Bachelor of Science (BS), Ohio University, 2022, Neuroscience

Lithium, a therapeutic first introduced in 1952, is still considered the gold standard for treatment of bipolar disorder. Although many new medications have been discovered over the years, lithium is the only therapeutic used exclusively for bipolar disorder-- indicating a separate mechanism of action from antipsychotic and antidepressive treatments (Kaidanovich-Beilin et al., 2011). Genetic and pharmacological studies have shown that lithium's therapeutic efficacy is at least partially due to inhibition of glycogen synthase kinase-3 beta, or GSK3b (Kaidanovich-Beilin et al., 2011). Development of small molecule therapeutics to target GSK3b with better drug like qualities such as higher affinity and selectivity can lead to advancements in our understanding and treatment of bipolar disorder. This study investigates the structure activity relationship of small molecule GSK3b inhibitors through ab initio calculations, protein kinase sequence alignment, and computational docking studies. We found that although the ATP binding pocket is highly conserved, a proline residue unique to GSK3 may contribute to selectivity of small molecule inhibitors. In addition, small molecule inhibitors may show increased selectivity for GSK3b through preferential SH-π interactions with cysteine residue 199. Finally, docking studies suggest that GSK3b may be too dynamic or ligand-specific for predictive docking. Overall, this work has improved understanding of how small molecule inhibitors may utilize the GSK3b ATP binding pocket and their potential to treat bipolar disorder.

Committee: Jennifer Hines (Advisor) Subjects: Neurosciences

Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2019, College of Sciences and Health Professions

Ribonuclease L (RNase L) mediates interferon (IFN) function during viral infection and cell proliferation. RNase L deficient mice are heavier and have more lipid accumulation in the liver tissues compared to wide type mice under the same condition, suggesting that RNase L might mediate lipid homeostasis. The first part of the dissertation focuses on investigating the role of RNase L in lipid homeostasis. By using RNase L gene knockout mice with C57BL/6 background, we found a novel role of RNase L in lipid homeostasis through upregulating the rate-limiting enzymes that are essential for fatty acid and cholesterol syntheses. The excess amount of lipids is synthesized continuously, which leads to the development of Non-Alcoholic Fatty Liver Disease (NAFLD). Tissue distribution reveals that RNase L is highly expressed in the lung and other organs. However, the physiological roles of RNase L in the lung are largely unknown. In the second part of the dissertation, we found that lipopolysaccharide (LPS)-induced acute lung injury (ALI) was remarkably intensified in mice deficient RNase L compared to wild type mice under the same conditions. Furthermore, we found that RNase L mediated the TLR4 signaling pathway and regulated the expression of various pro- and anti-inflammatory genes in the lung tissue and blood. Most importantly, RNase L function in macrophages during LPS stimulation may be independent of the 2-5A system. These findings demonstrate a novel role of RNase L in the immune response via an atypical molecular mechanism. In the third part of the dissertation, an LC-MS/MS assay was developed and fully validated for the rapid quantitative measurement of GSK3 inhibitors, a promising therapeutic treatment for acute myeloid leukemia (AML). This assay has been applied for the measurement of GSK3 inhibitors in mice plasma and can be used for the preclinical and clinical study of the drugs.

Committee: Aimin Zhou (Committee Chair); David Anderson (Committee Member); Mekki Bayachou (Committee Member); Yana Sandlers (Committee Member); Bin Su (Committee Member); Yuping Wu (Committee Member) Subjects: Analytical Chemistry; Biochemistry; Biology; Molecular Biology

PHD, Kent State University, 2018, College of Arts and Sciences / Department of Biological Sciences

Glycogen synthase kinase 3 (GSK3) is a highly conserved protein-serine kinase regulating key cellular functions. In mammals GSK3 is expressed as two isoforms, GSK3a and GSK3ß, encoded by distinct genes. The catalytic domains of the two isoforms are 98% identical. In most tissues the two isoforms are functionally interchangeable, except in the developing embryo where GSK3ß is essential. One functional allele of either of the two isoforms is sufficient to maintain normal tissue functions. That is, 25% of the total GSK3 catalytic activity appears to be sufficient to maintain normalcy in cells and tissues. We have previously shown that both GSK3 isoforms are present in bovine, primate, and mouse sperm and suggested that they may play a role in epididymal initiation and regulation of sperm motility. Using genetic approaches, here we have tested requirement for each of the two GSK3 isoforms in testis and sperm. Both GSK3a and GSK3ß are expressed at high levels in testis coincident with the onset of spermatogenesis. Mice harboring a conditional knock out of GSK3ß in developing germ cells in testis are normal and fertile. By contrast, conditional knock out of GSK3a in developing testicular germ cells results in male infertility. Mice lacking one allele each of GSK3a and GSK3ß, i.e. heterozygous for both isoforms, are fertile. Despite overlapping expression and localization of the two isoforms in testis, GSK3ß does not substitute for loss of GSK3a. GSK3a is essential and irreplaceable in testis and sperm. Loss of GSK3a impairs sperm hexokinase activity resulting in low ATP levels. Low ATP and net adenine nucleotide levels in caudal sperm lacking GSK3a resemble immature caput epididymal sperm. Changes in the association of the protein phosphatase PP1¿2 with known protein interactors, which occurs during sperm maturation in the epididymis, is impaired in sperm lacking GSK3a. Localization of GSK3a is predominant in the principal piece suggesting this protein kinase binds t (open full item for complete abstract)

Committee: Srinivasan Vijayaraghavan PhD (Committee Chair); Douglas Kline PhD (Committee Member); Gary Koski PhD (Committee Member); Hamza Balci PhD (Committee Member); Songping Huang PhD (Committee Member) Subjects: Biochemistry; Biology; Cellular Biology; Molecular Biology

PHD, Kent State University, 2018, College of Arts and Sciences / Department of Biological Sciences

Cyclic-AMP plays an important role in sperm motility and activation. The actions of cAMP in sperm involve Protein Kinase A mediated protein phosphorylation. The crucial roles of sperm cAMP and PKA have also been demonstrated using genetic approaches. Understanding how a cAMP act to sustain sperm motility and fertility requires identification of the protein substrates of PKA. Identification of substrates of specific protein kinases and determination of changes in phosphorylation of these substrates during sperm motility initiation and fertilization will be a major advance in understanding signaling mechanisms underlying male gamete function. In our study we are using a novel chemical-genetic approach to identify proteins which are phosphorylated by PKA during sperm motility. In this approach the structurally conserved ATP-binding pocket in Protein Kinase A is genetically modified (analog-sensitive-Protein Kinase A) to generate mutant allele, this mutant protein will in addition to ATP, can also utilize specific ATP analogs. The mutation replaces a conserved amino acid with a bulky side chain with a smaller residue (alanine or glycine) creates a “gap” or an enlarged ATP binding pocket. The engineered ``gap'', located in the active site of the enzyme where the N6 amino group on the purine moiety of ATP is positioned, allows binding of not only ATP but also structurally modified ATP analogs with substitutions at the N6 position, such as N6-(benzyl)-ATP. Only the as-mutant kinase, but not the wild type kinase, can use N6-substituted ATP analogs as phosphate donors. Thus, only substrates of the as-mutant kinase are labeled by the ATP analogs. Another recent advance in this approach was the development of an affinity tagging technique where the ATP-analog labeled proteins can be recognized by specific antibodies. In this approach following labeling of substrates with N6-(benzyl)-ATP-¿S, the thio-phosphate group on the polypeptides is alkylated by para-nitrobenzylmesylate (open full item for complete abstract)

Committee: Srinivasan Vijayaraghavan PHD (Committee Chair); Douglas Kline PHD (Committee Member); Gary Koski PHD (Committee Member); Sanjaya Abeysirigunawardena PHD (Committee Member); Aaron Jasnow PHD (Committee Member) Subjects: Biochemistry; Biology; Cellular Biology; Molecular Biology

Doctor of Philosophy (PhD), Ohio University, 2013, Molecular and Cellular Biology (Arts and Sciences)

Atherosclerosis, a progressive inflammatory disease, is a leading cause of cardiovascular related death worldwide. Uptake of oxidized-low density lipoprotein (ox-LDL) by monocytes/macrophages plays a critical role in the pathogenesis of atherosclerosis. The importance of macrophages in the development of an atherosclerotic plaque has been well documented. Wnt5a, a secreted glycoprotein, is highly present in atherosclerotic lesions, and is known to be upregulated in other inflammatory diseases like rheumatoid arthritis and sepsis. Interleukin-6 (IL-6), an inflammatory cytokine secreted by activated macrophages, is also known to be expressed in these lesions. However, to date, the exact source of Wnt5a in these atherosclerotic lesions has not been established. The above observations motivated us to determine whether ox-LDL stimulated macrophages are an important source of Wnt5a, and can its increased expression be used as a marker for diagnostic identification of atherosclerosis. Using quantitative real time-polymerase chain reaction (RT-PCR), we demonstrated that lipopolysaccharide (LPS) and ox-LDL, but not native-LDL, induces Wnt5a and IL-6 mRNA expression in human monocyte-derived macrophages. Furthermore, we found increased expression of Wnt5a protein in human atherosclerotic serum samples as compared to control serum samples using an enzyme-linked immunosorbent assay (ELISA), suggesting a possible diagnostic/prognostic approach for the detection of atherosclerosis. Combined, the results of this study indicate that Wnt5a plays an active role in the pathophysiology of atherosclerosis. Glycogen synthase kinase (GSK3), a ubiquitously expressed multifunctional serine/threonine kinase, participates in a multitude of cellular and physiological processes, ranging from cell cycle, both cell death and survival, transcription, translation, microtubule stability and beta catenin Wnt signaling. Given its constitutive expression and diversity of putative substrates, GS (open full item for complete abstract)

Committee: Douglas Goetz (Advisor) Subjects: Biomedical Research; Cellular Biology; Molecular Biology