Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2017, College of Sciences and Health Professions

The area of biosample analysis encompasses a very broad range of assays which support the clinical and nonclinical studies. Biosample analysis is used to provide a quantitative or qualitative measure of the active drug and/or its metabolite(s) in the biological matrix for the purpose of pharmacokinetics, toxicokinetics, bioequivalence, and exposure-response (pharmacokinetics /pharmacodynamics) studies. Due to the significance of pharmacological analysis, sensitive, reproducible and robust analytical methods are critically needed for pharmacological studies of the biosamples. A bioanalytical method mainly contains two components I) Sample preparation II) detection of the compound. Therefore, the main aims of this thesis are the development of quantitative and qualitative analytical methods for the target compounds using LC-MS(/MS) and development of accelerated sample preparation for high throughput sample analysis for DNA and proteins. In this dissertation, a brief review on the method rationale, workflow of the method development, sample preparation methods, instrumentations and analytical method validation, are discussed in Chapter 1. Also, research projects were discussed and the techniques used in the experiments for this thesis were reviewed. As so, chapter II and III were mainly focused on the accelerated sample preparation methods for the high throughput sample analysis of DNA and proteins respectively, where the sample preparation time was significantly reduced from hours to minutes, which are suitable for qualitative and quantitative analysis of DNA and proteins. In Chapter IV, a systematic study on the structural characterization of the model glycoprotein Human IgG was described. In chapter V successful development of LC-MS method was developed for the determination of Oxygen -18 isotope enrichment in the phosphate samples in the positional isotope exchange reactions to study the reversibility of certain enzymatic reactions was described. Success (open full item for complete abstract)

Committee: Yan Xu Ph.D. (Committee Chair); David Anderson Ph.D. (Committee Member); Bin Su Ph.D. (Committee Member); Aimin Zhou Ph.D. (Committee Member); Petru Fodor Ph.D. (Committee Member) Subjects: Chemistry

Master of Science in Biomedical Engineering, Cleveland State University, 2016, Washkewicz College of Engineering

Cancers in which epigenetic changes, such as hypermethylation of DNA, lead to drug resistance cause the cancer to become unresponsive to existing chemotherapeutic treatments. The epigenetic drug – 5-aza-2'-deoxycytidine (decitabine, DAC) – is a potent hypomethylating agent, but its effect is transient due to its instability. Previous studies have shown that loading DAC into nanogel significantly enhances its antiproliferative effect (compared to DAC in solution) in drug-resistant breast cancer cells (MCF-7/ADR). Further, the previous studies demonstrated changes in the membrane lipid profile of resistant cells following treatment with DAC either as solution or in nanogels. The objective of the present study was to compare the stability of DAC as solution and DAC encapsulated in nanogel, determine the effect of duration of DAC and DAC-nanogel pretreatment on the antiproliferative activity of subsequent chemotherapeutic agent addition, and to visualize and quantify the effect of DAC and DAC-nanogel pretreatment on uptake of poly dl-lactide co-glycolide (PLGA)-based nanoparticle in MCF-7/ADR cells. An increase in the stability of DAC when encapsulated in nanogel could be a mechanism contributing to the sustained effect of DAC. DAC-nanogel's sustained effect and its effectiveness at altering the membrane lipid profile to reduce resistance could cause a longer DAC-nanogel pretreatment time to increase the antiproliferative effect of subsequent chemotherapeutic agent addition. Additionally, the membrane lipid profile altering effects of DAC and DAC-nanogel could cause DAC and DAC-nanogel pretreatment to increase uptake of nanoparticles in MCF-7/ADR cells. The stability of DAC was assessed in mouse plasma at physiological conditions using mass spectrometry. DAC in solution was found to be less stable than DAC in nanogel. The effect of durations of 3-days and 5-days of DAC-nanogel treatments on the subsequent efficacy of chemotherapeutic agent, paclitaxel was assessed in MC (open full item for complete abstract)

Committee: Vinod Labhasetwar PhD (Advisor); Joanne Belovich PhD (Committee Member); Chandra Kothapalli PhD (Committee Member) Subjects: Biomedical Engineering; Biomedical Research; Oncology

Doctor of Philosophy, The Ohio State University, 2014, Molecular, Cellular and Developmental Biology

Acute myeloid leukemia (AML) is a biologically complex neoplastic disease of the hematopoietic system, characterized by an uncontrolled proliferation of malignant myeloid precursors leading to bone marrow failure at the clinical level. Today, the majority of AML patients fail to achieve long-term survival. Thus, new therapeutic approaches are needed. MicroRNAs (miRs), short noncoding RNAs that regulate the expression of their target mRNA-encoded proteins, are involved in tumorigenesis. We demonstrated that deregulated miR-29b and miR-181a in AML patients were associated with worse outcome. Moreover, AML patients with a higher pre-treatment level of miR-29b respond better to a hypomethylating agent, decitabine; and patients with higher miR-181a have longer survival under cytarabine/daunorubicine-based chemotherapy. Thus, increasing the levels of these miRs prior to the respective treatments may be beneficial. However, free synthetic miRs are easily degraded in the bio-fluid and have limited cellular uptake. To overcome this problem and explore miR-based therapy, our research focused on three major aims: (1) to develop a novel nanocarrier suitable for delivering miRs into AML cells; (2) to deliver miR-29b and assess the antileukemic activity; and (3) to investigate the role of miR-181a in AML, unravel the mechanism, and perform therapeutic evaluation via nanocarrier-delivered miR-181a. For aim 1, since AML cells overexpress transferrin receptor on their surface, we formulated novel transferrin (Tf) targeted anionic lipid based nanoparticles (NP) encapsulating miR mimic and demonstrated low toxicity and high efficiency. For aim 2, following miR-29b-nanoparticle treatment, we showed a significant increase in intracellular miR-29b levels and downregulation of its known targets. This resulted in decreased leukemia growth and improved survival in an AML mouse model. Furthermore, we showed that pretreatment with miR-29b nanoparticles improved the antileukemic activity of de (open full item for complete abstract)

Committee: L. James Lee (Advisor); Guido Marcucci (Advisor); Robert Lee (Committee Member); Natarajan Muthusamy (Committee Member) Subjects: Biomedical Engineering; Cellular Biology; Molecular Biology

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

Leukemia is the most common blood cancer and is characterized by the increased expansion of abnormal blood cells. Important insights into the pathogenesis of this disease have led to the development of a number of anti-leukemia drugs including nucleoside analogues, such as 5-azacytidine (5-AzaC), decitabine and aracytidine, and ribonucleotide reductase inhibitor such as GTI-2040. 5-Azacytidine and decitabine are hypomethylating agents that induce DNA demethylation, resulting in reactivation of hypermethylation-associated silencing of tumor suppressor genes. GTI-2040 is a 20-mer oligonucleotide inhibiting the expression of ribonucleotide reductase subunit 2 mRNA, an enzyme that has been found to be over-expressed in most cancers. Aracytidine is widely used as important cytostatic drug in the treatment for acute myelogenous leukemia. In order to support the mechanistic studies and potential combination treatment of these anti-cancer drugs, a non-radioactive, sensitive and specific LC-MS/MS method has been developed to quantify intracellular NTP and dNTP pools in cell matrices. A significant decrease in dCTP and dATP levels has been observed following GTI-2040 treatment in human leukemia MV411 cells. More importantly, GTI-2040 was found to down-regulate R2 mRNA and protein levels in a dose dependent manner. In order to evaluate the combination treatment effect of GTI-2040 and aracytidine, a sensitive HPLC method has been developed to determine the intracellular aracytidine triphosphate (Ara-CTP) level. A significant increase in intracellular Ara-CTP level has been observed after pretreatment of GTI-2040 in vitro. Further pharmacokinetics / pharmacodynamics (PK/PD) modeling and simulation of GTI-2040 and aracytidine in the cell exhibited the increase of intracellular Ara-CTP level by >2 fold. In order to characterize pharmacokinetic profile of 5-azacytidine in patients with hematologic malignancies, a simple, non-radioactive, sensitive and specific high-performance HPLC (open full item for complete abstract)

Committee: Kenneth Chan PhD (Advisor); Guido Marcucci PhD (Committee Member); Duxin Sun PhD (Committee Member); Robert Snapka PhD (Committee Member) Subjects: Biomedical Research; Chemistry; Oncology; Pharmaceuticals; Pharmacology