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Turning Density Functional Theory Calculations Into Molecular Mechanics Simulations : Establishing The Fluctuating Density Model For Rna Nucleobases, Christopher A. Myers
Turning Density Functional Theory Calculations Into Molecular Mechanics Simulations : Establishing The Fluctuating Density Model For Rna Nucleobases, Christopher A. Myers
Legacy Theses & Dissertations (2009 - 2024)
Molecular mechanics (MD) simulations and density functional theory (DFT) have been the backbone of computational chemistry for decades. Due to its accuracy and computational feasibility, DFT has become the go-to method for theoretically predicting interaction energies, polarizability, and other electronic properties of small molecules at the quantum mechanical level. Although less fundamental than DFT, molecular mechanics (MM) algorithms have been just as influential in the fields of biology and chemistry, owing their success to the ability to compute measurable, macroscopic quantities for systems with tens of thousands to hundreds of thousands of atoms at a time. Nevertheless, MD simulations would …
Fluorescent Labelling Of Ribosomal Rna In Live Mammalian Cells, Amir Agharezaee
Fluorescent Labelling Of Ribosomal Rna In Live Mammalian Cells, Amir Agharezaee
Legacy Theses & Dissertations (2009 - 2024)
This thesis describes a bio-orthogonal chemistry-based approach for new methods of fluorescence labelling of RNA in live HeLa cells via an IEDDA reaction of TCO with tetrazine. The key step of the approach is synthesis of two nucleobase analogues which were modified with TCO and a masked 5′-phosphate group using aryl phosphonamidite chemistry. Synthesis of tubercidin and uridine analogues were considered in this investigation for incorporation into RNA in live HeLa cells. Synthetic approach was successful at installing the handle containing TCO to tubercidin and uridine to make compounds 12 and 21 respectively (the synthesis of both of which are …
Mechanisms Underlying Pre- And Postnatal Development Of The Mouse Vomeronasal Organ, Raghu Ram Katreddi
Mechanisms Underlying Pre- And Postnatal Development Of The Mouse Vomeronasal Organ, Raghu Ram Katreddi
Legacy Theses & Dissertations (2009 - 2024)
The Vomeronasal organ (VNO) is a specialized olfactory sensory organ located in the ventral region of the nasal cavity in rodents. The vomeronasal epithelium (VNE) of rodents is composed of 2 major types of vomeronasal sensory neurons (VSNs): 1) VSNs distributed in the apical VNE regions that express vomeronasal type-1 receptors (V1Rs) and the G protein subunit Gi2, and 2) VSNs in the basal territories of the VNE that express vomeronasal type-2 receptors (V2Rs) and the G subunit Go. Besides these two neuronal types, VNE also accommodate a third non-neuronal cell type called Sustentacular cells that lie anatomically above apical …
Ampa And Kainate Receptor-Potentiating Rna Aptamers, Janet L. Lynch
Ampa And Kainate Receptor-Potentiating Rna Aptamers, Janet L. Lynch
Legacy Theses & Dissertations (2009 - 2024)
Glutamate receptors act to bring about excitatory transmission in the central nervous system. The receptors are divided into two groups: ionotropic and metabotropic glutamate receptors. Ionotropic glutamate receptors are ion channels which are activated by an agonist such as glutamate or kainate. The main receptors in the ionotropic glutamate receptor family are the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA) receptors. In the central nervous system ionotropic glutamate receptors are found both pre- and postsynaptically. It has been found that most AMPA and NMDA receptors are postsynaptic receptors while the kainate receptors can be pre- or postsynaptic. Underactivity of these …
Rna Modification Landscape And Its Contribution To Egg Production, Ian Thomas Rapisarda
Rna Modification Landscape And Its Contribution To Egg Production, Ian Thomas Rapisarda
Legacy Theses & Dissertations (2009 - 2024)
Germline stem cells differentiate into mature egg or sperm cells that go through the process of fertilization which gives rise to all sexually reproducing organisms. During this process of differentiation, germ cells undergo a switch from mitosis to meiosis that allows for proper development and specification of the future gamete. The mechanisms that facilitate this shift from mitosis to meiosis, however, are not well understood. To gain insight into this process, we used Drosophila oogenesis as our model. To identify what RNA modifications are present during oogenesis we genetically enriched for each stage of development and performed mass spectrometry. We …
Sensing Ribonuclease H Activity With Dna Nanoswitches, Ruju Trivedi
Sensing Ribonuclease H Activity With Dna Nanoswitches, Ruju Trivedi
Biological Sciences
Ribonuclease H (RNase H) is a damage-repair protein and ribonuclease that specifically catalyzes the hydrolysis of RNA in an RNA/DNA duplex and breaks down RNA/DNA junctions. It plays an important role in a variety of biological processes including DNA replication, DNA repair, and transcription. It is also pivotal in anti-HIV drug development and the analysis of cellular processes and has been shown to be a potential therapeutic target for various neoplastic diseases. This thesis discusses a unique assay based on DNA nanoswitches to detect RNase H levels and activity. The assay is based on conformational changes of DNA nanoswitches in …
Launching The Next Generation : Transcriptional Regulation During Oogenesis, Alicia K. Mccarthy
Launching The Next Generation : Transcriptional Regulation During Oogenesis, Alicia K. Mccarthy
Legacy Theses & Dissertations (2009 - 2024)
Germ cells give rise to gametes and link generations by passing genetic information from parent to offspring. Gametes arise from, in many sexually reproducing organisms, germline stem cells (GSCs) which are set aside early during development. GSCs have an amazing capacity to undergo self-renewal to give rise to a pool of undifferentiated cells, while also differentiating to generate specialized germ cells such as haploid gametes. Upon female GSC differentiation, mitotically dividing germ cells can initiate meiosis, and mature within a follicle. During maturation, the specified oocyte is provided with a trust fund of RNAs and proteins for the next generation …
Development Of Dual Functional Dna/Rna Nanostructures For Drug Delivery, Vibhav Amit Valsangkar
Development Of Dual Functional Dna/Rna Nanostructures For Drug Delivery, Vibhav Amit Valsangkar
Legacy Theses & Dissertations (2009 - 2024)
In addition to the traditional biochemical functions, DNA and RNA have been increasingly studied as building blocks for the formation of various 2D and 3D nanostructures. DNA has emerged as a versatile building block for programmable self-assembly. DNA-based nanostructures have been widely applied in biosensing, bioimaging, drug delivery, molecular computation and macromolecular scaffolding. A variety of strategies have been developed to functionalize these nanostructures. The major advantage is that DNA is a very stable molecule and its base-pairing properties can be easily utilized to control and program the formation of desired nanostructures. In addition, some of these DNA/RNA nanostructures have …
Development Of Small Molecule Antibiotics Against A Conserved Rna Gene Regulatory Element In Gram-Positive Bacteria, Ville Yrjö Petteri Väre
Development Of Small Molecule Antibiotics Against A Conserved Rna Gene Regulatory Element In Gram-Positive Bacteria, Ville Yrjö Petteri Väre
Legacy Theses & Dissertations (2009 - 2024)
Bacterial infections and the rise of antibiotic resistance, especially multidrug resistant strains, have generated a clear need for discovery of novel therapeutics. Most antibiotics in use today are derivatives of previous antibiotics to which resistance mechanisms already exist, and traditionally they have a single target: either a protein or rRNA. Gram-positive bacteria regulate the expression of several essential genes or operons using a mechanism called the T-box. The T-box is a structurally conserved riboswitch-like gene regulator in the 5’-untranslated region (UTR) of numerous essential genes of Gram-positive bacteria. T-boxes are stabilized by cognate, unacylated tRNA ligands, allowing the formation of …
Exploration Of Nucleic Acid Hydrogran Bonding Using Molecular Mechanics And Dft Calculations, Simi Kaur
Exploration Of Nucleic Acid Hydrogran Bonding Using Molecular Mechanics And Dft Calculations, Simi Kaur
Biological Sciences
Many recent theoretical and experimental techniques have been developed to probe the structurefunction relationships of complex biomolecules. The roles of RNAs are dependent upon various intricate structural motifs and interactions, including hairpins, pseudoknots, long range territory contacts, bulges and internal loops, that are not easily captured by these methods. We had previously developed an enhanced replica exchange molecular dynamics method that incorporated secondary structure information in the form of distance restraints in order to effectively overcome kinetic barriers and sample conformational space. In several structures, restrained RNA base pairs near large bulges displayed a preference for stacking over hydrogen bonding …
Computational Modeling As A Tool For Designing Ligands And Receptors, Waqas S. Awan
Computational Modeling As A Tool For Designing Ligands And Receptors, Waqas S. Awan
Legacy Theses & Dissertations (2009 - 2024)
Computational methods can be used for a wide range of applications, especially regarding DNA and RNA. Interactions such as sugar torsions, receptor-ligand interactions, ligand docking/drug docking, receptor modeling, and drug design are excellent candidates for computational analysis and in silico experiments. The use of molecular dynamics software (GROMACS) coupled with molecular design software (MOE) produce insights that may have been otherwise difficult to assess. All these problems are academic in nature but have practical uses outside of academia. Understanding alternate linkages can lead to antibiotic assays to address potential superbug epidemics. Modeling DNA superstructures can provide insight into how large …
Significance Of Rna 2'-5' Linkage And Metal-Ion Mediated Base Pairs, Fusheng Shen
Significance Of Rna 2'-5' Linkage And Metal-Ion Mediated Base Pairs, Fusheng Shen
Legacy Theses & Dissertations (2009 - 2024)
It has been known that the template-directed RNA chemical replication reaction produces mixture of backbones containing both 3’-5’ and 2’-5’ linkages. This backbone heterogeneity has been a significant problem in studying the emergence of RNA World from the prebiotic chemistry. However, very recently, it is reported that FMN binding aptamer and a hammerhead ribozyme are still able to retain considerable functions in the presence of certain 2’-5’ linkages, indicating that RNA backbones may be quite flexible and this backbone heterogeneity problem may not be as severe as originally thought. This finding also brings two related important questions: First, how does …
Examining The Mechanisms Of Nucleic Acid Structural Rearrangements Using Nanospray Ionization Mass Spectrometry, Botros Toro
Examining The Mechanisms Of Nucleic Acid Structural Rearrangements Using Nanospray Ionization Mass Spectrometry, Botros Toro
Legacy Theses & Dissertations (2009 - 2024)
RNA’s diverse gene regulatory functions are tied to its ability to adopt and rearrange between an ensemble of three-dimensional structures. This concept is illustrated by the process of genome dimerization in HIV-1 and other retroviruses, which is mediated by the dimerization initiation site (DIS) of viral RNA. This essential stem-loop domain establishes a metastable kissing complex (KC) intermediate that seeds the structural rearrangements necessary to stabilize genome dimerization. Most approaches applied to study RNA structure provide us with a snapshot of RNA at equilibrium, leaving key details on dynamics concealed. This thesis explored the merits of nanospray ionization mass spectrometry …
Transcriptional Regulation Of Dksa In E. Coli, Daniel Thomas Woods
Transcriptional Regulation Of Dksa In E. Coli, Daniel Thomas Woods
Legacy Theses & Dissertations (2009 - 2024)
DksA is a global transcription factor that binds RNAP directly to regulate the expression of many genes and operons, including ribosomal RNA, in a ppGpp-dependent or ppGpp–independent manner. It is also involved in facilitating the process of DNA replication by removing stalled transcription elongation complexes that could block the progress of the replication fork. In addition, DksA is important for colonization, establishment of biofilms, and pathogenesis. In order to sustain these various functions, an adequate level of cellular DksA is required. This work tested the hypothesis that the E. coli dksA is substantially regulated at the level of transcription. Using …
Circular Rna : A Review Of History, Diseases, And Diagnostic Potential, Daniel Conley
Circular Rna : A Review Of History, Diseases, And Diagnostic Potential, Daniel Conley
Legacy Theses & Dissertations (2009 - 2024)
Abstract
Technology Development For Detection Of Circulating Disease Biomarkers From Liquid Biopsies Using Multifunctional Nanomaterials, Mustafa Balcioglu
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 …
Mass Spectrometric Analysis And Machine Learning Enable Microorganism Classification Based On Rna Posttranscriptional Modifications, Colin Christopher Aldrich
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 …
The Role Of The Mediator Transcriptional Co-Activator Complex And Promoter Dependence In Ty1 Retrotransposition In Saccharomyces Cerevisiae, Alicia Salinero
The Role Of The Mediator Transcriptional Co-Activator Complex And Promoter Dependence In Ty1 Retrotransposition In Saccharomyces Cerevisiae, Alicia Salinero
Legacy Theses & Dissertations (2009 - 2024)
Retrotransposons are mobile genetic elements that replicate via an RNA intermediary and constitute a significant portion of most eukaryotic genomes. Saccharomyces cerevisiae has been invaluable to retrotransposon research due to the presence of an active retroelement known as Ty1. The mobility of Ty1 is regulated both positively and negatively by numerous host factors, including several subunits of the Mediator transcriptional co-activator complex. The Mediator core complex is organized into genetically and structurally defined head, middle, and tail modules, along with a transiently associated kinase module. We show that with the exception of the kinase module, deletion of non-essential subunits from …
The Investigation Of Dna And Rna Structural Differences Using Ultra High Performance Liquid Chromatography, Evanna Lerouge, Maria Basanta-Sanchez, Srivathsan V. Ranganathan
The Investigation Of Dna And Rna Structural Differences Using Ultra High Performance Liquid Chromatography, Evanna Lerouge, Maria Basanta-Sanchez, Srivathsan V. Ranganathan
Anthropology
DNA and RNA chromatography is extensively used for nucleic acid analysis. To better understand the chromatographic mechanisms by which DNA and RNA oligonucleotides are separated, ion pair reverse-pair ultra-high performance liquid chromatography (IP RP UHPLC) methods were developed. 11mer and 12mer DNA and RNA oligonucleotides of various compositions were used during this study. The first part of this study analyzed 11mer DNA and RNA oligonucleotides to better understand the chromatographic separations of DNA and RNA. The results gathered through the IP RP UHPLC analysis of these oligonucleotides demonstrated the existence of structural features that affect the chromatographic separations of DNA …
Novel Nmr Based Technologies To Study Macromolecular Structures, Subhabrata Majumder
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 …
Rna Aptamers For Molecular Chaperones Hsp27 And Hsp90, Sathishkumar Kumar Munusamy
Rna Aptamers For Molecular Chaperones Hsp27 And Hsp90, Sathishkumar Kumar Munusamy
Legacy Theses & Dissertations (2009 - 2024)
Hsp90 and Hsp27 are members of the heat shock protein family of chaperones that perform multiple roles in cellular maintenance through protein folding and inhibition of apoptosis. They are abundantly expressed in cells and are over-expressed during conditions of stress. Hsp90 requires ATP for its chaperone function while Hsp27 self-associates into higher order oligomers enclosing its substrate. Their ability to interact with other proteins or with themselves lies at the heart of their mechanisms. The specific consequences of each of their interactions on global cellular health have not yet been fully discovered. The sheer diversity of proteins that interact with …
Mass Spectrometry-Based Rnomics : Global Surveys Of Ribonucleotide Modifications As Possible Indicators Of Cell Identity, Epigenetic, Metabolic And Pathological State, Rebecca Erin Rose
Mass Spectrometry-Based Rnomics : Global Surveys Of Ribonucleotide Modifications As Possible Indicators Of Cell Identity, Epigenetic, Metabolic And Pathological State, Rebecca Erin Rose
Legacy Theses & Dissertations (2009 - 2024)
Discovery of the regulatory roles of non-protein coding RNAs (ncRNAs), once considered “junk” or “transcriptional noise”, has prompted a reassessment of the significance of the multifaceted activities of RNA in cellular response, which can result in cell transformation and diseased states. This reassessment, however, can only be accomplished if techniques are developed to capture, characterize and quantify the more than 100 covalent post-transcriptional modifications (PTMs) that adorn natural RNAs. Undoubtedly, the creation of high-throughput platforms such as next-generation sequencing and RNA-seq have provided unparalleled accuracy and sensitivity, however, they rely on strand amplification procedures that are blind to the known …
Molecular Dynamics Simulations Of Small Systems To Improve Base Pairing Parameters Of The Amber-99 Force Field, Angelo Christopher Setaro
Molecular Dynamics Simulations Of Small Systems To Improve Base Pairing Parameters Of The Amber-99 Force Field, Angelo Christopher Setaro
Legacy Theses & Dissertations (2009 - 2024)
Simulations of biochemical systems are being increasingly used to further our understanding of those systems. In broad strokes, chemical simulations can be broken into two categories, those that are rooted in a quantum mechanical approach and those that are classically rooted. Molecular dynamics is one such classical method, which propagates motion of a chemical system via repeatedly solving Newton’s Laws of motion. This approach is less computationally intensive than the quantum mechanical methods and allows for the simulation of systems of tens of thousands of atoms, if not more.
Rna Aptamer Mediated Manipulation Of The 70 Kilodalton Heat Shock Protein Chaperone Machinery, Deepak Thirunavukarasu
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 …
The Structural Heterogeneity And Dynamics Of Base Stacking And Unstacking In Nucleic Acids, Ada Anna Sedova
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 …
Probing Secondary And Tertiary Rna Folding Using Force And Temperature, William Stephenson
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 …
2'-O-Methyl Substitutions In The Yeast Telomerase Pseudoknot And Their Effects On Telomerase Activity, Katelyn Mae Jasper
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 …
From Whole-Cell To Compartment- And Class-Specific Analysis Of Rna Post-Transcriptional Modifications, Ryan Quinn
From Whole-Cell To Compartment- And Class-Specific Analysis Of Rna Post-Transcriptional Modifications, Ryan Quinn
Legacy Theses & Dissertations (2009 - 2024)
The genomic structure of the human immunodeficiency virus type 1 (HIV-1) has proven difficult to examine by traditional high-resolution techniques. Structural probing and in silico modeling have been turned to the task instead. Computer models are only as robust as the data used to create them. A number of techniques were explored in this investigation to expand the utility of in silico modeling as it relates to HIV-1: the study of nucleoside modifications, and the probing of in vivo conformations of the 5'-untranslated region (5'-UTR) by cross-linking and affinity capture.
Mutational Analysis Of An Rna Aptamer, Deblina Biswas
Mutational Analysis Of An Rna Aptamer, Deblina Biswas
Legacy Theses & Dissertations (2009 - 2024)
Aptamers are oligonucleotide molecules (DNA or RNA) that are selected against specific target proteins, nucleic acids or small molecules by using the well established technique of SELEX (Systemic Evolution of Ligands by Exponential Enrichment). Their ability to bind with high affinity and specificity has opened up a wide arena of scientific applications. Aptamers have been shown to have many potential therapeutic and bioanalytical contributions such as in the treatment of diseases like cancer, cardiovascular and other major ailments. The aim of this dissertation is to study and analyze double transversion involving 4 bases in a RNA aptamer molecule: MinB4-au-flipS clamp …
Ultraviolet Resonance Raman Spectroscopy For Characterization Of Rna Structure And Thermodynamics, Joseph Dustin Handen
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 …