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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
Science fiction works can reflect the relationship between science and society by telling a story set in the future of ethical implications or social consequences of scientific advancement. This thesis investigated how the concept of reproduction is depicted in popular science fiction works. I reviewed and analyzed four popular science fiction works that all showed fear over the government controlling our choices in reproductive technology. The analysis of my thesis can be used as a way to understand how we view the ideas and the consequences of reproductive technology through concepts of reproduction. These perspectives and ideas as a reflection of society's concerns as we discuss the future of the ethics and politics of reproductive technology and reproductive issues.
ContributorsKim, Grace Dayoung (Author) / Maienschein, Jane (Thesis director) / Ellison, Karin (Committee member) / Hurlbut, Ben (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
This project focuses on techniques contemporary American poets use in their work. Ten different poetry collections are analyzed for dominant writing styles and techniques, which I then apply to my own poems, concentrating on modeling that particular poet. I then reflect on those poems through an evaluation of my writing process, how those techniques were implemented, and how they affected the poem. In addition to these reviews and reflections, I also wrote three articles about the literary community and what I've learned from my interactions in that community. All these materials are organized into a website, which shows the connections between the different writings via links and menus. Creating this website brings all the materials together to demonstrate my growth as a poet, writer, and designer. This heavy focus on poetry and analysis has helped sharpen my critical thinking skills and has better prepared me for a career in design and journalism.
ContributorsHansen, Elizabeth Sarah (Author) / Murphy, Patricia (Thesis director) / Savard, Jeannine (Committee member) / Barrett, The Honors College (Contributor) / Department of English (Contributor) / Hugh Downs School of Human Communication (Contributor) / Walter Cronkite School of Journalism and Mass Communication (Contributor)
Created2015-05
Description
This thesis examines contemporary cinematic adaptations of the Ovidian Pygmalion story. The films Blade Runner (1981), Lars and the Real Girl (2007), Ruby Sparks (2012), and Her (2013) are analyzed. This thesis seeks to understand why this particular myth is so resonant in today's popular culture and what this relevance reveals about modern society. The roles of female subjugation, sexualization, and relationship with technology will be major areas of concern. Research includes film criticism, Ovidian scholarship, and new advances in computer technology.
ContributorsStory, Sara Katherine (Author) / Corse, Taylor (Thesis director) / Ellis, Lawrence (Committee member) / Barrett, The Honors College (Contributor) / Department of English (Contributor)
Created2015-05
Description
The Intercellular Adhesion Molecule-1 (ICAM-1, known as CD54) is a cell surface type I transmembrane glycoprotein with a molecular weight of 85 to 110 kDa. The primary function of ICAM-1 is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 is used as a receptor for various pathogens such as rhinoviruses, coxsackievirus A21 and the malaria parasite Plasmodium falciparum. ICAM-1 contains five immunoglobulin (Ig) domains in its long N-terminal extracellular region, a hydrophobic transmembrane domain, and a small C-terminal cytoplasmic domain. The Ig domains 1-2 and Ig domains 3-4-5 have been crystallized separately and their structure solved, however the full ICAM-1 structure has not been solved. Because ICAM-1 appears to be important for the mediation of cell-to-cell communication in physiological and pathological conditions, gaining a structural understanding of the full-length membrane anchored ICAM-1 is desirable. In this context, we have transiently expressed a plant-optimized gene encoding human ICAM-1 in Nicotiana benthamiana plants using the MagnICON expression system. The plant produced ICAM-1 is forming aggregates according to previous data. Thus, the current extraction and purification protocols have been altered to include TCEP, a reducing agent. The protein was purified using TALON metal affinity resin and partially characterized using various biochemical techniques. Our results show that there is a reduction in aggregation formation with the use of TCEP.
ContributorsPatel, Heeral (Author) / Mor, Tsafrir (Thesis director) / Mason, Hugh (Committee member) / Kannan, Latha (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Medical Humanities is a growing field and much scholarship focuses on the promotion of empathy in professionals. I argue incorporation of literature is crucial as it develops critical thinking skills that guard against the dangers of collective thought. A Foucauldian analysis of three literary works, my own creative non-fiction short story, William Carlos Williams' "The Use of Force," and Alexander Solzhenitsyn's Cancer Ward offer the student perspective, the doctor perspective and the institutional perspective, respectively, and subversive undertones offer an example of the analytical thought developed in humanities education by challenging assumptions and elucidating implicit power relations in the medical institution.
ContributorsBlock, Courtney Samantha (Author) / Lussier, Mark (Thesis director) / Fox, Cora (Committee member) / Barrett, The Honors College (Contributor) / Department of English (Contributor)
Created2015-05
Description
Membrane proteins located within or as attachments to the cell membrane play critical roles in many essential cellular functions and host-pathogen interactions. Knowledge of the structure and function of membrane proteins in pathogenic species can allow for the development of specific vaccines and therapeutic agents against the pathogen. Francisella tularensis is an intracellular pathogen that is the causative agent of the severe, life-threatening infection, tularemia, in humans and other small mammals. F. tularensis is prevalent within the environment and is a potential bioterrorism agent due to its high virulence and its ability to be spread easily as an aerosol. The CapBCA membrane protein complex has been identified as a virulence factor of F. tularensis. This project, derived from the Membrane Proteins in Infections Diseases (MPID) Project, aims to successfully express the membrane proteins CapBCA, which are crucial to the pathogenic properties of F. tularensis. To accomplish this goal, methods for in vivo recombinant expression and purification of membrane proteins are in the process of being developed. The expression of the CapA component has been successful for some time, therefore, the goal of this study is to develop an approach toward recombinant in vivo membrane protein expression of both the CapB and CapC components of the CapBCA membrane protein complex. In this study, the CapB and CapC components were expressed for the first time in vivo through the use of the novel MPID vector, pelB-MBP. The expression of the CapB and CapC components will allow for large-scale expressions to commence with the end goal of determining the crystal structures of the individual proteins or the complex. Ultimately, it is hoped that knowledge of these molecular structures can lead to the development of a vaccine or other therapeutic agents against this pathogen.
ContributorsTrimble, Kelli Lauren (Author) / Fromme, Petra (Thesis director) / Hansen, Debra (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Film, Dance and Theatre (Contributor)
Created2015-05
Description
This project examines the literary figures of Tristan and Isolde, looking to see how each character is portrayed, how their portrayals change through time, and takes a socio-cultural perspective in attempts to explain why these portrayals were used, and why they changed. Three different versions of the Tristan and Isolde story from three different time periods were used: Le Morte Darthur by Sir Thomas Malory from the 1400's, Idylls of The King by Lord Alfred Tennyson from the 1800's, and the film Tristan + Isolde distributed by 20th Century Fox in the mid 2000's. For each version of the story, the primary text or film, along with secondary sources, were used to determine how each character was portrayed. This was done by examining Tristan and Isolde's physical appearances, stations in life, actions, and personality/tone. These portrayals from each version were then compared with portrayals from the other versions to determine what changes had occurred. Finally, secondary textual information was used to examine the culture in which each version was originally published, specifically examining such socio-cultural changes that could explain why the previously determined portrayals of Tristan and Isolde were used and why they differ from versions of these characters from different time periods. The results of this study found that some characteristics of Tristan and Isolde's portrayals do not change through time. Specifically, their physical appearances and stations in life are, for the most part, fixed. Tristan is always a handsome, strong, and noble knight/warrior while Isolde is always a beautiful and delicate princess. Other characteristics, such as personality/tone and actions do change drastically from one version to the next in accordance with the changing culture in which the authors and audience members lived.
ContributorsKupsch, Mary Francis (Author) / Newhauser, Richard (Thesis director) / Haggins, Bambi (Committee member) / Barrett, The Honors College (Contributor) / School of Human Evolution and Social Change (Contributor) / Department of English (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
In my thesis paper, I examine the gothic elements found in classical gothic fairy tales from European and Japanese tradition, particularly those works by the Brothers Grimm and Yei Theodora Ozaki. By examining the principle gothic elements that are unique to both stories, and further analyzing the commonalities of story, plot, and other major tropes, a better understanding of the message meant to be imparted and other cultural nuances can be ascertained. Gothic literature creates an atmosphere of gloom and suspense, toying with concepts of dread and darkness by employing Gothic elements such as shadows, the supernatural, sinister buildings, and strong-willed villains, all of which affect the rational mind in an irrational way. Fairytales freely use such tropes to their advantage, playing with the many fears of children, while simultaneously painting an idealistic fantasy world. The degree of usage and the application of gothic elements is closely examined in the Grimm works, "Hansel and Gretel," and "The Robber Bridegroom," as well as the Japanese tales, "The Goblin of Adachigahra,""Kintaro the Golden Boy" and "The Monkey and the Crab." These stories have been chosen due for their usage of animal tricksters, themes of control, and aspects of isolation, supernatural entities, and substantial gothic imagery. The gothic elements of death, sinister older women, the supernatural, fears of abandonment, and cunning animals are akin to both Western and Eastern tales, while the concept of gothic setting and the type of monsters prepared to feast on men is significantly different for both cultures, similar lessons are intended to be gleaned by children from these tales, with the intention of generally producing positive results \u2014 while the means differ, the message is strikingly similar, yet there remain cultural differences in terms of central themes and character traits.The effect of re-introducing the darker, gothic elements of traditional fairy tales into modern literature and retellings of the original narratives has been profound.Today, whether it has been at the bequest of the public or simply a new-age movement by modern cinema audience for the "gritty and realistic," fairy tales are returning to their former gothic forms. "Snow White and The Huntsman" is one example of a film which has gone this route, opting for a more gothic, classic telling rather than the chip, cheery, rosy cheeked Disney versions. There is a tendency for most media nowadays to be far less censored and fantastical, aiming for a more realistic, grittier approach \u2014 this bleeds into film and literature likewise, and thus children are impacted by this shift as well. Children seem to be able to handle more, perhaps desensitized at younger and younger ages by the products of our widely consumerist society, or perhaps due to parents raising their children in such a way so that the darkness that tinges these tales doesn't disturb and derail but rather, emphasizes their meaning of teaching certain lessons. Tales such as these are still valuable, and will continue to be so long as we seek a reality greater than our own, where the evil of the world is wiped away, and we all live happily ever after.
ContributorsMoschonas, Jerasimos Theodore (Author) / Ellis, Lawrence (Thesis director) / Hoyt, Heather (Committee member) / Barrett, The Honors College (Contributor) / Department of English (Contributor)
Created2015-05