PhD, University of Cincinnati, 2004, Medicine : Pathobiology and Molecular Medicine

Cardiovascular disease, including stroke and atherosclerosis, remains a major cause of morbidity and mortality in the United States due to consumption of a high-fat “Western” diet. A major indication of these disease states is excess lipid deposition, particularly cholesterol, in the arterial walls. It has been well documented that high levels of circulating high-density lipoprotein (HDL) and its main protein component apolipoprotein AI (apoA-I) are associated with lowered risk of cardiovascular disease. The protective effects of HDL are thought to be mediated by a process called reverse cholesterol transport - in which HDL takes up excess cholesterol from peripheral tissues and transports it to the liver for excretion in the bile. It is now widely accepted that the interaction of apoA-I with the cell membrane protein ATP-binding cassette transporter AI (ABCAI) is critical for the formation of nascent HDL particles. Since sphingomyelin maintains a preferential interaction with cholesterol in membranes, the breakdown of sphingomyelin may regulate the availability of cellular cholesterol utilized by ABCAI. Furthermore, the catabolite of sphingomyelin, ceramide, is a potent signaling molecule and may play an important role in ABCAI regulation or function. The following study examines the potential contribution of sphingolipids in ABCAI-mediated cholesterol removal from the cell. It was discovered that treatment with C2-ceramide enhances cholesterol release to apoA-I. This effect appeared to becaused by an increase in cellular ABCAI content with enrichment at the cell surface. These findings may lead to new ways to increase cellular ABCAI and further promote cholesterol removal from regions of excess cholesterol such as the atherosclerotic lesion.

Committee: Dr. William Davidson (Advisor) Subjects:

Master of Science, The Ohio State University, 2011, Evolution, Ecology and Organismal Biology

Lipids of the stratum corneum (SC), the outer layer of the epidermis of birds and mammals, provide a barrier to water vapor diffusion through the skin. The SC of birds consists of flat dead cells, called corneocytes, and two lipid compartments: an intercellular matrix and a monolayer of covalently bound lipids (CBL) attached to the outer surface of corneocytes. We previously found two classes of sphingolipids, ceramides and cerebrosides, covalently bound to corneocytes in the SC of house sparrows and that these were associated with cutaneous water loss (CWL). In this study, we collected adult and nestling house sparrows from Ohio and nestlings from Saudi Arabia, acclimated them to either a high or low humidity environment, and measured their rates of CWL. We also collected natural populations of nestlings from Ohio and Saudi Arabia from 2 days after hatching until they fledged, and measured their CWL rates. We then evaluated the composition of the CBL of the SC using thin layer chromatography. We found that CBL development differed between habitats, but that lipid density generally increased with age. CBL profile did not exhibit phenotypic plasticity with acclimation and was mostly the same between habitats. CWL appears to be functionally related with the interactions of CBL classes as a whole, and this may be associated with age. Finally, we found that house sparrows have a very diverse range of CBLs in their SC, including free fatty acids and cholesteryl esters, and we propose a new model for CBL organization.

Committee: Joseph Williams (Committee Chair); David Denlinger (Committee Member); W. Mitch Masters (Committee Member) Subjects: Biology; Ecology; Physiology; Zoology

Doctor of Philosophy, The Ohio State University, 2007, Integrated Biomedical Science

Cellular accumulation of Sphingosine 1-phosphate (S1P) stimulates cellular motility, invasion, proliferation, adhesion, and angiogenesis. S1P tranduces these cellular effects by signaling through one of a family of high affinity G-protein coupled receptors that include EDG-1/S1P1, EDG-5/S1P2, EDG-3/S1P3, EDG-6/S1P4, and EDG-8/S1P5. Upon receptor stimulation, unique and preferential G-protein signaling pathways are activated to varying degrees to elicit these cellular responses. Enzymatic production of S1P is a result of the activity of sphingosine kinase (SK). Expression of this protein has been shown to correlate to poor glioblastoma multiforme (GBM) patient prognosis. This deadly neoplasm is diagnosed through histologic criteria that include significant neovascularization, high mitotic index, and diffuse infiltrative appearance. Although progress has been made in the treatment of many cancers in recent years, the dismal patient prognosis for GBM patients has not. Mo lecular based therapies targeting the infiltrative nature of this tumor could ultimately improve patient outcome. This work focuses on GBM malignant behavior as influenced by S1P receptor subtype signaling. All the individual receptor subtypes were found to have unique influences over in vitro GBM proliferative, migratory, adhesive, and invasive responses. The S1P2 receptor subtype displayed novel S1P-induced pro-invasive, but anti-migratory effects, which were mediated through increased adhesion to the extracellular matrix. Invasiveness was promoted through CCN1, uPA, and uPAR, which have all been correlated to GBM tumorigenesis. S1P induced expression of CCN1 through S1P1-2 promoted invasion that was limited with antibody in a spheroid invasion assay. Enhanced activation of SK with receptor subtype S1P1 overexpression resulted in endogenous upregulation of uPA and uPAR expression as well as uPA activity. Additionally, antibody targeting uPA also limited total invasiv e distance of spheroids. The inva (open full item for complete abstract)

Committee: James Van Brocklyn (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2007, Integrated Biomedical Science

Human cytomegalovirus (HCMV) is a beta-herpes virus which can cause serious disease and even death in congenitally-infected infants and in immunocompromised individuals or immunosuppressed transplant recipients. Sphingolipids are structural components of cell membranes that can act as critical mediators of cell signaling. An unexplored area of research is whether HCMV modulates sphingolipids and their signaling pathways. Our data show that HCMV infection results in increased accumulation and activity of sphingosine kinase (SphK) within different cell types and occurs during early times of infection. Measuring the levels of transcripts encoding key enzymes of the sphingolipid metabolic pathway during HCMV infection revealed a temporal regulation of both synthetic and degradative enzymes of this pathway. Using mass spectrometry, we were able to generate a sphingolipidomic profile of HCMV-infected cells, which suggests an enhancement of de novo sphingolipid synthesis at early times of infection. This was followed by a decrease in the levels of several sphingolipids at 48 hrs, correlating with the upregulation of degradative enzymes. Then by knocking down SphK1 expression with siRNA, we showed that this enzyme may function within HCMV infected cells to sustain levels of the immediate early (IE) transactivator, IE1. Later, we show evidence suggesting that de novo sphingolipid biosynthesis is necessary for the production of optimal levels of infectious virus progeny, but has an intermediate product which suppresses the levels of IE1 protein accumulation. Through exogenous addition of dhSph, an intermediate of de novo sphingolipid synthesis, we show that this lipid can inhibit HCMV protein accumulation. dhS1P treatment, however, results in increased accumulation of HCMV proteins, although only at early times during infection. Moreover, pretreatment of cells with dhS1P or S1P prior to infection results in reduced accumulation of HCMV gene products, thus indicating a time-de (open full item for complete abstract)

Committee: James Van Brocklyn (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2004, Food Science and Nutrition

Prostate cancer is the second leading cause of cancer death in men, and diet is thought to play a role in the development of this disease. The “Westernized” diet, rich in red meat and dairy products, is associated with increased prostate cancer risk. Despite the negative perception of dairy products, milk contains several components, such as whey proteins and sphingomyelin (SM), which may play a role in prevention and/or treatment of prostate cancer. Whey proteins are a cysteine-rich protein source, and consumption of these proteins can elevate plasma concentrations of the antioxidant glutathione (GSH). Elevation of prostate GSH may reduce inflammation that is associated with cancer development. SM is a phospholipid that can induce apoptosis in cancer cells, and consumption of SM is associated with reduced incidence of colon cancer in mice. It is hypothesized that SM may also limit the proliferation of prostate cancer cells. The present studies were designed to examine the role of whey proteins and SM in prostate cells to determine if there is a potential role for these dairy components in prostate cancer prevention and treatment. First, the effects of oxidative stress in non-cancerous prostate cells was examined. Next, we examined the elevation of GSH in non-cancerous prostate cells by hydrolyzed whey proteins. Finally, the antiproliferative effects of SM in human prostate cancer cells were studied. The results of the present study revealed that bovine pituitary extract, a supplement used in the growth medium of the non-cancerous prostate cells, provides significant protection against oxidant-induced cell damage and may represent a confounding variable when studying oxidative stress in cell lines requiring this supplement. Hydrolyzed whey proteins elevated GSH concentrations in non-cancerous prostate cells by 64% and this GSH elevation protected the cells against oxidant-induced cell death. Exogenous SM significantly reduced prostate cancer cell proliferation by 17 (open full item for complete abstract)

Committee: W. Harper (Advisor) Subjects: Health Sciences, Nutrition

Doctor of Philosophy, Case Western Reserve University, 1993, Physiology and Biophysics

The role of sphingolipids in mediating the action of platelet-derived growth factor (PDGF) has been investigated in the vascular smooth muscle-derived A7r5 cell line. L-cycloserine (2 mM), an inhibitor of sphingolipid synthesis, caused time-dependent inhibition of (3H) serine incorporation into (3H) sphingomyelin in A7r5 cells. PDGF-AB (10 ng/ml), PDGF-BB (10 ng/ml) or sphingosine (10 uM) independently stimulated (3H) thymidine incorporation into DNA of A7r5 cells. L-cycloserine (2 mM) inhibited stimulation of DNA synthesis by both PDGF-AB and PDGF-BB. L-cycloserine (2 mM, 16 h) did not affect the ability of PDGF or sphingosine to increase intracellular free calcium ( (Ca2+) i) in A7r5 cells loaded with the fluorescent indicator fura-2. Measurement of adenine nucleotide levels in A7r5 cell extracts by reverse-phase high-performance liquid chromatography indicated that treatment with L-cycloserine did not adversely affect cellular metabolism. To determine directly whether PDGF activates sphingolipid metabolism, A7r5 cells were labeled with (3H) serine for 48 h and then treated with PDGF-AB (10 ng/ml) for 1 h. Sphingolipids were separated by thin-layer chromatography and quantified by liquid scintillation counting. PDGF-AB stimulated an increase in (3H) sphingosine from 25.5 ± 3.0 to 37.5 ± 4.1 cpm/ug protein and a concomitant decrease in (3H) ceramide from 24.3 ± 3.2 to 18.5 ± 2.9 cpm/ug protein. Exogenous sphingosine (10 uM, 6 min) stimulated an increase in (32P) lysophosphatidic acid but had no effect on (32P) lysophosphatidylcholine, suggesting specificity of sphingosine action. In A7r5 cells labeled with 1-O- (3H) alkyl-2-lyso-sn-glycero-3-phosphocholine, exogenous sphingosine stimulated a dose-dependent increase in (3H) phosphatidic acid. Exogenous sphingosine produced a dose-dependent increase in (32P) sphingosine-1-phosphate, indicating that A7r5 cells have an active sphingosine kinase. These data suggest that the PDGF-stimulated increase in (Ca2+) i is not su (open full item for complete abstract)

Committee: Mark Kester (Advisor) Subjects: Biology, Cell