biology

PhD, University of Cincinnati, 2008, Medicine : Pathobiology and Molecular Medicine

Fibrinogen, a plasma protein central to clot formation, has long been considered to play a role in inflammation and immunity. Fibrin(ogen) interactions with various immune cells have been heavily investigated; lacking is an understanding of the protein's influence on dendritic cell activity. Results from Chapter 2 demonstrate that fibrinogen initiates human dendritic cell production of inflammatory cytokines and chemokines. Adsorbed fibrin(ogen) has increased stimulatory capacity over fibrin(ogen) free in solution, indicating the bound protein, acting through the ß2-integrins, is the active species. Adsorbed fibrin(ogen) also stimulates the focal accumulation of dendritic cells. This is likely due to ß2-integrin-mediated chemotaxis of dendritic cells toward released chemokines and fibrin(ogen) degradation fragments. Because studies suggested adsorbed fibrinogen might be exploited to enhance vaccine adjuvanticity, fibrinogen-coated olive oil droplets were investigated as vaccine adjuvant. Results from Chapter 3 demonstrate the importance of surface in adjuvant activity, and the possible use of olive oil droplets as a safe and effective vaccine adjuvant. Having investigated the interactions between fibrinogen-coated particles and human dendritic cells, Chapter 4 describes the existence of a not yet recorded phenomenon: extracellular transport of cell-sized particles by dendritic cells. Results presented in that chapter not only demonstrate particles can be carried extracellularly by dendritic cells, but also that the process can be directed. Adsorbed fibrin(ogen) appears to enhance particle/dendritic cell interactions mediated through the ß2-integrins. The influence of adsorbed fibrin(ogen) on dendritic cells provides new knowledge into the protein's involvement in initiating inflammatory and immune responses, knowledge that may be applied to the development of new therapeutics to treat and prevent disease.

Committee: Gregory Retzinger MD,PhD (Committee Chair); Alison Weiss PhD (Committee Member); George Deepe MD (Committee Member); Judith Rhodes PhD (Committee Member); Philip Howles PhD (Committee Member) Subjects: Immunology; Pathology

Bachelor of Science (BS), Ohio University, 2011, Chemistry

There were over 207,000 new cases of invasive breast cancer and nearly 40,000 deaths from the disease in the U.S. in 2010. It is, therefore, critical to develop a better understanding of the disease process. Invasive cancer is marked by the loss of integrity of the basement membrane of a tissue, a specialized form of extracellular matrix (ECM) that normally separates a tissue from its surroundings; however, a complex, dynamic interaction involving many overlapping signaling pathways exists between breast tissue and its neighboring ECM. Invasion is initiated when the tumor cells have increased adhesion to the ECM, which is mainly modulated by interactions with the integrin family of adhesion proteins on the surface of the tumor cells. Each integrin heterodimer has a different adhesive affinity for each of the ECM proteins, namely fibronectin, vitronectin, collagen I, and collagen IV. This project examined the relative expression of integrins following exposure to ionizing radiation, which is commonly used for cancer therapy. Ionizing radiation forms free radicals and reactive oxygen species, which then cause damage within the cell. Although the exact mechanism is unknown, the hypothesis of this study was that exposure to high dose ionizing radiation would change the expression of the integrins and change integrin-mediated adhesion to the ECM proteins. Results showed that in the MDA-MB-231 cell line, ionizing radiation induces significant changes in both integrin expression and integrin-mediated adhesion to fibronectin, laminin, and collagens I and IV. After gathering further evidence from this and other cell lines, this information could potentially be used to optimize treatment for patients with invasive cancers.

Committee: Shiyong Wu PhD (Advisor) Subjects: Biochemistry; Biomedical Research; Cellular Biology; Chemistry

Doctor of Philosophy, The Ohio State University, 2010, Molecular, Cellular and Developmental Biology

Maintaining genomic integrity is essential for cellular viability. Alteration of bases can occur via exposure to reactive oxygen species. There exist cellular repair mechanisms to correct aberrant bases which can lead to single strand breaks. The primary repair mechanism the cell employs for damaged bases is Base Excision Repair (BER). The steps of BER involve identifying and removing the damaged base with a DNA glycosylase, incising the DNA backbone and processing the DNA ends by an endonuclease, filling the abasic site by DNA polymerase, and sealing the DNA backbone by DNA ligase. While the steps of BER have been well characterized, there is still much to investigate about how BER is regulated. Studies have suggested that BER proteins undergo post-translational modification. Additionally, proteins not previously shown to participate in BER are being recognized as regulating BER through protein localization, turnover, or interactions. The purpose of this thesis was to identify and characterize novel modulators of BER proteins and activity. The first modulator described is β1 integrin engagement. It was previously described that integrin engagement can reduce 3'OH and phospho-H2A.X formation. In Chapter 2, the effects of β1 integrin-engagement on reducing total DNA breaks in murine lung endothelial cells (MLECs) will be discussed. β1 integrin-engagement did not reduce the total number of breaks in DNA after genotoxic insult, therefore the effect of integrin-engagement on processing of DNA breaks was examined. Apurinic/Apyrimidinic Endonuclease 1 (APE1) is the primary endonuclease responsible for incising the DNA at an abasic site. In Chapter 3, the effect of integrin-engagement on APE1 activity and interaction with X-Ray Cross Complementing Repair Protein 1 (XRCC1) will be discussed. APE1 and XRCC1's interaction stimulates APE1 activity. Integrin-engagement in the presence of H2O2-induced damage reduced APE1-XRCC1 interaction and APE1 activity. This provides a p (open full item for complete abstract)

Committee: Dale Hoyt PhD (Advisor); Tsonwin Hai PhD (Committee Member); Michael Ostrowski PhD (Committee Member); Mark Parthun PhD (Committee Member) Subjects: Cellular Biology; Molecular Biology

Master of Science (MS), Ohio University, 2003, Chemical Engineering (Engineering)

Leukocyte adhesion to the endothelium is an important component of the inflammatory response. The adhesion process is a multi-step cascade that is mediated by ligands expressed on leukocytes and complementary receptors expressed on endothelial cells. The ligand-receptor pairs involved in leukocyte adhesion to the endothelium can be classified into four main groups: selectins, immunoglobulin superfamily, integrins, and cadherins. It is known that leukocyte expressed Mac-1 (an α M β 2 integrin) plays an important role in this adhesion process, but the identification of the counter receptor for Mac-1 on the endothelium remains controversial. Several studies suggest that ICAM-1 is a counter receptor for Mac-1. There are data both supporting and refuting this conjecture. Previous studies in our lab using Mac-1 coated microspheres strongly suggest that ICAM-1 is not involved in Mac-1 microsphere adhesion to human umbilical vein endothelial cells (HUVEC). Therefore, we hypothesize that HUVEC express an as yet unidentified receptor for Mac-1. The goal of this thesis is to investigate the feasibility of using two different techniques to isolate this novel receptor for Mac-1: immunoprecipitation and Phosphatidylinositol Phospholipase C (PI-PLC) approach. The first approach would be to immuoprecipitate the novel receptor from HUVEC lysates using Mac-1 coated microspheres. In this approach, the surface proteins on the HUVEC would be biotinylated to help discriminate surface proteins from intercellular proteins and a detergent would be used to solubilize the membrane bound proteins. In the present study the conditions for optimal biotinylation of HUVEC were established and the maximal detergent level that can be used before the presence of the detergent desorbs proteins from microspheres was determined. The results indicate that incubation of HUVEC in Hanks Balanced Salt Solution containing biotin for 5 minutes at 4 °C results in significant biotinylation of the HUVEC while havi (open full item for complete abstract)

Committee: Douglas Goetz (Advisor) Subjects: Engineering, Biomedical

Doctor of Philosophy (PhD), Ohio University, 2002, Chemical Engineering (Engineering)

The circulatory system consists of blood flowing through an intricate network of blood vessels, whose inner lining is composed of endothelial cells, collectively known as the vascular endothelium. Blood is composed of formed elements (blood cells and platelets) suspended in a saline solution containing dissolved proteins and other solutes. Molecular interactions between receptors expressed on the vascular endothelium and ligands expressed on the formed elements play a critical role in a variety of physiological and pathological processes. Well-known examples include, leukocyte recruitment and platelet deposition at sites of tissue injury, tumor metastasis and atherosclerosis. Since this adhesion occurs in the fluid dynamic environment of circulation, it is paramount to study vascular adhesion from both a biological and an engineering standpoint. There is a distinct possibility that differences in the diameters of formed elements affect their adhesion and consequently, their function. This motivates a study into the role of particle diameter in receptor-ligand mediated adhesion. Our results clearly demonstrate that adhesion is strongly dependent on particle size and provide experimental proof for mathematical models linking particle size to adhesion. The selectin and integrin families of cell adhesion molecules play a key role in orchestrating leukocyte recruitment to sites of inflammation. In a separate study, we examine molecular interactions between the leukocyte integrin Mac-1 (CD11b/CD18), and endothelial cells under flow. Our results reveal that Mac-1 coated microspheres adhere to endothelial cells via E-selectin and an additional mechanism that perhaps involves a yet unidentified endothelial receptor. Motivated by a desire to further the understanding of leukocyte recruitment and hematopoietic progenitor cell (HPC) entry into bone marrow, numerous studies have focused on identifying ligands for E-selectin on a specific HPC cell line, namely HL60 cells. These p (open full item for complete abstract)

Committee: Douglas Goetz (Advisor) Subjects: Engineering, Chemical

Master of Sciences, Case Western Reserve University, 2009, Pathology

When T cells adhere to the ECM, under certain circumstances they will polarize and exhibit the morphology of a migrating cell. We are investigating three specific questions: (a) What environmental signals modulate T cell cytoskeletal reorganization, especially vimentin, the intermediate filament? (b) Which Rho GTPase is involved in vimentin polarization on discrete ECM ligands, such as collagen type I and fibronectin? (c) What is the mechanism and function of vimentin reorganization upon engagement of distinct ECM ligands? Our preliminary findings demonstrate that (a) certain but not all inside-out and outside-in signals lead to integrin activation that results in T cell cytoskeletal rearrangement, particularly vimentin polarization, on ECM ligands. (b) Rho GTPases differentially modulate vimentin polarization when T cells adhere to distinct ECM ligands. This work sets a base for further investigation of the potential role vimentin plays in lymphocyte migration and signaling.

Committee: Nicholas Ziats PhD (Committee Chair); Alan Levine PhD (Advisor); Steven Eppell PhD (Committee Member); Clive Hamlin PhD (Committee Member) Subjects: