Pharmacology & Toxicology Theses and Dissertations (UMKC)
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The items in this collection are the theses and dissertations written by students of the Division of Pharmacology & Toxicology. Some items may be viewed only by members of the University of Missouri System and/or University of Missouri-Kansas City. Click on one of the browse buttons above for a complete listing of the works.
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Item Delivery of sting agonist using lipid nanoparticles and discovery of anti-TIGIT peptides for cancer therapy(2023) Shaji, Sherin George; Cheng, Kun (Professor); Yao, XiaolanOver the past decades, cancer treatment has significantly advanced with the approval of new therapeutic entities aimed at treating this disease. However, these advancements have not universally benefitted all patients or different cancers due to the complexity of the tumor microenvironment found in these malignancies. Therefore, it has become crucial to understand the molecular and cellular mechanisms that cancer cells use to evade the immune system of their hosts in order to discover effective therapeutic interventions. Tumors can be classified based on their immunogenicity into two types: cold and hot tumors. Cold tumors like pancreatic cancer, glioblastoma, prostate cancer, and ovarian cancer exhibit a high number of immune suppressive cells including regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSC), and cancer-associated fibroblasts (CAFs). Additionally, they often lack significant infiltration of immune cells like T cells. On the other hand, hot tumors contain T cells and other immune cells within the tumor microenvironment. However, despite the presence of these immune cells, cancer cells utilize various mechanisms to render the immune cells, especially T cells, dysfunctional, thereby evading the immune system. One significant mechanism involves immune checkpoints, where cancer cells manipulate the interaction between a receptor on T cells and a ligand on cancer cells. This manipulation often involves the cancer cells overexpressing their specific ligands, leading to a phenomenon where T cells become exhausted and unable to effectively eliminate tumor cells. Cold tumors can be treated by converting them into hot tumors and then removing the brakes by inhibiting the receptor-ligand interaction. This dissertation focuses on two main research objectives related to the interventions mentioned above. The first research aims to deliver a STING agonist into a cold tumor using lipid nanoparticles. This delivery is intended to provoke an immune response that leads to the elimination of tumor cells. STING pathway agonism has shown promise in innate immune signaling to tune the tumor microenvironment toward an immunogenic phenotype by promoting immune cell infiltration, especially CD8+ T cells, in various types of cancer. The second research involves the discovery of anti-TIGIT cyclic peptides using phage biopanning to block the interaction between the receptor TIGIT and its high-affinity ligand, CD155. In clinical studies, inhibiting this pathway within the adaptive immune system has demonstrated the ability to reverse the exhaustion of T- and NK cells, thereby restoring their functional ability to elicit cytotoxic activity against tumor cells. Chapter 1 introduces the dissertation research, presenting the statement of problems and outlining the research objectives. Chapter 2 provides an in-depth literature review on pancreatic cancer, the STING-2’3’-cGAMP pathway activation, and TIGIT-CD155 checkpoint inhibition for cancer immunotherapy. Chapter 3 involves the development and characterization of 2’3 cGAMP lipid nanoparticles (cGAMP-LNP) using various in-vitro and in-vivo techniques. These biodegradable cGAMP-LNP were prepared by encapsulating 2’3' -cGAMP within a lipid-based system containing an ionizable lipid, LHHK, and co-lipids. The cGAMP-LNP were found to have higher cellular uptake, improved IRF activation, and endosomolytic activity compared to free cGAMP in various cell-based assays. The cGAMP-LNP demonstrated significant inhibition of pancreatic cancer growth in a mouse model. Furthermore, these LNPs displayed a good safety profile in both in-vitro and in-vivo testing. These results suggest that cGAMP-LNP is a promising therapeutic entity for cold tumors like pancreatic cancer. In Chapter 4, the dissertation illustrates the discovery of anti-TIGIT cyclic peptides using the phage display technique. A unique cyclic peptide library developed in our laboratory was utilized to identify cyclic peptides using a solution-based biopanning procedure. These peptides were selected for their ability to bind to the TIGIT protein and block the TIGIT-CD155 interaction. The blocking efficiency was analyzed using an in-vitro protein-based assay, and two peptides, CSCP-7, and CSCP-16, showed the highest blocking of TIGIT-CD155 interaction. Moreover, alanine scanning revealed the amino acids responsible for the blocking activity of the CSCP-16 peptide.Item Identification and assessment of peptides and single domain antibodies for cancer therapy(2023) Kandel, Sashi; Cheng, Kun (Professor)The discovery of immune checkpoint inhibitors has revolutionized cancer therapy. These immune checkpoint inhibitors suppress tumor growth by immune checkpoint blockade. This approach involves blocking inhibitory immune checkpoint pathways that are believed to play a crucial role in cancer progression. Immune checkpoint blockade has shown remarkable and enduring clinical responses across a broad spectrum of cancers. The most studied immune checkpoints are CTLA4, PD1, TIM3, BTLA, and LAG-3. Our first research work is focused on discovering peptide drugs to block the interaction of immune checkpoint protein TIM3 with its ligand Gal9, i.e., the TIM3/Gal9 pathway. Interaction of TIM3 with Gal9 inhibits immune cell functions and aids in tumor cells evasion from the immune system. Therefore, the goal of the research project is to block the interaction of TIM3/Gal9 pathway which helps to enhance the immune response against cancers. The cyclic peptide phage library was screened against TIM3 using a novel biopanning procedure. The peptides discovered were screened for their binding affinity and blocking efficiency. Unfortunately, no peptides were found to effectively bind to and block TIM3, prompting a discussion about the challenges in peptide drug discovery against the TIM3/Gal9 pathway. Similarly, the second project also aims to block the other immune checkpoint pathway i.e BTLA/HVEM pathway. First, we worked to develop peptides and then switched to single domain antibody (also known as nanobody or VHH) inhibitors that could bind to BTLA protein, thus preventing the binding of HVEM to BTLA helping to restore the immune response against cancers. We applied a novel biopanning technique to screen a sdAb phage library against BTLA and identified five promising candidates. Among them, sdAb Nb-E1, with a KD of approximately 312 nM and an IC₅₀ of about 616 nM, displayed significant binding affinity and blocking efficiency. It also demonstrated anti-tumor activity in a preliminary study on C57BL/6 mice bearing colon cancer. Another attractive approach for eliminating cancer cells is targeted therapy. This approach involves treating cancer by specifically targeting receptors overexpressed by tumor cells or molecules that are unique to a specific cancer type. Targeted therapy can be used to disrupt cancer cell proliferation or metastasis, thus offering therapeutic advantages in the treatment of various types of cancers. As CD44 isoforms are widely expressed in a wide range of cancers, targeting CD44 for targeted cancer therapy stands out as a promising approach in the development of novel therapeutics. Therefore, our goal is to discover CD44 binding peptide so that it can be conjugated with other therapeutic agents for targeted therapy. CD44-targeting cyclic peptides were identified through biopanning. The obtained peptides were screened for their binding activity towards CD44. Some peptides showed binding affinity to CD44. Moving forward, additional experiments should be conducted to further confirm the binding affinity of these newly discovered peptides.Item Alcohol/HIV-Induced Neurodeficit and Circumvention by Neuroprotective Agent(2023) Schwartz, Daniel Christopher; Wang, JianpingAlcohol Use Disorder (AUD) remains a major problem in the United States, with usage varying between acute (binge) and chronic (heavy usage) staging. Alcohol Use Disorders affect 14.5 million people, with 9 million men and 5.5 million women. In the case of alcohol abuse, the effect of alcohol has been very well studied on the fetus but understanding of the chronic effect of alcohol abuse on neurotoxicity in the adult population, especially with comorbid conditions, such as Wernicke’s and Korsakoff’s Encephalopathy, remains more limited. Alcohol use and infection with the Human Immunodeficiency Virus-1 (HIV-1) in the United States is relatively common, with 30 to 60 % of these individuals having AUDs. These patients are exposed to different viral proteins that are known to be neurotoxic in the central nervous system. The combined effects of alcohol induced neurotoxicity with HIV-induced neurotoxicity are the interest of the current project. We have utilized the HIV-1 transactivator of transcription (Tat) protein as a model for HIV infection in an animal model. This is a widely accepted model that has been used to study aspects of HIV infection. Through use of this animal model, the work demonstrates that chronic exposure to alcohol and Tat creates a deficit in neurocognitive function with concomitant changes in receptors, cytokines and other inflammatory substances and molecules. This is important to keep in mind about changes in receptors and cytokines because the levels and changes can lead to the perpetuation of that neurodeficit. In this model, animals were treated with alcohol for 12 weeks. Tat is introduced by use of a transgenic animal line that has been treated the same way as control animals. The animals were subjected to a behavioral battery that tested different types of memory, anxiety, and motor function. We have found that treated animals exhibited additive, meaning the effects of the substances are combined, synergistic, meaning the substances work in concert to cause drug effect, or antagonistic, meaning the substances work against each other to cause no drug effects, which ultimately created an increased neurodeficit. Importantly, this work has also identified sex differences that are evident with treatment by HIV-1 Tat and alcohol, and these may be clinically relevant for treatment of patients with HIV-1 infection combined with alcohol abuse. Understanding sex difference or how drugs and other xenobiotics affect the individual is important because of development of different treatment strategies. This work evaluated the use of peroxisome proliferator activated receptor (PPAR) agonist on its ability to help alleviate the neurodeficit caused by Tat. The animals completed the described behavior battery above allowed us to identify the drug effects and potential changes in sex difference. In Chapter 1, we introduce the pharmacology and toxicology of alcohol, exposure plans and types of toxicity studies, HIV-1 Tat and HIV Associated Neurocognitive Disorders, and PPAR γ agonist pharmacology and toxicology. We also provide a detailed effort to link all of these topics together to further the study described below. In Chapter 2, we propose and carry out a two-pronged study to demonstrate prolonged exposure to alcohol in a controlled setting. We also examine synaptic protein analysis to ascertain the neurodeficit incurred from prolonged exposure to alcohol. In Chapter 3, we propose and carry out a three-pronged study to investigate prolonged exposure to alcohol, as well as to HIV-1 Tat. We also investigate how the resulting neurodeficit changes cytokine and receptors, and at how drug effect influences these changes. In Chapter 4, we propose and carry out a three-pronged study to demonstrate the neuroprotective properties of PPAR γ agonist, Rosiglitazone. We investigate the efficacy of this treatment using an animal study, and further investigate the effects on receptors and cytokines. We also evaluate the type of drug effect rosiglitazone exerts in the system. In Chapter 5, we conclude by giving an overview of our results. We also provide some commentary on the use of personalized, precision medicine approach to help with treatment of HIV-1 and alcohol abuse in populations. Further studies using animal models and three different clinical trial options for expand the goals of this work that are ultimately proposed.Item A Method for Identification of Pancreatic Cancer through Methylation Signatures in Cell-Free DNA(2022) Miller, Neil Andrew; Wyckoff, Gerald J.Pancreatic cancer has high mortality rates in comparison to other cancers due to limited treatment options and challenges in detecting the disease. Early diagnosis is difficult; successful outcomes are directly tied to detection of the cancer before it can spread throughout the body. Evaluation of circulating cell-free DNA (cfDNA), specifically, detection of circulating tumor DNA (ctDNA), is being explored as an approach for non-invasive ‘liquid biopsy’ that can be deployed widely and cost-effectively to screen for early signs of disease. DNA methylation signatures found in cfDNA can serve as a biomarker for detection of cancer. Previous efforts to detect pancreatic cancer using cfDNA showed limited sensitivity for detection. This manuscript describes work to develop a method for early detection of pancreatic cancer in circulating cell-free DNA using publicly available data from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). The work includes the development of methods for simulation of samples and cell-free DNA data at multiple ctDNA concentrations as well as a pilot implementation and evaluation of a machine learning model for automated classification of cfDNA samples. Chapter 1 introduces background of pancreatic cancer, cfDNA, liquid biopsy, DNA methylation and DNA methylation in cancer as well as a review of previous efforts to develop early detection applications. Chapter 2 describes the identification of DNA methylation markers that distinguish pancreatic tumor from normal pancreas and blood using publicly available data from the TCGA and GEO. In addition, the chapter outlines the development of a machine learning model to classify samples as tumor or normal based on these markers. Chapter 3 gives an overview of the challenges of genetic data simulation and the development of a novel tool, Heisenberg, for simulating DNA methylation data and cfDNA methylation data. This chapter also illustrate the use of Heisenberg to simulate normal blood samples and pancreatic cancer cfDNA samples. Chapter 4 describes the development of a neural network classification model for detection of pancreatic tumor in different concentrations of cfDNA using simulated samples. The chapter also reports the detection performance of the model using different model training strategies.Item Amphetamine-Induced Toxicity in Astrocytes and its Implications in the Pathogenesis of Psychiatric Disorders(2022) Chaudhari, Nitish; Wang, JianpingAmphetamine-type stimulants (ATS), such as amphetamine (AMPH) and methamphetamine (METH), cause long-term toxic effects on the dopaminergic neurons in the brain leading to the development of several psychiatric disorders. The mechanisms underlying ATS neurotoxicity are unclear, but oxidative stress has been implicated. Most studies on ATS have focused on neurons. However, in recent years, the role of glial cells, particularly astrocytes, in ATS pathogenesis has received considerable focus. Yet, little work has been done to study gene expression changes in ATS-treated astrocytes as a part of the ATS neurotoxicity cascade. Therefore, these studies aimed to examine the contribution of gene expression in astrocytes to ATS toxicity and to identify novel gene targets for treating ATS-induced psychiatric disorders. In the first chapter, we studied the effects of repeated METH exposure on astrocyte transcriptome using microarray. METH induced differential expression of 1,819 genes in astrocytes. Furthermore, METH significantly dysregulated biological pathways, molecular functions, cellular components, and transcriptional regulators involved in the cell cycle, DNA replication and repair, apoptosis, and phagocytosis. These results suggested that METH-induced gene expression changes play a critical role in METH-induced cell cycle arrest, defective DNA repair, and increased apoptosis. In the second chapter, we investigated the effect of METH treatment on the expression of integrins in astrocytes and the involvement of integrins in METH-induced apoptosis. We found that METH significantly decreased the expression of 10 integrin subunits expressed in astrocytes and that integrin subunit β1 is involved in METH-induced apoptosis in astrocytes. In the third chapter, we utilized a population-level study to determine the prevalence of psychiatric disorders in METH-abusing individuals using the Cerner Health Facts database. Our results showed a significantly higher prevalence of anxiety, depression, bipolar disorder, psychosis, and schizophrenia among METH-abusing individuals compared to the general population. We then identified common gene expression signatures by comparing gene expression changes from our microarray data and independent microarray datasets from patients with anxiety, depression, and schizophrenia retrieved from the gene expression omnibus (GEO) database. In conclusion, we demonstrated that ATS-induced gene expression alterations are involved in developing neurotoxicity and psychiatric disorders in ATS-abusing users.