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- Creators: School of Molecular Sciences
- Status: Published
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
Volunteer motivation and satisfaction were assessed at Project CURE, a nonprofit that collects, sorts, tests, and ships donated medical supplies and equipment to healthcare facilities in developing countries. This research was the result of a yearlong partnership between the Community Action Research Experiences (CARE) program and Project CURE. Volunteers at Project CURE were surveyed (N=147) after completing a volunteer session to assess their motivation for volunteering, satisfaction with their experience, and any recommendations they had for improving the volunteer program. Five categories of motivating factors were assessed and it was found that the Values and Understanding categories were the strongest motivating factors. Overall, volunteers rated their experience highly, but the results indicated a number of small changes that Project CURE could make to better meet volunteers' needs, and better communicate the impact of volunteers' work, which could pave the way to increases in the numbers of volunteer hours Project CURE receives and increased quality of volunteer work.
ContributorsStepanek, Rachel (Author) / Reesing, Amy (Thesis director) / Dumka, Larry (Committee member) / School of Molecular Sciences (Contributor) / School of Human Evolution and Social Change (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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
The goal of the International Rescue Committee (IRC, created by Albert Einstein in 1933) is to serve those “whose lives and livelihoods are shattered by conflict and disaster to survive, recover, and gain control of their future,” (6), by providing victims of humanitarian crises with health care, education, and counseling. The IRC of Phoenix branch holds this same mission through the services it provides to the refugees of the Phoenix area. One important need that is not currently met by the IRC of Phoenix is the special health care needs of pregnant refugee women. The IRC of Phoenix is seeking funds to initiate a new “Prenatal Care Program” to meet the needs of the 40 or so pregnant refugees who come to our area each year.
This new program will build upon the existing programs currently provided by the IRC of Phoenix, including support in areas of resettlement, finance, community integration, and health. The objectives of this new prenatal-focused program are to serve the needs of pregnant refugee women by providing the physical and emotional support they need through partnerships with hospitals (such as Saint Joseph’s Hospital) and organizations (such as the Refugee Women’s Health Clinic of Phoenix).
The target audience includes women who seek refuge in the Phoenix area in the midst of a pregnancy, or with the intention to become pregnant and who are receiving other services from the IRC of Phoenix. This grant will fund a prenatal care caseworker position for as long as there are incoming funds and pregnant refugee women in Phoenix. The prenatal care caseworker’s duties include:
● Monitoring the overall health of pregnant refugee women assisted by the IRC of Phoenix
● Expanding access to prenatal care for pregnant refugee women
● Connecting pregnant refugee women with necessary information on healthy pregnancies and introducing them to childcare programs that the IRC of Phoenix currently provides
● Creating and maintaining strong relationships with hospitals and health care facilities
● Accompanying pregnant refugee women to medical appointments as needed
● Participating in all other duties necessary to ensure the safe pregnancy of refugee women under the care of the IRC of Phoenix
To evaluate the success of the program, the IRC of Phoenix will monitor the number of pregnant refugee women seeking help and monitor health to see how the women are being served and the number of these women who are fully served based on the above objectives. The required grant money needed each year amounts to $40,000. A multi-year commitment of at least five years is expected. This funding represents the annual salary of the newly hired caseworker, and the IRC of Phoenix will be covering administration costs, supplies, and equipment.
This new program will build upon the existing programs currently provided by the IRC of Phoenix, including support in areas of resettlement, finance, community integration, and health. The objectives of this new prenatal-focused program are to serve the needs of pregnant refugee women by providing the physical and emotional support they need through partnerships with hospitals (such as Saint Joseph’s Hospital) and organizations (such as the Refugee Women’s Health Clinic of Phoenix).
The target audience includes women who seek refuge in the Phoenix area in the midst of a pregnancy, or with the intention to become pregnant and who are receiving other services from the IRC of Phoenix. This grant will fund a prenatal care caseworker position for as long as there are incoming funds and pregnant refugee women in Phoenix. The prenatal care caseworker’s duties include:
● Monitoring the overall health of pregnant refugee women assisted by the IRC of Phoenix
● Expanding access to prenatal care for pregnant refugee women
● Connecting pregnant refugee women with necessary information on healthy pregnancies and introducing them to childcare programs that the IRC of Phoenix currently provides
● Creating and maintaining strong relationships with hospitals and health care facilities
● Accompanying pregnant refugee women to medical appointments as needed
● Participating in all other duties necessary to ensure the safe pregnancy of refugee women under the care of the IRC of Phoenix
To evaluate the success of the program, the IRC of Phoenix will monitor the number of pregnant refugee women seeking help and monitor health to see how the women are being served and the number of these women who are fully served based on the above objectives. The required grant money needed each year amounts to $40,000. A multi-year commitment of at least five years is expected. This funding represents the annual salary of the newly hired caseworker, and the IRC of Phoenix will be covering administration costs, supplies, and equipment.
ContributorsBatty, Rebecca (Co-author) / Gerais, Reem (Co-author) / Weitz, Rose (Thesis director) / Faurel, Lucile (Committee member) / Department of Information Systems (Contributor) / School of Accountancy (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
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
The Community Action Research Experiences program integrates the research and teaching mission of Arizona State University by providing services to the community by fostering professional and leadership development of students. It is hoped that the results of the collaborations with CARE will serve to further an organization's goals and effectiveness. VALLEYLIFE (VL) is a non-profit organization striving to help people with disabilities. VL develops Action Programs for each of its clients, whom they call members, to improve their independent or social skills. Examples of programs that members may work on include tasks such as computer training, visual arts, or writing. VALLEYLIFE lacked the data to evaluate if the developed and implemented Action Programs are properly carried out by the staff in ways that are beneficial to members. Given the problem, this research project sought to conduct a process evaluation of the staff regarding their implementation of the Action Programs. This involved observations of employee-member interactions in performing the Action Programs and an interview of staff measuring their preparedness and confidence in performing the program and their feelings of the programs and how things are run. This research provided the following implications to VALLEYLIFE. VL might consider performing periodic observations and reviews of the program implementation to monitor quality. VL may consider involving staff in program development and revision to create programs that better serve members. VL may consider generating ideas for how they may cooperate when a peer is struggling to keep up with events that happen through the day in the interest of better serving the members. Overall, employees are doing well as they are efficient in carrying out the written programs during program time. They are comfortable with what they are doing, use time effectively, and do their best to help the members. There is always room for improvement however and by considering some of the implications mentioned, VALLEYLIFE and their employees may be able to take action that may hold potential for further improvements in effectiveness.
ContributorsAbalos, Cherylene Sales (Author) / Bradley, Robert (Thesis director) / Dumka, Larry (Committee member) / Goldblatt, Lois (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / T. Denny Sanford School of Social and Family Dynamics (Contributor)
Created2013-05
Description
Research has examined the many motivations of international volunteers (voluntourists), but there is limited research about how volunteers are reached, as well as differing perceptions between travelers who have and have not traveled before. This study examines the preferences and perspectives of college-age, western backpackers. The general terms "backpacker" and "traveler" are used throughout the paper for simplicity, but it is important to note that these backpackers are specifically from the college-age, western demographic. First, the study addresses which recruitment avenues are the most successful, as well as which avenues could be utilized to increase the number of foreign, short-term volunteers. In addition, this study examines the differences between backpacker perceptions - specifically the differences in potential volunteering motivations and concerns. Data was collected through an anonymous online survey distributed to self-identified travelers between the ages of 18 and 25 in the United States and travel destinations in Vietnam and India. According to traveler responses, personal recommendations and hotels/hostels are important resources when making travel plans. Despite the importance of both resources, personal recommendations drew more travelers to volunteer than hostels/hotels (none of the travelers surveyed learned about their last volunteer opportunity through a hostel), revealing a potential avenue of recruitment. A small number of organizations have reported successfully utilizing the hostel-partnership model, which implies that successful partnerships are possible. Further, potential motivations to volunteer were similar between those who have and those who have not volunteered, however, potential concerns between the two groups differed. Those who had volunteered before reported to be considerably more concerned about adherence to cultural norms, as well as communication barriers, while those who had not volunteered were much more concerned about safety. These findings lead to several theoretical implications for nonprofits with respect to utilizing hostels for volunteer recruitment, as well as addressing concerns of those who have volunteered before differently from those who have not.
ContributorsWorkman, Hunter (Co-author) / Pfeiffer, Nicholaus (Co-author) / Wang, Lili (Thesis director) / Salamone, Damien (Committee member) / Louis, Arulraj (Committee member) / School of Human Evolution and Social Change (Contributor) / School of Molecular Sciences (Contributor) / Department of Economics (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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
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