2018 UMKC Dissertations - Freely Available Online
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Item The Effect of Participatory Budgeting on the Provisioning Process(University of Missouri -- Kansas City, 2018) Bateman, George Robert Jr; Forstater, Mathew, 1961-The hypothesis of this dissertation is that as more of us become actively engaged in deliberations concerning local economic/political issues, the happier, more tolerant, and more understanding we can become. This is a philosophical dissertation because PB’s potential benefits are researched by examining the works of relevant American social philosophers who wrote about these benefits. The research uses scientific methodology to examine social policy, as advocated by John Dewey. Foster’s theory of institutional adjustment explains what is needed for PB to realize its potential benefits. Dewey’s insight on internal deliberation explains how individuals can change to think of themselves as part of the community, to meet one of Foster’s three principles of change. The Iroquois Confederation is an example of an egalitarian democratic society with an oral constitution. The earliest settlers in New England are reviewed to learn about their Congregationalist policy, which empowered each church congregation to govern themselves. Next, Thomas Jefferson’s motivation for his ward system proposal seems to have been directed toward increasing public happiness though public participation. Then, three transcendentalist writers are analyzed. Ralph Waldo Emerson, Henry David Thoreau, and Walt Whitman advocated the power of self-reflection to teach people that they have unlimited potential, the importance of using one’s voice and the importance of equality. Next is consideration of the pragmatic political thought of John Dewey and of C. Wright Mills. Dewey believed that improving the methods of public communication was a key to improving democracy, which should also help people become more tolerant. Mills, like Dewey, believed that small publics could help individuals as well as the community. The vision of participation of the Port Huron Statement inspired social movements in the 1960s. Alfred Schutz studied how people can come to understand each other through face-to-face communication. And finally, Robert Putnam’s social capital is explored to learn why people get more done together than separately. Finally, two suggestions are made for PB in NYC, to test the hypothesis of this dissertation: First, improving public deliberation within PB, and second, suggesting PB reach out to additional social justice organizations. This could help PB grow, which should help the participants as well as our political system.Item Development of Targeted siRNA Therapeutics for Triple Negative Breast Cancer and Liver Fibrosis(University of Missouri -- Kansas City, 2018) Zhao, Zhen; Cheng, Kun (Professor)The objective of this dissertation is to develop various systems to deliver small interfering RNA (siRNA) for the treatment of triple negative breast cancer (TNBC) and liver fibrosis. siRNAs targeting vascular endothelial growth factor (VEGF) and IκB kinase ε (IKBKE) were used for treating TNBC, while siRNA targeting poly(rC) binding protein 2 (PCBP2) was used for treating liver fibrosis. In Chapter 1, we briefly introduced the background about TNBC and liver fibrosis. We also presented the Statement of the Problems and Objectives. In Chapter 2, we reviewed the molecular mechanisms and potential treatments for TNBC and liver fibrosis. In Chapter 3, we developed a poly(ethyleneimine) (PEI) conjugated biodegradable multiblock polymer to form nanocomplexes with VEGF siRNA for TNBC treatment. The nanocomplex is able to deliver the VEGF siRNA into TNBC cells with a high transfection efficiency and low cytotoxicity. In vitro activity studies showed that the siRNA nanocomplexes significantly inhibit migration and invasion of TNBC cells. More importantly, the VEGF siRNA nanocomplex efficiently inhibit tumor growth in vivo and successfully downregulate VEGF expression in the tumor. These results suggested that VEGF siRNA is a promising anti-tumor agent for TNBC therapy, and the PEI 1800 conjugated bio-degradable multiblock polymer is a promising system to deliver siRNAs to TNBC cells. In Chapter 4, we developed a CD44-targeting, cholesterol modified cationic peptide nanocomplex to co-deliver IKBKE siRNA and cabazitaxel (CTX) for TNBC therapy. IKBKE siRNA significantly inhibits the proliferation, migration, and invasion of TNBC cells but has no apoptosis-inducing effect. In vivo studies also indicated that IKBKE siRNA can inhibit TNBC tumor growth. CD44-targeting CHA/CP/siRNA/CTX nanocomplex showed the synergistic effect of IKBKE siRNA and cabazitaxel on the inhibition of invasiveness and growth of TNBC tumors with an enhanced CD44 specific targeting effect. CHA/CP/siRNA/CTX nanocomplexes also exhibited a significant antitumor effect through IKBKE siRNA and cabazitaxel in vivo. Thus, IKBKE siRNA may be a promising anti-tumor agent for TNBC therapy, and co-delivery of IKBKE siRNA and cabazitaxel through a CD44-targeting nanocomplex is a potential strategy for TNBC treatment. Moreover, this multifunctional delivery system can also provide a good option for combined gene therapy and chemotherapy. In Chapter 5, we prepared three neutravidin-based siRNA nanocomplexes with different targeting ligands to deliver PCBP2 siRNA to hepatic stellate cells (HSCs). Insulin-like growth factor 2 receptor (IGF2R) is overexpressed in activated HSCs and therefore can be utilized for HSC-specific drug delivery. Compared to vitamin A and cholesterol, the IGF2R-specific peptide exhibited the highest targeting effect to human LX-2, rat HSC-T6 cell line, and activated primary rat HSCs. Accordingly, the IGF2Rspecific peptide coupled nanocomplex demonstrated higher silencing activity of PCBP2 and better inhibition on the migration of activated HSCs. The IGF2R-specific peptide coupled nanocomplex also showed the highest uptake in the liver and lowest uptake in the lung and kidney of the rats with CCl4-induced liver fibrosis.Item Evaluation of the Anti-Fibrotic Activity of PCBP2 siRNA in Primary Hepatic Stellate Cells and Discovery of Anti-PD-L1 Peptide and Nanobody for Immunotherapy(University of Missouri -- Kansas City, 2018) Liu, Hao; Cheng, Kun (Professor)There are two major research objectives in this dissertation. Our laboratory recently discovered a small interfering RNA (siRNA) that silences the poly (rC) binding protein 2 (PCBP2) gene. It is hypothesized that silencing of the PCBP2 gene in hepatic stellate cells (HSCs) leads to the reversal of the accumulated type I collagen in fibrotic liver. The first research objective is therefore to evaluate whether the PCBP2 siRNA can reverse the alcohol- and cytokine-induced profibrogenic effects on HSCs. The second research objective is to discover programmed death ligand 1 (PD-L1) specific peptides and nanobodies using phage display. These PD-1/PD-L1 checkpoint inhibitors can be potentially used as immunotherapy agents for cancer. Chapter 1 introduces the background of the dissertation research and presented the Statement of the Problems and Objectives. Chapter 2 reviews the mechanisms of liver fibrogenesis and cancer immunotherapy using checkpoint inhibitors. Chapter 3 details our investigation of the anti-fibrotic effect of the PCBP2 siRNA in rat primary HSCs and HSC-T6 cells. The α-complex protein-2 (αCP2), encoded by the PCBP2 gene, is responsible for the accumulation of type I collagen in fibrotic liver. In this dissertation, we aimed to silence the PCBP2 gene using a siRNA to reverse the alcohol- and cytokines-induced pro-fibrogenic effects on HSCs. Primary rat HSCs and the HSC-T6 cell line was used as fibrogenic models to mimic the initiation and perpetuation stages of fibrogenesis, respectively. Our laboratory recently discovered a PCBP2 siRNA, which can efficiently silence the expression of αCP2 and reduce the stability of type I collagen mRNA in HSC-T6 cells. In this dissertation, we investigated the effects of PCBP2 siRNA on cell proliferation and migration. Expression of type I collagen in primary HSCs was analyzed using quantitative real-time PCR and western blot. In addition, we evaluated the effects of PCBP2 siRNA on apoptosis of the HSCs. Our results showed that PCBP2 siRNA reversed the alcohol- and cytokine-induced multiple pro-fibrogenic effects on primary rat HSC and HSC-T6 cells. The PCBP2 siRNA also reversed the alcohol- and cytokine-induced accumulation of type I collagen as well as cell proliferation and migration. Moreover, the combination of LY2109761, a TGF-β1 inhibitor, and PCBP2 siRNA exhibited a synergistic inhibition effect on the accumulation of type I collagen in HSCs. We therefore concluded that silencing of PCBP2 using siRNA could be a potentially therapeutic strategy for alcoholic liver fibrosis. Chapter 4 discusses our discovery of several anti-PD-L1 peptides using a phage display peptide library. PD-L1 is overexpressed on a variety of cancer cells, and programmed cell death protein 1 (PD-1) is expressed on T cells. The interaction between PD-L1 and PD-1 negatively regulates the immune responses of T cells, leading to escape of cancer cells from the attack of T cells. Blocking the PD-L1/PD-1 interaction is therefore a promising strategy to treat cancers by restoring the immune activity of T cells. The FDA has approved several monoclonal antibodies targeting PD-1 or PD-L1 for various cancer immunotherapies. However, large size (150 kDa) of antibodies limits their tumor penetration, especially in solid tumors. There has been a growing interest in developing low-molecular weight checkpoint inhibitors, such as peptides, in the past few years. Using a novel phage biopanning, we discovered several PD-L1-specific peptide antagonists to block the PD-1/PD-L1 interaction. The peptide candidate CLP002 exhibited the highest binding affinity to PD-L1 with an equilibrium dissociation constant (KD) of 366 nM. The apparent KD values of CLP002 to PD-L1-positive human cancer cell lines MDA-MB231 and DU145 are 212.9 nM and 184.1 nM, respectively. CLP002 efficiently blocked 85% of the PD-1/PD-L1 interactions with an IC₅₀ of 2.17 µM. We verified that CLP002 restored T cell proliferation and prevented T cell apoptosis in vitro, when T cells were co-cultured with cancer cells. More importantly, we demonstrated that CLP002 is highly specific for PD-L1, thus minimizing its potential off-target effects in the body. According to the results of the in vivo anti-tumor activity study, the CLP002 peptide inhibited tumor growth and increased the survival of the CT26 tumor-bearing mice, suggesting the CLP002 peptide represents a promising drug candidate for cancer immunotherapy. Chapter 5 describes our work towards discovering anti-PD-L1 nanobodies for cancer immunotherapy using a phage display of single-domain antibody (sdAb) library. Nanobody (also named sdAb or VHH) is an antibody fragment composed of a single variable domain of the heavy chain on heavy chain only antibody. Nanobody is the smallest antibody fragment (14 kDa) that maintains the similar antigen-binding affinity. We discovered seven anti-PD-L1 nanobodies that blocked the PD-1/PD-L1 interaction. Among them, the nanobody CLV3 showed the highest binding affinity to PD-L1 with a KD value of 12.37 nM. The nanobody CLV3 also exhibited the highest blockade of the PD-1/PD-L1 interaction with an IC50 of 32.3 nM. CLV3 inhibited tumor cell proliferation by binding to PD-L1 on the cancer cell surface and blocking the PD-1/PD-L1 interaction between cancer cells and T cells. Antitumor activity of the nanobody CLV3 was evaluated in a CT26 xenograft mouse model. The nanobody CLV3 significantly inhibited tumor growth and increased the survival of the tumor-bearing mice. The nanobody CLV3 is therefore a promising PD-L1 inhibitor that can be used for cancer immunotherapy.Item Establishing Metronidazole as a Novel Biomarker of CYP2A6 Activity(University of Missouri -- Kansas City, 2018) Stancil, Stephani Lili Almai; Melchert, Russell B.CYP2A6 is a polymorphically expressed enzyme with variation associated with smoking behavior, cessation success, lung cancer risk, and differential drug exposure to numerous medications including anti-infectious and chemotherapeutic agents. Current CYP2A6 probes that could provide insight a priori into variability are limited due to safety, accessibility, general applicability and/or enzyme specificity. Therefore, the purpose of this research was to develop a novel biomarker to understand variability in CYP2A6 activity in humans. A step-wise approach was utilized and included evaluation of the ability of metronidazole and nicotine (current gold standard) to modulate CYP2A6 activity and expressionin vitro, validation of a novel analytical method to determine the concentrations of metronidazole and 2-hydroxymetronidazole in human plasma, and finally, comparison of metronidazole to nicotine (via metabolite/parent ratio) for use as a CYP2A6 phenotyping probe in humans. Metronidazole and nicotine had minimal effects on CYP2A6 expression or activity in vitro at therapeutically relevant concentrations and are predicted to not lead to meaningful clinical impact. Using a low volume of human plasma (10 µL), metronidazole and 2-hydroxymetronidazole were simultaneously quantitated by a novel, validated UPLCMS/MS method that is among the most sensitive to date. The metronidazole probe measure (2-hydroxymetronidazole/metronidazole ratio in plasma) proved well-tolerated, highly specific for CYP2A6, and robust with a wide window of use. This novel probe measure was also able to dichotomize individuals based on genotype-predicted phenotype in a way that mirrored the nicotine probe measures. In summary, this work establishes the metronidazole probe measure as a novel biomarker of CYP2A6 variability in humans, thus providing a tool to understand human diversity and potentially, improve health outcomes in a diverse pool of individuals. Future studies evaluating the use of metronidazole as a probe of CYP2A6 activity in sub-populations of humans would be useful to further investigate the performance of this tool in special populations at risk for poor health outcomes.Item Role of Redox-Active Trace Metal (RATM) Oxidants in the Regulation of Toll-Like Receptor 4 (TLR4) Signaling-Mediated Inflammatory Phenotype in Synovial Fibroblasts(University of Missouri -- Kansas City, 2018) Alsousi, Asmaa; Igwe, Orisa JohnWhile the involvement of oxidant stress in rheumatoid arthritis (RA) has been suspected, the role of redox-active trace metals (RATM) as exogenous pro-oxidants in the pathogenesis of RA has not yet been investigated. Evidence suggests that oxidant-induced Toll-like receptor 4 (TLR4) activation plays a significant role in initiating “sterile” inflammation. Here, we investigate for the first time the role of RATM-induced oxidant stress in the molecular mechanism of the pathogenesis of RA. Potassium peroxychromate (PPC) (Cr⁺⁵), cuprous chloride (Cu⁺), and ferrous chloride (Fe⁺²) RATM agents were used as exogenous sources of reactive species. LPS-EK as a TLR4 specific agonist was used as a positive control for TLR4 activation. Given the importance of synovial fibroblasts in the development of RA, HIG-82, a rabbit fibroblast like-synoviocytes (FLS) cell line, was used as a model system in the studies proposed in this dissertation research. The expression of TLR4 in HIG-82 was confirmed by quantitative PCR (RT-PCR) and Western blots. Intracellular reactive oxygen species (iROS) production was visualized and quantified by fluorescence imaging microscopy and flow cytometry (FC), respectively. Activation of TLR4 signaling pathway was determined by measuring the expression of TLR4 and the downstream signaling proteins. Either ELISA kits or FC quantified levels of TNF-α, interleukin (IL-1β), and HMGB1 (as pro-inflammatory agents), and IL-10 (as an anti-inflammatory mediator) released into the culture medium. Proliferation index of FLS and examination of the effects of RATM on apoptosis and autophagy-related protein levels were quantified by FC and Western blots. We found that (1) RATM induced iROS production, which was attenuated by pretreatment.with antioxidants (2) Similar to TLR4 specific agonist LPS-EK, RATM significantly increased the activity of TLR4, which was blocked by pretreatment with TLR4 signaling inhibitor (CLI 095). (3) To our surprise, RATM increased proliferation of FLS and protected cells against apoptosis through activation of autophagy which is in agreement with the pathophysiological changes that occur in active RA. (4) RATM exogenous RS activate TLR4-mediated different down-stream signaling cascades that lead to an increased production of pro- and anti inflammatory mediators in FLS, and (5) Further studies reveal that RATM exogenous RS treatment increased the expression of all three major MAPK; (Extracellular signal-regulated protein kinase (ERK), the c-Jun N-terminal Kinase (JNK), and P38 MAPK pathways). Moreover, RATM concurrently increased the expression of AP-1 nuclear protein through TLR4 stimulation. Taken together, our findings indicate that TLR4 has mediated RATM induced inflammatory phenotype through AP-1 pathway activation in synovial fibroblasts. Therefore, oxidant stress through TLR4 activation may initiate and propagate inflammatory processes that maintain many chronic diseases. The design of dual-functioning antioxidants possessing both metal chelating and oxidative stress scavenging properties will be an essential milestone in pharmacotherapy and could help us live free of many chronic diseases. For the first time, we present evidence that supports a connection between exogenous and endogenous reactive species in enhancing inflammatory phenotype in synovial fibroblasts which is likely responsible for the initiation, propagation, and maintenance of RA.