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- Creators: Barrett, The Honors College
- Creators: Lake, Douglas
Description
The Founders lab is a year-long program that gives its students an opportunity to participate in a unique team-based, experiential Barrett honors thesis project to design and apply marketing and sales strategies, as well as business and financial models to start up and launch a new business. This honors thesis project focuses on increasing the rate of vaccination outcomes in a country where people are increasingly busy (less time) and unwilling to get a needle through a new business venture that provides a service that brings vaccinations straight to businesses, making them available for their employees. Through our work with the Founders Lab, our team was able to create this pitch deck.
ContributorsZatonskiy, Albert (Co-author) / Hanzlick, Emily (Co-author) / Gomez, Isaias (Co-author) / Byrne, Jared (Thesis director) / Hall, Rick (Committee member) / Silverstein, Taylor (Committee member) / Department of Finance (Contributor) / Department of Information Systems (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
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
Abstract Development of a Vaccine for Immunization Against Smallpox and Anthrax Jason Maurice Cameron Biological weapons are often considered to be the most dangerous weapons of mass destruction because of there potential to infect huge numbers of people, who may then in turn infect others who were not even present at the point of initial impact. Among the most feared biological weapons are those that contain smallpox and anthrax because of these diseases' high rates of both infection and death. For this reason, the development of a vaccine that immunizes the receivers against both smallpox and anthrax would be great progress. This study seeks to develop such a vaccine by constructing a recombination plasmid that will introduce new genes that combat anthrax into the strain of vaccinia virus (VV), the virus used to vaccinate against smallpox. This study includes a highly detailed analysis of the various processes used to attempt this recombination and proposes plans further research into the subject.
ContributorsCameron, Jason (Author) / Stout, Valerie (Thesis director) / Jacobs, Bert (Committee member) / Hogan, Genevieve (Committee member) / Barrett, The Honors College (Contributor)
Created2003-05
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
Patients who are infected with the human immunodeficiency virus (HIV) and who remain adherent to their highly active antiretroviral treatment (HAART) regimen are likely to achieve good virologic control over significant periods of time. Children who start with a low CD4 percentage (below 15%) are associated with adverse clinical outcomes and the risk of never increasing their CD4 counts to normal functioning levels. While adherent adult HIV patients have been studied frequently, this was a retrospective chart study that aimed to describe the immune reconstitution pattern for up to 16 years in virologically controlled pediatric patients who had been or who are currently being treated at the Bill Holt Clinic at Phoenix Children's Hospital. In the preliminary study, 35 patients met criteria for inclusion and three years later for this extension study 7 more were added while 17 of the initial patients were followed further because they have remained in care and virologically controlled. All 28 patients who achieved 5 years of viral suppression were >25% CD4. All 8 patients who achieved 12 years of viral suppression were >31% CD4. All patients who achieved 16 years of viral suppression were >41% CD4. After 12 years, the 8 patients who maintained viral suppression all had absolute CD4 counts of over 600 cells and additionally each had CD4/CD8 ratios greater than 1. Overall, the data shows immune system normalization for up to 16 years, although CD4/CD8 ratios improved but never completely normalized. Some limitations include a small sample size and missing data points due to laboratory testing errors or the lack of technology in different countries to test for CD8 cells. These findings suggest that children who remain adherent to HAART can experience ongoing immune healing for up to 16 years. This may provide additional incentive to providers and caretakers to encourage adherence and maximize long-term immune competence in HIV positive children.
ContributorsRichards, Anne Elizabeth (Author) / Wachter, Rebekka (Thesis director) / Blattman, Joseph (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Human Evolution and Social Change (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
Identifying immunoreactive cytotoxic T lymphocytes (CTLs) by current technologies (cytokine secretion, intracellular cytokine, ELISPOT, and MHC tetramer assays) is often difficult when probing for multiple target antigens. CTLs activate and induce apoptosis of pathogenic cells when T-cell receptors (TCRs) specifically bind to antigenic peptides and major histocompatibility complexes (pMHCs) presented on the target cell’s surface. Flow cytometric MHC class I tetramer assays allow for the direct quantification and sorting of most CD8+ T lymphocytes whose TCRs recognize bound peptides, regardless of effector function. Class I tetramers are traditionally produced using BL21-DE3 E. coli expression, denaturation and folding in vitro, which is technically challenging, time-consuming, and low-throughput. We are developing an assay amenable to rapid, high-throughput screening of peptide libraries to characterize and quantitate antigen-specific CTLs in peripheral blood mononuclear cells (PBMCs). Baculovirus expression systems, utilizing host eukaryotic chaperones and isomerases, are capable of producing soluble, properly-folded protein complexes with high yields. The HLA-A*0201 heavy chain and beta-2-microglobulin genes were cloned into pIEx baculovirus expression vectors. Recombinant HLA-A*0201 and β2m viruses were synthesized using the BacMagic-3 DNA/pIEx method and transfected into Spodoptera frugiperda (Sf9) cells, and protein expression was confirmed by Western blot. To prepare T cells for testing, PBMCs from a healthy HLA-A2+ donor were collected and pulsed with DMSO control or CEF peptide pool (a mixture of CMV-, EBV-, and Flu-specific HLA class I epitopes). After 5 days, the CD8+ and CD8- fractions were sorted by MACS-based magnetic separation, and the frequency of FluM1-specific lymphocytes in the CD8+ populations was determined (0.1% of DMSO control vs. 0.772% of CEF-pulsed cells) using a commercial tetramer. We are optimizing HLA-A*0201 and β2m baculovirus co-infection ratios and evaluating the efficiency of intracellular MHC folding.
ContributorsRoesler, Alexander Scott (Author) / Anderson, Karen (Thesis director) / Blattman, Joseph (Committee member) / School of Molecular Sciences (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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
Description
In the United States, a dispute has arisen over the safety and need for vaccination, particularly in regard to compulsory vaccination laws. New outlets and social media sites publish countless reports about the dangers of vaccines or of known adverse reactions as well as imagined or unproven worries. Individuals' rights to choose to get vaccinated or allow their children to be vaccinated comes to direct conflict with measures needed to protect communities from preventable viral diseases. The controversy surrounding vaccines is not new, nor necessarily are the fundamental reasons for skepticism. Looking back through the history of vaccines as a medical tool, the evolution of the controversy can be observed taking place with each new historical context, scientific development, and social conditions. Despite scientific research and assurances of vaccine safety, opposition and unease about vaccination appear to take Looking individually at the development and distribution of the smallpox (variola virus), polio (poliovirus) and human papilloma virus(HPV) vaccines, concerns regarding the violation of personal rights, safety of vaccines themselves, and social stigmas and connotations surrounding vaccines can be seen to evolve and change. Due to the way doubt can manifest in different ways over time, it may be impossible to fully end the vaccine debate. However, nderstanding the sociological factors behind anti-vaccine sentiment may allow healthcare professionals to work with concerned people with a particular care to address these visceral and sometimes irrational fears surrounding vaccination.
ContributorsStevens, Luke Christian (Author) / Jacobs, Bertram (Thesis director) / Washo-Krupps, Delon (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2014-05