This collection includes both ASU Theses and Dissertations, submitted by graduate students, and the Barrett, Honors College theses submitted by undergraduate students. 

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Description
Transient Receptor Potential (TRP) ion channels are a diverse family of nonselective, polymodal sensors in uni- and multicellular eukaryotes that are implicated in an assortment of biological contexts and human disease. The cold-activated TRP Melastatin-8 (TRPM8) channel, also recognized as the human body's primary cold sensor, is among the few

Transient Receptor Potential (TRP) ion channels are a diverse family of nonselective, polymodal sensors in uni- and multicellular eukaryotes that are implicated in an assortment of biological contexts and human disease. The cold-activated TRP Melastatin-8 (TRPM8) channel, also recognized as the human body's primary cold sensor, is among the few TRP channels responsible for thermosensing. Despite sustained interest in the channel, the mechanisms underlying TRPM8 activation, modulation, and gating have proved challenging to study and remain poorly understood. In this thesis, I offer data collected on various expression, extraction, and purification conditions tested in E. Coli expression systems with the aim to optimize the generation of a structurally stable and functional human TRPM8 pore domain (S5 and S6) construct for application in structural biology studies. These studies, including the biophysical technique nuclear magnetic spectroscopy (NMR), among others, will be essential for elucidating the role of the TRPM8 pore domain in in regulating ligand binding, channel gating, ion selectively, and thermal sensitivity. Moreover, in the second half of this thesis, I discuss the ligation-independent megaprimer PCR of whole-plasmids (MEGAWHOP PCR) cloning technique, and how it was used to generate chimeras between TRPM8 and its nearest analog TRPM2. I review steps taken to optimize the efficiency of MEGAWHOP PCR and the implications and unique applications of this novel methodology for advancing recombinant DNA technology. I lastly present preliminary electrophysiological data on the chimeras, employed to isolate and study the functional contributions of each individual transmembrane helix (S1-S6) to TRPM8 menthol activation. These studies show the utility of the TRPM8\u2014TRPM2 chimeras for dissecting function of TRP channels. The average current traces analyzed thus far indicate that the S2 and S3 helices appear to play an important role in TRPM8 menthol modulation because the TRPM8[M2S2] and TRPM8[M2S3] chimeras significantly reduce channel conductance in the presence of menthol. The TRPM8[M2S4] chimera, oppositely, increases channel conductance, implying that the S4 helix in native TRPM8 may suppress menthol modulation. Overall, these findings show that there is promise in the techniques chosen to identify specific regions of TRPM8 crucial to menthol activation, though the methods chosen to study the TRPM8 pore independent from the whole channel may need to be reevaluated. Further experiments will be necessary to refine TRPM8 pore solubilization and purification before structural studies can proceed, and the electrophysiology traces observed for the chimeras will need to be further verified and evaluated for consistency and physiological significance.
ContributorsWaris, Maryam Siddika (Author) / Van Horn, Wade (Thesis director) / Redding, Kevin (Committee member) / School of Molecular Sciences (Contributor) / Department of English (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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Description
Higher plant Rubisco activase (Rca) is a stromal ATPase responsible for reactivating Rubisco. It is a member of the AAA+ protein superfamily and is thought to assemble into closed-ring hexamers like other AAA+ proteins belonging to the classic clade. Progress towards modeling the interaction between Rca and Rubisco has been

Higher plant Rubisco activase (Rca) is a stromal ATPase responsible for reactivating Rubisco. It is a member of the AAA+ protein superfamily and is thought to assemble into closed-ring hexamers like other AAA+ proteins belonging to the classic clade. Progress towards modeling the interaction between Rca and Rubisco has been slow due to limited structural information on Rca. Previous efforts in the lab were directed towards solving the structure of spinach short-form Rca using X-ray crystallography, given that it had notably high thermostability in the presence of ATP-γS, an ATP analog. However, due to disorder within the crystal lattice, an atomic resolution structure could not be obtained, prompting us to move to negative stain electron microscopy (EM), with our long-term goal being the use of cryo-electron microscopy (cryo-EM) for atomic resolution structure determination. Thus far, we have screened different Rca constructs in the presence of ATP-γS, both the full-length β-isoform and truncations containing only the AAA+ domain. Images collected on preparations of the full-length protein were amorphous, whereas images of the AAA+ domain showed well-defined ring-like assemblies under some conditions. Procedural adjustments, such as the use of previously frozen protein samples, rapid dilution, and minimizing thawing time were shown to improve complex assembly. The presence of Mn2+ was also found to improve hexamer formation over Mg2+. Calculated class averages of the AAA+ Rca construct in the presence of ATP-γS indicated a lack of homogeneity in the assemblies, showing both symmetric and asymmetric hexameric rings. To improve structural homogeneity, we tested buffer conditions containing either ADP alone or different ratios of ATP-γS to ADP, though results did not show a significant improvement in homogeneity. Multiple AAA+ domain preparations were evaluated. Because uniform protein assembly is a major requirement for structure solution by cryo-EM, more work needs to be done on screening biochemical conditions to optimize homogeneity.
ContributorsHernandez, Victoria Joan (Author) / Wachter, Rebekka (Thesis director) / Chiu, Po-Lin (Committee member) / Redding, Kevin (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Description
Antiviral lectins are potential candidates for future therapies against enveloped viruses like HIV due to their ability to recognize and bind glycans displayed on their surface. Cyanovirin-N (CVN), a lectin that specifically recognizes mannose-rich moieties, serves as a useful model for studying these glycan-recognition mechanisms. This study seeks to improve

Antiviral lectins are potential candidates for future therapies against enveloped viruses like HIV due to their ability to recognize and bind glycans displayed on their surface. Cyanovirin-N (CVN), a lectin that specifically recognizes mannose-rich moieties, serves as a useful model for studying these glycan-recognition mechanisms. This study seeks to improve CVN's glycan-binding affinity by conjugating a boronic acid functional group to the N-terminus via N-terminal specific reductive alkylation by way of a benzaldehyde handle. However, large discrepancies were observed when attempting to confirm a successful conjugation, and further work is necessary to identify the causes and solutions for these issues.
ContributorsDiep, Tristan H (Author) / Ghirlanda, Giovanna (Thesis director) / Redding, Kevin (Committee member) / Mills, Jeremy (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
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Description
The FoF1 ATP synthase is a molecular motor critical to the metabolism of virtually all life forms, and it acts in the manner of a hydroelectric generator. The F1 complex contains an (αβ)3 (hexamer) ring in which catalysis occurs, as well as a rotor comprised by subunit-ε in addition to

The FoF1 ATP synthase is a molecular motor critical to the metabolism of virtually all life forms, and it acts in the manner of a hydroelectric generator. The F1 complex contains an (αβ)3 (hexamer) ring in which catalysis occurs, as well as a rotor comprised by subunit-ε in addition to the coiled-coil and globular foot domains of subunit-γ. The F1 complex can hydrolyze ATP in vitro in a manner that drives counterclockwise (CCW) rotation, in 120° power strokes, as viewed from the positive side of the membrane. The power strokes that occur in ≈ 300 μsec are separated by catalytic dwells that occur on a msec time scale. A single-molecule rotation assay that uses the intensity of polarized light, scattered from a 75 × 35 nm gold nanorod, determined the average rotational velocity of the power stroke (ω, in degrees/ms) as a function of the rotational position of the rotor (θ, in degrees, measured in reference to the catalytic dwell). The velocity is not constant but rather accelerates and decelerates in two Phases. Phase-1 (0° - 60°) is believed to derive power from elastic energy in the protein. At concentrations of ATP that limit the rate of ATP hydrolysis, the rotor can stop for an ATP-binding dwell during Phase-1. Although the most probable position that the ATP-binding dwell occurs is 40° after the catalytic dwell, the ATP-binding dwell can occur at any rotational position during Phase-1 of the power stroke. Phase-2 of the power stroke (60° - 120°) is believed to be powered by the ATP-binding induced closure of the lever domain of a β-subunit (as it acts as a cam shaft against the γ-subunit). Algorithms were written, to sort and analyze F1-ATPase power strokes, to determine the average rotational velocity profile of power strokes as a function of the rotational position at which the ATP-binding dwell occurs (θATP-bd), and when the ATP-binding dwell is absent. Sorting individual ω(θ) curves, as a function of θATP-bd, revealed that a dependence of ω on
θATP-bd exists. The ATP-binding dwell can occur even at saturating ATP concentrations. We report that ω follows a distinct pattern in the vicinity of the ATP-binding dwell, and that the ω(θ) curve contains the same oscillations within it regardless of θATP-bd. We observed that an acceleration/deceleration dependence before and after the ATP-binding dwell, respectively, remained for increasing time intervals as the dwell occurred later in Phase-1, to a maximum of ≈ 40°. The results were interpreted in terms of a model in which the ATP-binding dwell results from internal drag at a variable position on the γε rotor.
ContributorsBukhari, Zain Aziz (Author) / Frasch, Wayne D. (Thesis director) / Allen, James P. (Committee member) / Redding, Kevin (Committee member) / School of Molecular Sciences (Contributor) / Department of Physics (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
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Description
The goal of this investigation was to perform a correlational analysis of the intelligence mindsets, motivational background, and significance of gender identity as factors driving student success. 42 students enrolled in Computer Science and Engineering (CSE) 110: Principles of Programming with Java completed a modified Scientific Measurement Questionnaire (SMQ), a

The goal of this investigation was to perform a correlational analysis of the intelligence mindsets, motivational background, and significance of gender identity as factors driving student success. 42 students enrolled in Computer Science and Engineering (CSE) 110: Principles of Programming with Java completed a modified Scientific Measurement Questionnaire (SMQ), a survey instrument designed to study the previously mentioned factors. This survey was modeled on a similar survey administered by Dr. Ian Gould to students enrolled in his Organic Chemistry course at Arizona State University. Following the development of a scoring system to generate quantifiable data, it was determined that students in this course displayed a greater inclination towards beliefs in malleable intelligence and in an intrinsic locus of control as opposed to a belief in static intelligence and an external locus of control. Students exhibited a multi-faceted approach in responding to the questions in the motivational background section, indicating that there were no distinctively dominating factors driving student motivation. Instead, it was observed that students generally derived motivation from these factors in a synergistic fashion. Responses to questions regarding gender indicated that while students believed that the way they were perceived by others was significantly influenced by their gender, the notion of gender identity played little to no role in their overall personal identity and self-schema. As the study was designed to offer insight into the role of gender identity and the population discrepancies within the course, it is important to note that the findings suggest gender identity is not a primary factor of concern with regard to student performance. While the data acquired suggested potential trends in student mindsets, a notable limitation of the scope of the project was the undersized sample population.
ContributorsLevinthal, Ryan (Co-author) / Santos, Cedric (Co-author) / Gould, Ian (Thesis director) / Redding, Kevin (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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Description
Acyl Carrier Protein (ACP) is a small, acidic protein that plays an essential role in fatty acid synthesis by elongating fatty acid chains. ACP was isolated from an extract of a modified strain of Synechocystis sp. PCC 6803 that contains a thioesterase and from which the acyl-ACP synthetase has been

Acyl Carrier Protein (ACP) is a small, acidic protein that plays an essential role in fatty acid synthesis by elongating fatty acid chains. ACP was isolated from an extract of a modified strain of Synechocystis sp. PCC 6803 that contains a thioesterase and from which the acyl-ACP synthetase has been deleted. Using ammonium sulfate precipitation to isolate a crude protein fraction containing ACP, immunoblot analysis was performed to determine relative amounts of free and acylated-ACP in the cell. The nature of fatty acids attached to ACP was determined by creating butylamide derivatives that were analyzed using GC/MS. Immunoblot analysis showed a roughly 1:1 ratio of acylated ACP to free ACP in the cell depending on the nutritional state of the cell. From GC/MS data it was determined that palmitic acid was the predominate component of acyl groups attached to ACP. The results indicate that there is a significant amount of acyl-ACP, a feedback inhibitor of early steps in the fatty acid biosynthesis pathway, in the cell. Moreover, the availability of free ACP may also limit fatty acid biosynthesis. Most likely it is necessary for ACP to be overexpressed or to have the palmitic acid cleaved off in order to synthesize optimal amounts of lauric acid to be used for cyanobacterial biofuel production.
ContributorsWu, Sharon Gao (Author) / Vermaas, Willem (Thesis director) / Redding, Kevin (Committee member) / School of Sustainability (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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Description
With a quantum efficiency of nearly 100%, the electron transfer process that occurs within the reaction center protein of the photosynthetic bacteria Rhodobacter (Rh.) sphaeroides is a paragon for understanding the complexities, intricacies, and overall systemization of energy conversion and storage in natural systems. To better understand the way in

With a quantum efficiency of nearly 100%, the electron transfer process that occurs within the reaction center protein of the photosynthetic bacteria Rhodobacter (Rh.) sphaeroides is a paragon for understanding the complexities, intricacies, and overall systemization of energy conversion and storage in natural systems. To better understand the way in which photons of light are captured, converted into chemically useful forms, and stored for biological use, an investigation into the reaction center protein, specifically into its cascade of cofactors, was undertaken. The purpose of this experimentation was to advance our knowledge and understanding of how differing protein environments and variant cofactors affect the spectroscopic aspects of and electron transfer kinetics within the reaction of Rh. sphaeroides. The native quinone, ubiquinone, was extracted from its pocket within the reaction center protein and replaced by non-native quinones having different reduction/oxidation potentials. It was determined that, of the two non-native quinones tested—1,2-naphthaquinone and 9,10- anthraquinone—the substitution of the anthraquinone (lower redox potential) resulted in an increased rate of recombination from the P+QA- charge-separated state, while the substitution of the napthaquinone (higher redox potential) resulted in a decreased rate of recombination.
ContributorsSussman, Hallie Rebecca (Author) / Woodbury, Neal (Thesis director) / Redding, Kevin (Committee member) / Lin, Su (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12
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Description

Hybrid metalloproteins incorporating synthetic organometallic active sites within a protein scaffold are being researched as viable catalysts for the production of hydrogen fuel. Our group and others have shown that the incorporation of cobalt protoporphyrin IX in cytochrome b₅₆₂ yields artificial enzymes that reduce protons to molecular hydrogen in the

Hybrid metalloproteins incorporating synthetic organometallic active sites within a protein scaffold are being researched as viable catalysts for the production of hydrogen fuel. Our group and others have shown that the incorporation of cobalt protoporphyrin IX in cytochrome b₅₆₂ yields artificial enzymes that reduce protons to molecular hydrogen in the presence of photoinductive light and photosensitizers. Using random mutagenesis via error-prone PCR we have created a library of mutants to use in directed evolution to optimize hydrogen catalysis, though a challenge in this project is that testing individual variants by gas chromatography is not feasible on a large scale. For this reason, we are developing a gasochromic, hydrogen assay that is based on the interaction of molecular hydrogen with tungsten trioxide with a palladium catalyst. Initially, results show this assay to be qualitatively accurate between trials; however, its application in screening remains a challenge.

ContributorsGutierrez, Elijah (Author) / Ghirlanda, Giovana (Thesis director) / Mills, Jeremy (Committee member) / Redding, Kevin (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor)
Created2022-05
Description

The incidence of childhood obesity has become increasingly prevalent in the United States in recent years. The development of obesity at any age, but especially in adolescence, can have lasting negative effects in the form of cardiometabolic disease, increased incurred healthcare costs, and potential negative effects on quality of life.

The incidence of childhood obesity has become increasingly prevalent in the United States in recent years. The development of obesity at any age, but especially in adolescence, can have lasting negative effects in the form of cardiometabolic disease, increased incurred healthcare costs, and potential negative effects on quality of life. In recent years, a rising trend of obesity, in both adults and adolescents, has been observed in lower income and ethnic groups. Increased adiposity can be influenced by modifiable factors -(physical activity, caloric intake, or sleep) or by non-modifiable factors (ethnicity, genetic predispositions, and socioeconomic status). The influence of these factors can be observed in individuals of all ages, including infants. A common indicator of the development of childhood obesity is rapid weight gain (RWG) within an infant’s first year of life. The composition of the gut microbiome can act as a predictor for RWG and the development of childhood obesity. Infants are exposed to an immense microbial load when they are born and their gut microbiome is continually diversified through their method of feeding and the subsequent introduction to solid foods. While currently understudied, it is understood that cultural and socioeconomic factors influence the development of the gut microbiome, which is further explored in this analysis. The DNA from 51 fecal samples from infants ranging from 3 weeks to 12 months in age was extracted and sequenced using next-generation sequencing, and the resulting sequences were analyzed using QIIME 2. Results from alpha-diversity and beta-diversity metrics showed significant differences in the gut microbiome of infants when comparing groups based on baby race/ethnicity, household income, and mom’s education. These findings suggest the importance of sociodemographic characteristics in shaping the gut microbiome and suggest the importance of future studies including diverse populations in gut microbiome work.

ContributorsGallello, Chloe (Author) / Whisner, Corrie (Thesis director) / Petrov, Megan (Committee member) / Redding, Kevin (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor)
Created2023-05
Description

Oceanic life is facing the deleterious aftermath of coral bleaching. To reverse the damages introduced by anthropological means, it is imperative to study fundamental properties of corals. One way to do so is to understand the metabolic pathways and protein functions of corals that contribute to the resilience of coral

Oceanic life is facing the deleterious aftermath of coral bleaching. To reverse the damages introduced by anthropological means, it is imperative to study fundamental properties of corals. One way to do so is to understand the metabolic pathways and protein functions of corals that contribute to the resilience of coral reefs. Although genomic sequencing and structural modeling has yielded significant insights for well-studied organisms, more investigation must be conducted for corals. Better yet, quantifiable experiments are far more crucial to the understanding of corals. The objective is to clone, purify, and assess coral proteins from the cauliflower coral species known as Pocillopora damicornis. Presented here is the pipeline for how 3-D structural modeling can help support the experimental data from studying soluble proteins in corals. Using a multi-step selection approach, 25 coral genes were selected and retrieved from the genomic database. Using Escherischia coli and Homo sapiens homologues for sequence alignment, functional properties of each protein were predicted to aid in the production of structural models. Using D-SCRIPT, potential pairwise protein-protein interactions (PPI) were predicted amongst these 25 proteins, and further studied for identifying putative interfaces using the ClusPro server. 10 binding pockets were inferred for each pair of proteins. Standard cloning strategies were applied to express 4 coral proteins for purification and functional assays. 2 of the 4 proteins had visible bands on the Coomassie stained gel and were able to advance to the purification step. Both proteins exhibited a faint band at the expected migration distance for at least one of the elutions. Finally, PPI was carried out by mixing protein samples and running in a native gel, resulting in one potential pair of PPI.

ContributorsHuang, Joe (Author) / Klein-Seetharaman, Judith (Thesis director) / Fromme, Petra (Committee member) / Redding, Kevin (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor)
Created2023-05