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Background: High risk types of human papillomavirus (HPV) are known to cause cancer, including cervical (99%) and oropharyngeal cancer (70%). HPV type 16 is the most common subtype. Three antigens that are critical for integration or tumor progression are E2, E6 and E7. In this study, we developed a systematic

Background: High risk types of human papillomavirus (HPV) are known to cause cancer, including cervical (99%) and oropharyngeal cancer (70%). HPV type 16 is the most common subtype. Three antigens that are critical for integration or tumor progression are E2, E6 and E7. In this study, we developed a systematic approach to identify naturally-processed HPV16-derived HLA class I epitopes for immunotherapy development. Methods: K562 cells, which lack HLA expression, were transduced with each HPV16 antigen using lentivirus and supertransfected with HLA-A2 by nucleofection. Stable cell lines expressing each antigen were selected for and maintained throughout the investigation. In order to establish a Gateway-compatible vector for robust transient gene expression, a Gateway recombination expression cloning cassette was inserted into the commercial Lonza pMAX GFP backbone, which has been experimentally shown to display high transfection expression efficiency. GFP was cloned into the vector and plain K562 cells were transfected with the plasmid by nucleofection. Results: Expression of K562-A2 was tested at various time points by flow cytometry and A2 expression was confirmed. Protein expression was shown for the transduced K562 E7 by Western blot analysis. High transfection efficiency of the pMAX_GFP_Dest vector (up to 97% GFP+ cells) was obtained 48 hours post transfection, comparable to the commercial GFP-plasmid. Conclusion: We have established a rapid system for target viral antigen co-expression with single HLA molecules for analysis of antigen presentation. Using HPV as a model system, our goal is to identify specific antigenic peptide sequences to develop immunotherapeutic treatments for HPV-associated cancers.
ContributorsVarda, Bianca Marie (Author) / Anderson, Karen (Thesis director) / Borges, Chad (Committee member) / Krishna, Sri (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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Description
Protein AMPylation is a recently discovered and relatively unstudied post-translational modification (PTM). AMPylation has previously been shown to play an important role in metabolic regulation and host pathogenesis in bacteria, but the recent identification of potential AMPylators across many species in every domain of life has supported the possibility that

Protein AMPylation is a recently discovered and relatively unstudied post-translational modification (PTM). AMPylation has previously been shown to play an important role in metabolic regulation and host pathogenesis in bacteria, but the recent identification of potential AMPylators across many species in every domain of life has supported the possibility that AMPylation could be a more fundamental and physiologically significant regulatory PTM. For the first time, we characterized the auto-AMPylation capability of the human protein SOS1 through in vitro AMPylation experiments using full-length protein and whole-domain truncation mutants. We found that SOS1 can become AMPylated at a tyrosine residue possibly within the Cdc25 domain of the protein, the Dbl homology domain is vital for efficient auto-AMPylation activity, and the C-terminal proline-rich domain exhibits a complex regulatory function. The proline-rich domain alone also appears to be capable of catalyzing a separate, unidentified covalent self-modification using a fluorescent ATP analogue. Finally, SOS1 was shown to be capable of catalyzing the AMPylation of two endogenous human protein substrates: a ubiquitous, unidentified protein of ~49kDa and another breast-cancer specific, unidentified protein of ~28kDa.
ContributorsOber-Reynolds, Benjamin John (Author) / LaBaer, Joshua (Thesis director) / Borges, Chad (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2014-05
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Description
In this thesis, glycan nodes, the basic subunits of complex biological sugars, were studied to determine the reproducibility of gas chromatography-mass spectrometry (GC/MS) based methylation analysis of whole blood plasma by normalization using an internal standard of heavy permethylated glycans. Glycans are complex biological sugars that have a variety of

In this thesis, glycan nodes, the basic subunits of complex biological sugars, were studied to determine the reproducibility of gas chromatography-mass spectrometry (GC/MS) based methylation analysis of whole blood plasma by normalization using an internal standard of heavy permethylated glycans. Glycans are complex biological sugars that have a variety of applications in the human body and will display aberrant compositions when produced by cancerous cells. Thus an assay to determine their composition can be used as a diagnostic tool. It was shown that the assay may have potential use, but needs further refinement to become an improvement over current methods by analyzing the results of ratio-determination and replicate experiments.
ContributorsMiyasaki, Tyler Takeo (Author) / Borges, Chad (Thesis director) / Van Horn, Wade (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Chemical Engineering Program (Contributor)
Created2015-05
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Description
Western diets, high in dietary fat and red meat, are associated with hyperglycemia and weight gain, symptoms that promote insulin resistance and diabetes. Previous studies have shown that elevated glucose promotes glycation of circulating proteins such as albumin, which is thought to lead to hyperglycemia complications. It was hypothesized that

Western diets, high in dietary fat and red meat, are associated with hyperglycemia and weight gain, symptoms that promote insulin resistance and diabetes. Previous studies have shown that elevated glucose promotes glycation of circulating proteins such as albumin, which is thought to lead to hyperglycemia complications. It was hypothesized that diets with no meat consumption (pesco-vegetarian and lacto-vegetarian) would reduce protein glycation, in comparison to a diet with meat. Forty six healthy adult omnivorous subjects were randomized into one of three groups and instructed to either consume red meat (i.e. meat) or poultry twice per day (control), eliminate meat and increase fish consumption (pesco-vegetarian), or adopt a vegetarian diet devoid of fish, meat or poultry (lacto-vegetarian) for four weeks. Fasting plasma samples were collected from participants at baseline and after 4 weeks of the dietary intervention. Plasma glucose concentrations were measured using a commercially available kit. Percent glycated albumin was measured on a separate aliquot of plasma by mass spectrometry. Plasma glucose concentrations were significantly increased following 4-weeks of pesco-vegetarian diet (P=0.002, paired t-test). Neither the lacto-vegetarian (P=0.898) or the control diet (P=0.233) affected plasma glucose concentrations. Despite the significant increase in plasma glucose following a pesco-vegetarian diet, no change in percent glycated albumin was observed (P>0.50, ANOVA). These findings may indicate a protective effect of the pesco-vegetarian diet on protein glycation in the presence of elevated plasma glucose and suggest the need for additional studies to examine the link between increased fish consumption and glucose regulation.
ContributorsRaad, Noor (Author) / Sweazea, Karen (Thesis director, Committee member) / Borges, Chad (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
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Description

This paper discusses the theoretical approximation and attempted measurement of the quantum <br/>force produced by material interactions though the use of a tuning fork-based atomic force microscopy <br/>device. This device was built and orientated specifically for the measurement of the Casimir force as a <br/>function of separation distance using a

This paper discusses the theoretical approximation and attempted measurement of the quantum <br/>force produced by material interactions though the use of a tuning fork-based atomic force microscopy <br/>device. This device was built and orientated specifically for the measurement of the Casimir force as a <br/>function of separation distance using a piezo actuator for approaching and a micro tuning fork for the <br/>force measurement. This project proceeds with an experimental measurement of the ambient Casmir force <br/>through the use of a tuning fork-based AFM to determine its viability in measuring the magnitude of the <br/>force interaction between an interface material and the tuning fork probe. The ambient measurements <br/>taken during the device’s development displayed results consistent with theoretical approximations, while<br/>demonstrating the capability to perform high-precision force measurements. The experimental results<br/>concluded in a successful development of a device which has the potential to measure forces of <br/>magnitude 10−6 to 10−9 at nanometric gaps. To conclude, a path to material analysis using an approach <br/>stage, alternative methods of testing, and potential future experiments are speculated upon.

ContributorsMulkern, William Michael (Author) / Wang, Liping (Thesis director) / Kwon, Beomjin (Committee member) / Mechanical and Aerospace Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
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Description

This paper investigates near-field thermal radiation as the primary source of heat transfer between two parallel surfaces. This radiation takes place extremely close to the heated surfaces in study so the experimental set-up to be used will be done at the nanometer scale. The primary theory being investigated is that

This paper investigates near-field thermal radiation as the primary source of heat transfer between two parallel surfaces. This radiation takes place extremely close to the heated surfaces in study so the experimental set-up to be used will be done at the nanometer scale. The primary theory being investigated is that near-field radiation generates greater heat flux that conventional radiation governed by Planck’s law with maximum for blackbodies. Working with a phase shift material such as VO2 enables a switch-like effect to occur where the total amount of heat flux fluctuates as VO2 transitions from a metal to an insulator. In this paper, the theoretical heat flux and near-field radiation effect are modeled for a set-up of VO2 and SiO2 layers separated by different vacuum gaps. In addition, a physical experimental set-up is validated for future near-field radiation experiments.

ContributorsSluder, Nicole (Author) / Wang, Liping (Thesis director) / Wang, Ropert (Committee member) / Mechanical and Aerospace Engineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
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Description
Dielectrophoresis is a separations strategy that has the potential to separate small amounts of different proteins from each other. The forces at play in the channel used for dielectrophoresis are electroosmotic flow (EOF), electrophoresis (EP), and dielectrophoresis (DEP). EOF is the force exerted on liquid from an applied potential (1).

Dielectrophoresis is a separations strategy that has the potential to separate small amounts of different proteins from each other. The forces at play in the channel used for dielectrophoresis are electroosmotic flow (EOF), electrophoresis (EP), and dielectrophoresis (DEP). EOF is the force exerted on liquid from an applied potential (1). EP is the force exerted on charged particles in a uniform electric field (2). DEP is the force exerted on particles (charged and uncharged) in a non-uniform electric field (3). This experiment was focused on the testing of a new microfluidic device to see if it could improve the focusing of proteins in dielectrophoresis. It was predicted that the addition of a salt bridge would improve focusing by preventing the ions created by the electrolysis of water around the electrodes from interacting with the proteins and causing aggregation, among other problems. Control trials using the old device showed that electrolysis was likely occurring and was the causal agent for poor outcomes. After applying the electric potential for some time a pH front traveled through the channel causing aggregation of proteins and the current in the channel decreased rapidly, even while the voltage was held constant. The resistance in the channels of the control trials also slightly decreased over time, until the pH shift occurred, at which time it increased rapidly. Experimental trials with a new device that included salt bridges eliminated this pH front and had a roughly linear increase of current in the channel with the voltage applied. This device can now be used in future research with protein dielectrophoresis, including in the potential differentiation of different proteins. References: 1) Electroosmosis. Oxford Dictionary of Biochemistry and Molecular Biology. 2. Oxford University Press: Oxford, England. 2006. 2) Electrophoresis. Oxford Dictionary of Biochemistry and Molecular Biology. 2. Oxford University Press: Oxford, England. 2006. 3) Dielectrophoresis. Oxford Dictionary of Biochemistry and Molecular Biology. 2. Oxford University Press: Oxford, England. 2006.
ContributorsHayes, Katelyn Donna (Author) / Hayes, Mark (Thesis director) / Borges, Chad (Committee member) / School of Life Sciences (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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Description

Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which

Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.

ContributorsDeb, Arpan (Author) / Johnson, William (Author) / Kline, Alexander (Author) / Scott, Boston (Author) / Meador, Lydia (Author) / Srinivas, Dustin (Author) / Martin Garcia, Jose Manuel (Author) / Dorner, Katerina (Author) / Borges, Chad (Author) / Misra, Rajeev (Author) / Hogue, Brenda (Author) / Fromme, Petra (Author) / Mor, Tsafrir (Author) / ASU Biodesign Center Immunotherapy, Vaccines and Virotherapy (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Life Sciences (Contributor) / Biodesign Institute (Contributor) / School of Molecular Sciences (Contributor) / Applied Structural Discovery (Contributor) / Personalized Diagnostics (Contributor)
Created2017-02-22
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Description
The rise in community-associated methicillin-resistant Staphylococcus aureus (MRSA) infections and the ability of the organism to develop resistance to antibiotics necessitate new treatment methods for MRSA. Geopolymers (GPs) are cheap, porous materials that have demonstrated adsorptive capabilities. In this study, GPs were investigated for their ability to adsorb whole MRSA

The rise in community-associated methicillin-resistant Staphylococcus aureus (MRSA) infections and the ability of the organism to develop resistance to antibiotics necessitate new treatment methods for MRSA. Geopolymers (GPs) are cheap, porous materials that have demonstrated adsorptive capabilities. In this study, GPs were investigated for their ability to adsorb whole MRSA cells and MRSA secreted proteins [culture filtrate proteins (CFPs)] as a complementary method of controlling MRSA infections. GPs have been synthesized with variable pore sizes (meso/macro scale) and further modified with stearic acid (SA) to increase surface hydrophobicity. Four GPs (SA-macroGP, macroGP, SA-mesoGP, and mesoGP) were incubated with whole cells and with CFPs to quantify GP adsorption capabilities. Following MRSA culture incubation with GPs, unbound MRSA cells were filtered and plated to determine cell counts. Following CFP incubation with GPs, unbound CFPs were separated via SDS-PAGE, stained with SYPRO Ruby, and analyzed using densitometry. Results indicate that macroGP was the most effective at adsorbing whole MRSA cells. Visual banding patterns and densitometry quantitation indicate that SA-mesoGP was the most effective at adsorbing CFP. Ultimately, GP-based products may be further developed as nonselective or selective adsorbents and integrated into fibrous materials for topical applications.
ContributorsGanser, Collin (Co-author, Co-author) / Haydel, Shelley E. (Thesis director) / Seo, Don (Committee member) / Borges, Chad (Committee member) / School of Earth and Space Exploration (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
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Description
In the development of personalized medicine and many other clinical studies, biospecimen integrity serves as the prerequisite for not only the accurate derivation of patient- and disease-specific molecular data from biological specimens but the meaningful downstream validation of biomarkers. However, a large number of preanalytical variables may influence the quality

In the development of personalized medicine and many other clinical studies, biospecimen integrity serves as the prerequisite for not only the accurate derivation of patient- and disease-specific molecular data from biological specimens but the meaningful downstream validation of biomarkers. However, a large number of preanalytical variables may influence the quality of biospecimens in an undesired way and ultimately render the samples unsuitable for molecular analysis. The limited ability to directly reduce discrepancies caused by preanalytical variables gives rise to the need for development and retrospective application of appropriate tests for assessment of biospecimen integrity. Nevertheless, the most standard approaches to assessing biospecimen integrity involve nontrivial procedures. Thus, the need for quality control tools or tests that are readily applicable and can produce results in a straightforward way becomes critical. As one of the major ex vivo biomolecular degradation mechanisms, oxidation that occurs when blood plasma and serum samples are exposed to thawed states during storage and processing is hard to forestall and detect. In an attempt to easily detect and monitor the degree of oxidation, the technique of Fluorescence Resonance Energy Transfer (FRET) was examined to determine whether this concept could be employed to monitor exposure of samples to thawed conditions when controlled by spontaneous oxidative disulfide bonding. The intended mode of usage was envisioned as a fluorescence liquid being stored in a separate compartment but within the same test tube as archived plasma and serum samples. This would allow the assessment of sample integrity by direct visualization of fluorescence under a hand-held black light. The fluorescent dynamic range as well as kinetic control of the reaction were studied. While the addition of Cu(II) proved to facilitate excellent dynamic range with regard to fluorescence quenching, the kinetics of the reaction were too rapid for practical use. Further investigation revealed that the fluorescence quenching mechanism might have actually occurred via Intramolecular Charge Transfer (ICT) rather than FRET mediated by oxidative disulfide bond formation. Introduction of Cu(II) via copper metal slowed fluorescence quenching to the point of practical utility; facilitating demonstration that storing at room temperature, refrigerating or freezing the samples delayed fluorescence quenching to different extents. To establish better kinetic control, future works will focus on establishing controlled, thoroughly understood kinetic release of Cu(II) from copper metal.
ContributorsZhang, Zihan (Author) / Borges, Chad (Thesis director) / Emady, Heather (Committee member) / Williams, Peter (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12