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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

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
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Description
We examined the evolutionary morphological responses of Drosophila melanogaster that had evolved at constant cold (16°), constant hot (25°C), and fluctuating (16° and 25°C). Flies that were exposed to the constant low mean temperature developed larger thorax, wing, and cell sizes than those exposed to constant high mean temperatures. Males

We examined the evolutionary morphological responses of Drosophila melanogaster that had evolved at constant cold (16°), constant hot (25°C), and fluctuating (16° and 25°C). Flies that were exposed to the constant low mean temperature developed larger thorax, wing, and cell sizes than those exposed to constant high mean temperatures. Males and females both responded similarly to thermal treatments in average wing and cell size. The resulting cell area for a given wing size in thermal fluctuating populations remains unclear and remains a subject for future research.
ContributorsAdrian, Gregory John (Author) / Angilletta, Michael (Thesis director) / Harrison, Jon (Committee member) / Rusch, Travis (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
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Description
Three populations of experimentally evolved Drosophila melanogaster populations made up of high temperature (H, constant 25 ᵒC), low temperature (C, constant 16 ᵒC) and temporal homogeneity (T, environment changes between 16 ᵒC and 25 ᵒC) were prepared and assayed to determine difference in citrate synthase activity. Between the three groups,

Three populations of experimentally evolved Drosophila melanogaster populations made up of high temperature (H, constant 25 ᵒC), low temperature (C, constant 16 ᵒC) and temporal homogeneity (T, environment changes between 16 ᵒC and 25 ᵒC) were prepared and assayed to determine difference in citrate synthase activity. Between the three groups, the results were inconclusive: the resulting reaction rates in units of nmol min-1mgfly-1 were 81.8 + 20.6, 101 + 15.6, and 96.9 + 25.2 for the hot (H), cold (C), and temporally homogeneous (T) groups, respectively. We conclude that the high associated variability was due to a lack of control regarding the collection time of the experimentally evolved Drosophila.
ContributorsBelohlavek, David (Author) / Angilletta, Michael (Thesis director) / Francisco, Wilson (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
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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

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
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Description
Bee communities form the keystone of many ecosystems through their pollination services. They are dynamic and often subject to significant changes due to several different factors such as climate, urban development, and other anthropogenic disturbances. As a result, the world has been experiencing a decline in bee diversity and abundance,

Bee communities form the keystone of many ecosystems through their pollination services. They are dynamic and often subject to significant changes due to several different factors such as climate, urban development, and other anthropogenic disturbances. As a result, the world has been experiencing a decline in bee diversity and abundance, which can have detrimental effects in the ecosystems they inhabit. One of the largest factors that impacts bees in today's world is the rapid urbanization of our planet, and it impacts the bee community in mixed ways. Not very much is understood about the bee communities that exist in urban habitats, but as urbanization is inevitably going to continue, knowledge on bee communities will need to strengthen. This study aims to determine the levels of variance in bee communities, considering multiple variables that bee communities can differ in. The following three questions are posed: do bee communities that are spatially separated differ significantly? Do bee communities that are separated by seasons differ significantly? Do bee communities that are separated temporally (by year, interannually) differ significantly? The procedure to conduct this experiment consists of netting and trapping bees at two sites at various times using the same methods. The data is then statistically analyzed for differences in abundance, richness, diversity, and species composition. After performing the various statistical analyses, it has been discovered that bee communities that are spatially separated, seasonally separated, or interannually separated do not differ significantly when it comes to abundance and richness. Spatially separated bee communities and interannually separated bee communities show a moderate level of dissimilarity in their species composition, while seasonally separated bee communities show a greater level of dissimilarity in species composition. Finally, seasonally separated bee communities demonstrate the greatest disparity of bee diversity, while interannually separated bee communities show the least disparity of bee diversity. This study was conducted over the time span of two years, and while the levels of variance of an urban area between these variables were determined, further variance studies of greater length or larger areas should be conducted to increase the currently limited knowledge of bee communities in urban areas. Additional studies on precipitation amounts and their effects on bee communities should be conducted, and studies from other regions should be taken into consideration while attempting to understand what is likely the most environmentally significant group of insects.
ContributorsPhan, James Thien (Author) / Sweat, Ken (Thesis director) / Foltz-Sweat, Jennifer (Committee member) / School of Music (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
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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

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
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Description
The college textbook is the most commonly required component of almost any college course, regardless of a student's academic discipline. Professors often expect students to have access to the textbook and to use it to complete assigned readings. Textbooks often contain features that are designed to facilitate active reading, or

The college textbook is the most commonly required component of almost any college course, regardless of a student's academic discipline. Professors often expect students to have access to the textbook and to use it to complete assigned readings. Textbooks often contain features that are designed to facilitate active reading, or critical engagement with the information being read, to enhance learning of the material. However, students often do not read or prioritize reading the textbook. Students who do read, tend not to read the textbook as intended or use many of the features designed to promote active reading and enhanced learning of the material. Educational studies of textbooks tend to focus on aspects related to topics more relevant to publishers or professors with less research on aspects of the textbook applicable to students at the college level. The purpose of this study is to evaluate students' textbook use and their attitudes toward the textbook in an introductory biology course. Results of this study indicate students hold positive attitudes toward their textbook in an introductory biology course and majority of students do not use components meant to facilitate active learning. Although students report completing assigned readings, students may actually be reading select portions of what is assigned in using the textbook to prepare for exams. These results suggest that students may only be using their textbook to enhance their understanding of materials they expect to be tested on. The findings of this study help to understand the role of the textbook from the perspective of the student and provide insight for improving textbook design and use in science courses at the college level.
ContributorsRudolph, Alexia Marion (Author) / Vanmali, Binaben (Thesis director) / Chen, Ying-Chih (Committee member) / Yoho, Rachel (Committee member) / Department of Psychology (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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Description

Lyme disease is a common tick-borne illness caused by the Gram-negative bacterium Borrelia burgdorferi. An outer membrane protein of Borrelia burgdorferi, P66, has been suggested as a possible target for Lyme disease treatments. However, a lack of structural information available for P66 has hindered attempts to design medications to target

Lyme disease is a common tick-borne illness caused by the Gram-negative bacterium Borrelia burgdorferi. An outer membrane protein of Borrelia burgdorferi, P66, has been suggested as a possible target for Lyme disease treatments. However, a lack of structural information available for P66 has hindered attempts to design medications to target the protein. Therefore, this study attempted to find methods for expressing and purifying P66 in quantities that can be used for structural studies. It was found that by using the PelB signal sequence, His-tagged P66 could be directed to the outer membrane of Escherichia coli, as confirmed by an anti-His Western blot. Further attempts to optimize P66 expression in the outer membrane were made, pending verification via Western blotting. The ability to direct P66 to the outer membrane using the PelB signal sequence is a promising first step in determining the overall structure of P66, but further work is needed before P66 is ready for large-scale purification for structural studies.

ContributorsRamirez, Christopher Nicholas (Author) / Fromme, Petra (Thesis director) / Hansen, Debra (Committee member) / Department of Physics (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
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Description

Studies of animal contests often focus solely on a single static measurement of fighting ability, such as the size or the strength of the individual. However, recent studies have highlighted the importance of individual variation in the dynamic behaviors used during a fight, such as, assessment strategies, decision making, and

Studies of animal contests often focus solely on a single static measurement of fighting ability, such as the size or the strength of the individual. However, recent studies have highlighted the importance of individual variation in the dynamic behaviors used during a fight, such as, assessment strategies, decision making, and fine motor control, as being strong predictors of the outcome of aggression. Here, I combined morphological and behavioral data to discover how these features interact during aggressing interactions in male virile crayfish, Faxonius virilis. I predicted that individual variation in behavioral skill for decision making (i.e., number of strikes thrown), would determine the outcome of contest success in addition to morphological measurements (e.g. body size, relative claw size). To evaluate this prediction, I filmed staged territorial interactions between male F. virilis and later analyzed trial behaviors (e.g. strike, pinches, and bout time) and aggressive outcomes. I found very little support for skill to predict win/loss outcome in trials. Instead, I found that larger crayfish engaged in aggression for longer compared to smaller crayfish, but that larger crayfish did not engage in a greater number of claw strikes or pinches when controlling for encounter duration. Future studies should continue to investigate the role of skill, by using finer-scale techniques such as 3D tracking software, which could track advanced measurements (e.g. speed, angle, and movement efficiency). Such studies would provide a more comprehensive understanding of the relative influence of fighting skill technique on territorial contests.

ContributorsNguyen, Phillip Huy (Author) / Angilletta, Michael (Thesis director) / McGraw, Kevin (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description

Due to what is known as the “biologically desert fallacy” and the pervasive westernized ideal of wilderness that has influenced widespread American Conservation culture for millennia, urban areas have long been deemed as areas devoid of biodiversity. However, cities can contribute significantly to regional biodiversity and provide vital niches for

Due to what is known as the “biologically desert fallacy” and the pervasive westernized ideal of wilderness that has influenced widespread American Conservation culture for millennia, urban areas have long been deemed as areas devoid of biodiversity. However, cities can contribute significantly to regional biodiversity and provide vital niches for wildlife, illuminating the growing awareness that cities are crucial to the future of conservation and combating the global biodiversity crisis. In terms of the biodiversity crisis, bats are a relevant species of concern. In many studies, different bat species have been broadly classified according to their ability to adapt to urban environments. There is evidence that urban areas can filter bat species based on traits and behavior, with many bats possessing traits that do not allow them to live in cities. The three broad categories are urban avoiders, urban adapters, or urban exploiters based upon where their abundance is highest along a gradient of urban intensity. A common example of an urban exploiter bat is a Mexican Free-tailed bat, which can thrive and rely on urban environments and it is found in the Phoenix Metropolitan area. Bats are important as even in urban environments they play vital ecological roles such as cactus pollination, insect management, and seed dispersal. Bat Crazy is a thesis project focused on urban enhancement and the field of urban biodiversity. The goals of this thesis are to observe how bio-conscious urban cities that work to promote species conservation can serve as a positive tool to promote biodiversity and foster community education and engagement for their urban environment.

ContributorsKaiser, Nicole (Author) / Senko, Jesse (Thesis director) / Angilletta, Michael (Committee member) / Lynch, John (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor)
Created2023-05