Chemistry & Chemical Biology Department Theses and Dissertations

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DNA Recognition and Cleavage by Phenyl-Benzimidazole Modified Gly-Gly-His-Derived Metallopeptides
(2010-04-08T16:20:15Z) Wang, Tianxiu; Long, Eric C. (Eric Charles); Blacklock, Brenda J.; Naumann, Christoph A.
Metallopeptides of the general form M(II)∙Gly1-Gly2-His induce DNA strand scission via minor groove interactions. This peptide system can serve as a nucleic acid-targeted cleavage agent – either as an appendage to other DNA binding agents, or as a stand alone complex. In an effort to further our knowledge of DNA recognition and cleavage, a novel series of phenyl-benzimidazole modified Gly-Gly-His-derived metallopeptides was synthesized via solid phase methods and investigated. The new systems allow the formation of additional contacts to the DNA minor groove through the incorporation of a DNA binding phenyl-benzimidazole moiety, thus strengthening the overall binding interaction and further stabilizing the metal complex-DNA association. In addition, how Lys side chains and an amidinium group influence the efficiency of DNA cleavage was also studied. DNA cleavage studies suggested that the phenyl-benzimidazole-modified Gly-Gly-His metallopeptides possess enhanced DNA cleavage abilities. In particular, when amidines are placed on the benzimidazole moieties, these moieties appeared to play an important role in increasing the DNA cleavage activity of the metal complex, most likely through an enhanced electrostatic attraction to the DNA.
Solid-Phase Synthesis of N-Carboxyalkyl Unnatural Amino Acids
(2011-03-09) Fischer, Lindsey Gayle; O'Donnell, Martin J.; Minto, Robert E.; Scott, William L.
A novel route has been developed for the solid-phase synthesis of N-carboxyalkyl unnatural amino acids as potential metalloprotease inhibitors. The key step involves a nitrogen alkylation of resin-bound amino acids with -bromoesters. Alkylation of the benzophenone imine of glycine on Wang resin was used to introduce unnatural amino acid side chains onto the resin-bound glycine. The benzyl -bromoesters [BrCH(R2)CO2Bn], starting materials for the C-N bond construction, were prepared in solution by diazotization of naturally-occurring amino acids to form the -bromoacids, followed by benzylation of the carboxylic acid to form the benzyl -bromoesters. N-Alkylation of the resin-bound, unnatural amino acids with the benzyl -bromoesters and subsequent cleavage from resin gave the benzyl ester monoacid intermediates. Exploration of reverse-phase cyano-silica gel chromatography and preparative liquid chromatography provided effective purification of the benzyl ester intermediates. Hydrolysis of the analytically pure benzyl ester monoacids afforded clean products as the diacids. The two points of variation introduced through the two on-resin alkylation steps, C-alkylation of the benzophenone imine of glycine and N-alkylation with the benzyl -bromoesters, allow for the combinatorial synthesis of a library of target compounds.
Evaluation of the Odor Compounds Sensed by Explosive-Detecting Canines
(2011-03-09) Lotspeich, Erica H.; Goodpaster, John V. (John Vincent); Siegel, Jay A.; Deo, Sapna K.
Trained canines are commonly used as biological detectors for explosives; however, there are some areas of uncertainty that have led to difficulties in canine training and testing. Even though a standardized container for determining the accuracy of explosives-detecting canines has already been developed, the factors that govern the amount of explosive vapor that is present in the system are often uncertain. This has led to difficulties in comparing the sensitivity of canines to one another as well as to analytical instrumentation, despite the fact that this container has a defined headspace and degree of confinement of the explosive. For example, it is a common misconception that the amount of explosive itself is the chief contributor to the amount of odor available to a canine. In fact, odor availability depends not only on the amount of explosive material, but also the explosive vapor pressure, the rate with which the explosive vapor is transported from its source and the degree to which the explosive is confined. In order to better understand odor availability, headspace GC/MS and mass loss experiments were conducted and the results were compared to the Ideal Gas Law and Fick’s Laws of Diffusion. Overall, these findings provide increased awareness about availability of explosive odors and the factors that affect their generation; thus, improving the training of canines. Another area of uncertainty deals with the complexity of the odor generated by the explosive, as the headspace may consist of multiple chemical compounds due to the extent of explosive degradation into more (or less) volatile substances, solvents, and plasticizers. Headspace (HS) and solid phase microextraction (SPME) coupled with gas chromatography/mass spectrometry (GC/MS) were used to determine what chemical compounds are contained within the headspace of an explosive as well as NESTT (Non-Hazardous Explosive for Security Training and Testing) products. This analysis concluded that degradation products, plasticizers, and taggants are more common than their parent explosive.
Biochemical applications of DsRed-monomer utilizing fluorescence and metal-binding affinity
(2011-03-09) Goulding, Ann Marie; Deo, Sapna K.; Oh, Kyungsoo; Davidson, Amy; Simpson, Garth
The discovery and isolation of naturally occurring fluorescent proteins, FPs, have provided much needed tools for molecular and cellular level studies. Specifically the cloning of green fluorescent protein, GFP, revolutionized the field of biotechnology and biochemical research. Recently, a red fluorescent protein, DsRed, isolated from the Discosoma coral has further expanded the pallet of available fluorescent tools. DsRed shares only 23 % amino acid sequence homology with GFP, however the X-ray crystal structures of the two proteins are nearly identical. DsRed has been subjected to a number of mutagenesis studies, which have been found to offer improved physical and spectral characteristics. One such mutant, DsRed-Monomer, with a total of 45 amino acid substitutions in native DsRed, has shown improved fluorescence characteristics without the toxic oligomerization seen for the native protein. In our laboratory, we have demonstrated that DsRed proteins have a unique and selective copper-binding affinity, which results in fluorescence quenching. This copper-binding property was utilized in the purification of DsRed proteins using copper-bound affinity columns. The work presented here has explored the mechanism of copper-binding by DsRed-Monomer using binding studies, molecular biology, and other biochemical techniques. Another focus of this thesis work was to demonstrate the applications of DsRed-Monomer in biochemical studies based on the copper-binding affinity and fluorescence properties of the protein. To achieve this, we have focused on genetic fusions of DsRed-Monomer with peptides and proteins. The work with these fusions have demonstrated the feasibility of using DsRed-Monomer as a dual functional tag, as both an affinity tag and as a label in the development of a fluorescence assay to detect a ligand of interest. Further, a complex between DsRed-Monomer-bait peptide/protein fusion and an interacting protein has been isolated taking advantage of the copper-binding affinity of DsRed-Monomer. We have also demonstrated the use of non-natural amino acid analogues, incorporated into the fluorophore of DsRed-Monomer, as a tool for varying the spectral properties of the protein. These mutations demonstrated not only shifted fluorescence emission compared to the native protein, but also improved extinction coefficients and quantum yields.
Instrumental and Statistical Methods for the Comparison of Class Evidence
(2011-03-09) Liszewski, Elisa Anne; Goodpaster, John V. (John Vincent); Siegel, Jay A.; Deo, Sapna K.
Trace evidence is a major field within forensic science. Association of trace evidence samples can be problematic due to sample heterogeneity and a lack of quantitative criteria for comparing spectra or chromatograms. The aim of this study is to evaluate different types of instrumentation for their ability to discriminate among samples of various types of trace evidence. Chemometric analysis, including techniques such as Agglomerative Hierarchical Clustering, Principal Components Analysis, and Discriminant Analysis, was employed to evaluate instrumental data. First, automotive clear coats were analyzed by using microspectrophotometry to collect UV absorption data. In total, 71 samples were analyzed with classification accuracy of 91.61%. An external validation was performed, resulting in a prediction accuracy of 81.11%. Next, fiber dyes were analyzed using UV-Visible microspectrophotometry. While several physical characteristics of cotton fiber can be identified and compared, fiber color is considered to be an excellent source of variation, and thus was examined in this study. Twelve dyes were employed, some being visually indistinguishable. Several different analyses and comparisons were done, including an inter-laboratory comparison and external validations. Lastly, common plastic samples and other polymers were analyzed using pyrolysis-gas chromatography/mass spectrometry, and their pyrolysis products were then analyzed using multivariate statistics. The classification accuracy varied dependent upon the number of classes chosen, but the plastics were grouped based on composition. The polymers were used as an external validation and misclassifications occurred with chlorinated samples all being placed into the category containing PVC.
Studies in pressurized Planar Electrochromatography
(2011-08-19) Woodward, Scott D.; Muhoberac, Barry; Nurok, David; Sardar, Rajesh
This thesis describes separations performed by Pressurized Planar Electrochromatography (PPEC), which is a chromatographic method developed at IUPUI. In PPEC the mobile phase is driven by electroosmotic flow, while the system is pressurized to allow temperature control. This results in a highly efficient chromatographic system that has several attractive attributes including the ability to separate multiple samples simultaneously. The first three chapters of the thesis describe the relationship of PPEC to other forms of chromatography, the theoretical background of PPEC, the PPEC apparatus, including the plate holders used, and the different manipulations involved in preparing a plate for a PPEC run. The fourth chapter describes two short studies. The first demonstrates that a very fast separation of steroids on a high efficiency sorbent layer can be effected by PPEC. This is illustrated by the separation of six steroids in three minutes on a Superspher layer, with an efficiency of over 100,000 plates per meter. The second study attempted to improve the efficiency of separation by imposing a temperature gradient. The study was not successful, possibly due to Joule heating within the layer overriding the temperature gradient. The final chapter of the thesis describes two different studies on separating peptides by PPEC. The first study was performed on a bonded C18 sorbent layer that was treated with Brij-35, which is a non-ionic surfactant that prevents irreversible adsorption of the peptides to the sorbent surface while allowing electroosmotic flow. The variables involved in preparing the plates by soaking in a Brij-35 solution were investigated as well as the variables for PPEC (temperature, pressure, electrical potential, and mobile phase composition and pH). It was possible to separate six peptides in eight minutes using this approach. The second study used monolithic sorbent layers prepared by Dr. Frantisek Svec of Lawrence Berkeley National Laboratory. Separations were by conventional PPEC on charged monoliths and by electrophoresis on neutral monoliths. The same variables for PPEC, listed in the above paragraph, were investigated for the monolith study. It was possible to separate six peptides in two minutes on neutral monoliths and in one minute on negatively charged monoliths.
Design of Biomembrane-Mimicking Substrates of Tunable Viscosity to Regulate Cellular Mechanoresponse
(2012-03-20) Minner, Daniel Eugene; Naumann, Christoph A.; Long, Eric C. (Eric Charles); Suter, Daniel; Shah, Kavita
Tissue cells display mechanosensitivity in their ability to discern and respond to changes in the viscoelastic properties of their surroundings. By anchoring and pulling, cells are capable of translating mechanical stimuli into a biological response through a process known as mechanotransduction, a pathway believed to critically impact cell adhesion, morphology and multiple cellular processes from migration to differentiation. While previous studies on polymeric gels have revealed the influence of substrate elasticity on cellular shape and function, a lack of suitable substrates (i.e. with mobile cell-substrate linkers) has hindered research on the role of substrate viscosity. This work presents the successful design and characterization of lipid-bilayer based cell substrates of tunable viscosity affecting cell-substrate linker mobility through changes in viscous drag. Here, two complementary membrane systems were employed to span a wide range of viscosity. Single polymer-tethered lipid bilayers were used to generate subtle changes in substrate viscosity while multiple, polymer-interconnected lipid bilayer stacks were capable of producing dramatic changes in substrate viscosity. The homogeneity and integrity of these novel multibilayer systems in the presence of adherent cells was confirmed using optical microscopy techniques. Profound changes in cellular growth, phenotype and cytoskeletal organization confirm the ability of cells to sense changes in viscosity. Moreover, increased migration speeds coupled with rapid area fluctuations suggest a transition to a different migration mode in response to the dramatic changes in substrate viscosity.
Instrumental and Chemometric Analysis of Automotive Clear Coat Paints by Micro Laser Raman and UV Microspectrophotometry
(2012-07-19) Mendlein, Alexandra Nicole; Siegel, Jay A.; Goodpaster, John V. (John Vincent); Li, Lei
Automotive paints have used an ultraviolet (UV) absorbing clear coat system for nearly thirty years. These clear coats have become of forensic interest when comparing paint transfers and paint samples from suspect vehicles. Clear coat samples and their ultraviolet absorbers are not typically examined or characterized using Raman spectroscopy or microspectrophotometry (MSP), however some past research has been done using MSP. Chemometric methods are also not typically used for this characterization. In this study, Raman and MSP spectra were collected from the clear coats of 245 American and Australian automobiles. Chemometric analysis was subsequently performed on the measurements. Sample preparation was simple and involved peeling the clear coat layer and placing the peel on a foil-covered microscope slide for Raman or a quartz slide with no cover slip for MSP. Agglomerative hierarchical clustering suggested three classes of spectra, and principal component analysis confirmed this. Factor loadings for the Raman data illustrated that much of the variance between spectra came from specific regions (400 – 465 cm-1, 600 – 660 cm-1, 820 – 885 cm-1, 950 – 1050 cm-1, 1740 – 1780 cm-1, and 1865 – 1900 cm-1). For MSP, the regions of highest variance were between 230 – 270 nm and 290 – 370 nm. Discriminant analysis showed that the three classes were well-differentiated with a cross-validation accuracy of 92.92% for Raman and 91.98% for MSP. Analysis of variance attributed differentiability of the classes to the regions between 400 – 430 cm-1, 615 – 640 cm-1, 825 – 880 cm-1, 1760 – 1780 cm-1, and 1860 – 1900 cm-1 for Raman spectroscopy. For MSP, these regions were between 240 – 285 nm and 300 – 370 nm. External validation results were poor due to excessively noisy spectra, with a prediction accuracy of 51.72% for Raman and 50.00% for MSP. No correlation was found between the make, model, and year of the vehicles using either method of analysis.
Regulating Lipid Organization and Investigating Membrane Protein Properties in Physisorbed Polymer-tethered Membranes
(2012-08-07) Siegel, Amanda P.; Naumann, Christoph A.; Minto, Robert; Thompson, David H. Pai; Ritchie, Kenneth
Cell membranes have remarkable properties both at the microscopic level and the molecular level. The current research describes the use of physisorbed polymer-grafted lipids in model membranes to investigate some of these properties on both of these length scales. On the microscopic scale, plasma membranes can be thought of as heterogenous thin films. Cell membranes adhered to elastic substrates are capable of sensing substrate/film mismatches and modulating their membrane stiffness to more closely match the substrate. Membrane/substrate mismatch can be modeled by constructing lipopolymer-enriched lipid monolayers with different bending stiffnesses and physisorbing them to rigid substrates which causes buckling. This report describes the use of atomic force microscopy and epimicroscopy to characterize these buckled structures and to illustrate the use of the buckled structures as diffusion barriers in lipid bilayers. In addition, a series of monolayers with varying bending stiffnesses and thicknesses are constructed on rigid substrates to analyze changes in buckling patterns and relate the experimental results to thin film buckling theory. On the molecular scale, plasma membranes can also be thought of as heterogeneous mixtures of lipids where the specific lipid environment is a crucial factor affecting membrane protein function. Unfortunately, heterogeneities involving cholesterol, labeled lipid rafts, are small and transient in live cells. To address this difficulty, the present work describes a model platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains into which transmembrane proteins are incorporated (αvβ3, and α5β1integrins). This flexible platform enables the use of confocal fluorescence fluctuation spectroscopy to quantitatively probe the effect of cholesterol concentrations and the binding of native ligands (vitronectin and fibronectin for αvβ3, and α5β1) on protein oligomerization state and on domain-specific protein sequestration. In particular, the report shows significant ligand-induced integrin sequestration with a low level of dimerization. Cholesterol concentration increases rate of dimerization, but only moderately. Ligand addition does not affect rate of dimerization in either system. The combined results strongly suggest that ligands induce changes to integrin conformation and/or dynamics without inducing changes in integrin oligomerization state, and in fact these ligand-induce conformational changes impact protein-lipid interactions.
Luminescence-Based MicroRNA Detection Methods
(2012-08-27) Cissell, Kyle A.; Deo, Sapna K.; Long, Eric C. (Eric Charles); Simpson, Garth; Mao, Chengde
MicroRNAs (miRNA) are short, 18-24 nucleotide long noncoding RNAs. These small RNAs, which are initially transcribed in the nucleus, are transported into the cell cytoplasm where they regulate protein translation either through direct cleavage of mRNA, or indirect inhibition through binding to mRNA and disrupting the protein translation machinery. Recently, miRNAs have gained much attention due to their implication in numerous diseases and cancers. It has been found that heightened or lowered levels of miRNA in diseased cells vs. healthy cells are linked to disease progression. It is therefore immensely important to be able to detect these small molecules. Current detection methods of Northern blotting, microarrays, and qRT-PCR suffer from drawbacks including low sensitivity, a lack of simplicity, being semi-quantitative in nature, time-consuming, and requiring expensive instruments. This work aims to develop novel miRNA technologies which will address these above problems. Bioluminescent labels are promising alternatives to current methods of miRNA detection. Bioluminescent labels are relatively small, similar in size to fluorescent proteins, and they emit very intense signals upon binding to their substrate. Bioluminescent labels are advantageous to fluorescent labels in that they do not require an external excitation source, rather, the excitation energy is supplied through a biochemical reaction. Therefore, background signal due to excitation is eliminated. They also have the advantage of being produced in large amounts through bacterial expression. Four miRNA detection methods are presented which utilize luminescence-based methods. Three employ Renilla luciferase, a bioluminescent protein, and one is based on fluorescence. The presented methods are capable of detecting miRNA from the picomole (nanomolar) level down to the femtomole (picomolar) level. These methods are rapid, sensitive, simple, and quantitative, can be employed in complex matrices, and do not require expensive instruments. All methods are hybridization-based and do not require amplification steps.

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