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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
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
In our exponentially expanding world, the knowledge of a group versus that of an individual is more relevant than ever. Social insects have evolved to rely on the information from the collective, and in the case of harvester ants, their choice revolves around the best seeds to collect.
The objective of this experiment is to study a colony’s seed preference following previous exposure to a seed type in the seed harvester ant Pogonomyrmex californicus. It was hypothesized that foragers would demonstrate a measurable preference for the seed type they had previously experienced over the novel seed type. The cuticular hydrocarbon profile is suspected to be an influence in the foragers’ seed selection. Following an incubation period with the designated seed type, a series of preference trials were conducted over the course of two days for two experiments in which each colony fragment was given a seed pile with a 1:1 ratio of niger and sesame, after which any seeds moved off the seed pile were determined to be chosen, as well as if the workers were observed moving the seeds off the pile from the video recordings. Using video recordings, the seed selections of individual foragers were also tracked. The results partially support the hypothesis, however, in some cases, the ants did not collect enough seeds for the preference to be significant, and not all colony fragments had preferences that lined up with what they had previously experienced according to their treatment. Familiarity with the hydrocarbon profile of the seed type the colony had experienced is a possible proximal explanation for why colonies had seed preferences that aligned with their treatment, the seed they were designated to experience. Due to the low quantity of seeds collected during preference trials, seed preference amongst individual foragers remains unclear due to many different foragers selecting a seed during only one trial, with very few foragers returning to forage for seeds over the course of the experiment.
The objective of this experiment is to study a colony’s seed preference following previous exposure to a seed type in the seed harvester ant Pogonomyrmex californicus. It was hypothesized that foragers would demonstrate a measurable preference for the seed type they had previously experienced over the novel seed type. The cuticular hydrocarbon profile is suspected to be an influence in the foragers’ seed selection. Following an incubation period with the designated seed type, a series of preference trials were conducted over the course of two days for two experiments in which each colony fragment was given a seed pile with a 1:1 ratio of niger and sesame, after which any seeds moved off the seed pile were determined to be chosen, as well as if the workers were observed moving the seeds off the pile from the video recordings. Using video recordings, the seed selections of individual foragers were also tracked. The results partially support the hypothesis, however, in some cases, the ants did not collect enough seeds for the preference to be significant, and not all colony fragments had preferences that lined up with what they had previously experienced according to their treatment. Familiarity with the hydrocarbon profile of the seed type the colony had experienced is a possible proximal explanation for why colonies had seed preferences that aligned with their treatment, the seed they were designated to experience. Due to the low quantity of seeds collected during preference trials, seed preference amongst individual foragers remains unclear due to many different foragers selecting a seed during only one trial, with very few foragers returning to forage for seeds over the course of the experiment.
ContributorsNewton, Natalie Nicole (Author) / Fewell, Jennifer (Thesis director) / Steven, Pratt (Committee member) / Ioulia, Bespalova (Committee member) / School of Art (Contributor) / School of Life Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Coffee is an important link between the United States and Latin America and an important part of Latin America’s culture and economy. This paper looks at the similarities and differences between coffee organizations in Colombia, Ecuador, Peru, and Guatemala. Colombia has the strongest coffee organizations with the most political power. Guatemala and Peru, to a lesser extent, have well organized and powerful organizations that make up their industry. However, Ecuador has a significantly less organized organization. At their core, each country has a similar structure. There is one organization on the national level that watches out for the industry as a whole. Underneath that, there are smaller, often regional organizations made up of cooperatives pooling their resources for export. They function in similar ways as the national organizations, but have less reach. At the bottom, there are individual cooperatives and independent farmers. These cooperatives do not have much reach or connection to international markets.
ContributorsChabin, James Edward (Author) / Janssen, Marco (Thesis director) / Taylor, Keith (Committee member) / School of Sustainability (Contributor) / School of International Letters and Cultures (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
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
Description
Studies of cooperation remain an important aspect in understanding the evolution of social cues and interactions. One example of cooperation is pleometrosis, an associative behavior of forming a colony with two unrelated, fertile queens. However, most ant species display haplometrosis, the founding of a colony by a single queen. In these associations, the queen typically rejects cooperation. In populations of Pogonomyrmex californicus, both pleometrosis and haplometrosis exists. It is not clear how associative -metrosis became a practiced behavior since haplometrotic queens tend to fight. However, as fighting in pleometrotic queens became less frequent, this induces benefit, in terms of cost savings, in having associative behaviors. The hypothesis tested was nest excavation of pleometrotic queens show sociality, while haplometrotic queens show association independence. Isolated pleometrotic queens (P) showed low excavation rate at 2.72cm2/day, compared to the rate when the task was shared in (PP) nests, 4.57cm2/day. Nest area of the (P) queens were also affected during days 3 and 4 of the experiment, where there was presence of nest area decrease. Furthermore, the excavation session of (P) was the only one determined as significant between all other nests. Although the (P) queens have low values, they eventually reach a similar point as the other nests by day 6. However, the lack of haste in excavation leads to longer exposure to the elements, substituting the risk of losing cuticles in excavation for the risk of predation. For the haplometrotic queens, nests of (H) and (HH) displayed no significant difference in excavation values, leading to having social effect in their association.
ContributorsGabriel, Ian Paulo Villalobos (Author) / Fewell, Jennifer (Thesis director) / Pratt, Stephen (Committee member) / Bespalova, Ioulia (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
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 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
Description
In order to survive, species must regulate their intake of nutrients. In desert leafcutter ant colonies, acquisition of nutrients is not only important for maintaining the health of the colony, but also for the survival of a fungus which the ants cultivate and then consume. This multi-trophic, symbiotic relationship is relatively unique to leafcutter ants and interesting to researchers due to the complexity of how the individual foragers supply nutrients to both the colony and the fungus. The objective of this experiment is to study foraging rates and variation in macronutrient preference among foragers from the same colony of the desert leafcutter ant Acromyrmex versicolor. This study asks if individual foragers vary in their preference of protein to carbohydrate ratios when compared to the overall nutrient content of the colony, and how do these individuals respond as the nutrient content of the available diets increasingly deviates from the previously determined nutritional intake target ratio between 1 Protein:6.3 Carbohydrates to 1 Protein:7.5 Carbohydrates. It was hypothesized that foragers express individualized nutritional preferences that in aggregate balance colony macronutrient consumption, and the number of individuals collecting the diets would decrease as the available nutritional diets deviated away from the colony-level intake target of approximately 1P:7C. The results show trends that support the hypothesis that the number of foraging instances and the number of foragers who exhibit individualized preference towards a certain protein to carbohydrate ratio is highest when the colony is presented with diets that are closest to the colony nutritional intake target.
ContributorsGlovsky, Taylor Ella (Author) / Fewell, Jennifer (Thesis director) / Harrison, Jon (Committee member) / School of Life Sciences (Contributor) / Sanford School of Social and Family Dynamics (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Biomarkers are the cornerstone of modern-day medicine. They are defined as any biological substance in or outside the body that gives insight to the body's condition. Doctors and researchers can measure specific biomarkers to diagnose and treat patients, such as the concentration of hemoglobin Alc and its connection to diabetes. There are a variety of methods, or assays, to detect biomarkers, but the most common assay is enzyme-linked immunosorbent assay (ELISA). A new-generation assay termed mass spectrometric immunoassay (MSIA) can measure proteoforms, the different chemical variations of proteins, and their relative abundance. ELISA on the other hand measures the overall concentration of protein in the sample. Measuring each of the proteoforms of a protein is important because only one or two variations could be biologically significant and/or cause diseases. However, running MSIA is expensive. For this reason, an alternative plate-based MSIA technique was tested for its ability to detect the proteoforms of a protein called apolipoprotein C-III (ApoC-III). This technique combines the protein capturing procedure of ELISA to isolate the protein with detection in a mass spectrometer. A larger amount of ApoC-III present in the body indicates a considerable risk for coronary heart disease. The precision of the assay is determined on the coefficient of variation (CV). A CV value is the ratio of standard deviation in relation to the mean, represented as a percentage. The smaller the percentage, the less variation the assay has, and therefore the more ability it has to detect subtle changes in the biomarker. An accepted CV would be less than 10% for single-day tests (intra-day) and less than 15% for multi-day tests (inter-day). The plate-based MSIA was started by first coating a 96-well round bottom plate with 2.5 micrograms of ApoC-III antibody. Next, a series of steps were conducted: a buffer wash, then the sample incubation, followed by another buffer wash and two consecutive water washes. After the final wash, the wells were filled with a MALDI matrix, then spotted onto a gold plate to dry. The dry gold target was then placed into a MALDI-TOF mass spectrometer to produce mass spectra for each spot. The mass spectra were calibrated and the area underneath each of the four peaks representing the ApoC-III proteoforms was exported as an Excel file. The intra-day CV values were found by dividing the standard deviation by the average relative abundance of each peak. After repeating the same procedure for three more days, the inter-day CVs were found using the same method. After completing the experiment, the CV values were all within the acceptable guidelines. Therefore, the plate-based MSIA is a viable alternative for finding proteoforms than the more expensive MSIA tips. To further validate this, additional tests will need to be conducted with different proteins and number of samples to determine assay flexibility.
ContributorsTieu, Luc (Author) / Borges, Chad (Thesis director) / Nedelkov, Dobrin (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2017-12
Description
Brown adipose tissue (BAT) is thought to be important in combating obesity as it can expend energy in the form of heat, e.g. thermogenesis. The goal of this study was to study the effect of injected norepinephrine (NE) on the activation of BAT in rats that were fed a high fat diet (HFD). A dose of 0.25 mg/kg NE was used to elicit a temperature response that was measured using transponders inserted subcutaneously over the BAT and lower back and intraperitoneally to measure the core temperature. The results found that the thermic effect of the BAT increased after the transition from low fat diet to a high fat diet (LFD) yet, after prolonged exposure to the HFD, the effects resembled levels found with the LFD. This suggests that while a HFD may stimulate the effect of BAT, long term exposure may have adverse effects on BAT activity. This may be due to internal factors that will need to be examined further.
ContributorsSion, Paul William (Author) / Herman, Richard (Thesis director) / Borges, Chad (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05