Matching Items (15)
Filtering by
- All Subjects: Genetics
- Creators: School of Molecular Sciences
- Member of: Barrett, The Honors College Thesis/Creative Project Collection
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
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
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
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 goal of this project was to design and create a genetic construct that would allow for <br/>tumor growth to be induced in the center of the wing imaginal disc of Drosophila larvae, the <br/>R85E08 domain, using a heat shock. The resulting transgene would be combined with other <br/>transgenes in a single fly that would allow for simultaneous expression of the oncogene and, in <br/>the surrounding cells, other genes of interest. This system would help establish Drosophila as a <br/>more versatile and reliable model organism for cancer research. Furthermore, pilot studies were <br/>performed, using elements of the final proposed system, to determine if tumor growth is possible <br/>in the center of the disc, which oncogene produces the best results, and if oncogene expression <br/>induced later in development causes tumor growth. Three different candidate genes were <br/>investigated: RasV12, PvrACT, and Avli.
ContributorsSt Peter, John Daniel (Author) / Harris, Rob (Thesis director) / Varsani, Arvind (Committee member) / School of Molecular Sciences (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
The transcriptome of an organism is a collection of the various messenger RNAs that the genes of an organism produce. As the level of gene expression is different between different tissues of an organism, understanding the transcriptome serves as a way to better understand the differences between the functions and abilities of tissues and cells in an organism. This understanding of the transcriptome can aid further research in targeted disease treatments and indentifying new biomarkers. This study aims to gather the transcriptome from various tissues of the organism Daphnia pulex. This will be done by using a combination of single cell RNA sequencing (scRNA-seq), which involves the isolation and sequencing of single cells, and single nuclei RNA sequencing (snRNA-seq), which involves the isolation and sequencing of single nuclei. Here we show the viability of isolating single cells and single nuclei from various Daphnia pulex tissues using different techniques and enzymes including trypLE, trypsin EDTA, accutase, etc by using microscopy and automatic cell counting. The results show that each tissue is best isolated using different techniques.
ContributorsShahriari, Ryan (Author) / Lynch, Michael (Thesis director) / Ye, Zhiqiang (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor)
Created2023-05
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
The non-native mosquito Aedes aegypti has become a common nuisance in Maricopa county. Associated with human settlement, Ae. aegypti is known to reproduce in standing water sources both indoors and outdoors, within vessels such as tires, flowerpots, and neglected swimming pools (Jansen & Beebe, 2010). Ae. aegypti and the related Ae. albopictus are the primary vectors of the arboviral diseases chikungunya, Zika, yellow fever and dengue. Ae. aegypti tends to blood feed multiple times per gonotrophic cycle (cycle of feeding and egg laying) which, alongside a preference for human blood and close association with human habitation, contributes to an increased risk of Ae. aegypti borne virus transmission (Scott & Takken, 2012). Between 2010-2017, 153 travel-associated cases of dengue were reported in the whole of Arizona (Rivera et al., 2020); while there have been no documented locally transmitted cases of Aedes borne diseases in Maricopa county, there are no apparent reasons why local transmission can’t occur in the future via local Aedes aegypti mosquitoes infected after feeding from travelling viremic hosts. Incidents of local dengue transmission in New York (Rivera et al., 2020) and Barcelona (European Center for Disease Control [ECDC], 2019) suggest that outbreaks of Aedes borne arbovirus’ can occur in regions more temperate than the current endemic range of Aedes borne diseases. Further, while the fact that Ae. aegypti eggs have a high mortality rate when exposed to cold temperatures limits the ability for Ae aegypti to establish stable breeding populations in temperate climates (Thomas, Obermayr, Fischer, Kreyling, & Beierkuhnlein, 2012), global increases in temperature will expand the possible ranges of Ae aegypti and Aedes borne diseases.
ContributorsHon, Ruiheng (Author) / Paaijmans, Krijn (Thesis director) / Bond, Angela (Committee member) / Angilletta, Michael (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Immediate early genes (IEGs) are rapidly activated in response to an environmental stimulus, and most code for transcription factors that mediate processes of synaptic plasticity, learning, and memory. EGR3, an immediate early gene transcription factor, is a mediator of biological processes that are disrupted in patients with schizophrenia (SCZ). A microarray experiment conducted by our lab revealed that Egr3 also regulates genes involved in DNA damage response. A recent study revealed that physiological neuronal activity results in the formation of DNA double-stranded breaks (DSBs) in the promoters of IEGs. Additionally, they showed that these DSBs are essential for inducing the expression of IEGs, and failure to repair these DSBs results in the persistent expression of IEGs. We hypothesize that Egr3 plays a role in repairing activity- induced DNA DSBs, and mice lacking Egr3 should have an abnormal accumulation of these DSBs. Before proceeding with that experiment, we conducted a preliminary investigation to determine if electroconvulsive stimulation (ECS) is a reliable method of inducing activity- dependent DNA damage, and to measure this DNA damage in three subregions of the hippocampus: CA1, CA3, and dentate gyrus (DG). We asked the question, are levels of DNA DSBs different between these hippocampal subregions in animals at baseline and following electroconvulsive stimulation (ECS)? To answer this question, we quantified γ-H2AX, a biomarker of DNA DSBs, in the hippocampal subregions of wildtype mice. Due to technical errors and small sample size, we were unable to substantiate our preliminary findings. Despite these shortcomings, our experimental design can be modified in future studies that investigate the role of Egr3 in activity-induced DNA damage repair.
ContributorsKhoshaba, Rami Samuel (Author) / Newbern, Jason (Thesis director) / Gallitano, Amelia (Committee member) / Marballi, Ketan (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
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