Biochemistry Commons^™

Student Understanding Of Dna Structure–Function Relationships Improves From Using 3d Learning Modules With Dynamic 3d Printed Models, Michelle E. Howell, Christine S. Booth, Sharmin M. Sikich, Tomáš Helikar, Rebecca Roston, Brain A. Couch, Karin Van Dijk

Department of Biochemistry: Faculty Publications

Understanding the relationship between molecular structure and function represents an important goal of undergraduate life sciences. Although evidence suggests that handling physical models supports gains in student understanding of structure–function relationships, such models have not been widely implemented in biochemistry classrooms. Three-dimensional (3D) printing represents an emerging cost-effective means of producing molecular models to help students investigate structure–function concepts. We developed three interactive learning modules with dynamic 3D printed models to help biochemistry students visualize biomolecular structures and address particular misconceptions. These modules targeted specific learning objectives related to DNA and RNA structure, transcription factor-DNA interactions, and DNA supercoiling dynamics. …

Gq Noncanonical Roles In Translational Regulation, Brett Demarco

Electronic Theses and Dissertations

This study investigates protein nucleic acid interactions, focusing on G-quadruplex (GQ) forming DNA/RNA in human disease. GQ structures are formed in DNA/RNA, when four guanine residues form planar tetrads stabilized by Hoogsteen base pairing, that stack forming a GQ structure stabilized by potassium ions. These GQ structures are targeted by the arginine glycine-glycine (RGG) RNA-binding domain. Fragile X mental retardation protein (FMRP), a translation regulator protein implicated in the fragile X syndrome, has an RGG domain and has been previously shown to interact with neuronal GQ forming messenger RNA (mRNA). We have investigated three neuronal FMRP mRNA targets that we …

Detection Of Local Steroidogenic Enzyme Gene Expression In Brown Anoles, Ada Spahija

Honors Program Theses

The endocrine system in vertebrates responds to stress by releasing steroid hormones, mainly glucocorticoids (GC), which increase blood glucose levels to supply key organs and muscles with energy needed for survival. Steroid hormones are synthesized via an enzymatic pathway that converts cholesterol into either GCs, androgens, or estrogens in a step-wise manner. The adrenal cortex is known to produce GCs, but evidence suggests that individual organs can also produce steroid hormones de novo in response to stress. This study aims to quantify gene expression of four steroidogenic enzymes, encoded by CYP19A1, CYP17A1, StAR, and HSD17ß3 genes, via qRT-PCR to determine …

Technology Development For Detection Of Circulating Disease Biomarkers From Liquid Biopsies Using Multifunctional Nanomaterials, Mustafa Balcioglu

Legacy Theses & Dissertations (2009 - 2024)

Despite the advance health care, devastating health conditions such as cancer and infectious diseases that affect populations worldwide are too often not diagnosed until morbid symptoms become apparent in the late phase. Obtaining an early and accurate diagnosis that reveal a hidden lethal threat before the disease becomes complicated may dramatically reduce the severity of its impact on the patient’s life and increase the probability of survival. For example, in the case of ovarian cancer, which is the fifth most common malignancy and the fifth leading cause of cancer mortality in women in the US, the 5-year relative survival is …

Circular Rna : A Review Of History, Diseases, And Diagnostic Potential, Daniel Conley

Legacy Theses & Dissertations (2009 - 2024)

Abstract

Split Deoxyribozyme Probe For Efficient Detection Of Highly Structured Rna Targets, Sheila Raquel Solarez

Honors Undergraduate Theses

Transfer RNAs (tRNAs) are known for their role as adaptors during translation of the genetic information and as regulators for gene expression; uncharged tRNAs regulate global gene expression in response to changes in amino acid pools in the cell. Aminoacylated tRNAs play a role in non-ribosomal peptide bond formation, post-translational protein labeling, modification of phospholipids in the cell membrane, and antibiotic biosynthesis. ^[1] tRNAs have a highly stable structure that can present a challenge for their detection using conventional techniques. ^[2] To enable signal amplification and lower detection limits, a split probe - split deoxyribozyme (sDz or BiDz) probe, which …

Structure Of The Picornavirus Replication Platform: A Potential Drug Target For Inhibiting Virus Replication, Meghan Suzanne Warden

Chemistry & Biochemistry Theses & Dissertations

Picornaviruses are small, positive-stranded RNA viruses, divided into twelve different genera. Members of the Picornaviridae family cause a wide range of human and animal diseases including the common cold, poliomyelitis, foot and mouth disease, and dilated cardiomyopathy. The picornavirus genome is replicated via a highly conserved mechanism involving a presumed cloverleaf structure located at the 5’ noncoding region of the virus genome. The 5’ cloverleaf consists of three stem loops (B, C, and D) and one stem (A), which interact with a variety of virus and host cell proteins during replication. In this dissertation, human rhinovirus serotype 14 (HRV-14) SLB …

Uncovering The Identity And Metabolism Of Bacterial Coa-Rna, Joseph R. Spangler

Dissertations

Coenzyme A is an indispensable molecule in all known life with roles in metabolism, gene regulation, and macromolecule synthesis. As CoA is derived from RNA itself, it’s incorporation into RNA by in vitro methods has proven useful in research probing the origin of life based on the RNA World theory. The discovery in contemporary bacteria of RNA modified with CoA, however, provided an unexpected twist to previously well-characterized bacterial systems. The identity of sequences associated with CoA-RNA has been elusive since their discovery in 2009 based on the difficulties in isolation while maintaining RNA quality. The aim of this study …

Mass Spectrometric Analysis And Machine Learning Enable Microorganism Classification Based On Rna Posttranscriptional Modifications, Colin Christopher Aldrich

Legacy Theses & Dissertations (2009 - 2024)

RNA post-transcriptional modifications (PTMs) are dynamic features that can be up- or down-regulated by the health and metabolic state of a cell. These covalent modifications are installed and removed on RNA nucleosides by enzymes controlled by the activation and deactivation of specific genes. The goal of this research was to demonstrate that RNA PTMs can serve as a unique feature for the classification/identification of microorganisms. We utilized a scheme based on electrospray ionization mass spectrometry (ESI-MS) to obtain global PTM profiles from total RNA extracted from various microorganisms in optimal growth conditions as well as Salmonella typhimurium (S. typhimurium) spiked …

Group I Intron Internal Guide Sequence Binding Strength As A Component Of Ribozyme Network Formation, Elizabeth Satterwhite, Jessica Anne Mellor Yeates, Niles Lehman

Chemistry Faculty Publications and Presentations

Origins-of-life research requires searching for a plausible transition from simple chemicals to larger macromolecules that can both hold information and catalyze their own production. We have previously shown that some group I intron ribozymes possess the ability to help synthesize other ribozyme genotypes by recombination reactions in small networks in an autocatalytic fashion. By simplifying these recombination reactions, using fluorescent anisotropy, we quantified the thermodynamic binding strength between two nucleotides of two group I intron RNA fragments for all 16 possible genotype combinations. We provide evidence that the binding strength (K_D) between the 3-nucleotide internal guide sequence …

Interaction Of Spliceosomal U2 Snrnp Protein P14 With Its Branch Site Rna Target, William Perea Vargas

Dissertations, Theses, and Capstone Projects

Newly transcribed precursor messenger RNA (pre-mRNA) molecules contain coding sequences (exons) interspersed with non-coding intervening sequences (introns). These introns must be removed in order to generate a continuous coding sequence prior to translation of the message into protein. The mechanism through which these introns are removed is known as pre-mRNA splicing, a two-step reaction catalyzed be a large macromolecular machine, the spliceosome, located in the nucleus of eukaryotic cells. The spliceosome is a protein-directed ribozyme composed of small nuclear RNAs (snRNA) and hundreds of proteins that assemble in a very dynamic process. One of these snRNAs, the U2 snRNA, is …

The Foundations Of Network Dynamics In An Rna Recombinase System, Jessica Anne Mellor Yeates

Dissertations and Theses

How life originated from physical and chemical processes is one of the great questions still unanswered today. Studies towards this effort have transitioned from the notion of a single self-replicating entity to the idea that a network of interacting molecules made this initial biological leap. In order to understand the chemical kinetic and thermodynamic mechanisms that could engender pre-life type networks we present an empirical characterization of a network of RNA recombinase molecules. We begin with 1-, 2-, and 3-molecular ensembles and provide a game theoretic analysis to describe the frequency dependent dynamics of competing and cooperating RNA genotypes. This …

Stabilin-Mediated Cellular Internalization Of Phosphorothioate-Modified Antisense Oligonucleotides (Asos), Colton M. Miller, Aaron J. Donner, Emma K. Blank, Andrew W. Egger, Brianna M. Kellar, Punit P. Seth, Edward N. Harris

UCARE: Research Products

Introduction: Antisense oligonucleotides (ASOs) are short chemically modified oligonucleotides (5-7.4 kDa) that can produce a pharmacological effect by binding to RNA and affecting intermediary metabolism. Over 35 phosphorothioate (PS) ASOs are at various stages of clinical development for use as therapeutic agents and pharmacological tools. Antisense therapy is a progressing area of research, as these small strands of nucleotide oligomers can be produced to silence genes that aggravate chronic disorders or infections. An important distinction for ASOs compared to DNA is the substitution of the phosphodiester (PO) backbone with the PS modification. This sulfur substitution allows for these polar polyanionic …

Messenger Rna Transport And Translation Regulated By The 3' Utrs Of Dendritic Mrnas And Abnormal Alternative Splicing Of Neuroligin1 In The Fmr1 Ko Mouse Hippocampus, Tianhui Zhu

Dissertations, Theses, and Capstone Projects

Fragile X Syndrome (FXS) is one of the most commonly inherited mental retardations. It is caused by the loss of functional fragile X mental retardation protein (FMRP). Loss of functional FMRP is the most widespread single-gene cause of autism. The most prominent phenotype of FXS patients is an IQ ranging from 20 to 70. FMRP is an RNA binding protein, widely expressed in almost all tissues and highly expressed in brain. As a RNA binding protein, 85-90 % of FMRP in the brain is associated with polyribosomes. Approximately 4 % of total mRNA is associated with FMRP, which functions in …

The Development Of Peptide Ligands To Target H69 Rrna, Danielle Nicole Dremann

Wayne State University Dissertations

ABSTRACT

THE DEVELOPMENT OF PEPTIDE LIGANDS TO TARGET H69

by

DANIELLE NICOLE DREMANN

December 2015

Advisor: Prof. Christine S. Chow

Major: Chemistry (Biochemistry)

Degree: Doctor of Philosophy

In the development of peptide ligands to target H69, SPPS and ESI MS was used to determine if 1) peptides could bind to modified H69 and 2) if increased affinity for the target RNA could be enhanced with modification. An alanine and arginine scan was synthesized and tested for this determination. Selected peptides were then tested using biophysical techniques such as circular dichroism and isothermal titration calorimetry. An assay was also designed to …

Platination Kinetics: Insight Into Rna-Cisplatin Interactions As A Probe For Rna Microenvironments, Gayani Dedduwa-Mudalige

Wayne State University Dissertations

RNAs are crucial for many cellular functions. Thus, studying ligand-RNA interactions and their dynamics in response to changes in the surrounding environment is important. In spite of the well-known DNA coordination, current research also indicates cisplatin binding to RNA. Kinetic studies of rRNA platination reactions are largely unexplored. This research was conducted to achieve two objectives. First, a broad kinetic study was carried out to investigate the cisplatin-rRNA interactions. The structure, function, and ligand interactions depend on RNA microenvironments. Second, the application of platination kinetics as a tool to interrogate RNA electrostatic environments was explored.

Three model rRNA hairpins from …

The Role Of Rnai In Downregulation Of Physcomitrella Patens Defense Genes And The Preliminary Steps Of Peg Mediated Transformation Of Mosses, Adam Beswick

Undergraduate Honors Thesis Collection

When attacked by a pathogen, the moss Physcomitrella patens will undergo both a hypersensitive response (HR) as well as a systematic acquired resistance (SAR) response. The SAR response turns on genes that ready the plant for future pathogen attacks. The hormone jasmonic acid (JA) is one of the products of SAR response in P. patens. In this study interference RNA (RNAi) was used to decrease gene expression of the allene oxide cyclase (ACe) gene, a key gene in the production pathway of JA. Using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), RNAi was found to significantly lower ACC gene levels …

The Structural Heterogeneity And Dynamics Of Base Stacking And Unstacking In Nucleic Acids, Ada Anna Sedova

Legacy Theses & Dissertations (2009 - 2024)

Base stacking provides stability to nucleic acid duplexes, and base unstacking is involved in numerous biological functions related to nucleic acids, including replication, repair, transcription, and translation. The patterns of base stacking and unstacking in available nucleic acid crystal structures were classified after separation into their individual single strand dinucleotide components and clustering using a k-means-based ensemble clustering method. The A- and B-form proximity of these dinucleotide structures were assessed to discover that RNA dinucleotides can approach B-form-like structures. Umbrella sampling molecular dynamics simulations were used to obtain the potential of mean force profiles for base unstacking at 5'-termini for …

Rna Aptamer Mediated Manipulation Of The 70 Kilodalton Heat Shock Protein Chaperone Machinery, Deepak Thirunavukarasu

Legacy Theses & Dissertations (2009 - 2024)

Protein quality control involves refolding of damaged proteins and facilitating degradation of irreparable proteins. Understanding the protein quality control mechanism is critical, since defects in it has been implicated in a number of age-related diseases like neurodegenerative diseases and also in cancer. A vast network of molecular chaperones and proteolytic systems collaborate to maintain protein quality control. The 70 kilodalton Heat shock protein (Hsp70) is a highly conserved and ubiquitous chaperone, which interacts with a variety of protein substrates including newly synthesized polypeptides, unfolded, partially misfolded and native proteins to maintain protein quality control. Hsp70 chaperone function is coupled to …

Novel Nmr Based Technologies To Study Macromolecular Structures, Subhabrata Majumder

Legacy Theses & Dissertations (2009 - 2024)

Nuclear Magnetic Resonance Spectroscopy (NMR) is one of the principle tools in structural biology to probe macromolecular structures and interactions. The atomic resolution afforded by this technique has been widely used to probe protein-protein, and protein-ligand interactions in-vitro. However, the natural milieu of the proteins is the living cell and the cellular cytoplasm is extremely heterogeneous. The NMR studies of folded protein in-cell, till now, have been limited by non-specific interactions of the cytosol. This thesis outlays a general methodology to study protein structure/interactions inside the living cells using NMR. In a closely related objective, it also describes the use …

Coprinus Comatus Cap Inhibits Adipocyte Differentiation Via Regulation Of Pparγ And Akt Signaling Pathway, Hyoung Joon Park, Jisoo Yun, Hong-Duck Kim, Chung-Kil Won, Gon-Sup Kim, Jae-Hyeon Cho

NYMC Faculty Publications

This study assessed the effects of Coprinus comatus cap (CCC) on adipogenesis in 3T3-L1 adipocytes and the effects of CCC on the development of diet-induced obesity in rats. Here, we showed that the CCC has an inhibitory effect on the adipocyte differentiation of 3T3-L1 cells, resulting in a significant decrease in lipid accumulation through the downregulation of several adipocyte specific-transcription factors, including CCAAT/enhancer binding protein β, C/EBPδ, and peroxisome proliferator-activated receptor gamma (PPARγ). Moreover, treatment with CCC during adipocyte differentiation induced a significant down-regulation of PPARγ and adipogenic target genes, including adipocyte protein 2, lipoprotein lipase, and adiponectin. Interestingly, the …

Cloning And Expression For The Future Characterization Of The Air2 Protein, Emily Sue Frampton

Undergraduate Honors Capstone Projects

Air2 is a eukaryotic protein involved in multiple biological processes including protein-protein interactions as well as RNA binding. Air2 plays a critical role in RNA quality control and also helps regulate post-translational modification of various proteins. Although previous studies have revealed information regarding Air2's roles within a cell, the molecular and structural basis for Air2 function is unclear. Using a codon-optimized version of the Air2 gene, various constructs were created that improved the expression and solubility of Air2. Additionally a co-expression complex of Air2 with a PRMTI mutant, K13S, was made to obtain the Air2 protein with a native binding …

Primary Microrna Processing Assay Reconstituted Using Recombinant Drosha And Dgcr8., Ian Barr, Feng Guo

Natural Sciences and Mathematics | Faculty Scholarship

In animals, the Microprocessor complex cleaves primary transcripts of microRNAs (pri-miRNAs) to produce precursor microRNAs in the nucleus. The core components of Microprocessor include the Drosha ribonuclease and its RNA-binding partner protein DiGeorge critical region 8 (DGCR8). DGCR8 has been shown to tightly bind an Fe(III) heme cofactor, which activates its pri-miRNA processing activity. Here we describe how to reconstitute pri-miRNA processing using recombinant human Drosha and DGCR8 proteins. In particular, we present the procedures for expressing and purifying DGCR8 as an Fe(III) heme-bound dimer, the most active form of this protein, and for estimating its heme content.

Probing Secondary And Tertiary Rna Folding Using Force And Temperature, William Stephenson

Legacy Theses & Dissertations (2009 - 2024)

RNA folding is the process whereby a single stranded RNA molecule assumes its three-dimensional functional conformation. Along with the protein folding problem, the RNA folding problem remains as one of the great unsolved problems in biophysics. Generally RNA folding occurs in a hierarchical manner whereby the sequence of an RNA (primary structure) determines which regions will form helical segments (secondary structure) before further rearrangement and base pairing of secondary structure motifs (tertiary structure). Due to the intimate connection between structure and function within molecular biology, increased familiarity with the thermodynamic and kinetic factors that govern RNA folding will permit the …

Partitioning The Fitness Components Of Rna Populations Evolving In Vitro, Carolina Diaz Arenas, Niles Lehman

Chemistry Faculty Publications and Presentations

All individuals in an evolving population compete for resources, and their performance is measured by a fitness metric. The performance of the individuals is relative to their abilities and to the biotic surroundings – the conditions under which they are competing – and involves many components. Molecules evolving in a test tube can also face complex environments and dynamics, and their fitnessmeasurements should reflect the complexity of various contributing factors as well. Here, the fitnesses of a set of ligase ribozymes evolved by the continuous in vitroevolution system were measured. During these evolution cycles there are three different catalytic …

2'-O-Methyl Substitutions In The Yeast Telomerase Pseudoknot And Their Effects On Telomerase Activity, Katelyn Mae Jasper

Legacy Theses & Dissertations (2009 - 2024)

Telomerase is a ribonucleoprotein (RNP) complex that synthesizes telomeric repeats at the ends of linear chromosomes to form the DNA–protein complexes known as telomeres. Telomeres protect the ends of chromosomes from degradation during replication due to the end-replication problem. When replication occurs, gaps are created at the beginning of the lagging and leading strands that result in the loss of a small amount of DNA at every replication cycle. By adding thousands of copies of telomeric repeats, the repeats are lost in the replication process and not precious genetic information. The telomerase RNA varies drastically among different species both in …

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 …

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 …

Rna Oxidation Adducts 8-Ohg And 8-Oha Change With Aβ42 Levels In Late-Stage Alzheimer's Disease, Adam M. Weidner, Melissa A. Bradley, Tina L. Beckett, Dana M. Niedowicz, Amy L.S. Dowling, Sergey V. Matveev, Harry Levine, Mark A. Lovell, M. Paul Murphy

Sanders-Brown Center on Aging Faculty Publications

While research supports amyloid-β (Aβ) as the etiologic agent of Alzheimer's disease (AD), the mechanism of action remains unclear. Evidence indicates that adducts of RNA caused by oxidation also represent an early phenomenon in AD. It is currently unknown what type of influence these two observations have on each other, if any. We quantified five RNA adducts by gas chromatography/mass spectroscopy across five brain regions from AD cases and age-matched controls. We then used a reductive directed analysis to compare the RNA adducts to common indices of AD neuropathology and various pools of Aβ. Using data from four disease-affected brain …

Stomatin-Like Protein 2 Binds Cardiolipin And Regulates Mitochondrial Biogenesis And Function., Darah Christie, Caitlin D Lemke, Isaac M Elias, Luan A Chau, Mark G Kirchhof, Bo Li, Eric H Ball, Stanley D Dunn, Grant M Hatch, Joaquín Madrenas

Biochemistry Publications

Stomatin-like protein 2 (SLP-2) is a widely expressed mitochondrial inner membrane protein of unknown function. Here we show that human SLP-2 interacts with prohibitin-1 and -2 and binds to the mitochondrial membrane phospholipid cardiolipin. Upregulation of SLP-2 expression increases cardiolipin content and the formation of metabolically active mitochondrial membranes and induces mitochondrial biogenesis. In human T lymphocytes, these events correlate with increased complex I and II activities, increased intracellular ATP stores, and increased resistance to apoptosis through the intrinsic pathway, ultimately enhancing cellular responses. We propose that the function of SLP-2 is to recruit prohibitins to cardiolipin to form cardiolipin-enriched …