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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
With the new independence of adulthood, college students are a group susceptible to adopting unsupported, if not harmful, health practices. A survey of Arizona State University undergraduate students (N=200) was conducted to evaluate supplement use, trust in information sources, and beliefs about supplement regulation. Of those who reported using supplements, college students most frequently received information from friends and family. STEM majors in fields unrelated to health who were taking a supplement were found to be less likely to receive information about the supplement from a medical practitioner than those in health fields or those in non-STEM majors (-26.9%, p=0.018). STEM majors in health-related fields were 15.0% more likely to treat colds and/or cold symptoms with research-supported methods identified from reliable sources, while non-health STEM and non-STEM majors were more likely to take unsupported cold treatments (p=0.010). Surveyed students, regardless of major, also stated they would trust a medical practitioner for supplement advice above other sources (88.0%), and the majority expressed a belief that dietary supplements are approved/regulated by the government (59.8%).
ContributorsPerez, Jacob Tanner (Author) / Hendrickson, Kirstin (Thesis director) / Lefler, Scott (Committee member) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Department of Physics (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Structure is a critical component in drug development. This project supports antibody- facilitated structure determination for the following eleven membrane proteins: the human histamine and dopamine G protein-coupled receptors (HRH4 and DRD2) involved in a wide variety of pathologies such as allergies, inflammation, asthma, pain along with Parkinson's and schizophrenia respectively, the human cystic fibrosis transmembrane conductance regulator (CFTR), the human NaV1.8 voltage-gated sodium ion channel, the human TPC2 two-pore channel, the SARS virus proteins 3a, E and M, the MERS virus protein E and M, and the malarial chloroquine resistance transporter (PfCRT). Serum antibodies against these proteins were generated by genetic immunization, and both in vitro and in vivo expressed membrane proteins were created to characterize the serum antibodies. Plasmid clones were generated for genetic immunization, in vitro protein expression, and in vivo expression (HEK293T transfection). Serum antibodies were generated by genetic immunization of mice by gene gun. Genetic immunization promotes an immune response that allows for the generation of antibodies in the absence of purified protein. In vitro expression was accomplished through the novel technique: in vitro translation with hydrophobic magnetic beads (IVT-HMB). Transfections were performed using the HEK293T cell line to express the protein in vivo. The generated protein was then used in gel electrophoresis and silver stain and/or Western blot analyses to identify and visualize the proteins. These expressed proteins will allow for forthcoming characterization of the generated antibodies. The resulting antibodies will in turn enable structure determination of these important membrane proteins by co-crystallization.
ContributorsDrotar, Beniamin (Author) / Fromme, Petra (Thesis director) / Hansen, Debra T. (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
G protein-coupled receptors, or GPCRs, are receptors located within the membrane of cells that elicit a wide array of cellular responses through their interactions with G proteins. Recent advances in the use of lipid cubic phase (LCP) for the crystallization of GPCRs, as well as increased knowledge of techniques to improve receptor stability, have led to a large increase in the number of available GPCR structures, despite historic difficulties. This project is focused on the histamine family of receptors, which are Class A GPCRs that are involved in the body’s allergic and inflammatory responses. In particular, the goal of this project was to design, express, and purify histamine receptors with the ultimate goal of crystallization. Successive rounds of optimization included the use of recombinant DNA techniques in E.coli to truncate sections of the proteins and the insertion of several fusion partner proteins to improve receptor expression and stability. All constructs were expressed in a Bac-to-Bac baculovirus expression system using Sf9 insect cells, solubilized using n-Dodecyl-β-D-Maltoside (DDM), and purified using immobilized metal affinity chromatography. Constructs were then analyzed by SDS-Page, Western blot, and size-exclusion chromatography to determine their presence, purity, and homogeneity. Along with their expression data from insect cells, the most stable and homogeneous construct from each round was used to design successive optimizations. After 3 rounds of construct design for each receptor, much work remains to produce a stable sample that has the potential to crystallize. Future work includes further optimization of the insertion site of the fusion proteins, ligand screening for co-crystallization, optimization of purification conditions, and screening of potential thermostabilizing point mutations. Success in solving a structure will allow for a more detailed understanding of the receptor function in addition to its vital use in rational drug discovery.
ContributorsCosgrove, Steven Andrew (Author) / Liu, Wei (Thesis director) / Mills, Jeremy (Committee member) / Mazor, Yuval (Committee member) / W. P. Carey School of Business (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
U.S. border colonias, otherwise known as Disadvantaged Unincorporated Communities, are rural settlements along the U.S. Mexico border with substandard housing conditions. Colonia residents often face inadequate access to necessities such as appropriate shelter, septic and sewer systems, and potable water. Water insecurity in colonias poses a particularly difficult challenge for residents who require clean water not only for consumption, but also household use in sanitation and hygienic practices. As of 2015, an estimated 30% of over five million US colonia residents lack access to clean drinking water, resulting in health complications and unsanitary living conditions. Preliminary health data collected indicates that due to water insecurity, colonia residents are more likely to contract gastrointestinal disease, be exposed to carcinogenic compounds from contaminated water, and experience psychosocial distress. Yet more comprehensive research needs to be conducted to understand the full breadth of the public health issue. A scoping review on water insecurity in colonias has not been completed before and could be beneficial in informing policymakers and other stakeholders on the severity of the situation while advising possible solutions.
ContributorsZheng, Madeleine (Author) / Wutich, Amber (Thesis director) / Brewis, Alexandra (Committee member) / Kavouras, Stavros (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor)
Created2021-12
Description
Transient receptor potential (TRP) channels are a superfamily of ion channels found in plasma membranes of both single-celled and multicellular organisms. TRP channels all share the common aspect of having six transmembrane helices and a TRP domain. These structures tetramerize to form a receptor-activated non-selective ion channel. The specific protein being investigated in this thesis is the human transient receptor potential melastatin 8 (hTRPM8), a channel activated by the chemical ligand menthol and temperatures below 25 °C. TRPM8 is responsible for cold sensing and is related to pain relief associated with cooling compounds. TRPM8 has also been found to play a role in the regulation of various types of tumors. The structure of TRPM8 has been obtained through cryo-electron microscopy, but the functional contribution of individual portions of the protein to the overall protein function is unknown.
To gain more information about the function of the transmembrane region of hTRPM8, it was expressed in Escherichia coli (E. coli) and purified in detergent membrane mimics for experimentation. The construct contains the S4-S5 linker, pore domain (S5 and S6 transmembrane helices), pore helix, and TRP box. hTRPM8-PD+ was purified in the detergents n-Dodecyl-B-D-Maltoside (DDM), 16:0 Lyso PG, 1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphoglycerol (LPPG), and 14:0 Lyso PG, 1-Myristoyl-2-hydroxy-sn-glycero-3-phosphoglycerol (LMPG) to determine which detergent resulted in a hTRPM8-PD+ sample of the most stability, purity, and highest concentrations. Following bacterial expression and protein purification, hTRPM8-PD+ was studied and characterized with circular dichroism (CD) spectroscopy to learn more about the secondary structures and thermodynamic properties of the construct. Further studies can be done with more circular dichroism (CD) spectroscopy, planar lipid bilayer (BLM) electrophysiology, and nuclear magnetic resonance spectroscopy (NMR) to gain more understanding of how the pore domain plus contributes to the activity of the whole protein construct.
To gain more information about the function of the transmembrane region of hTRPM8, it was expressed in Escherichia coli (E. coli) and purified in detergent membrane mimics for experimentation. The construct contains the S4-S5 linker, pore domain (S5 and S6 transmembrane helices), pore helix, and TRP box. hTRPM8-PD+ was purified in the detergents n-Dodecyl-B-D-Maltoside (DDM), 16:0 Lyso PG, 1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphoglycerol (LPPG), and 14:0 Lyso PG, 1-Myristoyl-2-hydroxy-sn-glycero-3-phosphoglycerol (LMPG) to determine which detergent resulted in a hTRPM8-PD+ sample of the most stability, purity, and highest concentrations. Following bacterial expression and protein purification, hTRPM8-PD+ was studied and characterized with circular dichroism (CD) spectroscopy to learn more about the secondary structures and thermodynamic properties of the construct. Further studies can be done with more circular dichroism (CD) spectroscopy, planar lipid bilayer (BLM) electrophysiology, and nuclear magnetic resonance spectroscopy (NMR) to gain more understanding of how the pore domain plus contributes to the activity of the whole protein construct.
ContributorsMorelan, Danielle Taylor (Co-author) / Morelan, Danielle (Co-author) / Van Horn, Wade (Thesis director) / Chen, Julian (Committee member) / Luu, Dustin (Committee member) / Dean, W.P. Carey School of Business (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-12
Description
Over the last century, the Latino population in the United States has increased dramatically. Like most ethnic groups, Latinos highly value their culture and bring aspects of it with them when they come to the United States. One such aspect of Latino culture is the use of traditional medicine. As the Latino population in the United States continues to grow, it is important that physicians and future physicians understand how the use of and belief in traditional medicine within different Latino populations can affect the healthcare experience for both provider and patient. Many physicians lack this knowledge and therefore are unsure how to proceed when confronted with these situations; in order to remedy this issue, this project seeks to propose and demonstrate a potential course that would be intended to inform pre-medical and pre-health students about traditional medicine in different Latin American countries so that they will be better prepared.
In this 3-credit course, students will gain awareness and understand the importance of Latino traditional medical practices from the perspective of future medical professionals. Students will learn about concepts such as folk illnesses and traditional religious practices within different Latino populations and will discover how these cultural beliefs can affect a patient’s attitude and cooperation in the medical office.
Through study of the traditional medicines of Puerto Rico, Mexico, and Cuba, students will be exposed to new concepts that will allow them to gain a broader understanding of their future patients, which will allow them to provide the best possible care as a physician. Students will reflect on the importance of having respect for a patient’s cultural beliefs in the medical profession, regardless of their knowledge of Spanish, so that they will be best equipped to handle these situations within the United States and abroad.
In this 3-credit course, students will gain awareness and understand the importance of Latino traditional medical practices from the perspective of future medical professionals. Students will learn about concepts such as folk illnesses and traditional religious practices within different Latino populations and will discover how these cultural beliefs can affect a patient’s attitude and cooperation in the medical office.
Through study of the traditional medicines of Puerto Rico, Mexico, and Cuba, students will be exposed to new concepts that will allow them to gain a broader understanding of their future patients, which will allow them to provide the best possible care as a physician. Students will reflect on the importance of having respect for a patient’s cultural beliefs in the medical profession, regardless of their knowledge of Spanish, so that they will be best equipped to handle these situations within the United States and abroad.
ContributorsIncha, Carmen (Author) / Estévez, Dulce (Thesis director) / Oberstein, Bruce (Committee member) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Transient receptor potential (TRP) channels are a diverse family of polymodally gated nonselective cation channels implicated in a variety of pathophysiologies. Two channels of specific interest are transient receptor potential melastatin 8 (TRPM8) and transient receptor potential vanilloid 1 (TRPV1).
TRPM8 is the primary cold sensor in humans and is activated by ligands that feel cool such as menthol and icilin. It is implicated to be involved in a variety of cancers, nociception, obesity, addiction, and thermosensitivity. There are thought to be conserved regions of structural and functional importance to the channel which can be identified by looking at the evolution of TRPM8 over time. Along with this, looking at different isoforms of TRPM8 which are structurally very different but functionally similar can help isolate regions of functional interest as well. Between TRP channels, the transmembrane domain is well conserved and thought to be important for sensory physiology. To learn about these aspects of TRPM8, three evolutionary constructs, the last common primate, the last common mammalian, and the last common vertebrate ancestor TRPM8 were cloned and subjected to preliminary studies. In addition to the initial ancestral TRPM8 studies, fundamental studies were initiated in method development to evaluate the use of biological signaling sequences to attempt to force non-trafficking membrane protein isoforms and biophysical constructs to the plasma membrane. To increase readout for these and other studies, a cellular based fluorescence assay was initiated. Eventual completion of these efforts will lead to better understanding of the mechanism that underlie TRPM8 function and provide enhanced general methods for ion channel studies.
Beyond TRPM8 studies, an experiment was designed to probe mechanistic features of TRPV1 ligand activation. TRPV1 is also a thermosensitive channel in the TRP family, sensing heat and vanilloid ligands like capsaicin, commonly found in chili peppers. This channel is also involved in many proinflammatory interactions and associated with cancers, nociception, and addiction. Better understanding binding interactions can lead to attempts to create therapeutics.
TRPM8 is the primary cold sensor in humans and is activated by ligands that feel cool such as menthol and icilin. It is implicated to be involved in a variety of cancers, nociception, obesity, addiction, and thermosensitivity. There are thought to be conserved regions of structural and functional importance to the channel which can be identified by looking at the evolution of TRPM8 over time. Along with this, looking at different isoforms of TRPM8 which are structurally very different but functionally similar can help isolate regions of functional interest as well. Between TRP channels, the transmembrane domain is well conserved and thought to be important for sensory physiology. To learn about these aspects of TRPM8, three evolutionary constructs, the last common primate, the last common mammalian, and the last common vertebrate ancestor TRPM8 were cloned and subjected to preliminary studies. In addition to the initial ancestral TRPM8 studies, fundamental studies were initiated in method development to evaluate the use of biological signaling sequences to attempt to force non-trafficking membrane protein isoforms and biophysical constructs to the plasma membrane. To increase readout for these and other studies, a cellular based fluorescence assay was initiated. Eventual completion of these efforts will lead to better understanding of the mechanism that underlie TRPM8 function and provide enhanced general methods for ion channel studies.
Beyond TRPM8 studies, an experiment was designed to probe mechanistic features of TRPV1 ligand activation. TRPV1 is also a thermosensitive channel in the TRP family, sensing heat and vanilloid ligands like capsaicin, commonly found in chili peppers. This channel is also involved in many proinflammatory interactions and associated with cancers, nociception, and addiction. Better understanding binding interactions can lead to attempts to create therapeutics.
ContributorsShah, Karan (Author) / Van Horn, Wade (Thesis director) / Neisewander, Janet (Committee member) / Biegasiewicz, Kyle (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / School of Molecular Sciences (Contributor, Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
As of March 2020, there were over 112,400 patients actively waiting on the United States national organ transplant waitlist with only about 3,300 donors1. Although transplantation is an effective treatment for end-stage organ failure, the access to a procedure will vary depending on national regulations, cost of health care, extensive screening processes, and the availability of organs2. Organ shortage is a worldwide problem, and the growing insufficiency has resulted patients becoming too for ill or dying while waiting3. Due to the varying wait times and costs of procedures, some patients have begun to outsource their own transplantation through international transactions, also known as transplant tourism2. The 2004 World Health Assembly resolution recognized these trades as a significant health policy issue, while also acknowledging the inability of national health care systems to meet the needs of patients4. To address this issue, a proposal will be made such that all live kidney and liver donors will be compensated $22,500 and $12,150 respectively through a cost-neutral scheme based on annual healthcare expenditures per organ that would be eliminated by a transplant. With this proposal, it is suggested that the organ transplant waitlist would not only be significantly reduced, but potentially eliminated, and the crisis of organ shortage would be defeated.
ContributorsMartin, Starla (Author) / Kingsbury, Jeffrey (Thesis director) / Edmonds, Hallie (Committee member) / School of Molecular Sciences (Contributor) / School of Human Evolution & Social Change (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
The Founder’s lab is a year-long program that gives students an opportunity to participate in a unique team-based, experiential Barrett honors thesis project to design and apply marketing and sales strategies, as well as business and financial models to start up and launch a new business. This Barrett honors thesis project focuses on increasing the accessibility of health and wellness programs for small businesses and their employees through a customizable and easily implemented third party program that encourages employee retention.
ContributorsSharifi, Megan (Author) / Chiarello, Allyssa (Co-author) / Germer, Brendan (Co-author) / Kwapiszeski, Jacob (Co-author) / Byrne, Jared (Thesis director) / Larson, Wiley (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor) / Watts College of Public Service & Community Solut (Contributor)
Created2022-05