Doctor of Philosophy, Case Western Reserve University, 2018, Pathology

Dynamic ubiquitination and phosphorylation are crucial in tuning immune cell homeostasis. This is most evident in genetic inflammatory disease, where a single gene, such as NOD2, can have gain or loss of function in signaling to NF-κB. Both result in a granulomatous inflammatory disease. Polymorphisms in NOD2 have long been implicated in Crohn's disease, Early Onset Sarcoidosis, and Blau Syndrome. To better understand immune signaling in inflammation, we examine kinase subunits of the major innate immune signaling hub, the IκB Kinase (IKK) Signalosome. The kinase subunits within the IKK Signalosome, IKKα and IKKβ, relay inflammatory inputs from cytokine receptors, ER stress receptors, and pathogen recognition receptors, alike. Upon activation, IKKs not only phosphorylate members of the IκB (Inhibitor of NF-κB) family to activate Rel-homology transcription factors, but IKKs also have other protein substrates. For example, immuno-regulatory proteins such as deubiquitinases CYLD and A20 are IKK substrates whose activities are modulated by IKK phosphorylation. In an effort to better understand the signaling contributions of IKKs, we employed a dual bioinformatic and proteomic approach. We used IKK phospho peptide array data in a bioinformatic search to find immune-regulatory proteins targeted by IKKs. In this work, we narrow the candidate search to a family of HECT E3 ubiquitin ligases, and as the focus of this work, we identify ITCH as a novel substrate of the IKKs. Nothing was previously known about the function or consequence of IKK-phosphorylation on this HECT E3 ubiquitin ligase. Here, we discover a site of IKK phosphorylation on ITCH that lies within the enzymatic HECT ubiquitin ligase domain of the protein. Specifically, the site of phosphorylation is within the E2:E3 interaction domain. We use this insight to examine the functional mechanism of impairment using SPR to detect differences in binding. Here, I present evidence supporting a model in which IKKs phosph (open full item for complete abstract)

Committee: Derek Abbott (Advisor); George Dubyak (Committee Chair); Theresa Pizarro (Committee Member); Pamela Wearsch (Committee Member); Clive Hamlin (Committee Member); Xiaoxia Li (Committee Member); Brian Cobb (Committee Member) Subjects: Biomedical Research; Immunology; Pathology

PhD, University of Cincinnati, 2012, Medicine: Toxicology (Environmental Health)

IKKβ is a protein kinase critical for the transmission of inflammatory cytokine signals, such as TNFα, to activation of transcription factor NF-κB and regulation of gene expression. Genetic studies in mice, however, show that IKKβ is not only involved in inflammation, but also plays a pivotal role in tissue homeostasis and stress responses. Our previous studies show that IKKβ is essential for maintaining redox homeostasis, but the underlying mechanisms and biological consequences have not been understood. The central theme of this thesis is to investigate how IKKβ modulates homeostasis and what roles it plays in biological processes. The thesis is composed of three main components. First, mouse fibroblasts were used to investigate the molecular signatures and functions of IKKβ. I showed that genetic knockout of Ikkβ gene in fibroblasts reduced the expression of anti-oxidants thus increased the intracellular ROS level. The increased ROS in turn activated AP-1, leading to transcriptional activation of the TGFβ gene promoter and its expression, while increased TGFβ can act through NOX4 to further stimulate ROS production. Removal of IKKβ therefore triggered the autocrine amplification of the ROS-TGFβ loop that led to progressive ROS accumulation. The IKKβ-deficient fibroblasts ultimately displayed SMAD activation, leading to the overproduction of extracellular matrices and remodeling enzymes, accelerated cell motility, and enhanced markers for myofibroblast transformation and cellular senescence. These results suggested that IKKβ safeguarded the homeostasis of fibroblasts by repressing a ROS-AP-1-TGFβ autocrine regulatory loop. Second, the in vivo roles of IKKβ were assessed in keratocytes, the fibroblasts in corneal stoma of the eye. I generated mice with Ikkβ ablation specifically in corneal keratocytes. While the knockout mice had normal corneal appearance, suggesting that IKKβ was dispensable for corneal keratocytes development and maintenance, they exhibited agg (open full item for complete abstract)

Committee: Ying Xia PhD (Committee Chair); Winston Whei Yang Kao PhD (Committee Member); Richard Lang PhD (Committee Member); Chia-Yang Liu PhD (Committee Member); Alvaro Puga PhD (Committee Member) Subjects: Environmental Health

PhD, University of Cincinnati, 2009, Medicine : Toxicology (Environmental Health)

The IκB kinase β (IKKβ) is a critical component of the classical inflammatory signaling pathway that activates nuclear factor κB (NF-κB) and propagates inflammatory responses. Recent genetic studies in mice suggest that IKKβ also has physiological roles in the prevention of oxidative stress. The central hypothesis of this thesis is that the IKKβ-NF-κB signal transduction pathway is crucial for maintaining redox homeostasis and preventing excessive reactive oxygen species (ROS). We test this hypothesis by addressing the following pertinent questions. 1. Does the IKKβ-NF-κB pathway regulate redox homeostasis? Redox homeostasis is largely maintained by a group of antioxidant enzymes and small scavenging molecules, such as reduced glutathione (GSH) and metallothionein (MT). Our results show that genetic and pharmacological inactivation of the IKKβ-NF-κB pathway reduces the cellular GSH and MT1 levels. 2. What are the consequences of IKKβ inactivation? We show that the IKKβ-NF-κB pathway is required for protection against oxidative injury caused by a wide variety of oxidative stress inducers, including environmental toxic compounds, such as arsenic, and therapeutic anti-cancer drugs, such as cisplatin and taxol. Under stress conditions, cells deficient in IKKβ display a marked increase in ROS accumulation, which leads to activation of the MKK4-c-JUN N-terminal kinase (JNK) pathway, c-JUN phosphorylation and apoptosis. Hence, the IKKβ-NF-κB pathway maintains ROS scavenging capacity and serves as a critical biological checkpoint for cell fate determination in stress environment. 3. What are the mechanisms for the IKKβ-NF-κB pathway in GSH regulation? It has long been established that GSH is an important defense against the toxicity of oxidants, of which exposure is an everyday occurrence. Our results show that at least two mechanisms are involved in GSH regulation by IKKβ. First, IKKβ is needed for the optimal activities of the transcription factors NF-κB and (open full item for complete abstract)

Committee: Ying Xia PhD (Committee Chair); Howard Shertzer PhD (Committee Member); Mary Beth Genter PhD (Committee Member); Alvaro Puga PhD (Committee Member); Keith Jones PhD (Committee Member) Subjects: Toxicology

Master of Science, The Ohio State University, 2008, Biophysics

Tumor associated macrophages (TAMs) perform various task that are essential for tumor growth, angiogenesis, metastasis, and tumor invasion. Growth factors, chemokines, cytokines and the extra cellular matrix in the tumor microenvironments signal the TAMs to perform these tasks. The tumor microenvironment is not homogeneous and can be classified into several distinct regions such as sites of tumor invasion, perivascular, hypoxic and stromal regions. Each of these sites likely has different cytokines and signaling molecules in their microenvironment that might signal and direct the function of TAMs. The purpose of this project is to isolate and analyze TAMs from distinct tumor regions to gain a better understanding of the mechanism behind the specialized role of these TAMs. As a first step towards this goal, micro array analysis was conducted on flow sorted TAMs and developmental macrophages expressing yellow fluorescence protein (YFP). Micro array data from age matched developmental macrophages was compared to TAMS from early, premalignant stage of breast cancer, and late stage metastatic breast cancer to find the genes which were upregulated in TAMs versus developmental macrophages, and in late stage TAMs versus TAMs from the earlier premalignant stage of breast cancer. From this list of genes upregulated in TAMs, candidate cell surface macrophage markers that could be used to classify TAMs based on there association with one of the distinct tumor microenvironments was selected. These candidate markers will be used to visualize macrophages associated with different regions within the tumor microenvironment using immuno-staining of tumor sections. In preliminary work, CD16 was selected as one potential candidate gene that was upregulated late in tumor development compared to earlier stages. Immunostaining indicated CD16 was expressed on a set of macrophages near blood vessels near the tumors, but not in macrophages located within tumors. Further evaluation of these m (open full item for complete abstract)

Committee: Michael Ostrowski PhD (Advisor); Chuck Bell PhD (Committee Member) Subjects: Bioinformatics

Doctor of Philosophy, The Ohio State University, 2008, Biochemistry

The NF-κB signaling pathway regulates immune and inflammatory responses, cellular growth, survival, differentiation and development. In this study, the functions of IκB kinase (IKK)β in angiogenesis during mouse development were examined. Conditional disruption of the Ikkβ locus in endothelial cells using the well-characterized Tie2-Cre transgene resulted in embryonic lethality between E13.5 and E15.5. Examination of the mutant embryos revealed that, while deletion of Ikkβ occurred in endothelial cells throughout the embryo, only the vascular network in the fetal liver was affected. Disruption of the fetal liver vasculature was accompanied by decreased cell proliferation and increased apoptosis of hepatocytes, but hematopoiesis was not affected. Increased apoptosis was not observed outside of fetal liver in the mutant embryos. These results indicate that the IKKβ/NF-κB pathway plays a previously unappreciated role in development of the sinusoidal vasculature in the fetal liver and, additionally, that this pathway is critical in the crosstalk between endothelial cells and hepatocytes during mouse development. Breast cancer is the second leading cause of cancer-related mortality in women. It is still unclear which of the changes in breast tumors are likely to lead to invasion and metastasis at the molecular level. Angiogenesis and invasiveness, two major traits contributing to tumor aggressiveness, are partially regulated by NF-κB. In this project, we carried out in vivo approaches using PyMT transgenic mouse model of breast cancer to study the functions of NF-κB in tumorigenesis of breast cancer. Mammary gland epithelia-specific MMTV-Cre and macrophage-specific Lys-Cre were used to delete Ikkβ, respectively in these compartments to study functions of NF-κB in microenvironment of breast cancer. Pulmonary metastasis was reduced by 5.3-fold in IKKβF/F/PyMT/Lys-Cre animals. However, tumor incidence or tumor burden was not affected in both groups. Inefficient recombinati (open full item for complete abstract)

Committee: Michael Ostrowski (Advisor); Denis Guttridge (Other); Erich Grotewold (Other); Gustavo Leone (Other) Subjects: Biology

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:

PHD, Kent State University, 2008, College of Arts and Sciences / School of Biomedical Sciences

Ovarian cancer is the seventh most common cancer in women and the fourth most frequent cause of cancer death in US. Ovarian cancer has the highest mortality of all cancers of the female reproductive system, reflecting, in part, a lack of early symptoms and proven ovarian cancer screening tests. NFκB and β-catenin are two transcription factors important in regulating the expression of genes that control cell proliferation, cell survival and transformation. Our laboratory has found that the phosphatidylinositol 3' kinase (PI3K)/AKT/ IκB kinase (IKK) pathway aberrantly activates both NFκB and β-catenin in cancer. Our hypothesis is that aberrant activation of AKT and IKKs may cause the deregulation and inappropriate activation of both the NFκB and β-catenin signal transduction pathways, which may lead to the development of ovarian cancer by promoting cellular transformation, uncontrolled cell division and resistance to apoptosis. We used RNAi technology to specifically knockdown the expression of AKT1 and IKKβ and determined the effects of the loss of either AKT1 or IKKβ on the regulation of the NFκB and β-catenin signal transduction pathways as well as on the tumorigenic characteristics and chemotherapeutic resistance of SKOV-3 and A2780 ovarian cancer cell lines. Loss of either IKKβ or AKT1 significantly inhibited both the inappropriate basal and TNFα-induced NFκB transcriptional activity and loss of IKKβ reduced TNFα-induced NFκB DNA binding in the SKOV-3 and A2780 ovarian cancer cell lines. Also, IKKβ knockdown significantly inhibited both the basal and Wnt-stimulated β-catenin-dependent transcriptional activity in A2780 ovarian cancer cell line. The loss of AKT1 but not IKKβ inhibited anchorage independent growth in both the SKOV-3 and A2780 ovarian cancer cells. Knockdown of either AKT1 or IKKβ inhibited both the migration and invasion of the A2780 ovarian cancer cells, while causing only an inhibition of the invasive capacity in SKOV-3 ovarian cancer cells, witho (open full item for complete abstract)

Committee: Nywana Sizemore PhD (Advisor); Brent Bruot PhD (Committee Member); Jennifer Marcinkiewicz PhD (Committee Member); Gail Fraizer PhD (Committee Member); John R.D. Stalvey PhD (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2008, College of Science

The Rel/NF-κB family of inducible transcription factors are evolutionarily conserved structurally and functionally, from insects to humans. They are ubiquitously expressed in a number of mature cell types, playing a pivotal role regulating cell growth, differentiation and apoptosis. Under normal circumstances, the proliferation of new cells is tightly regulated, as is the programmed lifespan of most cells, occasionally however cells loose their responsiveness to growth control mechanisms, resulting in a tumor or neoplasm. Sustained or chronic inflammation has been linked to a number of pathological conditions that destroy tissue and facilitate neoplastic growth. NF-κB in part mediates the opposing signals of cell survival and cell death, associated with this response. We hypothesized that inhibition of NF-κB would inhibit tumor cell growth. A number of anti-neoplastic drugs like some pro-inflammatory cytokines can activate both the cellular apoptotic and pro-survival (via NF-κB) pathways. We demonstrate, in vitro, that the use of Nitric Oxide mitigates NF-κB activation and induces program cell death, when administered as an adjuvant with this subset of pro-inflammatory cytokines. Secondly, several front-line anti-cancer drugs activate NF-κB as a result of their mode of action, resulting in the survival of a resistant population of tumor cells. In order to abrogate this beneficial activity for the tumor cell we explore, both in vitro and in vivo, the use of Nitric Oxide to inhibit NF-κB, augmenting the efficacy of the anti-neoplastic drugs. Thirdly, we identify that the intracellular signaling kinase, Inositol Hexkisphosphate Kinase2 (IHPK2) , binds to a key component in the NF-κB signaling pathway, inhibiting NF-κB activity promoting apoptosis in tumor cells. Lastly, an in depth study investigating the major initiator of pro-inflammatory signaling during bacterial infection of cells by Salmonela sp. was in fact due to recognition of the bacterial protein flagellin b (open full item for complete abstract)

Committee: Joseph DiDonato PhD (Committee Co-Chair); Lily Ng PhD (Committee Co-Chair); Michael Kalafatis PhD (Committee Member); Aimin Zhou PhD (Committee Member); Shukla Girish PhD (Committee Member) Subjects: Biology; Chemistry; Molecular Biology

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