Doctor of Philosophy, The Ohio State University, 2004, Biophysics

Casein Kinase 1 (CK1) enzyme is one of the largest family of Serine/Threonine protein kinases. CK1 has a wide distribution spanning many eukaryotic families. In cells, its kinase activity has been found in various sub-cellular compartments enabling it to phosphorylate many proteins involved in cellular maintenance and disease pathogenesis. Tau is one such substrate whose hyperphosphorylation results in degeneration of neurons in Alzheimer's disease (AD). AD is a slow neuroprogessive disorder which is histopathologically characterized by Granulovacuolar degeneration bodies (GVBs) and intraneuronal accumulation of tau in Neurofibrillary Tangles (NFTs). The level of CK1 isoforms, CK1α , CK1δ and CK1ε has been shown to be elevated in AD. Previous studies of the correlation of CK1δ with lesions had demonstrated its importance in tau hyperphosphorylation. Hence we investigated distribution of CK1α and CK1ε with the lesions to understand if they would play a role in tau hyperphosphorylation similar to CK1δ. The kinase results were also compared with lesion correlation studies of peptidyl cis/trans prolyl isomerase (Pin1) and caspase-3. Our results showed that among the enzymes investigated, CK1 isoforms have the greatest extent of colocalization with the lesions. We have also investigated the distribution of CK1α with different stages of NFTs that follow AD progression. It was observed that CK1α follows AD progression, establishing the importance of CK1 isoforms in AD. Correlation of CK1 isoforms with tau pathology led us to investigate the presence of the isoforms in a muscle degenerative disorder, Inclusion Body Myositis (IBM) containing tau inclusions. CK1α was found in the tau inclusions of IBM, demonstrating the importance of CK1 isoforms in degenerative disorders in general. Since CK1 is established in both maintenance of the cell and pathogenesis of degenerative diseases, we investigated the regulation and protein substrate recognition of the kinase domain of the en (open full item for complete abstract)

Committee: Jeff Kuret (Advisor) Subjects:

Honors Theses, Ohio Dominican University, 2020, Honors Theses

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is composed of three domains: an N-terminal 4.1, Ezrin, Radixin, Moesin (FERM) domain, a kinase domain, and a C-terminal focal adhesion targeting (FAT) domain. FAK is activated by the signaling cascade of integrins which contact the extracellular matrix (ECM). This signaling activation by integrins allows FAK to control cell migration, proliferation, and survival through the phosphorylation of and interaction with nearby proteins (Src, Cas, Crc, paxillin, etc.), therefore making FAK a signaling scaffold. Dysfunctional FAK signaling can promote cancerous metastasis. Phosphatidylinositol-4, 5-bisphosphate (PIP2), a common lipid found in cell membranes, also serves as an activation factor that changes FAK conformation. FAK is naturally autoinhibited as its FERM-kinase domains are bound together. The mechanism behind FAK activation is still unclear. Current research shows interaction of FERM and kinase domains individually with PIP2, but further research is needed to determine the structural changes of the two domains simultaneously. The process to create a unique glutathione-s-transferase (GST) tagged plasmid vector including only the FERM and kinase domains through polymerase chain reaction (PCR) cloning as well as site-directed mutagenesis was begun, but not completed due to COVID-19 and related impacts. Once complete, this process will create a unique construct vector which will allow for further research into the activation of autoinhibited FERM and kinase domains by PIP2 binding.

Committee: Jessica Hall Ph.D. (Advisor); John Marazita Ph.D. (Committee Chair); William Nelson Ph.D. (Committee Member); Michael Dougherty Ph.D. (Committee Member) Subjects: Biology; Molecular Biology

Doctor of Philosophy, University of Toledo, 2021, Biology (Cell-Molecular Biology)

Mixed Lineage Kinase 3 (MLK3) activates mammalian MAPK signaling pathways in response to cytokines and stress stimuli. MLK3 is important for proliferation, migration and invasion of different types of human tumor cells. We observed that endogenous MLK3 was localized to both the cytoplasm and the nucleus in immortalized ovarian epithelial (T80) and ovarian cancer cells; and mutation of arginines 474 and 475 within a putative MLK3 nuclear localization sequence (NLS) resulted in exclusion of MLK3 from the nucleus. The Large Tumor Suppressor (LATS) Ser/Thr kinase regulates cell proliferation, morphology, apoptosis and mitotic exit in response to cell-cell contact. RNAi-mediated knockdown of LATS1 increased nuclear, endogenous MLK3 in T80 cells. LATS1 phosphorylated MLK3 on Thr477 which is within the putative NLS, and LATS1 expression enhanced the association between MLK3 and the adapter protein, 14-3-3. Thr477 is essential for MLK3-14-3-3 association and MLK3 retention in the cytoplasm; and a T477A MLK3 mutant had predominantly nuclear localization and significantly increased invasiveness of SKOV3 ovarian cancer cells. In SKOV3 cells, MLK3 binds to MLK4, and this association is regulated by osmotic stress. These results suggest that in the early response to stressful stimuli, MLK4-MLK3 binding is important for regulating MLK3 activity and MAPK signaling, and after prolonged periods of stress exposure, MLK4 and MLK3 proteins decline via CHIP-dependent degradation. This study identifies a novel link between the MAPK and Hippo/LATS1 signaling pathways. Our results reveal LATS1 as a novel regulator of MLK3 that controls MLK3 nuclear/cytoplasmic localization and MLK3-dependent ovarian cancer cell invasion and an important function for MLK4 in modulating MLK3 activity in stress responses.

Committee: Deborah Chadee Ph.D (Advisor); Malathi Krishnamurthy Ph.D (Committee Member); William Taylor Ph.D (Committee Member); Dayanidhi Raman Ph.D (Committee Member); John Bellizzi Ph.D (Committee Member) Subjects: Biology

Master of Science (MS), Ohio University, 2020, Biomedical Engineering (Engineering and Technology)

Glycogen synthase kinase 3 (GSK-3) is a highly active kinase. Loss of regulatory function of GSK-3 has been implicated in several high-profile diseases. Previously, several novel GSK-3 inhibitors have been developed by a research team at Ohio University. This thesis expands on the previous work by synthesizing 11 new inhibitors inspired by the previously studied compounds. These new compounds were analyzed in molecular and cell-based assays to determine their ability to inhibit GSK-3 and abate LPS-induced cytokine production. Specifically, the compounds were analyzed in molecular kinase assays by SelectScreen Services to determine their ability to inhibit both isoforms of GSK-3. The cell-based assays probed the ability of the compounds to inhibit LPS-induced TNF-α production in THP-1 differentiated macrophages. Through this process several new potent inhibitors of GSK-3 were identified and insights into the structure activity relationship was gained.

Committee: Douglas Goetz (Advisor) Subjects: Biomedical Engineering

Master of Science (MS), Wright State University, 2020, Pharmacology and Toxicology

The ATR protein kinase is activated in response to DNA damage and other forms of genotoxic stress caused by both environmental carcinogens and anti-cancer drugs. However, much of our understanding of ATR kinase function is limited to proliferating cells in which DNA replication stress is the primary signal for ATR activation and where the major regulatory targets of ATR signaling are proteins involved in DNA synthesis and cell cycle progression. Here we have used HaCaT keratinocytes maintained in a non-replicating, quiescent state in vitro to examine how cell killing by different genotoxic agents is impacted by cell growth status and by treatment with small molecule ATR kinase inhibitors. The genotoxins we examined included drugs from several classes of anti-cancer agents, including topoisomerase inhibitors (camptothecin, etoposide), alkylating agents (mitomycin C, temozolomide, and cisplatin), and compounds that interfere with RNA polymerase movement ((5-6-dichlorobenzimidazole 1-beta-D-ribofuranoside), actinomycin D). As expected, we find that quiescent cells are more resistant to the acute, lethal effects of these genotoxins than replicating cells. However, though we find that nearly all of these compounds led to the activation of ATR kinase signaling in the quiescent state, little-to-no effect of ATR kinase inhibitors was observed on quiescent cell viability. These results indicate that ATR can be activated in the absence of canonical replication stress and that its function does not significantly impact acute cell survival. To examine potential alternative functions for ATR signaling in quiescent cells, we then examined how ATR kinase inhibition impacted the activation of the translesion synthesis (TLS) pathway of DNA synthesis, which involves the use of specialized, potentially mutagenic DNA polymerases to fill in DNA repair gaps and complete DNA repair. Interestingly, we found that ATR kinase inhibition potentiated the activation of this pathway in response (open full item for complete abstract)

Committee: Michael G. Kemp Ph.D. (Advisor); Jeffrey B. Travers M.D., Ph.D. (Committee Member); Yong-jie Xu M.D., Ph.D. (Committee Member) Subjects: Pharmacology; Pharmacy Sciences; Toxicology

Doctor of Philosophy, The Ohio State University, 2019, Biomedical Sciences

The elucidation of more universal therapeutic targets is key to effectively combat the heterogeneity of breast cancer (BrCa). MicroRNAs (miRNAs) are small noncoding RNAs that are an abundant class of endogenous regulatory molecules, which act by targeting mRNAs for cleavage and/or translational repression. Continuing studies into BrCa genetics show that the various subtypes of BrCa are also affected by miRNA activity, which can even influence drug response. This prompted our investigation into the nuances of how miRNAs may influence BrCa behavior and response to treatment with targeted therapies, focusing on the proto-oncogene Polo-like Kinase 1 (PLK1). PLK1 plays an important role in the cell cycle, and is considered to be a proto-oncogene. Inhibiting PLK1 in has shown promise in reducing tumor volume and promoting tumor cell death in various cancers. A member of the miRNA-183 cluster, miRNA-183-5p, was found to bind to the 3' UTR of PLK1, with transiently-induced overexpression resulting in reduced expression levels of the active PLK1 protein. We also discovered that the miRNA-regulated reduction of PLK1 influenced the expression of other proteins in the PLK1 pathway, and affected cancer cell response to a PLK1-specific inhibitor, NMS-P937. The activity of miRNA-183-5p on PLK1 demonstrated an effect on cancer cell apoptosis following treatment with NMS-P937, suggesting a link with miRNA-183-5p expression and the efficacy of PLK1-specific inhibition in breast cancer cell lines. MiRNA-mediated regulation plays an important role in the initiation, progression and drug response of tumors. Understanding how miRNAs regulate PLK1 in breast cancer will improve our understanding of the PLK1 pathway, and whether this miRNA-directed regulation affects anti-PLK1 therapy. The work outlined here sets the stage for further inquiry into miRNA-governed pathways and their effects on drug response, something that should be considered carefully as putative targets are identified for (open full item for complete abstract)

Committee: Carlo Croce MD (Advisor); Larry Schlesinger MD (Committee Member); Kay Huebner PhD (Committee Member); William Carson MD (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology

Doctor of Philosophy, University of Toledo, 2018, Biology (Cell-Molecular Biology)

The cell cycle is regulated by checkpoints which ensure genomic integrity and proper cell division. We discovered a novel role of a multifunctional ser/thr kinase, glycogen synthase kinase 3 (GSK3), in regulation of the mitotic checkpoint. Our data show that in multiple cancer cell lines spindle toxin induced mitotic arrest is relieved by GSK3 inhibitors SB 415286 (SB), RO-81220 (RO) and lithium chloride. GSK3¿ knockout mammalian cells and GSK3¿ knockout mouse embryo fibroblasts (MEFs) show reduced mitotic index compared to wild type cells in the presence of Taxol. Mitotic arrest is dependent on the mitotic checkpoint complex (MCC), which is composed of Mad2, BubR1-Bub3 and Cdc20. Our data show that GSK3 targets the MCC in its mitotic regulation. Co-treatment of GSK3 inhibitors with spindle toxins showed decreased levels of Mad2, BubR1 and Bub1 localization at the kinetochores compared to spindle toxin only. MCC assembly also decreased with SB when treated with Taxol, compared to Taxol only. The data imply that GSK3¿ plays a role in maintaining the MCC. Additionally, time-lapse imaging of cells co-treated with Taxol and SB show a role for GSK3 in mitotic checkpoint strength regulation. Overexpression of GSK3 in mammalian and MEFs show an increase in mitotic index in the absence of any spindle toxins. The Wnt-signaling pathway and the PI3K/Akt signaling pathway are known upstream regulators of GSK3¿, negatively regulating GSK3¿ activity. Our data show that inhibiting Wnt-signaling and PI3K/Akt-signaling in the presence of Taxol, induces a longer mitotic arrest compared to Taxol alone. This suggests that GSK3¿ is regulated upstream by the Wnt- and the PI3K/Akt signaling arcs to control mitosis. Finally, our data implicated the NAD-dependent de-acetylase, Sirtuin2, as a possible downstream target of GSK3 in its mitotic regulatory role. Our observations indicate a novel regulator of the checkpoint and insight in connecting growth-signaling pathways with mitosis.

Committee: William Taylor Dr. (Advisor) Subjects: Biology; Cellular Biology; Molecular Biology

Master of Sciences, Case Western Reserve University, 2018, Physiology and Biophysics

Erythropoietin-producing human hepatocellular (Eph) receptors account for the majority of the membrane-bound receptor tyrosine kinase (RTK) family. Eph receptors have significant roles during embryonic development, cell maturation, and adulthood. Furthermore, studies have shown that drastic increase in the expression of Eph receptors is detected in the malignant proliferation of tumor cells in numerous cancers, such as melanoma, breast cancer and glioblastoma. Indeed, recently, oncogenic activities have been observed in non-canonical unliganded Ephrin type-A receptor 2 (EphA2). This project aims to probe the effects of phosphorylation on the intracellular domain interactions of EphA2 in solution. Results from this study indicate that deletion of the sterile a motif (SAM) domain leads to a strong binding affinity between kinase domains in solution. Upon oligomerization, less kinase activity is observed, compared to that of the monomeric state of the intracellular domain (ICD) of EphA2. Moreover, in this study, microscale thermophoresis (MST) is utilized to assess the binding interactions of EphA2 in complex with liposomes, a membrane-like environment. Upon the truncation of the SAM domain, binding of the intracellular domains of EphA2 to the membrane is abrogated. Data from this study will help to elucidate and further our understanding of the signaling mechanism of receptor tyrosine kinases and the regulatory role of phosphorylation.

Committee: Matthias Buck (Advisor); Sudha Chakrapani (Committee Member); Rajesh Ramachandran (Committee Member); Bingcheng Wang (Committee Member); William Schilling (Committee Chair) Subjects: Biophysics; Physiology

Master of Science in Biological Sciences, Youngstown State University, 2009, Department of Biological Sciences and Chemistry

Myosin light chain kinase, Myosin light chain phosphatase, and Rho kinase are three important regulatory proteins that are involved in regulating penile erection. This study focused on identifying these regulatory proteins in the resting (contracted) state of the corpus cavernosum. Rat corpus cavernous tissues were isolated and collected for protein analysis. Protein samples were then subjected to quantitative protein analysis, one dimensional gel electrophoresis, two dimensional gel electrophoresis and western blotting. The results clearly confirmed that all three regulatory proteins are expressed in this tissue. Future work will be carried out to identify these proteins during different phases of contraction and relaxation under a number of experimental conditions.

Committee: Gary Walker PhD (Advisor); Robert Leipheimer PhD (Committee Member); James Toepfer PhD (Committee Member) Subjects: Biology; Health Education; Molecular Biology

PhD, University of Cincinnati, 2005, Medicine : Molecular and Cellular Physiology

Regulation of epithelial cell motility requires coordinated actin rearrangement, yet the signaling pathways that lead to such modulation are not fully described. Previously we showed that protein kinase C (PKC) regulates actin remodeling in T84 intestinal epithelial cells but little is known regarding the role of PKC during cellular spreading. In subconfluent T84 cells, phorbol 12-myristate 13-acetate (PMA), which activates the conventional cPKC isoform PKCα and the novel nPKC isoforms PKCδ and PKCε of T84 cells, dramatically accelerated cell spreading with concurrent increase in tyrosine phosphorylation of the focal adhesion-associated proteins, Src, FAK and paxillin. These effects were abolished by the PKC inhibitor Go6850 and PKCδ-specific inhibitor rottlerin. In contrast, the cPKC-selective inhibitor Go6976 had no effect. Furthermore, siRNA-mediated down-regulation of PKCδ and PKCε, but not PKCα, attenuated cell spreading, implicating PKCδ and PKCε as the critical elements participating in regulation of epithelial cell spreading. Phosphorylation of FAK and paxillin by PMA was mediated by ligand-dependent transactivation of epidermal growth factor receptor (EGFr). Inhibition of EGFr signaling abolished PMA-induced phosphorylation of FAK and paxillin as well as cell spreading, indicating that EGFr transactivation plays a central role in mediating PMA-elicited cellular spreading. PMA caused PKCδ to translocate to the basal compartment, yet pretreatment with Go6850 or siRNA against PKCε redistributed PKCδ to the lateral membrane; Go6976, rottlerin, or siRNAs against PKCα or PKCδ were ineffective in preventing basal translocation of PKCδ. Inhibition of actin remodeling also redirected PKCδ to the lateral membrane, preventing PMA-induced phosphorylation of Src, FAK, and paxillin. These data demonstrate that actin remodeling by PKCε is critical for basal translocation of PKCδ and subsequent phosphorylation of focal adhesion targets, emphasizing the cooperation between (open full item for complete abstract)

Committee: Dr. Jeffrey Matthews (Advisor) Subjects: Biology, Cell

Doctor of Philosophy, The Ohio State University, 2012, Chemistry

Src-homology domains are small modular domains that recognize phosphotyrosine-containing proteins and couple activated protein kinases to intracellular signaling pathways. Since they often have overlapping functions, their SH2 domains often compete for binding to the same pY proteins. Determining their sequence specificities will help identify target proteins. Consequently, this will help understand the molecular basis for their cellular functions. Twenty-six kinase SH2 domains were screened against a phosphotyrosyl (pY) peptide library, and positive beads were sequenced by partial Edman degradation and mass spectrometry. The data revealed that the kinase family SH2 domains selected a class of pY peptides consisting of mostly hydrophilic and hydrophobic residues at the pY+1 and pY+3 positions, respectively. After validating their binding, the literature was searched to find known SH2 targets and their pY motifs. Seventeen SH2 domains from several different protein families were also purified, screened, and sequenced to determine their binding motifs. The majority of the SH2 domains had high selectivity at the pY+3 or pY+1 position with a few selecting for multiple peptide classes. For example Vav1 and Vav2 SH2 domains selected for three classes of peptides. These minor classes of peptides may be motifs of new protein targets that have not been identified yet. Some SH2 domains such as the Grb7 family, HSH2D, and Vav family had high selectivity of Asn at the +2 position but little selectivity at other positions. More subtle differences were observed between protein families at certain positions. For instance, SH2 domains from a family of GTPase signaling proteins preferred Pro at the +3 position, while SH2 domains from the PIK3 family preferred norleucine at the +3 position. Genetic disorders such as Noonan's syndrome and hematologic disorders such as juvenile myelomonocytic leukemia are caused by mutations in the SHP2 (PTPN11) gene. The mutations are mainly located (open full item for complete abstract)

Committee: Dehua Pei PhD (Advisor); Jennifer J. Ottesen PhD (Committee Member); Karin Musier-Forsyth PhD (Committee Member) Subjects: Biochemistry; Chemistry

Doctor of Philosophy, The Ohio State University, 2010, Integrated Biomedical Science Graduate Program

Chronic lymphocytic leukemia (CLL) has long been incorrectly labeled “the good leukemia” due to the age of onset and the low incidence of detectable symptoms. No leukemia; however, is “good,” and CLL is no exception. CLL is the most common type of adult leukemia in the United States and yet remains incurable by traditional therapies; this provides strong justification for developing additional types of therapeutics. Of particular interest are therapies that target signal transduction pathways essential to CLL cell survival mechanisms that are known to be aberrantly activated as these contribute not only to the inherent survival of transformed cells but also contribute to survival signaling received from the microenvironment. The focus of the work presented here is on altering kinase signaling in either the cancerous B-cell or the surrounding microenvironment to alter disease progression, duration, or state. Chapter one is a general introduction into B-cell biology and CLL: biology, diagnosis and treatment. The dependence of CLL cells on microenvironmental signaling is discussed with particular attention given to the bone marrow stroma and T-lymphocytes. In addition, key signaling pathways involved in B-cell survival and potential target kinases are discussed. Chapter two discusses the effects of inhibiting Phosphatidylinositol 3-Kinase (PI3-Kinase) for the treatment of CLL using a novel drug, CAL-101. We found that inhibition of PI3-Kinase signaling using a selective inhibitor resulted in not only direct cytotoxicity to CLL cells, but also altered the microenvironment in a way that was unfavorable to CLL cell survival. Chapter three discusses the effects of inhibition of B-cell receptor (BCR) signaling via inhibition of Tec family kinases (specifically BTK) using a novel drug, PCI-32765. Similarly to PI3-Kinase, we again found that inhibition of BCR signaling resulted in the induction of apoptosis in CLL cells and an alteration of microenvironmental signaling. Chap (open full item for complete abstract)

Committee: John Byrd MD (Advisor); Amy Johnson PhD (Committee Member); Virginia Sanders PhD (Committee Member); Michael Freitas PhD (Committee Member) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 2006, Molecular, Cellular, and Developmental Biology

The goal of the present studies was to localize two proteins known to be involved in regulation of cell proliferation and survival in specific cell populations in normal mouse skin, during multi-stage skin carcinogenesis, following skin injury, and during tumor angiogenesis. The proteins evaluated included activated Akt, as defined by phosphorylation of Akt at Serine-473 (pAkt) and mTOR, defined by phosphorylation of mTOR at Serine-2448 (pmTOR). Our laboratory previously identified a novel murine VEGF splice variant, VEGF205*, which was differentially expressed in mouse skin carcinomas, but not in normal skin. VEGF205* encodes for a truncated 145 amino acid polypeptide with a unique 7 amino acid carboxyl-terminal tail, YVGAAAV, that is significantly different from the carboxyl-terminal tail of mouse or human VEGF proteins previously identified. VEGF205* stimulated a PI3-K-dependent-increases in pAkt and pmTOR in human vascular endothelial cells, which were significantly higher than pAkt and pmTOR levels induced following incubation of endothelial cells with VEGF120. Immunochemical staining analysis as well as triple color immunofluorescence were used in combination with confocal microscopy to evaluate the presence of pAkt and pmTOR in keratinocyte stem cells (KSC) located within a specific niche in hair follicles defined as “the bulge”, as identified by expression of markers of mouse KSC, CD34 and K15. The location of CD34+/K15+ KSC remained restricted to the bulge niche within hair follicles during multi-stage skin carcinogenesis and cutaneous wound healing. Our results also provide the first evidence for the presence of pAkt and pmTOR in CD34+/K15+ KSC localized to the ORS niche of the bulge region in normal skin, during multi-stage skin carcinogenesis, as well as during wound healing. Using the highly invasive and metastatic human Hs578T breast tumor cell line and the slow growing and non-invasive human MCF-7 breast tumor cell line, the present studies also compa (open full item for complete abstract)

Committee: Fredika Robertson (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2004, Plant Biology

Long alpha-helical coiled-coil proteins are involved in a variety of organizational and regulatory processes in eukaryotic cells. In contrast to yeast and animals, only few such proteins have been investigated in plants. Here, two plant long coiled-coil proteins were investigated in detail, MAR-binding filament-like protein1 (MFP1) and the putative Arabidopsis homolog of Tpr (translocated promoter region). MFP1 is a nuclear-encoded, long coiled-coil protein that is targeted to plastids. It accumulates to comparable levels in all tissues of Arabidopsis which contain green chloroplasts, regardless of age and organ identity, but is much less abundant in roots of both light-grown and dark-grown seedlings. MFP1 protein accumulation parallels chloroplast development during the greening of seedlings shifted from dark to light, suggesting that MFP1 expression is regulated in a tissue-specific and light-dependent manner. MFP1 is localized in chloroplasts both in suspension culture cells and in leaves, and it is associated with the stromal side of thylakoid membranes of mature chloroplasts. It is co-purified with nucleoids, suggesting a function at the interface of the chloroplast genome and the photosynthetic membranes. MFP1 comprises a major DNA-binding activity in Arabidopsis chloroplasts and binds to several regions of the chloroplast DNA with equal affinity. Several thylakoid proteins are phosphorylated by a protein kinase CKII-like activity, and the alpha subunit of a chloroplast-located CKII has recently been identified as a component of the chloroplast transcription complex. Chloroplast-localized MFP1 is phosphorylated in vivo, and in vitro by CKII and phosphorylation inhibits its DNA-binding activity. A tandem CKII site in the DNA-binding domain of MFP1 was identified which is involved in the phosphorylation-dependent loss of DNA-binding activity. These features of MFP1 make CKII-dependent phosphorylation a possible mode of regulating the DNA-binding activity of the (open full item for complete abstract)

Committee: Iris Meier (Advisor) Subjects: Biology, Botany

Doctor of Philosophy (PhD), University of Toledo, 2011, College of Medicine

Borrelia burgdorferi (Bb) is a tick-borne bacterium from the family Spirochaetes that is the causative agent for Lyme disease. These bacteria are notable for their ability to evade host defenses and persist extra-cellularly, even though infection elicits potent innate and adaptive immune responses. We previously demonstrated that host interleukin 10 (IL-10), an anti-inflammatory cytokine important for controlling excess inflammation, plays an important role in suppressing the immune-clearance of Bb. We hypothesize antigen-presenting cells (APCs) such as bone-marrow macrophages (BMM) and dendritic cells (BMDC) produce high-levels of IL-10 immediately upon recognition of Bb and this dysregulated IL-10 level subsequently suppresses the elicitation of pro-inflammatory mediators by the APCs against Bb. We also hypothesize that the production of IL-10 by APCs such as BMMs utilizes signaling pathways that are distinct from Bb-elicited pro-inflammatory mediators. Our results demonstrated that both cultured BMM and BMDCs rapidly produce IL-10 upon Bb-stimulation and this IL-10 suppressed the production of pro-inflammatory cytokines (e.g. IL-12), chemokines, reactive oxygen species, phagocytosis, and surface marker upregulation. Our data also indicate that IL-10 production by BMMs in response to Bb is dependent on surfaceToll-like receptor 2 (TLR2) yet independent of Bb phagocytosis/internalization. While most Bb-elicited pro-inflammatory mediators are also TLR2-dependent, they require that Bb be internalized. Bb-elicited IL-10 production by BMMs is dependent on signaling pathways involving both phosphotidylinositol-3 kinase (PI3-kinase) and mitogen-activating protein kinase (MAP kinase). On the other hand, the elicitation of most pro-inflammatory responses from BMMs by Bb is independent of both PI3-kinase and MAP kinase. Overall, our findings indicate that Bb stimulates APCs to produce dysregulated IL-10 through unique signaling pathways from those that produce inflammatory (open full item for complete abstract)

Committee: R. Mark Wooten (Advisor) Subjects: Immunology; Microbiology

MS, University of Cincinnati, 2024, Medicine: Biomedical Research Technology

Neurofibromatosis type I (NF1) is an autosomal dominant disorder caused by loss of function mutations in the NF1 gene that encodes the neurofibromin protein. Neurofibromin is a GTPase activating protein (GAP) that acts as a negative regulator of the RAS/mitogen-activated protein kinase (MAPK; RAS-RAF-MEK-ERK) signaling cascade. The clinical phenotype of NF1 is characterized by a predisposition to peripheral nerve sheath tumors that can be locally destructive and have potential for malignant transformation. Targeted gene therapy that rescues neurofibromin function could provide a method for tumor prevention and treatment. Adeno-associated virus (AAV) represents an appealing vector for gene therapy; however, its small packaging capacity prevents delivery of the entire neurofibromin gene. This paper describes the generation and in vitro validation of AAV-compatible transgenes that modulate MAPK signaling up- or downstream of RAS or affect other signaling pathways perturbed in NF1. It was found that all versions of the GRD (GAP-related domain) reduced growth factor stimulated ERK phosphorylation, proliferation of NF1-null immortalized human Schwann cells and Nf1-null primary mouse Schwann cells, and mouse Schwann cell precursor (SCP) sphere formation relative to YFP control vectors. These effects were dose-dependent and acted primarily by reducing proliferation rather than inducing cell death. Dominant negative KRAS and HRAS were also efficacious in ERK phosphorylation, proliferation, and SCP sphere formation assays.

Committee: John Robinson Ph.D. (Committee Chair); Anne-Karina Perl Ph.D. (Committee Member); Ronald Waclaw Ph.D. (Committee Member) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 2024, Biomedical Sciences

Heart failure (HF) is a complex heterogeneous syndrome characterized by altered left ventricular ejection function and impacts over six million individuals in the United States. Among cancer survivors, cardiovascular mortality due to HF is prevalent.1,2 Despite improvements in the medical and surgical management of HF, mortality rates remain high, with only modest improvements in survival during the past decade. Proper systolic heart function requires coordinated activation and force transmission throughout the myocardium. Cardiomyocytes have developed intricate molecular mechanisms to control both electrical and mechanical functions in response to physiological and pathological stressors. Therefore, active targets for diagnostic and therapeutic approaches for HF are currently centered on pathways that influence both the electrical and structural function of cardiomyocytes. The work described herein explores the mechanisms underlying electrical and structural dysfunction in tyrosine kinase inhibitor (TKI) cardiotoxicity and βII-spectrin (Sptbn1) deficient HF. Genetic or acquired (i.e., drug-induced cardiotoxicity) changes in signaling pathways responsible for electrical and structural homeostasis can lead to the development of lethal arrhythmias and HF. Several chemotherapeutic agents, including TKIs, are associated with arrhythmia and HF in patients with or without preexisting cardiovascular disease. It has been postulated that TKIs may induce HF by causing direct myocardial damage, culminating in reduced cardiac inotropy, lusitropy, and chronotropy. However, the mechanisms by which these phenomena develop or contribute to ventricular arrhythmias and cardiac dysfunction are incomplete. We first describe that pazopanib, a second-generation TKI, alters cardiomyocyte excitability in patients using a retrospective chart review and recapitulated these findings in two mouse models using surface electrograms. Cellular and computational model studies revealed th (open full item for complete abstract)

Committee: Sakima Smith (Advisor); Brandon Biesiadecki (Committee Member); Mark Ziolo (Committee Member); Jill Rafael-Fortney (Committee Member); Thomas Hund (Committee Member) Subjects: Cellular Biology; Molecular Biology; Physiology

Doctor of Philosophy, University of Toledo, 2023, Medicinal Chemistry

CD4+ T cells play a pivotal role in generating and regulating effective immune responses against invading pathogens by orchestrating intracellular calcium (Ca2+) signaling through the synthesis of Ca2+ signaling secondary messengers such as nicotinic acid adenine dinucleotide phosphate (NAADP), cyclic adenosine 5-diphosphate ribose (cADPR), and inositol 1,4,5 triphosphate (IP3). These signaling events significantly impact downstream immune responses. In contrast, in the tumor microenvironment, upregulation of Ca2+ signals are associated with cancer cell proliferation, malignancy, and immune response suppression. NAADP being the most potent secondary messenger, its signaling pathway remains elusive and controversial. NAADP is believed to bind to accessory proteins such as Jupiter microtubule-associated homolog 2 (JPT2) and an Sm-like protein Lsm 12. These are thought to influence the poorly characterized 'two-pore channels' (TPCs) in the endo-lysosomal compartments of cells. Notably, previous studies using PF-543, a sphingosine kinase-1 (SphK-1) inhibitor, effectively diminished NAADP-mediated Ca2+ signals in sea urchin eggs, and U2OS cells, warranting further investigation of the involvement of accessory proteins, TPCs, and SphK-1 in this pathway. To shed light on JPT2's role in influencing T cell function, we conducted experiments to investigate its impact on T cell activation events. Our findings indicate that the knockdown of JPT2 by siRNA did not affect early T cell activation events, such as the phosphorylation of tyrosine kinases LCK and ZAP-70, but instead influenced Ca2+ release and later events, such as the phosphorylation of the critical transcription factor NF- κB, a process which is well-known to be Ca2+ dependent and responsible for T cell proliferation and differentiation. Additionally, our investigation into a library of PF-543 analogs on murine T cells and sea urchin eggs revealed a correlation between SphK-1 inhibition and Ca2+ release. Our re (open full item for complete abstract)

Committee: Katherine A. Wall (Committee Chair); James T. Slama (Committee Member); Amit K. Tiwari (Committee Member); David Giovannucci (Committee Member); Zahoor Shah (Committee Member) Subjects: Molecular Biology; Pharmacy Sciences

Master of Science (MS), Wright State University, 2023, Biochemistry and Molecular Biology

Extracellular signal related kinase 3 (ERK3) is one of the atypical mitogen activated protein kinases (MAPK). It is expressed ubiquitously and plays a role in a variety of cellular processes, including cell growth and differentiation. ERK3's role in promoting migration and invasion in various cancers has been well established. ERK3 is upregulated in non-small cell lung cancers (NSCLCs) and has been shown to promote NSCLC tumor growth and progression. However, the regulation of ERK3 in lung cancers remains largely unclear. A recent study indicates that ERK3 phosphorylation at S189, an indicator of ERK3 activity, is upregulated by KRAS in NSCLCs. KRAS is one of the most commonly mutated oncogenes in lung cancers. To study the KRAS dependent regulation of ERK3, knockdown of KRAS was performed and it resulted in a remarkable reduction in ERK3 phosphorylation as well as total ERK3 protein level confirming the regulation of ERK3 by KRAS. Upon knockdown of KRAS a significant reduction of ERK3 mRNA level was observed indicating that KRAS regulates ERK3 at transcriptional level. Further, we found that the regulation of ERK3 by KRAS may be through the transcription factor c-Jun, that is well-known to be activated by KRAS signalling. Our data indicates that c-Jun positively regulates ERK3 transcription in LUAD cell lines. Further, we have found that KRAS upregulates c-Jun activating phosphorylations in LUAD cells, suggesting that KRAS regulates ERK3 through c-Jun. Given the discrepancy regarding the role of ERK3 in NSCLC cell growth reported in previous studies, we have thoroughly investigated the role of ERK3 in cell growth by stable knockdown of ERK3 using shRNA targeting different regions of ERK3 mRNA, as well as by using ERK3 inhibitors in a variety of NSCLS cell lines. While knockdown of ERK3 via targeting the coding region did not affect cell proliferation, targeting the 3'UTR of ERK3 or treatment with ERK3 inhibitors reduced the proliferation of LUAD cells.

Committee: Weiwen Long Ph.D. (Advisor); Kwang-Jin Cho Ph.D. (Committee Member); Michael Craig Ph.D. (Committee Member) Subjects: Biochemistry; Molecular Biology

Doctor of Philosophy, Case Western Reserve University, 2023, EECS - Computer and Information Sciences

This dissertation presents a comprehensive approach to advancing proteomics and under-studied proteins in network biology, emphasizing the development of reliable algorithms, fair evaluation practices, and accessible computational tools. A key contribution of this work is the introduction of RoKAI, a novel algorithm that integrates multiple sources of functional information to infer kinase activity. By capturing coordinated changes in signaling pathways, RoKAI significantly improves kinase activity inference, facilitating the identification of dysregulated kinases in diseases. This enables deeper insights into cellular signaling networks, supporting targeted therapy development and expanding our understanding of disease mechanisms. To ensure fairness in algorithm evaluation, this research carefully examines potential biases arising from the under-representation of under-studied proteins and proposes strategies to mitigate these biases, promoting a more comprehensive evaluation and encouraging the discovery of novel findings. Additionally, this dissertation focuses on enhancing accessibility by developing user-friendly computational tools. The RoKAI web application provides a convenient and intuitive interface to perform RoKAI analysis. Moreover, RokaiXplorer web tool simplifies proteomic and phospho-proteomic data analysis for researchers without specialized expertise. It enables tasks such as normalization, statistical testing, pathway enrichment, provides interactive visualizations, while also offering researchers the ability to deploy their own data browsers, promoting the sharing of findings and fostering collaborations. Overall, this interdisciplinary research contributes to proteomics and network biology by providing robust algorithms, fair evaluation practices, and accessible tools. It lays the foundation for further advancements in the field, bringing us closer to uncovering new biomarkers and potential therapeutic targets in diseases like cancer, Alzheimer' (open full item for complete abstract)

Committee: Mehmet Koyutürk (Committee Chair); Mark Chance (Committee Member); Vincenzo Liberatore (Committee Member); Kevin Xu (Committee Member); Michael Lewicki (Committee Member) Subjects: Bioinformatics; Biomedical Research; Computer Science