Chemistry Commons^™

Mass Spectrometric Analysis Of Environmental Contaminants, Protein Structure And Expression, Ian E. Atkinson

ETD Archive

There are a wide array of sciences at our disposal to further our understanding of the roles of toxins in the environment and their effects on living organisms. Of these disciplines, the field of mass spectrometry offers a powerful analytical means to identify compounds in varying and complex sample media. The presence of altered expressions of biomolecules upon exposure to toxins, the effects that these toxins may have on biomolecule structure and the identity of toxins in the sample all can be determined by mass spectrometry. In this study mass spectrometric analysis has been applied to environmental toxicological problems. First, …

Factor Va Directs Catalysis By Factor Xa During Prothrombin Activation, Michael Anthony Bukys

ETD Archive

Hemostasis occurs through the controlled activation and inactivation of clotting factors resulting in the arrest of bleeding without blockage to the vasculature, while thrombosis is the undesired formation of vascular blood clots, which adversely affects millions of people annually. Clotting factors circulate in blood as inactive zymogens, which are proteolytically activated in response to vascular injury, assembled into enzymatic complexes and in turn activate additional coagulation factors culminating in the production of thrombin from the enzymatic complex prothrombinase. The prothrombinase complex is composed of the activated enzymatic component factor Xa (fXa) complexed to the activated cofactor portion factor Va (fVa) …

A Case In Kinase 2 Inhibitor Is A Potent Anti-Cancer Drug Candidate, Alieta Ciocea

ETD Archive

Cancer is so widespread and lethal that it can be considered the biggest health problem of our century. Cancer drug development is a real challenge. Efforts outlined in this work were directed towards showing solid evidence that casein kinase 2 inhibitors can be used as a starting point for the development of potential cancer therapy. We first studied the effect of a specific CK2 inhibitor, DMAT, in a malignant megakaryoblastic leukemia cell line (MEG-01). Treated cells grew at a significantly lower rate than non-treated cells. Apoptosis was induced by DMAT in MEG-01 cells, dose and time dependent. When stimulated with …

Development Of Oligonucleotide Microarray For High Throughput Dna Methylation Analysis, Xiaopeng Li

ETD Archive

DNA methylation is a key event regulating gene expression. DNA methylation analysis plays a pivotal role in unlocking association of epigenetic events with cancer. However, simultaneous evaluation of the methylation status of multiple genes is still a technical challenge. Microarray is a promising approach for high-throughput analysis of the methylation status at numerous CpG sites within multiple genes of interest. In this dissertation study, we conducted a systematic study to examine the use of microarray methods for methylation analysis. First, a robust universal microarray was established with more flexible in design and content, and potential cost saving over commercial arrays. …

Technologies For Proteomic And Genomic Biomarker Analysis, Yiding Liu

ETD Archive

In the first part of this dissertation, we systematically validated the application of molecular weight cut-off ultrafiltration in separation and enrichment of low-molecular-weight peptides from human serum. Under optimized conditions, both free-phase and bound LMW peptides could be separated and enriched. The method proved to be highly efficient and reproducible coupled with MALDI-TOF MS proteomic pattern analysis. Three marker peaks were found to be eligible for distinguishing normal and ovarian cancer samples. A novel organic solvent precipitation method coupled with enzymatic deglycosylation was also developed for biomarker detection from human serum. This method allowed us to generate reproducible free-phase peptide …

Inhibition Of The Nf-Kb Signaling Pathway And Its Effects On Apoptosis And Cancer, Joseph A. Lupica

ETD Archive

The Rel/NF-kB family of inducible transcription factors are evolutionarily conserved structurally and functionally, from insects to humans. They are ubiquitously expressed in a number of mature cell types, playing a pivotal role regulating cell growth, differentiation and apoptosis. Under normal circumstances, the proliferation of new cells is tightly regulated, as is the programmed lifespan of most cells, occasionally however cells loose their responsiveness to growth control mechanisms, resulting in a tumor or neoplasm. Sustained or chronic inflammation has been linked to a number of pathological conditions that destroy tissue and facilitate neoplastic growth. NF-kB in part mediates the opposing signals …

High Temperature Volatility And Oxidation Measurements Of Titanium And Silicon Containing Ceramic Materials, Quynhgiao N. Nguyen

ETD Archive

Titanium (Ti) and silicon (Si) containing materials are of high interest to the aerospace industry due to its high temperature catability, strength, and ligh weight. A continuous exterior oxide layer is desirable to reduce the oxidation rate of these two materials. At high temperatures, water vapor plays a key role in the volatility of materials including oxide surfaces. This study first evaluated several hot-pressed Ti and Si-containing compositions at high temperatures as a function of oxidation environments to determine the volatile hydoxy! species using the transpiration method. The water content ranged from 0-76 mole and the oxygen content range was …

Modeling The Human Prothrombinase Complex Components, Tivadar Orban

ETD Archive

Thrombin generation is the culminating stage of the blood coagulation process. Thrombin is obtained from prothrombin (the substrate) in a reaction catalyzed by the prothrombinase complex (the enzyme). The prothrombinase complex is composed of factor Xa (the enzyme), factor Va (the cofactor) associated in the presence of calcium ions on a negatively charged cell membrane. Factor Xa, alone, can activate prothrombin to thrombin however, the rate of conversion is not physiologically relevant for survival. Incorporation of factor Va into prothrombinase accelerates the rate of prothrombinase activity by 300,000-fold, and provides the physiological pathway of thrombin generation. The long-term goal of …

Synthesis And Characterization Of Acylphosphine Oxide Photoinitiators, Hany F. Sobhi

ETD Archive

Recently, polymerization science has assumed an increasing relevance in many applications, including orthopedic and dental biomaterials. Current technologies are based on the use of photochemically reactive systems, which are suited to absorb light to produce primary radical species capable of initiating polymerization. Acylphosphine oxides and acylphosphonates are known to be very effective photoinitiators for various unsaturated polymeric systems. The high reactivity observed was ascribed to the phosphinyl and phosphinoyl radicals generated. Because of the poor qualities of other initiators, we examined new routes to synthesize acylphosphine oxide initiators to achieve compatibility with aqueous media and improved performance. The preparation and …

Mass Spectrometry-Based High Throughput Approach For Identification Of Molecular Modification Of Oxidative Process In Respiratory Diseases, Wei Song

ETD Archive

Eosinophil peroxidase (EPO) and myeloperoxidase (MPO) have been implicated in generating reactive species and promoting oxidative modifications in numerous diseases. The detection and identification of potential pathways has been proven extremely challenge due to evanescence nature of these reactive species. An alternative approach to study the involvement of oxidative modification is to detect and quantify the stable molecular fingerprints, like oxidized tyrosine species, in biological matrices. Previously reported analytical methods for quantifying oxidized amino acids have typically been limited by: low sensitivity, specificity and the failure of detection potential oxidation products generated during sample handling. Using GC/MS in combination with …

Study Of 3'-Untranslated Region Of Inducible Nitric Oxide Synthase And Identification Of Other Targets Of Gaitpathway, Sirisha Vadlamani

ETD Archive

Macrophages and other leukocytes play a vital role during inflammation. IFN-Îđ which is secreted by Th1 cells activates monocyte-macrophages and this activation leads to induction as well as suppression of certain genes expressed during inflammation. This suppression of genes may be potentially vital for resolution of inflammation. Upon IFN-Îđ stimulation, a Gamma-activated inhibitor of translation complex (GAIT) is formed, which binds to 3'-UTR of target mRNAs thereby silencing their translation. VEGF-A and ceruloplasmin are two proteins whose translation is suppressed by this pathway. The GAIT complex proteins have been identified as ribosomal protein L13a, glutamyl-prolyl tRNA synthatase (EPRS), NS-1 associated …

Spectroscopy Is Applied Quantum Mechanics. Part 2: The Quantum Revolution, David W. Ball

Chemistry Faculty Publications

The article presents several theories relative to the nature of matter. The quantum theory of German theoretical physicist Max Planck proposes that the energy of a vibrating atom was related to its frequency. Albert Einstein's quantum theory of light hypothesizes that light itself had an energy that was directly proportional to its frequency. Danish physicist Niels Bohr develops an equation that could help explain the lines that appeared in the spectrum of the hydrogen atom.

Suppression Of Aromatase In Human Breast Cells By A Cyclooxygenase-2 Inhibitor And Its Analog Involves Multiple Mechanisms Independent Of Cyclooxygenase-2 Inhibition, Bin Su, Edgar S. Diaz-Cruz, Serena Landini, Robert W. Brueggemeier

Chemistry Faculty Publications

Previous studies have demonstrated that cyclooxygenase-2 (COX-2) inhibitor NS-398 decrease aromatase activity at the transcript level in breast cancer cells. However, N-Methyl NS-398, which does not have COX-2 inhibitory activity but has very similar structure to NS-398, decreases aromatase activity and transcription in MCF-7 and MDA-MB-231 breast cells to the same extent as NS-398. This suggests that NS-398 decrease aromatase expression in breast cancer cells via other mechanism(s). Further investigations find that both compounds only decrease aromatase activity stimulated by forskolin/phorbol ester at the transcript level in both breast cancer cell lines and in breast stromal cells from patients. They …

Optimization Of Non-Natural Nucleotides For Selective Incorporation Opposite Damaged Dna, Diana Vineyard, Xuemei Zhang, Alison Donnelley, Irene Lee, Anthony J. Berdis

Chemistry Faculty Publications

The promutagenic process known as translesion DNA synthesis reflects the ability of a DNA polymerase to misinsert a nucleotide opposite a damaged DNA template. To study the underlying mechanism of nucleotide selection during this process, we quantified the incorporation of various non-natural nucleotide analogs opposite an abasic site, a non-templating DNA lesion. Our kinetic studies using the bacteriophage T4 DNA polymerase reveal that the π-electron surface area of the incoming nucleotide substantially contributes to the efficiency of incorporation opposite an abasic site. A remaining question is whether the selective insertion of these non-hydrogen-bonding analogs can be achieved through optimization of …

Ovarian Cancer G Protein–Coupled Receptor 1, A New Metastasis Suppressor Gene In Prostate Cancer, Lisam Shanjukumar Singh, Michael Berk, Rhonda Oates, Zhenwen Zhao, Haiyan Tan, Ying Jiang, Aimin Zhou, Kashif Kirmani, Rosemary Steinmetz, Daniel Lindner, Yan Xu

Chemistry Faculty Publications

Background

Metastasis is a process by which tumors spread from primary organs to other sites in the body and is the major cause of death for cancer patients. The ovarian cancer G protein–coupled receptor 1 (OGR1) gene has been shown to be expressed at lower levels in metastatic compared with primary prostate cancer tissues.

Methods

We used an orthotopic mouse metastasis model, in which we injected PC3 metastatic human prostate cancer cells stably transfected with empty vector (vector-PC3) or OGR1-expressing vector (OGR1-PC3) into the prostate lobes of athymic or NOD/SCID mice (n = 3–8 mice per group). Migration of PC3 …

Aromatase And Cox In Breast Cancer: Enzyme Inhibitors And Beyond, Robert W. Brueggemeier, Bin Su, Yasuro Sugimoto, Edgar S. Diaz-Cruz, Danyetta D. Davis

Chemistry Faculty Publications

Aromatase expression and enzyme activity in breast cancer patients is greater in or near the tumor tissue compared with the normal breast tissue. Complex regulation of aromatase expression in human tissues involves alternative promoter sites that provide tissue-specific control. Previous studies in our laboratories suggested a strong association between aromatase (CYP19) gene expression and the expression of cyclooxygenase (COX) genes. Additionally, nonsteroidal anti-inflammatory drugs (NSAIDs) and COX selective inhibitors can suppress CYP19 gene expression and decrease aromatase activity. Our current hypothesis is that pharmacological regulation of aromatase and/or cyclooxygenases can act locally to decrease the biosynthesis of estrogen and may …

The Use Of Non-Natural Nucleotides To Probe Template-Independent Dna Synthesis, Anthony J. Berdis, David Mccutcheon

Chemistry Faculty Publications

The vast majority of DNA polymerases use the complementary templating strand of DNA to guide each nucleotide incorporation. There are instances, however, in which polymerases can efficiently incorporate nucleotides in the absence of templating information. This process, known as translesion DNA synthesis, can alter the proper genetic code of an organism. To further elucidate the mechanism of template-independent DNA synthesis, we monitored the incorporation of various nucleotides at the “blunt-end” of duplex DNA by the high-fidelity bacteriophage T4 DNA polymerase. Although natural nucleotides are not incorporated at the blunt-end, a limited subset of non-natural indolyl analogues containing extensive π-electron surface …

1,4-Di-9-Anthrylbutane, Mustafa Arslan, Erol Asker, John Masnovi, Ronald J. Baker

Chemistry Faculty Publications

In the title compound, C₃₂H₂₆, the molecule has an inversion centre at the mid-point of the central C—C bond. Weak intermolecular C—H...π interactions help to stabilize the crystal structure.

1,3-Bi-9-Anthrylpropane, Mustafa Arslan, Erol Asker, John Masnovi, Ronald J. Baker

Chemistry Faculty Publications

The title compound, C₃₁H₂₄, with three molecules in the asymmetric unit. The crystal packing is mainly stabilized by weak C—H⋯π inter­actions in addition to van der Waals forces.

Molecular Mechanism Of Incorporation Of Factor Va Into Prothrombinase, Melissa Barhoover

ETD Archive

The mainstay of the blood coagulation cascade is the formation of the fibrin clot, catalyzed by the serine protease, thrombin. The prothrombinase complex catalyzes the activation of prothrombin to thrombin and, is composed of the enzyme, factor Xa, and the protein cofactor, factor Va, in the presence of divalent metal ions associated on a membrane surface. The enzyme, factor Xa, alone can activate prothrombin by two sequential proteolytic cleavages at Arg271 and Arg320 resulting in the intermediates, Fragment 1.2 and Prethrombin 2. The overall rate of this reaction is not compatible with survival. On the other hand, the incorporation of …

Characterization Of A Type Ii Metallothionein From Helianthus Annuus Using Recombinant Dna Techniques, Sridhar Bhogavalli

ETD Archive

Metallothioneins (MTs) are ubiquitous low molecular weight, cysteine rich proteins with a pronounced affinity for metal ions with d10 configuration such as Cu, Cd and Zn. These heavy metals form metal-thiolate clusters with cysteine side chains. In contrast to the vertebrate forms, knowledge about the properties of members of the plant metallothionein family is still scarce. We describe here a method of isolation and purification of metallothionein from the plant Helianthus annuus, performed by affinity chromatography using glutathione-agarose column. In this study, alignment of gene sequence of the isolated H. annuus metallothionein cDNA with known MTs showed that it belongs …

Characterization Of Polymorphic And Blended Drugs By Physical-Analytical Properties, Rajgopal Munigeti

ETD Archive

The polymorphs of Tolbutamide and Nifedipine were differentiated by measuring their unique physical properties. Differential Scanning Calorimetry (DSC) and Dielectric Analysis (DEA) verified the variations in these compounds with different crystal structures but the same chemical constituents. The crystalline-amorphous content of the polymorphs were determined based on a new DEA protocol. Solubility (w/w ) and spectroscopic methods clearly distinguished these drugs and compared favorably with the photomicrography of the polymorphs. Powder X-ray Diffraction (PXRD) analysis succintly showed differences in the various types of polymorphs. Scanning Electron Microscopy (SEM) supported the structural distinction and solubility. The FTIR spectra of all the …

Characterizatin Of A Type Ii Metallothionein From Helianthus Annuus Using Recombinant Dna Techniques, Sridhar Bhogavalli

ETD Archive

Metallothioneins (MTs) are ubiquitous low molecular weight, cysteine rich proteins with a pronounced affinity for metal ions with d10 configuration such as Cu, Cd and Zn. These heavy metals form metal-thiolate clusters with cysteine side chains. In contrast to the vertebrate forms, knowledge about the properties of members of the plant metallothionein family is still scarce. We describe here a method of isolation and purification of metallothionein from the plant Helianthus annuus, performed by affinity chromatography using glutathione-agarose column. In this study, alignment of gene sequence of the isolated H. annuus metallothionein cDNA with known MTs showed that it belongs …

Role Of 2-5a-Dependent Rnase-L In Senescence And Longevity, J. B. Andersen, X. L. Li, C. S. Judge, Aimin Zhou, B. K. Jha, S. Shelby, L. Zhou, Robert H. Silverman, B. A. Hassel

Chemistry Faculty Publications

Senescence is a permanent growth arrest that restricts the lifespan of primary cells in culture, and represents an in vitro model for aging. Senescence functions as a tumor suppressor mechanism that can be induced independent of replicative crisis by diverse stress stimuli. RNase-L mediates antiproliferative activities and functions as a tumor suppressor in prostate cancer, therefore, we examined a role for RNase-L in cellular senescence and aging. Ectopic expression of RNase-L induced a senescent morphology, a decrease in DNA synthesis, an increase in senescence-associated -galactosidase activity, and accelerated replicative senescence. In contrast, senescence was retarded in RNase-L-null fibroblasts compared with …

Tumour Suppressor Function Of Rnase L In A Mouse Model, Wendy Liu, Shu Ling Liang, Hongli Liu, Robert Silverman, Aimin Zhou

Chemistry Faculty Publications

RNase L is one of the key enzymes involved in the molecular mechanisms of interferon (IFN) actions. Upon binding with its activator, 5′-phosphorylated, 2′-5′ oligoadenylates (2-5A), RNase L plays an important role in the antiviral and anti-proliferative functions of IFN, and exerts proapoptotic activity independent of IFN. In this study, we have found that RNase L retards proliferation in an IFN-dependent and independent fashion. To directly measure the effect of RNase L on tumour growth in the absence of other IFN-induced proteins, human RNase L cDNA was stably expressed in P-57 cells, an aggressive mouse fibrosarcoma cell line. Three clonal …

Interaction Energy Decomposition In Protein–Protein Association: A Quantum Mechanical Study Of Barnase–Barstar Complex, Abdessamad Ababou, Arjan Van Der Vaart, Valentin Gogonea, Kenneth M. Merz Jr.

Chemistry Faculty Publications

Protein–protein interactions are very important in the function of a cell. Computational studies of these interactions have been of interest, but often they have utilized classical modelling techniques. In recent years, quantum mechanical (QM) treatment of entire proteins has emerged as a powerful approach to study biomolecular systems. Herein, we apply a semi-empirical divide and conquer (DC) methodology coupled with a dielectric continuum model for the solvent, to explore the contribution of electrostatics, polarization and charge transfer to the interaction energy between barnase and barstar in their complex form. Molecular dynamic (MD) simulation was performed to account for the dynamic …

Reactivation Pathway Of The Hydrogenase H-Cluster: Density Functional Theory Study, Stefan Motiu, Daniela Dogaru, Valentin Gogonea

Chemistry Faculty Publications

This work puts forth a reaction pathway for the reactivation of exogenous ligand inhibited H-cluster, the active site of Fe-only hydrogenases. The H-cluster is a dimetal complex, Fe–Fe, with the metal centers bridged by di(thiomethyl)amine. Exogenous ligands, H2O, and OH−, are bound to the distal iron (Fed). Density functional theory (DFT) calculations on the native and ruthenium-modified H-cluster have been performed using the B3LYP functional with 6-31+G** and 6-311+G** basis sets. We have ascertained that there is a thermodynamically favorable pathway for the reactivation of the OH− inhibited H-cluster, which proceeds by an initial protonation of the Fed–OH− complex. The …

A Control Switch For Prothrombinase: Characterization Of A Hirudin-Like Pentapeptide From The Cooh Terminus Of Factor Va Heavy Chain That Regulates The Rate And Pathway For Prothrombin Activation, Michael A. Bukys, Paul Y. Kim, Michael E. Nesheim, Michael Kalafatis

Chemistry Faculty Publications

Membrane-bound factor Xa alone catalyzes prothrombin activation following initial cleavage at Arg(271) and prethrombin 2 formation (pre2 pathway). Factor Va directs prothrombin activation by factor Xa through the meizothrombin pathway, characterized by initial cleavage at Arg(320) (meizo pathway). We have shown previously that a pentapeptide encompassing amino acid sequence 695-699 from the COOH terminus of the heavy chain of factor Va (Asp-Tyr-Asp-Tyr-Gln, DYDYQ) inhibits prothrombin activation by prothrombinase in a competitive manner with respect to substrate. To understand the mechanism of inhibition of thrombin formation by DYDYQ, we have studied prothrombin activation by gel electrophoresis. Titration of plasma-derived prothrombin activation …

(E,E)-1,3-Bis[9,10-Dihydro-9-Nitro-10-(Trinitro­Meth­Yl)-9-Anthr­Yl]Propane, Mustafa Arslan, Erol Asker, John Masnovi, Ronald J. Baker

Chemistry Faculty Publications

The title compound, C₃₃H₂₄N₈O₁₆, was obtained as a product of the photoreaction between 1,3-di-9-anthrylpropane and tetra­nitro­methane. The mol­ecule occupies a special position on a twofold axis. The trinitro­methyl and nitro substituents on the 9,10-dihydro­acridine system are E oriented.

A 2:1 Complex Of 1,3-Bis­(9h-Carbazol-9-Yl)Propane And Tetra­Chloro-P-Benzoquinone (P-Chloranil), Mustafa Arslan, Randolph B. Krafcik, John Masnovi, Ronald J. Baker

Chemistry Faculty Publications

In the title electron donor–acceptor complex, C₂₇H₂₂N₂·0.5C₆Cl₄O₂, the p-chloranil mol­ecule lies on a crystallographic inversion center, which is located at the center of the benzene ring. In the crystal structure, one p-chloranil mol­ecule lies above and below the central rings of each donor group of two neighboring 1,3-bis­(9H-carbazol-9-yl)propane mol­ecules, with a ring-centroid separation of 3.444 (1) Å. The angle between the planes of the stacking rings of the carbazole and p-chloranil mol­ecules is 3.4 (2)°.