Biochemistry Commons^™

Towards The Total Synthesis Of Thioviridamide: Thiyl Radical Approach To The Beta-Thioenamide Linkage Formation, Jung-Hoon Kang

Theses and Dissertations

We developed an approach to the β-thioenamide linkage contained in the S-(2-aminovinyl)cysteine (avCys) residue of thioviridamide.1,2 Kinetic and thermodynamic control of radical additions of thiols to ynamides were studied for the formation of β-thioenamide linkage. Thiyl radicals are electrophilic and ynamides are electron-rich alkynes. This complementary polarity of the radical and acceptor increases the likelihood of a successful radical addition reaction. Because little is known about these types of compounds (β-thioenamides), we were unsure what kinds of yields and stereoselectivities (cis vs. trans) to expect. The adduct stability is another issue to consider. Fortunately, under typical radical addition conditions, the …

Biochemical Characterization Of Two Yeast Paralogous Proteins Mth1 And Std1, Satish Pasula

Dissertations

Glucose is the most abundant monosaccharide and preferred carbon and energy source for most cells. Many organisms have evolved sophisticated means to sense glucose and respond to it appropriately. The budding yeast, Saccharomyces cerevisiae senses glucose through two transmembrane proteins, Snf3 and Rgt2. In the presence of extracellular glucose Snf3 and Rgt2 generate an intracellular signal that leads to the degradation of Mthl and Stdl, thereby inducing the expression of hexose transporter genes (EXT) by inhibiting the function of Rgtl, a transcriptional repressor of HXT genes. Mthl and Stdl are degraded via the Yckl/2 Kinase-SCFGrrl-26S proteasome pathway triggered by the …

Novel Xanthine And Oxanine Dna Glycosylase Activities In Yeast And Mammalian Systems, Liang Dong

All Dissertations

DNA that stores genetic information is frequently damaged in cells. The DNA bases carrying an exocyclic amino group [adenine (A), guanine (G), cytosine (C)] encounter deamination even under physiological conditions. Xanthine (X) and the newly discovered oxanine (O) are derived from deamination of guanine; they are potentially cytotoxic and mutagenic lesions. However, in yeast and eukaryotes, studies on the enzymatic repair of these lesions are limited.
In the yeast, Schizosaccharomyces pombe thymine-DNA glycosylase (Spo TDG) is homologous to human thymine-DNA glycosylase (hTDG), an enzyme that removes thymine from T/G pair. It was reported that Spo TDG contains uracil DNA glycosylase …

Dna Deamination Repair Enzymes In Bacterial And Human Systems, Rongjuan Mi

All Dissertations

DNA repair enzymes and pathways are diverse and critical for living cells to maintain correct genetic information. Single-strand-selective monofunctional uracil DNA glycosylase (SMUG1) belongs to Family 3 of the uracil DNA glycosylase superfamily. We report that a bacterial SMUG1 ortholog in Geobacter metallireducens (Gme) and the human SMUG1 enzyme are not only uracil DNA glycosylases (UDG), but also xanthine DNA glycosylases (XDG). Mutations at M57 (M57L) and H210 (H210G, H210M, H210N) can cause substantial reductions in XDG and UDG activities. Increased selectivity is achieved in the A214R mutant of Gme SMUG1 and G60Y completely abolishes XDG and UDG activity. Most …

Photocycloaddition Of Allenes, Crystal Ward

Theses and Dissertations

For the past ten years the Fleming research group has been involved in the research of allene compounds. We have explored the synthesis of allenes as well as several reactions involving allenes, such as oxidation and nitrene addition. Recently, we have explored the synthesis and photochemistry of allenic alcohols tethered to silicon. There are literature examples using allenes in synthesis, but very few examples exist using allenes in photochemical cycloaddition reactions. We have found that a diisopropylallenyloxy(cinnamyloxy)silane derivative undergoes [2+2] photochemical cycloaddition to produce a cyclobutylsilane product when irradiated for 60-75 minutes.

Polymer Supported Optical Biosensors, Ignacio Joseph Garcia

Electronic Theses and Dissertations

The use of various polymer supports adhering phospholipid multibilayers to an internal reflection element have been investigated. The polymer supports studied range from polystyrene (PS), and 100 nm wide PS nanospheres to triethylaminated poly(vinyl benzyl chloride) (PVBC). The PS nanospheres showed the most promise as an adhesion layer since they appear to be the most robust with repeated washings. They also appear to stabilize their adjacent phospholipid multibilayer by increasing the lipid melting temperature. The triethylaminated DVBC also provided an increase of lipid melting temperature, but not quite to the same degree as the PS nanospheres. The cooperativity of such …

I. Thermodynamics And Magnetism Of Cu2ocl2 Ii. Repairs To Microcalorimeter The "2"S Are Subscripts, And The Second 2 Is Preceded By A Lower Case L, Not A One, Thomas J. Parry

Theses and Dissertations

Adiabatic calorimetry provides accurate and precise specific heat (Cp) data. From this data, thermodynamic functions may be calculated. Cu2OCl2, melanothallite, became of interest as part of a study of a particular thermochemical cycle. The experimental specific heat data and the calculated thermodynamic functions are reported here. Free energies of formation, calculated from the thermodynamic functions, suggest the particular cycle of interest with this compound as an intermediate is not feasible; uncertainty as to the accuracy of CuO and CuCl2 data used in the calculations indicate further study may be necessary. Upon collection of the specific heat data, an antiferromagnetic transition …

A Radical Conjugate Addition Approach To The Total Synthesis Of Celogentin C, Steven G. Capps

Theses and Dissertations

The synthesis of five chiral DBFOX (dibenzofuran-oxazoline) ligands with either aryl or benzyl substituents will be presented. The requisite amino alcohols were obtained with high enantioselectivity either commercially (DBFOX/Bn), via Sharpless asymmetric aminohydroxylation (DBFOX/Nap, DBFOX/t-BuPh, DBFOX/Pip), or via phase-transfer catalyzed asymmetric alkylation (DBFOX/MeNap). These ligands, complexed with Mg(NTf2)2, were used as Lewis acid promoters of enantioselective radical conjugate additions to α/β-unsaturated nitro-amides/esters. A summary of these results is presented and discussed. These findings led us to believe that our initial binding model between metal, ligand, and substrate was flawed. Thus, we figured that if we started with a functionality known …

Regulators Of G-Protein Signaling, Rgs13 And Rgs16, Are Associated With Cxcl12-Mediated Cd4+ T Cell Migration, Lijin Xia

Theses and Dissertations

Chemokines are important chemical signals that guide lymphocyte movement within the immune system and promote the organization and functions of germinal centers (GCs) in the secondary lymphoid tissues. Previous studies have shown that GC T cells exhibit high expression of chemokine receptor 4, CXCR4, but that these cells are unable to migrate to the ligand for this receptor, the chemokine CXCL12. This “migratory paralysis” to CXCL12 was found to be correlated with the expression of two Regulators of G-protein Signaling, RGS13 and RGS16 in the GC T cells. The objective of my research was to determine whether RGS13 and RGS16 …

A Fluorescent Photoinduced Electron Transfer (Pet) Sensor For Cations With A Separate Pet Channel To Suppress Proton Signals, Supun Pathirana

Theses, Dissertations and Culminating Projects

This thesis presents A-((10-((2-methoxy-4-methylphenoxy)methyl)anthracen-9- yl)methyl)(pyridin-2-yl)-A-((pyridin-2-yl)methyl)methanamine, 1, a photoinduced electron transfer (PET) sensor that acts as a fluorescent ‘Off-On’ switch in the presence of Zn ions and not sensitive to protons.

Spectroscopic Study Of Compressible Mobile Phase And Stationary Phase Behavior In Chromatography, Lawrence R. Baker

Theses and Dissertations

Raman spectroscopy, laser-induced fluorescence, and sum-frequency generation (SFG) spectroscopy are used to investigate the behavior of compressible mobile phases and stationary phases under a variety of chromatographic conditions. Efforts to understand and optimize separations employing compressible mobile phases have been limited by a lack of understanding of the mobile phase density gradient. Mobile phase compressibility leads to gradients in linear velocity and solute retention and affects separation speed and efficiency, especially in packed columns. This work describes on-column density measurement of CO2, a common carrier fluid for SFC and SGC, in packed capillary columns using Raman microspectroscopy. On-column detection by …

Genetic Analysis Of The Role Of Smpb In Establishing The Reading Frame On Tmrna, Talina Christensen Watts

Theses and Dissertations

Ribosomes translate the genetic information encoded by mRNA into proteins. Defective mRNAs can cause stalling of translating ribosomes. The molecule tmRNA (transfer-messenger RNA) rescues stalled ribosomes in eubacteria. Together with its protein partner SmpB, tmRNA mimics a tRNA by entering the ribosomal A site and linking an alanine residue to the growing polypeptide chain. The ribosome then abandons the defective mRNA template and resumes translation on tmRNA, adding ten more amino acids to the nascent polypeptide. As a result of tmRNA action, stalled ribosomes are released and recycled, the defective mRNA is destroyed, and the aborted protein product is tagged …

Ion Permeation Through Membrane Channels: Molecular Dynamics Simulations Studies, Morad Mustafa

Theses and Dissertations

Molecular dynamics simulation was used to study ion permeation through different membrane proteins embedded in a lipid bilayer (DMPC) with different saline solutions. The potential of mean force (PMF) for ion transport was obtained by umbrella sampling simulations. A revised MacKerell force field for tryptophan residues was studied using gramicidin A (gA) channel as a test model. The revised force field contribution to the Na+ PMF was consonant with the prediction from the experimental results, but in stark contrast to the prediction of the CHARMM force field, version 22, for the tryptophan side-chain. A new grid-based correction map algorithm by …

Influence Of Carbonate On The Binding Of Carboplatin To Dna, Robert S. Sorokanich

Honors Capstone Projects - All

The reaction of aged carboplatin (reaction of carboplatin in 24 mM NaHCO₃ for 45 h, 37^o C, pH 8.6) with pBR322 DNA for 0 ≤ r ≤ 2.8, where r = [drug]/[DNA-bp], in 24 mM HEPES buffer, pH 7.4, for 24 h followed by agarose gel electrophoresis showed DNA mobility changes consistent with unwinding closed circular DNA. However, identical experiments conducted in a two buffer system, 24mM HEPES plus 24mM carbonate, showed no DNA mobility changes, indicating that carbonate blocks formation of the 1,2-intrastrand crosslink on DNA. Studies with aged carboplatin and with cisplatin carried out with 2.0 …

Expressing A Mammalian Signaling Protein In E. Coli, Soyika A. Richardson

Honors Capstone Projects - All

Much of what is currently known about GPCR structure was based on X-ray crystallographic measurements of rhodopsin. Furthermore, due to its inexpensiveness and the availability of simple methods to purify it in large quantities from cow eyes, bovine rhodopsin has been successfully used in obtaining diffraction-quality three-dimensional crystals. In our experiment, the bovine rhodopsin protein was cloned into the commercially available E. coli expression plasmid, pBAD TOPO^®, using forward and reverse PCR primers designed in our lab. Plasmid DNA from these cells was purified in order to analyze the direction of the inserted gene. The purified plasmid DNA …

The Ttc7Fsn/Fsn Mutation Results In Hyperactivation Of Lymphocytes And Overproduction Of Il-4 Leading To The Development Of Systemic Autoimmunity, Beth Lindroth Hill

Electronic Theses and Dissertations

Ttc7^fsn/fsn mice exhibit systemic autoimmunity characterized by hyperactivated B cells, increased interleukin-4, autoantibodies, kidney disease and reduced lifespan. Because the pathology is similar to systemic lupus erythematosus, Ttc7^fsn/fsn mice are a useful model with which to study early events that lead to autoimmune disease. Although the Ttc7^fsn mutation has been identified the gene function is unknown. The phenotype of Ttc7^fsn/fsn mice mimics the Th2 autoimmunity of the IL-4 transgenic (Erb et al 1997). It was previously unknown whether the over-production of IL-4 was an intrinsic defect of Ttc7^fsn/fsn lymphocytes that led to autoimmunity, or, whether …

Optimization Of Expression And Purification Of Acetoacetyl-Coa Thiolase From Sunflower, Anthony Muriithi Maina

Theses, Dissertations and Culminating Projects

The [beta]-oxidation system in sunflower (Helianthus annuus L.) cotyledons is distinguished by the existence of two different thiolase isoforms, Thiolase I (acetoacetyl-CoA thiolase, EC 2.3.1.9 AACT) and Thiolase II (3-oxoacyl-CoA thiolase, EC 2.3.1.16 OACT). Glyoxysomal AACT is the last enzyme in the p-oxidation of fatty acids in plant glyoxysomes. Glyoxysomal AACT has been successfully cloned, expressed and purified from sunflower cotyledons (Dyer et al., 2006). In this paper we investigate the optimal conditions for both expression and purification. These include the choice of vector, the growth conditions and the purification parameters for the maximum possible yield of AACT protein.

Identification Of Metallo-Ss-Lactamase Inhibitors Via Enzyme Assay And Hplc-Ms Monitoring, Peggy Ann Sanchez

Theses, Dissertations and Culminating Projects

The vast overuse of antibiotics along with extrachromosomal gene insertion between different bacterial species has led to the Darwinian consequence of antibiotic resistance. Amongst the contributing factors to the rise of bacterial resistances are the production of ß-lactamases, which pose a major impediment to treatment with ß-lactam drugs such as penicillins and carbapenems. SPM-1 is a metallo-ß-lactamase (MBL) that confers antibiotic resistance to bacteria through zinc-mediated hydrolysis of ß-lactams. Bacteria that express the SPM-1 metalloenzyme show reduced sensitivity to ß-lactam drugs. One method to defeat this form of resistance is to administer a ß-lactam antibiotic with a potent ß-lactamase inhibitor …

Hypochlorous Acid Mediated Damage Of Chromatin, Joseph Insugn Kang

Loma Linda University Electronic Theses, Dissertations & Projects

Hypochlorous acid (HOCl), the primary product of activated neutrophils at sites of inflammation, can damage both DNA and associated histone proteins. HOCl damage to cytosine bases in DNA generates the chlorination damage products 5-chlorocytosine (ClC) and 5-chlorouracil (ClU) as well as the oxidation damage products 5- hydroxycytosine (HOC) and 5-hydroxyuracil (HOU). Histone damage by HOCl can be measured by quantitation of 3-chlorotyrosine and 3,5-dichlorotyrosine, stable and unique markers of protein damage caused by HOCl. The studies presented here investigate how and where these damage products occur and what implications they might have in the correlation between chronic inflammation and carcinogenesis. …

Lipid Aldehydes Are Substrates And Transcriptional Regulators Of Cytochromes P450., Immaculate Amunom

Electronic Theses and Dissertations

4-hydroxy-2-nonenal (4-HNE) and propene-2-al (acrolein) are highly reactive á,â-unsaturated aldehydes. 4-HNE and acrolein are generated in vivo as products of lipid peroxidation. These aldehydes are implicated in the onset of several diseases including atherosclerosis and neurodegenerative disease. They also react with nucleophilic cellular macromolecules including proteins, DNA and phospholipids. Some of these reactions in vivo lead to inhibition of enzyme activities, depletion of glutathione and oxidative stress. Because of their pathophysiological relevance, attention has focused during the last decade on the ability of mammals to metabolize these lipid aldehydes. The purpose of this study was therefore to determine the role …

Toxic Gene Expression In Escherichia Coli Using Trpl As A Repressor, Ian Primm

Honors Theses

No abstract provided.

The Regulation Of Endothelial And Neuronal Nitric Oxide Synthase Expression In The Preglomerular Vessels By The Increase In Intracellular Calcium Concentration, Stephanie Thomas Montgomery

Chemistry & Biochemistry Theses & Dissertations

This purpose of this study was to examine the regulation of endothelial and neuronal nitric oxide synthase expression in the preglomerular vessels of mature and immature porcine kidneys by the increase in intracellular calcium concentration. Angiotensin II can increase intracellular calcium concentrations in two ways; either through the mobilization of intracellular calcium stores or through the influx of calcium from the extracellular fluid through calcium channels. The aim of this study was to determine the effects of a calcium control, a calcium-free control, L- and T-type voltage-gated calcium channel blockers, an internal calcium store release agent, KCI, and EDTA on …

Characterizing The Dynamics And Functional Role Of Site-Specific Phosphorylation Of G Protein-Coupled Receptors, John M. Busillo

Full-Text Theses & Dissertations

Phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) and the subsequent recruitment of arrestin is a well-established paradigm that initiates the process known as desensitization. However, an emerging theme in GPCR regulation is the possibility of differential regulation dictated by the phosphorylation pattern elicited by the different members of the GRK family. Therefore, we have used small interfering RNA-mediated knock down of the GRKs and arrestins in an attempt to better understand how phosphorylation regulates the activity and signaling of the M3 muscarinic acetylcholine receptor (M3 mAChR) and CXCR4, two receptors endogenously expressed in HEK293 cells.

Using a …

Urine Protein Electrophoresis: Comparison Of Agarose Gel And Capillary Zone Electrophoretic Techniques., Anne-Marie Curtin

Theses

Proteinuria is the excess excretion of protein in the urine and is found in a variety of conditions including renal diseases and neoplastic conditions such as multiple myeloma (MM). The investigation of proteinuria type through the use of electrophoresis is a useful and widely used non-invasive diagnostic tool. The aim of this study was to compare various agarose gel and capillary zone electrophoresis techniques in order to establish a method choice for the separation of protein in routine laboratories. Comparisons were made based on urine monoclonal peak quantification and protein fractions detected. An examination of a capillary zone electrophoresis (CZE) …

Development Of An Automated Method For Csf Bilirubin Measurement., Helen Caverley

Theses

Subarachnoid haemorrhage (SAH) is a potentially fatal condition which can be difficult to accurately diagnose. Patients often present with a second bleed which can be more damaging than the initial bleed. Diagnosis of SAH centres on computed tomography (CT) scans. However, 2% of patients positive for SAH will present as negative on CT scans. It is recommended that these patients have lumbar puncture and the CSF is analysed for bilirubin. The presence of bilirubin is suggestive of SAH. Spectrophotometric scanning involves expensive equipment which is often not available in routine settings. A need exists for a robust, inexpensive, automated method …

Gender Bias Of Cardiovascular Risk In Caucasian Type Ii Diabetics: A Haemostatic Explanation?, William Quirke

Theses

People with type II diabetes are disproportionately affected by cardiovascular disease compared to those without diabetes. The relative risk of cardiovascular disease caused by type II diabetes is higher in females than in males. This risk has not been fully explained by traditional risk factors and attention has turned to the possible role of haemostatic abnormalities in explaining the excess cardiovascular morbidity and mortality among diabetic females. We examined specific parameters of the coagulation (factor VII, factor VIII, von Willebrand factor, fibrinogen) and fibrinolytic (plasminogen activator inhibitor- 1, thrombin-activatable fibrinolysis inhibitor) system in 113 Irish Caucasian type II diabetics to …

The Role Of Protein Disulfide Isomerase (Pdi) In Oxidative Folding, Veronica Gonzalez

Open Access Theses & Dissertations

Protein Disulfide Isomerase is a highly evolved oxidoreductase enzyme that has the capability of oxidizing, reducing, and isomerizing disulfides in substrate. PDI is organized into several domains denoted a, b, b', a' and c. These domains are believed to have different functions but all must be present for full PDI activity. In this manuscript we recorded the ability of PDI b' to catalyze oxidative folding in fully reduced RNase A. We also examined competition between thiol-disulfide shuffling and conformational folding by PDI. This competition creates a rough effect of a highly efficient enzyme in two different substrates: RNase A and …

Degenerate Codon Mutagenesis And Cloning Of G-Protein Coupled Receptor Gene Fusion, Michael J. Sofolarides

Seton Hall University Dissertations and Theses (ETDs)

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Involvement Of Inflammation In Tau Pathology And Mechanisms That Drive Neurodegeneration In Alzheimer's Disease, Lisette T. Arnaud

Dissertations, Theses, and Capstone Projects

Two of the hallmarks of neurodegenerative disorders, such as Alzheimer's disease (AD), are (1) the appearance of proteinaceous deposits in inclusion bodies containing aggregates of ubiquitinated proteins and (2) activated microglia and astrocytes surrounding the diseased neurons. In Alzheimer's disease, the intracellular inclusion bodies are known as neurofibrilary tangles (NFT). The mechanisms leading to inclusion body formation and their role in the progression of neurodegeneration are still largely unknown.

Many of the proteins that accumulate in inclusion bodies depend on the ubiquitin/proteasome pathway (UPP) for their degradation. This pathway is responsible for the bulk (∼80%) of intracellular protein degradation. Because …

Stable Isotope Dynamics In Summer Flounder Tissues, With Application To Dietary Assessments In Chesapeake Bay, Andre Buchheister

Dissertations, Theses, and Masters Projects

Stable isotope techniques were applied to summer flounder, Paralichthys dentatus, in Chesapeake Bay to elucidate the relative importance of different prey groups on the growth and productivity of this species. Prior to field application, a laboratory diet-shift study was conducted to evaluate methodological assumptions and obtain necessary isotopic parameters. Specifically, the goals of the laboratory study were to 1) determine isotopic turnover rates and fractionations of δ13C and δ15N in liver, whole blood, and white muscle and 2) estimate the relative importance of growth and metabolic processes on isotopic turnover. Groups of captive juvenile summer flounder (130-255mm total length) were …