Doctor of Philosophy, The Ohio State University, 2007, Animal Science

Failure to terminate the inflammatory response results in chronic inflammation that may lead to disease or cancer, especially in epithelial cells. We explored mammary epithelial cells as a model to identify mechanisms of anti-inflammatory activity in epithelia alone in the absence of immune cells. Bacterial endotoxin (ET) added to SCp2 mammary secretory epithelial cells: (1) induced both interleukin-6 (IL-6) secretion and nitric oxide (NO) production, but with unexpected delay in expression of mRNA for iNOS compared to IL-6; and (2) NFκB activation by 1 h after ET application (post-ET) that was transient for NFκB/p65 but persisted for NFκB/p50. Selective inhibition of NFκB activation by Wedelolactone reduced ET-induced expression of IL-6 mRNA and protein but not iNOS mRNA or NO production, suggesting differences in ET-induced IL-6 and iNOS regulation via NFκB activation. Serum supplementation but not soluble extracellular matrix (EHS) enhanced ET-induced IL-6 mRNA expression and protein secretion without affecting iNOS mRNA expression or NO production, confirming the different modes of regulation of IL-6 and iNOS expressions. Culturing SCp2 cells on a confluent monolayer of SCg6 mouse mammary myoepithelial cells increased IL-6 secretion dramatically even in the absence of ET, with ET treatment further increasing IL-6 secretion but having little effect on induction of NO production over that for SCp2 cells alone; showing importance of microenvironment and cell-cell interaction in regulation of inflammation and likely its link to cancer in epithelia. ET-induced inflammation in SCp2 cells was used to screen and identify anti-inflammatory fractions of methanol extracts of wild Lebanese Centaurea ainetensis, used in Lebanese folk medicine to treat inflammatory diseases. A partially purified solid phase (SPE columns) methanolic elution fraction of C. ainetensis followed by methanol gradient elution on reverse phase HPLC chromatography (RP-HPLC) strongly inhibited ET-induc (open full item for complete abstract)

Committee: Floyd Schanbacher (Advisor); Charles Brooks (Other); James DeWille (Other); Joy Pate (Other) Subjects:

Doctor of Philosophy in Biomedical Sciences (Ph.D.), University of Toledo, 2010, College of Medicine

Phospholipid transfer protein (PLTP) is critically important for reverse cholesterol transport (RCT), and its expression level and activity increase when mice are fed a high fat diet. RCT is the process by which accumulated cholesterol from the blood vessel walls, peripheral tissues and macrophages is transported back to the liver for excretion. Interestingly, topoisomerase I inhibitors used in chemotherapy have been shown in our laboratory to dose dependently induced PLTP expression in both in vivo and in vitro studies. Since PLTP transports phospholipid as well as cholesterol into high density lipoprotein (HDL), we asked whether elevated PLTP levels might increase the transfer of drugs into HDL via RCT, thus increasing tumor cells resistance to the drug. However, we found that camptothecin, topoisomerase I inhibitor, does not accumulate in HDL or in other lipoprotein subfractions, thus ruling out the possibility of PLTP mediating the transfer of camptothecin into HDL for liver metabolism. The limonoid prieurianin, like topoisomerase I inhibitors, has also been shown to dose dependently increase PLTP mRNA and proteins levels, and here we show that prieurianin causes weight loss by reducing food intake in morbidly obese mice and in mice on high-calorie diet. Additionally, prieurianin is anti-adipogenic and (i) inhibits the proliferation and differentiation of preadipocytes into adipocytes, and (ii) induces either dedifferentiation or delipidation of mature adipocytes. Gene expression profiling showed that prieurianin suppresses the expression of a number of genes involved in fat metabolism, and inhibits the transcriptional activity of the adipogenesis master regulators including the CCAAT/enhancer binding proteins (C/EBPs) and the peroxisome proliferator-activated receptor gamma (PPARγ).

Committee: Knew-Voon Chin Ph.D. (Committee Chair); Sonia Najjar Ph.D. (Committee Member); Sandrine Pierre Ph.D. (Committee Member); Cynthia Smas M.D.Sc. (Committee Member); Ivana de la Serna Ph.D. (Committee Member); Manohar Ratnam Ph.D. (Committee Member) Subjects: Biology

Doctor of Philosophy in Biomedical Sciences (Ph.D.), University of Toledo, 2009, College of Medicine

RKIP (Raf Kinase Inhibitor Protein) was first identified as an inhibitor of Raf1 kinase inthe Raf-MEK-ERK signaling pathway. It can interact with Raf1 and MEK and disrupt the interaction of Raf1 and MEK by competing with their binding. Since RKIP is a very abundant protein, it must have additional targets other than Raf-MEK-ERK pathway. Indeed, RKIP was also found to play a pivotal role in NF-κB pathway. NF-κB pathways are ubiquitous pathways that exist in many different tissues. NF-κB pathway is involved in different biological processes including inflammation and immune responses. NF-κB gets activated by phosphorylation and subsequent degradation of IκB in response to stimulation. Our studies show that RKIP interacts with multiple components of canonical NF-κB pathway in response to IL-1β stimulation. RKIP affected IκB degradation. Overexpression of RKIP inhibited IκB degradation, whereas downregulation of RKIP also inhibits IκB degradation. The inhibitory effect of IκB degradation in RKIP knockdown cells can be rescued by restoring RKIP. The effect of RKIP on IκB degradation was concentration dependent. The mode of action of RKIP on IκB degradation was consistent with the properties described for a scaffolding protein. A scaffolding protein's main function is to bring other proteins together and allow them to interact. The signaling of the scaffolding cascade is dependent on the concentration of the scaffold protein. Both low and high concentration of scaffold protein will disrupt the interaction of the components of the scaffolding signaling cascade. Only intermediate concentration of scaffolding protein will allow for the optimal association of the different components in the signaling cascade, thus leads to the optimal signaling. One major feature of scaffolding protein is that it can interact with different components of the signaling cascade. Our studies show that RKIP interacts with TRAF6 and TAK1 of the canonical NF-κB pathway in both overexpression condit (open full item for complete abstract)

Committee: Kam Yeung Ph.D. (Committee Chair); Z. Kevin Pan Ph.D. (Committee Member); Dorothea Sawicki Ph.D. (Committee Member); Zi-Jian Xie Ph.D. (Committee Member); Ivana de la Serna Ph.D. (Committee Member) Subjects: Health; Molecular Biology

Doctor of Philosophy, Case Western Reserve University, 2009, Pathology

IRAK4 is a member of the IL-1 receptor (IL-1R) associated kinase family and has been shown to play an essential role in Toll-like receptor (TLR)-mediated signaling. We recently generated IRAK4 kinase-inactive knock-in mice to examine the role of the kinase activity of IRAK4 in TLR-mediated signaling pathways. The IRAK4 kinase-inactive knock-in mice were completely resistant to LPS- and CpG-induced shock, due to impaired TLR-mediated induction of pro-inflammatory cytokines and chemokines. Two parallel IL-1-mediated signaling pathways have been uncovered for TLR/IL-1R-mediated NFκB activation: TAK1-dependent and MEKK3-dependent, respectively. The TAK1-dependent pathway leads to IKKα/β phosphorylation and IKKβ activation, resulting in classical NFκB activation through IκBα phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKγ phosphorylation and IKKα activation, resulting in NFκB activation through dissociation of phosphorylated IκBα from NFκB without IκBα degradation. The IRAK4 kinase-inactive mutant failed to mediate the IL-1R-TLR-induced TAK1-dependent NFκB activation pathway, but mediated IL-1-induced TAK1-independent NFκB activation. While inactivation of the IRAK4 kinase activity did not affect the levels of TLR/IL-1R-mediated NFκB activation, a reduction of LPS-, R848- and IL-1-mediated mRNA stability contributed to the reduced cytokine and chemokine production in bone marrow (BM)-derived macrophages from IRAK4 kinase-inactive knock-in mice. The Type I interferon (IFN) production mediated by TLR7- and TLR9 was abolished in plasmacytoid dendritic (pDC) cells isolated from IRAK4 knock-in mice. In addition, influenza virus-induced production of IFNs in pDCs was also dependent on IRAK4 kinase activity. Taken together, our results indicate that IRAK4 kinase activity plays a critical role in TLR-dependent immune responses.

Committee: Neil Greenspan MD PhD (Committee Chair); Xiaoxia Li PhD (Advisor); George Stark PhD (Committee Member); Clifford Harding MD PhD (Committee Member); Clive Hamlin PhD (Committee Member) Subjects: Immunology

Doctor of Philosophy, Case Western Reserve University, 2009, Pharmacology

Protein S-glutathionylation is a reversible post-translational modification that is important in redox signal transduction and cellular defense against oxidative stress. S-glutathionylation occurs when a cysteine residue forms a mixed disulfide with glutathione. Glutaredoxin 1 is an enzyme that catalyzes the specific and efficient reversal of glutathionylation (de-glutathionylation). Roles for Grx1 in diabetic retinopathy, a disease characterized by oxidative stress and inflammation, had not been explored prior to the current study. Grx activity was found to be increased in retinae from diabetic rats. Incubation of rat retinal Muller glial cells (rMC-1) in normal glucose medium (5 mM) or diabetic-like glucose medium (25 mM, high glucose) led to corresponding increases in Grx content and activity. High glucose also led to increased nuclear translocation of NFkappaB and production of ICAM-1 (intercellular adhesion molecule-1), a transcriptional product of NFkappaB and known pro-inflammatory mediator in diabetic retinopathy. To evaluate the role of Grx1 in mediating these changes, Grx1 in rMC-1 cells was upregulated in normal glucose medium via infection with an adenoviral Grx1 construct (Ad-Grx1). Ad-Grx1 treatment led to increased NFkappaB activity and ICAM-1 production. Treatment of rMC-1 cells in high glucose medium with siRNA targeted to Grx1 prevented the increase in Grx1 and coincidentally blocked the increase in ICAM-1. The site of regulation was localized to the cytoplasm, and IkappaB kinase (IKK) is a master cytosolic regulator of NFkappaB activation. Inhibition of IKK activity abrogated the increase in ICAM-1 induced by high glucose or by Ad-Grx1. Conditioned medium from the rMC-1 cells over-expressing Grx1 was added to fresh cultures of rMC-1 cells and induced Grx1 and ICAM-1 (autocrine regulation). Similarly, Grx1 and ICAM-1 were elevated in retinal endothelial cells cultured in conditioned medium from the rMC-1 cells over-expressing Grx1 (paracrine regula (open full item for complete abstract)

Committee: Anthony Berdis PhD (Committee Chair); John Mieyal PhD (Advisor); Michael Maguire PhD (Committee Member); Timothy Kern PhD (Committee Member) Subjects: Cellular Biology; Molecular Biology; Pharmacology

Doctor of Philosophy, Case Western Reserve University, 2007, Pathology

Activation of NFkB requires the inhibitor of NFkB (IkB) kinase (IKK) complex, which comprises two functional kinases, IKKalpha and IKKbeta, and the scaffolding protein IKKgamma. We previously identified several interferon (IFN)-gamma-stimulated genes (ISGs) that are not induced in mouse embryonic fibroblast cells (MEFs) doubly -null for the expression of IKKalpha and IKKbeta, and we show here that IFN-gamma-induced transcription of IKK-dependent ISGs requires IKKbeta but not IKKalpha. Through microarray studies, we identified IKKbeta-dependent ISGs in both MEFs and RAW 264.7 macrophages. Many have both kB and ISRE elements, the latter of which bind IRF proteins, in their promoters. Although the IFN-gamma-induced expression of the IKKbeta-dependent gene ip-10 is sensitive to the super-repressor of NFkB and requires the p65 subunit of NFkB, IFN-gamma does not activate NFkB. Thus, a distinct subset of IRF-activated IFN-gamma-dependent genes requires some components of a normal NFkB activation pathway but not actual activation of NFkB in response to IFN-gamma. IFN-gamma activates IRF1 to bind the ISRE of ip-10. In IKKbeta-null cells, IRF1 activation and binding to DNA are slightly weaker and the ability of IRF1 to transactivate a synthetic promoter composed of tandem copies of the ip-10 ISRE element is impaired. IFN-gamma and IL-1 strongly synergize to induce ip-10. However, except at 1 h, the binding of IRF1 to the ip-10 ISRE in response to IFN-gamma is not enhanced by IL-1, and NFkB activation in response to IL-1 is not affected by IFN-gamma . IRF1-null cells show impaired ip-10 induction in response to IFN-gamma compared to wild-type cells. However, synergy between IFN-gamma and IL-1 is unaffected. The results presented provide new evidence of how NFkB components function in the induction of a specific subset of ISGs, and they provide mechanistic information about how IRF1 and NFkB cooperate to activate these promoters.

Committee: George Stark (Advisor) Subjects: Biology, Molecular