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- All Subjects: Health
- All Subjects: Immunology
- Creators: School of Life Sciences
- Status: Published
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
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
Efforts to quantify the diversity of the T cell repertoire have generally been unsuccessful because not all factors accounting for diversity have been considered. In order to get an accurate representation of the T cell repertoire, one must incorporate analysis of germline gene diversity, diversity from somatic recombination, joining diversity from N- and P- nucleotides, and TCR chain pairing diversity. Because of advances in high-throughput sequencing techniques, estimates have been able to account for diversity from TCR genes. However the ability to account for chain pairing diversity has been more difficult. In order to do so, single cell sorting techniques must be employed. These techniques, though effective, are time consuming and expensive. For this reason, no large-scale analyses have been done on the immune repertoires using these techniques. In this study, we propose a novel method for linking the two TCR chain sequences from an individual cell. DNA origami nanostructure technology is employed to capture and bind the TCRγ and TCRδ chain mRNA inside individual cells using probe strands complementary to the C-region of those sequences. We then use a dual-primer RT and ligation molecular strategy to link the two sequences together. The result is a single amplicon containing the CDR3 region of the TCRγ and TCRδ. This amplicon can then be easily PCR amplified using sequence specific primers, and sequenced. DNA origami nanostructures offer a rapid, cost-effective method alternative to conventional single cell sorting techniques, as both TCR mRNA can be captured on one origami molecule inside a single cell. At present, this study outlines a proof-of-principle analysis of the method to determine its functionality. Using known TCRγ and TCRδ sequences, the DNA origami and RT/PCR method was tested and resulting sequence data proved the effectiveness of the method. The original TCRγ and TCRδ sequences were linked together as a single amplicon containing both CDR3 regions of the genes. Thus, this method can be employed in further research to elucidate the γδ T cell repertoire. This technology is also easily adapted to any gene target or cell type and therefore presents a large opportunity to be used in other immune repertoire analysis and other immunological studies (such as the rapid identification and subsequent production of antibodies).
ContributorsPoindexter, Morgan Elizabeth (Author) / Blattman, Joseph (Thesis director) / Yan, Hao (Committee member) / Schoettle, Louis (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Cancer remains one of the leading killers throughout the world. Death and disability due to lung cancer in particular accounts for one of the largest global economic burdens a disease presents. The burden on third-world countries is especially large due to the unusually large financial stress that comes from late tumor detection and expensive treatment options. Early detection using inexpensive techniques may relieve much of the burden throughout the world, not just in more developed countries. I examined the immune responses of lung cancer patients using immunosignatures – patterns of reactivity between host serum antibodies and random peptides. Immunosignatures reveal disease-specific patterns that are very reproducible. Immunosignaturing is a chip-based method that has the ability to display the antibody diversity from individual sera sample with low cost. Immunosignaturing is a medical diagnostic test that has many applications in current medical research and in diagnosis. From a previous clinical study, patients diagnosed for lung cancer were tested for their immunosignature vs. healthy non-cancer volunteers. The pattern of reactivity against the random peptides (the ‘immunosignature’) revealed common signals in cancer patients, absent from healthy controls. My study involved the search for common amino acid motifs in the cancer-specific peptides. My search through the hundreds of ‘hits’ revealed certain motifs that were repeated more times than expected by random chance. The amino acids that were the most conserved in each set include tryptophan, aspartic acid, glutamic acid, proline, alanine, serine, and lysine. The most overall conserved amino acid observed between each set was D - aspartic acid. The motifs were short (no more than 5-6 amino acids in a row), but the total number of motifs I identified was large enough to assure significance. I utilized Excel to organize the large peptide sequence libraries, then CLUSTALW to cluster similar-sequence peptides, then GLAM2 to find common themes in groups of peptides. In so doing, I found sequences that were also present in translated cancer expression libraries (RNA) that matched my motifs, suggesting that immunosignatures can find cancer-specific antigens that can be both diagnostic and potentially therapeutic.
ContributorsShiehzadegan, Shima (Author) / Johnston, Stephen (Thesis director) / Stafford, Phillip (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12
Description
Viral infections are a significant cause of disease in humans. While some viral diseases have been eliminated, many more continue to infect millions. Viral infections are challenging to treat because viruses use host cell machinery to replicate, so it is difficult to develop drugs that can target viruses. Normally, the host’s immune system is capable of destroying the virus, but during chronic infections it becomes exhausted and T cells lose their effector functions necessary for the clearance of the virus. IL-2 can help relieve this exhaustion, but causes toxicity to the body. In mice infected with chronic LCMV, IL-2 administration causes death due to pulmonary hemorrhage. CD4 deficient mice were infected with chronic LCMV and then dosed with IL-2 and survived, but mice that were deficient for CD8 T cells died, indicating that toxicity was mediated by CD8 T cells. CD8 T cells can kill infected host cells directly by producing perforin, or can produce cytokines like IFN-γ and TNF to further activate the immune system and mediate killing. Mice that were deficient in perforin died after IL-2 administration, as well as mice that were deficient in IFN-γ. Mice deficient in TNF, however, survived, indicating that TNF was mediating the toxicity in response to IL-2. There are two different receptors for TNF, p55 and p75. p55 is known as TNFR1 and has been implicated in apoptosis of virally infected cells. P75 is known as TNFR2 and is associated more with inflammation in response to infection. My hypothesis was that if TNFR2 was knocked out, infected mice would survive IL-2 dosing. When single knockouts of TNFR1 and 2 were used in an experiment however, it was found that either receptor is capable of mediating toxicity, as both experimental groups failed to survive. This is relevant to current IL-2 therapies because there is no way to eliminate a single receptor in order to reduce toxicity. Further studies exploring the anti-viral capabilities of IFN-γ are suggested.
ContributorsJarvis, Jordan Alisa (Author) / Blattman, Joseph (Thesis director) / Denzler, Karen (Committee member) / McAfee, Megan (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05
ContributorsSavarese, Erica (Author) / Robert, Jason (Thesis director) / Hurlbut, Ben (Committee member) / Verrelli, Brian (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description
In 2004, the South Korean geneticist Woo-Suk Hwang published what was widely regarded as the most important research result in biotechnology of the year. In the prestigious American journal Science, he claimed that he had succeeded in cloning a human blastocyst, an embryo in its early stages (Hwang et al. 2004). A year later, in a second Science article, he made the earth-shattering announcement that he had derived eleven embryonic stem cell lines using his cloning technique (Hwang et al. 2005). The international scientific community was stunned. American scientists publicly fretted that President George W. Bush‘s 2001 executive order limiting federal funding for stem-cell research in the United States had put American bioscience behind the Koreans‘ (Paarlberg 2005). These breakthroughs offered potential solutions to immune system rejection of transplanted organs and possible cures for diseases such as rheumatoid arthritis, Parkinson‘s, Down‘s syndrome, and paralysis (Svenaeus 2007). However, within a year, Hwang was exposed as a fraud who had faked his results and pressured his female colleagues to donate eggs without informed consent. Despite protests against his methods from Korean religious and nongovernmental organizations, Hwang had used his prestige to ignore his ethical obligations. The Korean government, too, was slow to investigate Hwang and to subject his work to appropriate regulation.
ContributorsClay, Anne (Author) / Hurlbut, James (Thesis director) / Maienschein, Jane (Committee member) / Marchant, Gary (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2012-12
Description
The NCAA recently declared sickle cell trait (SCT) to be a risk factor for sudden illness and death among student athletes. Fetal hemoglobin (HbF) concentration in adults is negatively correlated with disease severity in sickle cell anemia, although its effect on SCT is not fully understood and the concentration is found to have high variability across populations. Two single nucleotide polymorphisms (SNPs) at the human beta globin gene cluster, rs7482144 and rs10128556, contribute to the heritable variation in HbF levels and are associated with increased HbF concentrations in adults. A sample population of NCAA football student athletes was genotyped for these two polymorphisms, and their allele frequencies were compared to those of other populations. The minor allele of both polymorphisms had allele frequencies of 0.091 in the sample population, which compared closely with other populations of recent African heritage but was significantly different from European populations. The results of this study will be included in a larger study to predict whether these among other polymorphisms can be used as markers to predict susceptibility to heat-related emergencies in NCAA student athletes with SCT, although the small sample size will delay this process until participation in the study increases. Since both rs7482144 and rs10128556 exhibit high levels of linkage disequilibrium, and as their contributions to the heritable variability of HbF concentrations tend to differ greatly between populations of different ancestry, further investigations should be aimed at distinguishing between the effects of each SNP in African American, European, and other populations represented in NCAA football before conclusions can be drawn as to their practical use as genetic markers of heat susceptibility in student athletes with SCT.
ContributorsGrieger, Ryan Wayne (Author) / Stone, Anne C. (Thesis director) / Rosenberg, Michael (Committee member) / Madrigal, Lorena (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
Protein is an essential macronutrient in the human diet, but the source of this protein has both human health and environmental impacts. Health complications can result from protein deficiency, but the practices by which protein sources are raised, grown, or harvested have environmental consequences, potentially reducing biodiversity, essential habitat, and crucial stocks of natural resources. Terrestrial cultivation encroaches on natural habitats and consumes resources inefficiently, while overfishing has greatly depleted wild fishery stocks. These environmental factors, along with concerns about nutrients, contaminants and the ethics of animal protein has led to confusion about weighing the risks and benefits associated with alternative sources of protein. Providing consumers \u2014 and policy makers \u2014 with a comprehensive account of major protein sources and their impacts in an understandable form is crucial to reducing environmental degradation and improving human health. Here I provide a general framework to compare the health and environmental impacts of livestock, seafood, and plant protein, and illustrate the application of this framework with case studies for each of these categories.
ContributorsGeren, Sarah Lindsey (Author) / Gerber, Leah (Thesis director) / Smith, Andrew (Committee member) / Minteer, Ben (Committee member) / Barrett, The Honors College (Contributor) / School of Human Evolution and Social Change (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
The transition from high school to college is, for many, a drastic change in lifestyle, social networks, and dietary choices. The prevalence of obesity in college students has been steadily increasing. Freshmen weight gains have been associated with a decrease in fruits and vegetables and an increase in unhealthy items such as desserts, alcohol, and late night snacking after dinner. A survey of college students was constructed to gauge students' perceptions of nutrition how these perceptions influenced dietary practices and behaviors. Survey results indicated that awareness of nutrition and health does not translate to dietary practices, aligning with results from previous studies. Several sex differences were noted in regards to dietary choices and perceptions, knowledge seeking behavior, and sources of information. While there were some similarities, it is clear from the results obtained that men and women have different approaches and thoughts with regard to nutrition. The results showed that college students who actively seek our nutritional information are more likely to do so in the form of social media or Internet sources. This study could be useful for those planning on conducting college-based nutritional programs in that the results indicate patterns and trends that should be taken into consideration in order for a successful nutrition intervention
ContributorsKeahon, Gabriela Estrada (Author) / Jehn, Megan (Thesis director) / Williams, Deborah (Committee member) / Barrett, The Honors College (Contributor) / School of Human Evolution and Social Change (Contributor) / School of Life Sciences (Contributor) / School for the Science of Health Care Delivery (Contributor)
Created2015-05