Biochemistry, Biophysics, and Structural Biology Commons^™

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

Genetic Control Of A Central Pattern Generator: Rhythmic Oromotor Movement In Mice Is Controlled By A Major Locus Near Atp1a2, Steven J. St. John, John D. Boughter Jr, Megan K. Mulligan, Kenichi Tokita, Lu Lu, Detlef H. Heck, Robert W. Williams

Faculty Publications

calreticulin, Animals, Chromosome Mapping, Mammalian Chromosomes, Gene Expression Regulation, Genetic Linkage, Genome-Wide Association Study. Inbred C57BL Mice, Inbred DBA Mice, Quantitative Trait Loci, Sodium-Potassium-Exchanging ATPase/genetics, Atp1a2 protein, Sodium-Potassium-Exchanging ATPase, feeding behavior, drinking behavior, mice, central pattern generator, genetic control

Cell Cycle Arrest Associated With Anoxia-Induced Quiescence, Anoxic Preconditioning, And Embryonic Diapause In Embryos Of The Annual Killifish Austrofundulus Limnaeus, Camie Lynn Meller, Robert Meller, Roger P. Simon, Kristin M. Culpepper, Jason E. Podrabsky

Biology Faculty Publications and Presentations

Embryos of the annual killifish Austrofundulus limnaeus can enter into dormancy associated with diapause and anoxia-induced quiescence. Dormant embryos are composed primarily of cells arrested in the G1/G0 phase of the cell cycle based on flow cytometry analysis of DNA content. In fact, most cells in developing embryos contain only a diploid complement of DNA, with very few cells found in the S, G2, or M phases of the cell cycle. Diapause II embryos appear to be in a G0-like state with low levels of cyclin D1 and p53. However, the active form of pAKT is high during diapause II. …

Microfluidic Impedance Spectroscopy As A Tool For Quantitative Biology And Biotechnology, Ahmet C. Sabuncu, Jie Zhuang, Juergen F. Kolb, Ali Beskok

Mechanical & Aerospace Engineering Faculty Publications

A microfluidic device that is able to perform dielectric spectroscopy is developed. The device consists of a measurement chamber that is 250 μm thick and 750 μm radius. Around 1000 cells fit inside the chamber assuming average quantities for cell radius and volume fraction. This number is about 1000 folds lower than the capacity of conventional fixtures. A T-cell leukemia cell line Jurkat is tested using the microfluidic device. Measurements of deionized water and salt solutions are utilized to determine parasitic effects and geometric capacitance of the device. Physical models, including Maxwell-Wagner mixture and double shell models, are used to …

Microfluidic Separation Of Live And Dead Yeast Cells Using Reservoir-Based Dielectrophoresis, Saurin Patel, Daniel Showers, Pallavi Vedantam, Tzuen-Rong Tzeng, Shizhi Qian, Xiangchun Xuan

Mechanical & Aerospace Engineering Faculty Publications

Separating live and dead cells is critical to the diagnosis of early stage diseases and to the efficacy test of drug screening, etc. This work demonstrates a novel microfluidic approach to dielectrophoretic separation of yeast cells by viability. It exploits the cell dielectrophoresis that is induced by the inherent electric field gradient at the reservoir-microchannel junction to selectively trap dead yeast cells and continuously separate them from live ones right inside the reservoir. This approach is therefore termed reservoir-based dielectrophoresis (rDEP). It has unique advantages as compared to existing dielectrophoretic approaches such as the occupation of zero channel space and …

Protein Structure Networks, Lesley H. Greene

Chemistry & Biochemistry Faculty Publications

The application of the field of network science to the scientific disciplines of structural biology and biochemistry, have yielded important new insights into the nature and determinants of protein structures, function, dynamics and the folding process. Advancements in further understanding protein relationships through network science have also reshaped the way we view the connectivity of proteins in the protein universe. The canonical hierarchical classification can now be visualized for example, as a protein fold continuum. This review will survey several key advances in the expanding area of research being conducted to study protein structures and folding using network approaches.

Expansion Dating: Calibrating Molecular Clocks In Marine Species From Expansions Onto The Sunda Shelf Following The Last Glacial Maximum, Eric D. Crandall, Elizabeth J. Sbrocco, Timery S. Deboer, Paul H. Barber, Kent E. Carpenter

Biological Sciences Faculty Publications

The rate of change in DNA is an important parameter for understanding molecular evolution and hence for inferences drawn from studies of phylogeography and phylogenetics. Most rate calibrations for mitochondrial coding regions in marine species have been made from divergence dating for fossils and vicariant events older than 1-2 My and are typically 0.5-2% per lineage per million years. Recently, calibrations made with ancient DNA (aDNA) from younger dates have yielded faster rates, suggesting that estimates of the molecular rate of change depend on the time of calibration, decaying from the instantaneous mutation rate to the phylogenetic substitution rate. aDNA …

Protein Folding By 'Levels Of Separation': A Hypothesis, Lesley H. Greene, Terri M. Grant

Chemistry & Biochemistry Faculty Publications

The protein folding process has been studied both computationally and experimentally for over 30 years. To date there is no detailed mechanism to explain the formation of long-range interactions between the transition and native states. Long-range interactions are the principle determinants of the tertiary structure. We present a theoretical model which proposes a mechanism for the acquisition of these interactions as they form in a modified version of ‘degrees of separation’, that we term ‘levels of separation’. It is based on the integration of network science and biochemistry. (C) 2012 Federation of European Biochemical Societies.

Examination Of The Neurochemical Mechanisms That Mediate Nicotine Withdrawal In Adolescent And Adult Rats, Luis Alberto Natividad

Open Access Theses & Dissertations

Introduction: The mechanisms that mediate nicotine withdrawal are presently unclear and age group differences in the neurochemical effects of withdrawal have been largely unexplored. Previous studies in our laboratory demonstrated that adult rats display a decrease in extracellular levels of dopamine in the nucleus accumbens (NAcc) during nicotine withdrawal and this decrease is reduced in adolescent rats (Natividad et al., 2010). The goal of this dissertation was to examine whether these age group differences in dopamine during withdrawal are mediated via excitatory and inhibitory mechanisms that modulate dopamine in the cell body region of the ventral tegmental area (VTA). Methods: …

Arachidonic Acid Signaling In Invasive And Non-Invasive Breast Cancer Cells, Debarshi Roy

Open Access Theses & Dissertations

Breast cancer is the second largest cause of cancer-related deaths in women all over the world. Epidemiological studies suggest that the consumption of high-fat diets can promote the incidence of breast cancers in both developed and developing countries. In particular, the lipid-rich diet contains arachidonic acid (AA, a C20:4 polyunsaturated fatty acid), which has been shown to be associated with tumor formation in breast tissues. Nevertheless, the actual mechanism by which AA induces the metastatic transformation and malignancy is not well understood. The goal of my dissertation, therefore, is to identify the molecules and unravel the pathways that participate in …

Use Of Methionine Sulfoximine To Dissect The Role Of Glutamine Synthetase And Glutamine In Progression Of Acute Liver Failure, Amruta Anil Jambekar

Wayne State University Dissertations

Methionine sulfoximine (MSO) is a modified amino acid and a well characterized irreversible inhibitor of glutamine synthetase (GS) enzyme. Glutamine is synthesized by GS enzyme and it is the most abundant amino acid in the body. Glutamine is required by immune cells for generation, propagation and maintenance of an immune response.

To induce acute liver failure (ALF) in mice, animals were given intraperitoneal injections of E. coli lipopolysaccharides (LPS) and sugar D-galactosamine (D-GalN). When these animals were pretreated with MSO, 80% of the animals were completely rescued from liver failure. Moreover, when we characterized the immune response generated during ALF …

The Role Of Cad,Flash And Fam129b In Cancer Cell Survial And Apoptosis, Song Chen

Wayne State University Dissertations

Apoptosis is a normal process in the human body. However, apoptosis is desregulated in cancer cells. Most cancer cells gain resistance to apoptosis, leading to uncontrolled proliferation. In this dissertation, we identified three proteins, associated with apoptosis pathway. 1) CAD, a large multifunctional complex that is invariably elevated in tumor cells, 2) FLASH, a large protein with multiple growth related functions and 3) FAM129B. We demonstrate that CAD could interact with FLASH by using yeast two hybrid, co-immunopreciptation and fluorescence microscopy. In addition, functional analysis using siRNA technology further indicated that CAD could co-operate with FLASH and play roles in …

Understanding The Gender-Based Mechanism Of Mso In Als Mice: A Metabolic Characterization Of The Sod1-G93a Mouse Model, Monica Ann Bame

Wayne State University Dissertations

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease characterized by motor neuron death and a corresponding loss of neuromuscular connections resulting in muscle atrophy. Patients become paralyzed shortly after symptom onset and typically die within one to five years of pulmonary complications. ALS is a relatively rare disease, with an overall incidence of approximately 2 in 100,000 people per year and a prevalence of about 5 in 100,000 people. It is typically associated with increasing age and has a slight male prevalence, with a male to female ratio of approximately 3:2. ALS is classified as either familial (the less …

Prevalence And Physiological Significance Of Gene Looping In Saccharomyces Cerevisiae, Banupriya Mukundan

Wayne State University Dissertations

My Ph.D. dissertation work is focused on studying the role of promoter-bound transcription initiation factors involved in gene looping. In this study we showed that the RNAP II subunit Rpb4 has a significant effect on termination of transcription. Gene looping is disrupted in the absence of Rpb4. Rpb4 shows a strong physical interaction with the Mediator subunit Srb5. Mediator subunit Srb5 crosslinked to the 5' and 3' ends of INO1 and CHA1 genes and is required for proper termination of transcription of these genes. Srb5 affected termination of transcription through its interaction with the CF1 complex. Srb5 interaction with the …

Cdk1 And Plk1 Mediate A Clasp2 Phospho-Switch That Stabilizes Kinetochore–Microtubule Attachments, Ana R. R. Maia, Zaira Garcia, Lilian Kabeche, Marin Barisic

Dartmouth Scholarship

Accurate chromosome segregation during mitosis relies on a dynamic kinetochore (KT)-microtubule (MT) interface that switches from a labile to a stable condition in response to correct MT attachments. This transition is essential to satisfy the spindle-assembly checkpoint (SAC) and couple MT-generated force with chromosome movements, but the underlying regulatory mechanism remains unclear. In this study, we show that during mitosis the MT- and KT-associated protein CLASP2 is progressively and distinctively phosphorylated by Cdk1 and Plk1 kinases, concomitant with the establishment of KT-MT attachments. CLASP2 S1234 was phosphorylated by Cdk1, which primed CLASP2 for association with Plk1. Plk1 recruitment to KTs …

Myricetin Inhibits Islet Amyloid Polypeptide (Iapp) Aggregation And Rescues Living Mammalian Cells From Iapp Toxicity, Casey Zelus, Ayano Fox, Anastasia Calciano, Bianca S. Faridian, Luiza A. Nogaj, David A. Moffet

Chemistry and Biochemistry Faculty Works

The aggregation of the amyloidogenic polypeptide IAPP (Islet Amyloid Polypeptide, amylin) is believed to play a direct role in the death of pancreatic β-islet cells in type II diabetes. Preventing the initial aggregation event of IAPP is one strategy for slowing, and possibly preventing, the progression of this disease. Here, we investigate myricetin’s potential as an inhibitor of IAPP aggregation. We show that myricetin prevented thioflavin T binding in a concentration dependent manner. Atomic force microscopy revealed that myricetin prevented fiber formation under rigorous conditions conducive to forming IAPP aggregates. Using an IAPP-EGFP (Enhanced Green Fluorescent Protein) protein construct, we …

Structural Basis Of Substrate Recognition In Thimet Oligopeptidase And Development Of Nanoparticles For Therapeutic Enzyme Delivery, Jonathan Mark Wagner

Theses and Dissertations--Molecular and Cellular Biochemistry

Neuropeptidases are responsible for degradation of signaling peptides in the central nervous system and periphery. Some neuropeptidases have also been shown to play a role as part of the cell’s hydrolytic machinery responsible for breaking down proteins and peptides into amino acids, and these enzymes therefore influence small peptide availability for antigen presentation. A better understanding of how neuropeptidases recognize their substrates could lead to therapeutics that modulate the activity of these important enzymes. Alternatively, re-engineering these enzymes to selectively hydrolyze undesirable peptides could make them attractive as therapeutics themselves. A key question in understanding the activity of these enzymes …

The Role Of Syntaxin And Tomosyn In Platelet Secretion, Shaojing Ye

Theses and Dissertations--Molecular and Cellular Biochemistry

Platelet secretion is important for hemostasis and thrombosis. The components released are also involved in atherosclerosis, inflammation, angiogenesis, and tumor growth. Though the exact mechanism(s) of platelet secretion is still elusive, accumulating evidence demonstrates that SNAREs (Soluble N-ethylmaleimide Sensitive Factor Associated Receptor) and their regulatory partners are critical for platelet exocytosis. Formation of a trans-bilayer complex composed of one v-SNARE (i.e. VAMPs) and two t-SNAREs (i.e. syntaxin and SNAP-25-type) is minimally required for membrane fusion. Regulatory proteins control the rate and specificity of the complex assembly. VAMP-8 and SNAP-23 (a SNAP-25-type t-SNARE) are clearly important; however, the …