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Breast cancer is the leading cause of cancer-related deaths of women in the united states. Traditionally, Breast cancer is predominantly treated by a combination of surgery, chemotherapy, and radiation therapy. However, due to the significant negative side effects associated with these traditional treatments, there has been substantial efforts to develop alternative therapies to treat cancer. One such alternative therapy is a peptide-based therapeutic cancer vaccine. Therapeutic cancer vaccines enhance an individual's immune response to a specific tumor. They are capable of doing this through artificial activation of tumor specific CTLs (Cytotoxic T Lymphocytes). However, in order to artificially activate tumor specific CTLs, a patient must be treated with immunogenic epitopes derived from their specific cancer type. We have identified that the tumor associated antigen, TPD52, is an ideal target for a therapeutic cancer vaccine. This designation was due to the overexpression of TPD52 in a variety of different cancer types. In order to start the development of a therapeutic cancer vaccine for TPD52-related cancers, we have devised a two-step strategy. First, we plan to create a list of potential TPD52 epitopes by using epitope binding and processing prediction tools. Second, we plan to attempt to experimentally identify MHC class I TPD52 epitopes in vitro. We identified 942 potential 9 and 10 amino acid epitopes for the HLAs A1, A2, A3, A11, A24, B07, B27, B35, B44. These epitopes were predicted by using a combination of 3 binding prediction tools and 2 processing prediction tools. From these 942 potential epitopes, we selected the top 50 epitopes ranked by a combination of binding and processing scores. Due to the promiscuity of some predicted epitopes for multiple HLAs, we ordered 38 synthetic epitopes from the list of the top 50 epitope. We also performed a frequency analysis of the TPD52 protein sequence and identified 3 high volume regions of high epitope production. After the epitope predictions were completed, we proceeded to attempt to experimentally detected presented TPD52 epitopes. First, we successful transduced parental K562 cells with TPD52. After transduction, we started the optimization process for the immunoprecipitation protocol. The optimization of the immunoprecipitation protocol proved to be more difficult than originally believed and was the main reason that we were unable to progress past the transduction of the parental cells. However, we believe that we have identified the issues and will be able to complete the experiment in the coming months.
ContributorsWilson, Eric Andrew (Author) / Anderson, Karen (Thesis director) / Borges, Chad (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Introduction: Human papillomavirus (HPV) infection is seen in up to 90% of cases of cervical cancer, the third leading cancer cause of death in women. Current HPV screening focuses on only two HPV types and covers roughly 75% of HPV-associated cervical cancers. A protein based assay to test for antibody biomarkers against 98 HPV antigens from both high and low risk types could provide an inexpensive and reliable method to screen for patients at risk of developing invasive cervical cancer. Methods: 98 codon optimized, commercially produced HPV genes were cloned into the pANT7_cGST vector, amplified in a bacterial host, and purified for mammalian expression using in vitro transcription/translation (IVTT) in a luminescence-based RAPID ELISA (RELISA) assay. Monoclonal antibodies were used to determine immune cross-reactivity between phylogenetically similar antigens. Lastly, several protein characteristics were examined to determine if they correlated with protein expression. Results: All genes were successfully moved into the destination vector and 86 of the 98 genes (88%) expressed protein at an adequate level. A difference was noted in expression by gene across HPV types but no correlation was found between protein size, pI, or aliphatic index and expression. Discussion: Further testing is needed to express the remaining 12 HPV genes. Once all genes have been successfully expressed and purified at high concentrations, DNA will be printed on microscope slides to create a protein microarray. This microarray will be used to screen HPV-positive patient sera for antibody biomarkers that may be indicative of cervical cancer and precancerous cervical neoplasias.
ContributorsMeshay, Ian Matthew (Author) / Anderson, Karen (Thesis director) / Magee, Mitch (Committee member) / Katchman, Benjamin (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
The purpose of this project was to identify proteins associated with the migration and invasion of non-transformed MCF10A mammary epithelial cells with ectopically expressed missense mutations in p53. Because of the prevalence of TP53 missense mutations in basal-like and triple-negative breast cancer tumors, understanding the effect of TP53 mutations on the phenotypic expression of human mammary epithelial cells may offer new therapeutic targets for those currently lacking in treatment options. As such, MCF10A mammary epithelial cells ectopically overexpressing structural mutations (G245S, H179R, R175H, Y163C, Y220C, and Y234C) and DNA-binding mutations (R248Q, R248W, R273C, and R273H) in the DNA-binding domain were selected for use in this project. Overexpression of p53 in the mutant cell lines was confirmed by western blot and q-PCR analysis targeting the V5 epitope tag present in the pLenti4 vector used to transduce TP53 into the mutant cell lines. Characterization of the invasion and migration phenotypes resulting from the overexpression of p53 in the mutant cell lines was achieved using transwell invasion and migration assays with Boyden chambers. Statistical analysis showed that three cell lines—DNA-contact mutants R248W and R273C and structural mutant Y220C—were consistently more migratory and invasive and demonstrated a relationship between the migration and invasion properties of the mutant cell lines. Two families of proteins were then explored: those involved in the Epithelial-Mesenchymal Transition (EMT) and matrix metalloproteinases (MMPs). Results of q-PCR and immunofluorescence analysis of epithelial marker E-cadherin and mesenchymal proteins Slug and Vimentin did not show a clear relationship between mRNA and protein expression levels with the migration and invasiveness phenotypes observed in the transwell studies. Results of western blotting, q-PCR, and zymography of MMP-2 and MMP-9 also did not show any consistent results indicating a definite relationship between MMPs and the overall invasiveness of the cells. Finally, two drugs were tested as possible treatments inhibiting invasiveness: ebselen and SBI-183. These drugs were tested on only the most invasive of the MCF10A p53 mutant cell lines (R248W, R273C, and Y220C). Results of invasion assay following 30 μM treatment with ebselen and SBI-183 showed that ebselen does not inhibit invasiveness; SBI-183, however, did inhibit invasiveness in all three cell lines tested. As such, SBI-183 will be an important compound to study in the future as a treatment that could potentially serve to benefit triple-negative or basal-like breast cancer patients who currently lack therapeutic treatment options.
ContributorsZhang, Kathie Q (Author) / LaBaer, Joshua (Thesis director) / Anderson, Karen (Committee member) / Gonzalez, Laura (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Description
Background: Measles virus (MV) infections are the main cause of vaccine-preventable death in children younger than 5 years. The World Health Organization (WHO) has estimated there are over 20 million cases of measles every year. Currently, diagnostic methods rely on enzyme immunoassays (EIA) to detect IgM or IgG Abs in serum. These commercial assays measure reactivity against the immunodominant N antigen and can have a false negative rates of 20-30%. Centralized testing by clinical labs can delay rapid screening in an outbreak setting. This study aims to develop a rapid molecular diagnostic assay to detect IgG reactive to five individual MV proteins representing 85% of the measles proteome. Methods: MV genes were subcloned into pANT_cGST vector to generate C-terminal GST fusion proteins. Single MV cistrons were expressed using in vitro transcription/translation (IVTT) with human cell lysate. Expression of GST-tagged proteins was measured using a sandwich ELISA for GST expression using relative light units (RLUs) as readouts. Single MV antigens were used as bait to determine the IgG-dependent reactivity in 12 serum samples obtained from immunized animals with previously determined neutralization titer (NT) and the correlation between NT and ELISA reactivity was determined. Results: Protein expression of five measles genes of interest, M, N, F, H, and L, was measured. L exhibited the strongest protein expression with an average RLU value of 4.34 x 10^9. All proteins were expressed at least 50% greater than control (2.33 x 10^7 RLU). As expected, reactivity against the N was the highest, followed by reactivity against M, F, H and L. The best correlation with NT titer was reactivity against F (R^2 = 0.62). Conclusion: These data indicate that the expression of single MV genes M, N, F, H, and L are suitable antigens for serologic capture analysis of measles immunity.
ContributorsMushtaq, Zuena (Author) / Anderson, Karen (Thesis director) / Reyes del Valle, Jorge (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Description
F2-isoprostanes are a series of prostaglandin-like compounds derived from the free radical-mediated lipid peroxidation of arachidonic acid, a polyunsaturated fatty acid that is ubiquitously expressed in cell membranes. F2-isoprostanes are biomarkers of oxidative stress, an imbalance between oxidants and antioxidants that can cause damage to DNA, proteins, lipids, and carbohydrates. Increased production of lipid peroxidation products have been implicated in the pathology of a number of conditions and diseases in humans. The objective of this thesis was to (1) optimize the LC/MS/MS F2-isoprostane method currently used in human samples for use in research animals and veterinary medicine, including the use of solid phase extraction, and (2) validate the optimized method in rodent and canine experimental studies. Our optimized method showed that Lyprinol treatment in dogs with osteoarthritis decreases F2-isoprostane levels nearly 2-fold. In addition, adjuvant alpha-tocopherol prevented tumor-induced increased F2-isoprostane levels. Finally, contrary to earlier studies using less specific ELISA F2-isoprostane methods, we demonstrate that unconditioned dogs benefit from low intensity exercise. Our data demonstrate successful optimization of the human LC/MS/MS F2-isoprostane method in rats and canines. Importantly, our results emphasize the need to use the more sensitive and specific LC/MS/MS method as compared to ELISA-based assays in order to distinguish the 15- and 5-series F2-isoprostanes, evidenced in particular by the two canine studies.
ContributorsCorrigan, Devin Connell (Author) / Redding, Kevin (Thesis director) / Anderson, Karen (Committee member) / Mustacich, Debbie (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Description
Background: Human papillomavirus (HPV) is the cause of 99.7% of cervical cancers. Research of cervical cancer has made this disease mostly curable in the developing world. Head and neck cancer, which is increasingly caused by HPV, still is associated with a mortality rate of 50,000 in the US annually. This study proposed to evaluate the biology of HPV-16 in head and neck tumors by using RT-qPCR to measure the RNA expression and its relation to physical status of the virus. Methods: This study was to develop an assay that uses RT-qPCR to determine the quantitative expression of HPV-16 RNA coding for proteins E1, E2, E4, E5, E6, and E7 in tumor samples. The assay development started with creation of primers. It went on to test the primers on template DNA through traditional PCR and then on DNA from HPV-16 positive cell lines, SiHa and CaSki, using RT-qPCR. This paper also describes the troubleshooting methods taken for the PCR reaction. Once the primers are verified, the RT-qPCR process can be carried out on RNA purified from tumor samples. Results: No primer sets have been confirmed to produce a product through PCR or RT-qPCR. The primer sequences match up correctly with known sequences for HPV-16 E1, E2, E4, E5, E6, and E7. RT-qPCR showed results consistent with the hypothesis. Conclusion: The RT-qPCR protocol must be optimized to confirm the primer sequences work as desired. Then primers will be used to study physical status and RNA expression in HPV-positive and HPV-negative head and neck tumor samples. This assay can help shed light on which proteins are expressed most in tumors of the head and neck and will aid in the development of future screening and treatment options.
ContributorsKhazanovich, Jakob (Author) / Anderson, Karen (Thesis director) / Mangone, Marco (Committee member) / Sundaresan, Sri Krishna (Committee member) / Barrett, The Honors College (Contributor)
Created2015-05
Description
Cancer poses a significant burden on the global health system and represents a leading cause of death worldwide. For late-stage cancers, the traditional treatments of chemotherapy, radiation, and surgery are not always viable, and they can pose unnecessary health risks to the patients. New immunotherapies, such as adoptive cell transfer, are being developed and refined to treat such cancers. T cell immunotherapies in particular, where a patient’s T cell lymphocytes are isolated and amplified to be re-infused into the patient or where human cell lines are engineered to express T cell receptors for the recognition of common cancer antigens, are being expanded on because for some cancers, they could be the only option. Constructing an optimal pipeline for cloning and expression of antigen-specific TCRs has significant bearing on the efficacy of engineered cell lines for ACT. Adoptive T cell transfer, while making great strides, has to overcome a diverse T cell repertoire – cloning and expressing antigen-specific TCRs can mediate this understanding. Having identified the high frequency FluM1-specific TCR sequences in stimulated donor PBMCs, it was hypothesized that the antigen-specific TCR could be reconstructed via Gateway cloning methods and tested for expression and functionality. Establishing this pipeline would confirm an ability to properly pair and express the heterodimeric chains. In the context of downstream applications, neoantigens would be used to stimulate T cells, the α and β chains would be paired via single-cell or bulk methods, and instead of Gateway cloning, the CDR3 hypervariable regions α and β chains alone would be co-expressed using Golden Gate assembly methods.
ContributorsHirneise, Gabrielle Rachel (Author) / Anderson, Karen (Thesis director) / Mason, Hugh (Committee member) / Hariadi, Hugh (Committee member) / School of Life Sciences (Contributor, Contributor) / School of Sustainability (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
miRNAs are short non-coding regulatory RNAs that have an important roles in a wide range of biological processes. Dysfunction of miRNA regulation has also been shown to occur in diseases such as cancer. Despite the widespread influence of miRNAs in these contexts, the vast majority of miRNA targets are poorly characterized. The aim of this research project was to gain a better understating of miRNA targeting by using the model organism C. elegans. In order to do this I adapted a novel high-throughput assay to detect miRNA targets for use with the C. elegans 3`UTRome. As a proof of principle I performed this assay on 96 C. elegans 3`UTRs using high-throughput techniques. The results revealed miRNA interactions with two predicted 3`UTR targets for the miRNA lin-4 and ten unpredicted targets. The results also corroborated previous findings that certain worm miRNAs require special modifications to be expressed in human cells.
ContributorsKotagama, Kasuen Indrajith Bandara (Author) / Mangone, Marco (Thesis director) / Anderson, Karen (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2013-12
Description
The focus of this project was to look at alternative treatments for endocrine resistant breast cancer (ERBC), which are breast cancers that have become resistant to hormone therapies such as Tamoxifen or aromatase inhibitors. The first part of this project involves investigating the relationship between histone de-acetylase inhibitor Vorinostat and Tamoxifen in MCF7 G11 cells, Tamoxifen resistant sub-clones, according to the PSOC Time grant. The second part involves targeting the androgen receptor (AR) in MCF7 sub-clones with AR antagonists, Bicalutamide and MDV3100, and investigating the possible usage of AR as a biomarker, due to over-expression of AR in ERBC, in accordance with the Mayo ASU Seed Grant.
The synergistic effects between Vorinostat and Tamoxifen observed through a phase II study on breast cancer patients resistant to hormone therapy may involve more than the modulation of ER-alpha to reverse Tamoxifen resistance in ERBC cells. RT-qPCR of genes expressed in Tamoxifen resistant cells, trefoil factor 1(TFF1) and v-myc avian myelocytomatosis viral oncogene homolog (MYC), were evaluated along with ESR1 and Diablo as a control. MYC was observed to have increased expression in the treated cells, whereas the other genes had a decrease in their expression levels after the cells were treated for 3 days with Vorinostat IC30 of 1 µM. As for targeting the AR, MCF7 Tamoxifen sensitive and resistant cells were not affected by the AR antagonists to determine an IC50. The cell viability for all MCF7 sub-clones only decreased for high concentrations of 5.56 µM - 50 µM in Bicalutamide and 16.67 µM – 50 µM of MDV1300. Furthermore, hormone depletion of MCF7 G11 Tamoxifen resistant sub-clones did not show a great response to DHT stimulation or the AR antagonists. In the RT-qPCR, the MCF7 G11 cells showed an increase in mRNA expression for ER, AR, and PR after 4 hours of treatment with estradiol. As for the DHT treatment, ER, AR, PR, and PSA had a minimal increase in the fold change, but the fold change in AR was less than in the estradiol treatment. The Mayo Clinic will investigate the possible usage of AR as a biomarker through immunohistochemistry.
The synergistic effects between Vorinostat and Tamoxifen observed through a phase II study on breast cancer patients resistant to hormone therapy may involve more than the modulation of ER-alpha to reverse Tamoxifen resistance in ERBC cells. RT-qPCR of genes expressed in Tamoxifen resistant cells, trefoil factor 1(TFF1) and v-myc avian myelocytomatosis viral oncogene homolog (MYC), were evaluated along with ESR1 and Diablo as a control. MYC was observed to have increased expression in the treated cells, whereas the other genes had a decrease in their expression levels after the cells were treated for 3 days with Vorinostat IC30 of 1 µM. As for targeting the AR, MCF7 Tamoxifen sensitive and resistant cells were not affected by the AR antagonists to determine an IC50. The cell viability for all MCF7 sub-clones only decreased for high concentrations of 5.56 µM - 50 µM in Bicalutamide and 16.67 µM – 50 µM of MDV1300. Furthermore, hormone depletion of MCF7 G11 Tamoxifen resistant sub-clones did not show a great response to DHT stimulation or the AR antagonists. In the RT-qPCR, the MCF7 G11 cells showed an increase in mRNA expression for ER, AR, and PR after 4 hours of treatment with estradiol. As for the DHT treatment, ER, AR, PR, and PSA had a minimal increase in the fold change, but the fold change in AR was less than in the estradiol treatment. The Mayo Clinic will investigate the possible usage of AR as a biomarker through immunohistochemistry.
ContributorsVorachitti, Merica (Author) / LaBaer, Joshua (Thesis director) / Anderson, Karen (Committee member) / Gonzalez, Laura (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-05
Description
Patients diagnosed with GBM, a highly migratory, heterogeneous, rapidly growing primary adult brain tumor are faced with a dismal prognosis. Recent research has shed light on cell-survival pathways that are induced after the DNA-damage response induced by TMZ. Autophagy, a major catabolic process meant to degrade damaged organelles and large misfolded proteins, has recently been shown to be activated by TMZ. However, a precise mechanism has not yet been determined. T98G cells treated with TMZ showed significant induction of unfolded protein response (UPR) markers such as GRP78, and LC3-II expression, indicating increased autophagosome formation. Additional experiments have used the autophagic inhibitor Bafilomycin A1 (Baf) to determine that autophagic flux is induced, supporting the conclusion that UPR induction by TMZ induces autophagy. Combination treatments with PI3K inhibitors PX-866 and BEZ235 with Baf as a means to shut down two critical mechanisms of GBM cell survival were explored in this research. PX-866 was found to inhibit autophagy, while BEZ235, was found to induce autophagy. The differential modulation of autophagy by these PI3K inhibitors offers new knowledge for utilizing more effective drug combinations to treat GBM and improve patient survival.
ContributorsSodoma, Andrej Michael (Author) / Anderson, Karen (Thesis director) / Hecht, Sidney (Committee member) / Nhan, Tran L. (Committee member) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05