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- Creators: Barrett, The Honors College
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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 vastly growing field of supercomputing is in dire need of a new measurement system to optimize JMRAM (Josephson junction magnetoresistive random access memory) devices. To effectively measure these devices, an ultra-low-noise, low cost cryogenic dipping probe with a dynamic voltage range is required. This dipping probe has been designed by ASU with <100 nVp-p noise, <10 nV offsets, 10 pV to 16 mV voltage range, and negligible thermoelectric drift. There is currently no other research group or company that can currently match both these low noise levels and wide voltage range. Two different dipping probes can be created with these specifications: one for high-use applications and one for low-use applications. The only difference between these probes is the outer shell; the high-use application probe has a shell made of G-10 fiberglass for a higher price, and the low-use application probe has a shell made of AISI 310 steel for a lower price. Both types of probes can be assembled in less than 8 hours for less than $2,500, requiring only soldering expertise. The low cost and short time to create these probes makes wide profit margins possible. The market for these cryogenic dipping probes is currently untapped, as most research groups and companies that use these probes build their own, which allows for rapid business growth. These potential consumers can be easily reached by marketing these probes at superconducting conferences. After several years of selling >50 probes, mass production can easily become possible by hiring several technicians, and still maintaining wide profit margins.
ContributorsHudson, Brooke Ashley (Author) / Adams, James (Thesis director) / Anwar, Shahriar (Committee member) / Materials Science and Engineering Program (Contributor) / W. P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
A series of mitochondria targeting probes was synthesized for the purpose of exploring the feasibility of a mitochondria targeting fluorescent sensor. Of the probes, the probe with a two carbon spacer showed the best co-localization from staining with the established MitoTracker Red® FM, indicating a potential development of the probe into mitochondria targeting sensor. However, cytotoxicity was observed for the probe with a six carbon spacer. Three additional mitochondria targeting fluorescent probes of longer spacer groups were synthesized, but the cytotoxicity was not observed to be as high as that of the probe with a two carbon spacer. The cytotoxicity was characterized to be that of caspase dependent cell death. To screen for a possible effect on apoptosis due to the mitochondrial probe, three fluorescent fusion proteins binding the anti-apoptotic proteins were designed and expressed. Each purified fusion protein was then incubated with the cytotoxic mitochondrial probe, and the mixture was isolated by running an affinity column. The fluorescence analysis of eluted fractions showed preliminary data of possible interaction between the protein and the mitochondrial probe.
ContributorsLee, Fred (Author) / Meldrum, Deirdre R. (Thesis director) / Tian, Yanqing (Committee member) / Zhang, Liqiang (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-12
Description
Although wind turbine bearings are designed to operate 18-20 years, in the recent years premature failure among these bearings has caused this life to reduce to as low as a few months to a year. One of the leading causes of premature failure called white structure flaking is a mechanism that was first cited in literature decades ago but not much is understood about it even today. The cause of this mode of failure results from the initiation of white etched cracks (WECs). In this report, different failure mechanisms, especially premature failure mechanisms that were tested and analyzed are demonstrated as a pathway to understanding this phenomenon. Through the use of various tribometers, samples were tested in diverse and extreme conditions in order to study the effect of these different operational conditions on the specimen. Analysis of the tested samples allowed for a comparison of the microstructure alterations in the tested samples to the field bearings affected by WSF.
ContributorsSharma, Aman (Author) / Foy, Joseph (Thesis director) / Adams, James (Committee member) / Barrett, The Honors College (Contributor) / Mechanical and Aerospace Engineering Program (Contributor)
Created2015-05
Description
As prices for fuel along with the demand for renewable resources grow, it becomes of paramount importance to develop new ways of obtaining the energy needed to carry out the tasks we face daily. Costs of production due to energy and time constraints impose severe limitations on what is viable. Biological systems, on the other hand, are innately efficient both in terms of time and energy by handling tasks at the molecular level. Utilizing this efficiency is at the core of this research. Proper manipulation of even common proteins can render complexes functionalized for specific tasks. In this case, the coupling of a rhenium-based organometallic ligand to a modified myoglobin containing a zinc porphyrin, allow for efficient reduction of carbon dioxide, resulting in energy that can be harnessed and byproducts which can be used for further processing. Additionally, a rhenium based ligand functionalized via biotin is tested in conjunction with streptavidin and ruthenium-bipyridine.
ContributorsAllen, Jason Kenneth (Author) / Ghirlanda, Giovanna (Thesis director) / Francisco, Wilson (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor)
Created2014-12
Description
Understanding glycosaminoglycans’ (GAG) interaction with proteins is of growing interest for therapeutic applications. For instance, heparin is a GAG exploited for its ability to inhibit proteases, therefore inducing anticoagulation. For this reason, heparin is extracted in mass quantities from porcine intestine in the pharmaceutical field. Following a contamination in 2008, alternative sources for heparin are desired. In response, much research has been invested in the extraction of the naturally occurring polysaccharide, heparosan, from Escherichia coli K5 strain. As heparosan contains the same structural backbone as heparin, modifications can be made to produce heparin or heparin-like molecules from this source. Furthermore, isotopically labeled batches of heparosan can be produced to aid in protein-GAG interaction studies. In this study, a comparative look between extraction and purification methods of heparosan was taken. Fed-batch fermentation of this E. coli strain followed by subsequent purification yielded a final 13C/15N labeled batch of 90mg/L of heparosan which was then N-sulfated. Furthermore, a labeled sulfated disaccharide from this batch was utilized in a protein interaction study with CCL5. With NMR analysis, it was found that this heparin-like molecule interacted with CCL5 when its glucosamine residue was in a β-conformation. This represents an interaction reliant on a specific anomericity of this GAG molecule.
ContributorsHoffman, Kristin Michelle (Author) / Wang, Xu (Thesis director) / Cabirac, Gary (Committee member) / Morgan, Ashli (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
A model has been developed to modify Euler-Bernoulli beam theory for wooden beams, using visible properties of wood knot-defects. Treating knots in a beam as a system of two ellipses that change the local bending stiffness has been shown to improve the fit of a theoretical beam displacement function to edge-line deflection data extracted from digital imagery of experimentally loaded beams. In addition, an Ellipse Logistic Model (ELM) has been proposed, using L1-regularized logistic regression, to predict the impact of a knot on the displacement of a beam. By classifying a knot as severely positive or negative, vs. mildly positive or negative, ELM can classify knots that lead to large changes to beam deflection, while not over-emphasizing knots that may not be a problem. Using ELM with a regression-fit Young's Modulus on three-point bending of Douglass Fir, it is possible estimate the effects a knot will have on the shape of the resulting displacement curve.
ContributorsSexton, Thurston Bryant (Author) / Takahashi, Timothy (Thesis director) / Jones, Donald (Committee member) / Barrett, The Honors College (Contributor) / Mechanical and Aerospace Engineering Program (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / School of Human Evolution and Social Change (Contributor)
Created2015-05
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
The following is a report that will evaluate the microstructure of the nickel-based superalloy Hastelloy X and its relationship to mechanical properties in different load conditions. Hastelloy X is of interest to the company AORA because its strength and oxidation resistance at high temperatures is directly applicable to their needs in a hybrid concentrated solar module. The literature review shows that the microstructure will produce different carbides at various temperatures, which can be beneficial to the strength of the alloy. These precipitates are found along the grain boundaries and act as pins that limit dislocation flow, as well as grain boundary sliding, and improve the rupture strength of the material. Over time, harmful precipitates form which counteract the strengthening effect of the carbides and reduce rupture strength, leading to failure. A combination of indentation and microstructure mapping was used in an effort to link local mechanical behavior to microstructure variability. Electron backscatter diffraction (EBSD) and energy dispersive spectroscopy (EDS) were initially used as a means to characterize the microstructure prior to testing. Then, a series of room temperature Vickers hardness tests at 50 and 500 gram-force were used to evaluate the variation in the local response as a function of indentation size. The room temperature study concluded that both the hardness and standard deviation increased at lower loads, which is consistent with the grain size distribution seen in the microstructure scan. The material was then subjected to high temperature spherical indentation. Load-displacement curves were essential in evaluating the decrease in strength of the material with increasing temperature. Through linear regression of the unloading portion of the curve, the plastic deformation was determined and compared at different temperatures as a qualitative method to evaluate local strength.
ContributorsCelaya, Andrew Jose (Author) / Peralta, Pedro (Thesis director) / Solanki, Kiran (Committee member) / Barrett, The Honors College (Contributor) / Mechanical and Aerospace Engineering Program (Contributor)
Created2015-05
Description
In this thesis, glycan nodes, the basic subunits of complex biological sugars, were studied to determine the reproducibility of gas chromatography-mass spectrometry (GC/MS) based methylation analysis of whole blood plasma by normalization using an internal standard of heavy permethylated glycans. Glycans are complex biological sugars that have a variety of applications in the human body and will display aberrant compositions when produced by cancerous cells. Thus an assay to determine their composition can be used as a diagnostic tool. It was shown that the assay may have potential use, but needs further refinement to become an improvement over current methods by analyzing the results of ratio-determination and replicate experiments.
ContributorsMiyasaki, Tyler Takeo (Author) / Borges, Chad (Thesis director) / Van Horn, Wade (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Chemical Engineering Program (Contributor)
Created2015-05