Master of Science, The Ohio State University, 1966, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 1961, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 1970, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 1966, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 1962, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 1966, Graduate School

Committee: Not Provided (Other) Subjects:

Doctor of Philosophy, The Ohio State University, 2000, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 1977, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 1969, Graduate School

Committee: Not Provided (Other) Subjects: Chemistry

Doctor of Philosophy, The Ohio State University, 1968, Graduate School

Committee: Not Provided (Other) Subjects: Health Sciences

Doctor of Philosophy, The Ohio State University, 1960, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 2014, Comparative and Veterinary Medicine

Chemotherapeutics remains a choice of treatment for several malignant diseases. However, selective cytotoxicity against cancer cells without compromising their normal counterparts pose a huge challenge for traditional drug design. Current therapies for chronic lymphocytic leukemia (CLL), the most prevalent adulthood leukemia in the western world are not curative rendering drug adverse effects and immunosuppression. Here we developed a novel non-immunosuppressive FTY720 derivative OSU-2S with potent cytotoxicity against leukemic B cells. OSU-2S induces activation of protein phosphatase 2A, phosphorylation and nuclear translocation of SHP1 S591 and deregulation of multiple cellular processes in CLL. Moreover, with relevant to CLL disease TCL1A expression that was identified to be down regulated in response to OSU-2S in the gene expression profile was independently confirmed to be significantly down regulated both at the mRNA and protein levels. Exposure of OSU-2S to unintended cells is expected to adversely affect physiological functions of these ubiquitous phosphatases. To selectively deliver OSU-2S to leukemic cells, we developed tumor antigen targeted delivery of immunonanoparticle carrying a OSU-2S (2A2-OSU-2S-ILP). 2A2-OSU-2S-ILP immunonanoparticles mediated selective cytotoxicity of CLL but not normal B cells through targeting receptor tyrosine kinase ROR1 expressed in leukemic but not normal B cells. Developing a novel spontaneous CLL mouse model expressing human ROR1 in all leukemic B cells, we demonstrate the therapeutic benefit of enhanced survival with 2A2-OSU-2S-ILP in-vivo. The newly developed non-immunosuppressive OSU-2S, its delivery using human CLL specific surface antigen ROR1 directed immunonanoparticles and the novel transgenic mouse model of CLL that expresses human ROR1 exclusively in leukemic B cell surface are highly innovative and can be applied to CLL and other ROR1+ malignancies including mantle cell lymphoma (MCL) and B-lineage acute lymph (open full item for complete abstract)

Committee: Natarajan Muthusamy DVM, PhD (Advisor); John Byrd MD (Committee Member); Ching-Shih Chen PhD (Committee Member); Cheryl London DVM, PhD (Committee Member) Subjects: Biomedical Research; Medicine; Nanotechnology; Therapy

Doctor of Philosophy, The Ohio State University, 2014, Chemistry

Protein phosphorylation is a post-translational modification controlled by two counteracting enzyme families, protein kinases and phosphatases. Protein phosphatases have been demonstrated to regulate many physiological pathways and exhibit distinct specificity in vivo. However, the factors determining their specificity are not well understood. In this study, the intrinsic specificity of various families of phosphatases has been explored utilizing combinatorial peptide library screening. The sequence specificity of eight classical-protein tyrosine phosphatases (PTPs) (PTPRA, PTPRB, PTPRD, PTPRO, PTP-PEST, PTP1B, SHP-1, and SHP-2) was determined. While PTPRA showed no selectivity, the other PTPs exhibited similar preferences for peptides containing acidic residues but disfavored basic ones. However, the enzymes differed in their level of selectivity and catalytic activity. Most of the classical-PTPs screened in this study also contains substrate recruiting/regulatory domains; it is likely that the in vivo PTP specificity is enhanced by the recruiting/regulatory domains. The sequence specificity of atypical dual-specificity phosphatases (DUSPs), which only contain a single catalytic domain, was examined to determine whether they exhibit more stringent specificity than the classical-PTPs. The screening of Vaccinia VH1-related (VHR) DUSP against pY-peptide libraries revealed two distinct classes of substrates. While class I peptide substrates are similar to the pY motifs derived from reported VHR protein substrates, the novel class II peptide substrates of the consensus (V/A)P(I/L/M/V/F)X1-6pY exhibit an alternative-binding mode to VHR, as suggested by site-directed mutagenesis and molecular modeling. ROBO1 and LASP1, which contain the class II consensus motif, were demonstrated to be VHR substrates in vitro. The haloacid dehydrogenase superfamily (HADSF) phosphatases have been shown to dephosphorylate a wide range of substrates including sequence with pY, phosphoseri (open full item for complete abstract)

Committee: Dehua Pei Professor (Advisor); Ross Dalbey Professor (Committee Member); James Cowans Professor (Committee Member) Subjects: Chemistry

Doctor of Philosophy, The Ohio State University, 2011, Chemistry

Protein tyrosine phosphatases (PTPs) hydrolyze phosphotyrosine (pY) back to tyrosine and inorganic phosphate, functioning in coordinate with protein tyrosine kinases (PTKs) to regulate a broad range of cell signaling processes. Once viewed as passive housekeeping enzymes, it has recently become apparent that PTPs exhibit substrate specificities in vivo. In this work, we have established a robust combinatorial peptide library screening method to determine the substrate specificities of any PTP. A novel high throughput enzyme-coupled assay has been developed, which for the first time enables the rapid screening of PTP activity and substrate specificities. Three major pY-containing peptide libraries were designed and synthesized on-bead to define the substrate specificities of the PTP domain at both N- and C- terminal sides of pY. The substrate specificity profiles of seven PTPs (PTP1B, TC-PTP, SHP1, SHP2, TULA-1, TULA-2, and VHR) were successfully obtained by the established screening method and validated by solution-phase enzyme kinetics. These PTPs exhibit different levels of sequence specificity and catalytic efficiency. The PTP1B catalytic domain has modest preference for acidic residues on both sides of pY, but disfavors basic residues at any position. TC-PTP has broader range of specificity. It prefers acidic residue N-terminal to pY, but exhibits tolerance for basic residues at the C-terminal side and N-terminal positions some distance from pY (-4 and -5). By contrast, SHP-1 and SHP-2 share similar but much narrower substrate specificities, with a strong preference for acidic and aromatic hydrophobic amino acids on both sides of pY. An efficient SHP-1/2 substrate generally contains two or more acidic residues on the N-terminal side and one or more acidic residue on the C-terminal side of pY but no basic residues. TULA-1 (UBASH3A/STS-2) and TULA-2 (p70/STS-1) represent a novel class of PTPs. Distinct from the classical PTPs, which utilize a conserved cysteine r (open full item for complete abstract)

Committee: Dehua Pei (Advisor); Karin Musier-Forsyth (Committee Member); Thomas Magliery (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, The Ohio State University, 2005, Molecular, Cellular, and Developmental Biology

Monocytes and macrophages express innate immune receptors such as FcgammaR and Toll-like receptor 4, which when engaged by their respective ligands; IgG immune complexes (ICs) and bacterial lipopolysaccharide (LPS), become activated to produce inflammatory mediators. While production of inflammatory mediators is critical for elimination of invading pathogens, this process must be tightly regulated to prevent collateral tissue damage. The goal of this thesis project is to analyze molecular mechanisms that regulate macrophage inflammatory responses to ICs and LPS. In the first part of this project we have analyzed the role of the inositol phosphatase SHIP-2 in FcgammaR-mediated macrophage responses and have established for the first time that SHIP-2 regulates these responses. During the course of these studies it was found that peripheral blood monocytes (PBM) express little to no SHIP-2. However, upon LPS treatment SHIP-2 expression is robust in PBM. A similar regulation of SHIP-1 was also found in PBM. These latter findings suggested a role for inositol phosphatases in LPS signaling. Hence, in the second part of this project we have examined the role of the inositol phosphatase SHIP-1 in LPS signaling. Our experiments established that SHIP serves to promote macrophage inflammatory responses. Thus macrophages from SHIP-deficient animals were hyporesponsive to LPS. A comparison of the signaling events in LPS-stimulated SHIP-sufficient and SHIP-deficient cells revealed that the serine/threonine kinase Akt was hyperactivated in SHIP-deficient macrophages. These results suggested a role for Akt in dampening pro-inflammatory responses to LPS. Therefore, in part three of this project we analyzed the role of Akt in LPS-induced cytokine responses. Here, using multiple independent experimental models we have demonstrated that Akt promotes the production of the anti-inflammatory cytokine IL-10 in response to LPS stimulation. Although Akt did not appear to have an effect on the (open full item for complete abstract)

Committee: Susheela Tridandapani (Advisor) Subjects: Biology, Molecular

PHD, Kent State University, 2012, College of Arts and Sciences / Department of Biological Sciences

Four isoforms of phospho-protein phosphatase 1 (PPP1), PPP1CA (α), PPP1CB (β), PPP1CC1 (γ1), PPP1CC2 (γ2) are derived from three genes in mammals (Ppp1ca, Ppp1cb, Ppp1cc). PPP1CC1 and PPP1CC2 are differentially spliced products of the Ppp1cc gene. All four isoforms are virtually identical (1-298 aa, approximately 90% identity) except at their extreme C-termini. While PPP1CC1, PPP1CA and PPP1CB are ubiquitous, PPP1CC2 is expressed largely in testis and is the sole PPP1 isoform present in spermatozoa. We have previously shown that sperm motility is inversely related to the activity of the protein phosphatase PPP1CC2. Furthermore, targeted disruption of Ppp1cc gene in mice, which eliminates both PPP1CC isoforms, results in male infertility due to lack of spermatogenesis suggesting an indispensible role for the proteins in spermatogenesis and sperm development. Mutant female mice are fertile and normal, presumably since PPP1CA or PPP1CB can substitute for the loss of PPP1CC isoforms. The goal of goal of this dissertation work was to identify the role of PPP1CC isoforms, PPP1CC1 and PPP1CC2 in spermatogenesis and male fertility. We wanted to determine if any one or both of the two PP1 isoforms are essential for spermatogenesis and sperm function. To this end we created transgenic mice lines eTg-G2 (γ2) and pTg-eG2 (γ2) expressing PPP1CC2 driven by either the endogenous promoter of Ppp1cc gene and by the testis specific Pgk2 promoter respectively. Based on in-silico and promoter assay studies a genomic fragment spanning 2.6 kb upstream of the transcription start site was identified as putative endogenous promoter and was thus subsequently used in these experiments. In addition, we also generated a third transgenic mouse line eTg-G1 (γ1) where PPP1CC1 transgene expression was driven by the same endogenous promoter fragment. Parallel to this, we used the Cre-Lox approach to generate germ cell conditional Ppp1cc gene knocdown mice to determine the somatic and germ cell requ (open full item for complete abstract)

Committee: Srinivasan Vijayaraghavan PhD (Committee Chair); Douglas W. Kline PhD (Committee Member); Yijing Chen PhD (Committee Member); Roger B. Gregory PhD (Committee Member); Soumitra Basu PhD (Other) Subjects: Biology; Biomedical Research; Cellular Biology; Developmental Biology; Molecular Biology

MS, Kent State University, 2010, College of Arts and Sciences / Department of Chemistry

Phosphatidyl inositides (PIs) are important regulators of cell signaling. Phosphoinositide 3-kinase (PI3-K)-activated signaling plays a key role in the development of cancer. Therefore various anticancer treatments target this pathway. The aim of this work was to develop an optimized method to separate fluorescent PIPs quickly and efficiently at room temperature using CE-LIF. The reason for the development of a method capable of separating the PIPs at room temperature was that, it could be used for single cell studies. In this regard, the effect of various cations on the separation of the PIs and their seven PIP derivatives was investigated. The effect of pH, temperature, voltage, as well as other buffer mixtures were also studied. Based on this developed method, the activity of PI3-K and PTEN enzymes in NIH 3T3 cells and MDA-MB 231 cells was also studied. The bioactivity of PIPs in these cells was investigated to ascertain whether preliminary CE studies would prove that they were able to be converted inside the cells into other phosphorylated derivatives. Lipid kinases and phosphatases play active roles in cell signaling. These have serious implications in many disease states. For example, accumulation of PIP3 leads to metastatic cancers. Therefore, inhibition studies were also performed to find out whether PI3-K inhibitors were able to block the conversion of PIP2 to PIP3. This is of particular interest in cell-based assays and research involving cancer drug development.

Committee: Simon M Mwongela PhD (Advisor); Songping Huang PhD (Committee Member); Bansidhar Datta PhD (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, Case Western Reserve University, 2006, Pathology

Activation of T cells through their T cell receptor (TCR) mediates the specificity of an immune response to antigens. The TCR signaling cascade involves the activation of protein tyrosine kinases with the resultant phosphorylation of cellular proteins, ultimately leading to new gene transcription, cytokine production and proliferation of T cells. Complex regulation of the TCR signaling cascade is not surprising since inappropriate cell activation could potentially lead to autoimmune diseases, inflammatory disorders and lymphoid malignancies. In an effort to understand the regulation of the TCR signaling cascade we studied the role of (1) TCR-zeta phosphorylation, (2) CD45 protein tyrosine phosphatase activity and the (3) balance between intracellular glutathione (GSH) and TCR-induced reactive oxygen species (ROS) in the tuning of the response of a T cell to activation. (1) TCR induced IL-2 production is dependent on the phosphorylation status of the æ chain of the TCR/CD3 complex induced by the actions of Src kinases upon TCR activation. In addition, TCR-zeta is constitutively phosphorylated in resting cells contributing to an inhibitory signaling environment. We demonstrate that phosphorylation of TCR-zeta is not entirely mediated by Src family kinases and propose the Tec family kinase, Itk, as a plausible candidate that mediates TCR-zeta phosphorylation in combination with Src kinases. (2) Varying the strength of TCR engagement by stimulation of T cells under different degrees of crosslinking lead to differences in the kinetics and duration of the TCR signaling cascade. We demonstrate that by varying the conditions of TCR engagement the activity of CD45 is affected, leading to Lck inhibition or activation, resulting in different TCR activation patterns. (3) TCR-induced production of H2O2 has been proposed to prolong the TCR signaling cascade since H2O2 reversibly inactivates PTPs. We extended this concept by proposing that elevated intracellular GSH should rapidly (open full item for complete abstract)

Committee: Alan Levine (Advisor) Subjects: