Biochemistry Commons^™

Protein Crystallization Tray Protocol Development, Brooke Connell

STAR Program Research Presentations

In the field of protein crystallography the transfer of the protein crystals from the crystallization plate/tray, where the crystals are initially grown, to the site of analysis is a difficult and lengthy process. A new crystallization tray has been designed and 3D printed to eliminate this intermediate step which is called crystal fishing. The crystallization tray allows crystals to be grown directly on grids that are attached to the pins that are used for analysis of the crystal. The efficacy of protein crystallization using the aforementioned tray was tested in order for a protocol to be developed for potential users …

Comparative Analysis Of In Situ Fibronectin Using Tof-Sims, Spi-Ms, And Dropdesi-Ms In A Microfluidic Reactor, Shannon Fasing, Xiao-Ying Yu, Juan Yao, Jiachao Yu

STAR Program Research Presentations

Fibronectin is an important biomolecule due to its role in cell differentiation, growth, kinesis, and adhesion. Such biological responses are mediated through membrane recognition and signaling; where fibronectin is found. Studying the outer molecular surface of fibronectin allows deeper insight into the microbiological reactions that occur during these processes. In situ mass spectrometry analysis in aqueous solution accurately represents fibronectin’s chemical components, made possible by a vacuum compatible microfluidic reactor, SALVI (System for Analysis at the Liquid Vacuum Interface). SALVI was paired with the analytical tools: time-of-flight secondary ion mass spectrometer (ToF-SIMS), single photon ionization mass spectrometer (SPI-MS) and drop …

Investigation Of An Energetic Coupling Between Ligand Binding And Protein Folding, Nathan W. Gardner

Open Access Dissertations

The cellular environment presents a protein with many small molecules with which it may interact. Many novel interactions between proteins and non-substrate metabolites are being uncovered through proteome-wide screens. The homodimeric Escherichia coli cofactor-dependant phosphoglycerate mutase (dPGM) was identified as an ATP binding protein in a proteome-wide screen, but dPGM does not use ATP for catalysis. This dissertation elucidates the effect of ATP and other non-substrate metabolites on dPGM. Initial investigations revealed a partially unfolded, monomeric intermediate of dPGM that forms during equilibrium unfolding. ATP binding was found to occur at the active site of dPGM and to be energetically …

Intestinal Cytoplasmic Lipid Droplets, Associated Proteins, And The Regulation Of Dietary Fat Absorption, Theresa M. D'Aquila

Open Access Dissertations

Dietary fat provides essential nutrients, contributes to energy balance, and regulates blood lipid concentrations. These functions are important to health, but can also become dysregulated and contribute to diseases such as obesity, diabetes, and cardiovascular disease. The small intestine absorbs dietary fat through an efficient multi step process of digestion, uptake, metabolism, and secretion or storage. When dietary fat is taken up by the absorptive cells of the small intestine, enterocytes, it can be secreted into circulation where it contributes to blood lipid levels or temporarily stored in cytoplasmic lipid droplets (CLDs). The objective of this dissertation is to investigate …

Curb-Cut Rain Garden Research, Patrick Jonker, David P. Warners

Student Papers and Reports

No abstract provided.

Parameterization Of Single-Scattering Albedo (Ssa) And Absorption Ångström Exponent (Aae) With Ec/Oc For Aerosol Emissions From Biomass Burning, Rudra P. Pokhrel, Nick L. Wagner, Justin M. Langridge, Daniel A. Lack, Thilina Jayarathne, Elizabeth A. Stone, Chelsea E. Stockwell, Robert J. Yokelson, Shane M. Murphy

Chemistry and Biochemistry Faculty Publications

Single-scattering albedo (SSA) and absorption Ångström exponent (AAE) are two critical parameters in determining the impact of absorbing aerosol on the Earth's radiative balance. Aerosol emitted by biomass burning represent a significant fraction of absorbing aerosol globally, but it remains difficult to accurately predict SSA and AAE for biomass burning aerosol. Black carbon (BC), brown carbon (BrC), and non-absorbing coatings all make substantial contributions to the absorption coefficient of biomass burning aerosol. SSA and AAE cannot be directly predicted based on fuel type because they depend strongly on burn conditions. It has been suggested that SSA can be effectively parameterized …

The Effect Of R382w Mutation On Citrus Paradisi Flavonol Specific 3-O-Glucosyltransferase, Kathleen King

Undergraduate Honors Theses

Flavonoids are a class of plant metabolites with C6-C3-C6 structure responsible for many biological functions, including coloration and defense. Citrus paradisi, grapefruit, contains a wide variety of flavonoids which are grouped by the extent of modification, examples of which are flavonols, flavones, and flavanones. A major modification is the addition of glucose by glucosyltransferases (GTs) to stabilize the structure and provide ease of transport. Glucosyltransferases can be highly substrate and regiospecific. With Cp3OGT, glucose is added at the 3-hydroxy position. This 3GT only accepts flavonols as its substrate; however, a Vitis vinifera (grape) 3-GT can accept both flavonols and …

Snp-Based High Density Genetic Map And Mapping Of Btwd1 Dwarfing Gene In Barley, Xifeng Ren, Jibin Wang, Lipan Liu, Genlou Sun, Chengdao Li, Hong Luo, Dongfa Sun

Publications

A high-density linkage map is a valuable tool for functional genomics and breeding. A newly developed sequence-based marker technology, restriction site associated DNA (RAD) sequencing, has been proven to be powerful for the rapid discovery and genotyping of genome-wide single nucleotide polymorphism (SNP) markers and for the high-density genetic map construction. The objective of this research was to construct a high-density genetic map of barley using RAD sequencing. 1894 high-quality SNP markers were developed and mapped onto all seven chromosomes together with 68 SSR markers. These 1962 markers constituted a total genetic length of 1375.8 cM and an average of …

Vascular Endothelial Growth Factor-A Gene Electrotransfer Promotes Angiogenesis In A Porcine Model Of Cardiac Ischemia, Anna A. Bulysheva, Barbara Hargrave, Nina Burcus, Cathryn G. Lundberg, Len Murray, Richard Heller

Bioelectrics Publications

This study aimed to assess safety and therapeutic potential of gene electrotransfer (GET) as a method for delivery of plasmid encoding vascular endothelial growth factor A (VEGF-A) to ischemic myocardium in a porcine model. Myocardial ischemia was induced by surgically occluding the left anterior descending coronary artery in swine. GET following plasmid encoding VEGF-A injection was performed at four sites in the ischemic region. Control groups either received injections of the plasmid without electrotransfer or injections of the saline vehicle. Animals were monitored for 7 weeks and the hearts were evaluated for angiogenesis, myocardial infarct size and left ventricular contractility. …

Heparin-Peptide Interactions, Jacqueline Anastasia Morris

Graduate Theses and Dissertations

Heparin is a polydispersed sulfated molecule that is part of the family called glycosaminoglycans found in the extracellular matrix and cell surfaces. This molecule is extremely important for the activation of proteins and protein-receptor interactions that are responsible for downstream cell signaling pathways. Heparin has been isolated from porcine intestine and used as an anticoagulant for the prevention of embolisms, heart thrombosis, and clotting during heart surgeries. This so-called miracle drug was in use until 2008, when isolated batches were found to be contaminated with other glycosaminoglycans similar to heparin. From 2008, there has been a dire need for a …

Enhancing Silymarin Fractionation Via Molecular Modeling Using The Conductor-Like Screening Model For Real Solvents, Emma C. Brace

Open Access Theses

The market for bio-based products from plant sources is on the rise. There is a global challenge to implement environmentally clean practices for the production of fuels and pharmaceuticals from sustainable resources. A significant hurdle for discovery of comparable plant-derived products is the extensive volume of trial-and-error experimentation required. To alleviate the experimental burden, a quantum mechanics based molecular modeling approach known as the COnductor-like Screening Model for Real Solvents (COSMO-RS) was used to predict the best biphasic solvent system to purify silymarins from an aqueous mixture. Silymarins are a class of flavonolignans present in milk thistle ( Silybum marianum …

Biochemical Changes In Animal Models Of Fetal Alcohol Spectrum Disorder, Christine E. M. Keller

Open Access Dissertations

Fetal alcohol spectrum disorder (FASD) is a completely preventable disease, that has profound effects on life-long health and function of the affected individual. Prevalence estimates of FASD in the United States indicate 33.5 per 1,000 live births are affected with this disorder (Roozen, 2016). FASD is caused by maternal ethanol intake during pregnancy. However, recommendations of the amounts of alcohol safe to drink during pregnancy are not established. Further, we lack a comprehensive understanding of the biochemical pathways modified in prenatal ethanol exposure. Biomarkers are also lacking. Our results demonstrate the vast array of biochemical pathways modified in the chronic …

Analysis Of The Intricacies Of Substrate Recognition Of High Mobility Group Proteins And Aminoacyl-Trna Synthetases Using Non-Cognate Substrates, Douglas Van Iverson Ii

Dissertations

The studies presented in section 1 (Chapters I-IV) focus on the design and development of nucleic acid four-way junctions (4WJs) to target a member of the high mobility group (HMG) proteins, the proinflammatory cytokine high mobility group box 1 protein (HMGB1). In the present study, hybrid PNA-DNA 4WJs based on a model DNA 4WJ were constructed to improve the thermal stability of 4WJs while maintaining strong binding affinity toward HMGB1. An electrophoretic mobility shift assay (EMSA) was used to examine the binding affinity of an isolated DNA binding domain of HMGB1, the HMGB1 b-box (HMGB1b), toward a set of PNA-DNA …

Loss Of Exogenous Androgen Dependence By Prostate Tumor Cells Is Associated With Elevated Glucuronidation Potential, Brenna M. Zimmer, Michelle E. Howell, Qin Wei, Linlin Ma, Trevor Romsdahl, Eileen G. Loughman, Jennifer E. Markham, Javier Seravalli, Joseph J. Barycki, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

Prostate epithelial cells control the potency and availability of androgen hormones in part by inactivation and elimination. UDP-glucose dehydrogenase (UGDH) catalyzes the NAD⁺-dependent oxidation of UDP-glucose to UDP-glucuronate, an essential precursor for androgen inactivation by the prostate glucuronidation enzymes UGT2B15 and UGT2B17. UGDH expression is androgen stimulated, which increases the production of UDP-glucuronate, and fuels UGT-catalyzed glucuronidation. In this study, we compared the glucuronidation potential and its impact on androgen-mediated gene expression in an isogenic LNCaP model for androgen dependent versus castration resistant prostate cancer. Despite significantly lower androgen-glucuronide output, LNCaP 81 castration resistant tumor cells expressed higher …

Investigating The Roles Of Δnp63 As A Suppressor Of Migration, Invasion, And Metastasis, Ramon E. Flores Gonzalez

Dissertations and Theses (Open Access)

Cancer is one of the leading causes of death and disease in the world. Considerable resources are spent to study and understand cancer, with the hope of developing new treatments and eventually cures that will help millions of people. Efforts to understand cancer are hindered by its inherent complexity and instability. Nonetheless, understanding the basics of tumor development and progression are the key to focused on studying the role of ΔNp63 in cancer, a p53 family member known to be involved in epithelial development, microRNA biogenesis, and stem cell maintenance. Using the strength of in vivo mouse models, we found …

Defining The Functions Of Usp22 And Usp44 In Regulation Of H2bub1 Levels, Xianjiang Lan

Dissertations and Theses (Open Access)

Aberrant levels of histone ubiquitination are involved in various human diseases including neurodegenerative disorders and cancers. Particularly, Histone H2B monoubiquitination (H2Bub1) is highly associated with gene regulation in both normal cells and diseases. Many deubiquitinases (mainly USPs) are defined to regulate global H2Bub1 levels. However, how these USPs are regulated and how they contribute to diseases are not well understood.

USP22, part of the deubiquitination module (DUBm) in the SAGA complex, is a well-defined regulator of H2Bub1 levels. ATXN7, another crucial subunit of the SAGA DUBm, is involved in a neurodegenerative disease, spinocerebellar ataxia type 7 (SCA7), due to a …

Two-Electron Quenching Of Dinuclear Ruthenium(Ii) Polypyridyl Complexes, Yinling Zhang

Graduate Theses and Dissertations

A bridging ligand 5,5’-Bi- 1,10-phenanthroline, diphen, was prepared using dichlorobis(triphenylphosphine)Ni(II), Ni(PPh3)2Cl2 as catalyst with a yield of 40%. Yellow cubic crystals were able to obtain from the good purity product for single crystal analysis. The torsion angle between the planes of the subunit phenanthrolines is about 66 degrees.

A dinuclear ruthenium (II) polypyridyl complex, (phen)2Ru(diphen)Ru(phen)24+, was synthesized by using polymeric ruthenium carbonyl compound as the entry point, diphen as the bridging ligand and 1,10-phenanthroline, phen, as the terminal legand. Brown needlelike crystals were precipitated from acetonitrile that were not suitable for single crystal diffraction.

The photochemistry of the dimer was …

Tackling Adverse Environment—Molecular Mechanism Of Plant Stress Response And Biotechnology Tool Development, Ning Yuan

All Dissertations

Abiotic and biotic stresses such as drought, salt, nutrition starvation, and pathogen infection are major factors threatening our agricultural production. With the rapidly increasing population and limited arable land area, genetic engineering of crops for new products with more stable and higher yield than conventional cultivars under adverse environment provides a powerful new tool for use in developing novel GMOs (Genetically Modified Organisms) to feed the large population in the immediate future. To develop novel GMOs with enhanced performance under adverse conditions, we need first to understand molecular mechanisms underlying plant stress response. To better understand how signaling transduction pathway …

Applications Of The Gst- Affinity Tag In The Purification And Characterization Of Proteins, Wibke Beatrice Kachel

Graduate Theses and Dissertations

With the latest innovations in biological sciences, large quantities of biologically active polypeptides as well as high throughput screening methods to quickly evaluate if these biomolecules potentially have therapeutic, diagnostic, or industrial purposes are required. The synthesis and purification of peptides and small proteins continue to be demanding as the production of high yields through chemical synthesis can involve large costs. On the other hand, there are only few examples of acquiring those biomolecules through cloning and expression in bacterial systems in form of recombinant fusion proteins. Glutathione S-Transferase (GST) is not only a very commonly used affinity tag to …

Investigation Of The Interaction Of Dimeric Ruthenium Complexes With Cytochrome B5, Christopher Dain Rupar

Graduate Theses and Dissertations

Photoreactive complexes to study the kinetics of electron transfer of proteins have been in use for a long time. It has always been speculated that complexes bind near the heme or the electron transfer reaction would not occur. But it is unkown exactly how the complex interacts with the protein. The structural, thermodynamic, and kinetic properties of rat liver microsomal cytochrome b5 were investigated when bound to ruthenium dimer complexes. Heteronuclear Single Quantum Coherence studies support a dynamic binding model of a dimer Ru complex bound near the protein’s heme involving residues H39, E44, G42, V61, G62, and H63. The …

Mechanism Of Rapid Electron Transfer Reactions Involving Cytochrome Bc1, Cytochrome C And Cytochrome Oxidase, Jeremy Erik Durchman

Graduate Theses and Dissertations

Electron transfer between mitochondrial proteins complexes represents the primary means by which living things acquire the requisite energy for survival. The coupling of electron transfer to proton translocation creates an electrochemical gradient that drives the synthesis of highly energetic compounds such as ATP. The purpose of these studies is to measure rates of electron transfer and elucidate the important governing factors in the redox events involving cytochrome bc1, cytochrome c and cytochrome oxidase. Using rapid initiation of redox events triggered by laser flash excitation of ruthenium compounds, and strategically monitoring unique spectral properties of these proteins in the visible region …

The San1 Ubiquitin Ligase Functions Preferentially With Ubiquitin-Conjugating Enzyme Ubc1 During Protein Quality Control, Rebeca Lea Ibarra

UNLV Theses, Dissertations, Professional Papers, and Capstones

Protein quality control (PQC) is a critical process wherein misfolded or damaged proteins are cleared from the cell to maintain protein homeostasis. In eukaryotic cells, the removal of misfolded proteins is primarily accomplished by the ubiquitin-proteasome system (UPS). In the UPS, ubiquitin-conjugating enzymes and ubiquitin ligases append poly-ubiquitin chains onto misfolded protein substrates signaling for their degradation. The kinetics of protein ubiquitylation are paramount since a balance must be achieved between the rapid removal of misfolded proteins versus providing sufficient time for protein chaperones to attempt refolding. To uncover the molecular basis for how PQC substrate ubiquitylation rates are controlled, …

Fulleretic Well-Defined Scaffolds: Donor–Fullerene Alignment Through Metal Coordination And Its Effect On Photophysics, Derek E. Williams, Ekaterina A. Dolgopolova, Danielle C. Godfrey, Evgeniya D. Ermolaeva, Perry J. Pellechia Dr., Andrew B. Greytak Prof. Dr., Mark Smith Dr., Stanislav M. Avdoshenko, Dr., Alexey A. Popov Dr., Natalia B. Shustova Prof. Dr.

Faculty Publications

Herein, we report the first example of a crystalline metal–donor–fullerene framework, in which control of the donor–fullerene mutual orientation was achieved through chemical bond formation, in particular, by metal coordination. The 13C cross-polarization magic-angle spinning NMR spectroscopy, X-ray diffraction, and time-resolved fluorescence spectroscopy were performed for comprehensive structural analysis and energy-transfer (ET) studies of the fulleretic donor–acceptor scaffold. Furthermore, in combination with photoluminescence measurements, the theoretical calculations of the spectral overlap function, Förster radius, excitation energies, and band structure were employed to elucidate the photophysical and ET processes in the prepared fulleretic material. We envision that the well-defined fulleretic donor–acceptor …

Identification Of Preferred Dna-Binding Sites For The Thermus Thermophilus Transcriptional Regulator Sbtr By The Combinatorial Approach Repsa, Michael W. Van Dyke, Matthew D. Beyer, Emily Clay, Jonathan L. Mcmurry

Faculty Articles

One of the first steps towards elucidating the biological function of a putative transcriptional regulator is to ascertain its preferred DNA-binding sequences. This may be rapidly and effectively achieved through the application of a combinatorial approach, one involving very large numbers of randomized oligonucleotides and reiterative selection and amplification steps to enrich for high-affinity nucleic acid-binding sequences. Previously, we had developed the novel combinatorial approach Restriction Endonuclease Protection, Selection and Amplification (REPSA), which relies not on the physical separation of ligand-nucleic acid complexes but instead selects on the basis of ligand-dependent inhibition of enzymatic template inactivation, specifically cleavage by type …

Domains Of Stip1 Responsible For Regulating Prpc-Dependent Amyloid-Β Oligomer Toxicity., Andrzej Maciejewski, Valeriy G Ostapchenko, Flavio H Beraldo, Vania F Prado, Marco A M Prado, Wing-Yiu Choy

Biochemistry Publications

Soluble oligomers of amyloid-beta peptide (AβO) transmit neurotoxic signals through the cellular prion protein (PrP(C)) in Alzheimer's disease (AD). Secreted stress-inducible phosphoprotein 1 (STIP1), an Hsp70 and Hsp90 cochaperone, inhibits AβO binding to PrP(C) and protects neurons from AβO-induced cell death. Here, we investigated the molecular interactions between AβO and STIP1 binding to PrP(C) and their effect on neuronal cell death. We showed that residues located in a short region of PrP (90-110) mediate AβO binding and we narrowed the major interaction in this site to amino acids 91-100. In contrast, multiple binding sites on STIP1 (DP1, TPR1 and TPR2A) …

Analysis Of The Stress - Inducible Carotenoid - Binding Protein Complex From Synechocystis Pcc 6803, Soumana Daddy Gaoh

Theses and Dissertations

Photosynthetic organisms experience continuous fluctuation in their environment, including changes in light conditions and nutrient availability. These organisms adjust their photosynthetic activity to balance light absorption with the energetic and growth requirements of the cell. This organization provides a mechanism to reduce the formation of potentially damaging reactive oxygen species that result from excessive illumination and excitation of pigment molecules. Cyanobacteria, probable progenitors of chloroplasts, are ancient photosynthetic prokaryotes that have adapted successfully to diverse and adverse environments. Their widespread occurrence and fascinating acclimation capability has rendered them the largest group of photosynthetic prokaryotes on our planet. How these organisms …

Quantifying Greenhouse Gas Flux Of Restored Vs. Unrestored Wetlands: A Case Study At Prairie Wolf Slough, Matthew Connors, Beth Lawrence

DePaul Discoveries

Wetlands provide ecological services such as cleansing the water supply, sequestering carbon, and providing habitat for wildlife, however wetland restoration often alters the greenhouse gas flux of the site. Our study aims to investigate the effects of wetland restoration on greenhouse gas flux at Prairie Wolf Slough. We did this by comparing greenhouse gas flux on matching hydric soil series from the restored wetland with an adjacent abandoned agricultural field. We measured known controls of greenhouse gas flux such as soil moisture and soil temperature. We found that there was no detectable methane and nitrous oxide flux at either site, …

Hepatitis C Virus Frameshift/Alternate Reading Frame Protein Suppresses Interferon Responses Mediated By Pattern Recognition Receptor Retinoic-Acid-Inducible Gene-I, Seung Bum Park, Scott Seronello, Wasima Mayer, David M. Ojcius

All Dugoni School of Dentistry Faculty Articles

Hepatitis C virus (HCV) actively evades host interferon (IFN) responses but the mechanisms of how it does so are not completely understood. In this study, we present evidence for an HCV factor that contributes to the suppression of retinoic-acid-inducible gene-I (RIG-I)-mediated IFN induction. Expression of frameshift/alternate reading frame protein (F/ARFP) from HCV -2/+1 frame in Huh7 hepatoma cells suppressed type I IFN responses stimulated by HCV RNA pathogen-associated molecular pattern (PAMP) and poly(IC). The suppression occurred independently of other HCV factors; and activation of interferon stimulated genes, TNFα, IFN-λ1, and IFN-λ2/3 was likewise suppressed by HCV F/ARFP. Point mutations in …

Identification Of Collagen Iv Associated Proteins In Drsophila Using Genetics And Mass Spectrometry, Mayank S. Kapadia

Masters Theses & Specialist Projects

Metastatic cancer cells invade and spread to other locations by disrupting the basement membrane (BM). The membrane plays a major role during the normal development of an organism as well. In order to understand the invasion mechanism it is important to know about the interactions occurring between the proteins of the BM during normal development. This study concentrates on isolating and identifying the major factors associated with collagen IV, a major component of BM, during the third instar larval development of Drosophila. Western blot and mass spectrometry analysis revealed that collagen IV associates with various growth factors, signaling molecules, and …

Synthetic Investigation On The Biomimetic Metal-Catalyzed Sulfoxidations And Photochemical Generation Of A Highly Reactive Ruthenium(V)-Oxo Porphyrin, Weilong Luo

Masters Theses & Specialist Projects

Catalytic oxidation plays a crucial role in current chemical and pharmaceutical industries which is also a leading technology for green chemical processes. In Nature, the ubiquitous cytochrome P450 enzymes can catalyze a wide variety of oxidation reactions with high efficiency and selectivity. Many transition metal catalysts are designed as the biomimetic model of cytochrome P450 enzymes. In this work, series of metalloporphyrins and metallocorroles have been successfully synthesized to investigate and develop catalytic selective oxidation of sulfides to sulfoxides.

Manganese(III) porphyrin complexes (2) and manganese(III) corrole complexes (6) with iodobenzene diacetate [PhI(OAc)2] as a mild oxygen source exhibited remarkable catalytic …