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High Temporal Resolution DNA-Flap Endonuclease 1 Interaction at the Single Molecule LevelHarris, Paul David 07 1900 (has links)
Numerous short flapped DNA structures are created during the semi-discontinuous replication. These toxic intermediates are quickly resolved to produce a fully intact duplex of replicated DNA. Structure specific nuclease are key to resolving these structures, and show a high degree of specificity for their cognate substrate structures while being essentially insensitive to nucleotide sequence. Herein I demonstrate through confocal based single molecule experiments that the 5’ structure specific nuclease Flap Endonuclease 1 (FEN1) achieves its substrate specificity by coupling the bending of DNA substrate with structuring of the active site in a way that non-cognate structures binding is significantly destabilized and enzymatic features are incapable of structuring in the absence of particular substrate features, in particular a single nucleotide 3’ flap the FEN1 induces in nearly all DNA substrates. Debate remained over whether DNA was bound via a conformational capture or induced fit mechanism, and so I proceed to investigate the dynamics of the DNA itself in solution. Conclusions about conformational capture or induced fit remain elusive, however I did determine that DNA structures are rigidified by charge repulsion, an effect lessened by the salt concentration, which functions to shield the negative charge of DNA from itself. Additionally unstacking of the DNA in nicked structures incurs a significant free energy penalty, which FEN1 overcomes by its hydrophobic wedge motif, lending credence to an induced fit mechanism.
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Investigating the Mutagenicity of Polycyclic Aromatic Compounds from the Athabasca Oil Sands Region in River Otters and a Mammalian Cell LineGyasi, Helina 27 May 2022 (has links)
Mining operations have led to an increase in polycyclic aromatic compound (PAC) concentrations in the Alberta oil sands area. However, the toxicity of most PACs and PAC mixtures is not well characterized. Some PACs and PAC mixtures are known to be mutagenic, though there is limited research on the genotoxicity of PACs from the Alberta oil sands to wildlife. This thesis tested the hypothesis that anthropogenic sources of PACs from the Alberta oil sands are mutagenic to wildlife. The objectives were: 1) to determine whether wildlife with increased exposure to PACs had increased mutations, and 2) to determine whether an anthropogenic source of PACs is mutagenic in a controlled lab setting. For the first objective, we used a single-molecule polymerase chain reaction (SM-PCR) assay to detect microsatellite mutations in river otters with differing liver tissue PAC concentrations in the Athabasca oil sand region (AOSR; Alberta, Canada). For objective two, an in vitro mammalian mutagenicity assay with the FE1 MutaMouse epithelial cell line (FE1) was used to determine the mutagenic potential of a bitumen extraction by-product, tailings pond bitumen. We found that PAC exposure in the AOSR was positively correlated with elevated microsatellite mutations in river otters. From the in vitro study, tailings pond bitumen extracts did not induce lacZ mutations in the FE1 cells. Differences in detection methods between the two assays and PAC profiles between the otter tissue and tailings pond bitumen are suspected reasons for contradictory results. Further investigation of the different sources and PAC profiles within the AOSR environment and wildlife food web will provide insights on what types of PACs are mutagenic. Cytotoxicity, observed following exposure to tailings pond bitumen extracts, also suggests other toxicity pathways should be considered when investigating the toxicity of bitumen from the AOSR. Overall, this thesis provided data on the potential mutagenicity of PACs in the AOSR, which can be used to elucidate potential molecular mechanisms of toxicity in wildlife exposed to oil processing contaminants.
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Using single molecule fluorescence to study substrate recognition by a structure-specific 5’ nucleaseRashid, Fahad 12 1900 (has links)
Nucleases are integral to all DNA processing pathways. The exact nature of substrate recognition and enzymatic specificity in structure-specific nucleases that are involved in DNA replication, repair and recombination has been under intensive debate. The nucleases that rely on the contours of their substrates, such as 5’ nucleases, hold a distinctive place in this debate. How this seemingly blind recognition takes place with immense discrimination is a thought-provoking question. Pertinent to this question is the observation that even minor variations in the substrate provoke extreme catalytic variance. Increasing structural evidence from 5’ nucleases and other structure-specific nuclease families suggest a common theme of substrate recognition involving distortion of the substrate to orient it for catalysis and protein ordering to assemble active sites.
Using three single-molecule (sm)FRET approaches of temporal resolution from milliseconds to sub-milliseconds, along with various supporting techniques, I decoded a highly sophisticated mechanism that show how DNA bending and protein ordering control the catalytic selectivity in the prototypic system human Flap Endonuclease 1 (FEN1). Our results are consistent with a mutual induced-fit mechanism, with the protein bending the DNA and the DNA inducing a protein-conformational change, as opposed to functional or conformational selection mechanism. Furthermore, we show that FEN1 incision on the cognate substrate occurs with high efficiency and without missed opportunity. However, when FEN1 encounters substrates that vary in their physical attributes to the cognate substrate, cleavage happens after multiple trials
During the course of my work on FEN1, I found a novel photophysical phenomena of protein-induced fluorescence quenching (PIFQ) of cyanine dyes, which is the opposite phenomenon of the well-known protein-induced fluorescence enhancement (PIFE). Our observation and characterization of PIFQ led us to further investigate the general mechanism of fluorescence modulation and how the initial fluorescence state of the DNA-dye complex plays a fundamental role in setting up the stage for the subsequent modulation by protein binding. Within this paradigm, we propose that enhancement and quenching of fluorescence upon protein binding are simply two different faces of the same process. Our observations and correlations eliminate the current inconvenient arbitrary nature of fluorescence modulation experimental design.
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Towards observing the encounter of the T7 DNA replication fork with a lesion site at the Single molecule levelShirbini, Afnan 05 1900 (has links)
Single-molecule DNA flow-stretching assays have been a powerful approach to study various aspects on the mechanism of DNA replication for more than a decade. This technique depends on flow-induced force on a bead attached to a surface-tethered DNA. The difference in the elastic property between double-strand DNA (long) and single-strand DNA (short) at low regime force allows the observation of the beads motion when the dsDNA is converted to ssDNA by the replisome machinery during DNA replication. Here, I aim to develop an assay to track in real-time the encounter of the bacteriophage T7 replisome with abasic lesion site inserted on the leading strand template. I optimized methods to construct the DNA substrate that contains the abasic site and established the T7 leading strand synthesis at the single molecule level. I also optimized various control experiments to remove any interference from the nonspecific interactions of the DNA with the surface. My work established the foundation to image the encounter of the T7 replisome with abasic site and to characterize how the interactions between the helicase and the polymerase could influence the polymerase proofreading ability and its direct bypass of this highly common DNA damage type.
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Étude à moyen-débit de la localisation d'ARNm dans les cellules humaines / Single molecule-based screening at medium throughtput of mRNA localization in human cellsTraboulsi, Abdel-Meneem 14 November 2017 (has links)
La localisation d’ARNm a été découverte en 1983 dans les ovocytes et les embryons des ascidies. Depuis, plusieurs exemples d'ARN localisés ont été trouvés dans de nombreux organismes, y compris les plantes, les levures, les champignons, les insectes, les poissons et les mammifères. Les ARNm localisés contribuent à de nombreuses fonctions biologiques, telles que le développement embryonnaire, la division cellulaire asymétrique, la migration cellulaire, la signalisation, la plasticité neuronale et plein d’autres ...Jusqu'à présent, quelques études ont analysé la localisation d’ARNm de manière systématique. Trois d'entre eux ont été effectués chez la drosophile pendant l'embryogenèse, l'oogenèse ou le stade larvaire et ont analysé environ 16000 ARN au total. Les deux autres études ont été réalisées dans des cellules de mammifères et ont analysé près de 1000 ARNm chacune. Ces études ont montré l'importance de la localisation d’ARNm dans les cellules humaines et son implication dans différents processus biologiques. L'objectif de ma thèse était donc d'augmenter le débit des techniques FISH à l’échelle de molécule unique (smFISH) et d'étudier la localisation d’ARNm dans les cellules HeLa de manière systématique.Une limitation de smFISH est le coût de sondes fluorescentes, qui limite le nombre d'ARNm qui peut être analysé. Par conséquent, j'ai développé un protocole alternatif dans lequel des sondes pour de nombreux gènes ont été synthétisées comme un pool d'oligonucléotides (40 par gène en moyenne, plus de 12000 au total). Les sondes spécifiques d’un ARNm donné ont ensuite été amplifiées par PCR et converties en simple brin par transcription in vitro. J'ai généré un protocole complet, à partir de la conception de la sonde et jusqu'à l'acquisition de l'image. Je me suis intéressé à l’étude des ARNm du cycle cellulaire. En effet, les gènes du cycle cellulaire ont été largement étudiés au niveau de la protéine, mais on sait peu de choses sur la localisation de leurs ARNm. Pendant la mitose, les cellules subissent d'importantes modifications morphologiques et la traduction locale pourrait être un moyen d'atteindre la localisation des protéines. Le screening sur ces ARNm est en cours.Parallèlement à ces expériences, j'ai réalisé des expériences de smFISH sur 100 gènes choisis au hasard et 50 régulateurs de la transition G2/M du cycle cellulaire, en utilisant un protocole de smFISH classique. Dans cette configuration, on disposait d'une collection de lignées cellulaires HeLa, dans laquelle chaque cellule contient un chromosome artificiel bactérien avec le gène d'intérêt marqué au GFP. Par conséquent, en utilisant des sondes qui s'hybridaient à la séquence GFP, je pourrais utiliser le même ensemble de sondes marquées pour étudier la localisation de tous les ARNm. Un autre avantage est que la localisation des protéines pourrait être évaluée simultanément. Mes résultats indiquent que 4 ARNm ont montré une localisation spécifique lors du screening de 100 gènes choisis d’une manière aléatoire et 15 ARNm parmi les 54 régulateurs de la transition G2 / M. Ces ARNm appartiennent à cinq classes de localisation: "blobs", qui sont des agrégats d'ARNm cytoplasmiques; «clusters», qui sont des zones de concentration locale élevée d'ARNm, mais où une molécule unique d’ARNm peut encore être résolu; «nuclear membrane », où les ARNm se concentrent autour de l'enveloppe nucléaire; "spindle", qui sont des ARNm accumulés sur l'appareil de division mitotique, “spots" qui sont des agrégats d'ARNm cytoplasmiques où une molécule unique d’ARNm ne peut pas être résolu, et qui sont plus grands que les blobs. La colocalisation entre l'ARNm et la GFP, qui suggère une traduction locale, n'a été trouvée que pour 1 ARNm.Ces screenings aléatoires et ciblés effectués à petite échelle montrent une fréquence et une diversité inattendues dans les modèles de localisation d’ARNm. Cela ouvre la voie pour effectuer des screenings à plus grande échelle. / MRNA localization was discovered in 1983 in ascidian oocytes and early embryos. Since then many examples of localized RNAs have been found in many organisms, including plants, yeast, fungi, insects, fish and mammals. Localized mRNAs contribute to many biological functions, such as embryonic patterning, asymmetric cell division, cell migration, signaling, neuronal plasticity and others…Until now, only few studies analyzed RNA localization in a systematic manner. Three of them were done in Drosophila, during embryogenesis, oogenesis or larval stage and analyzed around 16000 mRNAs in total. The two other studies were done in mammalian cells and analyzed nearly 1000 mRNAs each. These studies opened a door and raised questions regarding the importance of mRNA localization in human cells and its implication in different biological processes. The goal of my thesis was thus to increase the throughput of single molecule FISH techniques (smFISH) and to study mRNA localization in HeLa cells in a systematic manner.One limitation in smFISH is the cost of the fluorescent oligonucleotide probes, which limits the number of mRNAs that can be analyzed. Therefore, I developed an alternative protocol in which probes for many genes were synthesized as a pool of oligonucleotides (40 per gene in average, more than 12000 in total). Gene-specific probes were then amplified by PCR and converted into single strand by in vitro transcription. I generated a complete protocol, starting from probe design and up to image acquisition. I was interested in studying cell cycle genes. Indeed, cell cycle genes have been extensively studied at the protein level but little is known concerning the localization of their mRNAs. During mitosis, cells go through important morphological modifications and local translation could be a mean of achieving protein localization. This screen is ongoing.In parallel to these experiments, I performed a smFISH based screen on 100 randomly chosen genes and 50 regulators of the G2/M transition of the cell cycle, using a traditional smFISH protocol. In this set-up, I took advantage of a library of HeLa cell lines, in which each cell line contains a bacterial artificial chromosome with the gene of interest tagged with GFP. Therefore, using oligonucleotides hybridizing to the GFP sequence, I could use the same probe set to study the localization of all the tagged mRNAs. A further advantage is that protein localization could be assessed simultaneously. My results indicate that two mRNAs showed a specific localization when screening 100 random genes, and 16 mRNAs among the 50 regulators of the G2/M transition. These mRNAs belong to five localization classes: "blobs", which are cytoplasmic mRNA aggregates; "clusters", which are areas of high local mRNA concentration but where individual mRNA can still be resolved; "nuclear envelope", where mRNAs concentrate around the nuclear envelope; "spindle", which are mRNAs accumulating on the cell division apparatus during mitosis, “spots" which are cytoplasmic mRNA aggregates where individual mRNA can’t be resolved and are bigger than blobs. Interestingly, colocalization between mRNA and GFP, which suggests local translation, was only found for 1 mRNA.These random and targeted screens performed at small-scale show an unexpected frequency and diversity in mRNA localization patterns, therefore pointing to new functions related to this process. This will stimulate future studies aiming at performing screenings at a higher scale.
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Dynamics of Mismatch RepairBritton, Brooke Marie 05 October 2020 (has links)
No description available.
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MEASUREMENT AND MODULATION OF CHARGE TRANSPORT THROUGH SMALL BENZENE DERIVATIVESYasini, Parisa, 0000-0001-8072-6597 January 2021 (has links)
The incorporation of molecules as low-cost and stable structures in electronic circuits is a promising strategy to miniaturize electronic components. Although single-molecule electronics is still at an early phase, the investigation of charge transport through single molecules is fundamentally important to understand the relevant scientific concepts and technological applications. In this dissertation, we measured and modulated the charge transport perpendicular to the plane of small benzene derivatives. In contrast to the conventional strategy to link molecules to electrodes via anchoring groups, we used the electrode potential to control the geometry of molecules and to form the junctions through π-system-metal electrode interactions. Using a combination of electrochemical STM (EC-STM) imaging and STM-BJ methods, the measurement of charge transport through single, flat oriented tetrafluoroterephthalic acid (TFTPA) molecules on an electrified Au (111) electrode showed that, at potentials below the potential of zero-charge (pzc) of Au(111), the molecules lie flat on the electrode and form highly ordered structures. The conductance of TFTPA, along the axis perpendicular to the benzene plane, is 0.24 ± 0.04 G0, consistent with reports for other molecules oriented flat in the junction. The configuration dependent conductivity has been confirmed by first-principles non-equilibrium Green’s function computation performed by Professor John Perdew and Dr. Haowei Peng at Temple University. Hence, the electrochemical surface potential can be employed to control the orientation of molecules to access a new charge transport measurement axis.
Building on our previous results (Chapter 3), we studied charge transport through two fundamentally important molecules, tetracyanoquinodimethane (TCNQ) and tetrafluorotetracyanoquinodimethane (F4TCNQ) to determine the effect of molecule-electrode binding while maintaining the same core molecular structure. The findings show that on the negatively charged Au(111), the flat-oriented TCNQ and F4TCNQ molecules exhibit similar but high conductance of ~ 0.22 ± 0.01 G0 and 0.24 ± 0.01 G0, respectively. In addition to the high conductance, two peaks at 0.02 G0, and 0.05 G0 were detected for both molecules, assigned to the bidentate-bidentate and monodentate-bidentate configurations. Density functional theory (DFT) and non-equilibrium Green’s function (NEGF) calculations were performed by Professor Manuel Smeu and Dr. Stuart Shepard at Binghamton University to determine the conductance of four distinct molecular configurations. The results show how the orientation of molecules in the junction and the molecule-electrode denticity influence the molecular orbital offsets relative to the Fermi level and the consequent charge transport.
The electronic structure and charge transport through single molecules can be modulated using various functional groups. Interestingly, our previous findings (Chapters 3, and 4) showed that the conductance perpendicular to the plane of TFTPA and TCNQ/F4TCNQ was similar to the parent molecules (TPA and TCNQ). Thus, it appeared that fluorination did not significantly change charge transport properties perpendicular to the molecular plane. Building on our previous studies, we measured the conductance through mesitylene substituted with electron-withdrawing groups (e.g., NO2, Br) or with electron-donating groups (e.g., CH3) to determine if other groups might impact conductance. Our results showed that the conductance perpendicular to the molecular plane increases by introducing electron-withdrawing groups and decreases as electron-donating groups are introduced to the mesitylene molecule. Density functional theory (DFT) and non-equilibrium Green’s function (NEGF) calculations were performed to rationalize our experimental findings (By Professor Smeu and Dr. Stuart Shepard). We demonstrated that the changes in the conductance perpendicular to the molecular plane correlate well with the Hammett constant of the corresponding functional groups, indicating the importance of the nature and strength of chemical substituents on the degree of conductance modulations at least for mesitylene derivatives.
Following up on the modulation of charge transport through the intrinsic properties of molecules, we investigated the effect of solvent polarity on conductance of single molecules. Particularly, we focused on charge transport through dimethylaminobenzonitrile (DMABN), a molecule that shows unique behavior, such as noticeable bulk electronic modulations in response to the physical properties of the solvents in which the molecule is immersed, e.g., dual fluorescence in polar environment, due to the stabilized intramolecular charge transfer (TICT) state. Our charge transport results show that the conductance of DMABN in a polar solvent (acidified water) is ~ten times higher than the value observed in toluene (nonpolar solvent). The conductance of a molecule with no TICT properties shows no solvent polarity-dependent conductance, indicating that the intrinsic properties of DMABN (i.e., the TICT effect) play a critical role in the enhanced conductivity in the polar solvent. Molecular dynamics calculations (performed by Professor Manuel Smeu, and Dr. Stuart Shepard) suggest that the DMABN molecule can undergo internal rotation in the junction in polar solvents, result in a higher conductance compare to the planar geometry. Our results demonstrate that molecules exhibiting TICT properties can be promising candidates to design molecular devices with sensing and switching functionalities.
The findings of this dissertation, in combination with the calculations (via collaboration with computational experts), show that the intrinsic and extrinsic properties of junctions, e.g., the geometry of molecule within the junction, the charge transport axis, the molecule-electrode binding, the characteristics and electronic structure of the molecules investigated, and the physical properties of the environment, influence charge transport through single molecules. This fundamental understanding and the ability to control charge transport through single molecules may allow the design of practical devices, e.g., large-scale molecular architectures and circuits, molecular switches, and sensors. / Chemistry
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Mechanical stability evaluation of i-motif and G-quadruplex structures under diverse circumstancesDhakal, Soma Nath 25 April 2013 (has links)
No description available.
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SINGLE MOLECULE ANALYSIS AND WAVEFRONT CONTROL WITH DEEP LEARNINGPeiyi Zhang (15361429) 27 April 2023 (has links)
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<p> Analyzing single molecule emission patterns plays a critical role in retrieving the structural and physiological information of their tagged targets, and further, understanding their interactions and cellular context. These emission patterns of tiny light sources (i.e. point spread functions, PSFs) simultaneously encode information such as the molecule’s location, orientation, the environment within the specimen, and the paths the emitted photons took before being captured by the camera. However, retrieving multiple classes of information beyond the 3D position from complex or high-dimensional single molecule data remains challenging, due to the difficulties in perceiving and summarizing a comprehensive yet succinct model. We developed smNet, a deep neural network that can extract multiplexed information near the theoretical limit from both complex and high-dimensional point spread functions. Through simulated and experimental data, we demonstrated that smNet can be trained to efficiently extract both molecular and specimen information, such as molecule location, dipole orientation, and wavefront distortions from complex and subtle features of the PSFs, which otherwise are considered too complex for established algorithms. </p>
<p> Single molecule localization microscopy (SMLM) forms super-resolution images with a resolution of several to tens of nanometers, relying on accurate localization of molecules’ 3D positions from isolated single molecule emission patterns. However, the inhomogeneous refractive indices distort and blur single molecule emission patterns, reduce the information content carried by each detected photon, increase localization uncertainty, and thus cause significant resolution loss, which is irreversible by post-processing. To compensate tissue induced aberrations, conventional sensorless adaptive optics methods rely on iterative mirror-changes and image-quality metrics to compensate aberrations. But these metrics result in inconsistent, and sometimes opposite, metric responses which fundamentally limited the efficacy of these approaches for aberration correction in tissues. Bypassing the previous iterative trial-then-evaluate processes, we developed deep learning driven adaptive optics (DL-AO), for single molecule localization microscopy (SMLM) to directly infer wavefront distortion and compensate distortion near real-time during data acquisition. our trained deep neural network monitors the individual emission patterns from single molecule experiments, infers their shared wavefront distortion, feeds the estimates through a dynamic filter (Kalman), and drives a deformable mirror to compensate sample induced aberrations. We demonstrated that DL-AO restores single molecule emission patterns approaching the conditions untouched by specimen and improves the resolution and fidelity of 3D SMLM through brain tissues over 130 µm, with as few as 3-20 mirror changes.</p>
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Development Of An Efficient Molecular Single-electron Transport SpectroscopyGarrigues, Alvar 01 January 2013 (has links)
In this thesis I present a complete and detailed guide for the development process and fabrication of efficient single-electron transistors (SETs) and a better single-molecule magnets (SMMs) deposition yield. Starting from a commercial Si/SiO2 wafer I show the steps for the deposition of different layers to fabricate a SET as well as the improvements achieved in those for a completely functional SET device. The development process is based on a combination of optical lithography and e-beam lithography with metal deposition in ultra-high vacuum. The improvements involve a better conductance in the Al gate component, with a controlled formation of the superficial oxide layer and a faster feedback electromigration-induced breaking of Au nanowires for the creation of nanogaps at room temperature. The gate component is improved by increasing its thickness and exposing it to plasma oxidation for the complete oxidation of its surface. The nanowire breaking is realized at room temperature to make use of the surface tension of Au, which, after a previous feedback procedure, eventually opens the final gap in the nanowire. Finally, I demonstrate a new technique that allows increasing the yield of having a SMM connected in the nanowire gap. This new technique is based on monitoring the resistance of the broken nanowires during the SMM deposition from a controlled liquid solution at room temperature. When the resistance ( > GΩ for open gaps) drops to values below Mega-ohms (characteristic resistance of a molecule bridging the gap) for a number of nanowires in the chip, the device is then ready for low temperature measurements.
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