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1
CHOUDHARY, DHAWAL. « Studio a livello di singola molecola del folding, misfolding e aggregazione di proteine e dell’attività chaperonica della HSPB8 ». Doctoral thesis, Università degli studi di Modena e Reggio Emilia, 2020. http://hdl.handle.net/11380/1199862.
Texte intégralRésumé :
Negli ultimi decenni le pinze ottiche si sono rivelate una tecnica sperimentale estremamente efficace per eseguire studi di spettroscopia di forza a livello di singola molecola. In particolare, un’applicazione delle pinze ottiche che sta avendo una rilevanza biomedica sempre più importante è quella relativa allo studio dei processi di ripiegamento corretto (folding), non corretto (misfolding) e dell’aggregazione di proteine. Di forte rilevanza biomedica è anche la possibilità offerta dalle pinze ottiche di caratterizzare in grande dettaglio i meccanismi molecolari che mediano le interazioni tra due o più biomolecole, come ad esempio tra uno chaperone molecolare e il suo substrato. La rilevanza medica di questi studi deriva dal fatto che l'errato ripiegamento e l'aggregazione delle proteine sono processi deleteri, spesso associati a neurodegenerazione. Gli chaperoni molecolari si sono evoluti come strumento molecolare per combattere sia il misfolding che l’aggregazione proteica. Un funzionamento non corretto degli chaperoni molecolari spesso causa perdita di proteostasi e l’insorgenza di varie patologie umane. Il lavoro descritto in questa tesi spiega in maniera dettagliata l’approccio sperimentale utilizzato per utilizzare le pinze ottiche per lo studio del folding, misfolding e aggregazione di proteine. In particolare in questa tesi vengono descritti: i) i risultati di esperimenti mirati alla elucidazione del processo di ripiegamento corretto e non del sensore al calcio NCS-1 (Neuronal Calcium Sensor 1; ii) l'approccio sperimentale adottato per descrivere la dinamica strutturale e funzionale di vari chaperoni molecolari utilizzando le pinze ottiche e la microscopia a forza atomica; iii) recenti sviluppi tecnici che hanno ampliato le possibili applicazioni delle pinze ottiche in campo biologico; iv) i risultati di esperimenti mirati a far luce sui meccanismi molecolari che mediano l’attività chaperonica dello chaperone molecolare HSPB8. In quest’ultimi esperimenti abbiamo manipolato meccanicamente monomeri e tetrameri della Maltose Binding Protein (MBP) e analizzato i loro processi di folding, misfolding e aggregazione in presenza e assenza del HSPB8 wild-type e del suo mutante HSPB8-K141E. I nostri risultati dimostrano una forte attività antiaggregante (holdase activity) della HSPB8 che riduce significativamente l'aggregazione delle molecole di MBP e un’attività antiaggregante molto ridotta del mutante HSPB8-K141E. Inoltre, i nostri studi rivelano una inaspettata attività pro-folding (foldase activity) sia della forma mutata che di quella wild-type della HSPB8. Questi dati sperimentali evidenziano nuovi meccanismi di interazione tra HSPB8 e i suoi substrati e suggeriscono un ruolo fisiologico più complesso per questo chaperone molecolare di quanto precedentemente ipotizzato.Optical tweezers have evolved as an exemplary Single Molecule Force Spectroscopy (SMFS) technique over the past three decades. A distinct and bio medically relevant application of Optical Tweezers is their ability to observe directly at single molecule level the folding, misfolding and aggregation of protein molecules. Additionally the dynamic approach of Optical Tweezer setup also allows for the isolated study of interactions between two or more biomolecules, such as chaperone-protein interactions, in real time. The medical relevance of such studies stems from the fact that misfolding and aggregation of proteins are deleterious processes and have been linked to many neurodegenerative disorders. While molecular chaperones have evolved as an evolutionarily conserved sword and shield mechanism against such deleterious processes, wherein their holdase action acts as a shield preventing further aggregation of misfolded protein species and their foldase action acts as a sword and actively assists misfolded structure to regains their natively folded state. The dysfunction of this chaperone activity is also cytotoxic and can lead to loss of proteostasis. The present thesis dwells deeper in this specific application of Optical tweezer. The thesis will elaborate upon how optical tweezers can extract the mechanistic details of the folding and misfolding of protein molecules by reviewing the experiments performed on NCS-1 (Neuronal Calcium Sensor 1). It will also discuss the experimental approach taken by SMFS techniques like Optical Tweezers and AFM (Atomic Force Microscopy) to study the structural and functional dynamics of molecular chaperones. Furthermore, the thesis will explore the recent developments in Optical Tweezers and their biological applications. Finally, I describe the results of experiments we have carried out on the maltose binding protein to elucidate the mechanism of action of the chaperone HSPB8. We have mechanically denatured homotetramers of MBP as well as single MBP molecules and analyzed their folding and aggregation processes in the presence and absence of wild-type HSPB8 and its mutant form HSPB8-K141E/N. Our results reveal a strong holdase activity of wild type HSPB8, which either prevents completely the aggregation of denatured MBP molecules or allows the substrate to form only small and mechanically weak aggregates while this holdase activity is significantly suppressed in the mutant. Moreover, and importantly, a careful analysis of the data also discloses an unexpected foldase activity of both wild type and mutated forms of HSPB8, which guides the folding process of denatured MBP molecules into their native states. Our findings highlight new mechanisms of interaction between HSPB8 and its substrates and suggest a more complex physiological role for this chaperone than previously assumed.
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BUGLIONE, ENRICO. « Nanomeccanica per la Ricerca sul Cancro ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2021. http://hdl.handle.net/10281/304787.
Texte intégralRésumé :
Nonostante gli enormi passi avanti sulla comprensione dei meccanismi d’azione del cancro e nello sviluppo di farmaci antitumorali, la maggior parte delle forme di cancro è ancora incurabile. La ragione di tale insuccesso è radicata nella complessità di questa malattia, che è ancora poco conosciuta sia in fase di insorgenza, dovuta alla misregolazione di oncogeni, sia in fase di metastatizzazione delle cellule cancerose. Lo studio di tali caratteristiche da un punto di vista meccanico può fornire una visione differente dei meccanismi di azione del cancro ed aiutare a contestualizzarli all’interno di processi estremamente complessi.
L’insorgenza del cancro è dovuta ad una mancata regolazione del ciclo cellulare, che a sua volta dipende spesso da un’alterata espressione dei cosiddetti oncogeni. Ne è un esempio il proto-oncogene c-KIT, che codifica per un fattore di crescita delle cellule staminali. La regolazione della sua espressione è controllata dal suo promotore prossimale, la cui struttura è caratterizzata dalla presenza di 3 superstrutture tridimensionali complesse che si formano su uno dei due filamenti della sua sequenza, chiamate G-quadruplexes. Tali strutture sembrerebbero avere un ruolo nel riconoscimento del promotore da parte della polimerasi e nell’assemblaggio della macchina trascrizionale, ma la loro funzione non è ancora chiara. Lo studio della nanomeccanica di tali strutture a livello di singola molecola (grazie al Magnetic Tweezers) può fornire importanti informazioni sul loro ruolo e sulle modalità con cui regolano l’espressione dell’oncogene.
In seguito all’insorgenza del cancro, le cellule coinvolte subiscono forti cambiamenti strutturali: iniziano a dividersi velocemente e a migrare incontrollatamente. Entrambi questi processi richiedono un grande cambiamento nella struttura cellulare ed in particolare nella rigidità della cellula. La leucemia linfoide cronica (CLL) rappresenta un caso esemplare di tali capacità migratorie, non solo nell cellule cancerose, ma anche in quelle sane. Anche in questo caso, la nanomeccanica offre numerosi strumenti che permettono di studiare tali modifiche a livello di singola cellula, in particolare il microscopio a forza atomica (AFM) e il citofluorimetro a pressione deformante (RT-DC) offrono la possibilità di studiare le dinamiche cellulari in differenti condizioni e di osservare gli effetti dei farmaci di prima linea direttamente sulle cellule di pazienti malati.With the term cancer are intended many species of diseases having quite different properties from each other. Despite such vast differences, the mechanisms beyond the onset of any kind of cancer are very similar and can be classified in two main groups depending on their stage. The first is related to the dysregulation of particular genes (oncogenes), that results in an impairment of the cell cycle. The second concerns the ability of cancer cells to continuously divide and migrate through tissues, that results in a highly invasive potential. From a mechanical point of view, the investigation of such features can be crucial for a deeper understanding of cancer onset and progression as well as for the study of novel pharmacological treatments. The outbreak of cancer is caused by a deficiency in the regulation of the cell cycle which, in turn, often depends on an abnormal expression of oncogenes. It is the case of the proto-oncogene c-KIT, that encodes for a mast/stem cell growth factor receptor. Its regulation relies mainly on its promoter, which is constituted by 3 distinct three-dimensional DNA structures called G-quadruplexes (G4s). Those structures can be studied by means of nanomechanical tools such as Magnetic Tweezers, which can recognize folded G4s at single-molecule level, thus enabling to study their role in the regulation of the oncogene. After the onset of cancer, a generic cell undergoes mechanical changes: it divides quickly, and it starts migrating. Both phenomena require a modification in the cell structural phenotype, eventually modifying its rigidity. Chronic lymphocytic leukemia is a case in point: malignant B lymphocytes continuously traffic between peripheral blood and lymphoid tissues. Such frequent migrations require a change in the rigidity of cells. In this case, Atomic Force Microscopy can provide a nanomechanical approach allowing to measure the stiffness of single cells from patients with leukemia, which is slightly decreased if compared to rigidity of cells from healthy donors. This feature can also allow to observe the effect of targeted therapies on the cells, evaluating their effect from a mechanical point of view.
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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.
Texte intégralRésumé :
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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Murello, Anna. « La spettroscopia di forza basata sull'AFM nello studio dello spazio conformazionale e dei processi aggregativi di proteine prioniche ». Master's thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amslaurea.unibo.it/8878/.
Texte intégralRésumé :
Le malattie neurodegenerative sono caratterizzate da aggregazione proteica, dipendente dalla
perdita della usuale struttura fisiologica funzionale delle proteine coinvolte, a favore di conformazioni tossiche (patologiche). Il modello corrente descrive questi cambiamenti conformazionali come eventi rari e ritiene che non esista una sola conformazione patogena, ma che tali possibili conformazioni siano piuttosto eterogenee. La caratterizzazione di queste strutture è, di conseguenza, difficile con le tradizionali tecniche in bulk che permettono di studiare solo la conformazione media e non rendono possibile il riconoscimento delle caratteristiche dei conformeri individuali.
Lo sviluppo delle tecniche di singola molecola ha permesso di studiare in modo approfondito le conformazioni possibili. In questo lavoro la spettroscopia di forza di singola molecola basata sull'AFM viene applicata a PrP (proteina responsabile delle encefalopatie spongiformi trasmissibili). Si studiano gli equilibri conformazionali del monomero e quelli di costrutti oligomerici, allo scopo di caratterizzare gli step iniziali dei processi aggregativi.
Nel corso di questo lavoro di tesi è stato, in particolare, sviluppato un sistema di analisi dati, al fine di studiare in modo quantitativo le distribuzioni di eventi ottenute. Grazie a tale strumento è stato possibile riconoscere i segnali di unfolding della conformazione nativa del monomero e notare come essa sia presente anche in costrutti oligomerici, ad indicare come questo ripiegamento sia stabile anche in presenza di più monomeri ravvicinati. Si è osservato l'effetto del pH sulla stabilità di tale struttura, notando come pH acidi destabilizzino il ripiegamento nativo. Inoltre si è studiato il ruolo dell'orientazione dei monomeri nella formazione di strutture dimeriche.
Monomeri e oligomeri di PrP sono stati descritti come proteine parzialmente strutturate il cui panorama energetico contiene molti minimi locali, dando origine a parecchie conformazioni transienti.
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Rajagopal, Senthil Arun. « SINGLE MOLECULE ELECTRONICS AND NANOFABRICATION OF MOLECULAR ELECTRONIC DEVICES ». Miami University / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=miami1155330219.
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Sikor, Martin. « Single-molecule fluorescence studies of Protein Folding and Molecular Chaperones ». Diss., lmu, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-138521.
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Ökten, Zeynep. « Single molecule mechanics and the myosin family of molecular motors ». [S.l.] : [s.n.], 2006. http://www.diss.fu-berlin.de/2006/6/index.html.
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Zhao, Xiaotao. « The synthesis and single-molecule conductance of conjugated molecular wires ». Thesis, Durham University, 2014. http://etheses.dur.ac.uk/10634/.
Texte intégralRésumé :
The past decades have seen the fast development of electronic devices in the industrial sector. There is increasingly rapid growth in the demand for alternative electronic building blocks to compliment, and possibly replace, the conventional silicon-based products. Electronic devices based on organic molecules, especially those based on single molecules, receive intense studies both theoretically and experimentally. In this presented work, a new family of oligo(aryleneethenylene)s (OAE)s with molecular lengths (N…N distance) of ca. 2-6 nm were designed to investigate the length dependence of conductance at the single molecule level. X-ray molecular structures of OAEs with a molecular length up to 5.3 nm were successfully analysed and presented. Secondly, four groups of pyridyl terminated oligo(phenyleneethylene) (OPE) derivatives were studied for the quantum interference effects. A dramatic destructive quantum interference effect was observed which decreased the single molecule conductance by several orders of magnitude. Unsymmetrical molecules with only one anchor group were noticed to form π-π stacking between two molecules. Thirdly, amino terminated OPEs bearing various substituents on the central phenyl rings were explored to present the robustness of the central OPE backbone towards various functionalising substituents. Fourthly, diaryloligoynes with different anchor groups were synthesised and the single-molecule conductances were studied. The stability of the tetrayne compounds is discussed and X-ray crystal structures of the stable tetraynes are presented. Finally, pyridyl terminated OAE derivatives bearing an anthraquinone core were synthesised to investigate the charge transport through the central anthraquinone core, with special purpose of investigating quantum interference effects and the switching process of the central anthraquinone core.
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Lange, Jeffrey J. « Studies of molecular motions by fluorescence microscopy at single molecule and single fiber levels ». Diss., Manhattan, Kan. : Kansas State University, 2009. http://hdl.handle.net/2097/1629.
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Rüttinger, Steffen. « Confocal microscopy and quantitative single molecule techniques for metrology in molecular medicine ». [S.l.] : [s.n.], 2006. http://opus.kobv.de/tuberlin/volltexte/2007/1434.
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Sikor, Martin [Verfasser]. « Single-molecule fluorescence studies of Protein Folding and Molecular Chaperones / Martin Sikor ». München : Verlag Dr. Hut, 2012. http://d-nb.info/1020298650/34.
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Iancu, Violeta. « Single Molecule Switches and Molecular Self-Assembly : Low Temperature STM Investigations and Manipulations ». Ohio : Ohio University, 2006. http://www.ohiolink.edu/etd/view.cgi?ohiou1159980375.
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Schulze, Gunnar [Verfasser]. « Elementary processes in single molecule devices : electronic transport and molecular isomerization / Gunnar Schulze ». Berlin : Freie Universität Berlin, 2010. http://d-nb.info/102433547X/34.
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Goryaynov, Alexander G. « Molecular Size and Charge Effects on Nucleocytoplasmic Transport Studied By Single-Molecule Microscopy ». Bowling Green State University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1357278635.
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Sapra, K. Tanuj. « Single-Molecule Measurements of Complex Molecular Interactions in Membrane Proteins using Atomic Force Microscopy ». Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2007. http://nbn-resolving.de/urn:nbn:de:swb:14-1175696409847-74867.
Texte intégralRésumé :
Single-molecule force spectroscopy (SMFS) with atomic force microscope (AFM) has advanced our knowledge of the mechanical aspects of biological processes, and helped us take big strides in the hitherto unexplored areas of protein (un)folding. One such virgin land is that of membrane proteins, where the advent of AFM has not only helped to visualize the difficult to crystallize membrane proteins at the single-molecule level, but also given a new perspective in the understanding of the interplay of molecular interactions involved in the construction of these molecules. My PhD work was tightly focused on exploiting this sensitive technique to decipher the intra- and intermolecular interactions in membrane proteins, using bacteriorhodopsin and bovine rhodopsin as model systems. Using single-molecule unfolding measurements on different bacteriorhodopsin oligomeric assemblies - trimeric, dimeric and monomeric - it was possible to elucidate the contribution of intra- and interhelical interactions in single bacteriorhodopsin molecules. Besides, intriguing insights were obtained into the organization of bacteriorhodopsin as trimers, as deduced from the unfolding pathways of the proteins from different assemblies. Though the unfolding pathways of bacteriorhodopsin from all the assemblies remained the same, the different occurrence probability of these pathways suggested a kinetic stabilization of bacteriorhodopsin from a trimer compared to that existing as a monomer. Unraveling the knot of a complex G-protein coupled receptor, rhodopsin, showed the existence of two structural states, a native, functional state, and a non-native, non-functional state, corresponding to the presence or absence of a highly conserved disulfide bridge, respectively. The molecular interactions in absence of the native disulfide bridge mapped onto the three-dimensional structure of native rhodopsin gave insights into the molecular origin of the neurodegenerative disease retinitis pigmentosa. This presents a novel technique to decipher molecular interactions of a different conformational state of the same molecule in the absence of a high-resolution X-ray crystal structure. Interestingly, the presence of ZnCl2 maintained the integrity of the disulfide bridge and the nature of unfolding intermediates. Moreover, the increased mechanical and thermodynamic stability of rhodopsin with bound zinc ions suggested a plausible role for the bivalent ion in rhodopsin dimerization and consequently signal transduction. Last but not the least, I decided to dig into the mysteries of the real mechanisms of mechanical unfolding with the help of well-chosen single point mutations in bacteriorhodopsin. The monumental work has helped me to solve some key questions regarding the nature of mechanical barriers that constitute the intermediates in the unfolding process. Of particular interest is the determination of altered occurrence probabilities of unfolding pathways in an energy landscape and their correlation to the intramolecular interactions with the help of bioinformatics tools. The kind of work presented here, in my opinion, will not only help us to understand the basic principles of membrane protein (un)folding, but also to manipulate and tune energy landscapes with the help of small molecules, proteins, or mutations, thus opening up new vistas in medicine and pharmacology. It is just a matter of a lot of hard work, some time, and a little bit of luck till we understand the key elements of membrane protein (un)folding and use it to our advantage.
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Vonlanthen, David [Verfasser]. « Biphenyl-Cyclophanes : The Molecular Control over the Conductivity of Single-Molecule Junctions / David Vonlanthen ». München : Verlag Dr. Hut, 2010. http://d-nb.info/1009972952/34.
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Sapra, K. Tanuj. « Single-Molecule Measurements of Complex Molecular Interactions in Membrane Proteins using Atomic Force Microscopy ». Doctoral thesis, Technische Universität Dresden, 2006. https://tud.qucosa.de/id/qucosa%3A24922.
Texte intégralRésumé :
Single-molecule force spectroscopy (SMFS) with atomic force microscope (AFM) has advanced our knowledge of the mechanical aspects of biological processes, and helped us take big strides in the hitherto unexplored areas of protein (un)folding. One such virgin land is that of membrane proteins, where the advent of AFM has not only helped to visualize the difficult to crystallize membrane proteins at the single-molecule level, but also given a new perspective in the understanding of the interplay of molecular interactions involved in the construction of these molecules. My PhD work was tightly focused on exploiting this sensitive technique to decipher the intra- and intermolecular interactions in membrane proteins, using bacteriorhodopsin and bovine rhodopsin as model systems. Using single-molecule unfolding measurements on different bacteriorhodopsin oligomeric assemblies - trimeric, dimeric and monomeric - it was possible to elucidate the contribution of intra- and interhelical interactions in single bacteriorhodopsin molecules. Besides, intriguing insights were obtained into the organization of bacteriorhodopsin as trimers, as deduced from the unfolding pathways of the proteins from different assemblies. Though the unfolding pathways of bacteriorhodopsin from all the assemblies remained the same, the different occurrence probability of these pathways suggested a kinetic stabilization of bacteriorhodopsin from a trimer compared to that existing as a monomer. Unraveling the knot of a complex G-protein coupled receptor, rhodopsin, showed the existence of two structural states, a native, functional state, and a non-native, non-functional state, corresponding to the presence or absence of a highly conserved disulfide bridge, respectively. The molecular interactions in absence of the native disulfide bridge mapped onto the three-dimensional structure of native rhodopsin gave insights into the molecular origin of the neurodegenerative disease retinitis pigmentosa. This presents a novel technique to decipher molecular interactions of a different conformational state of the same molecule in the absence of a high-resolution X-ray crystal structure. Interestingly, the presence of ZnCl2 maintained the integrity of the disulfide bridge and the nature of unfolding intermediates. Moreover, the increased mechanical and thermodynamic stability of rhodopsin with bound zinc ions suggested a plausible role for the bivalent ion in rhodopsin dimerization and consequently signal transduction. Last but not the least, I decided to dig into the mysteries of the real mechanisms of mechanical unfolding with the help of well-chosen single point mutations in bacteriorhodopsin. The monumental work has helped me to solve some key questions regarding the nature of mechanical barriers that constitute the intermediates in the unfolding process. Of particular interest is the determination of altered occurrence probabilities of unfolding pathways in an energy landscape and their correlation to the intramolecular interactions with the help of bioinformatics tools. The kind of work presented here, in my opinion, will not only help us to understand the basic principles of membrane protein (un)folding, but also to manipulate and tune energy landscapes with the help of small molecules, proteins, or mutations, thus opening up new vistas in medicine and pharmacology. It is just a matter of a lot of hard work, some time, and a little bit of luck till we understand the key elements of membrane protein (un)folding and use it to our advantage.
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Edman, Lars. « Single molecule dynamics / ». Stockholm, 2000. http://diss.kib.ki.se/2000/91-628-4025-8/.
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Szumski, Douglas Stewart. « Single molecule spintronics ». Thesis, University of Bristol, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.535471.
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Davies, Eva Melari. « Single molecule microscopy ». Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-173355.
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Kirstein, Johanna, Christophe Jung, Christian Hellriegel et Christoph Bräuchle. « Single molecule spectroscopy ». Universitätsbibliothek Leipzig, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-196553.
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Aubin, Sheila Marie Josée. « Single-molecule magnets / ». Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1997. http://wwwlib.umi.com/cr/ucsd/fullcit?p9820882.
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Sikor, Martin [Verfasser], et Don [Akademischer Betreuer] Lamb. « Single-molecule fluorescence studies of Protein Folding and Molecular Chaperones / Martin Sikor. Betreuer : Don Lamb ». München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2011. http://d-nb.info/1019479078/34.
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Lillehei, Peter Thomas. « Single molecule mechanical testing ». Diss., Georgia Institute of Technology, 2001. http://hdl.handle.net/1853/31044.
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Sedghi, Gita. « Porphyrin single molecular wires ». Thesis, University of Liverpool, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.539493.
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Pugh, Sara Dorothy. « Single molecule protein folding ». Thesis, University of Leeds, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.441320.
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Haque, Md Firoze. « CONTROLLED DEPOSITION OF MAGNETIC MOLECULES AND NANOPARTICLES ON ATOMICALLY FLAT GOLD SURFACES ». Master's thesis, University of Central Florida, 2008. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2109.
Texte intégralRésumé :
In this thesis I am presenting a detailed study to optimize the deposition of magnetic molecules and gold nanoparticles in atomically flat surfaces by self-assembling them from solution. Epitaxially grown and atomically flat gold surface on mica is used as substrate for this study. These surfaces have roughness of the order one tenth of a nanometer and are perfect to image molecules and nanoparticles in the 1-10 nanometers range. The purpose of these studies is to find the suitable parameters and conditions necessary to deposit a monolayer of nano-substance on chips containing gold nanowires which will eventually be used to form single electron transistors by electromigration breaking of the nanowire. Maximization of the covered surface area is crucial to optimize the yield of finding a molecule/nanoparticle near the gap formed in the nanowire after electromigration breaking. Coverage of the surface by molecules/nanoparticles mainly depends on the deposition time and concentration of the solution used for the self-assembly. Deposition of the samples under study was done for different solution concentrations and deposition times until a self-assembly monolayer covering most of the surface area is obtained. Imaging of the surfaces after deposition was done by tapping-mode AFM. Analysis of the AFM images was performed and deposition parameters (i.e. coverage or molecule/particle size distribution) were obtained. The subjects of this investigation were a molecular polyoxometalate, a single-molecule magnet and functionalized gold nanoparticles. The obtained results agree with the structure of each of the studied systems. Using the optimized deposition parameters found in this investigation, single-electron transport measurements have been carried out. Preliminary results indicate the right choice of the deposition parameters.M.S.
Department of Physics
Sciences
Physics MS
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Täuber, Daniela, Mario Heidernätsch, Michael Bauer, Günter Radons, Jörg Schuster et Christian von Borczyskowski. « Single molecule tracking of the molecular mobility in thinning liquid films on thermally grown SiO 2 ». Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-191721.
Texte intégralRésumé :
Diffusion coefficients obtained from weighted mean square displacements along probe molecule trajectories within ultrathin liquid TEHOS films show a correlation with film thickness. By studying cumulative distributions obtained with a time resolution of 20 ms,
we could show that the diffusion is heterogeneous within our liquid films which consist of a few molecular layers only.
We detected two components of the diffusion process, a slower and a faster one. Thinning of the film due to evaporation caused a slowdown of the whole diffusion process. But this resulted not from a slowdown in the two contributing components itself. Instead their relative contributions changed in favor for the slow component. We conclude that there is no pronounced difference in the diffusion coefficients attributed to the molecular layers 3 to 5 vertically above the substrate, but with the loss of upper layers along with the thinning process the concentration of probe molecules in the near surface region containing only one or two molecular layers is increased.
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Täuber, Daniela, Mario Heidernätsch, Michael Bauer, Günter Radons, Jörg Schuster et Christian von Borczyskowski. « Single molecule tracking of the molecular mobility in thinning liquid films on thermally grown SiO 2 ». Diffusion fundamentals 11 (2009) 107, S. 1-11, 2009. https://ul.qucosa.de/id/qucosa%3A14081.
Texte intégralRésumé :
Diffusion coefficients obtained from weighted mean square displacements along probe molecule trajectories within ultrathin liquid TEHOS films show a correlation with film thickness. By studying cumulative distributions obtained with a time resolution of 20 ms,
we could show that the diffusion is heterogeneous within our liquid films which consist of a few molecular layers only.
We detected two components of the diffusion process, a slower and a faster one. Thinning of the film due to evaporation caused a slowdown of the whole diffusion process. But this resulted not from a slowdown in the two contributing components itself. Instead their relative contributions changed in favor for the slow component. We conclude that there is no pronounced difference in the diffusion coefficients attributed to the molecular layers 3 to 5 vertically above the substrate, but with the loss of upper layers along with the thinning process the concentration of probe molecules in the near surface region containing only one or two molecular layers is increased.
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Selvanathan, Pramila. « Photochromic switches for luminescence, plasmonic resonance, single molecule magnetic properties, and molecular wires for nano junctions ». Thesis, Rennes 1, 2016. http://www.theses.fr/2016REN1S145.
Texte intégralRésumé :
Ce travail est consacré à la synthèse et la caractérisation des commutateurs et des fils moléculaires incorporant l'unité et le ruthénium organométalliques fractions photochromiques. La première partie traite de lanthanides complexe Yb combiné avec l'unité et le ruthénium acétylure fractions photochromiques afin de moduler la luminescence avec l'aide de redox et de stimuli lumineux. Dans la deuxième partie explique la combinaison d'unités DTE photochromiques avec des fragments acétylures de ruthénium pour fixer sur la surface de nanoparticules métalliques afin d'affiner leur résonance plasmonique grâce à la modification de l'environnement de surface en utilisant la lumière et redox stimuli. La troisième partie décrit la préparation de complexes de lanthanides combinés avec une unité photochromique spiropyranne pour commuter les propriétés SMM des complexes via photoisomérisation de l'unité spiropyranne. Dans la dernière partie, nous présentons la synthèse de Oligo (phénylène éthylène) Les fils moléculaires avec différents noyaux centraux afin d'obtenir une variété de fil avec différents niveaux d'énergie HOMO-LUMO pour vérifier l'effet de l'épinglageThis work is devoted to the synthesis and characterization of novel molecular switches and wires incorporating photochromic unit and ruthenium organometallic moieties. The first part deals with lanthanide Yb complex combined with photochromic unit and ruthenium acetylide moieties in order to modulate the luminescence with the help of redox and light stimuli. In the second part explained the combination of photochromic DTE units with ruthenium acetylide moieties to attach on the surface of metal nanoparticles in order to tune their plasmonic resonance through the surface environment modification by using light and redox stimuli. The third part describes the preparation of lanthanide complexes combined with a spiropyran photochromic unit in order to switch the SMM properties of the complexes via photoisomerization of the spiropyran unit. In the last part, we report the synthesis of Oligo(phenylene ethylene) molecular wires with different central cores in order to obtain various wire with different HOMO-LUMO energy levels to check the effect of pinning
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Smith, Neil Ronald. « USING ELECTRON BEAM LITHOGRAPHY TO MAKE ELECTRODES FOR SINGLE MOLECULE ELECTRONICTS ». Miami University / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=miami1123213432.
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Xu, Kewei. « Single Molecule Studies of Diffusion Dynamics in Polymer Thin Films Near Tg ». Thesis, Georgia Institute of Technology, 2007. http://hdl.handle.net/1853/16233.
Texte intégralRésumé :
For polymers near the glass transition, the dynamics in some regions can be orders of magnitude different compared with the dynamics in other regions only a few nanometers away, so called spatial heterogeneity [1]. In this thesis, single molecule fluorescence microscopy as a powerful tool, was applied to study the spatially heterogeneous dynamics, both orientational and translational, within the polymer matrix near the glass transition temperature. With our total internal reflection fluorescence microscopy (TIRFM) methods, many individual fluorescent dye molecules embedded in the poly (isopropyl acrylate) (PIPA) thin films can be simultaneously excited. Their emission patterns are analyzed using our orientation determination methods [2] to give the true 3D orientational trajectories of the individual molecules. At Tg < T < 1.2 Tg, single molecule tracking was used to study the dye molecules translational diffusion. Results show that, below 1.1 Tg, the probe molecules are in the confined flow region [3]; at T > 1.1 Tg, the diffusion follows normal diffusion model; at T = 1.2 Tg, although the statistical results shows that normal random walk behavior is followed, the individual molecules still show different diffusion behaviors, clear evidence of the spatial heterogeneity that still exists at this temperature.
The second part of this thesis is a development of the 3-detector method to determine the 3D orientation of single molecules [4]. This method is based on the work proposed by Fourkas [4] in 2001. Results utilizing this experimental setup are compared with our emission pattern fitting methods. The results show that, with a little bit higher error range (10º in θ, 20º in φ), the 3-detector method can give agreeable orientation fittings, further more, with higher time resolution of < 10 ms. This 3-detector method is useful and can be applied to study the fast orientation dynamics in different systems.
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Stephenson, James David. « Structural analysis of the HIV-1 5' untranslated region RNA by single molecule FRET and molecular modelling ». Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610361.
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Maiuri, Paolo. « Single-cell and real-time analysis of transcription rates from integrated HIV-1 provirus ». Doctoral thesis, Scuola Normale Superiore, 2009. http://hdl.handle.net/11384/85935.
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Viral RNA biogenesis is a crucial step in the replication of retroviruses that require both the production of a genomic RNA as well as of translation templates. Cellular and viral factors concur in the biogenesis of RNA at the specific sub-nuclear chromatin site where the reaction takes place. The possibility of tracking viral RNA in living cells gives the unique possibility of measuring the kinetic parameters of RNA biogenesis as well as defining the dynamic recruitment of host and viral factors to the site of replication. In order to study the activation of HIV-1 gene expression from the integrated viral promoter we exploited a method that allows the visualization of newly transcribed RNA in living cells through the specific recognition of an RNA consensus sequence for the bacteriophage MS2 coat protein tagged with an autofluorescent protein. We observed that transcription of HIV-1 occurred in discrete foci within the nucleus of cells carrying several tandem arrays of the HIV-1 construct. These foci, representing newly transcribed RNA, co-localized with the viral Tat transactivator as well as members of the positive transcription elongation factor (P-TEFb) and RNA polymerase II (RNAPII). This experimental setting was used to measure the dynamic of HIV-1 RNA transcription in living cells. By fluorescence recovery after photobleaching (FRAP) we were able to demonstrate that following photobleaching the process reaches a steady state with a negligible immobile fraction allowing precise kinetic measurements of RNA polymerase elongation rates. We found that elongation proceeded at approximately 2 kb/min, and that 3'-end formation and release took another minute to complete. In addition we also analyzed the dynamic of RNAPII and the TAR:Tat:pTEFb complex at the site of HIV-1 transcription in living cells. Our data suggest that, while the residence time of RNAPII exceeds the time required for elongation through the viral template, the complex dissociates from the polymerase following transcription initiation, and may undergo subsequent cycles of association/dissociation. This approach was extended to the analysis of single integrated HIV- 1 transcription units by transduction of a HIV-based lentiviral vector into a human cell line and subsequent selection for Tat-induction from a latent state. Nascent RNAs from single integrated transcription units were detectable in living cells by MS2 RNA-tagging. At steady state a constant number of RNAs was measured at the transcription site corresponding to a minimal density of polymerases with negligible fluctuations over time both in space and intensity of the signal. Recovery of fluorescence after photobleaching of the transcription site was complete within seconds, much faster than what was observed previously. However, the necessity of taking into account also the diffusion of the tagged MS2 protein required the development of novel analytical tools. To this end we developed a model that describes each polymerase sliding along the DNA like the peak of a positive progressive traveling wave (TranWave) to predict the number of MS2 RNA repeats at the transcription site in function of time. The outcome of this approach and its following improvements are being discussed. This work provides for the first time a kinetic framework to analyze HIV-1 RNA biogenesis and RNA/protein dynamics in living cells.
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Snizhko, D. « Photone counting for single molecula acts registration ». Thesis, КрНУ, 2018. http://openarchive.nure.ua/handle/document/7522.
Texte intégralRésumé :
The proposed technic that included electrochemiluminescent amplification of the signal assists to obtain feedback on the interaction individual particles including organic molecules with an electrode. In the work the principal posibility to obtain a responce from a signal on ultramicroelectrodes is shown
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McHugh, Toni. « Single molecule mechanics of Kif15 ». Thesis, University of Warwick, 2015. http://wrap.warwick.ac.uk/77516/.
Texte intégralRésumé :
Kinesin-12 is a motor protein that has a role in the processes of mitotic spindle formation and maintenance. The human Kinesin-12, Kif15, has been shown to have some functional redundancy with Eg5, a Kinesin-5 that plays key roles in the formation of the bipolar spindle and is a potential target for anti-cancer drugs. Eg5 is thought to contribute to spindle formation by cross-linking and sliding microtubules, however little is known about the mechanism of Kif15. We have used laser tweezers to investigate the mechanical properties of Kif15 compared to those of kinesin-1. We have found that Kif15 is plus end directed and takes multiple steps along the microtubule without detaching. Full-length Kif15 walks faster and supports more load than full-length Eg5. Kif15 is less processive under load than kinesin-1, although it has a similar stall force. A second, diffusive, microtubule binding site in Kif15 supports processivity at zero load, and slows flyback following a detachment in the optical trap. The microtubule-associated protein, Tpx2, is necessary for the localisation of Kif15 to spindle microtubules. We find that Tpx2 binding arrests the motion of Kif15 and creates a stable binding state that resists both assisting and hindering loads. We also find evidence of a tail-mediated auto-inhibitory mechanism that creates a stable MT binding state and causes pausing during processive runs. C-terminal truncation of the Kif15 tail relieves this inhibition leading to faster overall stepping and abrogates the effects of Tpx2. We examined the detachment behaviour of Kif15 from microtubules, under assisting and hindering loads. We find that assisting loads cause single Kif15 and Kinesin-1 motors to detach from the microtubule more easily than hindering loads. Kif15 shows a much more asymmetric response to load in low levels of ATP than Kinesin-1, and both show more asymmetry than Eg5: previous work has shown that the behaviour of Eg5 does not change dramatically with differing loading directions. This has interesting implications for the roles of Kif15 and Eg5 motors in both parallel and anti-parallel microtubule bundles. Overall our data supports an in vivo mechanism for Kif15 that it distinct from that of Eg5. We investigated the load-dependent detachment of Kinesin-1 and Kif15 in millimolar concentrations of ADP, AMPPNP and micromolar concentrations of ATP. Kinesin-1 in ADP detached at low loads, and in AMPPNP at two different loads, both higher than in ADP. These two AMPPNP states of Kinesin1 likely corresponding to single and double headed microtubule binding, as proposed by Ishiwata and colleagues. Kif15 behaved broadly similarly. At micromolar ATP concentrations and hindering loads, both Kinesin-1 and Kif15 again showed two different high load detachment states. This is inconsistent with the model proposed by Ishiwata and possible modifications are discussed.
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Pánek, Dalibor. « Developments for single molecule studies ». Thesis, University of Strathclyde, 2010. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=16933.
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Single molecule fluorescence spectroscopy has attracted considerable attention over the past two decades. Measurement on a single entity provides an opportunity to avoid ensemble averaging which is always present in conventional bulk fluorescence measurements. This makes single molecule spectroscopy particularly interesting for biophysics and biochemistry where heterogeneous systems are often encountered. The general interest of this thesis is in studies of single immobilised molecules carried out at room temperature. One of t.he major issues of single molecule spectroscopy is finding a suitable immobilising medium. Inorganic silica matrices prepared by the sol-gel method have a great potential to provide a close- to-natural immobilising environment even for sensitive biomolecules and thus allow investigation of their natural behaviour on the most fundamental level. In order to be able to tailor both physical and chemical properties of the final gel, it is of great importance to develop reliable methods to control each stage of polymerisation. In one part of this thesis, applications of fluorescent probes to investigation of sol-gels properties, as well as monitoring the gel assembly process, are discussed. The thesis further presents studies of the genetically engineered glucose binding protein labelled with the environmentally sensitive dye badan. This system was developed in a search for an appropriate recognition-reporter unit to serve as a part of fluorescence-based sensor for continuous blood glucose monitoring. This labelled biomolecule represents an interesting subject for a single molecule study. Due to technical reasons however, single molecule spectroscopy could not be applied in this case. Therefore, conventional ensemble fluorescence spectroscopy methods were used to characterise behaviour of the labelled protein at different glucose concentrations. The last part of the thesis deals with instrumental aspects of single molecule imaging and spectroscopy. The aim of the work was to assess the applicability of a freshly installed commercial microscope a-SNOM (WITec GmbH) in single molecule fluorescence studies and at the same time to adopt the technique for future experiments in our research group.
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Lu, Siran. « Single molecule kinetic isotope effect ». Thesis, University of Oxford, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.526483.
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Li, Yang. « Single Molecule Spintronics and Friction ». Ohio University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou151561792063398.
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Larsson, Chatarina. « Single-molecule Detection in situ ». Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-98538.
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Yoo, Jae. « Polynuclear manganese single-molecule magnets / ». Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2000. http://wwwlib.umi.com/cr/ucsd/fullcit?p9988317.
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Sustarsic, Marko. « In vitro, in silico and in vivo studies of the structure and conformational dynamics of DNA polymerase I ». Thesis, University of Oxford, 2016. http://ora.ox.ac.uk/objects/uuid:ea317d58-00f7-4fc4-b71b-866b4becf0f7.
Texte intégralRésumé :
DNA polymerases are a family of molecular machines involved in high-fidelity DNA replication and repair, of which DNA polymerase I (Pol) is one the best-characterized members. Pol is a strand-displacing polymerase responsible for Okazaki fragment synthesis and base-excision repair in bacteria; it consists of three protein domains, which harbour its 5’-3' polymerase, 3’-5’ exonuclease and 5’ endonuclease activities. In the first part of the thesis, we use a combination of single-molecule Förster resonance energy transfer (smFRET) and rigid-body docking to probe the structure of Pol bound to its gapped-DNA substrate. We show that the DNA substrate is highly bent in the complex, and that the downstream portion of the DNA is partly unwound. Using all-atom molecular dynamics (MD) simulations, we identify residues in the polymerase important for strand displacement and for downstream DNA binding. Moreover, we use coarse-grained simulations to investigate the dynamics of the gapped-DNA substrate alone, allowing us to propose a model for specific recognition and binding of gapped DNA by Pol. In the second part of the thesis, we focus on the catalytically important conformational change in Pol that involves the closing of the ‘fingers’ subdomain of the protein around an incoming nucleotide. We make use of the energy decomposition method (EDM) to predict the stability-determining residues for the closed and open conformations of Pol, and test their relevance by site-directed mutagenesis. We apply the unnatural amino acid approach and a single-molecule FRET assay of Pol fingers-closing, to show that substitutions in the stability-determining residues significantly affect the conformational equilibrium of Pol. In the final part of the thesis, we attempt to study Pol in its native environment of the living cell. We make use of the recently developed method of internalization by electroporation, and optimize it for organically labelled proteins. We demonstrate the internalization and single-molecule tracking of Pol, and provide preliminary data of intra-molecular FRET in Pol, both at the single-cell and single-molecule levels. Finally, by measuring smFRET within an internalized gapped-DNA construct, we observe DNA binding and bending by endogenous Pol, confirming the physiological relevance of our in vitro Pol-DNA structure.
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Heß, Volkmar [Verfasser], Daniel [Akademischer Betreuer] Bürgler et Thomas [Akademischer Betreuer] Michely. « Scanning tunneling microscopy of single-molecule magnets and hybrid-molecular magnets : Two approaches to molecular spintronics / Volkmar Heß. Gutachter : Daniel Bürgler ; Thomas Michely ». Köln : Universitäts- und Stadtbibliothek Köln, 2016. http://d-nb.info/1099592941/34.
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Dekker, Cees. « The appeal of single-molecule and single-cell studies ». Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-179451.
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Dekker, Cees. « The appeal of single-molecule and single-cell studies ». Diffusion fundamentals 20 (2013) 9, S. 1, 2013. https://ul.qucosa.de/id/qucosa%3A13532.
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Mishra, Abhudaya. « Mixed-metal molecular complexes single-molecule nanomagnets and bioinorganic models of the water oxidizing complex of photosystem II / ». [Gainesville, Fla.] : University of Florida, 2006. http://purl.fcla.edu/fcla/etd/UFE0015608.
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Ericsson, Olle. « Biomolecular Analysis by Dual-Tag Microarrays and Single Molecule Amplification ». Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distributör], 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8475.
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Chen, Liang. « Single molecule and single particle studies of neuronal axonal transport / ». May be available electronically:, 2009. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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Lopez, Benjamin J. 1982. « Experimental realization of a feedback ratchet and a method for single-molecule binding studies ». Thesis, University of Oregon, 2010. http://hdl.handle.net/1794/11056.
Texte intégralRésumé :
xii, 112 p. : ill. (some col.)Biological molecular motors exist in an interesting regime of physics where momentum is unimportant and diffusive motion is large. While only exerting small forces, these motors still manage to achieve directed motion and do work. Brownian motors induce directed motion of diffusive particles and are used as models for biological and artificial molecular motors. A flashing ratchet is a Brownian motor that rectifies thermal fluctuations of diffusive particles through the use of a time-dependent, periodic, and asymmetric potential. It has been predicted that a feedback-controlled flashing ratchet has a center of mass speed as much as one order of magnitude larger than the optimal periodically flashing ratchet. We have successfully implemented the first experimental feedback ratchet and observed the predicted order of magnitude increase in velocity. We experimentally compare two feedback algorithms for small particle numbers and find good agreement with Langevin dynamics simulations. We also find that existing algorithms can be improved to be more tolerant to feedback delay times. This experiment was implemented by a scanning line optical trap system. In a bottom-up approach to understanding molecular motors, a synthetic protein-based molecular motor, the "tumbleweed", is being designed and constructed. This design uses three ligand dependent DNA repressor proteins to rectify diffusive motion of the construct along a DNA track. To predict the behavior of this artificial motor one needs to understand the binding and unbinding kinetics of the repressor proteins at a single-molecule level. An assay, similar to tethered particle motions assays, has been developed to measure the unbinding rates of these three DNA repressor proteins. In this assay the repressor is immobilized to a surface in a microchamber. Long DNA with the correct recognition sequence for one of the repressors is attached to a microsphere. As the DNA-microsphere construct diffuses through the microchamber it will sometimes bind to the repressor protein. Using brightfield microscopy and a CCD camera the diffusive motion of the microsphere can be characterized and bound and unbound states can be differentiated. This method is tested for feasibility and shown to have sufficient resolution to measure the unbinding rates of the repressor proteins.
Committee in charge: Dr. Raghu Parthasarathy, Chair; Dr. Heiner Linke, Research Advisor; Dr. Dan Steck; Dr. John Toner; Dr. Brad Nolan
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Koch, Jens. « Quantum transport through single molecule devices ». [S.l.] : [s.n.], 2006. http://www.diss.fu-berlin.de/2006/380/index.html.
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