Biochemistry, Biophysics, and Structural Biology Commons^™

Rapid And Noncontaminating Sampling System For Trace Elements In Global Ocean Surveys, Gregory A. Cutter, Kenneth W. Bruland

OES Faculty Publications

A system for the rapid and noncontaminating sampling of trace elements with volumes of up to 36 L per depth and including the dissolved and particulate phases has been developed for ocean sections that are a crucial part of programs such as International GEOTRACES. The system uses commercially available components, including an aluminum Seabird Carousel with all titanium pressure housings for electronics and sensors to eliminate zinc sacrificial anodes and holding twenty-four 12 L GO-FLO bottles, and a 7500 m, 14 mm Vectran conducting cable (passing over an A-frame with nonmetallic sheave) spooled onto a traction winch. The GO-FLO bottles …

Controls On Dissolved Cobalt In Surface Waters Of The Sargasso Sea: Comparisons With Iron And Aluminum, R. U. Shelley, Peter N. Sedwick, T. S. Bibby, P. Cabedo-Sanz, T. M. Church, R. J. Johnson, A. I. Macey, C. M. Marsay, E. R. Sholkovitz, S. J. Ussher, P. J. Worsfold, M. C. Lohan

OES Faculty Publications

Dissolved cobalt (dCo), iron (dFe) and aluminum (dAl) were determined in water column samples along a meridional transect (∼31°N to 24°N) south of Bermuda in June 2008. A general north-to-south increase in surface concentrations of dFe (0.3-1.6 nM) and dAl (14-42 nM) was observed, suggesting that aerosol deposition is a significant source of dFe and dAl, whereas no clear trend was observed. for near-surface dCo concentrations. Shipboard aerosol samples indicate fractional solubility values of 8-100% for aerosol Co, which are significantly higher than corresponding estimates of the solubility of aerosol Fe (0.44-45%). Hydrographic observations and analysis of time series rain …

Bio-Logic Builder: A Non-Technical Tool For Building Dynamical, Qualitative Models, Tomáš Helikar, Bryan Kowal, Alex Madrahimov, Manish Shrestha, Jay Pedersen, Kahani Limbu, Ishwor Thapa, Thaine Rowley, Rahul Satalkar, Naomi Kochi, John Konvalina, Jim A. Rogers

Department of Biochemistry: Faculty Publications

Computational modeling of biological processes is a promising tool in biomedical research. While a large part of its potential lies in the ability to integrate it with laboratory research, modeling currently generally requires a high degree of training in mathematics and/or computer science. To help address this issue, we have developed a web-based tool, Bio- Logic Builder, that enables laboratory scientists to define mathematical representations (based on a discrete formalism) of biological regulatory mechanisms in a modular and non-technical fashion. As part of the user interface, generalized ‘‘biologic’’ modules have been defined to provide users with the building blocks for …

Modes And Mechanisms Of Hfq Mediated Stress Regulation In Bacteria, Nilshad Nilam Salim

Wayne State University Dissertations

To survive bacteria must be able to respond to its ever-changing environmental conditions. sRNAs have been implicated in a variety of stress-response pathways that help bacterial systems modulate gene expression. The RNA binding protein Hfq facilities this process by, helping sRNA to base pair with its target mRNAs to initiate gene regulation. A common feature of Hfq-mediated gene regulation is the network-based organization where a single sRNA can control multiple messages to promote integrated response to stress. Current mechanistic models that are present to describe Hfq functions cannot explain the complexity at which Hfq performs gene regulation. In this work …

Drug Resistance Mechanisms And Drug Design Strategies For Human Immunodeficiency Virus And Hepatitis C Virus Proteases, Yong Wang

Wayne State University Dissertations

The antiviral drug development has improved steadily to treat the infections of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) which represent heavy public health burdens. The viral protease plays an indispensable role in viral maturation and therefore becomes one of the most important targets for drug design. Nine HIV-1 protease inhibitors and two HCV protease inhibitors have been developed and approved by the U.S. Food and Drug Administration. However, mutations in the protease decrease reduce the efficacy the drugs. In this study, the enzyme assays indicate that darunavir and tipranavir exhibit the most potent inhibition against the multi-drug …

Single Molecule Studies Of Rna-Target Interactions, Sharla Leann Wood

Wayne State University Dissertations

We have used FRET, single molecule spectroscopy, and several other biophysical techniques to study how the folding of RNA allows it to perform its various functions by recognizing and binding a target ligand. We have shown that the c-di-GMP riboswitch undergoes a large, global conformational change upon binding of the ligand. The folding dynamics of the c-di-GMP riboswitch upon Mg2+ binding help to pre-organize the aptamer for efficient ligand binding and ultimately efficient gene expression. We have also investigated two instances, a fluorophore-binding aptamer and molecular beacon, where the folding of RNA can be used to detect an analyte. We …

The Speed Of Sound And Attenuation Of An Iec Agar-Based Tissue-Mimicking Material For High Frequency Ultrasound Applications, Chao Sun, Stephen Pye, Jacinta Browne, Anna Janeczko, Bill Ellis, Mairead Butler, Vassilis Sboros, Adrain Thomson, Mark Brewin, Charles Earnshaw, Carmel Moran

Articles

No abstract provided.

Quantitative Reagent-Free Detection Of Fibrinogen Levels In Human Blood Plasma Using Raman Spectroscopy, Kelvin Poon, Fiona Lyng, Peter Knief, Orla L. Howe, Aidan Meade, James Curtin, Hugh Byrne, Joseph Vaughan

Articles

Fibrinogen assays are commonly used as part of clinical screening tests to investigate haemorrhagic states, for detection of disseminated intravascular coagulation and as a predictor of a variety of cardiovascular events. The Clauss assay, which measures thrombin clotting time, is the most commonly used method for measuring fibrinogen levels. Nevertheless, inconsistencies are present in inter-manufacturer reagent sources, calibration standards and methodologies. Automated coagulation analysers, which measure changes in optical density during the prothrombin time (PT-Fg), have found use in many hospitals. However, the PT-Fg method is found to give falsely elevated values due to varying choices of calibrants, reagents and …

Disrupting Cxcr2 Macromolecular Complex Pdz-Domain Interactions During Inflammatory Chemotaxis, Marcello Castelvetere

Wayne State University Theses

Neutrophils are the body's first responders to inflammation, being the most abundant white blood cell type in circulation and they quickly initiate an immune response through chemokine signaling. Inflammatory chemokines signal via their receptor CXCR2, which initiates an inflammatory response, recruiting leukocytes to sites of inflammation. Chemokine signaling is important for proper host protection, yet uncontrolled activity is responsible for a variety of pathological conditions: including rheumatoid arthritis, ischemia-reperfusion injury, arteriosclerosis, multiple sclerosis, psoriasis, inflammatory bowel disease, and allergic reactions.

In this report I show a CXCR2 macromolecular signaling complex exists in neutrophils, containing NHERF1 and PLCβ2. I also demonstrate …

Modulation Of Anti-Tumor Immune Response By Tgf-Β-Inducible Early Gene 1 (Tieg1), Andi Cani

Wayne State University Theses

Cancer immunotherapy has had limited clinical efficacy partly because regulatory T cells (Treg) suppress the immune response to tumor-associated antigens. Inducible regulatory T cells (iTreg), which are converted from naïve CD4 T cells by TGF-β, an abundant cytokine in the tumor microenvironment, may contribute to this immune suppression. Induction of Foxp3 by TGF-β is mediated by the transcription factor TIEG1 and abrogation of this protein prevents Foxp3 expression. We are testing the hypothesis that blockade of TIEG1 to prevent iTreg conversion will enhance immune response in DNA vaccination to the tumor associated antigen Her-2. Wild type and TIEG1 knockout mice …

The Spliceosomal Protein Prp8 Stabilizes A Compact Conformation Of The U2-U6 Complex, Subasinghe Appuhamilage Lemintha Imali Subasinghe

Wayne State University Theses

The spliceosome is a large, RNA-protein complex that catalyzes pre-mRNA splicing during mRNA maturation. The RNA components (small nuclear RNA; snRNAs) of the spliceosome have been well studied and are believed to be involved in the splicing catalysis. Although proteins are essential for splicing, they may not be directly involved in catalysis. Among hundreds of proteins, Prp8 is the only protein that interacts with all of the catalytically important snRNAs. Therefore, it is hypothesized that Prp8 may catalyze splicing either by directly participating in catalysis or by stabilizing the conformation of the catalytically active spliceosome. In order to test whether …

Opposing Roles Of The Μ-Opioid And Nociceptin/Orphanin Fq Receptors In Oligodendrocyte Development And Myelination, Allison Vestal-Laborde

Theses and Dissertations

While the classical function of myelin is to facilitate saltatory conduction, this membrane and the myelin-making oligodendrocytes (OLGs) are now recognized as regulators of plasticity and remodeling in the central nervous system (CNS). Thus, OLG maturation and myelination are highly vulnerable processes along CNS development. We previously showed that rat brain myelination is altered by perinatal exposure to buprenorphine, an opioid analogue in clinical trials for the treatment of pregnant opioid addicts. We now found that the in vivo effects on myelination could result from direct alteration in the balance between μ-opioid receptor (MOR) and nociceptin/orphanin FQ receptor (NOPR) activities …

Ultraviolet Resonance Raman Spectroscopy For Characterization Of Rna Structure And Thermodynamics, Joseph Dustin Handen

Legacy Theses & Dissertations (2009 - 2024)

Raman spectroscopy is a technique well suited for the study of biological molecules such as proteins, peptides, or RNAs. By utilizing an ultraviolet resonance Raman (UVRR) instrument, we are afforded a tremendous increase in sensitivity because of resonance enhancement. Additionally, this technique does not require any labeling. Moreover, this technique is better suited for studying biological systems than infrared absorption due to reduced interference from water. In this study, we apply techniques we have developed for the study of protein and peptide fibrillation to a model RNA homodimer. We demonstrate that UVRR spectroscopy is uniquely suited to monitoring the free …

A Study Of The Effects Of Inosine Incorporation Into Dna Due To Defects In Purine Biosynthesis In Escherichia Coli, Jonathan Spence Church

Legacy Theses & Dissertations (2009 - 2024)

Deamination of purine bases can result in the formation of xanthine and hypoxanthine which can be miscoding and mutagenic in DNA. There are several mechanisms for the introduction of deaminated bases into DNA including simple hydrolysis, nitrosative chemistry and through the action of deaminase enzymes. A fourth method was recently presented which describes how deaminated purines can be incorporated into DNA due to defects in purine biosynthesis. Using fluctuation analysis, spontaneous mutation rates were studied in bacterial mutants that were deficient in specific genes involved in purine biosynthesis and dNTP precursor pool maintenance, including purA (adenylosuccinate synthetase), guaA (GMP synthetase), …

Structural And Functional Characterization Of Dna Polymerase Ss Mutator Mutants, Sneha Rangarajan

Legacy Theses & Dissertations (2009 - 2024)

DNA Polymerase ß (polß) plays a crucial role in repairing damaged DNA in a process called Base Excision Repair (BER). BER is a major pathway of DNA repair, making this system absolutely vital for maintaining genomic integrity. Recent studies estimate 30% of human tumors to contain polß variants that led us to believe that there is a high degree of association between mutations in polß and cancer. In this pathway, after recognition and excision of the damaged base, the DNA is cleaved at an apurinic (AP) site by AP endonuclease leaving behind a 3' hydroxyl and 5' deoxyribose phosphate (dRP). …

Selection Of Chemically Modified Rna Aptamers Against The Glua2q Flop Ampa Receptor, Hyojung Seo

Legacy Theses & Dissertations (2009 - 2024)

The á-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors play an important role in neurotransmission and synaptic plasticity in the central nervous system. Excessive activity of these receptors has been implicated in some neurodegenerative diseases, such as Parkinson's disease, ischemic stroke and amyotrophic lateral sclerosis (ALS). AMPA receptor inhibitors are therefore drug candidates for potential treatment of these neurological disorders and diseases. The objective of my MS thesis work is to develop a stable aptamer so that it can be tested and used in vivo where the aptamer must be exposed to ribonucleases. To do this, I prepared a library containing RNAs with 2'-Fluoro …

Characterization Of The Calmodulin-Ryanodine Receptor Interaction By Cryo-Electron Microscopy, Xiaojun Huang

Legacy Theses & Dissertations (2009 - 2024)

Ryanodine receptor (RyR) is a key player in excitation-contraction coupling (E-C coupling). Calmodulin (CaM) is one of the important regulatory factors of RyR. Two mammalian RyR isoforms, RyR1 and RyR2, are highly enriched in skeletal and cardiac muscle, respectively. Apo-calmodulin weakly activates RyR1 but inhibits RyR2, whereas Ca2+-calmodulin inhibits both the isoforms. Previous cryo-electron microscopy studies showed distinctly different binding locations on RyR1 for the two states of calmodulin. However, recent studies employing fluorescence resonance energy transfer appeared to challenge these findings. In chapter 1, using cryo-electron microscopy, we have determined that a mutant calmodulin, which is incapable of binding …

The Role Of Chromatin And Cofactors In The Transcriptional Memory Effect Exerted In Saccharomyces Cerevisiae, Emily Leigh Paul

Legacy Theses & Dissertations (2009 - 2024)

Abf1 and Rap1 are functionally similar general regulatory factors (GRFs) found in Saccharomyces cerevisiae . Abf1, in its role as a transcriptional activator, exerts a memory effect on some genes under its control. This effect results in transcription levels remaining steady when Abf1 dissociates from its binding site in a conditional mutant. In contrast, Rap1 fails to elicit the same effect on its regulatory targets. Transcriptional memory effects have been observed in many fields of study, including immunology, cancer, and stem cells, and conservation of transcription machinery will allow studies in yeast to be applied to higher organisms.

Inhibition Of Glutamate Receptors By Constructing Bipartite Rna Aptamers, Jeffrey Hebert

Legacy Theses & Dissertations (2009 - 2024)

The relationship of excessive activity of AMPA-type glutamate receptors, and cell death, has long provided researchers a means of investigating neurodegenerative disorders, such as Parkinson's Disease (PD) and amyotrophic lateral sclerosis (ALS). Inhibitors of AMPA receptor channels, including chemical and nucleic acid molecules such as RNA aptamers, have served as potential therapeutic agents and treatment of neurodegenerative disorders. In this study, building bipartite aptamers to enhance inhibitory potency, as compared with a monomeric aptamer of AMPA receptor, is described. An enhanced potency is due, at least in part; to the proximity effect in bipartite structures or binding of a monomeric …

Sirtuin 6 : A Review Of Biological Affects And Potential Therapeutic Properties, Jade Beauharnois

Legacy Theses & Dissertations (2009 - 2024)

ABSTRACT: Sirtuins, possessing either histone deacetylase or mono-ribosyltransferase activity, regulate important biological pathways in bacteria, archaea and eukaryotes. The term Sir2 comes from the yeast 'silent mating-type information regulation 2' gene, a gene directing cellular regulation in yeast. Sirtuins have been implicated in aging, regulation of transcription, apoptosis and stress resistance, in addition to energy efficiency and metabolism. In mammals, a variety of sirtuin family members are genetically encoded to include sirtuins 1-7 (SIRT1-SIRT7). SIRT6 is a human sirtuin of interest in a variety of research areas, influencing the genomic instability, metabolic defects and degenerative pathologies associated with aging. Until …

Structure And Function Of Non-Coding Regulatory Rna Domains, Nakesha L. Smith

Legacy Theses & Dissertations (2009 - 2024)

Ribonucleic acid (RNA) has been found to be a very versatile molecule, exhibiting countless functions and can act as a catalyst in biochemical reactions. These functions have typically been attributed to the unique structures that it forms. Novel non-coding RNAs capable of regulating gene expression are still being discovered, and the scope of the RNA world is still being uncovered. The structure function relationship of two different types of non-coding RNA has been investigated: riboswitches and sxRNAs. UV-monitored thermal denaturation experiments, Nuclear Magnetic Resonance spectroscopy, native gel electrophoresis and an in vivo luciferase assay were used to investigate the structural …

The Role Of Ess1 In Survival, Morphogenetic Switching And Transcription In The Fungal Pathogen Candida Albicans, Dhanushki Poornima Samaranayake

Legacy Theses & Dissertations (2009 - 2024)

Candida albicans is a fungal pathogen that causes serious infections among immune-compromised patients and premature infants. C. albicans can become drug resistant, therefore, identifying new antifungal drug targets is an important goal. Here, we study a peptidyl-prolyl cis/trans isomerase called Ess1 as a potential drug target. Ess1 is conserved among pathogenic fungi, and therefore, potential inhibitors of Ess1 should display a broad spectrum of activity. We confirm that Ess1 is essential for growth in Candida albicans, but unlike the previously published find, deleting one copy of the C. albicans ESS1 gene did not affect morphogenetic switching. However, further reducing activity …

Mechanism Of Inhibition Of The Glua2 Receptors By N-3 Derivatives Of 2,3-Benzodiazepines With C-4 Methyl Group, Congzhou Wang

Legacy Theses & Dissertations (2009 - 2024)

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are one of the three subtypes of ionotropic glutamate receptors. AMPA receptors mediate fast synaptic neurotransmission in the central nervous system (CNS). Over-activation of calcium permeable AMPA receptors causes intracellular calcium overload, which leads to neurodegeneration and cell death. As such, AMPA receptors have been implicated in a number of neurological disorders and diseases, such as epilepsy, amyotrophic lateral sclerosis (ALS), and Parkinson's disease. 2,3-Benzodiazepine derivatives (or GYKI compounds) are a group of structurally similar compounds synthesized as inhibitors of AMPA receptors, and they have been used as potential drug candidates for the treatment of various …

Interactions Of The Human Immunodeficiency Virus Type 1 Nucleocapsid Protein With Non-Hiv-Derived Nucleic Acids : Implications For The Viral Replication Cycle, Abhijit Padmakar Jadhav

Legacy Theses & Dissertations (2009 - 2024)

During attempts to develop aptamers that bind to the nucleocapsid protein of HIV-1 (i.e. NCp7) with high affinity, a heretofore unreported property of the protein--the ability to mediate degradation of nucleic acids, was discovered. Using λ DNA as a model nucleic acid system, it was shown that NCp7-mediated degradation of nucleic acids is non-specific, depends on incubation time, the concentration of NCp7, and the presence of divalent and monovalent cations. It was further demonstrated that degradation can be abrogated if NCp7 is pre-incubated with NCp7-inhibitors. Lyophilization of NCp7 can induce irreversible changes in its secondary structure that result in loss …

Dna Repair Fidelity And Cancer : Structural And Kinetic Insights From Dna Polymerase Beta Mutator Variants, Chelsea Lynne Gridley

Legacy Theses & Dissertations (2009 - 2024)

DNA polymerases are essential for genome replication and DNA repair in all living organisms. Precise DNA replication is critical for the preservation of genomic stability. Any insult, endogenous/exogenous, to cellular DNA requires properly functioning repair polymerases. In eukaryotes, DNA polymerase beta, a small enzyme (39 kDa), plays an important role in DNA repair during the base excision repair pathway. Pol beta catalyzes the incorporation of nucleotides in small stretches (1-6 nucleotides) of damaged double-stranded DNA. Should gap-filling synthesis by pol beta be compromised, mutations in genomic DNA accumulate, which are frequently linked to human diseases, including cancers. For this reason, …

Amyloid Fibril Polymorphism : Structure, Supramolecular Chiraliy And Spontaneous Interconversion, Dzmitry Kurouski

Legacy Theses & Dissertations (2009 - 2024)

Specific protein aggregation has been linked to more than 25 severe human maladies including prion, Alzheimer's, and Parkinson's diseases. These important malfunctions are often referred to as 'conformational' disorders and result from the conversion of a normal isoform of a protein into a specific b-sheet rich polymeric amyloid form. This work elaborates a comprehensive characterization of amyloids and dedicated to the investigation of the fibril polymorphism using advanced microscopic tools, such as Atomic Force and Scanning Electron microcopies, together with several vibrational spectroscopy techniques, such as Raman, Infrared and Vibrational Circular Dichroism. A new type of protein folding-aggregation phenomenon, spontaneous …

Gld-1 Represses Its Puf Mrna Targets Prior To/At Initiation Of Translation In The C.Elegans Germline, Gautham Sarathy

Legacy Theses & Dissertations (2009 - 2024)

The C.elegans germline offers an ideal system to study posttranscriptional regulation of gene expression as it is a major mechanism through which the control over gene expression is achieved. GLD-1 (defective in GermLine Development) is a maxi-KH motif containing RNA binding protein that controls various aspects of germline development from decision over germcell proliferation vs. meiotic entry to the production of mature gametes suggesting that GLD-1 likely controls many mRNA targets.

Fret Studies Of Dna Binding By Lambda Cro Variants, Lei Wang

Electronic Theses and Dissertations

Proteins bind to DNA to form switches and circuits that control the flow of genetic information from DNA sequence to the physical components of cells and organisms. The dynamic performance of genetic switches depends on the coupling of the comprocesses of transcription and translation to protein-specific folding, assembly and binding reactions. For many model circuits protein dimerization and DNA binding are fast relative to the time scale of transcription and translation and thus amenable to modeling by statistical thermodynamics. In these cases, the distribution of proteins between monomers and dimers and between nonspecific and specific DNA complexes can be simply …

Investigation Of A 16s Rna Central Domain Pseudoknot, Jenna Marie Jasinski-Bolak

Wayne State University Theses

X-ray crystallography of the prokaryotic 30S ribosomal subunit revealed a myriad of complex RNA-RNA, RNA-protein, and protein-protein interactions. Among these are several phylogenetically conserved RNA pseudoknots. Pseudoknots are structurally and functionally diverse RNA secondary structures. They are generally formed by two short complimentary sequences separated by many bases of single stranded regions or loops. These relatively simple folds are often yield complex structures that are key components of functionally important conformational changes in RNA structure. One such pseudoknot is located in the central domain of the 16S rRNA.

The central domain pseudoknot is formed by Watson-Crick base pairing between G570-C866 …

Investigating The Glutamine-Trna (Glutamine) Synthesis Appartus Of The Human Pathogen Helicobacter Pylori, Nilesh Joshi

Wayne State University Theses

INVESTIGATING THE GLUTAMINE-TRNA (GLUTAMINE) SYNTHESIS APPARTUS OF THE HUMAN PATHOGEN HELICOBACTER PYLORI

by

NILESH JOSHI

August 2012

Advisor: Tamara L. Hendrickson

Major: Chemistry

Degree: Master of Science

Accurate protein biosynthesis is a vital process to all cellular life. Aminoacyl-tRNAs are at the heart of this process: A correctly formed aminoacyl-tRNA is critical for protein biosynthesis. Organisms have evolved many mechanisms to repair misacylated tRNAs before they cause errors in protein biosynthesis, thus maintaining the integrity of the genetic code. The human pathogen Helicobacter pylori (H. pylori) synthesizes Glu-tRNAGln as an intermediate to producing Gln-tRNAGln. This misacylated intermediate could cause lethal …