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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
Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disease characterized by progressive muscle loss and weakness. This disease arises from a mutation that occurs on a gene that encodes for dystrophin, which results in observable muscle death and inflammation; however, the genetic changes that result from dystrophin's dysfunctionality remain unknown. Current DMD research uses mdx mice as a model, and while very useful, does not allow the study of cell-autonomous transcriptome changes during the progression of DMD due to the strong inflammatory response, perhaps hiding important therapeutic targets. C. elegans, which has a very weak inflammatory response compared to mdx mice and humans, has been used in the past to study DMD with some success. The worm ortholog of the dystrophin gene has been identified as dys-1 since its mutation phenocopies the progression of the disease and a portion of the human dystrophin gene alleviates symptoms. Importantly, the extracted RNA transcriptome from dys-1 worms showed significant change in gene expression, which needs to be further investigated with the development of a more robust model. Our lab previously published a method to isolate high-quality muscle-specific RNA from worms, which could be used to study such changes at higher resolution. We crossed the dys-1 worms with our muscle-specific strain and demonstrated that the chimeric strain exhibits similar behavioral symptoms as DMD patients as characterized by a shortened lifespan, difficulty in movement, and a decrease in speed. The presence of dys-1 and other members of the dystrophin complex in the body muscle were supported by the development of a resulting phenotype due to RNAi knockdown of each component in the body muscle; however, further experimentation is needed to reinforce this conclusion. Thus, the constructed chimeric C. elegans strain possesses unique characteristics that will allow the study of genetic changes, such as transcriptome rearrangements and dysregulation of miRNA, and how they affect the progression of DMD.
ContributorsNguyen, Thuy-Duyen Cao (Author) / Mangone, Marco (Thesis director) / Newbern, Jason (Committee member) / Duchaine, Thomas (Committee member) / School of Social Transformation (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
microRNAs (miRNAs) are short ~22nt non-coding RNAs that regulate gene output at the post-transcriptional level. Via targeting of degenerate elements primarily in 3'untranslated regions (3'UTR) of mRNAs, miRNAs can target thousands of varying genes and suppress their protein translation. The precise mechanistic function and bio- logical role of miRNAs is not fully understood and yet it is a major contributor to a pleth- ora of diseases, including neurological disorders, muscular disorders, and cancer. Cer- tain model organisms are valuable in understanding the function of miRNA and there- fore fully understanding the biological significance of miRNA targeting. Here I report a mechanistic analysis of miRNA targeting in C. elegans, and a bioinformatic approach to aid in further investigation of miRNA targeted sequences. A few of the biologically significant mechanisms discussed in this thesis include alternative polyadenylation, RNA binding proteins, components of the miRNA recognition machinery, miRNA secondary structures, and their polymorphisms. This thesis also discusses a novel bioinformatic approach to studying miRNA biology, including computational miRNA target prediction software, and sequence complementarity. This thesis allows a better understanding of miRNA biology and presents an ideal strategy for approaching future research in miRNA targeting.
ContributorsWeigele, Dustin Keith (Author) / Mangone, Marco (Thesis director) / Katchman, Benjamin (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2014-12
Description
Cuticular hydrocarbons (CHCs) play a crucial role in social insect recognition systems. In this study we investigated mate choice in the red harvester ant, Pogonomyrmex barbatus. In Phoenix, this species has two lineages, J1 and J2, which look identical, but are genetically isolated. In the genetic caste determination (GCD) system workers and queens are determined by their genotype (i.e., workers develop from interlineage crosses, queens from intralineage crosses). As such, J1 and J2 lineages are dependent on each other in order for colonies to produce both workers and reproductive queens. Given their genetic isolation and interdependence, we hypothesized that the CHCs of alate males and queens are affected by lineage, and that differences in the CHC profile are used for mate recognition. We tested these hypotheses by analyzing the lineage distributions of actively mating pairs (n=65), and compared them with the overall distribution of male and female sexuals (n=180). We additionally analyzed the five most abundant CHC compounds for 20 of the actively mating P. barbatus alate male and queen pairs to determine how variable the two lineages are between each sex. We found that mating pair distributions did not significantly differ from those expected under a random mating system (�2= 1.4349, P= 0.6973), however, CHC profiles did differ between J1 and J2 lineages and sexes for the five most abundant CHC compounds. Our results show that random mating is taking place in this population, however given the differences observed in CHC profiles, mate recognition could be taking place.
ContributorsTula Del Moral Testai, Pedro Rafael (Co-author) / Cash, Elizabeth (Co-author) / Gadau, Juergen (Thesis director) / Liebig, Juergen (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
The Cannabis plant has historical roots with human beings. The plant produces compounds called cannabinoids, which are responsible for the physiological affects of Cannabis and make it a research candidate for medicinal use. Analysis of the plant and its components will help build a better database that could be used to develop a complete roster of medicinal benefits. Research regarding the cellular protein receptors that bind the cannabinoids may not only help provide reasons explaining why the Cannabis plant could be medicinally relevant, but will also help explain how the receptors originated. The receptors may have been present in organisms before the present day Cannabis plant. So why would there be receptors that bind to cannabinoids? Searching for an endocannabinoid system could help explain the purpose of the cannabinoid receptors and their current structures in humans. Using genetic technologies we are able to take a closer look into the evolutionary history of cannabinoids and the receptors that bind them.
ContributorsSalasnek, Reed Samuel (Author) / Capco, David (Thesis director) / Mangone, Marco (Committee member) / Stump, Edmund (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
A coincidence reporter construct, consisting of the p21-promoter and two luciferase genes (Firefly and Renilla), was constructed for the screening of drugs that might inhibit Olig2's tumorigenic role in glioblastoma. The reporter construct was tested using an Olig2 inhibitor, HSP990, as well as short hairpin RNA targeting Olig2. Further confirmatory analysis is needed before the reporter cell line is ready for high-throughput screening at the NIH and lead compound selection.
ContributorsCusimano, Joseph Michael (Author) / LaBaer, Joshua (Thesis director) / Mangone, Marco (Committee member) / Mehta, Shwetal (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-05
Description
I tested the hypothesis that in mature colonies of the seed harvester Pogonomyrmex californicus ant species, paired pleometrotic queens would produce workers more efficiently after a massive removal of their work force than haplometrotic queens, paired pleometrotic with haplometrotic queens, and single pleometrotic queens. I suggested that the paired pleometrotic queens would have an advantage of cooperating together in reproducing more workers quicker than the other conditions to make up for the lost workers. This would demonstrate a benefit that pleometrosis has over haplometrosis for mature colonies, which would explain why pleometrosis continues for P.californicus after colony foundation. After removing all but twenty workers for every colony, I took pictures and counted the emerging brood for 52 days. Analyses showed that the paired pleometrotic queens and the haplometrotic queens both grew at an equally efficient rate and the paired pleometrotic and haplometrotic queens growing the least efficiently. However, the results were not significant and did not support the hypothesis that paired pleometrotic queens recover from worker loss more proficiently than other social systems.
ContributorsFernandez, Marisa Raquel (Author) / Fewell, Jennifer (Thesis director) / Gadau, Juergen (Committee member) / Haney, Brian (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / Department of Psychology (Contributor)
Created2014-05
Description
Background: Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer deaths in females worldwide, accounting for 23% of all new cancer cases and 14% of all total cancer deaths in 2008. Five tumor-normal pairs of primary breast epithelial cells were treated for infinite proliferation by using a ROCK inhibitor and mouse feeder cells. Methods: Raw paired-end, 100x coverage RNA-Seq data was aligned to the Human Reference Genome Version 19 using BWA and Tophat. Gene differential expression analysis was completed using Cufflinks and Cuffdiff. Interactive Genome Viewer was used for data visualization. Results: 15 genes were found to be down-regulated by at least one log-fold change in 4/5 of tumor samples. 75 genes were found to be down-regulated in 3/5 of our tumor samples by at least one log-fold change. 11 genes were found to be up-regulated in 4/5 of our tumor samples, and 68 genes were identified to be up-regulated in 3/5 of the tumor samples by at least one-fold change. Conclusion: Expression changes in genes such as AZGP1, AGER, ALG11, and S1007 suggest a disruption in the glycosylation pathway. No correlation was found between Cufflink's Her2 gene-expression and DAKO score classification.
ContributorsHernandez, Fernando (Author) / Anderson, Karen (Thesis director) / Mangone, Marco (Committee member) / Park, Jin (Committee member) / Barrett, The Honors College (Contributor) / Department of Information Systems (Contributor)
Created2013-05
Description
Wolbachia is a genus of obligately intracellular bacterial endosymbionts of arthropods and nematodes, infecting up to 66% of all such species. In order to ensure its transmission, it may modify host reproduction by inducing one of four phenotypes: cytoplasmic incompatibility, feminization of genetic males, killing of male embryos, and induction of thelytokous parthenogenesis. This investigation was a characterization of the so-far unexamined Wolbachia infection of Pogonomyrmex ants. Five main questions were addressed: whether Wolbachia infection rates vary between North and South America, whether infection rates are dependent on host range, whether Wolbachia affects the caste determination of P. barbatus, whether infection rates in Pogonomyrmex are similar to those of other ants, and whether Wolbachia phylogeny parallels the phylogeny of its Pogonomyrmex hosts. Using PCR amplification of the wsp, ftsZ, and gatB loci, Wolbachia infections were detected in four of fifteen Pogonomyrmex species (26.7%), providing the first known evidence of Wolbachia infection in this genus. All infected species were from South America, specifically Argentina. Therefore, Wolbachia has no role in the caste determination of the North American species P. barbatus. Additionally, while it appears that the incidence of Wolbachia in Pogonomyrmex may be limited to South America, host range did not correlate with infection status. The incidence of Wolbachia in Pogonomyrmex as a whole was similar to that of invasive Solenopsis and Linepithema species, but not to Wasmannia auropunctata or Anoplolepis gracilipes, which retain Wolbachia infection in non-native locations. This suggests that there may be a parallel in Wolbachia infection spread in certain short-term models of species colonization and long-term models of genus radiation. Finally, there was no congruity between host and parasite phylogeny according to maximum likelihood analyses, necessarily due to horizontal transfer of Wolbachia between hosts and lateral gene transfer between Wolbachia strains within hosts.
ContributorsHarris, Alexandre Marm (Author) / Gadau, Juergen (Thesis director) / Martin, Thomas (Committee member) / Helmkampf, Martin Erik (Committee member) / Barrett, The Honors College (Contributor) / Computer Science and Engineering Program (Contributor) / School of Life Sciences (Contributor)
Created2014-05
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