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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 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
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 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
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 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
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 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
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). 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
Description
Mathematical epidemiology, one of the oldest and richest areas in mathematical biology, has significantly enhanced our understanding of how pathogens emerge, evolve, and spread. Classical epidemiological models, the standard for predicting and managing the spread of infectious disease, assume that contacts between susceptible and infectious individuals depend on their relative frequency in the population. The behavioral factors that underpin contact rates are not generally addressed. There is, however, an emerging a class of models that addresses the feedbacks between infectious disease dynamics and the behavioral decisions driving host contact. Referred to as “economic epidemiology” or “epidemiological economics,” the approach explores the determinants of decisions about the number and type of contacts made by individuals, using insights and methods from economics. We show how the approach has the potential both to improve predictions of the course of infectious disease, and to support development of novel approaches to infectious disease management.
ContributorsPerrings, Charles (Author) / Castillo-Chavez, Carlos (Author) / Chowell-Puente, Gerardo (Author) / Daszak, Peter (Author) / Fenichel, Eli P. (Author) / Finnoff, David (Author) / Horan, Richard D. (Author) / Kilpatrick, A. Marm (Author) / Kinzig, Ann (Author) / Kuminoff, Nicolai (Author) / Levin, Simon (Author) / Morin, Benjamin (Author) / Smith, Katherine F. (Author) / Springborn, Michael (Author) / Simon M. Levin Mathematical, Computational and Modeling Sciences Center (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Life Sciences (Contributor) / School of Human Evolution and Social Change (Contributor) / W.P. Carey School of Business (Contributor) / Economics (Contributor) / Julie Ann Wrigley Global Institute of Sustainability (Contributor)
Created2015-12-01
Description
Background
Seroepidemiological studies before and after the epidemic wave of H1N1-2009 are useful for estimating population attack rates with a potential to validate early estimates of the reproduction number, R, in modeling studies.
Methodology/Principal Findings
Since the final epidemic size, the proportion of individuals in a population who become infected during an epidemic, is not the result of a binomial sampling process because infection events are not independent of each other, we propose the use of an asymptotic distribution of the final size to compute approximate 95% confidence intervals of the observed final size. This allows the comparison of the observed final sizes against predictions based on the modeling study (R = 1.15, 1.40 and 1.90), which also yields simple formulae for determining sample sizes for future seroepidemiological studies. We examine a total of eleven published seroepidemiological studies of H1N1-2009 that took place after observing the peak incidence in a number of countries. Observed seropositive proportions in six studies appear to be smaller than that predicted from R = 1.40; four of the six studies sampled serum less than one month after the reported peak incidence. The comparison of the observed final sizes against R = 1.15 and 1.90 reveals that all eleven studies appear not to be significantly deviating from the prediction with R = 1.15, but final sizes in nine studies indicate overestimation if the value R = 1.90 is used.
Conclusions
Sample sizes of published seroepidemiological studies were too small to assess the validity of model predictions except when R = 1.90 was used. We recommend the use of the proposed approach in determining the sample size of post-epidemic seroepidemiological studies, calculating the 95% confidence interval of observed final size, and conducting relevant hypothesis testing instead of the use of methods that rely on a binomial proportion.
Seroepidemiological studies before and after the epidemic wave of H1N1-2009 are useful for estimating population attack rates with a potential to validate early estimates of the reproduction number, R, in modeling studies.
Methodology/Principal Findings
Since the final epidemic size, the proportion of individuals in a population who become infected during an epidemic, is not the result of a binomial sampling process because infection events are not independent of each other, we propose the use of an asymptotic distribution of the final size to compute approximate 95% confidence intervals of the observed final size. This allows the comparison of the observed final sizes against predictions based on the modeling study (R = 1.15, 1.40 and 1.90), which also yields simple formulae for determining sample sizes for future seroepidemiological studies. We examine a total of eleven published seroepidemiological studies of H1N1-2009 that took place after observing the peak incidence in a number of countries. Observed seropositive proportions in six studies appear to be smaller than that predicted from R = 1.40; four of the six studies sampled serum less than one month after the reported peak incidence. The comparison of the observed final sizes against R = 1.15 and 1.90 reveals that all eleven studies appear not to be significantly deviating from the prediction with R = 1.15, but final sizes in nine studies indicate overestimation if the value R = 1.90 is used.
Conclusions
Sample sizes of published seroepidemiological studies were too small to assess the validity of model predictions except when R = 1.90 was used. We recommend the use of the proposed approach in determining the sample size of post-epidemic seroepidemiological studies, calculating the 95% confidence interval of observed final size, and conducting relevant hypothesis testing instead of the use of methods that rely on a binomial proportion.
ContributorsNishiura, Hiroshi (Author) / Chowell-Puente, Gerardo (Author) / Castillo-Chavez, Carlos (Author) / Simon M. Levin Mathematical, Computational and Modeling Sciences Center (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Human Evolution and Social Change (Contributor)
Created2011-03-24
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 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
Description
Complex human controls is a topic of much interest in the fields of robotics, manufacturing, space exploration and many others. Even simple tasks that humans perform with ease can be extremely complicated when observed from a controls and complex systems perspective. One such simple task is that of a human carrying and moving a coffee cup. Though this may be a mundane task for humans, when this task is modelled and analyzed, the system may be quite chaotic in nature. Understanding such systems is key to the development robots and autonomous systems that can perform these tasks themselves.
The coffee cup system can be simplified and modeled by a cart-and-pendulum system. Bazzi et al. and Maurice et al. present two different cart-and-pendulum systems to represent the coffee cup system [1],[2]. The purpose of this project was to build upon these systems and to gain a better understanding of the coffee cup system and to determine where chaos existed within the system. The honors thesis team first worked with their senior design group to develop a mathematical model for the cart-and-pendulum system based on the Bazzi and Maurice papers [1],[2]. This system was analyzed and then built upon by the honors thesis team to build a cart-and-two-pendulum model to represent the coffee cup system more accurately.
Analysis of the single pendulum model showed that there exists a low frequency region where the pendulum and the cart remain in phase with each other and a high frequency region where the cart and pendulum have a π phase difference between them. The transition point of the low and high frequency region is determined by the resonant frequency of the pendulum. The analysis of the two-pendulum system also confirmed this result and revealed that differences in length between the pendulum cause the pendulums to transition to the high frequency regions at separate frequency. The pendulums have different resonance frequencies and transition into the high frequency region based on their own resonant frequency. This causes a range of frequencies where the pendulums are out of phase from each other. After both pendulums have transitioned, they remain in phase with each other and out of phase from the cart.
However, if the length of the pendulum is decreased too much, the system starts to exhibit chaotic behavior. The short pendulum starts to act in a chaotic manner and the phase relationship between the pendulums and the carts is no longer maintained. Since the pendulum length represents the distance between the particle of coffee and the top of the cup, this implies that coffee near the top of the cup would cause the system to act chaotically. Further analysis would be needed to determine the reason why the length affects the system in this way.
The coffee cup system can be simplified and modeled by a cart-and-pendulum system. Bazzi et al. and Maurice et al. present two different cart-and-pendulum systems to represent the coffee cup system [1],[2]. The purpose of this project was to build upon these systems and to gain a better understanding of the coffee cup system and to determine where chaos existed within the system. The honors thesis team first worked with their senior design group to develop a mathematical model for the cart-and-pendulum system based on the Bazzi and Maurice papers [1],[2]. This system was analyzed and then built upon by the honors thesis team to build a cart-and-two-pendulum model to represent the coffee cup system more accurately.
Analysis of the single pendulum model showed that there exists a low frequency region where the pendulum and the cart remain in phase with each other and a high frequency region where the cart and pendulum have a π phase difference between them. The transition point of the low and high frequency region is determined by the resonant frequency of the pendulum. The analysis of the two-pendulum system also confirmed this result and revealed that differences in length between the pendulum cause the pendulums to transition to the high frequency regions at separate frequency. The pendulums have different resonance frequencies and transition into the high frequency region based on their own resonant frequency. This causes a range of frequencies where the pendulums are out of phase from each other. After both pendulums have transitioned, they remain in phase with each other and out of phase from the cart.
However, if the length of the pendulum is decreased too much, the system starts to exhibit chaotic behavior. The short pendulum starts to act in a chaotic manner and the phase relationship between the pendulums and the carts is no longer maintained. Since the pendulum length represents the distance between the particle of coffee and the top of the cup, this implies that coffee near the top of the cup would cause the system to act chaotically. Further analysis would be needed to determine the reason why the length affects the system in this way.
ContributorsZindani, Abdul Rahman (Co-author) / Crane, Kari (Co-author) / Lai, Ying-Cheng (Thesis director) / Jiang, Junjie (Committee member) / Electrical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2019-12
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 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