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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 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
Description
The goal of our study is to identify socio-economic risk factors for depressive disorder and poor mental health by statistically analyzing survey data from the CDC. The identification of risk groups in a particular demographic could aid in the development of targeted interventions to improve overall quality of mental health in the United States. In our analysis, we studied the influences and correlations of socioeconomic factors that regulate the risk of developing Depressive Disorders and overall poor mental health. Using the statistical software STATA, we ran a regression model of selected independent socio-economic variables with the dependent mental health variables. The independent variables of the statistical model include Income, Race, State, Age, Marital Status, Sex, Education, BMI, Smoker Status, and Alcohol Consumption. Once the regression coefficients were found, we illustrated the data in graphs and heat maps to qualitatively provide visuals of the prevalence of depression in the U.S. demography. Our study indicates that the low-income and under-educated populations who are everyday smokers, obese, and/or are in divorced or separated relationships should be of main concern. A suggestion for mental health organizations would be to support counseling and therapeutic efforts as secondary care for those in smoking cessation programs, weight management programs, marriage counseling, or divorce assistance group. General improvement in alleviating poverty and increasing education could additionally show progress in counter-acting the prevalence of depressive disorder and also improve overall mental health. The identification of these target groups and socio-economic risk factors are critical in developing future preventative measures.
ContributorsGrassel, Samuel (Co-author) / Choueiri, Alexi (Co-author) / Choueiri, Robert (Co-author) / Goegan, Brian (Thesis director) / Holter, Michael (Committee member) / Sandra Day O'Connor College of Law (Contributor) / School of Molecular Sciences (Contributor) / School of Politics and Global Studies (Contributor) / Economics Program in CLAS (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
This study investigates how the patient-provider relationship between lesbian, gay, and bisexual women and their healthcare providers influences their access to, utilization of, and experiences within healthcare environments. Nineteen participants, ages 18 to 34, were recruited using convenience and snowball sampling. Interviews were conducted inquiring about their health history and their experiences within the healthcare system in the context of their sexual orientation. The data collected from these interviews was used to create an analysis of the healthcare experiences of those who identify as queer. Although the original intention of the project was to chronicle the experiences of LGB women specifically, there were four non-binary gender respondents who contributed interviews. In an effort to not privilege any orientation over another, the respondents were collectively referred to as queer, given the inclusive and an encompassing nature of the term. The general conclusion of this study is that respondents most often experienced heterosexism rather than outright homophobia when accessing healthcare. If heterosexism was present within the healthcare setting, it made respondents feel uncomfortable with their providers and less likely to inform them of their sexuality even if it was medically relevant to their health outcomes. Gender, race, and,socioeconomic differences also had an effect on the patient-provider relationship. Non-binary respondents acknowledged the need for inclusion of more gender options outside of male or female on the reporting forms often seen in medical offices. By doing so, medical professionals are acknowledging their awareness and knowledge of people outside of the binary gender system, thus improving the experience of these patients. While race and socioeconomic status were less relevant to the context of this study, it was found that these factors have an affect on the patient-provider relationship. There are many suggestions for providers to improve the experiences of queer patients within the healthcare setting. This includes nonverbal indications of acknowledgement and acceptance, such as signs in the office that indicate it to be a queer friendly space. This will help in eliminating the fear and miscommunication that can often happen when a queer patient sees a practitioner for the first time. In addition, better education on medically relevant topics to queer patients, is necessary in order to eliminate disparities in health outcomes. This is particularly evident in trans health, where specialized education is necessary in order to decrease poor health outcomes in trans patients. Future directions of this study necessitate a closer look on how race and socioeconomic status have an effect on a queer patient's relationship with their provider.
ContributorsRajan, Sindhu (Co-author) / Bradley, Arianna (Co-author) / Larson, Michelle (Co-author) / Alvarado, Aimee (Co-author) / Ingram-Waters, Mary (Thesis director) / Arnold, Michelle (Committee member) / School of Molecular Sciences (Contributor) / School of Social Transformation (Contributor) / T. Denny Sanford School of Social and Family Dynamics (Contributor) / College of Public Service and Community Solutions (Contributor) / School of Human Evolution and Social Change (Contributor) / School of Historical, Philosophical and Religious Studies (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Exploring Structure and Function of Human Cold Sensing Protein TRPM8 with ROSETTA Comparative Models
Description
Transient Receptor Potential (TRP) channels are a diverse class of ion channels notable as polymodal sensors. TRPM8 is a TRP channel implicated in cold sensation, nociception, and a variety of human diseases, including obesity and cancer. Despite sustained interest in TRPM8 since its discovery in 2001, many of the molecular mechanisms that underlie function are not yet clear. Knowledge of these properties could have implications for medicine and physiological understanding of sensation and signaling. Structures of TRP channels have proven challenging to solve, but recent Cryoelectron microscopy (Cryo-EM) structures of TRPV1 provide a basis for homology-based modeling of TRP channel structures and interactions. I present an ensemble of 11,000 Rosetta computational homology models of TRPM8 based on the recent Cryo-EM apo structure of TRPV1 (PDB code:3J5P). Site-directed mutagenesis has provided clues about which residues are most essential for modulatory ligands to bind, so the models presented provide a platform to investigate the structural basis of TRPM8 ligand modulation complementary to existing functional and structural information. Menthol and icilin appear to interact with interfacial residues in the sensor domain (S1-S4). One consensus feature of these sites is the presence of local contacts to the S4 helix, suggesting this helix may be mechanistically involved with the opening of the pore. Phosphatidylinositol 4,5-bisphosphate (PIP2)has long been known to interact with the C-terminus of TRPM8, and some of the homology models contain plausible binding pockets where PIP2 can come into contact with charged residues known to be essential for PIP2 modulation. Future in silico binding experiments could provide testable hypothesis for in vitro structural studies, and experimental data (e.g. distance constraints from electron paramagnetic resonance spectroscopy [EPR]) could further refine the models.
ContributorsHelsell, Cole Vincent Maher (Author) / Van Horn, Wade (Thesis director) / Wang, Xu (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Description
Home Base Initiative is a student-led venture project co-founded by Madison Sutton and Sonia Sabrowsky in January 2018. As an organization, Home Base Initiative addresses the problem of teen suicide by educating parents, teachers, and students about the research-backed mental health resources currently available to them and by implementing peer-based support programs in local high schools. With the belief that positive mental health habits are for everyone, not just individuals with a clinical diagnosis, Home Base Initiative aims to encourage positive conversations about mental health and to increase social and emotional resilience among adolescents to help them navigate the challenges in their lives. In addition to identifying the community problem our organization aims to solve, this document outlines the initial conception, development, and future outlook Home Base Initiative by describing the methods by which the organization has researched other like-minded programs, formed strategic partnerships with community members, piloted its peer-based program at a local high school, and established a foundation for future success as a student organization at Arizona State University. Currently, the Home Base Initiative team consists of 10 undergraduate students at ASU with diverse backgrounds and academic interests as well as credible mentors who are involved in the ASU Tillman Scholars Program, ASU Counseling Services, and The Courage Lab at ASU. We are united by our passion for supporting others’ mental health, and we are dedicated to playing an active role in the healthy development of our fellow community members through mental health advocacy and the facilitation of positive peer-to-peer interactions.
ContributorsSabrowsky, Sonia (Co-author, Co-author) / Sutton, Madison (Co-author) / Mokwa, Michael (Thesis director) / Eaton, John (Committee member) / School of Molecular Sciences (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
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 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.
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
Description
Na+/H+ antiporters are vital membrane proteins for cell homeostasis, transporting Na+ ions in exchange for H+ across the lipid bilayer. In humans, dysfunction of these transporters are implicated in hypertension, heart failure, epilepsy, and autism, making them well-established drug targets. Although experimental structures for bacterial homologs of the human Na+/H+ have been obtained, the detailed mechanism for ion transport is still not well-understood. The most well-studied of these transporters, Escherichia coli NhaA, known to transport 2 H+ for every Na+ extruded, was recently shown to bind H+ and Na+ at the same binding site, for which the two ion species compete. Using molecular dynamics simulations, the work presented in this dissertation shows that Na+ binding disrupts a previously-unidentified salt bridge between two conserved residues, suggesting that one of these residues, Lys300, may participate directly in transport of H+. This work also demonstrates that the conformational change required for ion translocation in a homolog of NhaA, Thermus thermophilus NapA, thought by some to involve only small helical movements at the ion binding site, is a large-scale, rigid-body movement of the core domain relative to the dimerization domain. This elevator-like transport mechanism translates a bound Na+ up to 10 Å across the membrane. These findings constitute a major shift in the prevailing thought on the mechanism of these transporters, and serve as an exciting launchpad for new developments toward understanding that mechanism in detail.
ContributorsDotson, David L (Author) / Beckstein, Oliver (Thesis advisor) / Ozkan, Sefika B (Committee member) / Ros, Robert (Committee member) / Van Horn, Wade (Committee member) / Arizona State University (Publisher)
Created2016
Description
The two chapters of this thesis focus on different aspects of DNA and the properties of nucleic acids as the whole. Chapter 1 focuses on the structure of DNA and its relationship to enzymatic efficiency. Chapter 2 centers itself on threose nucleic acid and optimization of a step in the path to its synthesis. While Chapter 1 discusses DNA and Uracil-DNA Glycosylase with regards to the base excision repair pathway, Chapter 2 focuses on chemical synthesis of an intermediate in the pathway to the synthesis of TNA, an analogous structure with a different saccharide in the sugar-phosphate backbone.
Chapter 1 covers the research under Dr. Levitus. Four oligonucleotides were reacted for zero, five, and thirty minutes with uracil-DNA glycosylase and subsequent addition of piperidine. These oligonucleotides were chosen based on their torsional rigidities as predicted by past research and predictions. The objective was to better understand the relationship between the sequence of DNA surrounding the incorrect base and the enzyme’s ability to remove said base in order to prepare the DNA for the next step of the base excision repair pathway. The first pair of oligonucleotides showed no statistically significant difference in enzymatic efficiency with p values of 0.24 and 0.42, while the second pair had a p value of 0.01 at the five-minute reaction. The second pair is currently being researched at different reaction times to determine at what point the enzyme seems to equilibrate and react semi-equally with all sequences of DNA.
Chapter 2 covers the research conducted under Dr. Chaput. Along the TNA synthesis pathway, the nitrogenous base must be added to the threofuranose sugar. The objective was to optimize the original protocol of Vorbrüggen glycosylation and determine if there were better conditions for the synthesis of the preferred regioisomer. This research showed that toluene and ortho-xylene were more preferable as solvents than the original anhydrous acetonitrile, as the amount of preferred isomer product far outweighed the amount of side product formed, as well as improving total yield overall. The anhydrous acetonitrile reaction had a final yield of 60.61% while the ortho-xylene system had a final yield of 94.66%, an increase of approximately 32%. The crude ratio of preferred isomer to side product was also improved, as it went from 18% undesired in anhydrous acetonitrile to 4% undesired in ortho-xylene, both values normalized to the preferred regioisomer.
Chapter 1 covers the research under Dr. Levitus. Four oligonucleotides were reacted for zero, five, and thirty minutes with uracil-DNA glycosylase and subsequent addition of piperidine. These oligonucleotides were chosen based on their torsional rigidities as predicted by past research and predictions. The objective was to better understand the relationship between the sequence of DNA surrounding the incorrect base and the enzyme’s ability to remove said base in order to prepare the DNA for the next step of the base excision repair pathway. The first pair of oligonucleotides showed no statistically significant difference in enzymatic efficiency with p values of 0.24 and 0.42, while the second pair had a p value of 0.01 at the five-minute reaction. The second pair is currently being researched at different reaction times to determine at what point the enzyme seems to equilibrate and react semi-equally with all sequences of DNA.
Chapter 2 covers the research conducted under Dr. Chaput. Along the TNA synthesis pathway, the nitrogenous base must be added to the threofuranose sugar. The objective was to optimize the original protocol of Vorbrüggen glycosylation and determine if there were better conditions for the synthesis of the preferred regioisomer. This research showed that toluene and ortho-xylene were more preferable as solvents than the original anhydrous acetonitrile, as the amount of preferred isomer product far outweighed the amount of side product formed, as well as improving total yield overall. The anhydrous acetonitrile reaction had a final yield of 60.61% while the ortho-xylene system had a final yield of 94.66%, an increase of approximately 32%. The crude ratio of preferred isomer to side product was also improved, as it went from 18% undesired in anhydrous acetonitrile to 4% undesired in ortho-xylene, both values normalized to the preferred regioisomer.
ContributorsTamirisa, Ritika Sai (Author) / Levitus, Marcia (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Windman, Todd (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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 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
Description
The purpose of the study was to examine the associations of protective (ethic identity, parent-child closeness) and risk (perceived discrimination, parent-child role reversal) factors with mental and behavioral health in 2nd generation Cambodian American (CA) young adults. A total of 66 participants who identified as being 2nd generation CA young adults aged 18-25 years old were recruited to participate in this cross-sectional. Reliable and valid measures were used to assess protective and risk factors and mental (depressive, anxiety, somatic symptoms) and behavioral health outcomes (alcohol and drug use). We used descriptive statistics to describe sample characteristics and study variables and conducted multiple regression analysis to examine the associations of factors with each of the 5 health outcomes. The findings suggested that peer discrimination was positively and significantly associated with depressive (β = 0.42, p = 0.023; R2 = 0.397) and somatic symptoms (β = 0.63, p = 0.000, R2 = 0.554). Father role-reversal was also found to be negatively and significantly associated with predicting CA young adults’ anxiety symptoms (β = -0.32, p = 0.005, R2 = 0.456).
Majority of the CA young adults have perceived racial/ethnic discrimination in the community. Furthermore, perceived discrimination has been positively associated with their depressive and somatic symptoms, suggesting a need to address racial/ethnic discrimination issues to promote positive mental health in this population. It is important for school/work personnel and healthcare providers to assess CA young adults’ discrimination experiences, and have the sufficient resources (e.g., education, support groups) to prevent negative consequences associated with discrimination.
Majority of the CA young adults have perceived racial/ethnic discrimination in the community. Furthermore, perceived discrimination has been positively associated with their depressive and somatic symptoms, suggesting a need to address racial/ethnic discrimination issues to promote positive mental health in this population. It is important for school/work personnel and healthcare providers to assess CA young adults’ discrimination experiences, and have the sufficient resources (e.g., education, support groups) to prevent negative consequences associated with discrimination.
ContributorsOu, Jason Heng (Author) / Chen, Angela Chia-Chia (Thesis director) / Sangalang, Cindy (Committee member) / Miroballi, Barbara (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12