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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

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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Brown adipose tissue (BAT) is thought to be important in combating obesity as it can expend energy in the form of heat, e.g. thermogenesis. The goal of this study was to study the effect of injected norepinephrine (NE) on the activation of BAT in rats that were fed a high

Brown adipose tissue (BAT) is thought to be important in combating obesity as it can expend energy in the form of heat, e.g. thermogenesis. The goal of this study was to study the effect of injected norepinephrine (NE) on the activation of BAT in rats that were fed a high fat diet (HFD). A dose of 0.25 mg/kg NE was used to elicit a temperature response that was measured using transponders inserted subcutaneously over the BAT and lower back and intraperitoneally to measure the core temperature. The results found that the thermic effect of the BAT increased after the transition from low fat diet to a high fat diet (LFD) yet, after prolonged exposure to the HFD, the effects resembled levels found with the LFD. This suggests that while a HFD may stimulate the effect of BAT, long term exposure may have adverse effects on BAT activity. This may be due to internal factors that will need to be examined further.
ContributorsSion, Paul William (Author) / Herman, Richard (Thesis director) / Borges, Chad (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
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For the past couple decades, there has been a continuous rise in obesity and Type II Diabetes which has been attributed to the rise in calorically dense diets, especially those heavy in fats. Because of its rising prevalence, accompanied health concerns, and high healthcare costs, detection and therapies for these

For the past couple decades, there has been a continuous rise in obesity and Type II Diabetes which has been attributed to the rise in calorically dense diets, especially those heavy in fats. Because of its rising prevalence, accompanied health concerns, and high healthcare costs, detection and therapies for these metabolic diseases are in high demand. Insulin resistance is a typical hallmark of Type II Diabetes and the metabolic deficiencies in obesity and is the main focus of this project. The primary purpose of this study is (1) detect the presence of two types of insulin resistance (peripheral and hepatic) as a function of age, (2) distinguish if diet impacted the presence of insulin resistance, and (3) determine both the short-term and long-term effects of caloric restriction on metabolic health. The following study longitudinally observed the changes in insulin resistance in high-fat fed and low-fat fed rodents under ad libitum and caloric restriction conditions over the course of 23 weeks. Fasting blood glucose, fasting insulin, body weight, and sensitivity of insulin on tissue were monitored in order to determine peripheral and hepatic insulin resistance. A high fat diet resulted in higher body weights and higher hepatic insulin resistance with no notable effect on peripheral insulin resistance. Caloric restriction was found to alleviate insulin resistance both during caloric restriction and four weeks after caloric restriction ended. Due to sample size, the generalizability of the findings in this study are limited. However, the current study did provide considerable results and can be viewed as a pilot study for a larger-scale study.
ContributorsZuo, Dana (Author) / Trumble, Benjamin (Thesis director) / Herman, Richard (Committee member) / Department of Psychology (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Description
With the influence of the Western Diet, obesity has become a rising problem in the country today. Western Diet is characterized by the overconsumption of processed food that is low in nutritional values and high in saturated fats. Study showed that every two out of three adults in the United

With the influence of the Western Diet, obesity has become a rising problem in the country today. Western Diet is characterized by the overconsumption of processed food that is low in nutritional values and high in saturated fats. Study showed that every two out of three adults in the United States are either overweight or obese. Being obese increase the risk of many other disease such as diabetes, cardiovascular disease and insulin resistance. Besides being a great health concern, obesity is also cause a great financial burden. Many efforts have been made to understand the defense against obesity and weight loss. The goal of this study was to understand the characterization of food intake and weight gain responses when imposed on a high-fat diet (HFD) using rats. It was predicted that weight gain would be dependent on energy intake and it would have a significant effect on adiposity compared to energy intake. Data showed that energy intake had high significance with adiposity whereas weight gain showed no significance. Also for the rats that were on HFD, the obesity-prone (OP) rats exhibited a great amount of weight gain and energy intake while the obesity-resistance (OR) rats showed a similar weight gain to the controlled group on low-fat diet (LFD) despite being hyperphagic. This suggests that OR is characterized by equal weight gain despite hyperphagia but this alone cannot explain the boy defense against obesity. More research is needed with a larger sample size to understand weight gain responses in order to fight against the epidemic of obesity.
ContributorsMao, Samuel (Author) / Herman, Richard (Thesis director) / Baluch, Page (Committee member) / Lamb, Timothy (Committee member) / WPC Graduate Programs (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Description
The FoF1 ATP synthase is a molecular motor critical to the metabolism of virtually all life forms, and it acts in the manner of a hydroelectric generator. The F1 complex contains an (αβ)3 (hexamer) ring in which catalysis occurs, as well as a rotor comprised by subunit-ε in addition to

The FoF1 ATP synthase is a molecular motor critical to the metabolism of virtually all life forms, and it acts in the manner of a hydroelectric generator. The F1 complex contains an (αβ)3 (hexamer) ring in which catalysis occurs, as well as a rotor comprised by subunit-ε in addition to the coiled-coil and globular foot domains of subunit-γ. The F1 complex can hydrolyze ATP in vitro in a manner that drives counterclockwise (CCW) rotation, in 120° power strokes, as viewed from the positive side of the membrane. The power strokes that occur in ≈ 300 μsec are separated by catalytic dwells that occur on a msec time scale. A single-molecule rotation assay that uses the intensity of polarized light, scattered from a 75 × 35 nm gold nanorod, determined the average rotational velocity of the power stroke (ω, in degrees/ms) as a function of the rotational position of the rotor (θ, in degrees, measured in reference to the catalytic dwell). The velocity is not constant but rather accelerates and decelerates in two Phases. Phase-1 (0° - 60°) is believed to derive power from elastic energy in the protein. At concentrations of ATP that limit the rate of ATP hydrolysis, the rotor can stop for an ATP-binding dwell during Phase-1. Although the most probable position that the ATP-binding dwell occurs is 40° after the catalytic dwell, the ATP-binding dwell can occur at any rotational position during Phase-1 of the power stroke. Phase-2 of the power stroke (60° - 120°) is believed to be powered by the ATP-binding induced closure of the lever domain of a β-subunit (as it acts as a cam shaft against the γ-subunit). Algorithms were written, to sort and analyze F1-ATPase power strokes, to determine the average rotational velocity profile of power strokes as a function of the rotational position at which the ATP-binding dwell occurs (θATP-bd), and when the ATP-binding dwell is absent. Sorting individual ω(θ) curves, as a function of θATP-bd, revealed that a dependence of ω on
θATP-bd exists. The ATP-binding dwell can occur even at saturating ATP concentrations. We report that ω follows a distinct pattern in the vicinity of the ATP-binding dwell, and that the ω(θ) curve contains the same oscillations within it regardless of θATP-bd. We observed that an acceleration/deceleration dependence before and after the ATP-binding dwell, respectively, remained for increasing time intervals as the dwell occurred later in Phase-1, to a maximum of ≈ 40°. The results were interpreted in terms of a model in which the ATP-binding dwell results from internal drag at a variable position on the γε rotor.
ContributorsBukhari, Zain Aziz (Author) / Frasch, Wayne D. (Thesis director) / Allen, James P. (Committee member) / Redding, Kevin (Committee member) / School of Molecular Sciences (Contributor) / Department of Physics (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
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Description
The two chapters of this thesis focus on different aspects of DNA and the properties of nucleic acids as the whole. Chapter 1 focuses on the structure of DNA and its relationship to enzymatic efficiency. Chapter 2 centers itself on threose nucleic acid and optimization of a step in the

The two chapters of this thesis focus on different aspects of DNA and the properties of nucleic acids as the whole. Chapter 1 focuses on the structure of DNA and its relationship to enzymatic efficiency. Chapter 2 centers itself on threose nucleic acid and optimization of a step in the path to its synthesis. While Chapter 1 discusses DNA and Uracil-DNA Glycosylase with regards to the base excision repair pathway, Chapter 2 focuses on chemical synthesis of an intermediate in the pathway to the synthesis of TNA, an analogous structure with a different saccharide in the sugar-phosphate backbone.
Chapter 1 covers the research under Dr. Levitus. Four oligonucleotides were reacted for zero, five, and thirty minutes with uracil-DNA glycosylase and subsequent addition of piperidine. These oligonucleotides were chosen based on their torsional rigidities as predicted by past research and predictions. The objective was to better understand the relationship between the sequence of DNA surrounding the incorrect base and the enzyme’s ability to remove said base in order to prepare the DNA for the next step of the base excision repair pathway. The first pair of oligonucleotides showed no statistically significant difference in enzymatic efficiency with p values of 0.24 and 0.42, while the second pair had a p value of 0.01 at the five-minute reaction. The second pair is currently being researched at different reaction times to determine at what point the enzyme seems to equilibrate and react semi-equally with all sequences of DNA.
Chapter 2 covers the research conducted under Dr. Chaput. Along the TNA synthesis pathway, the nitrogenous base must be added to the threofuranose sugar. The objective was to optimize the original protocol of Vorbrüggen glycosylation and determine if there were better conditions for the synthesis of the preferred regioisomer. This research showed that toluene and ortho-xylene were more preferable as solvents than the original anhydrous acetonitrile, as the amount of preferred isomer product far outweighed the amount of side product formed, as well as improving total yield overall. The anhydrous acetonitrile reaction had a final yield of 60.61% while the ortho-xylene system had a final yield of 94.66%, an increase of approximately 32%. The crude ratio of preferred isomer to side product was also improved, as it went from 18% undesired in anhydrous acetonitrile to 4% undesired in ortho-xylene, both values normalized to the preferred regioisomer.
ContributorsTamirisa, Ritika Sai (Author) / Levitus, Marcia (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Windman, Todd (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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Description
With a quantum efficiency of nearly 100%, the electron transfer process that occurs within the reaction center protein of the photosynthetic bacteria Rhodobacter (Rh.) sphaeroides is a paragon for understanding the complexities, intricacies, and overall systemization of energy conversion and storage in natural systems. To better understand the way in

With a quantum efficiency of nearly 100%, the electron transfer process that occurs within the reaction center protein of the photosynthetic bacteria Rhodobacter (Rh.) sphaeroides is a paragon for understanding the complexities, intricacies, and overall systemization of energy conversion and storage in natural systems. To better understand the way in which photons of light are captured, converted into chemically useful forms, and stored for biological use, an investigation into the reaction center protein, specifically into its cascade of cofactors, was undertaken. The purpose of this experimentation was to advance our knowledge and understanding of how differing protein environments and variant cofactors affect the spectroscopic aspects of and electron transfer kinetics within the reaction of Rh. sphaeroides. The native quinone, ubiquinone, was extracted from its pocket within the reaction center protein and replaced by non-native quinones having different reduction/oxidation potentials. It was determined that, of the two non-native quinones tested—1,2-naphthaquinone and 9,10- anthraquinone—the substitution of the anthraquinone (lower redox potential) resulted in an increased rate of recombination from the P+QA- charge-separated state, while the substitution of the napthaquinone (higher redox potential) resulted in a decreased rate of recombination.
ContributorsSussman, Hallie Rebecca (Author) / Woodbury, Neal (Thesis director) / Redding, Kevin (Committee member) / Lin, Su (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12
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Description

Over 40% of adults in the United States are considered obese. Obesity is known to cause abnormal metabolic effects and lead to other negative health consequences. Interestingly, differences in metabolism and contractile performance between obese and healthy weight individuals are associated with differences in skeletal muscle fiber type composition between

Over 40% of adults in the United States are considered obese. Obesity is known to cause abnormal metabolic effects and lead to other negative health consequences. Interestingly, differences in metabolism and contractile performance between obese and healthy weight individuals are associated with differences in skeletal muscle fiber type composition between these groups. Each fiber type is characterized by unique metabolic and contractile properties, which are largely determined by the myosin heavy chain isoform (MHC) or isoform combination that the fiber expresses. In previous studies, SDS-PAGE single fiber analysis has been utilized as a method to determine MHC isoform distribution and single fiber type distribution in skeletal muscle. Herein, a methodological approach to analyze MHC isoform and fiber type distribution in skeletal muscle was fine-tuned for use in human and rodent studies. In the future, this revised methodology will be implemented to evaluate the effects of obesity and exercise on the phenotypic fiber type composition of skeletal muscle.

ContributorsOhr, Jalonna Rose (Author) / Katsanos, Christos (Thesis director) / Tucker, Derek (Committee member) / Serrano, Nathan (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
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Description

Seven human subjects with body mass indices (BMIs) ranging from 19.4 kg/ m2 to 26.7 kg/ m2 and six human subjects with BMIs ranging from 32.1 kg/ m2 to 37.6 kg/ m2 were recruited and subjected to 45-minute bouts of acute exercise to look at the changes in the plasma

Seven human subjects with body mass indices (BMIs) ranging from 19.4 kg/ m2 to 26.7 kg/ m2 and six human subjects with BMIs ranging from 32.1 kg/ m2 to 37.6 kg/ m2 were recruited and subjected to 45-minute bouts of acute exercise to look at the changes in the plasma concentration of the dopamine metabolite homovanillic acid (HVA) in response to acute physical activity. Plasma HVA concentration was measured before exercise and during the last 10 minutes of the exercise bout via competitive ELISA. On average the optical density (OD) of the samples taken from lean subjects decreased from 0.203 before exercise to 0.192 during exercise, indicating increased plasma HVA concentration. In subjects with obesity OD increased from 0.210 before exercise to 0.219 during exercise, indicating reduced plasma HVA concentration. These differences in OD were not statistically significant. Between the lean group and the group with obesity no significant difference was observed between the OD of the plasma samples taken before exercise, but a significant difference (p = 0.0209) was observed between the ODs of the samples taken after exercise. This indicated that there was a significant difference between the percent changes in OD between the lean group and the group with obesity, which suggested that there may be a body weight-dependent difference in the amount of dopamine released in response to exercise. Because of the lack of significance in the changes in OD within the lean group and the group with obesity the results of this study were insufficient to conclude that this difference is not due to chance, but further investigation is warranted.

ContributorsYoder, Jordan Corinne (Author) / Katsanos, Christos (Thesis director) / Davies, Pauline (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
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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