2024-03-28T09:43:07Zhttps://thescholarship.ecu.edu/oai/requestoai:TheScholarship.intra.ecu.edu:10342/31782021-03-03T20:52:54Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Solvent-Controlled Switch of Selectivity between sp[superscript]2 and s[superscript]p3 C-H Bond Activation by Platinum (II)
Garner, Alexander William
Huo, Shouquan
Chemistry
Chemistry
Cyclometalation reactions have been studied intensely for the past few decades, especially those containing palladium. The factors that control the process of the C-H bond activation, however, are not yet completely understood. C-H bonds are ever-present in organic molecules, but the vast majority of them cannot be exploited for chemical reactions due to their inert and stable nature. Early attempts to activate these bonds led to very complicated mixtures of products, and therefore not an acceptable means of C-H activation due to poor selectivity. Controlling the selectivity of a reaction is one of the most important issues surrounding synthetic chemistry. It is generally recognized that aromatic C-H bonds are more likely to undergo activation by platinum complexes. However, recently it has been illustrated that there is a delicate balance between sp[superscript]2 and sp[superscript]3 C-H bond activation in a platinum (II) complex system.       In this study, the solvent-controlled switch of selectivity between sp[superscript]2 and sp[superscript]3 C-H bond activation in platinum (II) complex systems will be discussed. Ligands L1 through L3 were designed and synthesized to test the selectivity of cycloplatination of a reaction with potassium tetrachloroplatinate (II) in two different solvents, acetonitrile and glacial acetic acid. It was found that in each of the solvents used, a different isomer was produced from the complexation reaction. Reactions of L1 through L3 with potassium tetrachloroplatinate (II) in acetonitrile produced the sp[superscript]2 substituted isomer (1B-3B), while the same reaction performed in glacial acetic acid formed the sp[superscript]3 substituted isomer (1A-3A). It was determined through mechanistic studies that the sp[superscript]2 substituted isomer is a kinetically controlled product, while the sp[superscript]3 substituted isomer is a thermodynamically controlled product. Also, it was found that the ratio of products depends on time, where as more time goes by the thermodynamically stable product begins to predominate.   Other issues examined in this study were the side reactions that occurred during the complexation of ligands L2 and L3. These side products were due to C-C bond cleavage in L2 and C-N bond cleavage in L3. These side products were characterized and studied in their own right. Â
2011-02-03
2011-05-17
2011-02-03
2011-05-17
2010
Master's Thesis
http://hdl.handle.net/10342/3178
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/53362021-03-03T21:02:17Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Using Fluorescent Probes to Study the Location and Interaction of Disaccharides within Lipid Bilayers
Barker, Morgan Elizabeth
Kennedy, Anthony
Chemistry
Chemistry
Lipid
Bilayer
It is well known that certain organisms, such as the tardigrade, are capable of surviving extreme conditions including dehydration, freezing and / or oxygen deficiency. The tardigrades ability to withstand such extreme conditions is associated with a decrease in its internal water content and the excess production of a disaccharide known as trehalose. Studies have focused on better understanding this organism survival mechanism in order to help aid in the preservation of biological cells. Researchers have attempted to replicate this phenomenon in vitro through a freeze-drying technique known as lyopreservation and have shown trehalose to be an effective lyoprotecting agent. However, lyopreservation is very technically challenging so some studies have focused on trehalose has a potential cryoprotectant. Cryopreservation involves storing hydrated cells or other materials at very low temperatures in a frozen state. Unfortunately, trehalose did not show effectiveness as a cryoprotecting agent. This may be due to trehalose being a highly hydrophilic molecule which does not penetrate into the membrane to offer protection during freezing. Our research aims to investigate other disaccharides with varying hydrophilicity as potential cryoprotectants for hydrated cells. In order to study the effectiveness of certain disaccharides as cryoprotectants, liposomes were used as a model membrane system. The location and interactions of both sucralose and sucrose within these membranes were monitored through fluorescence and ultra-violet visible spectrophotometry using two specific fluorophores. The first of these, diphenylhexatriene, is a hydrophobic probe quenched by water that exhibits strong fluorescence when incorporated into a cell membrane. The second, merocyanine 540, is a negatively charged cyanine dye located slightly above the glycerol backbone of the phospholipids and is sensitive to any structural variations within the membrane. The data obtained with each respective probe indicates that the packing efficiency and polarity of the membrane is decreased in the presence of sucralose, but not in the presence of sucrose. We hypothesize that this is due to different hydrophobic properties of the sugars allowing sucralose to penetrate further into the bilayer while sucrose does not. This study indicates that sucralose has a significant effect on both the packing efficiency and the polarity of the bilayer and thus its usefulness as a potential cryoprotectant for biological cells is of great interest and needs to be investigated.
2016-05-25
2019-02-26
2016-05
2016-04-26
May 2016
2016-05-25
Master's Thesis
http://hdl.handle.net/10342/5336
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/34192021-03-03T20:53:00Zcom_10342_55com_10342_1col_10342_58
Identification of the Cu(II) Coordinating Residues in the Prion Protein by Metal-Catalyzed Oxidation Mass Spectrometry: Evidence for Multiple Isomers at Low Cu(II) Loadings
Srikanth, Rapole
Wilson, Jonathan
Burns, Colin Sanderson
Vachet, Richard W.
Cu(II) prion binding
Different stoichiometric loadings
Metal-catalyzed oxidation
Diethyl pyrocarbonate
While the Cu(II) binding sites of the prion protein have been well studied under Cu-saturation conditions, the identity of the residues involved in coordinating Cu(II) at low stoichiometries and the order in which the binding sites load with Cu(II), remain unresolved. In this study, we have used two mass spectrometry based methods to gather insight into Cu(II)-prion binding under different stoichiometric loadings of Cu(II). The first method uses metal-catalyzed oxidation reactions to site specifically modify the residues bound to Cu(II) in solution, and the second method determines Cu binding sites based on the protection of His from modification by diethyl pyrocarbonate when this residue binds Cu(II) in solution. For both methods, the residues that are labeled by these reactions can then be unambiguously identified using tandem mass spectrometry. Upon applying these two complementary methods to a construct of the prion protein that contains residues 23-28 and 57-98, several noteworthy observations are made. Coordination of Cu(II) by multiple His imidazoles is found at 1:1 and 1:2 PrP:Cu(II) ratios. Notably, there appear to be four to seven isomers of this multiple histidine coordination mode in the 1:1 complex. Furthermore, our data clearly show that His96 is the dominant Cu(II) binding ligand, as in every isomer His96 is bound to Cu(II). The individual octarepeat binding sites begin to fill at ratios of 1:3 PrP:Cu(II) with no clear preference for the order in which they load with Cu(II), although the His77 octarepeat appears to saturate last. The existence of several ‘degenerate’ Cu binding modes at low PrP:Cu(II) ratios may allow it to more readily accept additional Cu(II) ions, thus allowing PrP to transition from a singly Cu(II) bound state to a multiply Cu(II) bound state as a function of cellular Cu(II) concentration. Originally published Biochemistry, Vol. 47, No. 35, Sep 2008
2011-04-28
2011-05-17
2011-04-28
2011-05-17
2008-09-02
Article
Biochemistry; 47:35 p. 9258-9268
http://hdl.handle.net/10342/3419
PMC2730420
10.1021/bi800970m
en_US
http://pubs.acs.org/doi/abs/10.1021/bi800970m
Author notified of opt-out rights by Cammie Jennings prior to upload of this article.
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/49572021-03-03T20:56:55Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Electrochemical Detection of Anti-Biofilm Activity Using Unnatural Amino Acid-Containing Antimicrobial Peptides
Vinogradov, Sergey M.
Hvastkovs, Eli G.
Chemistry
Chemistry
Biology
Antimicrobial peptides
Biosensors
Bacterial infections are a significant health problem that can be detrimental to the human population. It is estimated that bacterial infections are responsible for billions of dollars’ worth of damages in the health care field alone, and numerous deaths annually. Bacterial infections can become so detrimental because they produce a structure called biofilm, which facilitates antibiotic resistance and is a major cause of chronic infections. In order to combat this threat, new anti-biofilm and antibiotic therapies are being developed and their efficiency must be tested. A series of antimicrobial peptides (AMP) containing unnatural Tic-Oic amino acids have been developed for this purpose.  Traditional methods such as biological assays are the standard by which antibiotics are judged, but they have their drawbacks, such as the lengthy test times and the costs associated with it and the reagents. Electrochemical biosensors can remedy some of those drawbacks by offering speed and cost benefits. Electrochemical biosensors consisting of Layer-by-Layer (LbL) modified electrodes were constructed. These sensors were fabricated to test the anti-biofilm activity of the aforementioned unnatural amino acid-containing antimicrobial peptides against a model of Pseudomonas aeruginosa or against the bacteria itself. P. aeruginosa is a common biofilm producing bacteria.  First, we employed alginate as one of the layers in our sensor as P. aeruginosa is known to produce this as its major biofilm component. We show that the penetration of the alginate layer by the AMP can be detected electrochemically utilizing a solution-phase redox active molecule that produces an increasing signal upon electrochemical reduction when the film becomes compromised. Biological assays are presented that provide some validation for the sensor, but elucidated a particular AMP as compared to the electrochemical alginate sensor.   Based on this slight disagreement between our electrochemical model and the biological assay, we employed sensors that featured directly immobilized P. aeruginosa PAO1 on the electrode surface. These bacteria are electroactive, which negated the need for an external redox active molecule, and allowed the monitoring of anti-biofilm activity via a signal decrease over time. The P. aeruginosa sensor showed more agreement with the biological assay, highlighting the same AMP as active toward biofilm degredation at low (<1 mircoM) concentrations. Overall, these electrochemical biosensors utilizing models of and actual P. aeruginosa have opened up new avenues to test for effectiveness of potential anti-biofilm agents toward biofilm forming bacteria. Â
2015-06-04
2017-08-24
2015
Master's Thesis
http://hdl.handle.net/10342/4957
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/76052021-08-01T08:01:54Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_123
Development of Green Subcritical Water Separation Technology
Doctor, Ninad
Yang, Yu
Chemistry
Subcritical Water Extraction
Subcritical Water Chromatography Vanillin and Coumarin Stability Danshen Subcritical Water Extraction Ibuprofen
Aspirin
Metformin HCl
Theoretically speaking, water under temperature and pressure conditions below its critical point is called subcritical water. However, the term “subcritical water� normally refers to hot water up to 374 C and pressure up to 218 atm. As the temperature increases, the hydrogen bonding between water molecules weakens, making water less polar at elevated temperatures. In chemical and pharmaceutical industry organic solvents are extensively used for extraction, chromatography, chemical analysis, and remediation of toxic chemicals. It requires a large amount of money not only for purchasing the expensive organic solvents but also properly disposing of their wastes. Subcritical water technology has shown a great potential in eliminating or minimizing the use of organic solvents in the processes mentioned above as it solely uses subcritical water as the solvent.
The objective of this research is to further develop the green subcritical water separation technology. Specific aims include organic analyte stability in subcritical water, subcritical water extraction and analysis of active pharmaceutical ingredients (APIs) from medicinal herbs, subcritical water chromatographic separation of APIs from cold drugs, development of thermally stable stationary phase materials, and destruction of toxic polychlorinated biphenyls (PCBs) under subcritical water conditions.
The stability of vanillin and coumarin in subcritical water was studied at 100-250 ï‚°C. Vanillin was stable up to 60 min at all temperatures tested. While coumarin was stable at temperatures up to 150 ï‚°C, it started to degrade at 200 and 250 ï‚°C after heating for 60 min. Vanillin and coumarin were also extracted from vanilla beans and whole Tonka beans using subcritical water at 100-200 ï‚°C. The vanillin quantity extracted increased from 10.7 mg per gram of vanilla beans (10.7 mg/g) at 100 ï‚°C to 19.9 mg/g at 200 ï‚°C. Similarly, the coumarin quantity extracted increased from 23.9 mg per gram of whole Tonka beans (23.9 mg/g) at 100 ï‚°C to 36.8 mg/g at 200 ï‚°C.
Due to effectiveness, wide availability, and low side effects and costs, the use of herbal medicine has increased all around the world. Danshen (salvia miltiorrhiza) is a Chinese medicinal herb used to treat patients suffering from stroke, angina, chronic liver disease or other diseases. Active pharmaceutical ingredients including protocatechualdehyde, ferulic acid and caffeic acid were extracted from Dan Shen using water at 75 to 150 ï‚°C. Our results show that the extraction efficiency for all three APIs is improved with increasing temperature.
To eliminate the use of organic solvents in high-performance liquid chromatography (HPLC), subcritical water chromatography (SBWC) methods were developed for aspirin and metformin. SBWC separation of metformin HCL and aspirin were achieved at 95 ï‚°C and 125 ï‚°C, respectively. The recovery for both pharmaceutical active ingredients obtained by SBWC is 99% in comparing with the stated content of each drug. The relative standard deviation is less than 1% for SBWC assays developed in this work. This level of accuracy and precision achieved by SBWC is the same as that resulted by the traditional HPLC analysis also carried out in this work.
For subcritical water chromatography separations, thermally stable stationary phases are critical. Since zeolite is superior to the traditional silica in thermal stability, a method was developed to synthesize organic-bonded zeolite through the substitution of silanol groups with C18. In FTIR-ATR experiment stretching band of CH2 and CH3 clearly revealed the presence of carbon chain after the modification. HPLC column was packed with C18 modified zeolite for the evaluation of chromatographic separation. Caffeine peak was detected at lower concentration at 272 nm wavelength. It also showed some separation between vanillin and coumarin at 284 nm. However, the HPLC column was unable to perform separation above 0.4 mL/min due to the small pore size, and it is one of the limitations of zeolite modified stationary phase.
Lastly, the destruction of PCB-118, PCB-156 and PCB-180 congeners under subcritical conditions has been investigated. The percent degradation of PCBs was mostly improved by increasing the heating time, concentration of hydrogen peroxide and sodium hydroxide in water. All three PCB congeners investigated were completely destroyed after heating in 0.2% sodium hydroxide at 350 °C for an hour.
2020-02-04
2021-08-01
2019-08
2019-08-19
August 201
2020-01-29
Doctoral Dissertation
http://hdl.handle.net/10342/7605
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/51852021-03-03T21:00:03Zcom_10342_55com_10342_1col_10342_58
Wurster's crown ligands
Sibert, John W.
Wurster's crown ligands comprise a macrocyclic ligand such as a crown ether in which a hetero atom is substituted with a 1,4-phenylenediamine group. The phenylenediamine group is covalently bound to the macrocyclic ligand by one or both of the amine nitrogens, the amine nitrogen thereby substituting for the hetero atom of the macrocyclic ligand. The resulting compounds are redox active. Methods of making and using the compounds are also disclosed.
2016-02-05
2016-02-05
2001-07-17
Patent
U.S. Patent No. 6,262,258
http://hdl.handle.net/10342/5185
oai:TheScholarship.intra.ecu.edu:10342/29172021-03-03T20:53:16Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Thermodynamic Investigation into the Binding Properties of Cardiac Troponin (Human and Bovine)
Haulsee, Zachary Merle
Spuches, Anne M.
Chemistry
Chemistry, Inorganic
Inorganic chemistry
Troponin is an integral protein in the mechanism of muscle contraction. In order to induce cardiac muscle contraction, Ca[superscript]2[superscript]+ must bind to the TnC subunit (calcium binding subunit) of troponin to begin a conformational change in the protein. The ATPase rate of myosin with actin present is cooperatively activated by Ca[superscript]2[superscript]+ and Myosin. Ca[superscript]2[superscript]+ greatly increases the rate of ATPase activity (18-fold) and decreases the concentration of actin needed for muscle contraction activity. Ca[superscript]2[superscript]+ binding to troponin induces a conformational change that leads to a process of muscle contraction [8].  The focus of our research has been to investigate thermodynamic binding properties of various divalent metals to the Troponin C subunit of the cardiac muscle protein using isothermal titration calorimetry. We have been able to successfully observe Ca[superscript]2[superscript]+ binding to the apo form of Bovine Cardiac TnC (BVCTnC) as well as the apo form of Human Cardiac TnC (HCTnC).   Familial Hypertrophic Cardiomyopathy, FHCM, is an autosomal dominant genetic disorder. FHCM causes an abnormal cardiac muscle contraction response in patients afflicted with the genetic mutations that result in the disorder. About 1 in 500 people, 0.2%, are afflicted with this disorder. There are many ways to approach treatment for this disease. A treatment that we have considered uses calcium sensitizing drugs. Calcium sensitizing drugs allow troponin to be more sensitive to the presence of calcium which induces cardiac muscle contraction [31].  Another focus of our research is to determine thermodynamic binding properties of calcium sensitizing drugs to troponin using isothermal titration calorimetry. Understanding the thermodynamic properties of drug-protein interaction can help reveal the mechanism of action by which the drug operates. These studies will lead to a better understanding of how calcium sensitizing drugs interact with troponin and determine their practicality in drug design for patients afflicted with familial hypertrophic cardiomyopathies. Â
2010-09-16
2011-05-17
2012-07-31
2010
Master's Thesis
http://hdl.handle.net/10342/2917
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/88392022-12-01T09:01:59Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
EXPLORING STUDENT EXPERIENCES ACROSS A LINKED SEQUENCE COURSE-BASED UNDERGRADUATE RESEARCH EXPERIENCE
Whiting, Brandon
Walker, Joi P
Chemistry
Course-based Undergraduate Research (CURE)
Focus Group
Theme
This study reports the effect a linked Course-based Undergraduate Research Experience (CURE) has on a students' understanding of research and the associated experiences they encountered. CUREs offer an alternative path to authentic research which can increase student access and potentially increase the overall diversity in a laboratory setting. The institution in this study has recently developed a linked-CURE sequence that bridges a lower-division organic chemistry laboratory with an upper-division quantitative analysis laboratory. Focus groups consisting of 3-5 students each were conducted with students participating in two cohorts of the CURE sequence during the last week of the academic term. Through the analysis of survey and interview data three themes were developed: working towards a common goal, developing a deeper understanding of the science content, and addressing obstacles that were encountered.
2021-01-05
2022-12-01
2020-12
2020-11-23
December 2
2020-12-18
Master's Thesis
http://hdl.handle.net/10342/8839
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/106842023-12-18T17:47:56Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Biophysical Investigation into the Protein Dynamics Governing the Allosteric Regulation of Plant and Animal 15-Lipoxygenases
Roberts, Daniella
Offenbacher, Adam R
Chemistry
Allostery
Protein Dynamics
Steady-State Kinetics
Hydrogen-Deuterium Exchange Mass Spectrometry
Lipoxygenases (LOXs) are a family of enzymes found in plants, animals, fungi, and bacteria that catalyze the per-oxidation of polyunsaturated fatty acids. In plants, LOXs are involved in growth, development, and defense against pathogenic attacks. There are also multiple isoforms present in humans, which have contradictory roles in the body. Specifically, human 15-LOX isoforms, 15-LOX-1 and 15-LOX-2, are involved in both homeostasis and pro-inflammatory pathways. In order to selectively target the activity of these enzymes, research has turned to allosteric regulation, which is the focus of this Thesis. Previously, the allosteric regulation of a model plant 15-LOX, soybean lipoxygenase-1 (SLO), has been characterized using hydrogen-deuterium exchange mass spectrometry (HDX-MS), revealing that the addition of the allosteric effector, oleyl sulfate (OS), alters a specific region of the enzyme. Herein, we used a combination of thermodynamic and biophysical techniques such as isothermal titration calorimetry and differential scanning calorimetry to investigate the allosteric regulation of SLO by OS. We present data which supports that the allosteric regulation of SLO by OS does not induce oligomerization or large-scale conformational changes and that the allostery is dynamically driven. We also employed HDX-MS to study the dynamics of 15-LOX-1 compared to previously collected data of 15-LOX-2 to reveal structural differences between the two isozymes that may explain their altered catalytic behavior.
2022-06-14
2022-05
2022-04-27
May 2022
2022-06-07
Master's Thesis
http://hdl.handle.net/10342/10684
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/50072021-03-03T20:58:24Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Synthesis Of Non-Natural Fmoc-Protected Amino Acids To Provide Novel Fluorescent Anion Probes And Their Incorporation Into Synthetic Peptides
Farrell, David Percy
Allen, William E.
Chemistry
Chemistry
Organic chemistry
Biochemistry
Anion binding
Computational chemistry
Membrane transport
Solid-phase peptide synthesis
Cystic fibrosis is a genetic disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CFTR serves to control the gradient of chloride and bicarbonate ions across the cellular membrane of epithelial tissues (e.g., mucosa, intestinal walls, and lungs). Excess chloride on the inside of epithelial cells causes mucus in the lungs to become very thick. This thickening is responsible for a patients' characteristic thick sputum, coughing, and trouble breathing. The thick mucus also creates an ideal environment for opportunistic bacteria like Pseudomonas aeruginosa and Mycobacterium tuberculosis to take residence and proliferate. Very little is known about the transmembrane structure of CFTR, but with current therapies a single mutation can shorten a person's life by about 50%.
Many researchers have attempted to bypass the CFTR protein and synthesize anion shuttle transporters or transmembrane pores. The research presented here focuses on the synthesis of anion-responsive amino acids, for eventual incorporation into membrane-spanning peptides. Amino acids 2.1 and 2.2 were successfully synthesized from commercially available Fmoc-Glu-OtBu and Fmoc-Asp-OtBu through the conversion of an acid azide into an isocyanate via a Curtis Rearrangement. An N-substituted-4-amino-naphthalimide was chosen as the chromophore due to its ability to absorb and emit light at a significantly longer wavelength than those produced by naturally occurring amino acids. The fluorescence provides an indirect insight into the binding strength of the novel amino acids, and eventually designer peptides, to various biologically relevant anions. The fluorescence can also be used by a researcher to learn more about a peptides concentration, location, microenvironment, structure, and mechanism of action. The presence of a fluorenylmethyloxycarbonyl (Fmoc) protecting group allows a researcher to use standard solid phase peptide synthesis protocols to produce either a known peptide or any imaginable variation. Fluorescent amino acids are useful tools in grasping a better understanding of protein channelopathies such as cystic fibrosis. One day, the use of synthetic peptide ion channels will be an effective therapy to alleviate the many symptoms and even the causes of death that afflicts the many patients suffering from channelopathies today.
2015-08-24
2015-08-24
2015
Master's Thesis
http://hdl.handle.net/10342/5007
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/110312022-09-07T07:16:09Zcom_10342_55com_10342_1col_10342_58
Subcritical Water Extraction of Salvia miltiorrhiza
Kapalavavi, Brahmam
Doctor, Ninad
Yang, Yu
Zhang, Baohong
active pharmaceutical ingredients
Salvia miltiorrhiza
reproduction
2022-09-06
2022-09-06
2021-03-15
Article
1420-3049
http://hdl.handle.net/10342/11031
10.3390/molecules26061634
en_US
oai:TheScholarship.intra.ecu.edu:10342/29142021-03-03T20:53:01Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Surfactant Assisted Electrospray Ionization Time of Flight Mass Spectrometry of Synthetic Polymers
Brooks, James L.
Romack, Timothy J.
Chemistry
Chemistry, Analytical
Analytical chemistry
Synthetic polymers are complex mixtures which often require extensive characterization to determine chemical composition and structure. Mass spectrometric techniques have greatly enhanced the quantity, as well as the quality, of data that can be used to characterize synthetic polymers. Electrospray Ionization Mass Spectrometry, ESI-MS, is capable of providing a wealth of information on biomolecules, inorganic compounds, and small synthetic polymer (molecular weight less than 3,000 g/mol). While ESI-MS has proven to be a powerful characterization technique for small synthetic polymers its tendency to generate multiply charged ions results in very complex data for higher molecular weight synthetic polymers and yields data that is too complicated for complete analysis. A means to simplify the data obtained for ESI-MS characterization of large synthetic polymers by reducing multiple charging and increasing signal to noise ratio of the singly charged analyte ions would be of substantial use.  Singly charged poly (methyl methacrylate) ions with weight average molecular weights 4,000, 8,000 and 12,000 g/mol were observed by adding a surfactant (cetyltrimethylammonium bromide) and changing critical ESI conditions. Altering these parameters, as well as others, has played an important role of reducing the overall charging that the analyte may gain during the desolvation process. Â
2010-09-16
2011-05-17
2010-09-16
2011-05-17
2010
Master's Thesis
http://hdl.handle.net/10342/2914
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/120882023-02-03T08:22:49Zcom_10342_55com_10342_1col_10342_58
Development of Transient Recombinant Expression and Affinity Chromatography Systems for Human Fibrinogen
Popovic, Grega
Kirby, Nicholas C.
Daub, Caroline E.
Offenbacher, Adam R.
Dement, Taylor C.
Belcher, Heather A.
Hudson, Nathan E.
fibrin
recombinant protein
affinity chromatography
2023-02-02
2023-02-02
2022-01-19
Article
1422-0067
http://hdl.handle.net/10342/12088
10.3390/ijms23031054
en_US
oai:TheScholarship.intra.ecu.edu:10342/128762023-06-05T14:06:31Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Towards Establishing Methods of Synthesis, Purification, and Structural Determination for Human Fibrinogen
Kirby, Nicholas Carter
Chemistry
Fibrinogen
Structure-function relationship
Transient transfection
Affinity chromatography
Cryogenic electron microscopy
Fibrinogen is a prevalent blood-based glycoprotein. In its polymerizable form, fibrin, it serves as the structural component of blood clots and helps to incorporate red blood cells, macrophages, and fibroblasts to the wound-site. Fibrinogen plays important roles in inflammation and tissue regeneration, regulating growth factor receptor pathway activation, scar formation, and immune response, yet many questions remain about fibrinogen’s structure-function link in these various pathophysiological states. Studies focused on improving expression systems for recombinant fibrinogen, and developing more rapid and robust techniques for purifying fibrinogen from complex media would provide a means by which to progress mutational studies of fibrinogen. Similarly, developing methods for high-resolution imaging of fibrinogen in its native, aqueous-based environment is necessary to establish proper structure-function relationships encompassing this complex 340 kDa glycoprotein. The work described in this thesis produced advancements in each of these crucial areas of fibrinogen research. Herein, chapter 2 reports, to our knowledge, the first methodology of transient recombinant human fibrinogen expression using suspension HEK Expi293TM cells. A highly selective and robust affinity-based approach was also developed for the rapid isolation of fibrinogen from a variety of complex media. Chapter 3 describes cryogenic electron microscopy and our process of obtaining a high-resolution, in-solution structure of human fibrinogen’s D region. This structure is currently the highest resolution structure of any fibrinogen domain obtained in the native, solution state and further supports evidence for fibrinogen’s intrinsic flexibility, expanding the textbook representations of fibrinogen as a rigid, rod-like structure. Lastly, future directions outlining fibrinogen’s role during pregnancy and unique fibrinogen-specific protofibril-like structures are discussed. In an appendix, I have included figures from an illustrated review journal article, which I participated in as a co-first author.
2023-06-05
2023-05
2023-05-04
May 2023
2023-06-02
Master's Thesis
http://hdl.handle.net/10342/12876
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/85612021-03-03T22:08:29Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Transthyretin Amyloidosis: Proteolytic cleavage accelerates G53A TTR misfolding and aggregation
Arreola, Jenette
Lim, Kwang Hun
The proteolytic cleavage of the peptide bond Lys48-Thr49 of the CD loop in TTR pathological mutants S52P and E51_S52dup was previously demonstrated to promote aggregation as a result of tetrameric structure destabilization. Type A and B fibrils were detected in vivo, suggesting alternative TTR misfolding and aggregation mechanisms. The main component of the fibrils was the residue 49-127 fragment. In the proceeding studies, the misfolding and aggregation of G53A TTR, whose mutation is nearby the K48-T49 peptide bond and also associated with TTR amyloidosis, was investigated in thepresence and absence of proteolytic agent, trypsin. Fragmented G53A TTR misfolded and aggregated via a similar mechanism as full-length TTR, but at a faster rate. Similar morphology was exhibited by fragmented and full-length G53A TTR oligomers, as revealed by TEM images, suggesting similar aggregation pathway. G53A TTR in the presence and absence of trypsin generated similar CD and FT-IR profiles, suggesting similar transthyretin morphology during the early and late stages of amyloidosis. Aggregation kinetics was accomplished by monitoring the optical density of G53A TTR in the presence and absence of trypsin and by comparing the ThT fluorescence signal produced. The differences in TTR solubility and ThT enhancement imply G53A TTR aggregation is promoted in the presence of trypsin. Oligomeric effects on mammalian cell line, SH-SY5Y, was probed employing the MTT assay, which determined G53A TTR was more toxic to the cells in the presence of trypsin.
2020-06-23
2020-06-23
5/1/2020
2020-06-22
Master's Thesis
http://hdl.handle.net/10342/8561
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/74342022-12-05T19:28:50Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Studying Collagen with PyrATS: Pyrene-Appended Trimeric Systems
Keever, Jared Matthew
Chemistry
Burns, Colin Sanderson
Hudson, Nathan E
Sargent, Andrew L
Spuches, Anne M
CD Spectrometry
Polyproline Helices
Stacking Interactions
Collagens are a family of triple-helical structural proteins that are ubiquitous in vertebrates. Improper folding of collagen can lead to disorders such as osteogenesis imperfecta, or "brittle bone disease." There is significant interest in understanding the factors that drive collagen folding and stability, but studying native collagens is difficult because they are hundreds of amino acids in length. This thesis describes a series of well-characterized (Pro-Hyp-Gly)7 model peptides which have been tagged at their N-termini with the fluorophore pyrene. When in close contact, pyrene units can form excimers that emit low-energy light. This allows for the study of several fundamental questions in collagen research using fluorescence spectroscopy, including concentration dependence, folding directionality, and local fraying, upon solutions that are significantly more dilute than those customarily used in circular dichroism (CD) experiments. Notably, for most of the peptides studied, there is agreement between the melting temperatures (Tm) obtained via fluorescence and CD techniques. In addition, the pyrene probes were found to provide a situational increase in thermal stability of triple helices.
2019-08-21
2020-12-01
2019-12
2019-07-30
December 2
2019-08-20
Master's Thesis
http://hdl.handle.net/10342/7434
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/42602021-03-03T20:54:53Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_123
Synthesis, structure, photophysics, and biological activity of platinum (II) complexes
Vezzu, Dileep Atchyuth Kumar
Huo, Shouquan
Chemistry
Chemistry
Chemistry, Inorganic
Oncology
Cancer
Cyclometalation
Cytotoxicity
Phosphorescence
Platinum (II) complexes
Tridentate
Inorganic chemistry
The synthesis, structure, and photophysical properties of luminescent platinum (II) complexes with different coordination patterns, (C^C*N^N), (N^C*N), (N^N*C) and (N^N^C) are reported, where "C^N or N^N" denotes a bidentate coordination to the platinum to form a five-membered metallacycle and "C*N" denotes a bidentate coordination to the platinum to form a six-membered metallacycle. Sixteen cyclometalated platinum complexes have been synthesized with different coordination patterns, which include six complexes with tridentate N^C*N cyclometalating ligands (13, 14, 15, 16, 17, and 18), five complexes with N^N*C cyclometalating ligands (19a, 19b, 20a, 20b, and 24), three complexes with N^N^C cyclometalating ligands (21a, 21b, and 29) and two complexes with tetradentate C^C*N^N cyclometalating ligands (33 and 34). The structures of the platinum complexes 13, 15, 16, 18, 19a, 19b, 20a, 21a, 21b, 24, 29, 33, and 39 were determined by single crystal X-ray diffraction. In platinum complexes with five-six membered metallacycle (15, 16, 18, 19a, 19b, 20a, 24, and 39), the square geometry of the complexes is improved when compared to that of platinum complexes with five-five membered metallacycle as the biting angle is increased. The tetradentate coordination (C^C*N^N), square planar geometry of complex 33 are revealed from its X-ray crystal structure. The DFT calculations have been carried out on complexes 13, 14, 15, 16, 17, 18, 33, and 34 to examine the molecular orbital character of the complexes, which are used in interpreting the electronic spectra of the complexes. The photophysical properties of the platinum complexes were studied and a majority of the complexes were highly emissive in solution at room temperature. Complex 13 exhibited the highest quantum yield (65%) among all of the complexes. Self quenching was not observed in a majority of the platinum complexes at lower concentrations. The cytotoxicity (ICâ‚…â‚€) of the complexes in three lung cancerous cell lines and one prostate cancer cell line were determined by MTT assay. The toxic platinum complexes induced the cell death by triggering apoptosis. The interactions of the platinum complexes with plasmid and calf thymus DNA were studied. Activation of caspase -7, PARP, and p53 were also observed in RV1 and HCC827 cell lines when treated with platinum complexes. Complexes 35, 37 and 38 were more potent than the clinically approved drug, cis-platin.
2013-08-24
2013
Doctoral Dissertation
http://hdl.handle.net/10342/4260
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/128822023-06-05T14:07:35Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
A Catalytic Approach to Cyclopentenones: Unexpected Challenges in Synthesizing Pent-2,4-dienals
English, Hunter Wade
Chemistry
cyclopentenones
hydroacylation
Transition metal catalysis has long been utilized to facilitate a number of chemical reactions outside the scope of traditional organic chemistry. Because of this, there has been a growing interest in the community in utilizing C-H bonds over C-X bonds as synthetic alternatives with better thermodynamic conditions (lower temperatures, pressures, energies, etc.) and forming C-C bonds. For example, rhodium-catalyzed hydroacylation can be used in the conversion of 4-pentenals to cyclopentanones. The mechanistic foundation of this reaction was applied to the synthesis of chiral 3-substituted indanones, unlocking a whole series of transformations towards the construction of biologically active molecules.
The Morehead group recently proposed an alternate synthesis of cyclopentenones via a rhodium-catalyzed intramolecular hydroacylation reaction of penta-2,4-dienals by the following reaction sequence: 1. Vilsmeier-Haack Formylation to form 3-bromo-2-enals, 2. Suzuki Cross-Coupling with vinylboronates, and 3. Hydroacylation. Indeed, all reaction steps were performed successfully starting from acetophenone and allylboronic acid pinacol ester, resulting in 4-methyl-3-phenyl-2-cyclopenten-1-one. However, some surprising results were obtained along the way. It was found that when 3-bromo-3-phenyl-2-propenal underwent Suzuki Cross-Coupling, an unexpected mixture of pent-2,4-dienal isomers was obtained.
To investigate this matter, a number of reaction conditions were examined in hopes of finding one with optimal yields and little to no isomerized pent-2,4-dienal product. In addition, a variety of cyclopentenones were synthesized to explore the steric effect in influencing the isomerization process. Some potential mechanistic details of this strange isomerization phenomenon along with results obtained from the synthesis will be reported and discussed.
2023-06-05
2023-05
2023-05-04
May 2023
2023-06-02
Master's Thesis
http://hdl.handle.net/10342/12882
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/91252022-05-01T08:01:55Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
DIRECT ELECTROCHEMICAL ANALYSIS OF THE REDOX ACTIVITY OF TRYPTOPHAN AND TYROSINE IN MODIFIED AZURINS: THE IMPACT OF THE PROTEIN ENVIRONMENT
Tyson, Kris Janell
Offenbacher, Adam R
Chemistry
SWV
PCET
ET
Azurin
protein environment
Proton-coupled electron transfer (PCET) is a biological process essential to life. It is imperative for respiration in animals as well as photosynthesis in plants. Long-range PCET is often facilitated by redox-active amino acids, such as tryptophan and tyrosine. While there are several examples in the literature for the involvement of these redox-active residues in PCET linked to biological catalysis, there has been a challenge in direct electrochemical efforts to resolve how the local protein environment controls PCET directionality. This thesis describes a protocol from which to directly test the reduction potentials of tyrosine and tryptophan radicals in a customizable protein environment. The model protein used for this study was azurin, a natural cupredoxin that natively contains two tyrosines and only one tryptophan, with the former mutated to phenylalanine to provide direct electrochemical detection of a single redox-active amino acid species. The reduction potentials of azurin with either tryptophan or tyrosine redox centers were monitored using the electrochemical technique square-wave voltammetry. Using this technique along with strategic protein engineering, it was found that the more solvent-exposed or polar mutants had a higher redox potential than those that were more solvent-excluded. These trends have biological importance as the difference in the reduction potentials between redox-active amino acid pairs is expected to control the thermodynamic driving force for PCET. This thesis details the impact of altering the surrounding protein environment, i.e. electrostatics, on the redox activity of tryptophan and tyrosine.
2021-06-15
2022-05-01
2021-05
2021-05-03
May 2021
2021-06-02
Master's Thesis
http://hdl.handle.net/10342/9125
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/76522022-12-05T19:29:35Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Platinum Catalyzed Synthesis of Alpha-Ketoester via C-H Functionalization
Javed, Erman
Huo, Shouquan
Chemistry
Morehead, Andrew
Christensen, Tim
Burns, Colin Sanderson
alpha-ketoester
C-H Functionalization
Alpha-ketoesters have proven to be useful in a variety of fields. They have found wide spread applications in pharmaceuticals, photochemistry, and biology. Additionally, they are of great interest in synthetic chemistry and are frequently used as a precursor to many useful organic compounds including alpha-keto acids, alpha-hydroxy acids, and alpha-amino acids. Numerous methods have been reported for synthesizing alpha-ketoesters but they all amount to a few notable drawbacks. Herein reported is a potentially more effective transition metal catalyzed reaction to synthesize alpha-ketoester via C-H functionalization. A series of ligands with structural modifications have been designed, synthesized and acylated to shed light on the scope and limitations of the reaction. An inexpensive and readily accessible reagent namely ethyl chlorooxoacetate was employed as the acylating reagent. Reaction conditions were optimized by screening various solvents and catalysts. A variety of solvents were found useful in this reaction, including chlorobenzene, benzonitrile, toluene, and m-xylene although the best results were obtained when chlorobenzene was used. The reaction showed great tolerance to both electron withdrawing and donating groups on the phenyl ring however some electronic effects were observed and it was found that the presence of electron withdrawing group on the phenyl ring decelerated the acylation reaction. Experimental results of the acylation reaction will be reported and the mechanistic implications of these results will be discussed.
2020-02-04
2020-12-01
2019-12
2019-12-16
December 2
2020-01-29
Master's Thesis
http://hdl.handle.net/10342/7652
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/69872022-12-12T17:56:22Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_123
Elucidating the Mechanisms of Transthyretin Aggregation in Transthyretin Amyloidosis
Dasari, Anvesh K. R.
Lim, Kwang Hun
Chemistry
Solid-state NMR
Accumulation of insoluble aggregates (amyloids) is a characteristic feature of many neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. Aggregation and deposition of Transthyretin (TTR) protein in tissues leads to transthyretin amyloidosis. Aggregation of wild-type (WT) TTR shown to affect the heart, causing senile systemic amyloidosis (SSA), while the two pathogenic mutants (V30M, and L55P) affect the peripheral nervous system. It is important to understand the misfolding/amyloid formation mechanism towards the development of therapeutic strategies. TTR is rich in the [beta]-sheet structure in which eight [beta]-strands are arranged in a [beta]-sandwich consisting of two [beta]-sheets (strands CBEF and DAGH). Previous solution NMR studies have shown that the amyloid precursor state of TTR is also rich in [beta]-sheets. Our solid-state NMR results indicated the presence of intact native-like [beta]-sheets in the amyloid state as well. But, the AB loop region appeared to be perturbed during the aggregation which could expose strand A for intermolecular associations. Solid-state NMR results of the mutant forms (V30M and L55P) also indicated the presence of native-like [beta]-sheets in the amyloid states but, with a distorted DA strand which further exposes strand A for more aggressive intermolecular interactions. We also reported the aggregation mechanism for early aggregates which are shown to be cytotoxic in the amyloid formation pathway. We also discovered that the hexamers of TTR are the building blocks of these early toxic aggregates.
2018-08-14
2019-02-26
2018-08
2018-07-24
August 201
2018-08-09
Doctoral Dissertation
http://hdl.handle.net/10342/6987
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/112722022-12-09T16:34:11Zcom_10342_55com_10342_1col_10342_58
Crystal Structures of Non‑Oxidative Decarboxylases Reveal a New Mechanism of Action with a Catalytic Dyad and Structural Twists
Zeug, Matthias
Iancu, Cristina V.
Choe, Jun-Yong
Markovic, Nebojsa
Tripp, Joanna
Oreb, Mislav
non‑oxidative decarboxylases
catalytic dyad
Hydroxybenzoic acids
2022-09-19
2022-09-19
2021
Article
2045-2322
http://hdl.handle.net/10342/11272
10.1038/s41598-021-82660-z
en_US
oai:TheScholarship.intra.ecu.edu:10342/31802021-03-03T20:53:01Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
WATER-SOLUBLE FLUORESCENT PH SENSORS BASED ON A DIARYLACETYLENE SCAFFOLD
McGrath, Jacqueline
Allen, William E.
Chemistry
Chemistry, Organic
Organic chemistry
Intracellular pH plays an important role in many biological processes: receptor-mediated signal transduction, enzymatic activity, cell growth and death, ion transport and homeostasis, calcium regulation, endocytosis, chemotaxis, and cell adhesion. Normal cell pH is around 7.40 and can range from 7.35-7.45 without any adverse effects. If it ranges more than 0.1-0.2 in either direction cardiopulmonary and neurologic problems can arise and in the case of extreme variations, death can result. Determination of pH in cells is of great importance and many methods exist for sensing pH; however, fluorescence is the most useful because of its nondestructive nature, high sensitivity, and specificity. We chose to use an intrinsic fluorescent probe in which the protonation site is integrated into the main chromophore. The systems were prepared by Sonogashira coupling of halopyridines with acetylenes. Protonation of the first class of pyridyl receptors synthesized occurred at a pH of 4 and was accompanied by a decrease in fluorescence. Protonation of the second class of pyridyl acceptor synthesized occurs near the pH of 5.5 and is accompanied by an increase in fluorescence intensity. Though water-soluble, model membrane transport studies show that the probes have a high affinity for non polar environments. Â
2011-02-03
2011-05-17
2012-03-28
2010
Master's Thesis
http://hdl.handle.net/10342/3180
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/31982021-03-03T20:58:15Zcom_10342_55com_10342_1col_10342_58
NMR characterizations of an amyloidogenic conformational ensemble of the PI3K SH3 domain
Ahn, Hee-Chul
Le, Yen T. H.
Nagchowdhuri, Partha S.
Derose, Eugene F.
Putnam-Evans, Cindy
London, Robert E.
Markley, John L.
Lim, Kwang Hun
Amyloids
PI3K SH3
NMR
Dynamics
Amyloidogenic intermediate
Long-range interactions
PRE
Amyloid formation is associated with structural changes of native polypeptides to monomeric intermediate states and their self-assembly into insoluble aggregates. Characterizations of the amyloidogenic
intermediate state are, therefore, of great importance in understanding the early stage of amyloidogenesis. Here, we present NMR investigations of the structural and dynamic properties of the acid-unfolded amyloidogenic intermediate state of the phosphatidylinositol 3-kinase (PI3K) SH3 domain—a model peptide. The monomeric amyloidogenic state of the SH3 domain studied at pH 2.0 (35°C) was shown to be substantially disordered with no secondary structural preferences. 15N NMR relaxation experiments indicated that the unfolded polypeptide is highly flexible on a subnanosecond
timescale when observed under the amyloidogenic condition (pH 2.0, 35°C). However, more restricted motions were detected in residues located primarily in the b-strands as well as in a loop in the native
fold. In addition, nonnative long-range interactions were observed between the residues with the reduced flexibility by paramagnetic relaxation enhancement (PRE) experiments. These indicate that the
acid-unfolded state of the SH3 domain adopts a partly folded conformation through nonnative longrange contacts between the dynamically restricted residues at the amyloid-forming condition. Originally published Protein Science, Vol. 15, No. 11, Nov 2006
2011-02-04
2011-05-17
2011-02-04
2011-05-17
2006-11
Article
Protein Science; 15:11 p. 2552-2557
http://hdl.handle.net/10342/3198
PMC2242406
10.1110/ps.062154306
en_US
http://onlinelibrary.wiley.com/doi/10.1110/ps.062154306/abstract;jsessionid=0F7196949CCE21C6D61089C8168C3A46.d03t04
Author notified of opt-out rights by Cammie Jennings
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/45272021-03-03T21:10:41Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Electrochemical Detection of Benzo[a]Pyrene-Induced DNA Damage at TP53 Oligomers : Impact of 5'-Methyl Cytosine and Bioactivation on the Genotoxicity Process
Trumbo, Caitlin M.
Hvastkovs, Eli G.
Chemistry
Chemistry
DNA houses the blueprint that dictates how an organism will develop. However, DNA features numerous reactive sites that can be attacked by chemicals and radiation, resulting in DNA damage and possibly mutations. Chemical products and other environmental toxins must be tested for genetic abnormalities due to exposure. Traditional DNA damage detection can be tedious, time-consuming, and cost prohibitive. Electrochemical methods to detect DNA damage offer remedies to these drawbacks. Simple and sensitive DNA hybridization sensors are widely used for DNA detection and studying biochemical processes at specific DNA sequences.   An electrochemical DNA hybridization sensor designed to detect DNA damage at hotspot TP53 gene sequences resulting from bioactivated benzo[a]pyrene (BP) will be discussed. TP53 codes for the p53 protein, and mutations at the studied genetic sequence have been shown to be prevalent in many different cancers. Double stranded DNA 21-mers were absorbed on gold electrodes followed by adsorption and saturation with a heme enzyme model, myoglobin. Myoglobin was activated using hydrogen peroxide and exposed to solutions of BP, which allowed BP to be oxidized into reactive metabolites. DNA damage was detected voltametrically by charting changes in square wave voltammetric signals due to a redox-active di-viologen derivative that has been shown to bind to DNA in a structure-specific manner. Aspects of the sensor optimization process will be discussed including how BP stereochemistry and epigenetic factors influence the voltammetry, the impact of myoglobin non-specific adsorption on the electrode surface, and numerous control reactions designed to show that bioactivated reactive metabolites were detected at the DNA sequence. Overall, the incorporation of enzymes with a DNA hybridization interface has opened up new possibilities to utilize more biologically relevant enzymes, such as cytochrome P450s, to study of important metabolic related DNA damage processes at specific gene sequences. Â
2014-08-28
2017-02-07
2014
Master's Thesis
http://hdl.handle.net/10342/4527
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/122062023-02-08T08:16:37Zcom_10342_7351com_10342_6421com_10342_55com_10342_1col_10342_11836col_10342_58
CRY–BARs: Versatile Light-Gated Molecular Tools for the Remodeling of Membrane Architectures
Wurz, Anna I.
Hughes, Robert M.
Bunner, Wyatt P.
Szatmari, Erzsebet M.
BAR (Bin, Amphiphysin, and Rvs) protein domains
membrane architectures
proteins
2023-02-07
2023-02-07
2022-08-18
Article
0021-9258
http://hdl.handle.net/10342/12206
10.1016/j.jbc.2022.102388
en_US
oai:TheScholarship.intra.ecu.edu:10342/112792022-12-09T16:34:02Zcom_10342_55com_10342_1col_10342_58
Identifcation of New GLUT2‑Selective Inhibitors Through in Silico Ligand Screening and Validation in Eukaryotic Expression Systems
Iancu, Cristina V.
Choe, Jun-Yong
Schmidl, Sina
Ursu, Oleg
Oreb, Mislav
Oprea, Tudor I.
Human glucose transporters (GLUTs)
GLUT2‑selective inhibitors
silico ligand screening
2022-09-20
2022-09-20
2021
Article
2045-2322
http://hdl.handle.net/10342/11279
10.1038/s41598-021-93063-5
en_US
oai:TheScholarship.intra.ecu.edu:10342/45282021-03-03T21:01:50Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Elucidating "Consumption" : Using Fluorescent Steroid Probes to Understand Host Cholesterol Utilization by Mycobacterium spp.
Wercholuk, Ashley N.
Allen, William E.
Chemistry
Chemistry, Biochemistry
Chemistry, Organic
Microbiology
Cholesterol--metabolism
Fluorescence
M. smegmatis
Naphthalimide
Synthesis
Biology, Microbiology
Organic chemistry
Biochemistry
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) disease, is the leading cause of death due to bacterial infection worldwide, claiming nearly two million lives each year. Often, the immune system cannot eradicate the bacteria and the disease progresses into a latent state characterized by the presence of granulomas, which are lesions formed in order to prevent bacterial dissemination. This state of infection can persist for years. Current research has shown that the utilization of host cholesterol by M. tuberculosis plays a crucial role in the tenacity of this organism during latent infection. Understanding the mechanisms of cholesterol acquisition and degradation could provide researchers with novel platforms for the strategic development of anti-mycobacterial drugs. In order to further understand the pathways employed by M. tuberculosis during cholesterol utilization, a series of fluorescently labeled cholesterol analogues were synthesized by tethering cholesterol chloroformate to 1,8-naphthalimide moieties via a carbamate linkage. The analogues and their 4-amino-1,8-naphthalimide precursors were found to be intensely fluorescent in nonpolar environments such as dichloromethane and liposomes comprised of POPC. Their in vitro effects were evaluated using the Mtb research model M. smegmatis. Bacterial growth curves were consistent with active incorporation and degradation of the fluorescently labeled cholesterol compounds, while inhibitory effects were observed for the unconjugated naphthalimide fluorophores. Current studies are focused on the isolation and identification of cholesterol/fluorophore metabolites to help elucidate the mechanism of uptake and decomposition of these compounds. Preliminary results of these metabolite experiments are discussed with relation to the observed effects on M. smegmatis growth.
2014-08-28
2016-05-11
2014
Master's Thesis
http://hdl.handle.net/10342/4528
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/53192021-03-03T21:02:48Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Quinone Formation via Ceric Ammonium Nitrate Oxidations of 2-Alkyl-1,4-dialkoxybenzenes
Simmons, Alexander Linwood
Love, Brian Ernest
Chemistry
Burns, Colin Sanderson
monoquinone
diquinone
ceric ammonium nitrate
chemistry
Quinones are cyclohexadiendiones that have a variety of uses ranging from medical applications to synthetic building blocks. Medicinal applications stem from the potent biological activity (e.g. antitumor and antibiotic) these compounds and some derivatives possess. The most common preparation method to access these compounds is oxidative demethylation of hydroquinone dimethyl ethers typically using ceric ammonium nitrate (CAN). Oxidation using CAN can yield a product mixture of the (mono)quinone and the symmetric dimeric quinone. Previous work in our group has resulted in the development of several protocols for altering the monoquinone to diquinone ratio by altering reaction conditions (e.g. substrate concentration, mode of addition, etc.). The current focus further explores manipulation of this ratio and reaction efficacy through substrate solubility and cerium coordination. We will discuss how ether linkages of various hydrophobicities and coordination modes change product outcome and if altering a single ether linkage or both linkages affect the product ratio.
2016-05-25
2017-02-07
2016-05
2016-05-03
May 2016
2016-05-25
Master's Thesis
http://hdl.handle.net/10342/5319
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/69622021-03-03T21:18:55Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Counting Protons: Using Isothermal Titration Calorimetry to Determine the Number of Protons Exchanged Upon Ca(II) and Cd(II) Binding to Wild-type HcTnC and Mutant C84A and C35A/C84A Proteins
Vang, Katie
Spuches, Anne M
Chemistry
ITC
human cardiac troponin c
calcium
cadmium toxicity
troponin c
calorimetry
EF hands
Maintaining a delicate balance of metal ions in the cell is crucial to the survival of many organisms. Divalent cadmium, Cd(II), is a non-essential metal ion that can disrupt this delicate balance and lead to deleterious effects. Cd(II) imparts its toxicity by mimicking essential metal ions such as Ca(II) and replacing these metals in various proteins. Many methods have been used to characterize both essential and toxic metal interactions with proteins. Isothermal Titration Calorimetry (ITC) is a powerful calorimetric method that measures the heat of a binding reaction. From a single titration experiment, one can obtain all thermodynamic parameters (Ka, [delta]H, [delta]G, and [delta]S) for a binding event. ITC will be used in this study to understand the thermodynamic driving forces that drive Cd(II) binding to Ca(II) binding proteins. The protein of interest is Human Cardiac Troponin C (HcTnC), an EF-hand protein that requires Ca(II) to regulate heart muscle contraction. In this study, ITC was used to determine the number of protons displaced upon Ca(II) and Cd(II) metal binding to the wild-type, C35A/C84A and C84A mutant proteins. These numbers are required to extract buffer-independent binding parameters which allow for direct comparison between metals and proteins. Most importantly, one can use the number of protons exchanged upon Cd(II) binding to the N-domain of both wild-type and C35A/C84A to shed light on the location of third Cd(II) ion in the N-domain. ITC experiments revealed that Ca(II) displaced a total of 1.1 (± 0.01) and 1.6 (± 0.8) protons in the C- and N-domain of both the C35A/C84A and C84A mutants, respectively. Upon the titration of Cd(II) into the C35A/C84A and C84A mutants, experimental results showed that in the C-domain, a total of 1.1 (± 0.03) and 1.1 (± 0.01) protons were displaced in each mutant respectively. In the N-domain, a total of 0.6 (± 0.1), 1.6 (± 0.4) and 0.7 (± 0.4) were displaced for the wild-type, C35A/C84A and C84A mutant respectively. It is important to see that the wild-type and C84A were within error of each other. Both the wild-type and C84A contain the cysteine in loop I, which gives indirect evidence that there is a Cd(II) binding to loop I. From the number of protons displaced, the buffer independent values for Ca(II) binding to the C35A/C84A and C84A mutants were determined. The buffer independent parameters (K, [delta]G, [delta]H, and T[delta]S) for Ca(II) binding to the C-domain in both mutants. Buffer independent enthalpy values were obtained for the N-domain of both mutants only. While buffer independent enthalpy values were obtained for Cd(II) binding to both the C- and N-domains of WT, C84A and C35A/C84A, further research is needed to conclude buffer independent binding parameters (K, [delta]G, T[delta]S) for Cd(II) binding to HcTnC and mutants because KITC was far too large to determine accurately.
2018-08-14
2020-08-01
2018-08
2018-07-20
August 201
2018-08-09
Master's Thesis
http://hdl.handle.net/10342/6962
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/43352021-03-03T20:53:32Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Oxidation of Isoprene to Methyl vinyl ketone and Methacrolein via Hydroxyl Radical : A Regenerative Mechanism for the Hydroxyl Radical
Howell, Richard
Bartolotti, Libero J.
Chemistry
Chemistry
Radical
Regeneration
Isoprene is an important molecule in atmospheric chemistry. Belonging to a class of molecules known as volatile organic compounds, it undergoes reactions with several different oxidizers in the atmosphere, including OH(hydroxyl radical). In 2005 the concentrations of isoprene, OH and a few other isoprene derivatives were measured above the Amazonian rain forest. OH was found to be in concentrations far above levels predicted by computer models. As a result, a mechanism involving a 1,5-H shift was proposed to help explain the high levels of OH. Four reaction pathways, two different conformations of two different oxidation products, for the oxidation of isoprene and regeneration of the hydroxyl radical involving a 1,5-H shift mechanism were studied using the M062X density functional and the maug-cc-PVTZ basis set. Â
2014-01-28
2014-01-28
2013
Master's Thesis
http://hdl.handle.net/10342/4335
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/40082022-12-12T17:51:07Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
IDENTIFICATION OF NOVEL CYTOTOXIC PROSTAGLANDIN METABOLITES PRODUCED IN ARACHIDONOYL ETHANOLAMIDE-TREATED TUMOREGENIC KERATINOCYTES
Thati, Drisheka
Danell, Allison Stokes
Chemistry
Chemistry
Pharmacology
Arachidonoyl ethanolamide (AEA) induces apoptosis in mouse tumorigenic keratinocytes (JWF-2 cells). Our previous data show that AEA is metabolized by COX-2 to pro-apoptotic J-series prostaglandins. COX-2 is an enzyme that is abundant in tumor cells but not in the normal epithelial cells surrounding the tumor. Thus, the pro-apoptotic J-series prostaglandins should be selectively synthesized in AEA-exposed tumor cells with elevated COX-2 expression. As such, AEA could be developed as a topical agent to treat non-melanoma skin cancer. The main goal of this project is to identify the specific J-series prostaglandins that are produced as a result of the metabolism of AEA by COX-2 using mass spectrometry. (ELISA analysis can detect J-series family prostaglandins but cannot distinguish between the individual J-series isoforms). Exogenous J-series prostaglandins were added to fresh cell culture medium, and the prostaglandins were extracted using solid phase extraction. Concentrated samples were then subjected to Liquid Chromatography/Electrospray Ionization /Mass Spectrometry (LC-ESI-MS) in negative mode for identification of J-series prostaglandin isoforms. Our data show good recovery of extracted species and acceptable resolution of these chemically similar standards. The AEA-treated cell culture medium and its control were extracted using the validated extraction protocol and analyzed using the method developed with LC-ESI-MS. The mass spectrum of AEA-treated, extracted and concentrated cell culture media clearly shows peaks at m/z ratio identified as the parent ion peak (M-H)� of ethanolamide conjugates of the J-series prostaglandins. The identification of the ethanolamide conjugates was confirmed by performing tandem mass spectrometry. It was also observed that with increased AEA concentration, the mass spectral intensity of the ethanolamide conjugates of J-series prostaglandins increased. The effect of COX-2 inhibitor and N-acetyl cysteine on the production of ethanolamide conjugates of J-series was also studied. The identification of the cytotoxic ethanolamide conjugates of J-series prostaglandins as the metabolites of AEA synthesized in tumor cells help us to determine the mechanism by which AEA induces apoptosis.
2012-09-04
2013-10-31
2012
Master's Thesis
http://hdl.handle.net/10342/4008
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/51842021-03-03T20:58:57Zcom_10342_55com_10342_1col_10342_58
Wurster's crown ligands
Sibert, John W.
Wurster's crown ligands comprise a macrocyclic ligand such as a crown ether in which a hetero atom is substituted with a 1,4-phenylenediamine group. The phenylenediamine group is covalently bound to the macrocyclic ligand by one or both of the amine nitrogens, the amine nitrogen thereby substituting for the hetero atom of the macrocyclic ligand. The resulting compounds are redox active. Methods of making and using the compounds are also disclosed.
2016-02-05
2016-02-05
2002-08-27
Patent
U.S. Patent No. 6,441,164
http://hdl.handle.net/10342/5184
oai:TheScholarship.intra.ecu.edu:10342/128512023-06-05T13:54:11Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Improving Prostamide Anti-Cancer Activity Through Derivatization and Micellar Delivery
Halatek, David James
Chemistry
Cancer Therapeutics
Cancer Treatments
Small Molecule
Cancer
Prostamide
Anandamide
Prostaglandin
J-series Prostaglandins
J-series
Colorectal cancer is the fourth most common cancer diagnosis per year as well as the fourth highest rate of death per year according to the Centers for Disease Control. Approximately 1/3rd of the diagnosed colorectal cancer cases per year will result in death. Prior research from our group has shown that the prostaglandin-ethanolamide 15-deoxy, D12,14 prostamide J2 (15d-D12,14-PMJ2) is selectively toxic to murine melanoma cells (B16F10) and murine colorectal cells (CT-26) both in vitro and in vivo and significantly reduces tumor growth. Further, 15d-D12,14-PMJ2 induces cell death in primary patient melanoma cells and thus may be a promising therapeutic. As 15d-D12,14-PMJ2 can be made by the condensation of ethanolamine with 15-deoxy, D12,14 prostaglandin J2 (15d-D12,14-PGJ2), we sought to test the cytotoxicity of other prostamide derivatives to determine the structural features required for activity. Based on prior results in a study of related prodrugs, 15d-D12,14-PMJ2-Arvanil was selected as the top candidate for testing anti-cancer activity. After testing in both a human and murine cell line, it was determined that 15d-D12,14-PMJ2-Arvanil was not as cytotoxic as 15d-D12,14-PMJ2. It is possible that the bulkier functional group on the α-chain of the prostamide prevents transport into the cell the same way or to the same degree 15d-D12,14-PMJ2 enters. In an effort to test this hypothesis, and to develop an improved means for systemic delivery for this class of hydrophobic prostamides, engineered micelles composed of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] and D-α-tocopherol polyethylene glycol 1000 succinate were investigated as drug carriers
2023-06-05
2023-06-05
2023-05
2023-04-28
May 2023
2023-06-02
Master's Thesis
http://hdl.handle.net/10342/12851
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/65182021-03-03T21:16:13Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
SYNTHESIS AND SPECTROSCOPIC PROPERTIES OF BOC- PROTECTED FLUORESCENT ARGININE
Marshall, Sarah Ruth
Allen, William E.
Chemistry
Fluorescence
Peptides
membrane transport
metal-catalyzed reactions
Small peptides capable of penetrating or spanning the hydrophobic core of lipid bilayers have many potential uses in medicine and in biochemical research. As the most basic of the amino acids that can comprise such peptides, arginine (pKa ~ 13.8) is one of the few naturally-occurring molecules capable of existing in a cationic form even when it is deep within the nonpolar membrane interior. In fact, an unambiguously neutral arginine inside of a membrane has never been reported. To allow for molecular-level tracking of peptide localization, a fluorescent arginine derivative (Boc-Arg*-OH) was prepared from commercially available backbone-protected arginine. Palladium(0)-catalyzed coupling of the side-chain to a 4-bromo-1,8-naphthalimide affords the derivative in yields of about 10%. The presence of the fluorophore lowers the pKa of the side-chain guanidinium group by several orders of magnitude, to 9.0 (± 0.1), allowing the derivative to access an electrically neutral protonation state that is not generally available to arginine itself. Fluorescence emission from the neutral form of Boc-Arg*-OH is red-shifted by ~100 nm relative to that of the positively-charged form, which appears in the 430-475 nm range depending on solvent polarity and H-bonding ability. Emission from the neutral form can be observed even under acidic conditions, suggesting that intermolecular excited state deprotonation, an uncommonly seen transfer of hydrogen ions between the fluorophore and solvent upon excitation, is occurring. Attempts to attach Boc-Arg*-OH at the N-terminal position of peptides like substance P (RPKPQQFFGLM-NH2) using solid-phase peptide synthesis were unsuccessful. This failure is attributed to a competing intramolecular cyclization that occurs when Boc-Arg*-OH is activated by agents like HBTU.
2018-01-23
2019-02-26
2017-08
2017-08-21
August 201
2018-01-22
Master's Thesis
http://hdl.handle.net/10342/6518
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/72862021-05-01T08:02:02Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
THE IMPACT OF GLYCOSYLATION ON ACTIVE SITE STRUCTURE AND ACTIVITY IN THE FUNGUS ENZYME MOLOX
Kostenko, Anastasiia
Offenbacher, Adam R
Chemistry
C-H activation
Lipoxygenases from pathogenic fungi belong to the lipoxygenase family that catalyze the C-H activation of polyunsaturated fatty acids to form diverse hydroperoxides. While the lipoxygenase catalytic domains are structurally and functionally similar, the fungal enzymes are decorated with N-linked glycosylations. MoLOX, a lipoxygenase from the fungus M. oryzae, is emerging as an important target for the devastating rice blast disease. Here we demonstrate for the first time that hydrogen transfer, associated with C-H cleavage of linoleic acid by MoLOX, occurs by a hydrogen tunneling mechanism. Using the temperature dependent kinetic isotope effect, [delta]Ea, as a kinetic reporter of tunneling efficiency, the loss of N-linked carbohydrates is linked to an increase in the activation energy for deuterium transfer, consistent with an impairment of the tunneling ready state. These results have important implications for MoLOX inhibitor design towards a potential 'treatment' of rice blast disease.
2019-06-12
2021-05-01
2019-05
2019-05-01
May 2019
2019-06-11
Master's Thesis
http://hdl.handle.net/10342/7286
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/29152021-03-03T20:53:16Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
A Molecular Dynamics Study into Annexin A1 Induced Membrane Binding and Aggregation
Donohue, Matthew P.
Li, Yumin
Chemistry
Chemistry, Physical
Physical chemistry
Annexins constitute a family of proteins that bind to anionic membranes in a reversible and calcium dependent manner through the unique architecture of their calcium binding sites. In addition, annexins with relatively large N-terminal domains have been identified to cause membrane aggregation and fusion. There is a contradiction between x-ray crystallography and cryo-EM studies as to the proposed mechanism of annexin-induced membrane aggregation.  Molecular dynamics simulations were performed in an effort to study the calcium dependent binding of annexin I to a phospholipid bilayer and to investigate the N-terminus as a possible second membrane binding site. Site specific mutations were created on the N-terminus to study the effects phosphorylation has on the tertiary structure of the protein. Simulation trajectories were analyzed in terms of non-bonded interaction energies of protein residues, root mean square deviations of the protein backbone, root mean square fluctuations of residues and nuclear distances between calcium ions and their oxygen ligands. Calcium coordination with lipid headgroups was observed in repeat IV of the core domain. Two lysine residues located in the N-terminus and speculated to be crucial to membrane aggregation displayed significant electrostatic   attractions to the phospholipid layer based on MM-PBSA calculations. This thesis will present a model for the mechanism of interaction between annexin A1 and membranes. Â
2010-09-16
2011-05-17
2012-03-28
2010
Master's Thesis
http://hdl.handle.net/10342/2915
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/60032022-12-12T17:51:00Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
DIFFERENTIATION OF J-SERIES PROSTAGLANDINS AS PRO-APOPTOTIC PRODUCTS OF CANCER CELL METABOLISM
Kobet, Robert A.
Danell, Allison Stokes
Chemistry
energy-resolved mass spectrometry
collision-induced dissociation
Cyclopentenone J-series prostaglandins and prostaglandin-ethanolamide conjugates have been shown to selectively induce endoplasmic reticulum stress-apoptosis in different cancer cell types. These lipids have been identified as the downstream products of COX-2 metabolism of arachidonic acid and arachidonoyl ethanolamide, respectively. While techniques such as ELISA and UV-Vis spectroscopy can detect these molecules based on their class, they fail to offer the specificity necessary to differentiate those prostaglandins that are isomeric in structure. The J-series prostaglandins PGJ2 and [Delta]12PGJ2 are structural isomers that differ in the position of a single double bond. The differentiation of these prostaglandins is an important step in the elucidation of the metabolic pathway mediated by COX-2 and the mechanism of action of the downstream products. Mass spectrometry has been shown to be a powerful method in the study of biological lipids due to the sensitivity and specificity it offers. Tandem mass spectrometry (MS/MS) offers the ability to isolate molecules of identical masses and obtain information regarding their individual fragmentation patterns as well as energetics and stability characteristics. The goal of this project is to differentiate the prostaglandin isomers PGJ2 and [Delta]12PGJ2 using a variety of MS/MS methods. MS/MS experiments were performed on prostaglandin standard solutions using a linear quadrupole-time of flight (Q-ToF) mass spectrometer and a quadrupole ion trap (QIT). An initial set of experiments aimed to identify product ions specific to either isomer using MS/MS and pseudo-MS3 on the Q-ToF and MSn analysis using the QIT. Our results indicate that neither method yielded product ions with significant intensity and reproducibility to be designated as specific to either isomer. Our focus then shifted to attempting to exploit differences in the energies of onset of some of the common product ions (energy-resolved mass spectrometry). Under controlled sample preparation and ESI source parameters, this method was also shown to be unsuccessful in providing information specific to either PGJ2 or [Delta]12PGJ2. These results are predicted to be the result of in-source isomerization of PGJ2 to the conjugated [Delta]12 isomer. In order to confirm unique fragmentation of the double bond isomers using the established LC-ESI-MS/MS method initially used to identify these metabolic products, offline derivatization methods will likely be necessary to fix the double bond position in order to prevent the in-source isomerization that is believed to have hindered our ability to differentiate these molecules.
2017-01-11
2019-02-26
2016-12
2016-12-16
December 2
2017-01-11
Master's Thesis
http://hdl.handle.net/10342/6003
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/51642021-03-03T20:58:28Zcom_10342_55com_10342_1col_10342_58
Biimidazole diamide anion binding agents
Allen, William E.
Causey, Corey
Biimidazole diamide compounds that are useful for binding anions such as sulfate are described, along with the use thereof for purposes such as extracting anions from a waste stream and/or detecting anions.
2016-02-05
2016-02-05
2006-07-18
Patent
U.S. Patent No. 7,078,528
http://hdl.handle.net/10342/5164
oai:TheScholarship.intra.ecu.edu:10342/74712021-03-03T21:23:26Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Computational Investigation of Calcium Binding Proteins Annexin A1 and Cardiac Troponin C
Lewis, Kimberly A
Li, Yumin
Chemistry
Annexin A1
Troponin
Mutation
Conformation Pathway
Calcium binding proteins are vital for many biological functions. Many undergo calcium induced conformation changes through sensing motifs, resulting in functional changes for the proteins. In this study computational investigations were performed for annexin A1 and cardiac troponin C (cTnC). Annexin A1 is known to induce membrane aggregation, and prior to interaction with membrane or proteins, must undergo specific calcium induced conformation change. This study focuses on investigating the conformation change pathway for annexin A1. Cardiac troponin C is involved in the calcium induced regulation of cardiac muscle contraction. This study investigates the impact of N-terminal mutations on the regulatory binding site for cTnC. The goal of this study is to use knowledge of these calcium binding proteins to determine the conformation pathway of annexin A1 and mutational effects of cTnC linked to cardiac diseases. Multiple nudged elastic band (NEB) method simulations were performed for annexin A1. The trajectories for the conformation change were generated and examined for the full length annexin A1 protein. Our results suggest that the N-terminal domain of annexin A1 is removed from repeat III of the core domain in a sliding motion. Previously, it has been unclear how the N-terminal removes itself from the core. The loop region of repeat III covering the N-terminal helix in the apo structure does not lift up allowing the N-terminal to swing out of the pocket. The process resembles a sliding motion, where the N-terminal pulls out from the bottom of the core domain. Our results also indicate that the folding of helix D in repeat III of the core domain folds in a two-step process, and during the conformation change calcium binding sites undergo secondary structure change. The results obtained using the NEB method provides an atomistic explanation for the complete conformation change pathway of annexin A1. Molecular dynamics simulations totaling 1,425 ns were performed for wild type, D65A, S69C, A8V, L29Q, and C84Y mutations of cTnC. These mutations included five point mutations in the N-terminal domain, two of which are located at calcium binding site II. The simulation trajectories were analyzed using MMPBSA/MMGBSA, RMSD, and distance analysis. Our results showed that D65A, S69C, and L29Q have a decrease in calcium binding affinity. The A8V and C84Y mutations had an increase in calcium binding affinity. The loss of calcium binding affinity is detrimental when mutation of anchoring residues in site II occur, such as D65 and E76. Overall, trends in the effect of calcium binding affinity for these mutations and calcium binding to site II show consistencies with experimental studies. Mutations in the N-terminal domain of cTnC did not have as strong as an effect on calcium binding site III and IV of the C-terminal domain. These binding sites have higher calcium binding affinity than site II and are not affected much by the mutations tested in this work. The mutational studies of cTnC are important to investigate due to their link with cardiac diseases. cTnC's involvement in the muscle contracting process is vital, and many of these mutations lead to cardiac diseases that need to be studied in more detail to understand the mechanistic effect they have on muscle contraction.
2019-08-22
2020-08-01
2019-08
2019-07-15
August 201
2019-08-19
Master's Thesis
http://hdl.handle.net/10342/7471
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/69562021-03-03T21:18:57Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Fluorescent Derivatives of Prostamides: Tools for Studying Anti-Cancer Activity
Stanley, Jordan Lynne
Allen, William E.
Chemistry
Naphthalimides
Prostamides
Fluorescence
The endocannabinoid arachidonoyl ethanolamide (AEA) and prostaglandins derived from it selectively induce apoptosis in tumorigenic cell lines due to overexpression of COX-2. It is likely that a novel J-series prostamide, 15-deoxy-[delta]12,14-prostaglandin J2-ethanolamide (15d-PMJ2), is the cytotoxic mediator of AEA-induced apoptosis. Conjugation of a fluorescent group to prostamides of interest may be helpful in determining localization and pharmacokinetic properties needed for clinical development. The synthetic flexibility and bright luminescence of naphthalimides make them attractive probes for this purpose. Previous studies in our labs focused on identifying substituents on naphthalimide that cause minimal interference with intracellular drug localization and metabolism. Those studies indicated that a 4-morpholino naphthalimide would be a suitable probe for conjugation based on its lack of cytotoxicity. Unfortunately, we found that this probe renders AEA inactive, presumably by preventing its cyclization into prostamides. We hypothesize that conjugation of the 4-morpholino naphthalimide to an already-cyclized metabolite, such as 15d-PMJ2, will allow the drug to retain its activity.
2018-08-14
2020-08-01
2018-08
2018-07-23
August 201
2018-08-09
Master's Thesis
http://hdl.handle.net/10342/6956
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/40722021-03-03T20:56:02Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_123
Elucidating the Mechanism of Action of Unnatural Amino Acid Containing Antimicrobial Peptides in Membrane Environments
Clark, Tiffany D.
Hicks, Rickey P.
Chemistry
Chemistry
Biophysics
Pharmaceutical sciences
Antimicrobial peptides
Force field parameterization
Membrane simulation
Micelle
Molecular dynamics
Unnatural amino acids
Organism resistance continues to develop to the currently available antimicrobial compounds necessitating the development of innovative new therapeutic compounds with different specificities and mechanisms of action that provide acceptable therapeutic indices. Unnatural amino acid containing antimicrobial peptides could provide a novel avenue for the development of therapies with improved efficacy and pharmacokinetics over natural amino acid containing peptides which are prone to protease degradation.  Molecular dynamics (MD) simulations of antimicrobial peptides containing unnatural amino acids have been performed using explicit water and multiple model membrane types in all-atom simulations. The structural properties of peptides were investigated using both the canonical and isothermal-isobaric ensembles to further understand the mechanism through which the collections of AMPs exert their in vitro activity.   Simulations with micelle membrane models were conducted at 300 K to correlate with experimental circular dichroism (CD) data showing the secondary structure the peptides adopt in the presence of an electrostatic membrane model. Analysis of the stabilized MD trajectory reflects peptide structural consistency with experimental data.  Simulations of the peptides with bilayer model membranes were conducted at the physiologically relevant 310 K to correlate with experimental cellular activity data which demonstrated the antimicrobial activity of the peptides without providing insight into the mechanism through which the activity was achieved. Long time scale simulations have noted distinct differences between bilayers in the presence of AMPs as compared to those without the peptide. Mixed bilayers with an anionic charge modeled bacterial membranes while a confluent zwitterionic bilayer modeled the mammalian membrane.  This research has demonstrated that force field parameters for unnatural amino acids can be derived from QM calculations. FF parameters derived from structures identified from a DFT approach have also been used to expand the AMBER ff03 force field. The FF parameters were able to able to model the interaction of the peptides which contain unnatural amino acids. The data is consistent with NMR data and further supported with CD spectroscopy. Â
2013-01-15
2015-02-02
2012
Doctoral Dissertation
http://hdl.handle.net/10342/4072
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/88092021-08-31T15:00:07Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
SCREENING AND CHARACTERIZATION OF BIOCATALYSTS FOR SYNTHESIS OF WIELAND MIESCHER KETONE: A VERSATILE SYNTHETIC INTERMEDIATE
Patel, Mitul P
Hughes, Robert
Chemistry
Lipases
Wieland Miescher Ketone
Biocatalysts derived from biological molecules such as proteins, RNA, and DNA have long been exploited for applications in synthetic chemistry. Lipases, a versatile class of biocatalysts, are known to exhibit significant promiscuity for non-native substrates in non-aqueous and mixed organic/aqueous solvents. While lipases are known to possess catalytic activity for a wide range of organic transformations, predicting which lipases will catalyze specific carbon-carbon bond forming reactions remains a significant challenge. In this study, the catalytic potential of a library of commercially available lipases was investigated by screening them for catalysis of the Robinson Annulation, a synthetically important carbon-carbon bond forming reaction. Specifically, the lipase library was screened for synthesis of the Wieland-Miescher Ketone, an important intermediate in the synthesis of biologically active compounds such as steroids and terpenoids, from methyl vinyl ketone and 2-methyl-1,3-cyclohexanedione. An optimized procedure is presented for Wieland-Miescher Ketone synthesis using a crude preparation of porcine pancreatic lipase (PPL), reporting on both yield and enantiomeric excess. Finally, commercially available lipases are often sold as crude preparations, containing many different proteins, and are often used by many researchers without further purification, potentially obscuring the identity of the catalytic species. Therefore, a methodology to isolate and characterize the active component(s) of these lipase formulations is described in this study.
2020-12-18
2020-12-18
2020-12
2020-12-07
December 2
2020-12-18
Master's Thesis
http://hdl.handle.net/10342/8809
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/52712021-03-03T21:01:49Zcom_10342_55com_10342_1col_10342_58
Calorimetric Investigation of Copper Binding in the N-Terminal Region of the Prion Protein at Low Copper Loading: Evidence for an Entropically Favorable First Binding Event
Gogineni, Devi Praneetha
Spuches, Anne M.
Burns, Colin Sanderson
2016-05-24
2016-05-24
2015-12
Article
Inorganic Chemistry; 54:2 p. 441-447
0020-1669
http://hdl.handle.net/10342/5271
pmc4303328
10.1021/ic502014x
http://www.ncbi.nlm.nih.gov/pubmed/25541747
oai:TheScholarship.intra.ecu.edu:10342/110632022-09-09T07:16:14Zcom_10342_55com_10342_1col_10342_58
Binding of Calcium and Magnesium to Human Cardiac Troponin C
Spuches, Anne Marie
Rayani, Kaveh
Seffernick, Justin
Li, Alison Yueh
Davis, Jonathan P.
Van Petegem, Filip
Solaro, R. John
Steffen Lindert, Steffen
Tibbits, Glen F.
calcium
magnesium
human cardiac troponin C
2022-09-08
2022-09-08
2021-02-03
Article
0021-9258
http://hdl.handle.net/10342/11063
10.1016/j.jbc.2021.100350
en_US
oai:TheScholarship.intra.ecu.edu:10342/122612023-02-11T08:16:30Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_123
Red Blood Cell Preservation Under Austere Conditions
Angermeier, Tori Marena
Kennedy, Anthony
Chemistry
Spuches, Anne
Hughes, Robert M
Scemama, Jean-Luc
red blood cell, hypothermic storage, sucralose
In the United States, the maximum approved preservation time for red blood cells under hypothermic storage conditions is 42 days when treated with an appropriate additive solution, namely AS-1. In this investigation, we analyze the possible protective properties of sucralose alone, in combination with, and as an added ingredient to the AS-1 preservation solution. Absorbance measurements were used to monitor the amount of free hemoglobin found in the supernatant for the variety of different treatments investigated. Blood treated with sucralose alone did not show to minimize hemolytic leakage as well as the current standard, whereas red cells treated with the modified AS-1 solution where sucralose replaced the mannitol constituent showed similar results to that of the current standard. Statistical analysis revealed that no significant difference between the standard AS-1 solution and the modified AS-1 solution was observed when all studies were integrated together. Osmotic fragility experiments were conducted to assess if the membrane flexibility was compromised due to the addition of sugar during incubation by quantifying the percent hemolysis at various salt concentrations. Analyses determining the concentration of salt required to induce 50% hemolysis, known as the IC50 value, were used to compare the treatments. Red cells treated with sucralose alone showed higher IC50 values, which is associated with red cell membrane rigidity. Lower IC50 values were observed for blood treated with the modified AS-1 solution, which showed no statistical significance in comparison to that of the standard AS-1 solution when all studies were considered. Potassium assays were conducted to measure potassium leakage as a function of time in storage. Blood samples treated with sucralose alone reached equilibrium more quickly, suggesting sucralose alone does not retard the leakage of potassium as well as the current standard. Red cells treated with the modified AS-1 solution showed similar results to the current standard as the rate of potassium leakage was the same. Cellular morphology studies were conducted to observe the morphological changes red blood cells undergo during extended preservation periods. Blood treated with sucralose alone reach undesirable morphologies more quickly when compared to the current standard, suggesting sucralose alone does not preserve the membrane as well as the standard. Red cells treated with the modified AS-1 storage solution showed to retard the progress to undesirable morphologies better than that of the standard AS-1 solution as the cells maintained discocyte and echinocyte structures for longer periods of time with no red cell fragmentation throughout the duration of time in storage.
2023-02-10
2022-12
2023-01-05
December 2
2023-01-31
Doctoral Dissertation
http://hdl.handle.net/10342/12261
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/88212022-12-05T15:01:45Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
HOW CHARGED RESIDUES INFLUENCE THE THERMAL STABILITY OF COLLAGEN: A STUDY WITH NATURAL AND NON-NATURAL AMINO ACIDS
Banzon, Patrick D.
Allen, William E.
Chemistry
Thermal Stability
Charge
Triple-helical collagens are key structural proteins in mammals. Their ubiquity and diverse functions drive our interest into understanding their behavior at a fundamental level. This thesis describes a reductionist approach using novel collagen-related peptides (CRPs), into which one or more electrical charges have been imparted at known positions. One series of CRPs includes fluorescent pyrene tags at their N-termini, directly adjacent to the charged residues lysine (Lys, K) and glutamic acid (Glu, E). When in close contact, the fluorophores form excimers that emit low-energy light. Monitoring of the excimer intensity shows that nucleation of collagen peptides is critically dependent on the charge location. Another series of CRPs features pH-independent (permanent) positive charge close to the peptide backbone, via a synthetic proline derivative called "Map." When in close contact, repulsion between Map residues overwhelms the natural tendencies of the peptides to fold. CD and fluorescence investigations into the thermodynamic and kinetic behaviors of these CRPs have been supplemented with computational analyses, to shed light on the deleterious role of charge in trimer formation.
2021-01-04
2021-06-01
2020-12
2020-09-16
December 2
2020-12-18
Master's Thesis
http://hdl.handle.net/10342/8821
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/51012021-03-03T20:59:12Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Investigating Intra- and Inter-Molecular Coevolution of Intrinsically Disordered Protein, Prothymosin-alpha
Biscardi, Brianna
Burns, Colin Sanderson
Chemistry
IDP
Intrinsically disordered
Prothymosin-alpha
Prothymosin-α (ProTα) is a small, highly acidic protein found in the nuclei of virtually all mammalian tissues. It belongs to a class of proteins known for their lack of a rigid three-dimensional structure called intrinsically disordered proteins (IDPs). ProTα has been shown to play essential roles in cell robustness. As an example, ProTα is involved in apoptosis, or programmed cell death by inhibiting apoptosome formation via binding Apaf1. This research focus is on detecting coevolution of ProTα and between ProTα and Apaf1 (ProTα-Apaf1 or ProTα-Apaf1 complex). Coevolution refers to correlated changes between pairs of interacting species to maintain or refine functional interaction. Coevolution can be defined at the molecular level as correlated sequence changes that occur to maintain a structural or functional interaction. Studying coevolution of ProTα and ProTα-Apaf1 may provide useful information such as structural contacts and specific residues necessary for complex formation. In this study, a pipeline for performing molecular coevolution studies was established at East Carolina University (ECU). This pipeline was used to analyze myoglobin, ProTα, and ProTα-Apaf1. Myoglobin has been a target of previous coevolutionary studies and was chosen to test the robustness of the pipeline developed in this study. Most of the coevolving residues that were found in myoglobin match closely with those detected in other work. ProTα, which has never been studied by way of coevolution, displays several coevolving residues involved in long range interactions or functionally important regions. These methods were also applied to ProTα- Apaf1 complex. Previous experimental studies using 1H-15N heteronuclear single quantum coherence (HSQC) NMR have revealed residues on ProTα necessary for interaction with Apaf1 however the residues on Apaf1 necessary for interaction with ProTα have not been resolved. Several residues of ProTα were found to have coevolution with Apaf1. Docking studies were performed to simulate binding between ProTα and Apaf1 at the sites detected in this study (ProTα: Thr8, Thr107; Apaf1: Ser1056, Asp1096). Six orientations of ProTα and Apaf1 were run for 9 nanoseconds (ns) and in each simulation, the two proteins did not drift apart from one another. This suggests that the residues detected by coevolution in this study may play a role in the interaction between ProTα and Apaf1.
2016-01-14
2016-01-14
1/13/16
Master's Thesis
http://hdl.handle.net/10342/5101
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/44902021-03-03T20:56:42Zcom_10342_55com_10342_1col_10342_58col_10342_72
Extraction of Salvia miltiorrhiza Using Subcritical Water
Biller, Jessica, J
Yang, Yu
Chemistry
Chemistry
Subcritical water
Chinese medicine
Salvia miltiorrhiza
Extraction (Chemistry)
Salvia miltiorrhiza, also known as Danshen, is an herb commonly used in Chinese medicine to treat cardiovascular and other diseases. As it has recently gained more recognition, this herb is currently being investigated for its anticancer properties. In traditional Chinese medicine, the patients extracted the prescribed herbs using boiling water, then consumed the extracts as medication. However, the extraction temperature of approximately 100 'C used in this traditional method may not be the most effective condition to remove the active pharmaceutical ingredients (APIs) from the herb. Previous studies show that higher temperatures can more efficiently extract anticancer agents, such as tanshinones, from Salvia miltiorrhiza. The goal of this study is to determine the most efficient subcritical water extraction temperature that extracts the APIs from the herb. The extraction process was performed at four different temperatures: 75 'C, 100 'C, 125 'C, and 150 'C. After the high-temperature water extraction, the APIs in the water extracts were analyzed using high-performance liquid chromatography (HPLC).
2014-08-06
2015-08-06
2014
Undergraduate Thesis
http://hdl.handle.net/10342/4490
oai:TheScholarship.intra.ecu.edu:10342/59312021-03-03T21:08:41Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Defining the Role of the C-Terminal Region of Troponin T by Analysis of a Series of Truncation Mutants
Johnson, Dylan James
Burns, Colin Sanderson
Chemistry
troponin
cardiomyopathy
TnT
Familial hypertrophic cardiomyopathy and other cardiovascular diseases result from mutations of any of the contractile proteins. Mutations within the actin binding regulatory complex of proteins, including tropomyosin (Tm) and the three subunits of the troponin (Tn) complex (TnI, TnC, and TnT), change the operation of the Ca2+ dependent 3-way switch that controls movement. The Δ14-TnT mutation, which is missing the last 14 residues of its C-terminus, is particularly important as it leads to hypertrophic cardiomyopathy and early sudden death. Our laboratory found that incorporation of Δ14-TnT into the regulatory complex stabilizes the open (M) state and removes the blocked (B) state from the actin state distribution. This suggests the last fourteen residues of the C-terminus of TnT are essential in maintaining the open state and the blocked state of the thin filament. This function had not previously been attributed to TnT.
Our lab aims to identify the key residues of TnT that are responsible for normal state distribution. This information will allow us to identify possible mechanisms of action of TnT and would facilitate the design of treatments of myopathies. We prepared truncation mutants of TnT that included Δ4, Δ6, Δ8, Δ10 and Δ14. We utilized two stopped flow kinetic assays and an ATPase assay to determine the effect of these deletions on the state distributions. Each assay supported the idea that successive deletions resulted in further diminished function. We conclude that all of the fourteen residues of the TnT C-terminus contribute to a similar extent to the function.
2016-08-26
2016-08-26
2016-05
2016-07-21
May 2016
2016-08-25
Master's Thesis
http://hdl.handle.net/10342/5931
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/47752021-03-03T20:55:57Zcom_10342_55com_10342_1col_10342_58col_10342_72
Identification of Damaged DNA Adducts from Exposure to Benzo[a]pyrene in the TP53 Gene
Taylor, Lea
Hvastkovs, Eli G.
Chemistry
Benzo[a]pyrene
DNA adducts
Benzo[a]pyrene is a carcinogen associated with tobacco smoke that can damage DNA after it is metabolized into highly reactive forms. Identifying the resulting DNA adducts can give greater insight into the mutations that frequently occur in lung cancer. This research aimed to identify damaged DNA adducts from exposure to benzo[a]pyrene in the TP53 gene. This gene codes for the p53 protein, which is frequently mutated in cancers. The oligomeric DNA sequence was exposed to myoglobin, benzo[a]pyrene, and hydrogen peroxide. Myoglobin acted as an heme enzyme mimic, and provided similar chemistry to bio-relevant cytochrome P450 enzymes that are involved in the metabolism of substances like benzo[a]pyrene. LC-MS was used to identify DNA adducts.
2015-04-29
2015-04-29
2015
Honors Thesis
Taylor, Lea. (2015). Identification of Damaged DNA Adducts from Exposure to Benzo[a]pyrene in the TP53 Gene. Unpublished manuscript, Honors College, East Carolina University, Greenville, NC.
http://hdl.handle.net/10342/4775
oai:TheScholarship.intra.ecu.edu:10342/44682022-12-12T17:51:24Zcom_10342_55com_10342_1col_10342_58col_10342_72
Insight Into the Location of DNA Xenobiotic Damage by Mass Spectrometry
Mehaffey, Megan R.
Danell, Allison Stokes
Chemistry
Mass spectrometry
DNA damage location
Xenobiotic
Genotoxicity
Damage to DNA by a bioactivated xenobiotic typically occurs at specific sites within the genome, called hotspots. An example of this is benzo[a]-pyrene (BP), a xenobiotic that enters the body via cigarette smoking. Hotspots at which the final BP metabolite, (+/-)-anti-benzo[a]-pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), adducts DNA oligomers have been electrochemically detected. Determination of the number of adducts formed on the DNA oligomer from exposure to the xenobiotic is necessary for further insight into the damage reaction. Analysis of the damaged DNA samples by Electrospray Ionization Mass Spectrometry (ESI-MS) allows for observation of structural features by separation of the ions present by mass. Using tandem mass spectrometry to obtain data, MS/MS spectra are interpreted for the damaged DNA and used to determine the location of the damage by observing a corresponding change in mass due to the reaction of DNA with the xenobiotic. Tandem mass spectrometry allows for the isolation of certain peaks in the spectrum to further separate and compare to similar spectra of undamaged DNA. Further collection of spectra is necessary before preliminary findings can be reported.
2014-08-06
2015-08-06
2014
Undergraduate Thesis
http://hdl.handle.net/10342/4468
oai:TheScholarship.intra.ecu.edu:10342/47942021-03-03T20:56:32Zcom_10342_55com_10342_1col_10342_58col_10342_72
Recognition of bacterial lipid headgroups by fluorescent crown ether-naphthalimides
Marshall, Sarah Ruth
Allen, William E.
Chemistry
Fluorophore
Membrane selectivity
The increasing incidence of antibiotic- resistant bacterial strains is a significant threat to human health. New antimicrobial mechanisms that feature reduced resistance potential are necessary to slow down the rapid evolution of bacteria and to develop more selective treatment. Large crown ethers are known to actively hydrogen bond with ammonium groups (R-NH3+). Such ammonium group binding can be used to molecularly recognize the terminal ammonium unit present in the bacterial membrane lipid, “POPE.� This selective binding would be advantageous in such bacterial lipids present in mammalian hosts, which express lipid “POPC,� which lack the N-H capable of crown interaction. A fluorescently labeled crown ether was synthesized by palladium- catalyzed cross coupling of a 4-bromonapthalimide with 1-aza-18-crown-6. The crown-naphthalimide conjugate is strongly luminescent in nonpolar, organic solvents like dichloromethane and 1-octanol, but is quenched in polar solution such as aqueous phosphate buffer. The integrated fluorescence intensity of the conjugate is approximately three times greater in the presence of POPC liposomes than POPE liposomes, suggesting that the compound may be able to discriminate between mammalian and bacterial cell membranes. While fluorescence spectrophotometry concurs with the original proposal of observable POPE lipid selectivity, other data revealed otherwise. DFT optimization treatments were run to foretell possible interactions and orientations of the desired molecules. The computational analysis predicts that the ammonium group of POPE favors strong hydrogen binding within the crown, however proton NMR and ESI-MS studies have so far not confirmed that this binding mode is operative.
2015-05-05
2015-05-05
2015
Honors Project
Marshall, Sarah. (2015). Recognition of bacterial lipid headgroups by fluorescent crown ether-naphthalimides.Unpublished manuscript, Honors College, East Carolina University, Greenville, N.C.
http://hdl.handle.net/10342/4794
oai:TheScholarship.intra.ecu.edu:10342/41002021-03-03T20:57:09Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Kinetic Modeling of Batch Reaction Processes
Joiner, David
Gemperline, Paul J.
Chemistry
Chemistry, Analytical
Batch reaction
Crystallization
Dissolution
Kinetic modeling
Slurries
Analytical chemistry
Kinetic models have been demonstrated to be useful in on-line batch monitoring systems. The ability to monitor a reaction in real time is invaluable to the production process of industrial and pharmaceutical products. However, it is not a commonly used technique, due to some of its limitations. Currently, a great deal of work has been done showing the ability of a kinetic model to accurately estimate the spectral profiles and concentrations of reaction systems as a function of time. Some models have even demonstrated the technique's ability to model the transitions of analytes during dissolution and crystallization processes, but little has been done to combine all of these processes into a flexible, robust modeling system that incorporates all of these processes simultaneously.  The goal of this work is three-fold: (1) to demonstrate the ability of a kinetic model to cohesively model dissolution, reaction, and crystallization processes, (2) accurately predict the spectral and concentration information produced by the reaction system, and (3), to accurately model an actual industrial slurry reaction using these same methods.  For first part of this work, an acetylsalicylic acid synthesis model system was chosen. This synthesis reaction contains all of the processes necessary to produce a cohesive model including dissolution of the salicylic acid reactant, simultaneous reaction of the reactants to form the product and side products, and subsequent crystallization and precipitation of the product. This work was performed using ATR-UV/Vis measurements to model changes in the solution phase of the reaction mixture and utilized HPLC measurements for validation of the results.  The second part of this work used the same techniques seen in part one and extended them to a complex industrial slurry model system. This section of the work was performed using NIR reflectance measurements to model the changes in the solid phase of the reaction mixture. This portion also used HPLC measurements for validation purposes.  The work done within these two sections demonstrates the ability of the kinetic model to operate in both in the solid and liquid state and using multiple spectroscopic methods. Â
2013-01-15
2015-02-02
2012
Master's Thesis
http://hdl.handle.net/10342/4100
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/87942021-08-31T15:25:57Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
DEVELOPMENT OF UNNATURAL SUBSTRATES AND TRYPTOPHAN AMINO ACIDS TO STUDY PROTON COUPLED ELECTRON TRANSFER IN ENZYMES
Ohgo, Kei
Offenbacher, Adam R
Chemistry
Proton Coupled Electron Transfer
Proton-coupled electron transfer plays an important role in substrate oxidation by C-H bond cleavage and long-range pathways associated with bioenergetics. This thesis is focused on the synthesis of unnatural substrates and tryptophan amino acids to study these effects in enzyme reactions. The functionalization of C-H bonds is an important chemical transformation, representing a challenge in the design of asymmetric organometallic catalysts to generate a range of organic molecules with diverse functional groups. Soybean lipoxygenase-1 is a model enzyme system that catalyzes C-H activation reactions. Unnatural and volume-filling fatty acid derivatives were designed to test with a mutant soybean lipoxygenase that has an expanded active site. The goal of this strategy is to develop new hydroperoxide-based products of long-chain aliphatic compounds. Fluorinated 5-hydroxytryptophan (Fn-5HOW) derivatives were synthesized on a large scale using a chemoenzymatic approach. These redox-active Fn-5HOW derivatives exhibit tyrosine-like proton-bound oxidation and are associated with the spectroscopic characteristics of neutral radicals that are easily distinguishable from natural aromatic amino acids. As a proof of concept, these unnatural amino acids have been incorporated into structured peptides and model proteins. These unnatural fluorinated 5HOW derivatives may act as reporters for tryptophan-mediated biological electron transport.
2020-12-18
2020-12-18
2020-12
2020-12-09
December 2
2020-12-18
Master's Thesis
http://hdl.handle.net/10342/8794
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/31792021-03-03T20:53:08Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Thermodynamic Investigations into Copper Binding in the N-Terminal Region of the Prion Protein at Low Copper Loadings
Gogineni, Devi Praneetha
Burns, Colin Sanderson
Chemistry
Chemistry
The prion protein (PrP) is a naturally occurring protein found at high levels in central nervous system (CNS). The misfolding of the PrP is responsible for neurodegenerative diseases called transmissible spongioform encephalopathies (TSE) that include mad cow disease, scrapie in sheep and goats, kuru and Creutzfelt-Jakob disease (CJD) in humans. The normal function of the PrP is still unknown but demonstrates high selectivity for copper (Cu[superscript]+2). The mature form of PrP consists of a highly unstructured N-terminal region (23-124). The copper binding region spans from residues 60 to 96 and contains four octarepeat segments, PHGGGWGQ, and a GGGTH segment. When fully copper loaded, each octarepeat binds to a copper and the fifth copper binding involves the GGGTH site. Although the molecular details of a fully Cu[superscript]+2 loaded state are well understood, very little is known about the low copper binding state of PrP. At low Cu[superscript]+2 occupancy there is a possibility of PrP cross-linking. This project aims at obtaining the thermodynamic profile of the prion copper complex at low copper loading state to determine the forces that drive the complex formation. The model peptides were generated using solid phase peptide synthesis; the thermodynamic studies were done using isothermal titration calorimetry (ITC) and supporting spectroscopic studies by circular dichroism (CD). Examination of the ITC titration data suggests an initial binding event where two PrP's are cross-linked by a single copper ion. ITC titrations were performed in both forward and reverse directions in order to examine the reversibility of the copper binding process. Fitting the ITC data with the existing models, i.e one sets of sites, two sets of sites or sequential binding, lead to unsatisfactory fits suggesting a more complex binding process. The hypothesized binding model will hopefully lead to good fits for the ITC data and will support the hypothesis that Cu[superscript]+2 is cross- linking PrP molecules at low equivalents of added Cu[superscript]+2 . Â
2011-02-03
2011-05-17
2011-02-03
2011-05-17
2010
Master's Thesis
http://hdl.handle.net/10342/3179
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/51952021-03-03T21:00:16Zcom_10342_55com_10342_1col_10342_58
Tissue lockable connecting structures
Yu, Chang
Harris, Glenn
Percutaneous skin access devices include a plurality of locked connecting units mounted to the exterior surface of an implantable medical object which, in position, is configured to penetrate the skin of a subject. The locked connecting units may be mounted directly onto the desired surface of the exterior of the device or may be held on a substrate sheet, which is mounted to the exterior surface of the device. In position, the locked connecting units engage with soft tissue which can include the skin to form a bio-junction layer which includes mechanical and bio-sealing connection between the device body and the soft tissue. The configuration at the bio-junction layer secures the medical object in location in the subject even for long-term indwelling applications in a manner, which inhibits soft tissue infection.The locked connecting units may be rigid or semi-rigid for longer-term indwelling applications, and semi-rigid and/or resilient for shorter term indwelling applications. The locked connecting units may take on the form of rings, hooks, or loops having aperture or gap width/length sizes of from about 0.2 4 mm. The rings, loops, or hooks may connect with any soft tissue including skin as well subcutaneous tissue. The rings, hooks, or loops may be released from the skin/tissue without requiring surgical cutting procedures.The locked connecting units may be configured as a semi-rigid mesh collar arranged about the primary body providing access to the subject such that it resides in the subject and engages with the skin (epidermal/dermal layer). The mesh collar can be described as a particular type of ring or loop structure as the mesh defines the gap provided in individual loop configurations. The mesh collar may be used alone, or in combination with the loops, rings, or hooks. A skin stop collar having increased rigidity may be disposed under the mesh collar.
2016-02-09
2016-02-09
2006-08-01
Patent
U.S. Patent No. 7,083,648
http://hdl.handle.net/10342/5195
oai:TheScholarship.intra.ecu.edu:10342/40032021-03-03T20:53:16Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Solvent and Solubility Effects on Quinone Ratios
Duffy, Brian
Love, Brian Ernest
Chemistry
Chemistry
Chemistry, Organic
Diquinone
Quinone
Synthesis
Organic chemistry
Monoquinones and diquinones are a biologically and chemically important class of compounds that can be found in numerous natural products such as: thymoquinone, oosporein, coenzyme Q, embelin and the respective dimer, biembelin. These 1, 4-benzoquinone derivatives exhibit prominent pharmacological applications such as antibiotic, anti-tumor, anti-malarial, anti-coagulant, and anti-convulsant activity.    Though quinones can be prepared by a variety of processes, they are most commonly synthesized through the treatment of hydroquinone dimethyl ethers with ceric ammonium nitrate (CAN). However, this can lead to an unpredictable mixture of monoquinone and diquinone as the products. This project has investigated the effect of solvent and substrate water solubility on monoquinone / diquinone product ratios with the aim of being able to consistently and predictably favor one (monoquinone or diquinone) over the other. Â
2012-09-04
2012-09-04
2012
Master's Thesis
http://hdl.handle.net/10342/4003
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/68132021-03-03T21:18:07Zcom_10342_7351com_10342_6421com_10342_55com_10342_1col_10342_7360col_10342_58
protaTETHER – a method for the incorporation of variable linkers in protein fusions reveals impacts of linker flexibility in a PKAc�GFP fusion protein
Norris, Jessica L.
Hughes, Robert M.
Linkers
protaTETHER
Protein
Protein engineering
Protein fusions
Restriction enzyme-free
Cloning
Copyright
© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. Article licensed under a Creative Commons Attribution License, CC BY 4.0, (https://creativecommons.org/licenses/by/4.0/).
Protein fusions are of fundamental importance in the study of cellular biology and the elucidation of cell signaling pathways, and the importance of linkers for the proper function of protein fusions is well documented in the literature. However, there are few convenient methods available to experimentalists for the systematic implementation of linkers in protein fusions. In this work, we describe a universal approach to the creation and insertion of focused linker libraries into protein fusions. This process, deemedprotaTETHER, utilizes reiterative oligomer design, PCR-mediated linker library generation, and restriction enzyme-free cloning methods in a straightforward, three-step cloning process. We utilize a fusion between the catalytic subunit of cAMP-dependent protein kinase A (PKAc) and green fluorescent protein (GFP) for the development of the protaTETHERmethod, implementing small linker libraries that vary by length, sequence, and predicted secondary structural elements. We analyze the impact of linker length and sequence on the expression, activity, and subcellular localization of the PKAc-GFP fusions, and use these results to select a PKAc-GFP fusion construct with robust expression and enzymatic activity. Based upon the results of both biochemical experiments and molecular modeling, we determine that linker flexibility is more important than linker length for optimal kinase activity and expression.
2018-07-02
2018-07-02
2018-03-14
Article
Norris, J. L., & Hughes, R. M. (2018). protaTETHER - a method for the incorporation of variable linkers in protein fusions reveals impacts of linker flexibility in a PKAc-GFP fusion protein. FEBS Open Bio, 8(6), 1029–1042. https://doi.org/10.1002/2211-5463.12414.
http://hdl.handle.net/10342/6813
https://doi.org/10.1002/2211-5463.12414
en_US
https://febs.onlinelibrary.wiley.com/doi/10.1002/2211-5463.12414
oai:TheScholarship.intra.ecu.edu:10342/68682022-11-11T14:51:29Zcom_10342_55com_10342_1col_10342_58col_10342_72
Membrane Localization of Pyrene-Trehalose Conjugates (PYRETs)
Hagwood, Abigail
Allen, William E.
Chemistry
trehalose
pyrene
membrane localization
hydration hypothesis
The disaccharide trehalose is found in many organisms that can survive in extremely cold or dry environments. According to the “hydration hypothesis,� trehalose interacts with the glycerol/phosphate regions of lipid bilayers and displaces water molecules, preventing ice crystals from puncturing the cell when frozen. In order to control the depth of trehalose penetration into membranes, we have attached the nonpolar fluorophore pyrene to trehalose via one-, three-, and five-carbon spacers. The conjugates, which we call PYRETs, are sufficiently soluble in ethanol-water to be purified by reversed-phase HPLC. Proton NMR showed low symmetry and integrations consistent with a single pyrene anchor per disaccharide unit. The effects of PYRETs on membrane dynamics is shown by fluorescence spectroscopy and differential scanning calorimetry. Long-term, PYRETs may be useful in cryopreservation of red blood cells.
2018-07-12
2020-05-01
2018-05
2018-05-01
May 2018
2018-07-03
Honors Thesis
http://hdl.handle.net/10342/6868
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/36632021-03-03T20:52:17Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
ELECTROCHEMICAL DETECTION OF BENZO[A]PYRENE METABOLITE DNA DAMAGE : IMPLICATIONS OF NUCLEOBASE SEQUENCE AND ADDUCT STEREOCHEMISTRY
Satterwhite, Jennifer Elizabeth
Hvastkovs, Eli G.
Chemistry
Chemistry
Adduct stereochemistry
Benzo[a]pyrene diol epoxide
Radical
Sequence-specific
Xenobiotic
Xenobiotics are chemical compounds introduced to living organisms that originate outside the body. A well-studied xenobiotic is benzo[a]pyrene (BP), a polyaromatic hydrocarbon (PAH) primarily introduced into the body via cigarette smoke as well as through environmental pollution. Once inside the body, BP is ultimately metabolized into electrophilic benzo[a]pyrene- 7,8-diol-9,10-epoxide (BPDE) that reacts with and damages nucleophilic biomaterial, including DNA. BPDE predominately targets specific genomic guanines at sites termed hotspots. BPDE hotspot damage can result in DNA mutations that alter key amino acids in proteins that are involved in cellular regulation, eventually leading to cancer. This process occurs regularly in vivo within the TP53 gene at selected codons. TP53 codes for the p53 protein, which is involved in cellular apoptosis. BPDE stereochemistry and cytosine methylation within hotspot codons are also important parameters that affect the eventual mutagenesis stemming from BPDE-guanine adducts.   Traditional detection of site-specific DNA damage can be expensive and time consuming. Electrochemical approaches can remedy these drawbacks. An electrochemical sensor was developed to detect DNA damage from (±)-anti-BPDE. A double stranded DNA (dsDNA) oligomer corresponding to a known hotspot sequence in the TP53 gene was immobilized on a gold electrode and then exposed to BPDE. Voltammetric measurements were then performed in the presence of electroactive C[subscript]12H[subscript]25V[superscript]2+C[subscript]6H[subscript]12V[superscript]2+C[subscript]12H[subscript]25 (V[superscript]2+ =4,4'-bipyridyl or viologen, C[subscript]12Viologen). BPDE exposure causes DNA structural changes through the formation of bulky adducts that influence the C[subscript]12Viologen-DNA interaction. These structural changes affect the resulting C[subscript]12Viologen voltammetry. At wild type TP53 sequences, BPDE exposure resulted in the emergence of a positive shifted C[subscript]12Viologen redox wave at -0.37 V. At an identical sequence containing 5-methylated cytosine at the hotspot location, two waves emerged at -0.37 V and -0.54 V. These redox signals were muted when DNA was exposed to alternate xenobiotics or alternate sequences were exposed to BPDE. Overall, this demonstrates sequence specific detection of DNA damage at this hotspot sequence. Differences in the voltammetric response suggest that the sensor is sensitive to the adduct stereochemistry.   Additional studies were performed to monitor the effects of BP activation. Activation includes chemical oxidation of BP through association with reactive oxygen species or metabolism. Initial studies focused on the reaction of BP with DNA in the presence of hydrogen peroxide. DNA exposed to this reaction cocktail provided C[subscript]12Viologen voltammetry that was similar to that caused by BPDE exposure, highlighted by a large SWV current increase at -0.37 V. Results were consistent with Fenton chemistry occurring in the damage buffer producing hydroxyl radicals from hydrogen peroxide and iron impurities. The hydroxyl radicals appear to activate BP producing a reactive species that results in BP-DNA adducts.   Overall, the data show a sequence-specific DNA hybridized sensor has the capability of providing hotspot and stereospecific genotoxicity information for benzo[a]pyrene derivatives. Â
2011-08-22
2013-08-31
2011
Master's Thesis
http://hdl.handle.net/10342/3663
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/44782021-03-03T20:55:42Zcom_10342_55com_10342_1col_10342_58col_10342_72
Investigation Into Intermolecular Interactions of Gd3N@C80(OH)n Using Fluorescence Spectroscopy
Purrman, Kyle C.
Rodriguez, Art A.
Chemistry
Intermolecular
Interactions
Metallofullerene
Fluorescence
Intensity
The aim of this experiment was to investigate the intermolecular interactions of the endohedral metallofulerene complex in water and various metal cation solutions. These interactions were observed using a methodology that is particularly sensitive to environmental changes surrounding the probe molecule, fluorescence spectroscopy. Changes in the intensity (I) of the fluorescence peak were monitored to indicate and characterize the nature of these intermolecular interactions. Certain types of interactions will enhance fluorescence while other modes will reduce fluorescence intensity. The results of this investigation are presented within this report.
2014-08-06
2015-08-06
2014
Undergraduate Thesis
http://hdl.handle.net/10342/4478
oai:TheScholarship.intra.ecu.edu:10342/76212021-12-01T09:01:54Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_123
Developing Novel Chemotherapeutics: A Structure-Activity Study of Anandamide Analogs and their Cytotoxic Profiles
Morris, Andrew
Burns, Colin Sanderson
Chemistry
Endocannabinoid
Skin Cancer
Colorectal Cancer
Chemotherapeutic
Prostaglandin
Many epithelial cancers have been shown to overexpress the enzyme cyclooxygenase-2 (COX-2), an enzyme responsible for both the metabolism of arachidonic acid (AA) to prostaglandins and arachidonoyl ethanolamine (AEA) to prostaglandin-ethanolamides (prostamides). AEA has demonstrated cytotoxicity in COX-2 overexpressing cancers via its metabolism to the novel J-series prostamide, 15d-PMJ2. Using what is known about how AEA induces cell death, derivatives of AEA were synthesized to investigate COX-2 metabolism, PGDS metabolism, FAAH degradation resistance, and the inherent cytotoxicity of their J-series prostaglandin analogs. A structure-activity relationship study was conducted with ten AEA derivatives to determine what modifications to the ethanolamide moiety improve anti-cancer activity in COX-2 overexpressing JWF2 tumorigenic keratinocytes and HCA-7 colorectal cancer cells. Important cytotoxic characteristics of AEA and AEA analogs were identified from the SAR study. Hydrogen bond accepting or donating ability seems to be the most important characteristic retained in cytotoxic AEA analogs. Distance between the amide bond and terminal hydroxyl moiety seems to be important, but no trend as to whether a shorter or longer carbon chain is apparent. Addition of an aryl group does not interfere with cytotoxicity and in fact improves it as long as a terminal hydroxyl group is retained. Two derivatives which displayed cytotoxicity similar to or greater than that of AEA, NAGly and arvanil, were selected for further studies exploring their cytotoxic and metabolic profiles. Arvanil (LC50 = 6.03 [micro]M) demonstrated greater cytotoxicity than NAGly (LC50 = 9.54 [micro]M) and our positive control AEA (LC50 = 9.39 [micro]M). Metabolism by COX-2 and PGDS to D-series and J-series prostaglandin analogs were responsible for arvanil's apoptotic anti-cancer activity. The novel J-series prostamide analog, 15d-PMJ2-arvanil, was identified for the first time as well as another metabolite, possibly an A-series prostamide analog, with identical mass-to-charge ratios and fragmentation patterns in the mass spectrum. These molecules will be synthesized, and their anti-cancer activity investigated in future studies. Collectively, these results suggest that structural modification of the ethanolamide moiety of AEA provides a means for improving both the selectivity and cytotoxicity of prodrugs resulting in formation of novel J-series prostamides.
2020-02-04
2021-12-01
2019-12
2020-01-06
December 2
2020-01-29
Doctoral Dissertation
http://hdl.handle.net/10342/7621
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/124112023-03-14T07:16:37Zcom_10342_55com_10342_1col_10342_58
Complementary Square Wave Voltammetry and Tandem Mass Spectrometry Analysis to Identify and Detect Compensatory Genomic Changes in Nematodes Due to Nickel (II) Exposure
LaFave, Elizabeth R.
Turner, Ryne
Schaaf, Nicholas J.
Hindi, Thekra
Rudel, David
Hvastkovs, Eli G.
SWV analysis
DNA damage
Nematode DNA
2023-03-13
2023-03-13
2022
Article
2666-0539
http://hdl.handle.net/10342/12411
10.1016/j.snr.2021.100070
en_US
oai:TheScholarship.intra.ecu.edu:10342/40072021-03-03T20:53:08Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
SYNTHESIS AND PHOTOPHYSICAL CHARACTERIZATION OF TRIDENTATE C^N*N PLATINUM (II) COMPLEXES and, INVESTIGATION OF SELECTIVITY IN C-H BOND ACTIVATION BY PLATINUM
Ravindranathan, Deepak
Huo, Shouquan
Chemistry
Chemistry
C-H bond activation
Complexes
Cyclometalation
Phosphorescence
The synthesis, structure, and photophysical properties of a series of novel, highly luminescent tridentate platinum complexes with general coordination geometry of (C^N*N)-PtL are reported, where "C^N" denotes a coordination of C and N to the platinum to form a five-membered metallacycle and "N*N" denotes a coordination of two N atoms to the platinum to form a six-membered metallacycle; L is a mono anionic ligand such as halides or acetylides. Compared to the known (C^N^N)-PtL type of complexes that were reported to emit with low quantum yields, the structural modification leads to dramatic improvements in phosphorescence efficiency. For example, new complexes (C^N*N)-PtL with L = hexylacetylide and phenylacetylide emitted intensely with quantum yields of 47% and 56%, respectively, latter of which is among the highest quantum yields reported so far for cyclometalated platinum (II) complexes. Selectivity in C-H bond activation by platinum and the exact mechanism of cycloplatination are issues that still remain unclear. A series of ligands which include sp²/sp³, primary/secondary sp³ C-H bonds, and aromatic/vinylic sp² C-H bonds with a carbon linker between the bipyridine and the carbon groups have been prepared. All ligands have been attempted for cycloplatination in glacial acetic acid and acetonitrile. All ligands produced the same sp² C-H bond activated complex in both solvents, which suggests that the linker atom does play a role on selectivity.
2012-09-04
2012-09-04
2012
Master's Thesis
http://hdl.handle.net/10342/4007
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/69292021-03-03T21:18:49Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Using Fluorescence and Infrared Spectroscopy to Analyze the Interaction of Disaccharides with Lipid Bilayers
Angermeier, Tori Marena
Kennedy, Anthony
Chemistry
lipid
It is well known that sugars, such as trehalose, dehydrate lipid membranes based upon the Water Replacement Hypothesis. Here, we have investigated the interaction between different sugars (sucralose, sucrose, and trehalose) and model phospholipid membrane 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) using fluorescence spectroscopy with the fluorescent probe Laurdan and infrared spectroscopy. For the fluorescence measurements, the addition of sugar molecules to the bilayer resulted in a decrease in the Generalized Polarization (GP) values, corresponding to an increase in polarity in the environment within the bilayer around the probe. A decrease in GP values of Laurdan also suggest the possibility of coexisting phases within the bilayer. Infrared spectroscopy was used to investigate the dynamics of the hydrophilic and hydrophobic regions of the membrane as the changes of the asymmetric phosphate group and fatty acid chain vibrational bands were monitored when introduced to sucrose and sucralose. These tests revealed that sucrose had no significant impact on the membrane in either region of the bilayer, while sucralose showed a significant impact on both regions of the bilayer. Overall, we have monitored the interaction of different sugar molecules with model DPPC membranes through fluorescence and infrared spectroscopy measurements to explore the polarity of the bilayer and the possible dynamic effects of sugars have on lipid membranes.
2018-08-14
2020-08-01
2018-08
2018-07-24
August 201
2018-08-09
Master's Thesis
http://hdl.handle.net/10342/6929
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/74672021-08-01T08:01:55Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
EXPLORATION INTO THE SCOPE AND MECHANISM OF THE PLATINUM-CATALYZED ACYLATION OF 2-(ARYLOXY)PYRIDINES
Guthrie, Jacob D
Huo, Shouquan
Chemistry
C-H Activation
Hammett Analysis
Transition metal catalysts have played a key role in direct C-H bond functionalization. However, one main drawback of these reactions is that oxidants and additives are often required to regenerate the active catalyst, oxidize the substrates, or promote the reaction. These needed reagents can often add significant cost and safety concerns to the synthesis. Recently the Huo group has discovered a unique platinum catalyzed acylation reaction to produce alpha-keto esters via C-H functionalization using ethyl chorooxoacetate, a cheap and readily accessible reagent. This reaction eliminated the need for any oxidants or additives and, more importantly, was free of any decarbonylation side reactions (see below). Further reaction optimization was performed to lower the catalyst loading. It was found that with the addition of potassium carbonate, strong acids that were produced during the C-H activation step (B) were neutralized thus allowing the catalyst to coordinate to the 1a more readily. The range of the overall synthesis was explored using different acylating agents. Finally, Hammett analysis was used to study the substituent effects of this reaction. Experimental results will be reported, and the significance of these findings will be discussed.
2019-08-22
2021-08-01
2019-08
2019-07-11
August 201
2019-08-19
Master's Thesis
http://hdl.handle.net/10342/7467
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/36622021-03-03T20:57:19Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
SEPARATION AND ANALYSIS OF PRESERVATIVES IN SKINCARE CREAMS BY HIGH TEMPERATURE LIQUID CHROMATOGRAPHY AND SUBCRITICAL WATER CHROMATOGRAPHY
Gujjar, Leena
Yang, Yu
Chemistry
Chemistry
Chemistry, Analytical
Cosmetics
High temperature liquid chromatography
Parabens
Preservatives
Subcritical water chromatography
Zirconia based column
Analytical chemistry
Preservatives are chemicals with antimicrobial activity commonly added to foods, pharmaceuticals and cosmetics in order to prolong products' shelf life and to protect the consumer from potential infection. Parabens, the most widely used preservatives worldwide are a family of alkyl esters of para-hydroxybenzoic acid. The most widely marketed para-hydroxybenzoic acid esters are methyl, ethyl, propyl and butyl parabens. Their microbial activity increases as the alkyl chain increases. Parabens are reported to have weak estrogen-like properties. According to Cosmetic Ingredient Review (CIR), limit up to 0.4% (single paraben) or up to 0.8% (mixtures of parabens) can be added to the cosmetic products. Therefore, to monitor the levels of preservatives in cosmetic products is important.  High-performance liquid chromatography (HPLC) is the most commonly used separation and analysis technique for the determination of preservative in skincare creams. HPLC involves a consumption of large quantities of organic solvents in the mobile phase. These HPLC organic solvents are toxic, expensive for purchasing as well as their proper disposal.   At ambient temperature, water is too polar to serve as a sole chromatographic solvent. Fortunately, at elevated temperatures and under moderate pressures, the polarity of water significantly decreases and liquid water behaves more like an organic solvent. Thus, high-temperature water can mimic organic solvent-water mixtures used in HPLC to achieve liquid chromatographic separation.  The goal of this research is to develop high temperature liquid chromatography (HTLC) and subcritical water chromatography (SBWC) methods for the separation and analysis of preservatives in skincare creams to either reduce or completely eliminate the consumption of the harmful organic solvents used in traditional HPLC. A ZirChrom®-DiamondBond-C18 column was used in this study to carry out the separation of preservatives. Preservatives studied include benzyl alcohol, 2-phenoxyethanol, methyl, ethyl, propyl, and butyl paraben. Quantitative analysis of preservatives in three Olay® skincare creams was achieved by HTLC and SBWC. The recoveries obtained by HTLC and SBWC are efficient. The major advantage of HTLC and SBWC techniques is the reduction or elimination of organic solvents used in traditional HPLC. Â
2011-08-22
2013-08-31
2011
Master's Thesis
http://hdl.handle.net/10342/3662
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/40042021-03-03T20:57:39Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Synthesis and Reactions of Novel Heteroarylzirconocene Reagents
Harris, Caleb F.
Huo, Shouquan
Chemistry
Chemistry
Cross-coupling
Heteroaromatic
Organometallic
Palladium-catalyzed
Zirconocene
The cross-coupling of metallated heteroaromatic species with aryl and heteroaryl halides is a powerful and highly desirable transformation in both the synthesis of pharmaceutical compounds as well as materials development. However, current methodology in this area has some limitations, particularly with organometallic reagents derived from 2-halopyridines, 2-haloquinolines, and other similar heteroaromatic compounds. Suzuki cross-coupling reactions of 2-pyridyl boronic acids or esters is often reported to be low yielding due to protodeborylation. In particular, the preparation of the corresponding heteroaromatic boronic reagents is often associated with low yields. Although Negishi cross-coupling reactions are often reported to be high yielding, the generation of heteroaromatic Negishi reagents require the use of expensive and user unfriendly Rieke® Zinc. The Stille reaction is the least desirable because of the highly toxic tin waste streams generated. Zirconium is a non-toxic, inexpensive, and abundant metal that exists primarily in the Zr(IV) oxidation state and its application in organic synthesis has been extensively studied. However, the preparation of heteroaromatic zirconocene reagents and their application in organic synthesis have not been reported. In 1986, Negishi reported the generation of a Zr(II) species, in situ, by reacting Cp₂Zr(IV)Cl₂ with 2 equivalents of n-BuLi followed by a [beta]-H abstraction process. We anticipated that the oxidative addition of a heteroaromatic halide to the Negishi reagent, "Cp₂Zr(II)", would generate the desired heteroaromatic zirconocene reagents. These novel reagents were successfully generated and used in palladium-catalyzed cross-coupling reactions.
2012-09-04
2012-09-04
2012
Master's Thesis
http://hdl.handle.net/10342/4004
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/49552021-03-03T20:56:45Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
A Calorimetric and Spectroscopic Study of the Interactions Between Model Lipid Membrane Bilayers and Simple Sugars
Pennington, Edward Ross
Kennedy, Anthony
Chemistry
Chemistry
Calorimetry
Dissaccharides
Infrared spectroscopy
Liposomes
Model membranes
It is well known that various organisms, such as the tardigrade, naturally use carbohydrates, particularly disaccharides, to help preserve their cell structural and functional integrity during freezing or dehydration. There have been many different studies that have focused on investigating how certain sugars may help to preserve the structural and functional integrity of biological cells throughout such extreme conditions. These studies have used lipids as a simple and effective alternative to studying how small molecules, such as disaccharides, interact with model lipid membrane bilayers. Lipid bilayers, also known as liposomes, have classically been used to study and better understand the structure of membrane bilayers since their phase behavior is very well understood. Therefore, liposomes are often used to investigate how small molecules interact with membrane bilayers, and how these interactions translate into physical, structural, and potential biological changes. Differential scanning calorimetry (DSC) and infrared spectroscopy were used to examine the thermotropic phase behavior of model liposome membranes in the presence of either sucrose or sucralose. The data indicates that membranes are dehydrated and form an interdigitated layer in the presence of sucralose, but not in the presence of sucrose. We hypothesize that this behavior is due to the different hydrophobic properties of the sugars and that sucralose may penetrate the bilayer whereas sucrose will not. Additionally, at higher concentrations of sucralose the data suggests that sucralose may hydrogen bond to the polar head groups, particularly to the phosphate region of the lipid molecule, in an effort to stabilize the membrane bilayer and Van de Waals forces upon interdigitation. This study demonstrates that sucralose has a significant effect on the phase behavior of liposomes, and its usefulness as a possible cryoprotecting agent for the preservation of biological cells and tissues is of great interest and needs to be comprehensively investigated. Â
2015-06-04
2020-01-23
2015
Master's Thesis
http://hdl.handle.net/10342/4955
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/88382022-12-01T09:01:59Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Identifying compensatory genomic changes due to nickel (Ni2+) exposure: electrochemistry and quantifying DNA damage
Turner, Ryne
Hvastkovs, Eli G
Rudel, David
Chemistry
Toxicity
Nickel is a toxic heavy metal that has been shown to cause adverse healthconditions in organisms. DNA damage has been observed in nematodes in response to nickel exposure. Nematodes originating from environments with high Ni concentrations have shown an increased resistance to Ni exposure. Recombinant inbred lines (RIAILS) have been created using a cross between a nematode originating from a Ni rich environment, with a nematode from an environment with low levels of Ni. In this study, RIAILs were grown on Ni treated plates and Ni absent plates. DNA from the different cultures were extracted and studied using electrochemistry and mass spectrometry. This study shows that RIAILs exhibit a varied response to Ni exposure. RIAILs with lower DNA damage may have advantageous genes reducing the amount of DNA damage in the organism in response to nickel exposure. Contrarily, nematodes with higher DNA damage may have inherited genes that make them more susceptible to Ni exposure. The varying response in DNA damage between RIAILs suggests there are multiple genes involved with Ni exposure. Future studies involving the outliers can lead to a better understanding of the mechanism(s) by which Ni causes DNA damage.
2021-01-05
2022-12-01
2020-12
2020-11-24
December 2
2020-12-18
Master's Thesis
http://hdl.handle.net/10342/8838
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/50472021-03-03T20:58:20Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Investigation Into The Scope, Limitations, And Mechanistic Aspects Of A Regioselective Acylation Of Cycloplatinated Complexes
Carroll, Jeffrey Samuel
Huo, Shouquan
Chemistry
Chemistry
Organic chemistry
Inorganic chemistry
Acylation
Cyclometalation
Functionalization
Platinum complexes
In the reaction of N-isopropyl-N-phenyl-2,2'-bipyridin-6-amine with potassium tetrachloroplatinate, the selective C-H bond activation was complicated by the low selectivity of sp² C-H bond activation in acetonitrile and low yield of sp³ C-H activation in acetic acid. The product resulting from the highly selective sp³ activation was collected in low yield due to competing side reactions. Following C[superscript]iPr-N bond dissociation, the platinum complex (2a, X = NH, R = H) was regioselectively acylated. It was later discovered that 2a could be regioselectively acylated by reacting with acetic anhydride in acetic acid. Furthermore, the acylated platinum complex (3a, X = NH, R = H) was also prepared in a cascade intramolecular cycloplatination-acylation reaction by reacting the organic ligand (1a, X = NH, R = H) with potassium tetrachloroplatinate in a mixture of acetic acid and acetic anhydride. A series of ligands with structural modifications have been designed, synthesized, cycloplatinated, and acylated to investigate the scope of this reaction, and to shed light on the mechanism of the acylation. The reaction showed great tolerance to various linker groups (X), as well as many electron donating/withdrawing groups (R) on the phenyl ring. Reaction conditions were optimized and a variety of solvents were useful in this reaction, including acetonitrile, benzonitrile, and 1,2-dichloroethane. Alternate electrophiles such as benzoyl chloride were also useful in the acylation reaction. Experimental results of the acylation reaction of a library of substrates (2) will be reported, and the mechanistic implications of these results will be discussed.
2015-08-24
2017-08-24
2015
Master's Thesis
http://hdl.handle.net/10342/5047
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/72552022-12-09T19:36:41Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Benzoate: The Quantitation of Soft Drinks Implicated in Obesity
Marechek, Jennifer
Danell, Allison Stokes
Collier, David N.
Chemistry
Health
Clinical Research
Obesity
HPLC
Chemistry
Research has shown that childhood obesity is a leading cause of diseases such as coronary artery disease, hypertension, type 2 diabetes, cancer, osteoarthritis, and many more. With approximately 17 percent of the United States (U.S.) pediatric population being obese, and approximately 35 percent overweight, it is important to assess the possible causes of obesity. Current research studies have proposed that an increased exposure to xenobiotic chemicals can lead to an increased risk of obesity amongst children. Benzoic acid is a common xenobiotic chemical that is of particular concern. Benzoic acid is a preservative that is added to naturally acidic fruit juices and soft drinks in the form of sodium benzoate, the sodium salt of benzoic acid. The concentration of benzoate salt in these beverages is being quantitated in order to determine daily exposure levels experienced by obese patients. A high-performance liquid chromatography (HPLC) method has been developed and validated for the quantitative analysis of benzoate salt found in various beverages. Using standard solutions, a correlation coefficient of 0.9999 was obtained over the measured calibration range of 0.0125 to 0.163 mg/mL. Samples classified as frequently consumed by overweight or obese children were obtained from local grocery stores, and were prepared by diluting each beverage sample in water proportionally to the concentration of benzoate salt present. The HPLC determination utilizes a phenyl hexyl column followed by UV detection at the absorption wavelength of 225 nm to confirm the presence of benzoic acid. All samples were filtered to remove particulate matter prior to the HPLC analysis. The method has been optimized to allow for the elution of sodium benzoate in less than 4 minutes. All validation requirements have been met, including an optimal flow rate and resolution, to ensure each peak was sufficiently separated, with minimal bandwidth, for accurate quantitation.
2019-06-12
2021-05-01
2019-05
2019-04-26
May 2019
2019-06-11
Master's Thesis
http://hdl.handle.net/10342/7255
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/50092021-03-03T20:58:39Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Thermodynamic and spectroscopic studies of Cd²� binding to the regulatory domain and full length human cardiac troponin C (HcTnC) : elucidating plausible Cd²� binding sites
Fulcher, Lindsay Michelle
Spuches, Anne M
Chemistry
Biochemistry
Inorganic chemistry
Bioinorganic chemistry
Circular dichroism
EF hands
HcTnC
Isothermal titration calorimetry
Toxic metals
Toxic metals such as cadmium (Cd²�) have been shown to bind to and interfere with various calcium (Ca²�) binding proteins including the regulatory protein cardiac troponin C (cTnC). Recent structural data has shown that Cd²� binds to both EF hand Ca²� binding loops in the regulatory N-domain of human cTnC including EF hand loop I, which normally does not bind metal. Although the data reveal two Cd²� ions bound to the protein, the binding constants and other thermodynamic parameters are not known. Therefore, the goal of this research project is to use Isothermal Titration Calorimetry (ITC) to obtain thermodynamic parameters such as K, [delta]H, [delta]G and [delta]S of Cd²� binding to both the full length and N-domain (amino acid residues 1-89) of HcTnC and compare the parameters to our previous data with Ca²�. Through our results we hope to shed light on potential mechanisms of cadmium toxicity.
2015-08-24
2017-08-24
2015
Master's Thesis
http://hdl.handle.net/10342/5009
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/86232021-03-03T22:08:36Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
GRADUATE TEACHING ASSISTANT FIDELITY OF IMPLEMENTATION IN INTRODUCTORY CHEMISTRY AND PHYSICS LABORATORIES: IMPACT ON SCIENCE PRACTICE PROFICIENCY
Smith-Joyner, Annalisa
Walker, Joi P
This study reports the fidelity of implementation of the Argument-Driven Inquiry (ADI) instructional model by graduate teaching assistants (GTAs) in introductory chemistry and introductory physics laboratories at East Carolina University (ECU). The ADI instructional model had been fully implemented in the General Chemistry I and II laboratories for several semesters, whereas General Physics I and II laboratories were observed during the first semester of course-wide implementation. An ADI-specific observation protocol was developed and used to document the facilitation techniques of two GTAs in each course for three investigations during Fall 2018 and Spring 2019. The ADI-specific observation protocol was used to determine if student-centered facilitation techniques, by guiding students through the process, or instructor-centered facilitation techniques (lecturing at the students and providing direct answers to questions) were used by the GTA. The ADI-specific observation protocol revealed that one of the GTAs in General Physics I provided primarily student-centered facilitation techniques while the other GTA provided primarily instructor-centered facilitation techniques during some of the stages of the ADI instructional model throughout the semester. The students' results on a practice-focused end-of-course laboratory practical exam were used to determine proficiency with science practices. There was not a significant difference in the mean scores on the end-of-course practice-focused laboratory practical exam in the General Chemistry I and II and General Physics II sections. These results indicate that for these three courses the facilitation techniques of the GTAs had minimal impact on the students' development of science practices. There was a significant difference in the mean scores by the students in General Physics I laboratories, which suggests that differences in facilitation techniques for this section impacted student proficiency with science practices.
2020-06-30
2020-06-30
5/1/2020
2020-06-22
Master's Thesis
http://hdl.handle.net/10342/8623
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/47972021-03-03T20:56:49Zcom_10342_55com_10342_1col_10342_58col_10342_72
Conservation of Archaeological Wood using Non-Reducing sugars
Tahira, Adeem
Kennedy, Anthony
Chemistry
Conservation
Archaeological wood
Sugars
In the conservation field there are many different thoughts on the best way to conserve waterlogged archaeological wood. The current method uses polyethylene glycol(PEG), a hydrophilic organic compound, to replace water within the wood. PEG, which is highly effective in the short term, is hygroscopic at low molecular weights and can reabsorb water leading to problems later on, such as the formation of acid within the wood and break down of PEG itself. These problems have proved especially difficult to deal with in the Vasa, a Swedish Viking ship, which is conserved with PEG. As a result conservators have been studying alternative treatments to PEG. Sugars such as sucrose have proven to be effective in the short term but can still reabsorb water, in addition to leaving crystalline deposits on the woods surface. Sucralose and trehalose, both analogs of sucrose, are non-reducing and less likely to reabsorb moisture. Our study evaluates the effective of these non-reducing sugars as a conservation practice. Tongue depressors serve as simple models for archaeological waterlogged wood. Samples are chemically degraded and then treated with varying concentrations of sugar solutions. Data collected from dimensional analysis, before and after drying, indicates that this method is effective in conserving degraded samples. Additional mechanical analysis shows that chemically degraded wood has increased elasticity and much lower mechanical strength, as is expected. Chemically degraded wood samples treated with sugars show improved mechanical properties, measured using a three point mechanical testing method. These results will demonstrate that non-reducing sugars are an effective consolidant for samples such as these and may prove to be more effective than PEG for improving the long-term stability of wooden artifacts.
2015-05-05
2016-05-11
2015
Honors Project
Tahira, Adeem. (2015). Conservation of Archaeological Wood using Non-Reducing Sugars.Unpublished manuscript, Honors College, East Carolina University, Greenville, N.C.
http://hdl.handle.net/10342/4797
oai:TheScholarship.intra.ecu.edu:10342/35902021-03-03T20:53:05Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
STABILITY OF POLYCYCLIC AROMATIC HYDROCARBONS AND BENZOIC ACID DERIVATIVES UNDER SUBCRITICAL WATER CONDITIONS
Lindquist, Edward J.
Yang, Yu
Chemistry
Chemistry, Analytical
Benzoic acid and derivatives
Degradation
Stability
Subcritical water
Analytical chemistry
The development of green environmental remediation, chromatography, and extraction techniques using subcritical water is the focus of our research group. The polarity of subcritical water can be manipulated by increasing its temperature in the range of 25 to 374 ºC while keeping it in the liquid state under moderate pressure. At elevated temperatures liquid water becomes less polar and behaves more like an organic solvent. The goal of this research is to determine the conditions under which certain polycyclic aromatic hydrocarbons (PAHs) and benzoic acid derivatives degrade in subcritical water. The stability of two PAHs (pyrene and naphthalene) and benzoic acid and three of its derivatives (anthranilic acid, syringic acid, and salicylic acid) under subcritical water conditions was investigated and the results are discussed in this thesis.  PAHs are pollutants widely formed as a result of incomplete combustion of organic materials. The effects of temperatures ranging from 200 to 350 ºC and heating times of 30 and 300 min on the degradation of pyrene and naphthalene in solutions of water and 3% hydrogen peroxide were determined. Our results show that PAHs can be degraded under subcritical water conditions, and thus, this technique may be applied to the environmental remediation of these pollutants.  Benzoic acid and its derivatives are found in medicinal herbs and other plants. While the extraction of these active ingredients from herbs using subcritical water is non-toxic and preferred, the decomposition of these compounds under subcritical water conditions has to be examined. The stability studies of this group of analytes were carried out at temperatures ranging from 50 to 250 ºC with heating times of 10 and 30 min. The degradation of the benzoic acid derivatives increased with rising temperature and additional heating time. The degradation products of benzoic acid and the three derivatives were identified and quantified by high performance liquid chromatography (HPLC) and confirmed by gas chromatography/mass spectrometry (GC/MS). Under subcritical water conditions anthranilic acid, syringic acid, salicylic acid, and benzoic acid underwent decarboxylation to form aniline, syringol, phenol, and benzene, respectively. Â
2011-06-24
2011-06-24
2011
Master's Thesis
http://hdl.handle.net/10342/3590
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/29162021-03-03T20:52:58Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Prothymosin-Alpha : Features of the protein sequence that contribute to the anti-HIV Activity
Garapati, Sri Ramya
Burns, Colin Sanderson
Chemistry
Chemistry, Biochemistry
Biochemistry
Prothymosin-alpha (ProT[alpha]) is a protein mainly located in the nucleus of the cells. Though its exact function is not known, it is believed to be involved in cell proliferation. The ability of this protein to inhibit HIV replication in macrophages cells was discovered recently. Apart from the full length protein, a peptide derived from central region of it (from residues 50-89) is shown to inhibit HIV in a dose dependent manner.  The central region of the protein (residues 50-89) has a combined total of 26 glutamic acid (Glu) and aspartic acid (Asp) residues which imparts high negative charge and acidity to the peptide. ProT[alpha] 50-89 peptide was synthesized to reconfirm its anti-HIV activity. Along with it three other peptide sequences (Polyglutamic acid, ProT[alpha](50-105) and AlexFluor488-ProT[alpha](51-89)) were synthesized in order to understand the features that give the protein its anti-HIV activity. They were synthesized to understand if high negative charge and entry of the peptide into the nucleus are required for protein's anti-HIV activity. All the sequences were synthesized using Solid Phase Peptide Synthesis coupled with a 9-Fluorenylmethoxycarbonyl (Fmoc) protecting strategy. Synthesis of ProT[alpha](50-89) was carried out on Microwave synthesizer whereas ProT[alpha](50-105) and the Polyglutamic acid sequence were synthesized using an automated synthesizer. During the synthesis of ProT[alpha](50-105) with a new protecting group strategy, we encountered unexpected formation of a glutarimide between residues 68 and 69 which was further resolved. Purification of these sequences was performed using Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) or Strong Anion Exchange Chromatography (SAX) and characterized with Electrospray Ionization-Mass Spectrometry (ESI-MS). Samples obtained from SAX contained salt and were desalted by dialysis for 6 hours.   Activity testing was done on two of the sequences. Results from these experiments reconfirmed the anti-HIV activity of ProT[alpha](50-89)N50W and also suggest that there is some sequence specificity to the peptide's ability to inhibit HIV replication in the post-integration step. Results demonstrating the importance of the NLS will also be discussed.
2010-09-16
2011-05-17
2010-09-16
2011-05-17
2010
Master's Thesis
http://hdl.handle.net/10342/2916
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/107452022-06-30T07:15:53Zcom_10342_55com_10342_1col_10342_58
Functional Expression of the Human Glucose Transporters GLUT2 and GLUT3 in Yeast Offers Novel Screening Systems for GLUT-Targeting Drugs
Schmidl, Sina
Tamayo Rojas, Sebastian A.
Iancu, Cristina V.
Choe, Jun-Yong
Oreb, Mislav
Glucose transport inhibitor
drug screening system
GLUT2
2022-06-29
2022-06-29
2021-02
Article
2296-889X
http://hdl.handle.net/10342/10745
10.3389/fmolb.2020.598419
en
oai:TheScholarship.intra.ecu.edu:10342/30072021-03-03T20:53:03Zcom_10342_55com_10342_1col_10342_58
DISNET: a Distributed Instrument System NETwork
Gemperline, Paul J.
Megargle, Robert
Dartt, Arthur
Slivon, Larry
Zadnik, Victor
Computer networks
Laboratory instruments
Chemistry laboratory
2010-11-17
2011-05-17
2010-11-17
2011-05-17
1984
Article
Journal of Automatic Chemistry; 6:1 p. 27-32
http://hdl.handle.net/10342/3007
PMC2547553
10.1155/S1463924684000067
en_US
http://www.ncbi.nlm.nih.gov/pmc/issues/172102/
Permission granted by author to Kent Nixon Myers to upload this article on 11/04/2010.
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/37372021-03-03T20:53:14Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
NMR Studies into the Potential Interactions of Fullerene C[subscript]60, with Tetraphenylporphyrin, H[subscript]2[TPP] and Some of its Derivatives
Obondi, Christopher Otara
Rodriguez, Art A.
Chemistry
Chemistry, Physical
Correlation time
C60
Relaxation time
Tetraphenylporphyrin
Physical chemistry
The curved [pi] surface of fullerene, C[subscript]60, shows a tendency to interact with other molecules, making it an interesting target for building supramolecular arrays. The interaction can be relative strong and may even exist in solutions, often leading to stable complexes. An important class of the macrocycles that interacts with fullerenes is the porphyrins. In our study, [superscript]1H NMR spectrometric studies have been done to gain information on the nature and the precise interaction site of fullerene 1, with tetraphenylporphyrin, H[subscript]2[TPP] 2, and para-substituted tetraphenylporphyrins, H[subscript]2[(p-X)[subscript]4TPP], where X= CN and OCH[subscript]3 in deuterated chlorobenzene-d[subscript]5(CBZ) and toluene-d[subscript]8. The porphyrin derivatives have been used to investigate any correlation in the interaction site due to changes in substituent. Relaxation rates of the pyrrole and phenyl hydrogen in the porphyrins were determined at temperatures of 288, 298 and 313 K for all the complexes in the presence and in the absence of fullerene, C[subscript]60. To study solvent effects on interactions, Toluene-d[subscript]8 and chlorobenzene-d[subscript]5 solvents were used. Overall, our data indicated that C[subscript]60 interacted with H[subscript]2[TPP] at both the pyrrole and phenyl hydrogen sites however, these intermolecular interaction are not long-lasting in chlorobenzene-d[subscript]5.   The porphyrin derivatives showed that the substituents have an effect on which site is preferred for interaction. For the case of H[subscript]2[(p-OCH[subscript]3)[subscript]4TPP], that has an electron -donating group, both phenyl and pyrrole hydrogen sites interacted with C[subscript]60 , but preferably at the pyrrole hydrogen at all the three temperatures(283, 298, and 313 K). In the case of H[subscript]2[(p-CN)[subscript]4TPP] with an electron-withdrawing group CN, our data indicates that at lower temperature there is a noticeable, but slight preference for the interaction of C[subscript]60 at pyrrole site but is quickly destroyed with an increase in temperature. Â
2012-01-18
2012-01-18
2011
Master's Thesis
http://hdl.handle.net/10342/3737
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/39092021-03-03T20:57:41Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Thermodynamic studies of Ca²� binding to human cardiac troponin C
Skowronsky, Rachel Ann
Spuches, Anne M.
Chemistry
Chemistry
Chemistry, Biochemistry
Chemistry, Inorganic
Calcium binding
Calorimetry
EF-hand proteins
Human cardiac troponin C
ITC
Thermodynamics
Inorganic chemistry
Biochemistry
Human Cardiac Troponin C (HcTnC) is an EF hand protein responsible for initiating contraction of the myocardium (heart muscle). The helix-loop-helix motif, characteristic of members of the EF-hand family, allows HcTnC to act as a Ca²� sensor and relays the calcium signal through the thin filament. The binding of Ca²� to the regulatory domain induces a change in HcTnC conformation which modifies subsequent protein-protein interactions. Mutations that alter the calcium sensitivity of HcTnC can lead to cardiomyopathies. One treatment for cardiomyopathies is the use of calcium sensitizing/desensitizing drugs which bind to HcTnC. A thorough understanding of the thermodynamic forces that drive calcium binding to HcTnC and allow it to act as a calcium sensor is crucial for future drug design. Isothermal titration calorimetry (ITC) is a quantitative technique which directly measures the heat of a binding reaction. Calorimetric measurements can be used to obtain the following thermodynamic parameters: binding constant (K), enthalpy (Delta H), stoichiometric ratio (n), enthalpy (Delta S), and Gibbs energy (Delta G) of a wide variety of biochemical reactions including Ca²� binding to proteins. Herein we report the first calorimetric study of Ca²� binding to human cardiac troponin C. Calcium binding isotherms obtained at 25 °°C and at pH 7.0 (10 mM 2-(N-morpholino)ethansulfonic acid (MES), 50 mM KCl) allowed us to obtain thermodynamic parameters for Ca²� ions binding to both the high and low affinity domains. Binding constants are consistent with those reported previously in the literature. Furthermore, binding to the low affinity N-domain was found to be endothermic and entropically driven, a result that is consistent with calcium binding to the regulatory domains of wheat germ calmodulin and the third site of skeletal troponin. Thermograms obtained at two additional temperatures, 10 °C and 37 °C, conducted under identical buffer conditions, allowed the change in heat capacity (Delta C[subscript]p) to be calculated from the slope of Delta H plotted against temperature. The enthalpies of binding for the two events exhibit a trend; as temperature increases the enthalpy becomes more favorable. ITC was also used to determine the thermodynamic parameters associated with Ca²� binding to an isolated N-domain of HcTnC (HcTnC�₋₈₉). Comparison of the N-domain of HcTnC and the isolated N-domain reveal a favorable free energy of calcium binding to the N-domain when isolated or attached to the C-domain. However, differences in the entropic and enthalpic contributions to the free energy of binding provide supporting evidence for the cooperativity of the N and C-domains.
2012-05-20
2013-07-01
2012
Master's Thesis
http://hdl.handle.net/10342/3909
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/19582021-03-03T20:52:59Zcom_10342_55com_10342_1col_10342_58
The implementation of a GC/MS data system using DISNET: a Distributed Instrument System NETwork
Gemperline, Paul J.
Megargle, Robert
GC/MS
DISNET
Distributed instrument system NETwork
Resource sharing
The primary goal of a computer network is to achieve resource sharing between several computers. Resource sharing is most easily performed at the application program level, with lower layers of software providing communications services. Application programs which make local data, peripherals, or processes available for shared use are defined as ’resource providers’. Application programs which require such resources from the
network are defined as ’resource consumers’. This distinction helps the system designer identify the qualities of resource providers and consumers necessary to address data communication problems in a straightforward manner, while ensuring the effort invested will provide satisfactory solutions. We have developed this resource sharing protocol for the Distributed Instrument System NETwork (DISNET) to provide an environment suitable for the development and growth of a distributed system for real-time instrument control, data acquisition, and computation in a laboratory environment.
2009-11-11
2011-05-17
2009-11-11
2011-05-17
1984
Article
The Journal of Automatic Chemistry; 6:4 p. 197-201
http://hdl.handle.net/10342/1958
PMC2547563
10.1155/S1463924684000389
en_US
Permission granted by author to Kent Nixon Myers to upload this article on 11/04/2010.
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/106922023-07-31T15:38:48Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Investigation of Molecular Mechanism of Transthyretin Oligomerization Associated with ATTR Amyloidosis
Coats, Matthew Fontaine
Lim, Kwang Hun
Chemistry
Transthyretin
Type A
SSA
FAC
FAP
Transthyretin amyloidosis (ATTR) has classically been diagnosed predominantly post-observation of amyloid fibril deposition on soft tissues within the body, however, recent studies have shown that oligomers which arise during the aggregation process are much more cytotoxic than their amyloid fibril counterparts. 2,6,7 Additionally, it has also been found that cleaved C-terminal fragments, within the peptide range of T49-E127, circulate in-vivo with these oligomers and amyloids, providing further insight into the vast complexity of amyloidogenic species circulating within patients. 5,11 Previous mechanistic studies of transthyretin misfolding and aggregation have shed light on the capability of the native protein to first dissociate into its constituent monomers, which are then capable of self-associating into dimeric species that subsequently form hexamers and eventually large cytotoxic oligomers. 2,6,9 Whilst searching for the specific amino acid sequences involved in amyloidosis within ATTR, it was found that mutations E92P, in the A91-T96 peptide chain, or T119W, in the T119-N124 peptide chain, were both able to completely inhibit aggregation. 23 This study seeks to examine the specific molecular mechanism behind transthyretin dimerization in misfolding pathways by introducing the E92P and T119W mutations directly into the H-H' and F-F' [beta]-strand of the native dimeric interface of TTR. Structural characterization profiles of non-native TTR species which arise during the aggregation process of wild-type TTR (WT-TTR), as well as those of the highly amyloidogenic mutations V30M and L55P were propagated under acidic and kinetically favorable conditions to induce aggregation while maintaining an observable timeline. The WT and mutant forms of TTR underwent similar aggregation processes, involving large concentrations of tetramers first dissociating into monomers, these misfolded monomers then form misfolded dimers, and at the peak of dimer formation high molecular weight oligomers begin to form. These observations indicate the importance of the misfolded monomer's ability to form into misfolded dimers with regards to the eventual formation of oligomeric species. In order to examine if the native dimeric interface sites, along the H-H' and F-F' [beta]-strand bonds, are integral parts of WT-TTR and L55P-TTR's ability to aggregate into misfolded dimeric species, proline substitutions were introduced into the E92 peptide within their F-strands as well as tryptophan substitutions into the T119 peptide within their H-strands. These two bulky peptide substitutions showed clear inhibition of non-native oligomeric species under acidic conditions as well as in samples which had been proteolytically cleaved at the L48-T49 peptide bond using trypsin digestion. As these mutations may be having a stabilizing effect on the native tetramer during the dissociation phase, rather than the intended inhibitory effect during the aggregation phase, leading to the observed decrease in oligomerization, a monomeric variant of TTR (m-TTR) was also studied in similar fashion to the WT-TTR and L55P-TTR trials. Similar to the previous wild-type and L55P studies, the inhibited m-TTR samples proved successful in eliminating dimerization. These results indicate that oligomerization of TTR is dependent upon the ability of its dissociated misfolded monomers to form into misfolded dimers, and by introducing large sidechains into locations within the dimeric interface, it is possible to eliminate the ability of these misfolded monomers to aggregate into larger amyloidogenic species.
2022-06-14
2022-11-01
2022-05
2022-04-27
May 2022
2022-06-07
Master's Thesis
http://hdl.handle.net/10342/10692
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/91422023-05-01T08:01:59Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
A Carbohydrate/Lipid Research Project for Organic and Analytical CURE Laboratories: Reactions and Recordkeeping
Hagwood, Abigail M
Allen, William E.
Walker, Joi P
Chemistry
glucose
MC540
Course-based Undergraduate Research (CURE)
chemical education
scientific notebooks
Course-based Undergraduate Research Experiences (or CUREs) are able to teach students laboratory skills while engaging them in novel research. The first part of this thesis aims to demonstrate the development of a feasible research project for implementation in organic chemistry II and quantitative analysis CURE courses. The goal of the research project was to develop effective, glucose-based cryoprotectants. While the synthesis and analysis of three tethered glucose molecules – glu4glu, glu8glu, and glu12glu – are shown, they were not expected to yield better cryoprotectants, but to form the basis of a research project for CURE students. The second part of this thesis aims to analyze the language and organization of CURE laboratory notebooks in comparison to traditional and undergraduate research notebooks to gain insight on the effectiveness of a CURE on engaging students in a research setting. The organization and language usage of CURE notebooks were found to align more closely with notebooks of experienced researchers than with notebooks of traditional (non-research based) teaching laboratories, suggesting that students learn the language of the scientific community more effectively through literature and discussion rather than through a laboratory manual.
2021-06-18
2023-05-01
2021-05
2021-05-06
May 2021
2021-06-02
Master's Thesis
http://hdl.handle.net/10342/9142
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/44632021-03-03T20:56:43Zcom_10342_55com_10342_1col_10342_58col_10342_72
The Duff Reaction: Researching A Modification
Lindler, Carson B.
Love, Brian Ernest
Chemistry
Chemistry
Chemistry, Organic
Duff reaction
Organic chemistry
Formylation reactions are used to synthesize aldehydes. The Duff reaction is a formylation reaction that uses phenol derivatives, hexamethylenetetramine, acetic acid, and acid to synthesis ortho substituted aromatic aldehydes. The Duff reaction is known for its low percent yield. In a previous project it was found that dimethyldichlorosilane increased the yield of 2-hydroxy-1-naphthaldehyde. This project verified that, but also determined that trimethylsilyl chloride achieved better yields. The modified Duff reaction was studied on 2-naphthol, 4-tert-butylphenol, 8-hydroxyquinoline, and butylated hydroxyanisole. 2-hydroxy-1-naphthaldehyde was synthesized relatively purely with an increased yield over the traditional Duff reaction. Attempted synthesis of 5-tert-butylsalicylaldehyde resulted in slightly higher percent yields over the traditional Duff reaction, but 1H NMR indicated that the yields for both, modified and traditional Duff reactions, were a mixture of 5-tert-butylsalicylaldehyde and starting material. Synthesis of 8-hydroxy-7-quinolinecarboxaldehyde was attempted. The yields were higher for the traditional Duff reaction than the modified reaction. 1H NMR indicated that the modified and traditional Duff reaction produced products that were so impure it was not possible to determine the desired product from the impurities. Synthesis of 3-(1,1-dimethylethyl)-2-hydroxy-5-methoxy-benzaldehyde resulted in relatively high yields for both the modified and traditional Duff reactions with the modified having a slightly higher yield. 1H NMR showed that the product was not entirely pure. The impure product and p-anisidine were used to synthesize 2-(1,1-dimethylethyl)-4-methoxy-6-[[(4-methoxyphenyl)imino]methyl]-phenol. The yields of the imine were higher for the reactions that originated from the modified reaction. 1H NMR showed that the imine formed was pure. Trimethylsilyl chloride was able to increase the yield for some of the compounds. The reason for the modest and variable yield improvement is unknown.
2014-08-06
2015-08-06
2014
Undergraduate Thesis
http://hdl.handle.net/10342/4463
oai:TheScholarship.intra.ecu.edu:10342/113072022-09-24T07:16:09Zcom_10342_55com_10342_1col_10342_58
Tau Induces Formation of α-synuclein Filaments with Distinct Molecular Conformations
Dasari, Anvesh K. R.
Lim, Kwang Hun
Hojjatian, Alimohammad
Sengupta, Urmi
Taylor, Dianne
Daneshparvar, Nadia
Yeganeh, Fatemeh Abbasi
Dillard, Lucas
Michael, Brian
Griffin, Robert G.
Borgnia, Mario
Kayed, Rakez
Taylor, Kenneth A.
α-Synuclein
cryo-EM
tau
2022-09-23
2022-09-23
2021
Article
0006-291X
http://hdl.handle.net/10342/11307
10.1016/j.bbrc.2021.03.091
en_US
oai:TheScholarship.intra.ecu.edu:10342/60002021-03-03T21:09:09Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Investigation of possible driving forces on counter-intuitive thermal collapse of intrinsically disordered proteins
Cormier, Zachary
Burns, Colin Sanderson
Chemistry
Intrinsically Disordered Proteins
Counter-Intuitive Thermal Collapse
Intrinsically disordered proteins are an emerging class of proteins that contain no secondary or tertiary structure. Through bioinformatics studies it has been predicted that a third of the human proteome consists of proteins containing intrinsic disorder. It was originally thought that a well-defined three dimensional structure was a prerequisite for function. However, this new class of proteins has been found to perform a variety of functions which has helped to establish new ways of classifying protein function. Intrinsically disordered proteins can undergo disorder-to-order transitions under different environmental factors. Of particular interest is a "turned out" response to increasing temperature. These proteins undergo a thermal collapse which is counter-intuitive to the traditional notion of protein folding and denaturation which states that under increasing temperature a protein should go into a more extended conformation. The reason for this counter-intuitive thermal collapse is still unknown. The goal of our research was to find the main driving force behind thermal collapse of intrinsically disordered proteins. Utilizing circular dichroism, the structure and transitions of four polypeptides (prothymosin-alpha (50-89)N50W, scrambled prothymosin-alpha (50-89), scrambled prothymosin-alpha (50-89) in 1M KF, and a polyE peptide) where analyzed under increasing temperatures of 25 [degrees]C to 80 [degrees]C to observe the possible effects of net charge, hydrophobicity, and sequence specificity on the thermal collapse of intrinsically disordered proteins. All peptides collapsed to similar degrees under increasing temperatures. The ionic strength condition appeared to be more collapsed than the non-salt condition indicating net charge as a major player. The hydrophobic effect also appears to play a major role in counter-intuitive collapse which may be attributed to the change in hydrophobicity of polar side chains.
2017-01-11
2018-01-23
2016-12
2016-08-18
December 2
2017-01-11
Master's Thesis
http://hdl.handle.net/10342/6000
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/67752021-03-03T21:17:51Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Detection of Environmental DNA Damage Using Electrochemical Sensors and Mass Spectrometry
Schaaf, Nicholas Joseph
Hvastkovs, Eli G.
Chemistry
Electrochemistry
Mass Spectrometry
Nickel exposure has been linked to a variety of health complications including instances of tumors and cancer in living organisms. Exposure to nickel can occur due to pollution from mining or industrial applications. Ingested nickel has been shown to be toxic and a promoter of DNA damage through direct and indirect interactions with DNA systems. Since the outcomes of nickel toxicity are well documented, it is advantageous to develop ways to assay the amount of DNA damage that has occurred after exposure and the mechanisms detailing how nickel interacts with DNA systems. Nematodes, primarily C. elegans and P. pacificus, have been used as model organisms to study the biological effects of environmental pollutants like nickel. Since the replication, transfer, and repair of DNA is a highly sophisticated and constantly evolving process, there is good reason to suspect that environment could play a key role in an organisms response to nickel. DNA from nematodes obtained in both high nickel and typical cosmopolitan environments was compared to discern any change in the amount of damage incurred from identical nickel concentrations provided in vitro. Genomic damage was assayed via electrochemical sensors prepared by incorporating DNA in a Layer-by-Layer (LbL) assembly. A variety of strains from Hawaii, New Zealand, England, and Pasadena were assayed after each had been exposed to increasing concentrations of nickel. Hawaii and New Zealand are environments with naturally high amounts of nickel due to volcanic activity. Our studies show a strong correlation between nickel exposure and DNA damage that is not only concentration dependent but influenced by the environmental conditions from which the nematodes originated. Mass spectrometry was also employed to highlight the types and increases in DNA nucleobase biomarkers that are used to characterize certain types of DNA damaging reactions. This provides specific information towards the way nickel alters DNA and how biological adaptation plays a role in genomic protection from exogenic toxins.
2018-05-25
2020-05-01
2018-05
2018-05-04
May 2018
2018-05-23
Master's Thesis
http://hdl.handle.net/10342/6775
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/113292022-09-27T07:16:37Zcom_10342_55com_10342_1col_10342_58
Collective ERK/Akt Activity Waves Orchestrate Epithelial Homeostasis by Driving Apoptosis-Induced Survival
Hughes, Robert M.
Gagliardi, Paolo Armando
Dobrzyński, Maciej
Jacques, Marc-Antoine
Dessauges, Coralie
Ender, Pascal
Blum, Yannick
Cohen, Andrew R.
Pertz, Olivier
epithelial homeostasis (EH)
apoptosis
ERK/Akt signaling dynamics
2022-09-26
2022-09-26
2021-06-21
Article
1534-5807
http://hdl.handle.net/10342/11329
10.1016/j.devcel.2021.05.007
en_US
oai:TheScholarship.intra.ecu.edu:10342/87812021-06-14T19:19:49Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_123
Modern and Classic Quantum Chemistry: Modeling Rhodium-Catalyzed Hydroacylation with Advanced Tools and the Development and Application of Software to Examine Simple Bonding Interactions in a Basis of Molecular Fragments
Shoopman, Thomas E
Sargent, Andrew L
In this dissertation, both modern and classic computational methods are utilized to examine two problems of interest in the field of chemistry. The first problem involves a complex rhodium-catalyzed hydroacylation reaction forms two ketone products. Interestingly and unexpectedly, the major product is the larger, more highly that structured ketone that results from dimerization. A detailed analysis of the reaction mechanism reveals that subtle, non-bonded dispersive interactions play a critical role in the reaction, and that a sophisticated application of the theory is required to reproduce the experimental findings. Specifically, the nudged elastic band and improved dimer methods are applied within the framework of density functional theory and a functional is employed that captures dispersive interactions to map out the reaction pathways. The second problem involves the classical analysis of simple bonding interactions within a test set of molecules. Computer software is developed to transform output from routine ab initio calculations in the atomic orbital basis into a basis of chemically meaningful fragment molecular orbitals. Molecular orbital diagrams are constructed for three prototypical systems: a simple inorganic complex to demonstrate bonding interactions between Lewis acids and bases, an alkene-coordinated squareplanar organometallic complex to highlight the driving force behind the preferred perpendicular orientation of the ligand, and a model of hemoglobin combining with small diatomic molecules to compare the binding characteristics of each.
2020-10-23
2020-10-23
8/5/2020
8/5/2020
Doctoral Dissertation
http://hdl.handle.net/10342/8781
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/113092022-09-24T07:16:10Zcom_10342_55com_10342_1col_10342_58
Kinetic and Structural Investigations of Novel Inhibitors of Human Epithelial 15-Lipoxygenase-2
Ohler, Amanda
Offenbacher, Adam R.
Tsai, Wan-Chen
Gilbert, Nathaniel C.
Armstrong, Michelle
Perry, Steven
Kalyanaraman, Chakrapani
Yasgar, Adam
Rai, Ganesha
Simeonov, Anton
Jadhav, Ajit
Standley, Melissa
Lee, Hsiau-Wei
Crew, Phillip
Iavarone, Anthony T.
Jacobson, Matthew P.
Neau, David B.
Newcomer, Marcia
Holman, Theodore R.
Human Epithelial 15-Lipoxygenase-2
Novel Inhibitors
Kinetic and Structural Investigations
2022-09-23
2022-09-23
2021
Article
0968-0896
http://hdl.handle.net/10342/11309
10.1016/j.bmc.2021.116349
en_US
oai:TheScholarship.intra.ecu.edu:10342/45262021-03-03T21:01:50Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
SPECTROSCOPIC INVESTIGATIONS OF BINDING MECHANISMS BETWEEN ANTIMICROBIAL PEPTIDES AND MODELS OF THE MEMBRANES OF PSEUDOMONAS AERUGINOSA AND KLEBSIELLA PNEUMONIAE
Chai, Hanbo
Hicks, Rickey P.
Chemistry
Chemistry
Antimicrobial peptides
Circular dichroism spectroscopy
Gram-negative bacteria
Lipopolysaccharides
Membrane models
The wide application of antibiotics goes back over sixty years to the first use of penicillin in the mid-1940s. Antimicrobial agents have well-documented activity and have played a significant role in defending against various bacterial infections. However, antibiotic resistance has never ceased to undermine the efficacy of those compounds and has become a severe threat to patients with serious infections. It is imperative to discover and develop antibacterial agents with novel action mechanisms to lower the chance of drug resistance. A wide series of compounds called "antimicrobial peptides" (AMPs) have been either discovered in the nature or synthesized in laboratories around the world. The advent of AMPs has brought a new hope in the fight against the rise of antibiotic-resistant organisms.  Far-UV Circular Dichroism (CD) spectroscopy and 1H NMR have been used to investigate the interactions of a series of synthetic, unnatural amino acid-containing AMPs with Lipopolysaccharide (LPS) isolated from drug resistant Gram-negative bacteria Pseudomonas aeruginosa and Klebsiella pneumoniae, along with various phospholipid compositions to better approximate the chemical makeup of the membranes of these two strains. The results showed that: (1) the binding interactions between the AMPs and the membranes are defined by the physicochemical properties of the peptide and the membrane model; (2) binding of these AMPs to the lipid A region (the innermost and phospholipid-like layer) of the LPS is stronger and dominant compared with the binding with the O-polysaccharide outer leaf moiety; (3) when different compositions of phospholipids were incorporated into the LPS to make a complete membrane model of the two strains, wavelength shifts in the CD spectra of the AMPs were observed that represents conformational changes of AMPs upon binding with the membrane model. Â
2014-08-28
2016-05-11
2014
Master's Thesis
http://hdl.handle.net/10342/4526
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/29182021-03-03T20:53:12Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Thermodynamic Investigation of Copper Binding to the Amyloid-Beta (A[beta]) Peptide
Sacco, Cristina
Spuches, Anne M.
Chemistry
Chemistry, Inorganic
Chemistry, Biochemistry
Inorganic chemistry
Biochemistry
Alzheimer's disease is a fatal and neurodegenerative disease with as many as 5.3 million Americans living with the disease. One of the characteristic hallmarks of Alzheimer's is the amyloid plaques that build up around the neurons. The aggregated amyloid-[beta] (A[beta]) peptide is one of the main components of the amyloid plaques found in individuals with Alzheimer's disease. Interestingly, high concentrations of metals (copper, zinc and iron) were found inside these amyloid plaques. The A[beta] peptide can be either 40 or 42 residues in length but studies have shown the metal binding site is within the first 16 residues. Copper interactions with A[beta] have been studied extensively, but there is no consensus on the coordinating ligands and binding affinity of metal to the peptide. The residues suspected in binding are the N-terminus (N-terminal amine or aspartate group), and the histidines in positions 6, 13, and 14.   The purpose of this research is to study the thermodynamics of copper binding to the A[beta] peptide using isothermal titration calorimetry (ITC). Our goal is to understand the thermodynamics of copper binding to A[beta] and give insight into the residues suspected in binding. We have studied copper binding to the shorter A[beta]16 and A[beta]28 along with Ac-A[beta]16 and Ac-A[beta]28 to determine the thermodynamic contribution from the N-terminus. We have observed a thermodynamic difference between the acetylated and nonacetylated forms of the peptide. We have also studied the thermodynamic contributions of the three histidine residues by studying copper binding to A[beta]28(H6A), A[beta]28(H13A), and A[beta]28(H14A). These studies provide information about the metal binding site of A[beta]. Understanding copper coordination and affinity to A[beta] may give insight regarding metal chelators that can be used for drug therapy. Â
2010-09-16
2011-05-17
2012-07-31
2010
Master's Thesis
http://hdl.handle.net/10342/2918
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/68752022-12-12T17:50:23Zcom_10342_55com_10342_1col_10342_58col_10342_72
Using LC/MS to Quantify Metabolites in Urine Samples Post Clinical Exposure to Benzoates in Beverages
Worthington, Cameron
Danell, Allison Stokes
Kew, Kimberly
Chemistry
Sodium benzoate, obesity, and mass spectrometry
Evidence has suggested that benzoate consumption has the potential to cause weight loss prevention. Benzoate is conjugated with glycine in the mitochondrial matrix to form hippurate which is eliminated in urine. IRB approval has been gained for clinical studies targeting a total of 20 overweight or obese young adults (18-30, BMI >25) from which urine samples were obtained before and after varying exposure to benzoate. Preliminary metabolite injections, instrument optimization, and calibration curve creation has been completed for benzoic acid and hippuric acid for LC/MS optimization. Primary injections resulted in identification of precursor and product ions of both metabolites. This also allowed for identification of ion transitions that will be used for quantification and qualification of the metabolites. The instrument was tuned to each ion, and mass spectrometer parameters were set and optimized. A standard calibration curve was created using developed LC methods for each metabolite.
2018-07-12
2018-07-12
2018-05
2018-05-01
May 2018
2018-07-03
Honors Thesis
http://hdl.handle.net/10342/6875
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/75952021-05-01T08:02:03Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Theoretical Probe to the Mechanism of Pt-catalyzed C-H Acylation Reaction: Possible Pathways for the Key Acylation Reaction of Platinacycles
Warden, Elizabeth Fae
Li, Yumin
Chemistry
Computational Chemistry
NEB
Theoretical Study
Platinum Catalyst
Nudged Elastic Band Method
Dimer
Transition State localization
QST2
Gaussian
C-H Activation
C-H Funtionalization
Transition metal-catalyzed C-H bond functionalization offers a variety of desirable transformations of simple hydrocarbons to more complex compounds with applications being applied in pharmaceuticals to material sciences. In 2017, Dr. Huo's research group reported a unique Pt-catalyzed acylation of 2-aryloxypyridines through direct C-H activation. The significance of this reported reaction is represented in the experiment occurring without any oxidants or additives; furthermore, presenting this relatively straight forward reaction mechanism which is initiated only in the presents of platinum. However, through computational analysis the key acylation step of the cyclometalated platinum complex has shown quite intriguing results. Utilization of the nudged elastic band (NEB) method followed by a min-mode optimization of the potential transition states has shown preference towards a nucleophilic substitution over the previously proposed electrophilic addition during the key acylation step. Theoretically, this DFT study at M062X/6-311g**/def2-TZVP-Pt level of theory was unable to simulate the initial formal electrophilic substitution reaction proposed as the arenium ion C could not be minimized. Therefore based on current results the preferred pathway is as follows: (1) nucleophilic substitution of the acyl chloride to form five-coordinate acylplatinum complex with a barrier of 21.7083 k cal/mol (B), followed by (2) 1,2-migration of the acyl group from the platinum to the metalated carbon to form a Pt-arene n-complex or platinacyclopropane with a barrier of 15.0713 kcal/mol (E). Lastly, (3) Re-aromatization of E leads to the acylated product P with a barrier of 35.3138 kcal/mol. Further probing and key mechanistic results will be presented.
2020-02-04
2021-05-01
2019-05
2019-08-23
May 2019
2020-01-29
Master's Thesis
http://hdl.handle.net/10342/7595
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/49562021-03-03T20:57:00Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Molecular Dynamics Studies of Point Mutations of Cardiac Troponin C and Annexin
Simpkins, Bradley
Li, Yumin
Chemistry
Chemistry
Annexin
Cardiac troponin
Molecular dynamics
Calcium binding proteins are biologically important for their ability to convert changes in calcium ion concentration to functional changes in proteins. All calcium binding proteins use a calcium sensing motif that changes the protein conformation and dynamics when associated with calcium ions. Dysfunction in calcium binding proteins has been linked to many diseases such as cancer and heart disease. In this study, models of mutated proteins were generated and Molecular Dynamics simulations were used to study Annexin and Cardiac Troponin C (cTnC). Annexin A1 is an important protein that is known to induce membrane aggregation, while cardiac Troponin C (cTnC) regulates cardiac muscle contraction. This study focuses on the impact of mutations on Annexin A1 and cTnC with the objective to gain insight of how these changes are made by mutations. The ultimate goal of this research is to use the knowledge of the effects of point mutations on calcium binding proteins dynamics to treat disease. Multiple MD simulations were performed for the full length wild type Annexin A1, the full length A1 mutations S27E and S27A, as well as the N-terminal peptide (AMVSEFLKQAWFIDNEEQEYIKTVKGS²�KGGPGSAVSPYPTFN) of wild-type A1 and mutations S27E and S27A. The MD simulation trajectories of about 350ns were generated and analyzed to examine the changes of the core domain calcium binding affinity, core domain and N-terminal domain structures, and N-terminal domain orientation. Our results indicated that S27A and S27E mutations caused little changes on the calcium-binding affinity of the core domain of A1. However, the S27A mutation made the N-terminal domain of A1 less helical, and made the N-terminal domain migrate faster toward the core domain; these impacts on A1 are beneficial or have no effect on the membrane aggregation process. On the contrary, the S27E mutation made the N-terminal domain of A1 more stable, and hindered the migration to the core domain; these changes on A1 are antagonistic for the membrane aggregation process. Our results using MD simulations provide an atomistic explanation for previous experimental observations that the S27E mutant showed a higher calcium concentration requirement and lower maximal extent of aggregation, while the wild-type and two mutants S27E and S27A required identical calcium concentrations for liposome binding. Molecular Dynamics simulations of about 145 ns total were performed for wild type cardiac troponin C and two Site II mutations, D65A and S69C. The simulation trajectories were analyzed using MMPBSA, MMGBSA, RMSF, RMSD, cluster analysis and various visualization programs. The results showed that the mutations caused a decrease in calcium binding affinity that is similar to what was shown in previous studies. The loss of calcium binding affinity can be attributed to a loss of binding at two residues, Ser 69 and Asp 73. Cross-correlation analysis shows that inter-domain interactions change dramatically when cTnC interacts with the other two subunits of cTn. Possible downstream effects of cTnC point mutations were seen through the changes of inter-domain flexibility, orientation and position from that of wild-type. The accessibility of cTnI to the hydrophobic binding pocket located within the N-terminal domain of cTnC was more restricted for the D65A and S69C cTnC mutants than wild-type cTnC. The restriction of cTnI to the switch segment suggests that the mutant cTnC proteins have a negative effect on the downstream function of cTn. The changes observed in this study on Site II point mutations provide insight into disease causing mechanisms of the D65A and S69C mutations.
2015-06-04
2015-06-04
2015
Master's Thesis
http://hdl.handle.net/10342/4956
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/106822023-11-21T19:26:58Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Defining Protein Motions the Comprise the Reaction Barrier in Human Epithelial 15-Lipoxygenase-2
Ohler, Amanda
Offenbacher, Adam R
Chemistry
protein motions
allosteric regulation
thermal activation
hydrogen deuterium exchange-mass spectroscopy
Proteins are dynamic in nature, with these motions playing a role in substrate binding and product release. Protein thermal motions have emerged as participating in the bond making/breaking steps of catalysis and by extension the rate enhancement observed in enzymes. A family of enzymes, known as lipoxygenases (LOXs), play a large role in growth and pathogenic defense in plants and homeostasis, cell signaling, and inflammation in humans. The regulation of LOX pro- and anti-inflammatory properties is thought to be controlled through allosteric interactions with small molecules, proteins, and membranes. For all organisms, LOXs oxidize polyunsaturated fatty acids through an often rate-limiting C-H activation step that proceeds through a tunneling mechanism. The activation energy barrier for this LOX reaction is expected to be related to the thermal fluctuations of the protein-substrate complex. How protein motions transfer heat from the surface to buried active sites remains an open question. Furthermore, the connection between thermal motions mediating allostery and the chemical step(s) are not well resolved. Recent studies on the model plant LOX, soybean lipoxygenase (SLO), have identified a solvent-exposed loop that is linked to the origins of a defined network for thermal activation that is distinct from the defined allosteric network. The human counterpart, human epithelial 15-lipoxygenase-2 (15-LOX-2), exhibits similar function but lacks some of these structural features found in SLO, thereby raising the question as to the evolution of structure and protein motions in these enzymes. In this thesis, biophysical methods, including temperature-dependent hydrogen deuterium exchange-mass spectrometry, X-ray crystallography, and differential scanning calorimetry, as well as enzyme kinetics are used to regionally define catalytically linked dynamics related to both allostery and chemical bond breaking step(s) of15-LOX-2 to further understand how thermal motions regulate lipoxygenase function.
2022-06-14
2022-05
2022-04-27
May 2022
2022-06-07
Master's Thesis
http://hdl.handle.net/10342/10682
en
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/18142021-03-03T20:54:32Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_124
Cu²� Binding to A[beta] peptides : Detailed Heat Capacity Studies Provide Structural Insight into Complex Formation
Gade, Sunitha
Spuches, Anne M.
Chemistry
Chemistry
Chemistry, Inorganic
Inorganic chemistry
Alzheimer's disease (AD), an irreversible, progressive and devastating neurodegenerative disease, is the most common cause of dementia. It is characterized by the extracellular amyloid plaques and the intracellular neurofibrillary tangles. Amyloid plaques are majorly constituted of A[beta] peptides, which are cleaved from the membrane bound amyloid precursor protein (APP). A[beta] peptides consist of either 40 or 42 amino acid residues. Elevated concentrations of copper, zinc, and iron have been measured in amyloid plaques and recent in vitro studies have shown that zinc and copper ions promote the aggregation of these peptides. Furthermore, it was found that high affinity metal ion chelators can be used in the dissolution of aggregated A[beta] peptides. Taken together, these studies show the important role metal ions play in AD.   In vitro studies have shown that aggregation of human A[beta] peptides increases due to the presence of Cu, but this effect is much less profound for rat A[beta] peptides. Previous studies indicate that Cu²â�º binding occurs within the first 16 amino acid residues of the full length A[beta]16, A[beta]28 and three variant peptides using isothermal titration calorimetry (ITC) and ACES buffer as a weak competing ligand. In this study, the binding of Cu²â�º to A[beta]16 was studied at three different concentrations of ACES buffer (20mM, 50mM, and 100mM). Buffer independent binding constants (K) are then extracted to test for ternary complex formation. According to the thermodynamic studies of Cu²â�º binding to A[beta]28 and A[beta]28 mutants, a model was proposed, which was further tested by obtaining the heat capacity ([delta]C[subscript]p) data for Cu²â�º binding to A[beta]16 A[beta]28 and A[beta]28 mutants. In addition, heat capacity ([delta]C[subscript]p) data for Cu²â�º binding to Rat A[beta]28 is obtained and compared to human A[beta]28. Taken together, this data will be used to shed light on why the rat peptides do not form fibrils. Â
2013-06-06
2015-06-05
2012
Master's Thesis
http://hdl.handle.net/10342/1814
East Carolina University
oai:TheScholarship.intra.ecu.edu:10342/45602021-03-03T21:10:43Zcom_10342_55com_10342_1com_10342_122col_10342_58col_10342_123
Fundamental and Applied Studies on Chromatographic Separation of Cold Drugs and Skincare Creams and Extraction of Salvia miltiorrhiza using Subcritical Water
Kapalavavi, Brahmam
Yang, Yu
Chemistry
Chemistry
Biology
Pharmaceutical sciences
C. elegans
Subcritical water chromatography (SBWC) and subcritical water extraction (SBWE) are two green techniques that use subcritical water as the sole solvent for separations, thus eliminating the use of toxic and expensive organic solvents. This dissertation research was mainly focused on the development of SBWC through both fundamental and applied studies. Fundamental studies include the solubility of parabens and stability of preservatives and stationary phases under subcritical water conditions. Solubility of parabens increased by 11 to 36 folds with temperature raise from 25 to 150 °C, but decreased at 200 °C due to degradation. A new approximation model developed in this work successfully estimated the solubility of parabens in subcritical water. The studies on the stability of preservatives in subcritical water revealed that the preservatives were stable up to 150 °C and there was approximately 10% degradation of preservatives at 200 °C. The stationary phase evaluation indicated that the Waters XBridge C18 and phenyl columns were stable for up to 30,000 column volume at 150 °C and the ZirChrom-DB-C18 column up to 14,250 column volume at 200 °C. Applied studies of SBWC were focused on separation and analysis of pharmaceuticals from cold drugs and niacinamide, preservatives, and sunscreens present in skincare products. Our best SBWC quantification results achieved in this work are in the range of 97.4 to 103.4% recoveries and RSDs less than 1.9%. A large number of replicate chromatographic runs and the comparison with high performance liquid chromatography results indicate that our SBWC methods for niacinamide and preservatives are quite accurate and precise. The Subcritical water extraction and traditional herbal decoction (THD) of Salvia miltiorrhiza were carried out and the herbal extracts were tested for cytotoxicity on Caenorhabditis elegans. In general, the concentration of anticancer agents obtained by SBWE increased by 4 to 18 folds when the temperature was raised from 75 to 150 °C. The concentration of tanshinones, important anticancer agents, obtained by SBWE at all four temperatures was higher than that of the THD. Similarly, the cytotoxicity tests revealed that the SBWE herbal extracts were more potent than the THD extracts.
2014-08-28
2017-02-07
2014
Doctoral Dissertation
http://hdl.handle.net/10342/4560
East Carolina University
etdms///col_10342_58/100