Matching Items (533)
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
Isotopic analyses of archaeological and modern materials are commonly used to reconstruct diet, climate, and habitat. This study analyzes 15 camelid samples from three sites (two archaeological, one modern) in South America to determine their carbon and nitrogen isotopic values to further explore the relationship between stable isotopes and environments. Camelid individuals in the modern site of Cuenca, Ecuador had a diet of almost entirely C3 vegetation, while those in Chen Chen, Peru had slightly higher values, still consistent with C3 plants. Those in the higher altitude site of Pumapunku, Bolivia had higher δ13C values than expected, indicating they may have been foddered with a mixed diet. These isotopic data indicate that vegetation, and therefore herbivore diets, are influenced by altitude. Additionally, it was found that a positive linear relationship exists between δ15N values and aridity of a site. Results indicate that aspects of the environment such as aridity are reflected in isotopic signatures. These results contribute to the increasing amount of data on isotopic variation in South American camelids, both modern and archaeological.
ContributorsSpencer, Katherine Clare (Author) / Knudson, Kelly (Thesis director) / Reed, Kaye (Committee member) / School of Human Evolution and Social Change (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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
This project was generated out of a desire to understand and explore a novel twist on a well-traversed route to happiness. I set out looking for a new perspective on fulfillment and found sustainable, everyday joy through gratitude. In doing so, I created a space where a group of people could practice and share gratitude as a community. Gratitude is familiar to most as a feeling, but putting intention behind gratitude turns it into an action, and even a virtue. In fact, Roman philosopher Cicero says, "Gratitude is not only the greatest of virtues, but the parent of all others." I created a Facebook community called Marvel at The Ordinary (MATO) applying principles rooted in the Theory of Change to express this greatest virtue. I found both success and earnest support from others in this novel approach to current gratitude practices. Defined by Dr. Robert Emmons, an expert in the science of gratitude, practicing gratitude is a two-step process: "(1.) affirming goodness in one's life, and (2.) recognizing that the sources of this goodness lie at least partially outside of the self." There is substantial research touting the worth of gratitude journaling, in fact, few things have been more repeatedly and empirically vetted than the connection between gratitude and overall happiness and well-being. Yet there is one facet ubiquitously overlooked in current gratitude research: what happens when gratitude journaling is shared with others? With anecdotal evidence, short-form interview analysis, thematic analysis of journaling lexicon, and a case study on the growth and engagement of Marvel at The Ordinary as a social movement, there is reason to believe that a social media-based community centered around gratitude may support and even enhance the practice of gratitude, which is typically practiced in isolation. It was also found that communities of this sort are highly sought after, based on the engagement within and growth of the Facebook group from 50 to 600+ members in a period of 2 months. MATO set out with the aspirations of creating a community which encourages others to gratitude journal, raising awareness about gratitude journaling, and building a community which fosters empathy, optimism, and awareness in an everyday sense. In each of these goals, overwhelming success was found.
ContributorsMccawley, Kevin William (Author) / Mokwa, Michael (Thesis director) / Huerta, Mark (Committee member) / Micevic, Vid (Committee member) / School of International Letters and Cultures (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Metalloporphyrins serve important roles in biological processes and in emerging technologies with applications to energy conversion. When electrochemically activated in solution, metalloporphyrins have the ability to catalyze the conversion of protons into hydrogen fuels. In this report, the synthesis and characterization of zinc, nickel, cobalt and copper analogs of 5,10,15,20-tetrakis(pentafluorophenyl) porphyrin (PF20) and 5,10,15,20-tetra-p-tolyporphyrin (TTP) are described. All target compounds are characterized with UV-Vis spectroscopy and MALDI-TOF mass spectrometry. The freebase porphyrins and non-paramagnetic metalloporphyrins are further characterized by proton nuclear magnetic resonance spectroscopy and all proton resonances are assigned. Electrochemical measurements show the reduction potential of the fluorinated phenyl substituted porphyrins is shifted to less negative values as compared to the reduction potential measured using non-fluorinated analogs. These results illustrate the ability to use fluorine as a synthetic tool for altering the electronic properties of metalloporphyrins. Further, these findings serve a critical role in choosing metalloporphyrin electrocatalysts with the appropriate energetic and optical properties for integration to semiconductors with applications to solar-to- fuels technologies.
ContributorsNanyangwe, Sylvia Kapya (Author) / Moore, Gary (Thesis director) / Van Horn, Wade (Committee member) / School of Criminology and Criminal Justice (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Recent evidence suggests that youth from affluent and upper-middle-class, white collar families are at high risk for maladaptive behaviors, such as aggression, rule breaking, and substance use. A major hypothesized underlying factor is lax parental discipline that involves low repercussions for errant behaviors such as substance use—also known as perceived parents’ “containment” of such behaviors. In this study, the focus is on multiple dimensions of perceived containment among parents and school authorities, in relation to both externalizing problems and drug use behaviors. These associations are examined in four different schools: two boarding schools and two day schools. Results show much stronger links with maladjustment for perceived containment by parents as opposed to perceived containment by school. The largest significant effects within the containment indices were found to be between parent containment of drug use and the levels of substance use behaviors reported by students. These effects were found across gender and all schools, indicating robust links. Overall, the most robust index studied was the perceived parental monitoring variables; monitoring effects were stronger than effects from any containment variables. Students who perceived the highest levels of parental monitoring exhibited the least amount of externalizing behaviors across all schools and genders. A possible explanation includes students perceiving that their parents monitor their behavior due to caring on the behalf of the parent, which then leads to fewer externalizing outcomes. These results suggest that the most effective means of decreasing substance use levels seem to lie within the parents of the students and not the schools.
ContributorsHudson, Johnny (Author) / Luthar, Suniya (Thesis director) / Glenberg, Arthur (Committee member) / Cavanaugh Toft, Carolyn (Committee member) / School of Molecular Sciences (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
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