Biochemistry Commons^™

Synthesis, Characterization, And Application Of High Surface Area, Mesoporous, Stabilized Anatase Tio2 Catalyst Supports, Rebecca Elizabeth Olsen

Theses and Dissertations

Nanomaterials have attracted substantial attention in the area of catalysis due to the unique properties they exhibit such as high surface areas, intricate pore networks and unique morphologies. TiO2 has attracted attention as a catalyst since the discovery of its high photocatalytic activity by Fuishima and Honda in 1972. Given its high thermal stability, low cost, low environmental impact, and versatility, TiO2 is a widely used commercial catalyst and catalyst support. TiO2 is used in many applications such as photocatalysis is also an excellent support material for noble metals in a number of oxidative synthesis and pollution-control reactions. Though TiO2 …

Nonlinear Spectroscopic Investigation Of Adsorption To C-18 Model Stationary Phase, Anthony D. Peterson

Theses and Dissertations

Reversed-phase liquid chromatography (RPLC) is a commonly used separation technique in chemistry. Nevertheless, the mechanistic interactions at the molecular level among the eluent, analyte, and the stationary phase are not fully understood. Because of this limited understanding, optimization of the separation must be done experimentally. Learning more about molecular interactions should aid in improving separations. We are currently using second-harmonic generation (SHG) spectroscopy to investigate how analytes adsorb to the surface. SHG is a spectroscopic technique that produces signal only at places of non-isotropic symmetry; this typically occurs at surfaces. SHG can be used to produce surface isotherms of test …

Formation And Analysis Of Zinc Oxide Nanoparticles And Zinc Oxide Hexagonal Prisms And Optical Analysis Of Cadmium Selenide Nanoparticles, Jared M. Hancock

Theses and Dissertations

In this dissertation, methods to synthesize ZnO are reported. First, zinc oxide nanoparticles were synthesized with small amounts of transition metal ions to create materials called dilute magnetic semiconductors (DMS). We employed a low temperature sol-gel method that produces ZnO nanoparticles of reproducible size and incorporates cobalt, nickel, and manganese ions into the nanoparticles. Conditions were controlled such that a range of amounts of Co, Ni, and Mn were incorporated. The incorporation was tracked by color changes in the white ZnO powder to blue for Co, green for Ni and yellow for Mn. XRD measurements showed the nanoparticles were on …

Evaluation Of Vdr-Coactivator Inhibitors Using Biochemical And Cell-Based Assays, Athena Marie Baranowski

Theses and Dissertations

ABSTRACT

EVALUATION OF VDR–COACTIVATOR INHIBITORS USING BIOCHEMICAL AND CELL–BASED ASSAYS

by

Athena Baranowski

The University of Wisconsin–Milwaukee, 2013

Under the Supervision of Dr. Alexander Arnold

The vitamin D receptor (VDR) is a ligand–dependent transcription factor, which belongs to the nuclear receptor superfamily. VDR–mediated gene regulation is governed by coregulators (coactivators and corepressors). VDR coregulator binding inhibitors (CBIs), which were discovered using high throughput screening (HTS), were evaluated using cell–based assays and biochemical assays to determine their ability to inhibit the interaction between VDR and steroid receptor coactivator–2 (SRC–2). Determining their ability to inhibit the VDR–SRC–2 interaction can lead to the …

Investigation Into The Effects Of Pegylation On The Thermodynamic Stability Of The Ww Domain, Sam S. Matthews

Theses and Dissertations

The covalent attachment of poly(ethylene glycol) (PEG) to a protein surface (known as PEGylation), has been demonstrated to increase the serum half-life of therapeutic proteins by reducing kidney clearance and immunogenicity and by protecting against proteolysis. Theses beneficial effects could be further enhanced if PEGylation consistently increased protein conformational stability (i.e. the difference in free energy between the folded and unfolded states). However, the effects of PEGylation on protein conformational stability are unpredictable; PEGylation has been reported to increase, decrease, or have no effect on the conformational stability of medicinal proteins.This thesis details the results of two studies aimed at …

Ferritin-Based Photo-Oxidation Of Biomass For Nanoparticle Synthesis, Bioremediation, And Hydrogen Evolution, Oscar Petrucci

Theses and Dissertations

The cell is the basic unit of all living organisms. It is an amazing machine capable of self-replicating, growing, and synthesizing and shuttling thousands of compounds. To perform all of these activities the cell needs energy. The original source of energy for all living beings is the Sun. The energy of the sun is collected by the autotrophs (mostly plants) through photosynthesis and stored in the chemical bonds of carbohydrates and lipids through carboxylic acid intermediates; animals use these compounds to obtain the energy for their cells. Most of the energy extracted by the cell comes from the citric acid …

Monodentate, Bidentate And Photocrosslinkable Thiol Ligands For Improving Aqueous Biocompatible Quantum Dots, Hiroko Takeuchi

Graduate Theses and Dissertations

Water-soluble Quantum Dots (QDs) are highly sensitive fluorescent probes that are often used to study biological species. One of the most common ways to render QDs water-soluble for such applications is to apply hydrophilic thiolated ligands to the QD surface. However, these ligands are labile and can be easily exchanged on the QD surface, which can severely limit their application. As one way to overcome this limitation while maintaining a small colloidal size of QDs, we developed a method to stabilize hydrophilic thiolated ligands on the surface of QDs through the formation of a crosslinked shell using a photocrosslinking approach. …

Obstacles And Solutions To Studying Functional Adhesives Using Vibrational Sum-Frequency Generation Spectroscopy, Angela Renee Andersen

Theses and Dissertations

Important aspects of adhesion occur at interfaces, including structures that may be different from those in the bulk materials. However, probing the orientation of molecules in functional adhesives poses a significant challenge because adhesive molecules are always located at a buried interface. The limited penetration depth of surface-specific analysis prohibits the study of buried interfaces using those techniques. The large quantity of bulk molecules relative to the adhesive molecules interacting at the interface results in the bulk signal swamping out adhesive signal in bulk analysis techniques. An interface-specific technique is required to study functional adhesives. One such technique that has …

Design, Synthesis, And Evaluation Of Molecular Inhibitors For Biologically Relevant Enzymes, Sarah Emma St. John

Open Access Dissertations

The work in this thesis details the design, synthesis, and biological evaluation of molecular inhibitors for the inhibition of biologically relevant enzymes. The first three chapters of this thesis concern the polyphenol resveratrol and its inhibition of the quinone reductase 2 (QR2) enzyme. The work on this subject resulted in the complete design, synthesis, biological and structural evaluation of a second generation library of resveratrol analogues. From this work we identified a novel resveratrol analogue that inhibits QR2 in a previously unknown binding orientation. The fourth chapter of this thesis details the de novo design of molecules for the inhibition …

Gas-Phase Covalent And Non-Covalent Ion/Ion Chemistry Of Biological Macromolecules, John Robert Stutzman

Open Access Dissertations

Gas-phase ion/ion chemistry involves the interaction of oppositely charged ions inside of the mass spectrometer. During this gas-phase chemistry, particle transfer (i.e., proton and electron) or synthesis can occur at rapid reaction rates. Particle transfer represents a mature area of ion/ion chemistry, while selective covalent modification represents a fairly new area of gas-phase chemistry. Gas-phase covalent chemistry is based on traditional solution phase organic chemistry.

The work demonstrated in this dissertation greatly involves gas-phase covalent and non-covalent Schiff base chemistry on peptide and protein ions. The reagent dianion, 4-formyl 1,3-benzene disulfonic acid, has been used to covalently modify unprotonated primary …

Development Of Isotags For Nmr Based Metabolite Profiling And Applications, Fariba Tayyari

Open Access Dissertations

NMR spectroscopy is a powerful analytical tool for both qualitative and quantitative metabolite profiling analysis. However, accurate quantitative analysis of biological systems especially using one dimensional NMR has been challenging due to signal overlap. In contrast, the enhanced resolution and sensitivity offered by chemoselective isotope tags have enabled new and enhanced methods for detecting hundreds of quantifiable metabolites in biofluids using NMR spectroscopy or mass spectrometry. In this thesis we show improved sensitivity and resolution of NMR experiments imparted by 15N and 13C isotope tagging which enables the accurate analysis of plasma metabolites. To date, isotope tagging has been used …

Synthesis Of Novel Isoprenoid Diphosphate Analogs As Chemical Tools To Investigate Protein Geranylgeranylation, Kayla Jo Temple

Open Access Dissertations

Many proteins require prenylation in order to be biologically functional. Some such proteins include the small Ras and Rho GTPase superfamilies, nuclear lamins A and B, and the kinesin motor proteins CENP-E and F. Prenyltransferase (PTase) inhibition is currently being explored as a possible treatment not only for cancer but for a wide variety of other diseases.

Clinical studies revealed that the effectiveness of farnesyltransferase inhibitors (FTIs) to treat Ras-dependent tumors is determined by which isoform of Ras is overactive. Unfortunately the majority of Ras-dependent tumors have a mutation in either the N- or K-Ras isoforms; both of these isoforms …

Size-Based Separation Of Bioparticles Using Planar Nanofluidic Devices, Jie Xuan

Theses and Dissertations

Nanofluidic devices are structures having at least one dimension in the submicron range, which is of the same order of magnitude as the sizes of biomolecules and bioparticles such as proteins and viruses. As a result, size-selective separations are important applications for nanofluidics. Well-defined micro or nano device structures fabricated via micromachining have greatly reduced sample consumption and enabled separations in a parallel fashion, promising significant speed and resolution advantages over conventional size separation techniques, such as gel electrophoresis and size exclusion chromatography. In collaboration with others, I have developed a size separation method using nanofluidic devices consisting of an …

Formation, Functionalization, Characterization, And Applications Of A Mixed-Mode, Carbon/Diamond-Based, Core-Shell Phase For High Performance Liquid Chromatography, Landon A. Wiest

Theses and Dissertations

My work has focused on a variety of different types of diamond-based, core-shell particles. These particles are formed with inert cores and poly(allylamine)/nanodiamond shells. Their intended purpose is to form an LC stationary phase that is stable from pH 1 – 14 and at elevated temperatures. At the beginning of my studies, the particles that had been made in the Linford laboratory were pH stable, but irregular and had poor mechanical stability. Since that time, I have worked to improve the particles by using more spherical zirconia and carbon cores, and I have improved their mechanical stability via chemical crosslinking …

Self-Assembled Dna Origami Templates For The Fabrication Of Electronic Nanostructures, Elisabeth Pound Gates

Theses and Dissertations

An important goal of nanoscience is the self-assembly of nanoscale building blocks into complex nanostructures. DNA is an important and versatile building block for nanostructures because of its small size, predictable base pairing, and numerous sequence possibilities. I use DNA origami to design and fold DNA into predesigned shapes, to assemble thin, branched DNA nanostructures as templates for nanoscale metal features. Using a PCR-based scaffold strand generation procedure, several wire-like nanostructures with varying scaffold lengths were assembled. In addition, more complex prototype circuit element structures were designed and assembled, demonstrating the utility of this technique in creating complex templates. My …

Zinc Chemical Biology: The Pursuit Of The Intracellular Targets Of Zinquin, Andrew Nowakowski

Theses and Dissertations

Fluorescent sensors have been a main microscopic tools used to understand Zn2+ physiology on a cellular level. The use of the fluorescent Zn2+ sensor Zinquin (ZQ) and its analogues have revealed that transient Zn2+ is a chief component in a variety of biochemical pathways. Yet, little work has been performed to validate the exact targets of Zinquin in a cellular environment. The goals of this investigation are to determine the types of Zinquin reactions that take place in the cell as well as the identities of its cellular targets.

It has been hypothesized that Zinquin reacts with free Zn2+ within …

The Role Of Metallothionein In Zinc Homeostasis, Kelly L. Summers

Electronic Thesis and Dissertation Repository

The structure of the unique metal-binding protein, metallothionein (MT), consists of two metal-thiolate-clustered binding domains; the β-domain binds up to three divalent metals and the α-domain binds four. The mechanisms through which the metals are bound and arranged into domains, as well as the function of MT in metal ion homeostasis, remains largely unknown. By utilizing electrospray ionization mass spectrometry (ESI MS) to identify each species, and by comparing the data with simulations, MT 1a was found to bind Zn²⁺ non-cooperatively. Through a competition experiment between MT and its individual domain peptides, MT was proposed to bind Zn^{2+ …}

Studies Toward The Synthesis Of Lyconadin A And Cranomycin, Brad M. Loertscher

Theses and Dissertations

Lyconadin A is a pentacyclic Lycopodium alkaloid isolated from the club moss Lycopodium companatum with anticancer activity. Our approach sought to incorporate a 7-exo–6-exo acyl radical cyclization cascade to access the bicyclo[5.4.0]undecane framework of lyconadin A. Our studies created methodology for the synthesis of 5-alkyl and 3,5-dialkyl-6-carbomethoxy-2-pyridones and sterically demanding epoxide substrates. These epoxide substrates underwent an unanticipated Payne rearrangement.Cranomycin is a potent antibiotic with antiprotozoal activity. Structurally it is a cyclopentane ring system with substitution at each carbon in the ring. Another interesting structural aspect is the existence of three contiguous quaternary stereocenters including two tertiary alcohols and a …

Developmental Signaling Requires Inwardly Rectifying K+ Channels In Drosophila Melanogaster, Giri Raj Dahal

Theses and Dissertations

Inwardly rectifying potassium (IRK/Kir) channels regulate intracellular K+ concentrations and membrane potential. Disruption of Kir2.1 causes dominantly inherited Andersen Tawil Syndrome (ATS). ATS patients suffer from cardiac arrhythmias, periodic paralysis, and cognitive impairment. These symptoms are consistent with current understanding of the role of ion channels in muscle cells and neurons. However, ATS symptoms also include craniofacial and digital deformities such as cleft palate, dental defects, wide set eyes, low set ears, and crooked or fused digits. These developmental defects were not consistent with current understanding of developmental signaling or previously described roles for ion channels. We found that phenotypes …

The Role Of Smpb In Licensing Tmrna Entry Into Stalled Ribosomes, Mickey R. Miller

Theses and Dissertations

Ribosomes translate the genetic information contained in mRNAs into protein by linking together amino acids with the help of aminoacyl-tRNAs. In bacteria, protein synthesis stalls when the ribosome reaches the 3'-end of truncated mRNA transcripts lacking a stop codon. Trans-translation is a conserved bacterial quality control process that rescues stalled ribosomes. Transfer-messenger RNA (tmRNA) and its protein partner SmpB mimic a tRNA by entering the A site of the ribosome and accepting the growing peptide chain. The ribosome releases the truncated mRNA and resumes translation on the tmRNA template. The open reading frame found on tmRNA encodes a peptide tag …

Investigation Of Water-Molecule Complexes And Their Catalytic Effect On Important Atmospheric Reactions, Taylor Scott Cline

Theses and Dissertations

This dissertation is a collection of works that investigates issues related to environmental chemistry. The first portion of this research explores the role of water vapor on the kinetics of important atmospheric reactions. Work is presented on the self-reaction of β-hydroxyethyl peroxy radical (β-HEP) and the catalytic increase in reaction rate by water vapor. β-HEP serves as a model system for investigating the possible role of water vapor in perturbing the kinetics and product branching ratio of atmospheric reactions of other alkyl peroxy radicals. The self-reaction rate coefficient of β-HEP was investigated between 276-296 K with 1.0 × 10^15 to …

Miniaturized Electrostatic Ion Beam Trap Mass Analyzer, Junting Wang

Theses and Dissertations

The electrostatic ion beam trap (EIBT) was designed by D. Zajfman during the previous decade. This ion trap combines many properties of the Fourier-transform ion cyclotron resonance (FTICR) mass analyzer and time-of-flight (TOF) mass analyzer. There are several advantages for the electrostatic ion beam trap. First, large mass-to-charge particles in an electrostatic field could be easier to analyze. Second, there is a folded flight path, which could make the mass analyzer smaller compared to conventional TOF mass analyzer. This principle of operation of this ion trap is analogous to an optical resonator. The ions are trapped in a voltage valley …

Identification And Differentiation Of Tier 1 Bacterial Agents Using Gas Chromatography-Mass Spectrometry, Dan Li

Theses and Dissertations

A simple method was developed for detection and differentiation of five Tier 1 bacterial agents, including Bacillus anthracis, Francisella tularensis, Yersinia pestis, Burkholderia pseudomallei and Burkholderia mallei as well as their closely related near neighbors by gas chromatography-mass spectrometry (GC-MS). Generally, different classes of compounds can be used as biomarkers for biowarfare agent detection, including nucleic acids (i.e., DNA or RNA), proteins (i.e., antibodies), carbohydrates (i.e., sugars), lipopolysaccharides, lipids (i.e., fatty acids) and small molecules. One-step thermochemolysis (TCM) was developed to provide GC-MS detectable biomarker signatures, including sugars, fatty acids and small molecules. Solid phase micro-extraction (SPME) was used for …

Bacterial Spores Remain Viable After Electrospray Charging And Desolvation, Sara Nielson Pratt

Theses and Dissertations

The electrospray survivability of B. subtilis spores and E. coli was tested in atmospheric mobility experiments. E. coli did not survive electrospray charging and desolvation, but B. subtilis did. Experimental conditions ensured that any surviving bacteria were charged, desolvated, and de-agglomerated. B. subtilis was also found to survive both positive and negative electrospray and subsequent introduction into vacuum conditions. Attempts were made to measure the charge distribution of viable B. subtilis spores using electrostatic deflection. From those experiments, it was found that either the spores do not become highly charged under the electrospray conditions used or only spores in a …

Isolation, Characterization And Synthesis Of Asthma Inducing Fungal Glycolipid And Analytical Method Development For Novel Antimicrobial Peptide Mimics, Vinod Chaudhary

Theses and Dissertations

NKT cells are an important part of human immune system and recognize a specific set of antigens called glycolipids. Only a handful of "natural" NKT cell antigens are known till date. Although NKT cells play a protective role against pathogenic organisms, imbalances in NKT cell functions are implicated in many diseases including asthma. Allergic asthma, a Th2 driven inflammation of airways, is primarily caused by inhalation of environmental allergens. In the last decade, inhaled allergen Aspergillus fumigatus has been under scrutiny for the presence of NKT cell antigens that might trigger asthma. We successfully isolated, characterized and synthesized a "natural" …

Synthesis And Immunological Evaluation Of Type I, Type Ii, And Gamma Delta Nkt Cell Antigens, Brian L. Anderson

Theses and Dissertations

The purpose of the immune system is to protect our bodies from infection. One way it accomplishes this task is through the presentation of foreign pathogens to NKT cells. After an antigen is presented to the T cell receptor, activated NKT cells quickly release soluble chemical signals, termed chemokines and cytokines, that modulate the response of the immune system. Due to the immunological relevance of NKT cell activation, we developed and synthesised non-natural analogs of immunostimulatory type I, II, and gamma delta NKT cell antigens. The immunological evaluations of these analogs resulted in identification of sulfatide as a gamma delta …

Quorum Sensing And Metabolism In Marine Environments, Amanda May

Doctoral Dissertations

Quorum sensing (QS) is a phenomenon that allows bacteria to communicate with each other. Small molecules known as autoinducers are synthesized and released by bacteria, and once enough members of the community are around to ensure survival, i.e. quorum, a phenotype, e.g. bioluminescence, is expressed. There are two types of QS molecules, intra- and inter-species.

S-4,5-Dihydroxy-2,3-pentanedione (DPD) is a byproduct of the activated methyl cycle which recycles methionine. This has led to the discussion as to whether DPD is a metabolic byproduct or is the interspecies signal as proposed previously. The detection and quantitation of DPD however, has not …

Towards The Use Of Time-Resolved X-Ray Crystallography In Mechanistic Studies Of Cytochrome C Nitrite Reductase From Shewanella Oneidensis, Matthew David Youngblut

Theses and Dissertations

A high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), and its characterization by a variety of methods, notably Laue crystallography, is reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein "Small Tetra-heme c" replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-C strain, generated approximately 20 mg crude ccNiR/L culture, compared with 0.5-1 mg/L for untransformed cells. Purified ccNiR exhibited nitrite and hydroxylamine reductase activities comparable to those of E. …

Spectroscopic Investigation Of The Interaction Of Fadh In Dna Photolyase With Uv-Damaged Dna, Kyle Luke Williams

Theses, Dissertations and Culminating Projects

Skin cancer is the most prevalent malignancy found in humans, with the diagnosis rate continuing to steadily increase. The primary cause of this disease is overexposure to harmful ultraviolet (UV) radiation from sunlight, which can induce damage to the nitrogenous bases in DNA via dimerization. The most prevalent UV-induced photoproducts in DNA are cyclobutane pyrimidine dimers (CPDs), most commonly between adjacent thymidines.

Organisms have implemented methods by which to repair these CPDs, the primary of which are nucleotide excision repair (NER) and photoreactivation by photolyases. Photolyases are blue-light activated flavoproteins that are more efficient at recognizing and repairing CPDs than …

Modeling Amyloid-Ss Self-Assembly : Stability Of On-Pathway Aggregate Formation, Andrew Kevin Mauro

Theses, Dissertations and Culminating Projects

Protein misfolding and concomitant aggregation towards amyloid formation is the underlying biochemical commonality among a wide range of human pathologies. Amyloid formation involves the conversion of proteins from their native monomeric states (intrinsically disordered or globular) to well-organized, fibrillar aggregates in a nucleation-dependent manner. Understanding the mechanism of aggregation is important not only to gain better insight into amyloid pathology but also to simulate and predict molecular pathways. One of the main impediments in doing so is the highly stochastic nature of interactions that complicates the development of meaningful insights. In this study, we have utilized a well-characterized intermediate along …