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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
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

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
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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

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
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Description
ABSTRACT:
The experiment was conducted to analyze the role of menaquinone (MQ) in heliobacteria’s reaction center (HbRC). Their photosynthetic apparatus is a homodimeric of type I reaction center (1). HbRC contains these cofactors: P800 (special pair cholorphyll), A0 (8-hydroxy-chlorophyll [Chl] a), and FX (iron-sulfur cluster). The MQ factor is bypassed during

ABSTRACT:
The experiment was conducted to analyze the role of menaquinone (MQ) in heliobacteria’s reaction center (HbRC). Their photosynthetic apparatus is a homodimeric of type I reaction center (1). HbRC contains these cofactors: P800 (special pair cholorphyll), A0 (8-hydroxy-chlorophyll [Chl] a), and FX (iron-sulfur cluster). The MQ factor is bypassed during the electron transfer process in HbRC. Electrons from the excited state of P800 (P800*) are transported to A0 and then directly to Fx. The hypothesis is that when electrons are photoaccumulated at Fx, and without the presence of any electron acceptors to the cluster, they would be transferred to MQ, and reduce it to MQH2 (quinol). Experiments conducted in the past with HbRC within the cell membranes yielded data that supported this hypothesis (Figures 4 and 5). We conducted a new experiment based on that foundation with HbRC, isolated from cell membrane. Two protein assays were prepared with cyt c553 and ascorbate in order to observe this phenomenon. The two samples were left in the glove box for several days for equilibration and then exposed to light in different intensity and periods. Their absorption was monitored at 800 nm for P800 or 554 nm for cyt c553 to observe their oxidation and reduction processes. The measurements were performed with the JTS-10 spectrophotometer. The data obtained from these experiments support the theory that P800+ reduced by the charge recombination of P800+Fx-. However, it did not confirm the reduction of P800+ done by cyt c553¬ which eventually lead to a net accumulation of oxidized cyt c553; instead it revealed another factor that could reduce P800+ faster and more efficient than cyt c553 (0.5 seconds vs several seconds), which could be MQ. More experiments need to be done in order to confirm this result. Hence, the data collected from this experiment have yet to support the theory of MQ being reduced to MQH2 outside the bacterial membranes.
ContributorsNguyen, Phong Thien Huynh (Author) / Redding, Kevin (Thesis director) / Van Horn, Wade (Committee member) / Wachter, Rebekka (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Description
Metalloporphyrins serve important roles in biological processes and in emerging technologies with applications to energy conversion. When electrochemically activated in solution, metalloporphyrins have the ability to catalyze the conversion of protons into hydrogen fuels. In this report, the synthesis and characterization of zinc, nickel, cobalt and copper analogs of 5,10,15,20-tetrakis(pentafluorophenyl)

Metalloporphyrins serve important roles in biological processes and in emerging technologies with applications to energy conversion. When electrochemically activated in solution, metalloporphyrins have the ability to catalyze the conversion of protons into hydrogen fuels. In this report, the synthesis and characterization of zinc, nickel, cobalt and copper analogs of 5,10,15,20-tetrakis(pentafluorophenyl) porphyrin (PF20) and 5,10,15,20-tetra-p-tolyporphyrin (TTP) are described. All target compounds are characterized with UV-Vis spectroscopy and MALDI-TOF mass spectrometry. The freebase porphyrins and non-paramagnetic metalloporphyrins are further characterized by proton nuclear magnetic resonance spectroscopy and all proton resonances are assigned. Electrochemical measurements show the reduction potential of the fluorinated phenyl substituted porphyrins is shifted to less negative values as compared to the reduction potential measured using non-fluorinated analogs. These results illustrate the ability to use fluorine as a synthetic tool for altering the electronic properties of metalloporphyrins. Further, these findings serve a critical role in choosing metalloporphyrin electrocatalysts with the appropriate energetic and optical properties for integration to semiconductors with applications to solar-to- fuels technologies.
ContributorsNanyangwe, Sylvia Kapya (Author) / Moore, Gary (Thesis director) / Van Horn, Wade (Committee member) / School of Criminology and Criminal Justice (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Description
This study was conducted to understand the reactivity of APE1 in repairing abasic sites associated with clustered DNA damages and to determine if the efficiency of APE1 enzyme is affected by the type of bases (purines or pyrimidines) neighboring the AP site. DNA damages are always occurring in living cells

This study was conducted to understand the reactivity of APE1 in repairing abasic sites associated with clustered DNA damages and to determine if the efficiency of APE1 enzyme is affected by the type of bases (purines or pyrimidines) neighboring the AP site. DNA damages are always occurring in living cells and if left uncorrected can lead to various problems such as diseases and even cell death. Cells are able to recognize and correct these DNA damages to prevent further damages to the genome, and the Base Excision Repair (BER) pathway is one of the mechanisms used in repairing DNA damages. A former student in the Levitus Lab, Elana Maria Shepherd Stennett, henceforth referred to as Elana worked on this project. She observed that the activity of the APE1 enzyme increased some when the base opposing the abasic site was changed from thymine (T) to adenine (A) while no difference was observed when the surrounding bases were changed. Thus, this experiment was conducted to further study the results she obtained and to possibly validate her findings. The AP sites used in this study are natural abasic sites created by UDG glycosylase enzyme from a double stranded uracil-containing DNA samples ordered from IDT technologies. Each reaction was carried out at physiological temperature (37degrees Celsius) and analyzed using polyacrylamide gel electrophoresis.
ContributorsOnyeabor, Moses Ekenedilichukwu (Author) / Levitus, Marcia (Thesis director) / Van Horn, Wade (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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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.

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
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Description
Transient receptor potential channels (TRP channels) are a family of ion channels that mediate a wide variety of sensations, including pain, temperature, and mechanosensation. Human phosphoinositide-interacting regulator of TRP (hPIRT) is a 15.5 kDa, relatively uncharacterized membrane protein that has been shown to modulate the activity of certain TRP channels

Transient receptor potential channels (TRP channels) are a family of ion channels that mediate a wide variety of sensations, including pain, temperature, and mechanosensation. Human phosphoinositide-interacting regulator of TRP (hPIRT) is a 15.5 kDa, relatively uncharacterized membrane protein that has been shown to modulate the activity of certain TRP channels and some other ion channels. hPIRT is also able to interact with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P¬2), a phospholipid that modulates the activity of many important signaling proteins, including TRP channels. Some information is already known about the structure of hPIRT: it contains a relatively unstructured N-terminus, two transmembrane helices, and a juxtamembrane region at the C-terminus that plays a role in binding PI(4,5)P2 and TRPV1. However, more detailed structural data about this molecule would be very informative in understanding how these interactions occur. In order to accomplish this, this thesis investigates the measurement of residual dipolar couplings (RDCs) in nuclear magnetic resonance spectroscopy (NMR) to refine the structure of hPIRT. RDCs are a measurable effect in NMR experiments caused by partial alignment of molecules solubilized in a weakly anisotropic medium. The resulting data set can be used to calculate bond angles within the protein relative to the axis of the external magnetic field, which will assist efforts to further constrain the structure of hPIRT.
ContributorsGowland, Samuel Luke Walker (Author) / Van Horn, Wade (Thesis director) / Mor, Tsafrir (Committee member) / Sisco, Nicholas (Committee member) / School of Life Sciences (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
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Description
Transient Receptor Potential Melastatin 8 (TRPM8) is a non-selective cation channel notable as a primary cold sensor in humans. It is also involved in a variety of (patho)physiological events including pain sensation, chronic cough, diabetes, obesity, and cancer. TRPM8 is modulated by a variety of stimuli including pH, temperature, cooling

Transient Receptor Potential Melastatin 8 (TRPM8) is a non-selective cation channel notable as a primary cold sensor in humans. It is also involved in a variety of (patho)physiological events including pain sensation, chronic cough, diabetes, obesity, and cancer. TRPM8 is modulated by a variety of stimuli including pH, temperature, cooling agents, voltage, lipid, and other proteins. However, the molecular mechanism underlining its function has not yet clear raising the need for isolated proteins to be well-characterized. Over 20 years, E. coli has been a heterologous expression system of interest due to its low cost and high yield. However, the lack of post-translational modifications and chaperone may cause a misfolding or affect protein function. Mammalian expression system addresses these drawbacks and is a good candidate for the functional study of complex human protein. Here I describe my research in optimizing the transfection, expression, and purification of the human TRPM8 from adherent Human Embryonic Kidney (HEK293) cells which can be used for small-scale studies including, but not limited to, planar lipid bilayer electrophysiology.
ContributorsNguyen, Hoang Phuong My (Author) / Van Horn, Wade (Thesis director) / Wang, Xu (Committee member) / Hilton, Jacob (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Computing and Informatics Program (Contributor) / Barrett, The Honors College (Contributor)
Created2017-12