Matching Items (54)
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- All Subjects: Biochemistry
- All Subjects: healthcare
- Creators: School of Molecular Sciences
- Member of: Barrett, The Honors College Thesis/Creative Project Collection
- 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
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
Description
Catastrophe events occur rather infrequently, but upon their occurrence, can lead to colossal losses for insurance companies. Due to their size and volatility, catastrophe losses are often treated separately from other insurance losses. In fact, many property and casualty insurance companies feature a department or team which focuses solely on modeling catastrophes. Setting reserves for catastrophe losses is difficult due to their unpredictable and often long-tailed nature. Determining loss development factors (LDFs) to estimate the ultimate loss amounts for catastrophe events is one method for setting reserves. In an attempt to aid Company XYZ set more accurate reserves, the research conducted focuses on estimating LDFs for catastrophes which have already occurred and have been settled. Furthermore, the research describes the process used to build a linear model in R to estimate LDFs for Company XYZ's closed catastrophe claims from 2001 \u2014 2016. This linear model was used to predict a catastrophe's LDFs based on the age in weeks of the catastrophe during the first year. Back testing was also performed, as was the comparison between the estimated ultimate losses and actual losses. Future research consideration was proposed.
ContributorsSwoverland, Robert Bo (Author) / Milovanovic, Jelena (Thesis director) / Zicarelli, John (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The primary objective of this project is to further the knowledge about SCL26 family of anion transporters. The goals of the experiment were to find the lowest sulfate concentration where the yeast without Sulp1 and Sulp2 is able to grow, but it grows very slowly, and to find a higher sulfate concentration where the yeast grows quickly, with or without the sulfate transporters. The lowest sulfate concentration where the yeast without the sulfate transporters is able to grow was determined to be 2-4 mM, however, this range can likely be refined by more quantitative analytical methods. At a sulfate concentration of 20 mM sulfate or higher, the yeast is able to grow quickly without high-affinity sulfate transporters. The next step in the project is to re-introduce the Sulp1 and Sulp2 genes into the yeast, so that growth in low and high sulfate conditions can be compared with and without the Sulp1 and Sulp2 proteins. The long-term goals of the project are to bring experience with yeast to Dr. Nannenga’s structural discovery lab, to determine if yeast sulfate transporters respond in the same way to drug candidates as human sulfate transporters, and to determine the structure of the proteins using cryo-electron microscopy.
ContributorsCall, Nicolas I (Author) / Nannenga, Brent (Thesis director) / Wang, Xuan (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The devastating 2014 Ebola virus outbreak in Western Africa demonstrated the lack of therapeutic approaches available for the virus. Although monoclonal antibodies (mAb) and other molecules have been developed that bind the virus, no therapeutic has shown the efficacy needed for FDA approval. Here, a library of 50 peptide based ligands that bind the glycoprotein of the Zaire Ebola virus (GP) were developed. Using whole virus screening of vesicular stomatitis virus pseudotyped with GP, low affinity peptides were identified for ligand construction. In depth analysis showed that two of the peptide based molecules bound the Zaire GP with <100 nM KD. One of these two ligands was blocked by a known neutralizing mAb, 2G4, and showed cross-reactivity to the Sudan GP. This work presents ligands with promise for therapeutic applications across multiple variants of the Ebola virus.
ContributorsRabinowitz, Joshua Avraam (Author) / Diehnelt, Chris (Thesis director) / Johnston, Stephen (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
The parameters of microwave-assisted acid hydrolysis (MAAH) and 1H NMR highly affect the quantitative analysis of protein hydrolysates. Microwave-induction source, NMR spectral resolution, and data analysis are key parameters in the nuclear magnetic resonance – amino acid analysis (NMR-AAA) workflow where errors accrue due to lack of an optimized protocol. Hen egg white lysozyme was hydrolyzed using an 800W domestic microwave oven for varying time points between 10-25 minutes, showing minimal protein hydrolysis after extended time periods. Studies on paramagnetic doping with varying amounts of gadolinium chloride for increased NMR resolution resulted in little T1 reduction in a majority of amino acids and resulted in significant line broadening in concentrations above 1µM. The use of the BAYESIL analysis tool with HOD suppressed 1H-NMR spectra resulted in misplaced template peaks and errors greater than 1% for 10 of 13 profiled amino acids with the highest error being 7.6% (Thr). Comparatively, Chenomx NMR Suite (v7.1) analysis resulted in errors of less than 1% for 9 of 13 profiled amino acids with a highest error value of 3.6% (Lys). Using the optimized protocol, hen egg white lysozyme C was identified at rank 1 with a score of 64 in a Gallus gallus species wide AACompIdent search. This technique reduces error associated with sample handling relative to previously used amino acid analysis (AAA) protocols and requires no derivatization or additional processing of the sample prior to analysis.
ContributorsJordan, Jacob Smith (Author) / Yarger, Jeffery (Thesis director) / Van Horn, Wade (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Integrin is a protein in cells that manage cell adhesion. They are crucial to the biochemical functions of cells. L 2 is one type of integrin. Its I domain is responsible for ligand binding. Scientists understand how Alpha L I domain binds Mg2+ at a pH of 7 but not in acidic environments. Knowing the specificity of integrin at a lower pH is important because when tissues become inflamed, they release acidic compounds. We have cloned, expressed, and purified L I-domain and using NMR analysis, we determined that wild type Alpha L I domain does not bind to Mg2+ at a pH of 5.
ContributorsALAM, RAHAT (Author) / Wang, Xu (Thesis director) / Podolnikova, Nataly (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Gle1 is an mRNP export mediator with major activity localized to the nuclear pore complex in eukaryotic cells. The protein's high preservation across vast phylogenetic distances allows us to approximate research on the properties of yeast Gle1 (yGle1) with those of human Gle1 (hGle1). Research at Vanderbilt University in 2016, which provides the research basis of this thesis, suggests that the coiled-coil domain of yGle1 is best crystallized in dicationic aqueous conditions of pH ~8.0 and 10\u201420% PEG 8000. Further exploration of crystallizable microconditions revealed a favorability toward lower pH and lower PEG concentration. Following the discovery of the protein's native crystallography conditions, a comprehensive meta-analysis of scientific literature on Gle1 was conducted on the association of Gle1 mutations with neuron disease.
ContributorsGaetano, Philip Pasquale (Author) / Foy, Joseph (Thesis director) / Dawson, T. Renee (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Predicting the binding sites of proteins has historically relied on the determination of protein structural data. However, the ability to utilize binding data obtained from a simple assay and computationally make the same predictions using only sequence information would be more efficient, both in time and resources. The purpose of this study was to evaluate the effectiveness of an algorithm developed to predict regions of high-binding on proteins as it applies to determining the regions of interaction between binding partners. This approach was applied to tumor necrosis factor alpha (TNFα), its receptor TNFR2, programmed cell death protein-1 (PD-1), and one of its ligand PD-L1. The algorithms applied accurately predicted the binding region between TNFα and TNFR2 in which the interacting residues are sequential on TNFα, however failed to predict discontinuous regions of binding as accurately. The interface of PD-1 and PD-L1 contained continuous residues interacting with each other, however this region was predicted to bind weaker than the regions on the external portions of the molecules. Limitations of this approach include use of a linear search window (resulting in inability to predict discontinuous binding residues), and the use of proteins with unnaturally exposed regions, in the case of PD-1 and PD-L1 (resulting in observed interactions which would not occur normally). However, this method was overall very effective in utilizing the available information to make accurate predictions. The use of the microarray to obtain binding information and a computer algorithm to analyze is a versatile tool capable of being adapted to refine accuracy.
ContributorsBrooks, Meilia Catherine (Author) / Woodbury, Neal (Thesis director) / Diehnelt, Chris (Committee member) / Ghirlanda, Giovanna (Committee member) / Department of Psychology (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Higher plant Rubisco activase (Rca) is a stromal ATPase responsible for reactivating Rubisco. It is a member of the AAA+ protein superfamily and is thought to assemble into closed-ring hexamers like other AAA+ proteins belonging to the classic clade. Progress towards modeling the interaction between Rca and Rubisco has been slow due to limited structural information on Rca. Previous efforts in the lab were directed towards solving the structure of spinach short-form Rca using X-ray crystallography, given that it had notably high thermostability in the presence of ATP-γS, an ATP analog. However, due to disorder within the crystal lattice, an atomic resolution structure could not be obtained, prompting us to move to negative stain electron microscopy (EM), with our long-term goal being the use of cryo-electron microscopy (cryo-EM) for atomic resolution structure determination. Thus far, we have screened different Rca constructs in the presence of ATP-γS, both the full-length β-isoform and truncations containing only the AAA+ domain. Images collected on preparations of the full-length protein were amorphous, whereas images of the AAA+ domain showed well-defined ring-like assemblies under some conditions. Procedural adjustments, such as the use of previously frozen protein samples, rapid dilution, and minimizing thawing time were shown to improve complex assembly. The presence of Mn2+ was also found to improve hexamer formation over Mg2+. Calculated class averages of the AAA+ Rca construct in the presence of ATP-γS indicated a lack of homogeneity in the assemblies, showing both symmetric and asymmetric hexameric rings. To improve structural homogeneity, we tested buffer conditions containing either ADP alone or different ratios of ATP-γS to ADP, though results did not show a significant improvement in homogeneity. Multiple AAA+ domain preparations were evaluated. Because uniform protein assembly is a major requirement for structure solution by cryo-EM, more work needs to be done on screening biochemical conditions to optimize homogeneity.
ContributorsHernandez, Victoria Joan (Author) / Wachter, Rebekka (Thesis director) / Chiu, Po-Lin (Committee member) / Redding, Kevin (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05