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- All Subjects: Cancer
- All Subjects: Microbiology
- Creators: Buetow, Kenneth
- Member of: Barrett, The Honors College Thesis/Creative Project Collection
- Resource Type: Text
Description
Background: Coccidioidomycosis (Valley Fever) is a respiratory disease that is caused by the soil-dwelling fungi Coccidioides immitis and Coccidioides posadasii. Because fungal glycosylation patterns are distinct from mammalian glycosylation patterns, we hypothesized that certain lectins (carbohydrate-binding proteins) might have differential binding properties to coccidioidal glycoproteins, and therefore serve as a tool for the purification and characterization of these glycoproteins from patient specimens. Materials and Methods: To identify potential Coccidioides-binding lectins, lectin-based immunohistochemistry was performed using a panel of 21 lectins on lung tissue from human patients infected with Coccidioides. Enzyme-Linked Immunosorbent Assays (ELISAs) were used to confirm and test candidate Coccidioides-binding lectins for their ability to bind to proteins from antigen preparations of laboratory-grown Coccidioides. Inhibition IHC and ELISAs were used to confirm binding properties of these lectins. SDS-PAGE and mass spectrometry were performed on eluates from coccidioidal antigen preparations run through lectin-affinity chromatography columns to characterize and identify lectin-binding coccidioidal glycoproteins. Results: Two GlcNAc-binding lectins, GSLII and sWGA, bound specifically to spherules and endospores in infected human lung tissue, and not to adjacent lung tissue. The binding of these lectins to both Coccidioides proteins in lung tissue and to coccidioidal antigen preparations was confirmed to have lectin-like characteristics. SDS-PAGE analysis of eluates from lectin-affinity chromatography demonstrated that GSLII and sWGA bind to coccidioidal glycoproteins. Mass spectrometric identification of the top ten lectin affinity-purified glycoproteins demonstrated that GSLII and sWGA share affinity to a common set of coccidioidal glycoproteins. Conclusion: This is the first report of lectins that bind specifically to Coccidioides spherules and endospores in infected humans. These lectins may have the potential to serve as tools for a better method of detection and diagnosis of Valley Fever.
ContributorsChowdhury, Yasmynn (Author) / Lake, Douglas (Thesis director) / Grys, Thomas (Committee member) / Magee, Mitchell (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of Human Evolution and Social Change (Contributor)
Created2015-05
Description
The long-term survival of patients with glioblastoma multiforme is compromised by the tumor's proclivity for local invasion into the surrounding normal brain. These invasive cells escape surgery and display resistance to chemotherapeutic- and radiation-induced apoptosis. We have previously shown that tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, can stimulate glioma cell invasion and survival via binding to the fibroblast growth factor-inducible 14 (Fn14) receptor and subsequent activation of the Rac1/NF-kappaB pathway. In addition, we have reported previously that Fn14 is expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Here we demonstrate that TWEAK can act as a chemotactic factor for glioma cells, a potential process to drive cell invasion into the surrounding brain tissue. Specifically, we detected a chemotactic migration of glioma cells to the concentration gradient of TWEAK. Since Src family kinases (SFK) have been implicated in chemotaxis, we next determined whether TWEAK:Fn14 engagement activated these cytoplasmic tyrosine kinases. Our data shows that TWEAK stimulation of glioma cells results in a rapid phosphorylation of the SFK member Lyn as determined by multiplex Luminex assay and verified by immunoprecipitation. Immunodepletion of Lyn by siRNA oligonucleotides suppressed the chemoattractive effect of TWEAK on glioma cells. We hypothesize that TWEAK secretion by cells present in the glioma microenvironment induce invasion of glioma cells into the brain parenchyma. Understanding the function and signaling of the TWEAK-Fn14 ligand-receptor system may lead to development of novel therapies to therapeutically target invasive glioma cells.
ContributorsJameson, Nathan Meade (Author) / Anderson, Karen (Thesis director) / Lake, Douglas (Committee member) / Tran, Nhan (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description
Cancer is one of the leading causes of death in the world and represents a tremendous burden on patients, families and societies. S. Typhimurium strains are specifically attracted to compounds produced by cancer cells and could overcome the traditional therapeutic barrier. However, a major problem with using live attenuated Salmonella as anti-cancer agents is their toxicity at the dose required for therapeutic efficacy, but reducing the dose results in diminished efficacy. In this project, we explored novel means to reduce the toxicity of the recombinant attenuated Salmonella by genetically engineering those virulence factors to facilitate maximal colonization of tumor tissues and reduced fitness in normal tissues. We have constructed two sets of Salmonella strains. In the first set, each targeted gene was knocked out by deletion of the gene. In the second set, the predicted promoter region of each gene was replaced with a rhamnose-regulated promoter, which will cease the synthesis of these genes in vivo, a rhamnose-free environment.
ContributorsBenson, Lee Samuel (Author) / Kong, Wei (Thesis director) / Martin, Thomas (Committee member) / Lake, Douglas (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor) / Center for Infectious Diseases and Vaccinology (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description
Pathway analysis helps researchers gain insight into the biology behind gene expression-based data. By applying this data to known biological pathways, we can learn about mutations or other changes in cellular function, such as those seen in cancer. There are many tools that can be used to analyze pathways; however, it can be difficult to find and learn about the which tool is optimal for use in a certain experiment. This thesis aims to comprehensively review four tools, Cytoscape, PaxtoolsR, PathOlogist, and Reactome, and their role in pathway analysis. This is done by applying a known microarray data set to each tool and testing their different functions. The functions of these programs will then be analyzed to determine their roles in learning about biology and assisting new researchers with their experiments. It was found that each tools holds a very unique and important role in pathway analysis. Visualization pathways have the role of exploring individual pathways and interpreting genomic results. Quantification pathways use statistical tests to determine pathway significance. Together one can find pathways of interest and then explore areas of interest.
ContributorsRehling, Thomas Evan (Author) / Buetow, Kenneth (Thesis director) / Wilson, Melissa (Committee member) / School of Life Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Bexarotene is a synthetic analog of 9-cis-retinoic acid and ligand for the retinoid X receptor which has a history of clinical success in the treatment of T-cell lymphoma. Bexarotene has also shown potential for treating a variety of other cancers, which we seek to explore in this project. The potential of bexarotene lies in its unique mechanisms and wide application, however, it has shown limited effectiveness thus far in the treatment of breast and lung cancer, with moderate levels of efficacy and symptoms such as cutaneous toxicity, hyperlipidemia, and hypothyroidism. For this project several analogs of bexarotene were synthesized with the intentions of making a more potent ligand that can be used to treat these carcinomas while minimizing harmful side effects. We were successful in synthesizing a large variety of analogs over the span of roughly two years, including iso-chroman derivatives of bexarotene and NEt-TMN, in addition to a new series of analogs of the reported NEt-TMN derivative. These analogs were analyzed via melting point determination and nuclear magnetic resonance (NMR) spectroscopy to confirm the molecular structure and determine purity, and it is our intent to continue with further testing of these compounds to determine their effectiveness as well as the side effects they are likely to cause with levels of toxicity. Recent studies suggest that continuing the analysis of these compounds and other rexinoids like the ones described herein is a worthwhile endeavor as similar rexinoids have shown in numerous assays to be more potent and less toxic in the treatment of cancers when compared with bexarotene.
ContributorsMoen, Grant Anthony (Author) / Wagner, Carl (Thesis director) / Deutch, Charles (Committee member) / School of Social and Behavioral Sciences (Contributor) / School of Mathematical and Natural Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Programmed cell death ligand-1 (PD-L1) is an overexpressed protein on many tumor cell types. PD-L1 is involved in normal immune regulation, playing an important role in self-tolerance and controlling autoimmunity. However, ligation of PD-L1 to PD-1 on activated T cells leads to tumor-mediated T cell suppression. Inhibiting the PD-1/PD-L1 pathway has emerged as an effective target for anti-tumor immunotherapies. Monoclonal antibodies (mAbs) targeting tumor-associated antigens such as PD-L1 have proven to be effective checkpoint blockades, improving therapeutic outcomes for cancer patients and receiving FDA approval as first line therapies for some cancers. A single chain variable fragment (scFv) is composed of the variable heavy and light chain regions of a mAb, connected by a flexible linker. We hypothesized that scFv proteins based on the published anti-PD-L1 monoclonal antibody sequences of atezolizumab and avelumab would bind to cell surface PD-L1. Four single chain variable fragments (scFvs) were constructed based on the sequences of these mAbs. PCR was used to assemble, construct, and amplify DNA fragments encoding the scFvs which were subsequently ligated into a eukaryotic expression vector. Mammalian cells were transfected with the scFv and scFv-IgG plasmids. The scFvs were tested for binding to PD-L1 on tumor cell lysates by western blot and to whole tumor cells by staining and flow cytometry analysis. DNA sequence analysis demonstrated that the scFv constructs were successfully amplified and cloned into the expression vectors and recombinant scFvs were produced. The binding capabilities of the scFvs constucts to PD-L1 protein were confirmed by western blot and flow cytometry analysis. This lead to the idea of constructing a CAR T cell engineered to target PD-L1, providing a possible adoptive T cell immunotherapy.
ContributorsPfeffer, Kirsten M. (Author) / Lake, Douglas (Thesis director) / Ho, Thai (Committee member) / Hastings, Karen (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Exosomes have been known to secrete an increased amount of miRNA and noncoding genes that are abnormally expressed in various cancer subtypes. Thus, they may be an early marker for pediatric cancer types that are more difficult to diagnosis without invasive techniques, and may also help identify progression of the disease. In the project, six types of pediatric cancer cell lines, along with their extracted exosomes, were analyzed and tested for different monoclonal antibodies through western blot analysis. The genes EWS-FLI1 and FGFR4 were also identified in some cancer cell lines through Reverse-Transcriptase Polymerase Chain Reaction analysis (RT-PCR). The results were indicative of similar protein markers being found in both the originating cells and their corresponding exosomes.
ContributorsKaur Bhinder, Harsimran (Author) / Lake, Douglas (Thesis director) / Azorsa, David (Committee member) / Barrett, The Honors College (Contributor)
Created2017-12
Big Data Network Analysis of Genetic Variation and Gene Expression in Individuals with Breast Cancer
Description
The advent of big data analytics tools and frameworks has allowed for a plethora of new approaches to research and analysis, making data sets that were previously too large or complex more accessible and providing methods to collect, store, and investigate non-traditional data. These tools are starting to be applied in more creative ways, and are being used to improve upon traditional computation methods through distributed computing. Statistical analysis of expression quantitative trait loci (eQTL) data has classically been performed using the open source tool PLINK - which runs on high performance computing (HPC) systems. However, progress has been made in running the statistical analysis in the ecosystem of the big data framework Hadoop, resulting in decreased run time, reduced storage footprint, reduced job micromanagement and increased data accessibility. Now that the data can be more readily manipulated, analyzed and accessed, there are opportunities to use the modularity and power of Hadoop to further process the data. This project focuses on adding a component to the data pipeline that will perform graph analysis on the data. This will provide more insight into the relation between various genetic differences in individuals with breast cancer, and the resulting variation - if any - in gene expression. Further, the investigation will look to see if there is anything to be garnered from a perspective shift; applying tools used in classical networking contexts (such as the Internet) to genetically derived networks.
ContributorsRandall, Jacob Christopher (Author) / Buetow, Kenneth (Thesis director) / Meuth, Ryan (Committee member) / Almalih, Sara (Committee member) / Computer Science and Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Medulloblastoma is the most common pediatric brain cancer and accounts for 20% of all pediatric brain tumors. Upon diagnosis, patients undergo tumor-resection surgery followed by intense chemotherapy and cerebrospinal irradiation (CSI) regimens. CSI therapy is highly toxic and poorly tolerated in pediatric patients and is known to cause long-term neurocognitive, endocrine, and developmental deficits that often diminish the quality of life for medulloblastoma patients. The development of targeted therapies is necessary for both increasing the chance of survival and reducing treatment-related morbidities. A potential therapeutic target of interest in medulloblastoma is the polyamine biosynthesis pathway. Polyamines are metabolites present in every living organism and are essential for cellular processes such as growth, survival, and differentiation. Recent studies have shown that polyamine production is dysregulated in several cancers, including brain cancers, and have highlighted polyamine biosynthesis as a potential cancer growth dependency. Dysregulated polyamine metabolism has also been linked to several oncogenic drivers, including the WNT, SHH, and MYC signaling pathways that characterize genetically distinct medulloblastoma subgroups. One way to target polyamine biosynthesis is through the inhibition of the rate-limiting enzyme ornithine decarboxylase with difluoromethylornithine (DFMO), an analog of the polyamine precursor ornithine. DFMO is well-tolerated in pediatric populations and exerts minimal toxicities, as shown through neuroblastoma clinical trials, and is a therapy of interest for medulloblastoma. While DFMO has been tested clinically in multiple cancers, few in vitro studies have been performed to understand the exact mechanisms of anti-proliferation and cytotoxicity. Our study screened two immortalized medulloblastoma cell lines, DAOY (SHH) and D283 (non-WNT/non-SHH), and three patient-derived medulloblastoma cell lines, SL00024 (SHH), SL00668 (non-WNT/non-SHH), SL00870 (Unknown subgroup), for DFMO sensitivity and profiled the immortalized medulloblastoma cell line metabolome to understand the interactions between inhibition of polyamine metabolism with other essential metabolic processes and tumor cell growth. We found that medulloblastoma cell lines are sensitive to DFMO and the adaptive response to DFMO in medulloblastoma may be caused by increased oxidative stress and free radical scavenging. Our study hopes to inform the use of DFMO as an anti-cancer therapy in medulloblastoma by understanding the drug’s single-agent anti-proliferative mechanisms.
ContributorsFain, Caitlyn (Author) / Buetow, Kenneth (Thesis director) / Pirrotte, Patrick (Committee member) / Pathak, Khyati (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor)
Created2024-05
Description
Hepatocellular carcinoma (HCC) is the most common type of liver cancer and has been shown to have genetic factors that contribute to cancer susceptibility. These genetic factors can be studied using Genome-Wide association studies (GWAS), which allow for the assessment of associations between specific biologic markers. Through GWAS, associations can be analyzed to identify genetic components that contribute to the onset of HCC. This study uses an extended version of Pathways of Distinction analysis (PoDA) to identify the subset of SNPs within the Antigen Presentation and Processing Pathway that distinguish cases from controls. Further analysis was performed to explore SNP-SNP association differences between HCC cases and controls using R-squared values and p-values. Three SNPs show significant inter-SNP associations in both HCC cases and controls. Additionally, 4 SNPs showed significant SNP-SNP associations exclusively in the control data set, possibly suggesting that control pathways have a greater degree of genetic regulation and robustness that is lost in carcinogenesis. This result suggests that these SNP associations may contribute to HCC susceptibility.
ContributorsAghili, Ardesher Joshua (Author) / Buetow, Kenneth (Thesis director) / Wilson Sayres, Melissa (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05