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Devaux, Floriane. "Synthesis and AFM-based single-molecule force spectroscopy of helical aromatic oligoamide foldamers." Thesis, Bordeaux, 2018. http://www.theses.fr/2018BORD0346.
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Les foldamères sont des architectures moléculaires synthétiques repliées, inspirées par les structures et les fonctions des biopolymères naturels. Le repliement est un processus sélectionné par la nature pour contrôler la conformation de sa machinerie moléculaire afin de réaliser des tâches chimiques ou mécaniques. Durant les dix dernières années de recherche sur les foldamères, des oligomères synthétiques, capables d'adopter des conformations repliées bien définies et prévisibles, comme des hélices, ont été proposés. Les progrès récents ont montré que la synthèse chimique par étapes et le design moléculaire basé sur un squelette oligoamide aromatique permettaient de produire des architectures moléculaires repliées de manière hélicoïdale. La forme du squelette et sa rigidité, des préférences conformationnelles locales, des interactions spécifiques entre monomères éloignés dans une séquence, ainsi que l'action de paramètres externes comme le solvant, ou la présence d'ions peuvent être combinés pour induire une tendance au repliement. Ces architectures sont remarquables car elles peuvent donner lieu à des motifs de repliement qui n'ont pas d'équivalent dans les structures des biopolymères naturels. Par exemple, des hélices dont le diamètre varie le long de la séquence, ou des hélices possédant un centre d'inversion du pas, des hélices en chevrons,... ont été rapportées. Alors que les structures de ces molécules hélicoïdales ont été abondamment caractérisées à l'état solide par cristallographie des rayons X, leur comportement en solution, et surtout le comportement dynamique, est très peu connu. Leurs propriétés mécano-chimiques sont quant à elles inconnues à ce jour. L'objectif du projet est de synthétiser différentes molécules synthétiques hélicoïdales de type oligoamide aromatique et d'obtenir une description détaillée de leur conformation dynamique en solution, ainsi que de leurs propriétés mécano-chimiques, par spectroscopie de force sur molécule unique basée sur l'AFMFoldamers are artificial folded molecular architectures inspired by the structures and functions of natural biopolymers. Folding is the process selected by nature to control the conformation of its molecular machinery to carry out chemical functions and mechanical tasks, such as en-zyme catalysis, duplication in nucleic acids, force generation,... During the last decade of research on foldamers, synthetic oligomers able to adopt well- defined and predictable folded conformations, such as helices, have been proposed. Recent progress has shown that stepwise chemical synthesis and molecular design based on aromatic oligoamide backbones enable to produce large helically folded molecular architectures. The shape and stiffness of the backbone, local conformational preferences, specific interactions between distant monomers in sequences, as well as the action of external parameters such as the solvent or the presence of ions, can be combine to induce folding tendency. A remarkable aspect of these architectures is that they can give rise to folded patterns that have no in natural counterparts biopolymer structures. For instance, helices whose diameter varies along the se-quence, helices possessing a handedness inversion centre, herringbone helices have been reported. While the structures of these helical molecules have been well characterized in the solid state by X-ray crystallography, much less is known about their dynamic behavior in solution. Their mechanochemical properties are unknown. The objective of the project is to synthesize various helical nanorchitectures based on an oli-goamide aromatic backbone and to obtain a detailed picture of their dynamical conformation in solution, as well as, their mechanochemical properties, by AFM-based single molecule force spectroscopy
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Kirstein, Johanna, Christophe Jung, Christian Hellriegel, and Christoph Bräuchle. "Single molecule spectroscopy." Universitätsbibliothek Leipzig, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-196553.
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Maity, Sourav. "Single Molecule Force Spectroscopy of CNGA1." Doctoral thesis, SISSA, 2014. http://hdl.handle.net/20.500.11767/3873.
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Single molecule force spectroscopy (SMFS) is known to be one of the most powerful tool to investigate the relation between structure and function in molecules and proteins. The possibility to work in aqueous conditions at a single molecular level opens up an extraordinary perspective to investigate rare events at a molecular level of biological systems. Over the past years Atomic Force Microscopy (AFM) based on SMFS has provided us information, that is either difficult or impossible to get from any other method. In spite of its advancements, SMFS has not been applied to many molecules of biological relevance for several reasons, such as problems with the biological samples, data analysis and other technical issues. Indeed, the development and improvement of SMFS is becoming is very important to study biological molecules and proteins in their natural environment.
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Iordanov, Iordan. "Structure and dynamics of the outer membrane protein A from Klebsiella pneumoniae : a joint NMR–SMFS–proteolysis and MS approach." Toulouse 3, 2012. http://thesesups.ups-tlse.fr/1602/.
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"KpOmpA est une protéine de la membrane externe de K. Pneumoniae. Elle fait partie de la famille des "outer membrane protein A", OmpA. KpOmpA est une protéine constituée de deux domaines: un domaine transmembranaire structuré en tonneau ß et une partie soluble, périplasmique. Le domaine transmembranaire de KpOmpA présente une homologie importante avec celle d'OmpA d'E. Coli dont la structure a été déterminée par cristallographie aux rayons X et spectroscopie RMN. OmpA d'E. Coli est responsable lors de la formation de biofilm. Elle a un rôle d'adhésine et d'invasine. Elle est la cible préférentielle du système immunitaire et est le récepteur de bactériophages. Il est admis que la plupart de ces fonctions sont dues aux boucles extracellulaires de ces protéines. Les différences majeures entre les protéines KpOmpA et OmpA d'E. Coli concernent les boucles extracellulaires de longueur plus importante dans le cas de KpOmpA. Elles jouent un rôle important au cours de l'activation des macrophages et des cellules dendritiques par la voie des récepteurs TLR2. Les boucles extracellulaires jouent un rôle essentiel au cours de l'activation du système immunitaire. Mieux définir la structure et la dynamique de ces boucles est d'une importance essentielle afin de mieux appréhender la fonctionnalité des boucles extracellulaires de KpOmpA. Les informations structurales connues actuellement (structure RMN déterminée dans le groupe IPBS RMN en 2009) ont été obtenus jusqu'à présent avec des échantillons de protéines recombinantes purifiées et repliées dans des micelles de détergent. Dans le présent travail, nous avons d'abord établi un protocole de reconstitution de la protéine dans une membrane phospholipidique et caractérisé nos échantillons par microscopie électronique. Des expériences de spectroscopie de force atomique sur molécule unique ont été réalisées pour caractériser le repliement de la protéine dans son environnement membranaire. Ces expériences suggèrent un nouveau rôle de KpOmpA au sein même de la membrane (collaboration D. Müller, ETH Zürich). Le domaine soluble périplasmique de la protéine a été exprimé indépendamment du domaine membranaire. Les premières expériences HSQC réalisées montrent une structuration de ce domaine. La structure de ce domaine par spectroscopie RMN est en cours de réalisation. Le comportement dynamique des boucles extracellulaires du domaine membranaire KpOmpA reconstitué dans des liposomes a été étudié par spectroscopie RMN à l'angle magique (MAS) et notamment par mesure des temps de relaxation. Nous avons montré que la dynamique intrinsèque de la protéine est indépendante de l'environnement (membrane vs micelle). Des expériences de protéolyse ménagée suivie par spectrométrie de masse (MALDI-TOF) ont été comparées avec les informations RMN afin d'évaluer plus précisément les niveaux de mobilité des différentes boucles extracellulaires. La préservation au cours de l'évolution des boucles extracellulaires semble lier à leur dynamique, ce qui suggère l'importance de ces boucles extracellulaires, en termes de séquence, longueur mais aussi de dynamique lors de la réponse immunitaire. "KpOmpA is a two-domain membrane protein from Klebsiella pneumoniae belonging to the outer membrane protein A (OmpA) family. It is composed of a transmembrane ß-barrel with 8 ß-strands and a C-terminal, soluble periplasmic domain. The transmembrane domain presents a significant homology with E. Coli OmpA whose three dimensional structure has been determined by X-ray crystallography and by NMR. The E. Coli homologue can function as an adhesin and invasin, participate in biofilm formation, act as both an immune target and evasin, and serves as a receptor for several bacteriophages. It is assumed that most of these functions involve the four protein loops that emanate from the protein to the exterior of the cell. The difference between KpOmpA and E. Coli OmpA is mostly concentrated in these extracellular loops which are larger in the case of KpOmpA. KpOmpA was shown to activate macrophages and dendritic cells through the TLR2 dependent pathway, and these larger loops are supposed to play a specific role in the interactions with the immune system. Thus the structure and dynamics of these loops is of prime functional significance. The currently available information in this regard, including the NMR structure determined in the IPBS NMR group in 2009, have been obtained so far with recombinant protein samples purified and refolded in detergent micelles. In the present work we first established a reconstitution protocol that allowed the incorporation of the membrane protein in the more native environment of the lipid bilayer and characterised our samples by electron microscopy. SMFS experiments were used to probe the reconstituted KpOmpA unfolding-refolding pathways, exploring the folding mechanisms for ß-barrel proteins and suggesting a novel role for OmpA in the bacterial membrane (in collaboration with the group of D. Müller, ETH Zürich). The C-terminal periplasmic domain of KpOmpA was expressed and purified as a separate product and the feasibility of its structure elucidation by NMR was demonstrated by obtaining a high quality HSQC spectrum. The dynamic behaviour of the extracellular portion of the KpOmpA membrane domain reconstituted in liposomes has been investigated by solid state MAS NMR relaxation experiments. We confirmed that the previously observed gradient of dynamic along the molecule axis is an intrinsic property of the protein. Limited proteolysis and MALDI-TOF experiments were coupled with the NMR information in order to assess more precisely the different mobility levels in the loops. Evolutional preservation of the different loops regions is related to their observed flexibility, pointing towards immunologically important, variable, dynamic and accessible loops sections
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Howard, John Brooks. "Double point contact single molecule absorption spectroscopy." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/31648.
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Thesis (Ph.D)--Physics, Georgia Institute of Technology, 2010.Committee Chair: Marchenkov, Alexei; Committee Member: Davidovic, Dragomir; Committee Member: Gole, James; Committee Member: Hunt, William; Committee Member: Reido, Elisa. Part of the SMARTech Electronic Thesis and Dissertation Collection.
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Gryte, Kristofer. "Analysis methods for single molecule fluorescence spectroscopy." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:148969c6-78aa-49c2-8f0e-2d5e5018fd98.
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This thesis describes signal analysis methods for single-molecule fluorescence data. The primary factor motivating method development is the need to distinguish single-molecule FRET fluctuations due to conformational dynamics from fluctuations due to distance-independent FRET changes. Single-molecule Förster resonance energy transfer (FRET) promises a distinct advantage compared to alternative biochemical methods in its potential to relate biomolecular structure to function. Standard measurements assume that the mean transfer efficiency between two fluorescent probes, a donor and an acceptor, corresponds to the mean donor-acceptor distance, thus providing structural information. Accordingly, measurement analysis assumes that mean transfer efficiency fluctuations entail mean donor-acceptor distance fluctuations. Detecting such fluctuations is important in resolving molecular dynamics, as molecular function often necessitates structural changes. A problem arises, however, in that factors other than donor-acceptor distance changes may induce mean transfer efficiency fluctuations. We refer to these factors as distance-independent FRET changes. We present analysis methods to detect distance-independent photophysical dynamics and to determine their correlation with distance-dependent FRET dynamics. First, we review a theory of photon statistics and show how we can use the theory to detect FRET fluctuations. Second, we extend the theory to alternating laser excitation (ALEx) measurements and demonstrate how fluorophore stoichiometry, a measure of fluorophore brightness, reports on distance-independent photophysical dynamics. Next, we provide a measure to determine the extent to which stoichiometry fluctuations account for FRET dynamics. Finally, we use a framework similar to the preceding along with recent advances in the theory of total internal reflection fluorescence (TIRF) microscopy FRET measurements to detect TIRF FRET fluctuations which occur on a timescale faster than the measurement temporal resolution. We validate our methods with simulations and demonstrate their utility in delineating RNA polymerase open complex conformational dynamics.
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Radiom, Milad. "Correlation Force Spectroscopy for Single Molecule Measurements." Diss., Virginia Tech, 2014. http://hdl.handle.net/10919/49677.
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This thesis addresses development of a new force spectroscopy tool, correlation force spectroscopy (CFS), for the measurement of the mechanical properties of very small volumes of material (molecular to µm³) at kHz-MHz time-scales. CFS is based on atomic force microscopy (AFM) and the principles of CFS resemble those of dual-trap optical tweezers. CFS consists of two closely-spaced micro-cantilevers that undergo thermal fluctuations. Measurement of the correlation in thermal fluctuations of the two cantilevers can be used to determine the mechanical properties of the soft matter, e.g. a polymeric molecule, that connects the gap between the two cantilevers. Modeling of the correlations yields the effective stiffness and damping of the molecule. The resolution in stiffness is limited by the stiffness of the cantilever and the frequency by the natural frequency of the cantilevers, but, importantly, the damping resolution is not limited by the damping of the cantilever, which has enabled high-resolution measurements of the internal friction of a polymer. The concept of CFS was originally presented by Roukes' group in Caltech [Arlett et al., Lecture Notes in Physics, 2007]; I developed the first practical versions of CFS for experimentation, and have used it in two applications (1) microrheology of Newtonian fluids and (2) single molecule force spectroscopy. To understand the correlation in thermal fluctuations of two cantilevers I initially validated the theoretical approach for analyzing correlation in terms of deterministic model using the fluctuation-dissipation theorem [Paul and Cross, PRL, 2004]. I have shown that the main advantages of such correlation measurements are a large improvement in the ability to resolve stiffness and damping. Use of CFS as a rheometer was validated by comparison between experimental data and finite element modeling of the deterministic vibrations of the cantilevers using the known viscosity and density of fluids. Work in this thesis shows that the data can also be accurately fitted using a simple harmonic oscillator model, which can be used for rapid rheometric measurements, after calibration. The mechanical properties of biomolecules such as dextran and single stranded DNA (ssDNA) are also described. CFS measurements of single molecule properties of ssDNA reveal the internal friction of the molecule in solution.Ph. D.
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Iljina, Marija. "Aggregation of alpha-synuclein using single-molecule spectroscopy." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/263216.
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The aggregation of alpha-synuclein (αS) protein from soluble monomer into solid amyloid fibrils in the brain is associated with a range of devastating neurodegenerative disorders such as Parkinson’s disease. Soluble oligomers formed during the aggregation process are highly neurotoxic and are thought to play a key role in the onset and spreading of disease. Despite their importance, these species are difficult to study by conventional experimental approaches owing to their transient nature, heterogeneity, low abundance and a remarkable sensitivity of the oligomerisation process to the chosen experimental conditions. In this thesis, well-established single-molecule techniques have been utilised to study the aggregation and oligomerisation of αS in solution.
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CORTI, ROBERTA. "Single molecule force spectroscopy of proteins and DNA." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2020. http://hdl.handle.net/10281/273770.
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Negli ultimi decenni, lo sviluppo di nuove tecniche di singola molecola ha creato le basi per nuovi paradigmi nel campo della biofisica. In particolare, la nanomanipolazione di singole biomacromolecole ha permesso la caratterizzazione meccanica di proteine e DNA, cercando di evidenziare la relazione fondamentale tra struttura e funzione biologica. Nel corso degli anni sono stati sviluppati diversi metodi di nanomanipolazione: tra questi, il Microscopio a Forza Atomica (AFM), il Magnetic Tweezers (MT) e il Flow-Stretching (F-S) accoppiato a fluorescenza. Queste tre tecniche sono state utilizzate per la caratterizzazione di singole biomacromolecole con tecniche di spettroscopia di forza a livello di singola molecola (SMFS) in quattro diversi progetti. In questa Tesi mi sono occupata principalmente di caratterizzare strutturalmente diverse proteine e correlarne la conformazione alla funzione biologica. In questa ottica, sono state studiate le diverse conformazioni strutturali di una proteina intrinsecamente disordinata (IDP) coinvolta nella malattia del Parkison’s (PD), chiamata alfa-synucleina (AS) e il riarrangiamento strutturale di una proteina coinvolta nel mantenimento strutturale cromosomico, la condensina, durante la condensazione di singoli filamenti di DNA. Inoltre, è stata studiata la struttura di un DNA-analogo sia caratterizzandone la stabilità termica sia attraverso studi di nanomeccanica in SMFS. Infine, è stata descritta una implementazione tecnica di un apparato di F-S accoppiato con illuminazione TIRF, per ottenere una rapida sostituzione di soluzioni durante esperimenti di microfluidica, ottenendo un setup particolarmente indicato per studi di interazione tra DNA e proteine. Durante lo studio della proteina AS in SMFS, il problema della mancanza di una struttura secondaria ben definita è stato risolto con l’impego di una singola poliproteina, contenente un modulo di AS umana. Sono state caratterizzati tre diversi stati conformazionali per AS, da uno stato totalmente destrutturato a una conformazione altamente strutturata. I riarrangiamenti conformazionali di AS sono stati studiati anche in presenza di gallato epigallocatechina (EGCG) e dopamina (DA). Una particolare attenzione è stata quindi riservata al confronto tra i risultati ottenuti da tecniche di SMFS e quelli precedentemente ricavati da analisi di spettroscopia di massa. La proteina AS è stata caratterizzata anche in presenza di tre diverse mutazioni puntuali legate al PD (A30P, A53T e E83A). Nel secondo progetto, un analogo del DNA (DAP) è stato caratterizzato sia con metodi di stabilità termica che di nanomeccanica, studiando il comportamento del DAP-DNA in presenza di forze esterne. L’estensione e la rigidità delle due molecole di DNA (DAP e WT) sono state caratterizzate a basse forze (AFM e MT) e alte forze (MT), dove è stata descritta la transizione di overstretching. Nel terzo progetto è stato studiato l’effetto della condensina sulla condensazione di singoli filamenti di DNA. In particolare, è stato effettuato uno studio di singola molecola per seguire, in tempo reale, la riduzione di estensione di DNA in presenza di condensina e ATP. Considerato che la maggior parte delle curve sono caratterizzate da improvvisi e ben evidenti salti durante la condensazione, sono stati sviluppati e validati due diversi algoritmi per l’identificazione automatica dei gradini. Infine, diverse celle fluidiche sono state testate nell’apparato di F-S, nell’ottica di studi di interazione tra proteine e DNA. Queste celle sono state caratterizzate sia in termini di rapidità di scambio di flussi laminari che di forza capaci di generare sul campione. Sono stati infine visualizzate singole molecole di DNA fluorescenti in presenza di flussi di diversa portata.In the last few decades, the constant development of novel single molecule techniques has created the basis for a new paradigm in the field of biophysics. Among all, the nanomanipulation of individual biomolecules revealed new insights into the mechanics of biological molecules, in particular proteins and DNA, improving the understanding of the fundamental relation between structural properties and biological functions. Therefore, several single-molecule nanomanipulation methods have been developed, including Atomic Force Microscopy (AFM), Magnetic Tweezers (MT) and Flow Stretching (F-S) coupled with fluorescence. All these technique were employed in this Thesis for the characterisation of biological macromolecules by single molecule force spectroscopy (SMFS). In this Thesis I focus mainly on several aspects of a few different proteins trying to depict a frame in which the strong link between proteins function and their structure can be clarified. With this aim, I study the conformational states of an intrinsically disordered protein (IDP) involved in Parkinson's Disease, the a-synuclein (AS) and the structural change driving the DNA compaction mediated by structural maintenance protein, the condensin. Finally, I present a structural study of a DNA-analogue by thermal shifting essays and single molecule experiments. I included also a technical implementation of a (F-S) combined with TIRF set up to promote the high-speed exchanging buffer for study protein DNA interactions. In the AS single molecule force spectroscopy (SMFS) study, I afford the problem of AS lacking of well defined structure by stretching and unfolding a single polyprotein containing the human AS by employing a SMFS approach. The analysis of the different unfolding pathways gives information about the structural conformation of the protein before the mechanical denaturation. The AS was found to assume three distinct conformational states ranging from a random coil to a highly structured conformation. Since ligands, such as Epigallocatechin-3-Gallate (EGCG) and Dopamine (DA), are known to affect the fibrillation process of AS, I used this single molecule technique to investigate the effect of EGCG and DA on the conformational ensemble of the WT AS. Moreover, knowing from several studies that the presence of point mutations, linked to familial PD, correlate with the gaining of structure and therefore with AS aggregation, I SMFS studies also on AS with three different single point mutations (A30P, A53T and E83A). A particular emphasis was given to the comparison between SMFS results and native mass spectrometry data for the conformational changes of AS in the presence of both DA and EGCG. In the following part, related to the DAP: diaminopurine-substituted DNA, a systematic comparison between a wild-type DNA and DAP DNA is performed, in terms of thermal stability and nanomechanical properties, measured at low and high forces. At low forces the DNA extension and bending rigidity were investigated, by using both MT and AFM, while at high forces the overstretching transition behaviour was explored. In the section related to condensin mediated DNA collapsing, I present a single-molecule MT study to measure, in real-time, the compaction of individual DNA molecules by the condensin complex in the presence of ATP. Since many compaction traces showed sudden distinct decreases in the DNA end-to-end length, I present and validate two different very conservative user-bias-independent step-finding algorithm to extract the size of these compaction steps. Finally, a DNA flow stretching implementation is presented. Briefly, several flow cells were tested to achieve a fast buffer exchange in both MT and F-S coupled with TIRF, in the frame of visualisation of DNA:proteins interactions. We validated our flow cells in term of boundary exchange and applied force. We also visualized fluorescent DNA molecules stretched in the presence of several flow rates.
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Yang, Shilong 1975. "Theoretical study of single-molecule spectroscopy and vibrational spectroscopy in condensed phases." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/30237.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2005.Includes bibliographical references (p. 267-279).
In this thesis, theoretical models and computer simulations are employed to study several problems of single-molecule spectroscopy and vibrational spectroscopy in condensed phases. The first part of the thesis concentrates on studying dynamic disorders probed by single molecule fluorescence spectroscopy. Event statistics and correlations of single-molecule fluorescence sequences of modulated reactions are evaluated for multi-channel model, diffusion-controlled reaction model, and stochastic rate model. Several event-related measurements, such as the on-time correlation and the two-event number density, are proposed to map out the memory function, which characterizes the correlation in the conformational fluctuations. A semiflexible Gaussian chain model is used to determine the statistics and correlations of single-molecule fluorescence resonant energy transfer (FRET) experiments on biological polymers. The distribution functions of the fluorescence lifetime and the FRET efficiency provide direct measures of the chain stiffness and their correlation functions probe the intra-chain dynamics at the single-molecule level. The fluorescence lifetime distribution is decomposed into high order memory functions that can be measured in single- molecule experiments. The scaling of the average fluorescence lifetime on the contour length is predicted with the semi-flexible Gaussian chain model and agrees favorably with recent experiments and computer simulations.
(cont.) To interpret the fluorescence measurements of the mechanical properties of double-stranded DNA, a worm-like chain model is used as a first-principle model to study double-stranded DNA under hydrodynamic flows. The second part of the thesis concentrates on nonperturbative vibrational energy relaxation (VER) effects of vibrational line shapes. In general, nonperturbative and non-Markovian VER effects are demonstrated more strongly on nonlinear vibrational line shapes than on linear absorption.
by Shilong Yang.
Ph.D.
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Tojira, Opas. "Single-molecule fluorescence spectroscopy : Implementation of alternating-laser excitation." Thesis, University of Leeds, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.531514.
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Shin, Yongdae. "Single molecule fluorescence spectroscopy of ClpXP-mediated substrate degradation." Thesis, Massachusetts Institute of Technology, 2009. http://hdl.handle.net/1721.1/50562.
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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2009.Includes bibliographical references (leaves 67-71).
Energy-dependent proteases, such as ClpXP, are responsible for the regulated destruction of proteins in prokaryotes and organelles of eukaryotes. AAA+ ATPases in these proteases recognize protein substrates and power their mechanical denaturation and subsequent translocation into a sequestered degradation chamber where polypeptide cleavage occurs. Here, we present the single molecule fluorescence assay for probing the interaction between the ClpXP enzyme and its substrates. A covalently crosslinked ClpX hexamer maintain functionally stable form at the low concentration of single molecule level. Surface passivation through polyethylene glycol (PEG) remove unwanted nonspecific binding of substrates, providing specific immobilization of ClpXP protease on the glass surface illuminated by total internal reflection fluorescence (TIRF). Cy3-labeled engineered substrates containing nondegradable GFP in the prescence of ATP[gamma]S form stable pre-engaged substrate-ClpXP complexes where the whole substrate degradation pathway, from unfolding to egress of degraded products, can be monitored without competing with dissociation or additional background characteristic of free labeled substrate in solution. We directly observe some terminal processes that are encountered by ClpXP at the end of substrate degradation process. It is also shown that GFP tail domain stably bind to the ClpX in the presence of ATP[gamma]S and even in the absence of ATP hydrolysis. With the developement of single molecule assay for AAA+ protease, we can expand our knowledge on the mechanism of this crucial motor protein family.
by Yongdae Shin.
S.M.
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Kastrup, Lars. "Fluorescence depletion by stimulated emission in single-molecule spectroscopy." [S.l. : s.n.], 2004. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB11513777.
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PONZELLINI, PAOLO. "Plasmonic nanopores for single molecule spectroscopy towards sequencing applications." Doctoral thesis, Università degli studi di Genova, 2019. http://hdl.handle.net/11567/939990.
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Viader, Godoy Xavier. "Biophysical properties of single-stranded DNA studied with single-molecule force spectroscopy." Doctoral thesis, Universitat de Barcelona, 2021. http://hdl.handle.net/10803/670920.
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In this thesis, single-molecule experiments using LOT are employed to extract accurate information about the thermodynamics and kinetics of various molecular systems, with special emphasis on the elastic properties of single-stranded DNA (ssDNA). The thesis is divided in three parts.
Part I provides a general description of the research field as well as the main theoretical framework for the basic concepts that will be developed in parts II and III. In Chapter 2 the miniTweezers and the experimental setup used throughout the thesis is described, as well as the physical basis of its working mechanisms, introducing the phenomenon of optical trapping. Chapter 3 contains a brief introduction of the biomolecules of study in this thesis, with an explanation of their historical discoveries, as well as their structure and function. The main focus of this chapter is on ssDNA, which is the main object of study of the thesis. Chapter 4 introduces the polymer models that are widely used in describing the elasticity of nucleic acids and proteins. Specifically, the Freely-Jointed Chain and Worm-Like Chain models are presented.
Part II deals with the elasticity of single-stranded DNA. This is the main part of the thesis, and it includes chapters 5-7. Chapter 5 is about the study of the elasticity of ideal ssDNA chain, i.e. the one that can be modelled as ideal polymers (presented in Chapter 4). The study of the elasticity of different DNA sequences is presented. The blocking-splint oligo technique is described, a experimental technique developed for studying the elasticity of short (tens of bases) DNA molecules. This study shows the need of using extensible models to succesfully describe ssDNA elasticity over a large range of forces, which explains the previous discrepancies on the elastic parameters obtained in different studies.
We also provide an explanation for the required extensibility of the model: a transition experienced at the nucleotide level: a change in DNA sugar pucker conformation. A simple two-states model is introduced and preeliminary results regarding its energetics are presented. The characterization of the ssDNA elasticity is central for the works developed in the following chapters.
Chapter 6 studies the stacking-unstacking transition for ssDNA, previously observed for certain sequences (mainly purine-rich ones). Several molecules, with different degrees of stacking, are studied by obtaining their force extension curves (FECs). A cooperative helix-coil model including heterogeneity is developed and used to fit the obtained FECs, allowing to obtain elastic parameters to describe the stacked chain. The salt dependence of the unstacking transition is also measured by studying two of the sequences by varying the salt concentration over two decades. The free energy of formation of dsDNA duplexes depends on the salt concentration. The obtained salt dependence on the stacking free-energy of ssDNA provides a possible explanation for the salt dependence of duplex formation.
Chapter 7 deals with the non-specific structures that arise at low forces and high salt concentration when pulling ssDNA molecules longer than $\sim 100$ bases. A helix-coil model with cooperativity is proposed and used to extract some mean-field characteristics of these structures. 8 different sequences are studied, characterizing their elasticity and deviation from the ideal elastic behaviour. The results for a $14$kb molecule for 3 decades of varying \ce{NaCl} and \ce{MgCl2} are also shown. All experimental FECs are fitted to the helix-coil model. The model can be used to predict the formation of secondary structures at zero force. A comparison between the predicted structures from the model and those obtained from Mfold is also investigated.
Part III contains two studies which also need of the correct determination of ssDNA elasticity. In Chapter 8, we study the interaction between the RecQ helicase from E. coli and DNA, i.e. how the RecQ unwinds double-stranded DNA molecules, releasing single-stranded DNA. We obtain some of its kinematic properties as well as study the entropy production of the system using the Fluctuation Theorem. In Chapter 9, the effect of DNA mismatches, i.e. non complementary base pairing, on the stability of DNA is studied. To do so, two types of experiments on several DNA sequences are performed: stretching and releasing the molecule by moving the optical trap (pulling experiments) and monitoring the folding/unfolding of the molecule passively (hopping experiments).En aquesta tesi hem realitzat experiments fent servir pinces òptiques per tal d’extreure informació precisa sobre les propietats termodinàmiques i cinètiques de diferents sistemes moleculars, posant especial èmfasi en les propietats elàstiques de la cadena simple d’ADN (ssDNA, pel seu acrònim en anglès). La tesi es troba dividida en tres parts. A la primera part s’introdueix de forma general el camp de recerca dels experiments de molècula única, així com s’expliquen els conceptes més bàsics que es desenvoluparan en les parts II i III. La configuració experimental emprada al llarg de tota la tesi, les pinces òptiques, s’introdueix al capítol 2. Per a fer-ho, s’expliquen els principis físics de funcionament de les pinces, que es basen en l’atrapament òptic. Breument, la focalització d’un feix de llum d’alta intensitat permet atrapar i exercir forces en micropartícules dielèctriques (pilotes fetes de plàstic de la mida d’un bacteri), que són recobertes químicament de manera que la molècula d’estudi pot estirar-se, de forma individual, repetides vegades. El capítol 3 conté una breu introducció a les biomolècules que apareixen en aquesta tesi, amb una breu explicació de la seva descoberta, així com la seva estructura i funció (íntimament relacionades). Ens centrem en la descripció de la ssDNA que és el principal objecte d’estudi de la tesi. Al capítol 4 s’introdueixen els models de polímers que s’empren habitualment per a descriure l’elasticitat d’àcids nucleics i proteïnes. En concret, es descriuen els models de la Freely-Jointed Chain i la Worm-Like Chain. La Part II tracta de l’elasticitat de la ssDNA, i inclou els capítols 5, 6 i 7. El capítol 5 es basa en la caracterització de l’elasticitat de la cadena ideal de ssDNA, és a dir, aquella que pot ser modelitzada pels polímers ideals introduïts en el capítol 4. S’estudia l’elasticitat de diferents seqüències de ssDNA, introduint un nou mètode experimental, blocking-splint oligo, per tal d’ampliar el rang de forces estudiat habitualment en molècules curtes (d’una longitud de desenes de bases) de ssDNA. L’estudi mostra la necessitat d’emprar models elàstics extensibles per a la correcte caracterització de l’elasticitat de ssDNA, que explica les discrepàncies existents entre els paràmetres elàstics trobats a la literatura. També hipotetitzem que l’extensibilitat del model pot ser explicada gràcies a la transició experimentada a nivell de nucleòtids: el canvi que experimenta la distància interfosfat de l’ADN es veu modificada segons quina sigui la configuració de l’anell de desoxiribosa. Tot i que és un fenomen molt més conegut en la cadena doble d’ADN, l’apilament-desapilament de bases també s’ha observat en certes seqüències de ssDNA (especialment les que són riques en contingut de purines). Al capítol 6 s’estudien quatre molècules amb un grau d’apilament diferent a partir de les seves corbes força-extensió (FECs). Es desenvolupa un model helix-coil (hèlix-cabdell) per tal d’ajustar les FECs, fet que permet d’obtenir, indirectament, les propietats elàstiques de la cadena apilada. També s’estudia la dependència d’aquesta transició variant la concentració de sal dels experiments en més de dos ordres de magnitud. A través d’aquests experiments, trobem una dependència amb la concentració de sal de l’energia lliure de formació de l’apilament de la ssDNA, fet que ens permet explicar, parcialment, la dependència que es troba en la literatura per la hibridació de la cadena doble d’ADN. El capítol 7 tracta de la formació d’estructures no específiques que apareixen a forces baixes i a concentració de sal alta per a molècules de ssDNA de més de ~100bases. Es proposa un model helix-coil amb cooperativitat per tal de caracteritzar propietats de camp mitjà de les estructures estudiades. S’estudien vuit seqüències diferents, entre 120 i ~14000 bases, i es caracteritza el seu desviament respecte de la corba elàstica ideal amb el model. També s’estudia la dependència de l’estructura secundària de la ssDNA en funció de la concentració de la sal. Analitzant experiments variant la concentració de MgCl2 i NaCl, aconseguim reproduir les FECs a partir de fer dependre els paràmetres del model amb la sal. Finalment, el model desenvolupat ens permet predir la formació d’estructura secundària a força zero (fet que no podem detectar directament a partir d’experiments d’espectroscopia de forces). Es comparen les previsions del model amb les trobades per Mfold, trobant una compatibilitat per als resultats per a molècules de de menys de 1000 bases. La darrera part se centra en col·laboracions que he fet durant a tesi i que necessiten una determinació precisa de les propietats elàstiques de la ssDNA. Al capítol 8 s’estudia la interacció entre l’helicasa del bacteri E. coli i l’ADN, que s’encarrega d’obrir la cadena doble d’ADN, alliberant ssDNA. S’extreuen les seves propietats cinètiques, com la velocitat de translocació – obtenim, independentment de la força aplicada, d’uns 50bp/s, d’acord amb la literatura –. També n’estudiem les seves propietats termodinàmiques, a partir del Teorema de Fluctuació. Finalment, al capítol 9 s’estudien els efectes de certs defectes en molècules d’ADN. A partir d’experiments fora de l’equilibri s’extrau la penalització que suposa per a la hibridització d’ADN la presència d’aquestes bases no complementàries (és a dir, que no són enllaços de A-T o G-C).
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Dunn, James Albert. "Single Molecule Characterization of Peptide/Hematite Binding." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1494014864020062.
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Duchi, Llumigusin Diego Armando. "Single-molecule studies of transcription initiation." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:fa5d7117-4270-4362-95f4-ce1c870f2921.
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Single-molecule Förster resonance energy transfer (smFRET) has emerged as an important tool for studying biological reactions. This thesis describes smFRET investigations into the mechanism of bacterial transcription initiation. We developed protocols to immobilize RNAP-DNA initiation complexes using vesicles and antibodies. We used these techniques to show that the transcription bubble conformation in immobilized complexes exhibits inter-molecular heterogeneity. We observed large FRET changes that we attribute to transcription bubble opening and closing dynamics. We found that σ70 region 3.2 (σR3.2) influences the kinetics of the bubble dynamics, which supports proposals that σR3.2 interacts with the transcription bubble template strand. We extended our investigations to RNA synthesis and were able to observe abortive initiation cycles directly. We observed RNAP pausing and backtracking for the first time in transcription initiation. We obtained data suggesting that σR3.2 stabilises short RNAs at the active centre and forms a barrier to the extension of RNAs longer than 5-nt in length. We extended our abortive initiation assay to observe signal changes that we attribute to promoter escape. Our data revealed the number of abortive cycles that occur prior to escape, the kinetics of promoter escape, and pausing events that may have some regulatory function. We investigated the conformational dynamics of the RNAP β clamp and observed dynamic conformational changes between clamp-open and clamp-closed states. Our work confirms proposals that the clamp remains stably closed once the open complex (RPO) is formed. We investigated what affect the antibiotics Myxopyronin and Lipiarmycin have on the clamp conformation. Our results revealed that Myxopyronin traps the clamp in a closed conformation, while Lipiarmycin traps it in an open conformation. Overall, we made a number of novel observations that we believe advance our understanding of the mechanism of transcription. We hope that the discoveries reported here will direct future research efforts into RNAP function.
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Kurtz, Andrea H. "Probing single-stranded DNA structure and conformational transitions with single-molecule fluorescence spectroscopy /." May be available electronically:, 2008. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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Dhital, Bharat. "Single-molecule interfacial electron transfer dynamics in solar energy conversion." Bowling Green State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1477997482545831.
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Bippes, Christian Alexander. "Investigation of biological macromolecules using atomic force microscope-based techniques." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2009. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-23734.
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The atomic force microscope (AFM) provides a powerful instrument for investigating and manipulating biological samples down to the subnanometer scale. In contrast to other microscopy methods, AFM does not require labeling, staining, nor fixation of samples and allows the specimen to be fully hydrated in buffer solution during the experiments. Moreover, AFM clearly compares in resolution to other techniques.
In general, the AFM can be operated in an imaging or a force spectroscopy mode. In the present work, advantage was taken of this versatility to investigate single biomolecules and biomolecular assemblies.
A novel approach to investigate the visco-elastic behavior of biomolecules under force was established, using dextran as an example. While a molecule tethered between a solid support and the cantilever tip was stretched at a constant velocity, the thermally driven oscillation of the cantilever was recorded. Analysis of the cantilever Brownian noise provided information about the visco-elastic properties of dextran that corresponded well to parameters obtained by alternative methods. However, the approach presented here was easier to implement and less time-consuming than previously used methods.
A computer controlled force-clamp system was set up, circumventing the need for custom built analogue electronics. A commercial PicoForce AFM was extended by two computers which hosted data acquisition hardware. While the first computer recorded data, the second computer drove the AFM bypassing the manufacturer's microscope control software. To do so, a software-based proportional-integral-differential (PID) controller was implemented on the second computer. It allowed the force applied to a molecule to be held constant over time. After tuning of the PID controller, response times obtained using that force-clamp setup were comparable to those of the recently reported analogue systems. The performance of the setup was demonstrated by force-clamp unfolding of a pentameric Ig25 construct and the membrane protein NhaA. In the latter case, short-lived unfolding intermediates that were populated for less than 10 ms, could be revealed.
Conventional single-molecule dynamic force spectroscopy was used to unfold the serine:threonine antiporter SteT from Bacillus subtilis, an integral membrane protein. Unfolding force patterns revealed the unfolding barriers stabilizing structural segments of SteT. Ligand binding did not induce new unfolding barriers suggesting that weak interactions with multiple structural segments were involved. In contrast, ligand binding caused changes in the energy landscape of all structural segments, thus turning the protein from a brittle, rigid into a more stable, structurally flexible conformation. Functionally, rigidity in the ligand-free state was thought to facilitate specific ligand binding, while flexibility and increased stability were required for conformational changes associated with substrate translocation. These results support the working model for transmembrane transport proteins that provide alternate access of the binding site to either face of the membrane.
Finally, high-resolution imaging was exploited to visualize the extracellular surface of Cx26 gap junction hemichannels (connexons). AFM topographs reveal pH-dependent structural changes of the extracellular connexon surface in presence of HEPES, an aminosulfonate compound. At low pH (< 6.5), connexons showed a narrow and shallow channel entrance, which represented the closed pore. Increasing pH values resulted in a gradual opening of the pore, which was reflected by increasing channel entrance widths and depths. At pH > 7.6 the pore was fully opened and the pore diameter and depth did not increase further. Importantly, coinciding with pore gating a slight rotation of the subunits was observed. In the absence of aminosulfonate compounds, such as HEPES, acidification did not affect pore diameters and depths, retaining the open state. Thus, the intracellular concentration of taurine, a naturally abundant aminosulfonate compound, might be used to tune gap junction sensitivity at low pH.
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Opfer, Jan. "Single-molecule force spectroscopy studies of integrin-mediated cell signaling." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-148393.
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Nieder, Jana Berit [Verfasser]. "Single-molecule spectroscopy on pigment-protein complexes / Jana Berit Nieder." Berlin : Freie Universität Berlin, 2011. http://d-nb.info/102593881X/34.
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Marshall, Addison Robert Lee. "Surface enhanced Raman spectroscopy for single molecule detection and biosensing." Thesis, University of Hull, 2017. http://hydra.hull.ac.uk/resources/hull:16553.
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The aim of this thesis is to design plasmonic nano-gaps capable of detecting materials down to sufficiently low concentrations such that single molecule characteristics are observed. We begin first, by discussing the theory of plasmonics. Then, we assess the recent literature on the subject to develop an understanding in the field of plasmonics and to describe the fundamental concepts behind how plasmonic nano-sensors operate. Also, this allows us to show where our research fits in. The aim of this thesis is to design plasmonic nano-gaps capable of detecting materials down to sufficiently low concentrations such that single molecule characteristics are observed. We begin first, by discussing the theory of plasmonics. Then, we assess the recent literature on the subject to develop an understanding in the field of plasmonics and to describe the fundamental concepts behind how plasmonic nano-sensors operate. Also, this allows us to show where our research fits in. The second area of research involves practical Surface Enhanced Raman Spectroscopy (SERS) experiments from our optimized nano-gaps. The nano-gaps were doped with the molecular dyes Rhodamine 6G and Crystal Violet at concentrations of 2x10−7 M. SERS measurements revealed differences in the relative intensities of their respective SERS peaks at low concentrations when compared to the SERS spectra measured from gaps doped the same dye at higher concentrations of 2x10−5 M. Time dependent SERS measurements showed that the SERS signal is stable over a long period of time, indicating the observed relative intensity changes are due to changes in molecular orientation from one gap to another, demonstrating that our optimized nano-gaps have single molecule sensitivity. When exciting at 532 nm, the 118 nm silver spheres used to form the nanogap with the silver film below were shown to enhance the Raman signal by 4:2x relative to the 200 nm silver nano-spheres, and up to 7:73x relative to the 60 nm silver nanospheres. When compared to our simulation results for the same structures excited with a Gaussian source with NA = 0:55, we showed the information collected from the Raman study correlated well with the theoretical data. Following our work investigating single molecule characterisation of fluorescent materials, we began looking at trace levels of a conjugated polymer (F8-PFB). The previous investigation had been from a purely electromagnetic enhancement perspective using a secondary polymer matrix buffer which was optically transparent in the region of interest for the Raman spectra of our target molecule. This polymer provided a barrier between the target material and the metallic nanostructure, thereby minimizing the potential of photo-induced chemical processes in the Raman signal. In this study, the material itself forms the basis of the cavity between the particle and the film below. This system classifies the single molecule regime via the observation of intensity blinking events, which are characteristic of Single Molecule SERS (SM-SERS). We also demonstrated the biosensing applications of our research, where nanoparticle clusters on a metallic film were used to produce spectra from bio-molecules undergoing conformation changes as a result of UV light exposure. The SERS spectra revealed decreased intensity from the Tryptophan (Trp) modes and appearance of disulphide bonds as time under UV light exposure progresses for lysozyme. Our final chapter shows that by using nanoparticles coupled to different substrates such as Distributed Bragg Reflectors (DBRs) and dielectric slabs, the hybrid modes improved the Quality-factor (Q-factor) of the scattering spectra. Therefore, these systems theoretically have great potential for refractive index sensing with high sensitivity to binding activity of molecular targets. The highest Q-factor of the systems we investigated was the 200 nm gold particle coupled to the 2 μm dielectric slab at 22:48, followed by the same particle deposited on a 700 nm stop-band DBR at 7:41.
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Fisher, Brent R. "Time resolved fluorescence of CdSe nanocrystals using single molecule spectroscopy." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/33649.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2005.Vita.
Includes bibliographical references.
A wide variety of spectroscopic studies of CdSe nanocrystals (NCs) are presented in this thesis, all studying some aspect of the temporal evolution of NC fluorescence tinder different conditions. In particular the methods of single molecule spectroscopy are used in many experiments allowing the behavior of individual NCs to be resolved from the blurring effect of averaging over the ensemble. Studies of the excited state lifetime of band edge fluorescence from single NCs reveal multiexponential relaxation dynamics that stem from fluctuations of non-radiative decay rates for the band edge exciton. Analysis of these fluctuations allows us to extract single exponential dynamics by sampling only "maximum-intensity" photons, and we find that this single exponential decay is remarkably uniform across a wide variety of NC samples and sizes. We also investigate luminescence from multiexciton (e.g. biexciton and triexciton) states of nanocrystals at both the ensemble and single NC level.
(cont.) Energy splittings, size and temperature dependencies, quantum yields and lifetimes of multiexciton states are measured and discussed. We show for the first time direct resolution of biexciton emission from single exciton emission using two different techniques, fluorescence line narrowing and single NC spectroscopy. We also study the non-classical light emission properties of single NCs and show how multiexciton emission leads to radiative quantum cascades of single photons in the emission of a single NC. Time resolved studies of fluorescence from NCs in solution environments conclude the thesis. The relationship between lifetime and quantum yield for non-homogeneous ensembles like NCs is studied in chapter 8. We show that a sub-population of non-luminescent nanocrystals can reduce the quantum yield of an ensemble of NCs even though the measured lifetime stays constant. A study of NCs in solution fluorescence correlation spectroscopy (FCS) is presented last. We find that FCS is a capable tool for distinguishing small differences in hydrodynamic radius of NCs in solution. We also find that blinking of NCs may have a significant impact on these FCS measurements.
(cont.) An appendix to this thesis presents a general summary the lifetime of various samples CdSe and CdTe NCs.
by Brent R. Fisher.
Ph.D.
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Khalil, Gamal Ezat Abdel-Razek. "Single-Molecule Spectroscopy of Fluorene and Thiophene-Based Organic Semiconductors." Thesis, University of Sheffield, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.521976.
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Lu, Maolin. "Single-Molecule Spectroscopy Studies of the Conformational Dynamics of Enzymes." Bowling Green State University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1415118092.
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Asano, Marie. "Design, synthesis and single molecule force spectroscopy of biosynthetic polypeptides." Thesis, Bordeaux, 2016. http://www.theses.fr/2016BORD0163/document.
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Le repliement des protéines est principalement gouverné par les interactions spécifiques des structures secondaires. 1, 2 Toutefois, il existe expérimentalement peu d’informations sur les propriétés mécaniques fondamentales des hélices α et des feuillets β isolées. Les recherches antérieures sur l'étude du déploiement des hélices sont peu concluantes 3-5 et à notre connaissance l'étude des propriétés mécaniques d'un feuillet β isolé, intramoléculaire est sans précédent. Les copolymères PEG114-b-poly(L-lysine)134-(2-pyridyl disulfure),PEG114-b-poly(L-lysine)-b-PEG114 et poly(L-acide glutamique)85-b-(2-pyridyldisulfure) été synthétisés et utilisés comme systèmes modèles pour tester les propriétés mécaniques des motifs secondaires de type hélice α et feuillet β. Les résultats obtenus se sont révélés être en bon accord avec les résultats théoriques obtenus en utilisant un modèle statistique basé sur AGAGIR 6. La différence de force de déroulement comparant les hélices de poly(L-Lysine) ≈ 30 pN et de poly(L-acide glutamique) ≈ 20 pN des copolymères diblocs a été attribuée à l'hydrophobicité différente des chaînes latérales. La plus grande hydrophobie dumotif lysine conduit à de plus grandes interactions entre les chaînes latérales qui empêchent les fluctuations aléatoires au sein de l’hélice, et conduisent à une stabilité supérieure de l'hélice α. Lorsque les expériences ont été conduites dans des conditions favorisant la solubilité des chaînes latérales de lysine, les interactions ont diminué à une force de ≈ 20 pN, similaire à la force des interactions observées pour le poly(L-acide glutamique). Nous supposons qu'un minimum de ≈ 20 pN est nécessaire pour rompre la liaison hydrogène en maintenant l'hélice α, car cette force a été obtenue dans des conditions où les interactions de la chaîne latérale étaient minimisées. La présence de plateaux de force constants et d'inflexions correspondantes démontre une force de dépliement indépendante de la longueur, qui supporte un mécanisme de déroulement tour-par-tour pour l'hélice. De plus, la plus grande hydrophobie des chaînes latérales a été suggérée non seulement pour stabiliser la structure en hélice, mais également pour inhiber la formation d'une structure de type β-turn métastable intermédiaire lorsque les forces entropiques dominent. Des études préliminaires ont été effectuées sur le système de PEG114-bpoly(L-Lysine)134-(2-pyridyl disulfure) après induction d’une transition - β par un traitement thermique dans des conditions basiques. Une inflexion à une force≈ 70 pN a été obtenue, ce qui suggère la formation d'une interaction de type feuillet β. Une stratégie bottom-up a ainsi été proposée avec succès, démontrant le potentiel d'utilisation de tels systèmes artificiels pour simplifier et modéliser des systèmes biologiques réels. La compréhension de ces modèles isolés plus simples aidera sans doute la compréhension de systèmes plus complexesProteins fold by the initial, preferential folding of secondarystructures 1, 2, however surprisingly little is known about the basic mechanicalproperties of isolated α-helices and β-sheets from an experimental standpoint.Previous investigations into studying the generic unfolding behaviour of α-heliceshave proved inconclusive 3-5, and to our knowledge the study of an isolated,intramolecular β-sheet is unprecedented.Bioinspired PEG114-b-poly(L-glutamic acid)85-(2-pyridyl disulphide),PEG114-b-poly(L-lysine)134-(2-pyridyl disulphide) and PEG114-b-poly(Llysine)134–b-PEG114 were designed, synthesized and utilized as model systems toprobe the mechanical properties of α-helix and β-sheet secondary motifs. Theobtained results were shown to be in good agreement with theoretical resultsobtained by utilizing a AGAGIR-based statistical mechanical model 6. Thedifference in unravelling force comparing the helices of poly(L-Lysine) ≈30 pNand poly(L-glutamic acid) ≈20 pN diblock copolymers was attributed to thediffering hydrophobicity of the side chains. The greater hydrophobicity of thelysine allowed greater interactions between the side chains and sterically hinderedrandom helix-coil fluctuations, which lead to a superior α-helix stability. Whenexperiments were conducted in conditions promoting the solubility of the lysineside chains, the interactions decreased to a force of ≈20 pN, similar to the force ofinteractions observed for the poly(L-glutamic acid). We infer that a minimum of≈20 pN is needed to rupture the hydrogen bonding maintaining the α-helix as thisforce was obtained in conditions where the side chain interactions wereminimized.The presence of constant force plateaus and corresponding inflectionsdemonstrates a length independent unfolding force, which supports a turn-by-turnunfolding mechanism for the α-helix.In addition, the greater hydrophobicity of the side chains was suggestedto not only stabilize the α-helix structure, but also to inhibit the formation of anintermediate metastable β-hairpin-like structure when entropic forces dominate.Preliminary studies were also conducted on the PEG114-b-poly(LLysine)134-(2-pyridyl disulphide) system after a α-β transition had been inducedby heat in basic conditions, where an inflection at a much higher force of ≈ 70 pNwas obtained suggesting the formation of a β-sheet interaction.A bottom-up, investigative strategy has thus been successfully proposeddemonstrating the potential of utilizing such artificial systems to simplify andexemplify real biological systems. The comprehension of these simpler isolatedmodels will no doubt aid the understanding of more complex systems
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Schüller, Verena. "DNA origami structures for applications in single molecule spectroscopy and nanomedicine." Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-157179.
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Psychogyiopoulou, Krystallia. "Synthesis, surface spectroscopy and single molecule conductance measurements of some metalloporphyrins." Thesis, University of Liverpool, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422991.
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Magness, Alastair. "Diagnosing cancer one cell at a time with single molecule spectroscopy." Thesis, Imperial College London, 2017. http://hdl.handle.net/10044/1/57501.
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Mapping protein expression heterogeneity in cancer at single cell resolution is essential for the understanding of disease progression, emergent drug resistance, and metastasis, but is a great technical challenge. Longitudinal monitoring of heterogeneity pertaining to biomarker expression may provide the necessary medical cues for the administering of personalised therapeutics. Studying molecular heterogeneity in the ultra-rare circulating tumour cells [CTCs] found in the blood of cancer patients is a greater challenge still, but success may yield deep insight into the nature of the metastatic cascade, and also provide the technologi-cal means of a non-invasive ‘liquid biopsy’. The MAC chip is a quantitative single molecule sensitive protein assay for the evalua-tion of protein copy number in single cells. In this thesis, we attempt the development of a new biomarker-targeting MAC chip assay for the breast cancer oncoprotein estrogen recep-tor alpha. We describe a series of improvements to the MAC chip assay architecture allow-ing multiplexed measurement of several proteins simultaneously, and improvements to analysis methods allowing for superior molecule counting. Building on the work of others in the field of circulating tumour cell isolation, we attempt the integration of the MAC chip analysis method into a multiple-stage device for the isolation and proteomic analysis of cir-culating tumour cells. Finally, using a multiplexed MAC chip device for the tumour sup-pressor protein p53 and its activated form phosphorylated at serine-15, we demonstrate for the first time that the MAC chip can be used to study protein expression heterogeneity in quasi-clinical samples. The patient-derived xenografts we use to perform this work are a key resource of clinically-relevant tumour material, and a model system directly analogous to primary patient biopsies, thus demonstrating the feasibility of translational single cell pro-teomics with the MAC chip system.
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Fisher, Aidan Antony Edward. "Colloidal synthesis, structural characterisation and single molecule spectroscopy of semiconducting nanocrystals." Thesis, University of Sussex, 2018. http://sro.sussex.ac.uk/id/eprint/73443/.
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Adhikari, Subhasis, and Frank Cichos. "Probe size dependent rotational dynamics in polymer by single molecule spectroscopy." Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-185732.
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Adhikari, Subhasis, and Frank Cichos. "Probe size dependent rotational dynamics in polymer by single molecule spectroscopy." Diffusion fundamentals 16 (2011) 77, S. 1-2, 2011. https://ul.qucosa.de/id/qucosa%3A13821.
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Yang, Darren. "Exploring Biomolecular Interactions Through Single-Molecule Force Spectroscopy and Computational Simulation." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493410.
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Molecular interactions between cellular components such as proteins and nucleic acids govern the fundamental processes of living systems. Technological advancements in the past decade have allowed the characterization of these molecular interactions at the single-molecule level with high temporal and spatial resolution. Simultaneously, progress in computer simulation has enabled theoretical research at the atomistic level, assisting in the interpretation of experimental results. This thesis combines single-molecule force spectroscopy and simulation to explore inter- and intra-molecular interactions. Specifically, we investigate the interaction between RecA and DNA to elucidate the underlying molecular mechanism of the DNA homologous recombination process. We also evaluate the stability of the von Willebrand Factor (vWF) A2 domain to determine the molecular origins of von Willebrand Diseases (vWD). This thesis also describes the development and application of a new single-molecule technique that combines the centrifuge force microscope (CFM) with DNA self-assembled mechanical switches to enable massively parallel repeating force measurements of molecular interactions.Engineering and Applied Sciences - Applied Physics
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Botha, Joshua Leon. "Using single molecule spectroscopy to study fast photoprotective processes in plants." Diss., University of Pretoria, 2016. http://hdl.handle.net/2263/60864.
Full textAbstract:
The fundamental mechanisms involved in photosynthesis provide an opportunity
to study physical principles that span over both classical and quantum scales. A
better understanding of these mechanisms will benefit the development of alternative
energy sources such as cheaper biofuel and more effective photovoltaics.
This dissertation describes the single molecule spectroscopy setup that was assembled
during my MSc-degree and the underlying theory required to understand
the technique, is discussed. The greatest part of the setup development involved
customised software development that performs the measurement. The code of
this software is briefly discussed. Thereafter the results of a series of single molecule
spectroscopy measurements of isolated light harvesting complex II (LHCII)
that undergo non-photochemical quenching (NPQ) are described. The fast, reversible,
energy-dependent component (qE) of NPQ is emulated by lowering the pH of
the solvent in which the complexes are diluted. Apart from fluorescence intensity
measurements, time correlated single photon counting is used to measure fluorescence
lifetimes, which serves as an indirect measurement of NPQ. It was found
that quenching could be taking place before the energy reaches the terminal emitter,
and a relationship between intermediate fluorescence states and high jumping
frequencies was established.Die fundamentele meganismes wat by fotosintese betrokke is skep 'n ideale geleentheid om beginsels te bestudeer wat oor beide klassieke en kwantumskale strek. 'n Beter verstaan van hierdie meganismes sal die ontwikkeling van alternatiewe energiebronne soos goedkoop biobrandstof en meer effektiewe fotovoltaïese selle bevorder. Hierdie verhandeling beskryf die enkelmolekuulspektroskopie-opstelling wat tydens my MSc-graad opgerig is en die onderliggende teorie wat nodig is om die tegniek te verstaan, word bespreek. Die grootste deel van die ontwikkeling van die opstelling het die ontwikkeling van toepassingsgerigte sagteware behels. Die kode van hierdie sagteware word oorsigtelik bepreek. Vervolgens word die resultate van 'n reeks enkelmolekuulspektroskopie-metings beskryf waartydens nie-fotochemiesedowing (NFD) in die geïsoleerde ligversamelingskompleks II (LHCII) van hoër plante bestudeer is. Die vinnige, omkeerbare, energie-afhanklike komponent (qE) van NFD is geëmuleer deur die pH van die oplossing waarin die komplekse opgelos is, te verlaag. Buiten metings van die fluoressensie-intensiteite is tydsgekorreleerde enkelfotontelling ook toegepas om fluoressensieleeftye te meet, wat as 'n indirekte meting van die mate van NFD dien. Die moontlikheid dat dowing plaasvind voordat die opwekkingsenergie die laagste energietoestand in die kompleks bereik, is ontdek en 'n verwantskap tussen intermediêre fluoressensietoestande en hoëfrekwensieskakeling word gelê.
Dissertation (MSc)--University of Pretoria, 2016.
Physics
MSc
Unrestricted
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Ye, Fangmao. "Single molecule studies of meso/macro porous silica materials and gradient films." Diss., Manhattan, Kan. : Kansas State University, 2009. http://hdl.handle.net/2097/1699.
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Vogelsang, Jan. "Advancing single-molecule fluorescence spectroscopy and super-resolution microscopy with organic fluorophores." Diss., kostenfrei, 2009. http://edoc.ub.uni-muenchen.de/11480/.
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Vongtragool, Suriyakan. "Frequency-domain magnetic resonance spectroscopy on the Mn12-acetate single-molecule magnet." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972905952.
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Klamecka, Kamila [Verfasser], and Heinrich [Akademischer Betreuer] Leonhardt. "Single-molecule force spectroscopy of biological complexes / Kamila Klamecka ; Betreuer: Heinrich Leonhardt." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2017. http://d-nb.info/1156851874/34.
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Korlann, You. "Methodology development and biological applications of single molecule fluorescence spectroscopy and microscopy." Diss., Restricted to subscribing institutions, 2008. http://proquest.umi.com/pqdweb?did=1694502711&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Frano, Kristen A. "Surface-Enhanced Raman and Single-Molecule Spectroscopy Studies of Fugitive Artists' Pigments." W&M ScholarWorks, 2015. https://scholarworks.wm.edu/etd/1539791830.
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Kirstein, Johanna, Christophe Jung, Christian Hellriegel, and Christoph Bräuchle. "Single molecule spectroscopy: translational and rotational diffusion of single fluorescent dyes in nano-structured porous materials." Diffusion fundamentals 2 (2005) 94, S. 1-2, 2005. https://ul.qucosa.de/id/qucosa%3A14431.
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Giri, Dipak. "Single-molecule spectroscopic studies of thin-film chemical gradients." Diss., Kansas State University, 2016. http://hdl.handle.net/2097/35225.
Full textAbstract:
Doctor of PhilosophyDepartment of Chemistry
Daniel A. Higgins
This dissertation describes the application of single molecule spectroscopy and tracking to investigations of the nanoscale properties of thin-film chemical gradients and the transport dynamics of molecules dispersed within and upon these gradients. Chemical gradients are surface bound materials that incorporate gradually changing chemical and/or physical properties. A continuous and gradual change in the properties of gradients are expected and often required for their intended applications, which range from directed growth of cell colonies to combinatorial materials science. In reality, such conditions are almost never met due to spontaneous demixing and dewetting processes that can lead to properties variations on microscopic length scales. A better understanding on the properties of chemical gradients on microscopic length scales will aid in the production of better engineered materials. Single molecule spectroscopy (SMS) allows for gradient properties to be probed on nanometer-to-micrometer length scales. In this dissertation, quantitative measurements of gradient polarity (i.e., dielectric properties) are made along a sol-gel derived thin film that incorporates a macroscopic polarity gradient. These measurements report on the microscopic heterogeneity of the gradient film, and point to the occurrence of phase separation of the polar and nonpolar components along the gradient. Single molecule tracking (SMT) provides an important means to examine the dynamics of molecular mass transport in thin films and on surfaces. In this dissertation, SMT is employed to study mass transport in thin water films condensed over monolayer wettability gradients under ambient environments. The results show that the rate and the mechanism of molecular transport depend on the surface wettability, and on the ambient relative humidity. Finally, wettability gradients have been broadly used to drive the transport of liquid droplets. In this dissertation, these applications are extended to achieve spontaneous stretching of DNA by the propulsion of liquid droplets along the gradient. Single molecule fluorescence imaging of DNA stretched along these gradients demonstrates that hydrophobic surfaces play an important role in DNA stretching. The study also shows the surface tension force acting at the gradient-droplet contact line (interface) to be responsible for DNA elongation and alignment. Overall, single molecule methods have been shown to be highly useful for better understanding the properties of chemical gradients as described in this dissertation.
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Wörmke, Stephan. "Single Molecule Spectroscopy on Native and Reconstituted Peridinin-Chlorophyll-Protein Light-Harvesting Complexes." Diss., lmu, 2008. http://nbn-resolving.de/urn:nbn:de:bvb:19-91182.
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Ott, Wolfgang Bernhard [Verfasser], and Hermann [Akademischer Betreuer] Gaub. "Single molecule force spectroscopy with biological tools / Wolfgang Bernhard Ott ; Betreuer: Hermann Gaub." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2017. http://d-nb.info/1175878677/34.
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Brown, Frank Leon Halet 1972. "Interactions of light with matter-- applications to single molecule spectroscopy and quantum control." Thesis, Massachusetts Institute of Technology, 1998. http://hdl.handle.net/1721.1/47482.
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Wörmke, Stephan. "Single molecule spectroscopy on native and reconstituted Peridinin-Chlorophyll-Protein light-harvesting complexes." kostenfrei, 2008. http://edoc.ub.uni-muenchen.de/9118/.
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Vongtragool, Suriyakan. "Frequency domain magnetic resonance spectroscopy on the Mn 12 -acetate single molecule magnet." [S.l. : s.n.], 2004. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB11513999.
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Kim, So Yeon. "Observing protein dynamics and conformational changes by ensemble and single-molecule fluorescence spectroscopy /." May be available electronically:, 2008. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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Chong, Michael. "Electrically driven fluorescence of single molecule junctions." Thesis, Strasbourg, 2016. http://www.theses.fr/2016STRAE022/document.
Full textAbstract:
Les propriétés optoélectroniques de jonctions moléculaires sont étudiées par microscopie à effet tunnel (STM). Premièrement, les structures moléculaires sont synthétisées sur une surface Au(111). Puis, par manipulation, nous soulevons et suspendons une molécule entre la pointe du STM et la surface d’or pour obtenir une jonction moléculaire. En appliquant une tension entre la pointe et l'échantillon, un courant est généré, ce qui conduit à l'excitation de la molécule. Ce processus est médié par des modes de plasmons de surface localisé de la pointe. Finalement, la molécule se désexcite de manière radiative et génère un signal de fluorescence. On utilise cette technique pour étudier deux systèmes moléculaires. Dans le premier, un émetteur (porphyrin) est suspendu dans la jonction grâce à des fils organiques (oligothiophène). Ce type de jonction génère une émission de lumière étroite dont la couleur est contrôlée en sélectionnant la structure chimique de l'émetteur. Le contrôle de la largeur du pic d’émission est obtenu en détachant progressivement l'unité émettrice de la surface. On observe aussi des pics vibroniques décalés vers le rouge qui fournissent une empreinte chimique de l’émetteur, et des pics décalés vers le bleu, signe d’une deséxcitation d’un exciton non-thermalisé. Le deuxième type de jonction est composé de nano-rubans de graphène (GNRs) dont la largeur et la structure de l’arrête sont définis avec une précision atomique. Une fois suspendu dans la jonction, les GNRs qui présentent une terminaison spécifique (terminaison C) montrent un spectre d’émission de lumière avec un pic principal et deux pics vibroniques décalés vers le rouge. Le pic principale est associé à une transition intra-ruban entre un état Tamm localisé et un état delocaliséThis thesis presents a study of the optoelectronic properties of molecular junctions performed by scanning tunneling microscopy (STM). First, the molecular structures are synthesized on a Au(111) surface. Then, by manipulation we lift and suspend a molecule between the tip of the STM and the gold surface, creating a single molecule junction. By applying a voltage bias between the tip and the sample, a current is generated, which leads to the excitation of the molecule. This process is mediated by the localized surface plasmon modes of the tip. Eventually, the molecule de-excites in a radiative way, generating a fluorescence signal. We use this technique to study two different molecular junctions. First, an emitting unit (fused-porphyrin) is suspended in the junction by means of organic linkers (oligothiophene). This type of junction generates a narrow-line emission of light whose color is controlled by selecting the chemical structure of the emitting unit. Moreover, control over the linewidth is obtained by progressively detaching the emitting unit from the surface. Also, we observe red-shifted vibronic features that provide a chemical fingerprint of the emitter, and blue- shifted vibronic features that are a sign of hot-luminescence. For the second type of junctions we use graphene nanoribbons (GNRs) of atomically precise width and edge structure. When lifted in the junction, GNRs with a specific type of termination (C-terminated) exhibit a light emission spectrum with a main peak and two red-shifted vibrational features. The main peak is associated to an intra-ribbon transition between a localized state (Tamm) and a delocalized state