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Gortari, Antu Nehuen. "Metasurfaces for bioimaging." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS416/document.
Full textAbstract:
Au cours des dernières années, des efforts importants ont été déployés pour développer des métasurfaces (MSs) électromagnétiques avec la possibilité de changer de manière abrupte les propriétés de la lumière. Ces avancées ont ouvert une nouvelle gamme de possibilités pour contrôler la lumière en utilisant des dispositifs optiques ultra-minces. Dans ce contexte, et plus spécifiquement dans le spectre visible, les applications en bio-imagerie s’avèrent particulièrement intéressantes. Une technique qui est particulièrement bien adaptée à l'étude de molécules proches d'une membrane cellulaire est la microscopie à fluorescence par réflexion interne (TIRFM), qui repose sur un champ évanescent d'excitation. Dans ce cas la lumière incidente est totalement réfléchie sur une interphase (typiquement verre/eau) en raison de son angle d'incidence élevé. À ce jour, la TIRFM est généralement mise en œuvre à l'aide d'objectifs volumineux de grande ouverture numérique et de petit champ de vision.Dans ce travail de thèse, nous réalisons de substrats pour la microscopie TIRF à base de métasurfaces constituées de réseaux périodiques de structures asymétriques fabriquées en dioxyde de titane (TiO2) sur du verre borosilicaté. Ces structures, aussi petites que 48 nm, ont été optimisées à l’aide de simulations numériques "Rigorous coupled-wave analysis” (RCWA) dans le but de coupler de 50 à 90% de la lumière incidente dans le premier ordre de diffraction avec des angles élevés (θ > 63deg). Le fait de pouvoir utiliser des objectifs de faible grossissement et d'avoir une grande zone de champ évanescent fournit des conditions TIRF uniques qui ne sont pas accessibles par les méthodes traditionnelles. De plus, ces structures sont compatibles avec la lithographie par nanoimpression UV, ce qui permet d’envisager une fabrication à bas coût et à grande échelle. Outre la conception, et la fabrication, dans cette thèse nous aboutissons à une preuve de principe de la microscopie TIRF basée sur des métasurfaces en milieu biologique en imageant notamment des membranes fluorescentes de cellules souches. Ces métasurfaces permettent ainsi l’implémentation TIRFM à contraste élevé et à faible photo-blanchissement compatible avec des microscopes à champ large peu coûteuxIn recent years there has been a significant effort to push electromagnetic metasurfaces with the ability to abruptly change light properties into visible wavelengths. These advancements have opened a new range of possibilities to reshape light using ultra-thin optical devices and there is one field that is starting to gather attention: bioimaging. One technique particularly well suited for the study of molecules near a cell membrane is Total Internal Reflection Fluorescence (TIRF) microscopy, which relies on an evanescence field created by light being totally internally reflected within a glass substrate due to its high incidence angle. As of today, TIRF is generally implemented using bulky high-NA, small field of view oil objectives.In this project we present the realization of metasurface-based TIRF microscopy substrates consisting of periodic 2D arrays of asymmetric structures fabricated in titanium dioxide on borosilicate glass. These patterns, as small as 48nm, were optimized through rigorous coupled-wave analysis to couple 50-90% of the incoming normally incident light into the first diffraction order, which outputs at an angle that suffices total internal reflection in water and eliminates the requirement for high NA objectives or prisms to achieve TIRF. Being able to utilize lower-magnification air objectives and having a large evanescence field area provide unique TIRF conditions not accessible by traditional methods. Additionally, these structures are compatible with soft UV nanoimprint lithography, for cost-effective scale production, to give TIRF’s high contrast, low photodamage and low photobleaching capabilities to inexpensive wide-field microscopes
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Burgess, Helen Jane. "A bioelectrochemical FRET switch : combined total internal reflection (TIRF) microscopy and electrochemistry." Thesis, University of Oxford, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437177.
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Uhlig, Katja. "Untersuchungen PEG-basierter thermo-responsiver Polymeroberflächen zur Steuerung der Zelladhäsion." Phd thesis, Universität Potsdam, 2010. http://opus.kobv.de/ubp/volltexte/2010/4778/.
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Moderne Methoden für die Einzelzellanalyse werden dank der fortschreitenden Weiterentwicklung immer sensitiver. Dabei steigen jedoch auch die Anforderungen an das Probenmaterial. Viele Aufbereitungsprotokolle adhärenter Zellen beinhalten eine enzymatische Spaltung der Oberflächenproteine, um die Ablösung vom Zellkultursubstrat zu ermöglichen. Verschiedene Methoden, wie die Patch-Clamp-Technik oder eine auf der Markierung extrazellulärer Domänen von Membranproteinen basierende Durchflusszytometrie können dann nur noch eingeschränkt eingesetzt werden. Daher ist die Etablierung neuer Zellablösemethoden dringend notwendig.
In der vorliegenden Arbeit werden erstmals PEG-basierte thermo-responsive Oberflächen erfolgreich für die Zellkultur eingesetzt. Dabei wird das zerstörungsfreie Ablösen verschiedener Zelllinien von den Oberflächen durch Temperatursenkung realisiert. Die Funktionalität der Oberflächen wird durch Variation der Polymerstruktur, sowie der Konzentration der Beschichtungslösung, durch Beschichtung der Oberflächen mit einem zelladhäsionsfördernden Protein (Fibronektin) und durch Adsorption zelladhäsionsvermittelnder Peptide (RGD) optimiert. Um den Zellablösungsprozess detaillierter zu untersuchen, wird hier zum ersten Mal der direkte Zellkontakt mit thermo-responsiven Oberflächen mittels oberflächensensitiver Mikroskopie (TIRAF) sichtbar gemacht. Mit dieser Technik sind die exakte Quantifizierung und die Analyse der Reduktion der Zelladhäsionsfläche während des Abkühlens möglich. Hierbei werden in Abhängigkeit von der Zelllinie Unterschiede im Zellverhalten während des Ablösens festgestellt: Zellen, wie eine Brustkrebszelllinie und eine Ovarzelllinie, die bekanntermaßen stärker mit ihrer Umgebung in Kontakt treten, vergrößern im Verlauf des Beobachtungszeitraumes den Abstand zwischen Zellmembran und Oberfläche, reduzieren jedoch ihre Zell-Substratkontaktfläche kaum. Mausfibroblasten hingegen verkleinern drastisch die Zelladhäsionsfläche. Der Ablösungsprozess wird vermutlich aktiv von den Zellen gesteuert. Diese Annahme wird durch zwei Beobachtungen gestützt: Erstens verläuft die Reduktion der Zelladhäsionsfläche bei Einschränkung des Zellmetabolismus durch eine Temperatursenkung auf 4 °C verzögert. Zweitens hinterlassen die Zellen Spuren, die nach dem Ablösen der Zellen auf den Oberflächen zurückbleiben. Mittels Kombination von TIRAF- und TIRF-Mikroskopie werden die Zelladhäsionsfläche und die Aktinstruktur gleichzeitig beobachtet. Die Verknüpfung beider Methoden stellt eine neue Möglichkeit dar, intrazelluläre Prozesse mit der Zellablösung von thermo-responsiven Oberflächen zu korrelieren.Modern methods for single-cell analysis are becoming increasingly sensitive. At the same time, requirements for the sample material are on the rise. Today, sample preparation of adherent cells usually includes steps of enzymatic treatment to digest surface proteins thus, inducing cell detachment from culture substrates. This strongly limits the application of different techniques like patch clamp or labelling of extracellular domains of membrane proteins for flow cytometry. Therefore, a new cell detachment method is urgently required. In the present work, new PEG-based thermo-responsive polymers are used for cell culture for the first time. Here, non-destructive detachment of different cell lines from polymer-coated surfaces is realised by controlled temperature reduction. The surface functionality is systematically optimised by varying the concentration of the coating solutions, by artificial surface coating of a cell adhesion-mediating protein (fibronectin) and by co-adsorption of a cell adhesion-mediating peptide (RGD). For detailed analysis of the cell detachment process, TIRF microscopy is used to directly visualise the cell contacts on the thermo-responsive surfaces. Using this technique allows both the quantification and analysis of the reduction of the cell adhesion area during sample cooling. Furthermore, for several cell lines, different behaviours in cell detachment are observed. Cells that have close contact to their substrate like MCF-7 breast cancer cell line and CHO-K1 ovary cells increase the distance between cell membrane and surface, but there is only little decrease of cell-substrate adhesion area. In contrast, L929 fibroblasts reduce the cell adhesion area drastically. Furthermore, the hypothesis that the cell detachment is an active process is shown by lowering the cell metabolism by temperature reduction to 4 °C and by the cell traces that are left behind after rinsing the surfaces. A combination of TIRAF and TIRF enables visualising the cell adhesion area and actin structures. Measuring both parameters simultaneously opens up new possibilities to correlate intracellular and cell detachment processes on thermo-responsive surfaces.
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Ries, Jonas. "Advanced Fluorescence Correlation Techniques to Study Membrane Dynamics." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2008. http://nbn-resolving.de/urn:nbn:de:bsz:14-ds-1219846317196-73420.
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Fluorescence Correlation Spectroscopy (FCS) is a powerful tool to measure important physical quantities such as concentrations, diffusion coefficients, diffusion modes or binding parameters, both in solution and in membranes. However, it can suffer from severe artifacts, especially in non-ideal systems. Here we develop several novel implementations of FCS which overcome these limitations and facilitate accurate and quantitative determination of dynamic parameters in membranes. Two-focus FCS with camera-detection allows for accurate and calibration-free determination of diffusion coefficients. Confocal FCS using a laser scanning microscope provides an unprecedented positioning accuracy which enabled us to study, for the first time with FCS, dynamics in bacterial membranes. Scanning FCS with a scan path perpendicular to the membrane plane allows to correct for instabilities permitting long measurement times necessary to study slow diffusion. It can easily be extended to measure calibration-free diffusion coefficients with two-focus scanning FCS and to quantify binding with dual color scanning FCS. Spectral crosstalk can be avoided effectively by using alternating excitation. Using this method we were able to perform measurements in systems previously not accessible with FCS, such as yeast cell membranes or membranes of living zebrafish embryos. Line-scan FCS with a scan path in the membrane plane uses the parallel acquisition along the line to increase the statistical accuracy and decrease the measurement times. Knowledge of the scan speed serves as an internal calibration, enabling accurate diffusion and concentration measurements within seconds, hardly affected by photobleaching. Both realizations of scanning FCS can be easily implemented with commercial laser scanning microscopes. Often, a fluorescence background around the membrane cannot be avoided. The high surface selectivity needed in this case can be achieved efficiently by using a novel objective for FCS, the supercritical angle objective, which produces a very flat and laterally confined detection volume. Another technique with similar surface selectivity is FCS with total internal reflection excitation (TIRFCS). Due to the lack of a correct model, the accurate analysis of TIR-FCS data was previously not possible. In this work we develop such a model, enabling quantitative measurements of membrane dynamics with TIR-FCS. The novel FCS techniques developed here will have a high impact on the use of FCS to address key questions in biological systems, previously inaccessible by other methodsFluoreszenz-Korrelations-Spektroskopie (FCS) ist eine mächtige Methode, um wichtige physikalische Parameter wie Konzentrationen, Diffusionskoeffizienten, Diffusionsarten oder Bindungsparameter in Lösung und in Modell- oder Zellmembranen zu bestimmen. In nichtidealen Systemen ist FCS fehleranfällig. In dieser Arbeit entwickeln wir mehrere neuartige Realisierungen von FCS, welche diese Fehlerquellen umgehen und die genaue und quantitative Messung dynamischer Parameter in Membranen ermöglichen. Zwei-Fokus FCS mit Kamera-Detektion erlaubt eine genaue und kalibrationsfreie Messung von Diffusionskoeffizienten. Konfokale FCS mit einem Laserscanningmikroskop besitzt eine bislang unerreichte Positionsgenauigkeit, welche uns erstmals dynamische Messungen in Bakterienmembranen mit FCS ermöglichte. Scanning FCS mit einem Scanweg senkrecht zur Membran ermöglicht eine Korrektur von Instabilitäten und damit lange Messzeiten, die zur Bestimmung langsamer Diffusionskoeffizienten notwendig sind. Eine Erweiterung zur kalibrationsfreien Messung von Diffusionskoeffizienten mit Zwei-Fokus Scanning FCS und von Bindungsparametern mit Zwei-Farben Scanning FCS ist einfach. Mit diesen Methoden konnten wir in Systemen messen, die bislang FCS nicht zugänglich waren, so in Hefezellmembranen oder in Membranen lebender Zebrafischembryonen. Line-scan FCS besitzt einen Scanweg parallel zur Membran. Die parallele Messung entlang der ganzen Linie führt zu einer deutlichen Verbesserung der Statistik und damit zu kurzen Messzeiten. Die Kenntnis der Scangeschwindigkeit dient einer internen Kalibration und erlaubt eine akkurate Bestimmung von Diffusionskoeffizienten und Konzentrationen innerhalb weniger Sekunden, kaum beeinflusst vom Bleichen von Fluorophoren. Beide Arten von Scanning FCS können mit einem kommerziellen Laserscanningmikroskop realisiert werden. Häufig kann bei FCS Messungen ein fluoreszierender Hintergrund nicht vermieden werden. Hier ist eine hohe Oberflächenselektivitiät nötig, welche effizient mit einem neuartigen Objektiv erreicht werden kann. Dieses Supercritical Angle-Objektiv erzeugt ein sehr flaches und lateral begrenztes Detektionsvolumen. Eine weitere Methode mit einer ähnlich guten Oberflächenselektivität ist FCS mit Anregung über totale interne Reflektion (TIR-FCS). Bislang war eine quantitative Analyse der TIR-FCS Daten kaum möglich, da keine ausreichend genaue theoretische Beschreibung existierte. In dieser Arbeit entwickeln wir ein akkurates Modell, welches quantitative Messungen mit TIR-FCS erlaubt. Die hier entwickelten neuartgien FCS-Techniken ermöglichen die Untersuchung biologischer Fragestellungen, welche bislang keiner anderen Methode zugänglich sind
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Nathwani, Bhavik Bharat. "Developing luminescent nanoprobes for labeling focal adhesion complex proteins and performing combined AFM-TIRF imaging of these conjugates." Texas A&M University, 2008. http://hdl.handle.net/1969.1/85904.
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Recent progress in the field of semiconductor nanocrystals or Quantum Dots (QDs)
has seen them find wider acceptance as a tool in biomedical research labs. As produced,
high quality QDs synthesized by high temperature organometallic synthesis, are coated
with a hydrophobic ligand. Therefore, they must be further processed to be soluble in
water and made biocompatible.
A process to coat the QDs with silk fibroin, a fibrous protein derived from the
Bombyx mori silk worm, is described. Following the coating process, the characterization
of size, optical properties and biocompatibility profile of these particle systems is
described. In addition, conjugation of the silk fibroin coated QDs to different labeling
proteins such as phalloidin and streptavidin is described.
Proteins on the surface of ovarian cancer cells (HeyA8) and of cytoskeletal
components participating in the formation of focal adhesion complex (FAC), such as F-actin
in endothelial cells (HUVECS) were labeled using the bio-conjugated QDs. Various imaging techniques such as epi-fluorescence, TIRF and AFM were used to
study the QD labeled cells. Overall the project has produced luminescent nanoprobes that
enable the study of FAC formation dynamics and potentially a better in vivo fluorescent
marker tool.
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Bethea, Tomika R. C. "Silica Colloidal Crystals as Porous Substrates for Total Internal Reflection Fluorescence Microscopy." Thesis, The University of Arizona, 2006. http://hdl.handle.net/10150/193371.
Full textAbstract:
In cell biology and chemistry, total internal reflection microscopy (TIRFM) has proven to be a useful technique that allows the probing of cellular processes with high-signal-to-noise ratio imaging. However, samples on solid substrates limit the accessibility to probe processes on extracellular membrane surface closest to the microscope objective. Colloidal crystals provide a porous alternative to the traditional solid substrates. Thin crystals exhibit optical properties similar to that of a fused silica coverslip allowing for TIRFM in the same manner as with a typical coverslip as demonstrated by the observance of Chinese hamster ovary cells with fluorescently labeled receptors on both types of substrates. Accessibility of the cell membrane closest to the substrate and the ability to probe fluorophore orientation information was observed by the binding of TIPP-cy5 to the human delta opioid receptor.
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Tassinari, Andrea 1991. "In vitro reconstitution of the collective activities of motor protein complexes." Doctoral thesis, Universitat Pompeu Fabra, 2020. http://hdl.handle.net/10803/670056.
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Tschmelak, Jens M. [Verfasser]. "New ultra-sensitive immunoassays for (Total Internal Reflectance Fluorescence) TIRF-based biosensors / Jens M Tschmelak." Aachen : Shaker, 2005. http://d-nb.info/1186578238/34.
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Faure, Laura. "La machinerie de motilité de Myxococcus xanthus : caractérisation d'une nouvelle famille de moteurs moléculaires dans l'enveloppe bactérienne." Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0011/document.
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Dans les cellules il existe deux grandes sources d’énergie : l’ATP et la force proton-motrice, produites au niveau du cytoplasme et de la membrane interne respectivement. La mise en place de processus actifs dans la membrane externe ou à la surface des bactéries à Gram négatif requière la présence de machineries protéiques transmettant les forces de leur lieu de production à leur lieu d’utilisation. Durant ma thèse j’ai étudié une de ces machines : la machinerie de motilité (Agl-Glt) de Myxococcus xanthus. Plus précisément, j’ai cherché à comprendre comment les composants de cette machine s’organisent pour permettre le déplacement d’une bactérie. J’ai montré que l’assemblage de la machinerie de motilité au pôle avant des cellules nécessite la formation d’une plateforme cytosolique sur laquelle vient se fixer la machine Agl-Glt. Sous l’action du moteur, le complexe interne de la machine se déplace en direction du pôle arrière en suivant une trajectoire hélicoïdale de main droite. Au niveau de la surface les protéines de membrane externe sont recrutées au niveau d’adhésions focales et permettent l’ancrage de la machinerie au substrat. Enfin, la transmission des forces de la membrane interne à la surface par la machinerie de motilité génère le déplacement des cellules selon une trajectoire hélicoïdale de main gauche. Finalement, cette étude a révélé l’existence d’une machine protéique de l’enveloppe dont l’activité repose sur l’association d’un moteur linéaire et du cytosquelette bactérien. De par l’homologie qu’il existe entre les systèmes il est possible de proposer que ce type de machines peut-être retrouvé associées à d’autres fonctions que la motilité cellulaireTwo energy sources are present in cells: the ATP and the Proton Motive Force, produced in the cytoplasm and inner membrane respectively. Active processes in the outer membrane or on the surface of Gram negative bacteria require the presence of a proteic machinery to transduce the forces from their production site, in the cytoplasm or inner membrane, to their usage site. During my thesis I have studied one of these machineries: the motility machinery (Agl-Glt) of Myxococcus xanthus. More precisely, I try to understand how the components of this transmembrane machinery interact with each other to promote cell motility. I have shown that the assembly of the motility machinery at the leading pole requires the formation of a cytoplasmic platform onto which the Agl-Glt machinery is going to nucleate. The inner-membrane motor complex moves intracellularly along a right-handed path in the cell and becomes stationary at focal adhesion sites on the surface through the connection of the motor to the outer membrane proteins of the complex. This powers the left-handed helical motion of the bacteria. Finally, this study reveals the existence of a dynamic transmembrane machinery which associates the bacterial cytoskeleton to a linear motor to promote cell movement. The homology between the systems tells us that this type of motor is likely to be found associate with other function than cell motility
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Nassereddine, Aya. "Surface patterning strategies to dissect T-Cell adhesion and actin organisation." Thesis, Aix-Marseille, 2019. http://www.theses.fr/2019AIXM0458.
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Pour une réponse immunitaire efficace, une interaction optimale entre les cellules T et les cellules présentatrices d'antigène (APC) est nécessaire. Elle se présente sous la forme d'un contact cellulaire à différentes échelles allant du moléculaire (1-10 nm) au cellulaire (1-10 micromètres). La liaison entre les récepteurs spéciaux des cellules T (TCR) et leurs ligands sur une APC entraîne une réorganisation moléculaire à plus grande échelle menant d'abord à la formation de micro-clusters de TCR, puis à une restructuration à l'échelle cellulaire de la membrane et du cytosquelette. La création d'un substrat artificiel avec des amas de ligands qui induisent à leur tour l'accumulation de TCR est un outil important pour comprendre le lien entre l'organisation du TCR et de son ligand, l'organisation du cytosquelette d'actine et l'impact de ces deux facteurs sur le comportement cellulaire global, notamment l'adhérence et la signalisation. Nous avons développé un nouveau substrat basé sur la nanotechnologie et utilisé une stratégie alternative basée sur l'auto-assemblage colloïdal pour montrer que le TCR est clairement groupé sur des points de 700 nm et non pas sur des points de 400 nm. L'actine est distribuée de manière homogène sous forme de réseau dans la plupart des cellules, mais dans quelques-unes d'entre elles, elle apparaît sous la forme de points co-localisés avec les amas de ligands. Une observation plus fine à l'aide de la microscopie de reconstruction optique aléatoire indique que les points peuvent en fait être des sites où des faisceaux d'actine se croisent pour former des nœuds non visibles à moindre résolutionFor an efficient immune response, an optimal interaction between T-cells and antigen presenting cells (APC) is required; it takes the form of a cell-cell contact involving different scales ranging from the molecular (1-10 nm) to the cellular (1-10 micrometer). The ligation of the special T cell receptors (TCR) to its ligands on an APC, leads to larger scale molecular reorganisation leading first to formation of TCR micro-clusters, and later to cell-scale restructuring of both the membrane and the cytoskeleton. Patterning an artificial substrate with ligand-clusters that in turn induce TCR-clustering is an important tool to understand the link between the organisation of TCR and its ligand, the organisation of the actin cytoskeleton and the impact of both on overall cell behavior including adhesion and signaling. We developed a new nanotechnology based substrate (ligand-dot size down to 250 nm) and also used an alternative strategy based on colloidal self-assembly (700 or 400 nm) to show that TCR is clearly clustered on 700 nm dots but not on smaller 400 nm dots. Actin is homogeneously distributed in the form of a network in most cells but in a few of them, it appears as dots that co-localize with the ligand clusters. Finer observation using stochastic optical reconstruction microscopy indicates that the dots may in fact be sites where actin bundles cross each other forming nodes that are not visible at lower resolution. This work confirms a close link between T cell receptor organisation and actin structure
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Morén, Björn. "Caveolae associated proteins and how they effect caveolae dynamics." Doctoral thesis, Umeå universitet, Institutionen för medicinsk kemi och biofysik, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-92500.
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Caveolae are a type of invaginated membrane domain that has been shown to be involved in several disease states, including lipodystrophy, muscular dystrophies and cancer. Several of these diseases are caused by the lack of caveolae or caveolae-related signaling deficiencies in the tissues in which the caveolar domain are abundant such as lung, adipose, muscle and their related endothelial cells. Caveolae are formed through the assembly of the membrane inserted protein caveolin, cholesterol and the recently described family of cavin proteins, which together form the caveolae coat. The work in this thesis focuses on understanding the protein components and mechanisms that control the biogenesis and dynamics of caveolae. We have found that the protein EHD2 is an important regulator and stabilizer of the caveolar domain at the cell membrane. EHD2 is a dimeric ATPase known to oligomerize into ring-like structures around lipid membranes to control their shape. We have characterized the domain interactions involved in the specific targeting and assembly of this protein at caveolae. We propose a stringent regulatory mechanism for the assembly of EHD2 involving ATP binding and switching of the EH domain position to release the N-terminus and facilitate oligomerization in the presence of membrane species. We show that loss of EHD2 in cells results in hyper- dynamic caveolae and that caveolae stability at the membrane can be restored by reintroducing EHD2 into these cells. In a study of the protein cavin-3, which is known to be an integral component of the caveolar coat, we showed that this protein is targeted to caveolae via direct binding to the caveolar core protein caveolin1. Furthermore, we show that cavin-3 is enriched at deeply invaginated caveolae and regulate the duration time of caveolae at the cell surface. In combination with a biochemical and cellbiological approach, the advanced fluorescence microscopy techniques, like Fluorescence Recovery After Photobleaching (FRAP), Total Internal Reflection microscopy (TIRF), combined with correlative Atomic Force Microscopy (AFM) have allowed us to characterize distinct caveolae-associated proteins and their respective functions at caveolae.
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Thi, Huong Phan. "Spatial Survival Models for Analysis of Exocytotic Events on Human beta-cells Recorded by TIRF Imaging." Doctoral thesis, Università degli studi di Padova, 2018. http://hdl.handle.net/11577/3427181.
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Exocytosis on beta-cells is one of the fundamental cellular processes that releases insulin-containing secretory granules to blood through the plasma membrane due to stimulus. Studying survival of granules on the plasma membrane and their spatial correlation within cells during the exocytosis is of great interest to researchers in biological and medical area, as it is closely related to the regulation of insulin level in blood. Data are a collection of TIRF images recorded from 8 human beta-cells, containing granules and syntaxin information. One of the main objectives of this thesis is to investigate the relationship between the survival rates of granules and syntaxin levels, while adjusting for spatial correlation among granules within cells.
To answer our specific biological problem, we propose a semiparametric proportional hazard model, where the baseline hazard function is estimated nonparametrically and a multivariate normal distribution is assumed for individual frailties. Hence, the clustering structure, as well as the spatial correlation between granules are modeled via the variance-covariance matrix of frailties. We firstly extend the penalized partial likelihood method and the Monte-Carlo EM method to estimate the parameters in the model. Then, we contribute a novel inferential approach based on pairwise likelihood, EM algorithm and quadrature approximation. We conduct simulations to validate and compare three approaches, hence the advantages and disadvantages for each approach are discussed. Finally, we apply our method to the exocytosis data and interpret the results.Nelle cellule beta, l'esocitosi è uno dei processi cellulari fondamentali che rilascia nel sangue granuli secretori contenenti insulina, i quali attraversano la membrana del plasma quando sono sotto stimolo. Lo studio del tempo di vita dei granuli ssati alla membrana, prima del loro distacco, il tasso di esocitosi dei granuli e di altri eventi correlati, e la loro correlazione spaziale all'interno delle cellule, sono aspetti di grande interesse per i ricercatori nel campo biomedico, poiche sono strettamente collegati alle disfunzioni del livello di insulina nel sangue. I dati consistono in un insieme di immagini di tipo TIRF registrate nel tempo su 8 cellule beta umane, le quali contengono molte informazioni sull'andamento e sulla posizione dei granuli, oltre che sui livelli di alcune proteine, come per esempio la sintassina. Uno degli scopi principali della tesi è quello di studiare la relazione tra il tasso degli eventi di scomparsa dei granuli dalla membrana e i livelli della sintassina, tenendo conto della correlazione spaziale tra i granuli all'interno di ciascuna cellula. Per rispondere al problema biologico sotto studio, nella tesi è stato proposto un nuovo modello semiparametrico, un modello spaziale gerarchico di sopravvivenza ad effetti misti ("frailty") per dati raggruppati in clusters, dove la funzione hazard di riferimento è stimata nonparametricamente ed è assunta una distribuzione multivariata Normale per il vettore degli effetti casuali individuali. La struttura dei clusters e la correlazione spaziale tra le unità statistiche, sono modellati tramite la matrice di varianza e covarianza degli effetti casuali. Inizialmente, la tesi ha esteso il metodo della verosimiglianza parziale penalizzata ed il metodo EM Monte-Carlo, adattandoli all'inferenza per il modello spaziale di sopravvivenza proposto. In seguito, per tale modello, è stato presentato un nuovo approccio inferenziale, il quale si basa sulla verosimiglianza a coppie, l'algoritmo EM e l'approssimazione basata sull'integrazione numerica. Sono stati condotti studi di simulazione per confrontare il comportamento dei tre approcci inferenziali, e sono stati discussi i vantaggi e gli svantaggi di ciascun approccio. Infine, il modello ed i metodi proposti sono stati applicati ai dati sull'esocitosi ed è stata fornita una possibile interpretazione biologica del fenomeno.
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Valon, Léo. "Contrôle Optogénétique de la Polarité Cellulaire." Thesis, Paris, Ecole normale supérieure, 2014. http://www.theses.fr/2014ENSU0008/document.
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Dans cette thèse, nous avons concentré notre étude sur les mécanismes qui génèrent la polarité cellulaire, en particulier dans le cas de la migration cellulaire. Malgré les derniers développements concernant l’observation de l’activité des RhoGTPases, les principes qui dictent la capacité des cellules à coordonner plusieurs modules de signalisation en parallèle ne sont toujours pas compris. L’optogénétique est un outil d’intérêt pour disséquer ces réseaux de signalisation à partir de la création d’une perturbation dont les caractéristiques spatiotemporelles sont contrôlées. Tout d’abord, à partir de la caractérisation des différents processus biophysiques en jeu, nous avons établi les relations quantitatives entre l’illumination et les gradients moléculaires que l’on induit. Nous avons déterminé qu’il est possible de créer des gradients subcellulaires avec une résolution spatiale de l’ordre de 5 μm et temporelle d’environ 3 minutes Ensuite, nous avons utilisé cette approche optogénétique pour contrôler l’activité de Cdc42, Rac1 et RhoA. Nous avons caractérisé les effets subcellulaires de l’activation de ces RhoGTPases en utilisant l’activité de membrane, les changements de forme cellulaire et leurs déplacements comme rapporteurs de la polarisation et de la migration. Nous avons ainsi montré qu’une activation locale de RhoGTPase permet la réorganisation interne des cellules jusqu’à générer un phénotype de migration.Enfin, nous avons caractérisé les effets d’une activation locale de RhoA sur différents acteurs moléculaires comme les points focaux d’adhésion, l’actine et les moteurs moléculaires myosines. Nous avons mesuré alors la dynamique de l’intégration des points focaux dans le cytosquelette et analysé la réponse du réseau d’acto-myosine au cours d’évènements de rétraction.Notre approche optogénétique couple le contrôle d’une perturbation à la mesure de la réponse cellulaire simultanément de manière directe et reproductible. Elle apporte une méthode pour contrôler la polarité cellulaire et une manière de disséquer des réseaux de signalisation à l’échelle subcellulaireIn this thesis we focus on the mechanisms that establish cell polarization, particularly during cell migration. Despite latest developments that enable visualization of RhoGTPases activity, the underlying principles dictating the cell’s ability to coordinates multiple signaling modules is still unclear. Optogenetic methods have been recognized as promising tools to dissect these intracellular signaling networks by allowing perturbations to be spatially and temporally controlled. We established the quantitative relationship between illumination patterns and the corresponding gradients of induced signaling activity through the characterization of the biophysical properties of CRY2/CIBN. We determined that it is possible to create subcellular gradients of recruited proteins of different shapes of choice up to spatial resolutions of 5μm and temporal ones of ca. 3 minutes.We applied the aforementioned optogenetic approach as a means to perturb the activity of cdc42, Rac1 and RhoA. We characterized the effects of subcellular activation of those RhoGTPases using membrane activity, cell shape changes and cell displacement as reporters of cell polarization and migration. We show that localized activation of RhoGTPases can trigger cellular organization and drive the cell into a migrating state.We also characterized the effects of local activation of RhoA on different cellular effectors as focal adhesion complexes, actin filaments and myosin molecular motors. We measured the dynamics of the newly formed focal adhesion complexes and the acto-myosin complex during retraction events.Altogether, our optogenetic methodology enables simultaneous measurement of the imposed perturbation and the cell response in a straightforward and reproducible way. It provides a quantitative way to control cell polarity and a step forward in the dissection of subcellular signaling networks
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Schwarz, Friedrich. "The role of 1D diffusion for directional long-range communication on DNA." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-99388.
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Many genetic processes require enzymes or enzyme complexes that interact simultaneously with distant sites along the genome. Such long-range DNA-enzyme interactions are important for example in gene regulation, DNA replication, repair and recombination. In addition many restriction enzymes depend on interactions between two recognition sites and form therefore a model system for studying long-range communications on DNA.
Topic of the present work are Type III restriction enzymes. For these enzymes the communication mechanism between their distant target sites has not been resolved and conflicting models including 3D diffusion, 1D translocation and 1D diffusion have been proposed. Also the role of ATP hydrolysis by their superfamily 2 helicase domains which catalyse functions of many enzyme systems is still poorly understood. To cleave DNA, Type III restriction enzymes sense the relative orientation of their distant target sites and cleave DNA only if at least two of them are situated in an inverted repeat. This process strictly depends on ATP hydrolysis. The aim of this PhD thesis was to elucidate this long-range communication.
For this a new single molecule assay was developed using a setup combining magnetic tweezers and objective-type total internal reflection fluorescence microscopy. In addition of being able to mechanically manipulate individual DNA molecules, this assay allows to directly visualize the binding and movement of fluorescently labelled enzymes along DNA.
Applying this assay to quantum dot labelled Type III restriction enzymes, a 1D diffusion of the enzymes after binding at their target sites could be demonstrated. Furthermore, it was found that the diffusion depends on the nucleotide that is bound to the ATPase domains of these enzymes. This suggested that ATP hydrolysis acts as a switch to license diffusion from the target site which leads to cleavage.
In addition to the direct visualization of the enzyme-DNA interaction, the cleavage site selection, the DNA end influence (open or blocked) and the DNA binding kinetics were measured in bulk solution assays (not part of this thesis). The experimental results were compared to Monte Carlo simulations of a diffusion-collision-model which is proposed as long-range communication in this thesis.
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BERRETTONI, CHIARA. "Design, implementation and characterization of an optoelectronic platform for the detection of immunosuppressants in transplanted patients by means of a microfluidic optical chip." Doctoral thesis, Università di Siena, 2017. http://hdl.handle.net/11365/1007099.
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The past few years have seen great interest and advances in scientific research on fluorescence-based microfluidic optical sensors for clinical/medical applications. In particular, the growing demand for point-of-care-testing (POCT) devices to be applied near the patient’s bed, has driven the development of simple to use, reliable and low cost microfluidic optical platforms.
The main objective of the research project presented in this thesis is the development of a novel fluorescence-based microfluidic optical chip for the simultaneous analysis of different analytes and its integration into a stand-alone POCT device. The work was undertaken in the framework of the EU project NANODEM (NANOphotonic Device for Multiple therapeutic drug monitoring - FP7-ICT-2011) that aims at developing a POCT device for the measurement of immunosuppressants in transplanted patients, characterized by a narrow therapeutic range and serious potential side effects.
The benefit of this device will be an optimized dosage of the therapeutic drugs to support patient management in a clinical environment. In particular, the system will accurately measure the patient blood drug free fraction, which is considered the active fraction in terms of both drug effect and toxicity. In order to reach the low limit of detection required by the clinicians and enable the detection of the therapeutic drug free fraction, a heterogeneous binding inhibition immunoassay has been developed which makes use of antibody-coated fluorescent and magnetic nanoparticles.
The microfluidic optical chip, which exploits total internal reflection fluorescence (TIRF) and fluorescence anisotropy, is constituted by an array of microfluidic channels whose surface is chemically modified with the analyte derivative. The excitation light, coming from an external source, is properly coupled and confined by total internal reflection into the optical waveguide constituting the chip, and is guided towards the sensing area. After binding with the analyte derivative immobilised on the microchannel surface, the fluorescent-magnetic nanoparticles, coated with the analyte-specific antibodies, can be excited by means of the evanescent field, which arises at the waveguide/chip surface, and the emitted fluorescence can be collected by means of large area photodiodes. A thorough study on the excitation and emission of a fluorophore near a dielectric interface was undertaken theoretically and experimentally in order to optimize the microfluidic chip design for the best fluorescence collection efficiency. Different materials have been also investigated and several chip prototypes have been realized and characterized. The final microfluidic optical chip consists of three different polymeric parts bonded together: a thin Zeonor foil (188 µm thick, R.I. = 1.53) which is used as excitation waveguide, a double side adhesive tape (140 µm thick, R.I. = 1.49), in which ten channels are structured by laser cutting and in which the heterogeneous inhibition binding immunoassay is performed, and a Zeonex slide (1 mm thick, R.I = 1.51). The developed microfluidic optical chip is capable of performing the simultaneous analysis of three different selected analytes (Tacrolimus, Cyclosporin A and Mycophenolic acid), each of them measured three times in three different channels with an additional channel for waste flowing.
One of the main challenges of the work is to make the microfluidic optical chip easy to use and easy to integrate with different functional elements into a POCT device. These elements include the optoelectronic system (for both the excitation and the detection of the fluorescent signal), the magnetic trapping system (for the attraction and confinement of magnetic and fluorescent nanoparticles within the sensing area), and the fluidic system (for the automatic management of samples and reagents). Specifically, for the optoelectronic excitation system, different laser sources and different optical arrangements for the in-coupling of the light into the excitation waveguide have been experimentally evaluated and tested. An optimized butt-coupling illumination system based on an optical fibre bundle has been finally developed and integrated with the microfluidic chip. As second step of integration, the optical acquisition system, constituted of amorphous large area silicon (a-Si:H) photodiodes, absorption optical filters and readout electronics, has been properly developed in collaboration with the partners of the project. In order to favour the interaction between the fluorescent magnetic nanoparticles and the sensing layer, two magnetic trapping strategies based on current line structures (magnetic coils) and on permanent magnets have been evaluated, and a permanent magnet platform has been properly designed to be finally coupled with the microfluidic optical chip. The automatic management of the fluidics is an essential requirement for the development of POCT devices: from this point of view a fluidic system has been optimized for the implementation of the heterogeneous assay into the channels of the chip. A laboratory version of the integrated system has been developed and it can be considered as a transition setup. The final NANODEM POCT device has been finally manufactured and its functionality has been evaluated.
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Mohammed, Asma Hadi. "An investigation of RNR regulation in fission yeast by confocal laser scanning FRET and near-TIRF microscopy." Thesis, University of Sussex, 2011. http://sro.sussex.ac.uk/id/eprint/7401/.
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For genome integrity, adequate levels of deoxyribonucleotide (dNTPs) are essential to maintain faithful DNA replication and repair via the regulation of ribonucleotide reductase (RNR). In the fission yeast, RNR is composed of two subunits: Cdc22 and Suc22. The importance of Spd1 (RNR inhibitor) in Cdc22-Suc22 complex formation has been demonstrated by imaging of S. pombe containing fluorescent protein (FP) modified RNR subunit proteins in the presence of Spd1 and absence of Spd1 cells using confocal laser scanning microscopy. To investigate further the significant role of Spd1 in the regulation of RNR, 41 mutants created by Nestoras group. We used fluorescence resonance energy transfer (FRET) by acceptor photobleaching to investigate the RNR subunit interaction and provide evidence for a new model for the role of Spd1 in RNR regulation. Different treatments such as HU, 4NQO and heat shock have been used to investigate the effect of radical scavenging on the inhibition of RNR activity and induced DNA damage on S. pombe cell viability to elucidate further the role of Spd1 in the regulation of RNR. Finally a novel imaging technique, near-total internal reflection microscopy has been developed and applied with dual-view detection. The technique has been applied to image, simultaneously, the donor CFP and acceptor YFP channels of the FP-tagged RNR complex in the wild-type S. pombe cells and perform FRET measurements that are consistent with the confocal fluorescence results. In conclusion, a new hypothesis for the role of Spd1 has been drawn from the results, which is that the inhibitory role of Spd1 mediates the Suc22-Cdc22 (R1-R2) interaction to form a FRET competent but immature and inactive RNR complex, while with Spd1 deleted RNR is clearly active in a conformation that lacks FRET.
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Lino, Aline Monteiro. "Síntese, caracterização, estudos fotofísicos e acompanhamento in situ da reação de formação do corante (E)-2-[3-[4-(difenilamina)-fenil]-1-(p-tolil)-alilideno] malononitrila por microscopia de fluorescência." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/75/75134/tde-31032016-140123/.
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Neste trabalho foi sintetizado o corante (E)-2-[3-[4-(difenilamina)-fenil]-1-(p-tolil)- alilideno]-malononitrila (DFTAM), a partir da reação de condensação entre 4- (difenilamino)-benzaldeído e 2- [1- (4- metilfenil)-etilideno]-malononitrila, com catálise básica de piperidina. O produto obtido foi purificado por cromatografia líquida de alta eficiência (HPLC) e caracterizado pelas técnicas de espectrometria de massas, ressonância magnética nuclear de 13C e 1H e espectroscopia no infravermelho com transformada de Fourier. Para estudar suas propriedades fotofísicas, espectros de absorção e emissão de fluorescência, decaimento de fluorescência e espectro de absorção de transientes foram feitos em diferentes solventes, variando-se a polaridade e viscosidade do meio. Duas bandas de absorção foram observadas, uma em 303 nm e outra em cerca de 490 nm, a qual apresentou deslocamento batocrômico com o aumento da polaridade do solvente. Para essa região de excitação a banda de emissão variou entre 517 e 630 nm, com o aumento da polaridade do meio. Os decaimentos de fluorescência mostraram duas componentes, uma na ordem de picossegundos e a outra de nanossegundos. Os experimentos de absorção de transientes apresentaram três espécies, uma mais longa (maior que 10 ms) e duas outras de cerca 2 e 22 μs. Surfactantes catiônicos, não iônico, e aniônico também foram usados para produzir micelas e fazer os experimentos já citados. Pôde-se observar que o corante interagiu com as micelas, melhorando sua fluorescência e aumentando o tempo de vida do estado singleto. Por fim, acompanhou-se in situ, através da técnica de microscopia TIRF, a reação de formação de DFTAM a nível single molecule com catalise básica de nanopartículas de MgO e lamínulas de vidro funcionalizadas com piperazina. Através da intermitência de fluorescência dos filmes feitos de ambas as amostras, observou-se a formação de moléculas do corante através de ciclos de catálise da piperazina.In this project the synthesis of (E) -2- [3- [4- (diphenylamine) phenyl] -1- (p-tolyl) - allylidene] -malononitrile (DFTAM) dye, from the condensation reaction between 4- (diphenylamino) benzaldehyde and 2- [1- (4-methylphenyl) ethylidene]-malononitrile using piperidine basic catalysis has been achieved. The dye was purified by high-performance liquid chromatography (HPLC) and characterized by mass spectrometry, nuclear magnetic resonance 13C and 1H and Fourier Transform infrared spectroscopy techniques. To study DFTAM photophysical properties, absorption and fluorescence emission spectra, fluorescence decay and transient absorption spectrum were recorded in solvents with different polarity and viscosity. Two absorption bands of DFTAM were observed, the first one at 303 nm was solvent independent while the second one at about 490 nm, had bathochromic shift with increasing polarity of the medium. In the visible region of excitation the maximum of the dye emission band observed varied between 517 and 630 nm, upon increasing solvent polarity. Fluorescence decays showed two distinct components, a fast one in picosecond time scale and a slow one in nanoseconds. Transient absorption experiments indicated the presence of three species with different lifetimes, one longer than 10 ms and the other two with lifetimes about 2 and 22 μs. Cationic, nonionic, anionic surfactants were also used to produce micelles for easy solubilization of DFTAM. It was observed that the dye interacted with the micelles, improving its fluorescence yield and lifetime. Finally, the DFTAM formation reaction was monitored in situby TIRF wide field microscopy technique at single molecule level. The basic catalysis was tested for MgO nanoparticles and glass surface functionalized with bound piperazine. Through the fluorescence intermittency time trace obtained from TIRF movies, the discrete formation of dye molecules was only observed in the case of piperazine catalytic cycles.
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Devauges, Viviane. "Microscopie de fluorescence résolue en temps et en polarisation pour le suivi d’interactions protéiques en neurobiologie." Thesis, Paris 11, 2011. http://www.theses.fr/2011PA112315/document.
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Le suivi des interactions entre protéines, localisées à la membrane plasmique ou à l’intérieur de cellules, a été réalisé au cours de cette thèse par imagerie de fluorescence et par l’analyse de processus dits de FRET (Forster Resonance Energy Transfer). Pour quantifier le FRET entre nos protéines d’intérêt, nous avons choisi le contraste de durée de vie de fluorescence car cette méthode est indépendante de la concentration et de l’intensité de fluorescence. Afin d’obtenir une résolution suffisante pour des problématiques neurobiologiques, un microscope TIRFLIM (Total Internal Reflection Fluorescence Lifetime Imaging Microscopy) avait préalablement été développé. Celui-ci nous permet de faire de l’imagerie en plein champ avec une résolution axiale sub-longueur d’onde. Ce dispositif a été calibré et optimisé au cours de cette thèse pour répondre au mieux à des problématiques biologiques. Différentes approches ont ainsi été testées dans le but de calibrer la profondeur de pénétration de l’onde évanescente. Des surfaces plasmoniques ont entre autres été utilisées pour augmenter la sélectivité axiale du montage. Notre microscope a été dédié à l’étude de l’effet du cholestérol sur l’interaction entre la protéine précurseur de l’amyloïde APP, protéine transmembranaire impliquée dans la maladie d’Alzheimer et une de ses enzymes de clivage BACE1. Nous avons ainsi effectué un suivi dynamique de l’effet du cholestérol sur l’interaction entre APP et BACE1 dans des cellules HEK-293 et dans des cultures primaires de neurones d’hippocampe d’embryons de rat, de la membrane plasmique à l’intérieur des cellules grâce à notre dispositif TIRFLIM. La mesure d’anisotropie de fluorescence résolue en temps a également été implémentée sur notre montage. Ces mesures résolues en temps et en polarisation ont permis de mesurer le temps de corrélation rotationnelle de fluorophores et de mettre en évidence de manière qualitative différents niveaux d’homodimérisation de protéines impliquées dans la maladie d’AlzheimerIn the framework of this thesis, we have used FRET (Forster Resonance Energy Transfer) as a mechanism to follow the interaction of proteins from the plasma membrane to the cytoplasm of cells. To quantify FRET, we have chosen Fluorescence Lifetime Imaging Microscopy (FLIM) since this method is independent of the concentration and intensity of the fluorophores. To have a good axial resolution, a TIRFLIM set-up (Total Internal Reflection Fluorescence Lifetime Imaging Microscopy) was developed and this allowed us to perform wide-field imaging with sub-wavelength axial resolution. This set-up was calibrated and optimized in order to answer biological questions. Different approaches were tested in order to measure the penetration depth of the evanescent field and especially plasmonic surfaces were used to further enhance the axial resolution. Our set-up was dedicated to the study of the effect of cholesterol on the interaction between the Amyloid Precursor Protein (APP), a transmembrane protein involved in Alzheimer Disease, and one of its cleaving enzyme (BACE1). We performed a dynamic tracking of APP and BACE1 proximity under the effect of cholesterol, in HEK-293 cells and primary cultures of embryonic rat hippocampal neurons, thanks to our TIRFLIM set-up.Time-resolved fluorescence anisotropy has been implemented on our set-up. This has enabled us to measure the rotational correlation time of fluorophores and to investigate quantitatively different states of homodimerization of proteins involved in Alzheimer’s disease
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Gelléri, Márton [Verfasser], Philippe I. [Akademischer Betreuer] Bastiaens, and Roland [Gutachter] Winter. "TIRF-anisotropy microscopy: homo-FRET and single molecule measurements / Márton Gelléri. Betreuer: Philippe I. Bastiaens. Gutachter: Roland Winter." Dortmund : Universitätsbibliothek Dortmund, 2013. http://d-nb.info/1104736411/34.
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Roy, William Arthur Jr. "A Single Molecule Study of G-quadruplex and Short Duplex DNA Structures." Kent State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=kent1470001158.
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Barbieri, Francesco. "INVESTIGATING THE FUNCTIONAL MORPHOLOGY OF IN SITU IFT TRAINS BY CORRELATIVE LIGHT-ELECTRON MICROSCOPY." Doctoral thesis, Università di Siena, 2018. http://hdl.handle.net/11365/1052316.
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Cilia and flagella are cellular organelles involved in several and crucial aspects for the cell, tissue and organs normal physiology. Cilia can be classified according to their ability to perform a movement or not. Eukaryotic unicellular organisms and particular cells of mostly evolved organisms, i. e. spermatozoa, have evolved motile cilia for the cell propulsion in liquid media. Ciliated epithelia direct the movement of fluids by the ciliary beating. The primary cilium is an immotile cilium present in all the so far studied metazoan. This organelle is ubiquitous in human somatic cells when they are in G0 and G1 phases. Primary cilium and motile cilia work as sensory antennae highly specialized in receiving and secreting molecular signals. Such signal pathways regulate many aspects of the cellular metabolism, the cellular life-cycle, and the eventual cellular differentiations. Thus, ciliary alterations trigger severe consequences in human health causing a group of diseases known as ciliopathies. Such alterations in ciliary integrity are generated also from mutations affecting peptides involved in the intra-flagellar transport system (IFT), a selective and specific system of flagellar proteins transport, delivery, and recycling in which are employed proteic complexes (IFT trains) able to move bi-directionally along the flagellar axoneme. IFT trains transport into the ciliary compartment all the components needed for a correct ciliary assembly and maintenance i.e. peptide and proteic complexes of the ciliary axoneme, and several membrane-associated receptors. Analysis directed to better understand all the IFT features are thus of primary importance. Many analyses have been performed by studying the green algae model organism Chlamydomonas reinhardtii. Studies on IFT dynamics were performed in vivo, lacking in high resolution imaging. Other studies oriented in the determination of IFT trains ultrastructural morphology, and 3D-modeling digital reconstruction were performed ex vivo by transmission electron microscopy (TEM) imaging. A very recent work by Stepanek and Pigino (2017) revealed new insights in IFT system by using the correlative light electron microscopy (CLEM), which combines in vivo dynamics observed by florescence microscopy with ex vivo high-resolution analysis by electron microscopy. This study tough providing interesting anew clues about IFT functioning, left other important questions open on the motility direction of the different IFT trains categories present in Chlamydomonas flagella. In the present thesis we aimed at providing deeper insights in IFT system by using CLEM, in order to correlate a specific function to a specific ultrastructural category of IFT train. The reported results highlighted “short narrow IFT trains” moving in both anterograde and retrograde directions along the axoneme, while “short wide IFT trains” and “long IFT trains” were observed to remain static during in vivo observation.
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Hastoy, Benoit. "Structure et dynamique fonctionnelle du domaine transmembranaire de la protéine SNARE VAMP2 lors de l’exocytose." Thesis, Bordeaux 1, 2011. http://www.theses.fr/2011BOR14467/document.
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Le maintien de l’homéostasie passe notamment par la sécrétion d’hormones provenant des cellules neuro-endocrines ou endocrines telles que les cellules chromaffines ou les cellules b pancréatiques. Par exemple, la régulation de la glycémie nécessite l’exocytose de l’insuline depuis les cellules b pancréatiques des îlots de Langerhans. Une famille de protéines membranaires est au cœur de la machinerie de fusion d’une vésicule avec la membrane plasmique. Ce groupe appelé, la famille des protéines SNARE est composé de trois protéines. VAMP2 est localisée à la membrane vésiculaire alors que syntaxine 1A et SNAP25 sont localisées à la membrane plasmique. Syntaxine 1A et VAMP2 ont un domaine transmembranaire alors que SNAP25 est reliée à la membrane par prénylation de résidus cystéine. Cette famille forme le complexe cytosolique SNARE décrit comme essentiel à l’exocytose. La structure et la fonction du complexe cytosolique ont été étudiées en profondeur et ont mené au modèle du « zipper ». Celui-ci décrit un enroulement progressif des domaines cytosoliques SNARE permettant l’apposition des membranes puis la fusion. Le rôle des domaines transmembranaires reste encore peu décrit. Pourtant, leur étude est nécessaire afin d’établir un modèle complet de la fusion membranaire par les protéines SNARE. Nous avons donc mené une étude alliant une analyse structurale dynamique à une analyse biologique pour déterminer l’importance du domaine transmembranaire de VAMP2 dans la sécrétion. L’analyse biologique représente donc le centre de ma thèse. Le système biologique utilisé est basé sur l’extinction de l’expression de la protéine VAMP2 endogène et l’expression concomitante d’une protéine VAMP2 mutée dans son domaine transmembranaire. Deux lignées cellulaires considérées comme des modèles dans l’étude de la sécrétion hormonale et du trafic vésiculaire ont servi de support à notre étude. Par des approches de microscopies (confocal, TIRF) et d’analyses biochimiques, nous avons observé les conséquences fonctionnelles des mutations ponctuelles, établis par mutagénèse dirigée, sur le trafic vésiculaire et sur la capacité des cellules à sécréter.Les mutations induites présentent différents effets cellulaires. Certaines bloquent la sortie de VAMP2 du réseau golgien alors que d’autres ont un effet important sur la sécrétion hormonale et plus précisément sur l’exocytose. Les études structurales ont permis de corréler ces effets avec une diminution de la flexibilité structurale dans le cas de la diminution de l’exocytose, ou avec une restriction à la conformation hélice alpha dans le cas du sorting. Ce projet pluridisciplinaire a pu mettre en avant le rôle biologique du domaine transmembranaire de VAMP2 au cours de l’exocytose probablement soutenue par la dynamique conformationelle unique observée par le versant structural du projetThe hormonal secretion plays a key role in the maintenance of homeostasis. For example, the maintenance of normoglycaemia requires insulin exocytosis from the pancreatic beta cells. The SNARE membrane family protein has been described as the core machinery of fusion between the vesicle containing hormones and the plasma membrane. This family consists of 3 different membrane proteins that are essential during exocytosis. VAMP2 is localized on the vesicle and Syntaxin 1A - on the plasma membrane. They both are transmembrane protein whereas SNAP25 is linked to the plasma membrane by palmitoylation. The SNAREs appear to be essential as they form the cytosolic SNARE complex to dock the vesicle to the plasma membrane. Even though the role of this cytosolic domain has been studied in depth, much less is known on the role of their transmembrane domain during the fusion. Their study remains necessary to establish a complete model of membrane fusion mediated by the SNARE proteins.Here, we have studied the behavior and the role of the SNARE transmembrane domain during exocytosis. In a multidisciplinary project, we have combined a structural approach with a biological study to evaluate the role of this domain. Using mutagenesis in the transmembrane domain of VAMP2 and a cellular system with a clean background, we have assessed the effect of mutations on the secretion and exocytosis in two different cell lines (INS1E and PC12). The biological system is based on the silencing of endogenous VAMP2 and reconstitution of the expression of VAMP2 wt or mutated in the transmembrane domain. Using biochemistry assay and TIRF microscopy we have shown that mutations in this domain can lead to a missorting of the Golgi apparatus or a reduction of the stimulated secretion and exocytosis. This effect can be correlated to a modification of the structural dynamics of this domain.The obtained results clearly demonstrate the role of the transmembrane domain of VAMP2 during exocytosis probably sustained by its unique structural dynamics observed by physico-chemistry
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Joos, Uta S. "Untersuchungen zum Adhäsions- und Migrationsverhalten eukaryotischer Zellen auf künstlichen Substraten." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2007. http://dx.doi.org/10.18452/15625.
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Der zerstörungsfreien Charakterisierung und Analyse von lebenden humanen Zellen über längere Zeiträume kommt zukünftig in Medizin und Biotechnologie eine zentrale Rolle zu. Für eine therapeutische Nutzung müssen die Zellen nach der Analyse unverändert und vital vorliegen. Ein Ansatz beruht auf der Analyse von nanoskopischen Zellrückständen, die von Zellen während der Migration hinterlassen werden, den Zellspuren. Diese spiegeln in repräsentativer Weise die Merkmale der Erzeugerzelle wider. Für die technische Nutzung muss der Entstehungsprozess reproduzierbar kontrolliert werden können. Im Zusammenhang wurde ein Versuchsaufbau zur Beobachtung der dynamischen Prozesse Adhäsion, Migration und substratnahe Organisation des Zytoskeletts von lebenden Zellen entwicklet, der hochauflösende Langzeitbeobachtungen mittels Totaler Interner Reflexions Fluoreszenz (TIRF-) Mikroskopie ermöglicht. Zur Auswertung wurde eine auf Falschfarben beruhende Darstellungsweise der dynamischen Prozesse entwickelt. Es konnte eine Korrelation der Eigenschaften der Zellspuren mit dem Adhäsions- und Migrationsverhalten, sowie dem Aufbau der substratnahen Bereiche des Zytoskeletts der Erzeugerzellen nachgewiesen werden. Ebenso wurde der Einfluss der oberflächenspezifischen Substrateigenschaften (Beschichtung oder topografische Strukturierung) auf die Zellspurablage gezeigt.Nondestructive characterisation and analysis of human living cells over a long period will be an important issue for medicin and biotechnology in the near future. In order to use the cells after analysis for therapeutical applications, the cells have to be unmodified and still alive after the analytical procedure. The analysis of nanoscopic cell residues, called cell traces, which are left behind during cell migration represent an appropriate approach. Attributes of the donor cell are shown by the cell trace characteristically. In order to use cell traces for biotechnological applications the formation and deposition of cell traces has to be repeatable. Thus, an experimental set up using Total Internal Fluorescence Microscopy (TIRF) has been established to observe the dynamic processes of cell adhesion, cell migration and the organisation of the cytoskeleton used therein. Using miscolours a new embodiment has been developed to evaluate dynamic processes. Cell adhesion, cell migration and the organisation of the actin cytoskeleton of the donor cells have been found to influence the attributes of cell traces. Further specific modified surfaces have been used to influence the deposition of cell traces. Effects have been shown for coated surfaces or surfaces with a topographic structure.
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Liang, Boying. "Studies of Ligand-Receptor Pairs Utilizing Polymerized Planar Supported Lipid Bilayers." Diss., The University of Arizona, 2013. http://hdl.handle.net/10150/311232.
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Artificial membranes composed of natural lipids are not stable when exposed to air/vacuum, surfactant, organic solvent, etc. Polymerizable lipids provide an opportunity to broaden the use of lipid membranes to study ligand-receptor pairs under harsh experimental conditions. This dissertation presents the utilization of polymerizable lipids in matrix assisted laser desorption and ionization-mass spectrometry (MALDI-TOF MS) for analysis of ligands bound to membrane receptors. This platform may be applied to rapid drug-screening for membrane receptors including transmembrane proteins. Bacterial toxins and their membrane receptors were used as model ligand-receptor pairs to demonstrate the feasibility of using polymerizable lipids to detect and identify ligands by MALDI-TOF MS. Cholera toxin B (CTB) was successfully detected bound to polymerized lipid membranes with incorporation of its membrane receptor, GM1, while no CTB was detected in non-polymerizable lipid membranes. This affinity capture platform based on poly(lipid) showed a high resistance to interferences. On-plate digestion of bound CTB was performed and 57% amino acid sequence coverage was achieved. Total internal reflection fluorescence microscopy (TIRF-M) was applied to compare CTB-GM1 binding affinity in polymerized and unpolymerized membranes. Under a static flow system, the binding between CTB and GM1 was found to be stronger in polymerized membranes than other membranes. However, the ligand concentration under a static flow system is not in excess and the apparent binding affinity is likely to be significantly different than the true value. The true binding affinity can be approached under a continuous flow system, however equilibration time was found to be too long to address experimentally. Membrane fluidity, which may be required to maintain the membrane receptor activity, is suppressed in poly(lipid) membranes compared to unpolymerized membranes. In order to maintain fluidity, a non-polymerizable lipid was mixed into a polymerized lipid. Fluorescence recovery after photobleaching (FRAP) data showed that fluidity of membrane composed of the mixed lipid was maintained compared to pure poly(lipid). Phase segregation of polymerized lipid and non-polymerizable lipid was detected by atomic force microscopy (AFM). CTB bound to GM1 in mixed lipid membranes was detected by MALDI-MS, indicating the mixed lipid membranes retain stability under MALDI-MS analysis conditions.
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Ming, Min [Verfasser], and Jens [Akademischer Betreuer] Rettig. "Simultaneous capacitance and TIRF measurements from lytic granule fusion in primary human cytotoxic T lymphocytes / Min Ming. Betreuer: Jens Rettig." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2015. http://d-nb.info/1065232608/34.
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Khanduja, Nimisha. "Processive Acceleration of Actin Barbed End Assembly by N-WASP." Diss., Virginia Tech, 2014. http://hdl.handle.net/10919/54933.
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Actin-based cell motility plays crucial roles throughout the lifetime of an organism. The dynamic rearrangement of the actin cytoskeleton triggers a plethora of cellular processes including cellular migration. Neural Wiskott Aldrich syndrome protein (N-WASP) is involved in transduction of signals from receptors on the cell surface to the actin cytoskeleton. N-WASP activated actin polymerization drives extension of invadopodia and podosomes into the basement layer. In addition to activating Arp2/3 complex, N-WASP binds actin filament barbed ends, and both N-WASP and barbed ends are tightly clustered in these invasive structures.
We used nanofibers coated with N-WASP WWCA domains as model cell surfaces and single actin filament imaging to determine how clustered N-WASP affects Arp2/3-independent barbed end assembly. Individual barbed ends captured by WWCA domains of N-WASP grew at or below their diffusion limited assembly rate. At high filament densities, overlapping filaments formed buckles between their nanofiber tethers and myosin attachment points. These buckles grew 3.4-fold faster than the diffusion-limited rate of unattached barbed ends. N-WASP constructs with and without the native poly-proline (PP) region showed similar rate enhancements. Increasing polycationic Mg2+ or Spermine to enhance filament bundling increased the frequency of filament buckle formation, consistent with a requirement of accelerated assembly on barbed end bundling.
Our preliminary data shows that tethered N-WASP construct containing one WH2 domain does not generate processive bundles or filament loops leading us to believe that tandem WH2 is required for processivity. We propose that this novel N-WASP assembly activity provides an Arp2/3-independent force that drives nascent filament bundles into the basement layer during cell invasion. Discovery of this bundle mediated unique pathway involved in invasion and metastasis will provide new targets for therapeutic development.Ph. D.
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Basset, Antoine. "Détection et caractérisation par des approches statistiques locales d'évènements dynamiques dans des séquences d'images : application à la fusion membranaire en microscopie TIRF." Thesis, Rennes 1, 2015. http://www.theses.fr/2015REN1S096/document.
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Notre travail de thèse porte sur la détection et la modélisation de configurations dynamiques dans des séquences d'images. Nous développons des approches statistiques locales sans apprentissage supervisé. Notre application principale est la microscopie de fluorescence, un outil fondamental de la biologie cellulaire moderne. Deux cas peuvent se présenter : 1. les objets étudiés n'interagissent pas, et les dynamiques individuelles peuvent être analysées indépendamment ; 2. les objets étudiés interagissent, et la dynamique à analyser est celle du groupe entier d'objets. En ce qui concerne les dynamiques individuelles, nous nous intéressons à des séquences d’images biologiques dans lesquelles des protéines évoluent au sein de la cellule. Plus précisément, nous étudions la fusion de vésicules à la frontière de la cellule, appelée membrane plasmique. Les vésicules sont des intermédiaires de transport qui véhiculent des molécules dans la cellule. À la fin du processus d’exocytose, la fusion des vésicules avec la membrane s’accompagne d’une diffusion desdites protéines. Les images sont acquises en microscopie de fluorescence par réflexion totale interne (TIRF). Afin de repérer les évènements de fusion, nous proposons une nouvelle méthode de détection de spots. Puis, nous modélisons les dynamiques des protéines et estimons les paramètres biophysiques associés dans les séquences TIRF. La dynamique de groupe, quant à elle, est notamment rencontrée dans les mouvements de tissus cellulaires, le développement embryonnaire ou dans d’autres domaines, comme les mouvements de foules dans des vidéos. Nous proposons une nouvelle méthode d’estimation du mouvement de groupe permettant de caractériser le mouvement à la fois de façon quantitative et qualitative. Elle est utilisée pour classifier le mouvement de groupe, retrouver les chemins principaux dans la scène et détecter des anomalies locales. Dans l'un ou l'autre cas d'étude, nous abordons les problématiques selon une démarche commune, essentiellement dirigée par les données et mettant en œuvre des tests statistiques. Par ailleurs, nous avons le souci de proposer des méthodes nécessitant le réglage d'un faible nombre de paramètres qui sont, de plus, peu sensibles ou calibrés avec des règles statistiques. Enfin, nous adoptons des approches locales, qui ont l’avantage d’être rapides, flexibles et peu sensibles aux variations de contexte, qu’elles soient spatiales (arrière-plan variable) ou temporelles (changement d’illumination globale comme le photoblanchiment en microscopie de fluorescence)In this thesis, we investigate statistical methods to detect, estimate and characterize dynamical events in image sequences. Our main focus is on fluorescence microscopy images, which represent a fundamental tool for cell biology. There are two cases : 1. Studied objects do not interact, and individual dynamics can be independently analyzed ; 2. Studied objects interact, and group dynamics must be analyzed as a whole. In the case of individual dynamics, our primary focus is on biological image sequences showing proteins evolving in a cell, and more precisely at the cell frontier named plasma membrane. Proteins transported in the cell by vesicles, are observed in total internal reflection fluorescence microscopy (TIRFM), an observation technique well adapted to plasma membrane dynamics analysis. At the end of the exocytosis process, vesicles fuse to the plasma membrane and release proteins, which then diffuse. We first propose a new spot detection method aimed at localizing fusion events. Then, we model the protein dynamics and estimate the biophysical parameters in TIRFM image sequences for further biological analysis. We also address the processing of image sequences at lower magnifications, that is, depicting groups of cells, instead of an isolated cell. We propose a method to jointly estimate quantitative and qualitative motion measurements. It is used to classify the group motion, recover principal paths followed in the scene, and detect localized anomalies. Since they are free of appearance model, the developed methods are quite general and also applied to other applications including crowd motion analysis in videos. Whether it is for spot detection, protein dynamics estimation or group motion analysis, a common approach is ubiquitous, however. First, statistical arguments are used to automatically infer the method parameters. Secondly, we rely on local approaches, which have the advantage of being computationally efficient. Local modeling handles spatially varying image statistics much more easily and more accurately than global modeling. Local approaches also allow neglecting contextual variations such as spatially varying background contrast or, in fluorescence microscopy, temporal fading known as photobleaching
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Macleod, Charlotte Victoria. "Investigating TLR-4 signalling in response to protein ligands." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/274540.
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Toll-like receptor (TLR)-4 is a pattern recognition receptor (PRR) that recognises the pathogen-associated molecular pattern (PAMP) lipopolysaccharide (LPS) produced by Gram-negative bacteria. LPS binds to Myeloid differentiation 2 (MD-2)/TLR-4 heterodimers, driving their dimerisation and inducing a conformational change of the intracellular TLR-4 toll/interleukin-1 receptor (TIR) domains. The adaptor protein Myeloid differentiation primary response gene 88 (MyD88)-adaptor-like (Mal)/TIR domain-containing adaptor protein (TIRAP) then binds to the TIR domains of TLR-4 and acts as a bridge for MyD88 which goes on to form the myddosome, a large protein complex of six to eight MyD88 molecules and four Interleukin-1 receptor- associated kinase (IRAK) 4 and four IRAK1/2 molecules. This triggers a signalling cascade which results in nuclear factor (NF)-κB transcription factor activation and production of pro-inflammatory effector molecules such as the cytokine Tumour Necrosis Factor (TNF)-α. Upon activation TLR-4 is also endocytosed where it interacts with a second set of adaptor proteins TIR-domain-containing adaptor- inducing interferon (IFN)-β (TRIF)-related adaptor molecule (TRAM) and TRIF to initiate the type I IFN response. How TLR-4 dimerisation results in the formation of the oligomeric myddosome is not fully understood, but it is possible that the stoichiometry of Mal/TIRAP may be important in the formation of this protein complex. The aim of my thesis was to determine the stoichiometry of Mal/TIRAP at the plasma membrane of immortalised bone marrow derived macrophages (iBMDMs) and whether this stoichiometry changes upon stimulation with different TLR-4 ligands. To investigate Mal/TIRAP stoichiometry I first developed a viral transduction experimental cell model to visualise fluorescently labelled Mal/TIRAP. Mal/TIRAP-/- iBMDMs were lentivirally transduced with a Mal/TIRAPHALO construct. The halotag was fluorescently labelled then the cells were stimulated with TLR-4 ligands, such as LPS, fixed at different time points, then imaged. Total internal reflection fluorescence (TIRF) microscopy was used to image the plasma membrane and photobleaching experiments performed to determine Mal/TIRAP stoichiometry. I developed a computer-based analysis pipeline to analyse the resulting photobleaching data. Under resting conditions, Mal/TIRAP is present at the plasma membrane in clusters of approximately ten Mal/TIRAP molecules per cluster. After five minutes of stimulation with 10 ng/ml LPS Mal/TIRAP redistributes into cluster sizes of approximately six, twelve and much larger. After ten and fifteen minutes stimulation with 10 ng/ml LPS the clusters return to the resting size of approximately ten Mal/TIRAP molecules per cluster with a few much larger clusters remaining present. This confirms the rapid time frame within which TLR-4 signalling occurs at the plasma membrane and is consistent with myddosome stoichiometry of six MyD88 molecules or proposed super myddosomes of twelve MyD88 molecules. The computer-based analysis pipeline developed can be used to analyse any protein of interest at the plasma membrane. Protein ligands have also been found to activate TLR-4; for example allergens, such as Fel d 1 and Der p 2, as well as endogenous damage associated molecular patterns (DAMPs), such as extracellular matrix (ECM) proteins, for example fragments of fibronectin and tenascin-C. The mechanism by which these proteins interact with TLR-4 and induce signalling is unclear. Proteins from the ECM (fragments FNIII1c, FNIII13-14, FNIII9-E and FNIII9-E-14 from fibronectin and the fibrinogen-like globe (FBG) domain of tenascin-C) were tested using a transient transfection assay in HEK293 cells and shown to activate TLR-4. In conclusion, I have developed new tools and methodology to investigate how TLR-4 signals in response to LPS and DAMPs in living cells. Whether DAMP- activated TLR-4 forms similar signalling complexes to those induced by LPS will form part of a future study.
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Soubies, Emmanuel. "Sur quelques problèmes de reconstruction en imagerie MA-TIRF et en optimisation parcimonieuse par relaxation continue exacte de critères pénalisés en norme-l0." Thesis, Université Côte d'Azur (ComUE), 2016. http://www.theses.fr/2016AZUR4082/document.
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Cette thèse s'intéresse à deux problèmes rencontrés en traitement du signal et des images. Le premierconcerne la reconstruction 3D de structures biologiques à partir d'acquisitions multi-angles enmicroscopie par réflexion totale interne (MA-TIRF). Dans ce contexte, nous proposons de résoudre leproblème inverse avec une approche variationnelle et étudions l'effet de la régularisation. Une batteried'expériences, simples à mettre en oeuvre, sont ensuite proposées pour étalonner le système et valider lemodèle utilisé. La méthode proposée s'est montrée être en mesure de reconstruire avec précision unéchantillon phantom de géométrie connue sur une épaisseur de 400 nm, de co-localiser deux moléculesfluorescentes marquant les mêmes structures biologiques et d'observer des phénomènes biologiquesconnus, le tout avec une résolution axiale de l'ordre de 20 nm. La deuxième partie de cette thèseconsidère plus précisément la régularisation l0 et la minimisation du critère moindres carrés pénalisé (l2-l0) dans le contexte des relaxations continues exactes de cette fonctionnelle. Nous proposons dans unpremier temps la pénalité CEL0 (Continuous Exact l0) résultant en une relaxation de la fonctionnelle l2-l0 préservant ses minimiseurs globaux et pour laquelle de tout minimiseur local on peut définir unminimiseur local de l2-l0 par un simple seuillage. Par ailleurs, nous montrons que cette relaxation éliminedes minimiseurs locaux de la fonctionnelle initiale. La minimisation de cette fonctionnelle avec desalgorithmes d'optimisation non-convexe est ensuite utilisée pour différentes applications montrantl'intérêt de la minimisation de la relaxation par rapport à une minimisation directe du critère l2-l0. Enfin,une vue unifiée des pénalités continues de la littérature est proposée dans ce contexte de reformulationexacte du problèmeThis thesis is devoted to two problems encountered in signal and image processing. The first oneconcerns the 3D reconstruction of biological structures from multi-angle total interval reflectionfluorescence microscopy (MA-TIRF). Within this context, we propose to tackle the inverse problem byusing a variational approach and we analyze the effect of the regularization. A set of simple experimentsis then proposed to both calibrate the system and validate the used model. The proposed method hasbeen shown to be able to reconstruct precisely a phantom sample of known geometry on a 400 nmdepth layer, to co-localize two fluorescent molecules used to mark the same biological structures andalso to observe known biological phenomena, everything with an axial resolution of 20 nm. The secondpart of this thesis considers more precisely the l0 regularization and the minimization of the penalizedleast squares criteria (l2-l0) within the context of exact continuous relaxations of this functional. Firstly,we propose the Continuous Exact l0 (CEL0) penalty leading to a relaxation of the l2-l0 functional whichpreserves its global minimizers and for which from each local minimizer we can define a local minimizerof l2-l0 by a simple thresholding. Moreover, we show that this relaxed functional eliminates some localminimizers of the initial functional. The minimization of this functional with nonsmooth nonconvexalgorithms is then used on various applications showing the interest of minimizing the relaxation incontrast to a direct minimization of the l2-l0 criteria. Finally we propose a unified view of continuouspenalties of the literature within this exact problem reformulation framework
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McCluskey, Kaley A. "How the lysine riboswitch folds." Thesis, University of St Andrews, 2015. http://hdl.handle.net/10023/8241.
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To respond to rapidly-changing stresses in their environment, bacterial cells must be able to sense a variety of chemical cues and respond to them by activating the relevant genes. The lysine riboswitch is a short RNA motif, located just upstream of a gene encoding a lysine biosynthesis protein, that suppresses the expression of that gene when sufficient lysine is present in the cell. It acts by binding a lysine monomer in a region called the aptamer, which in turn rearranges an adjacent domain called the expression platform, sequestering the ‘start' sequence of the gene and preventing it from being transcribed. In this thesis, the lysine riboswitch's ligand-binding transition is studied using single-molecule fluorescence microscopy, optical tweezers, and a hybrid optical force/fluorescence technique. Förster Resonance Energy Transfer (FRET) is used with a fluorescently-labeled aptamer to show that it has a previously-undescribed, partially-folded structural state with enhanced ligand affinity compared to the unfolded structure. The Mg²⁺ dependence of the transition between these states is shown to resolve existing debates in the literature about the sensitivity of the riboswitch. The kinetics of the folding transition are explored using FRET, optical force, and hybrid ‘Fleezers' to map the free energy landscape of ligand binding and show that the ligand itself promotes transitions into the aptamer's folded state, a so-called ‘induced fit' mechanism rare among riboswitches. Finally, high-resolution optical tweezers are used to explore the link between the aptamer's secondary structure (the sequence of paired nucleotides) and its tertiary structure (three-dimensional folding) to illuminate the role of ligand binding in gene regulation, which depends on the equilibrium between competing secondary structures. Hybrid biophysical techniques like optical force/fluorescence microscopy are shown to be indispensable for addressing all the states in the reaction pathways of complex biomolecules like riboswitches and for discriminating between multiple levels of structure formation and interaction with the environment. Not only do the results presented here shed light on the RNA folding problem, particularly the role of tertiary structure in determining the minimum-energy configuration of an RNA sequence, but they could have implications for biomedical research, as the lysine riboswitch has already been shown to be a potential target for next-generation antibiotics.
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Robson, Alex J. "Single particle tracking as a tool to investigate the dynamics of integrated membrane complexes in vivo." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:7769f80c-a56d-4513-9123-1d65ef8c9911.
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The last decade has seen substantial advances in single-molecule tracking methods with nano-metre level precision. A powerful tool in single-molecule tracking is fluorescence imaging. One particular application, total internal reflection microscopy, can capture biological processes at high contrast video rate imaging at the single-particle level. This thesis presents methodologically novel methods in analysing single particle tracking data. Presented here is an application of a Bayesian statistical approach that can discriminate between the different diffusive modes that appear with the presence of membrane architecture. This algorithm is denoted BARD; a Bayesian Analysis to Ranking Diffusion. These algorithms are applied to a total internal fluorescence microscopy based experimental data of a novel membrane probe in Escherichia coli. This probe is a plasmid expressed, non-native membrane integrating trans-membrane helix and thus acts as an ideal protein based probe under no specific native control. Two experiments were performed using a combination of varying helix probe size and growth temperature experiments effectively altering the transition temperature of the membrane. These data are suggestive of a passive partitioning of the helix protein into mobile and immobile domains that emerge from the underlying phase behaviour of the membrane.
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Fargier, Guillaume. "Comportement dynamique au centrosome de la tyrosine kinase Syk, un nouveau suppresseur de tumeur dans le sein : étude par microscopie à haute résolution." Montpellier 2, 2009. http://www.theses.fr/2009MON20241.
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Très étudiée pour son rôle dans la signalisation des immuno-récepteurs, la tyrosine kinase Syk agirait comme suppresseur de tumeur et de métastase dans l'épithélium mammaire. Le mécanisme de cette activité anti-oncogénique reste inconnu. En plus de la localisation cytoplasmique, Syk est localisée aux extensions membranaires et au centrosome où elle est catalytiquement active, avec une variation de la concentration au cours du cycle cellulaire. Les localisations de mutants de Syk dans la cellule dépendent du type du site tyrosine muté d'une part, et les effecteurs potentiels de Syk reconnus par analyse protéomique d'autre part, font penser à un code de phosphorylation qui dépendrait du résidu tyrosine activé pour cibler la kinase à des sites sub-cellulaires différents. Afin de caractériser la dynamique des échanges de Syk entre compartiments subcellulaires et surtout dans le centrosome, nous avons employé des approches d'imagerie à haute résolution sur cellules vivantes de cancer du sein transfectées par des formes sauvage ou mutantes de DsRed-Syk. L'approche de FRAP et l'utilisation d'une chimère photo-activable (PA-GFP-Syk) montrent que Syk est recrutée activement au centrosome, avec un τ de 18,54 ± 3,63 sec. L'utilisation d'inhibiteurs de polymérisation de microtubules ou du moteur moléculaire dynéine/dynactine, la visualisation par TIRF des déplacements de particules de Syk à la base des cellules, la co-localisation par immunofluorescence, l'observation confirmée par modélisation d'une dérive directionnelle de PA-GFP-Syk après activation, tout indique que cytosquelette microtubulaire et moteur moléculaire sont nécessaires pour le recrutement de Syk au centrosomeInitially studied for its role in immunoreceptor-mediated downstream signalling, the tyrosine kinase Syk acts like a tumor and metastasis suppressor within breast cancer cells. The mechanism of its anti-oncogenic activity remains, however, to be identified. In addition to its cytoplasmic localization, Syk is also visualized at plasma membrane extensions and at the centrosome in which it exhibits a catalytic activity and is tightly regulated along the cell cycle. Considering both the action sites of potential effectors as identified by proteomic approach and differently targeted DsRed-Syk following the tyrosine residue mutated, we hypothesize a phosphorylation code targeting the kinase at different sub-cellular compartments depending on the tyrosine residue activated. In order to determine whether a dynamic exchange occurs between the subcellular compartments, we applied different imaging techniques on living breast cancer cells transiently expressing wild-type and mutant fluorescent Syk chimeras. Fluorescence Recovery After Photobleaching (FRAP) with DsRed-Syk and photoactivatable GFP-Syk clearly evidenced rapid exchanges at the centrosomes with a recruitment τ of 18,54 ± 3,63 sec. Treatments affecting the microtubule skeleton or the molecular motor dynein, TIRF imaging of Syk clusters, antibody co-localization, directional drift of activated PA-GFP-Syk corroborated by mathematical modelling, together show that the tubulin cytoskeleton and the microtubule motor dynein/dynactin are necessary for Syk recruitment at the centrosome
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Valenta, Hana. "Live-cell investigation of the NADPH oxidase active state using fluorescent proteins and quantitative spectro-microscopies." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASF010.
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En cellules vivantes, les interactions dynamiques entre les protéines jouent un rôle clé dans la régulation de nombreuses voies de signalisation et événements biochimiques. C’est également le cas de la NADPH oxydase (NOX) des phagocytes, qui est une enzyme majeure du système immunitaire inné. Elle génère des anions superoxyde (O₂•⁻), précurseurs d’espèces réactives de l’oxygène (ROS), qui sont essentielles dans la lutte contre les infections microbiennes. La NADPH oxydase est un complexe protéique composé de six sous-unités ; deux protéines membranaires (NOX2 et p22phox) formant le centre catalytique, trois protéines cytosoliques (p67phox, p47phox et p40phox) et une petite GTPase Rac. Le mécanisme d’activation de la NADPH oxydase est basé sur l’assemblage de toutes les sous-unités cytosoliques avec les sousunités membranaire, où les interactions protéineprotéine jouent un rôle important. Un défaut d’activité de la NADPH oxydase cause une maladie granulomateuse septique chronique (CGD) caractérisée par des infections sévères et récurrentes. En revanche, des niveaux élevés de ROS contribuent aux maladies cardiovasculaires et neurodégénératives. Ainsi, l’activité de la NADPH oxydase doit être strictement régulée. Une meilleure compréhension de la machinerie de la NADPH oxydase au niveau moléculaire aidera à identifier les aspects clés de l’activité enzymatique et donc les potentielles cibles thérapeutiques. Le but de ma thèse était d’étudier l’état actif de la NADPH oxydase en utilisant des stratégies de microscopie à fluorescence en cellules vivantes. Pour détecter les interactions protéine-protéine, nous avons exploité le phénomène de transfert résonant d’énergie de type Förster (FRET) mesuré par imagerie de durée de fluorescence (FLIM). Étant donné que le phénomène de FRET a lieu uniquement entre des fluorophores proches spatialement (< 10 nm), il est approprié pour révéler les interactions à l’échelle nanométrique entre les sous-unités de la NOX étiquetées par des protéines fluorescentes (PFs) et il fournit également des informations sur la topologie du complexe enzymatique. Les approches de FRET-FLIM ont été réalisées soit avec des sous-unités séparées, soit avec une protéine de fusion chimérique appelée "Trimère". Le Trimère est composé des domaines essentiels des protéines cytosoliques p47phox, p67phox et Rac1, permettant une activité constitutive de la NADPH oxydase dans les cellules, sans avoir besoin d’un stimulant. Dans une première étape, nous avons travaillé avec les sous-unités individuelles étiquetés par les PFs dans les cellules de type fibroblastes ou dans des modèles de phagocytes. Nous avons comparé le PMA et l’acide arachidonique en tant qu’activateurs de la NADPH oxydase en termes de cinétique d’activation et de production de ROS. Les conditions expérimentales les plus convenables ont été explorées par la microscopie TIRF, qui permet d’exciter sélectivement des fluorophores situés près de la membrane plasmique et ainsi rend possible de suivre la formation du complexe actif de la NADPH oxydase en temps réel. Dans la deuxième partie de ma thèse, nous avons utilisé majoritairement le Trimère. Les expériences FRET-FLIM ont révélé que le Trimère forme des clusters dans la membrane plasmique. L’activité continue de la NADPH oxydase incité par le Trimère a été également examinée en termes de conséquences sur la physiologie des cellules vivantes. Nous avons montré que la production continue de ROS à long terme conduit à l’acidification du pH intracellulaire et déclenche l’apoptoseIn living cells, dynamic interactions between proteins play a key role in regulating many signaling pathways and biochemical events. It is also the case of the phagocyte NADPH oxidase (NOX), a key enzyme of the innate immune system. It generates superoxide anions (O₂•⁻), precursors of reactive oxygen species (ROS), such as hydrogen peroxide or hydroxyl radical that are critical for host responses to microbial infections. The NADPH oxidase is a protein complex composed of six subunits; two membrane proteins (NOX2 and p22phox) forming the catalytic core, three cytosolic proteins (p67phox, p47phox and p40phox) and a small GTPase Rac. The sophisticated activation mechanism of the NADPH oxidase relies on the assembly of all cytosolic subunits with the membrane-bound components, whereby proteinprotein interactions play an important role. Lack of the NADPH oxidase activity leads to chronic granulomatous disease (CGD) characterized by severe and recurrent infections. On the other hand, enhanced levels of ROS contribute to cardiovascular and neurodegenerative diseases. Thus, the NADPH oxidase activity needs to be tightly regulated in order to maintain physiological levels of ROS. Understanding the NADPH oxidase machinery at the molecular level will help to identify the key aspects of its enzyme activity and thereby potential therapeutic targets. The aim of my PhD project was to investigate the active state of the NADPH oxidase in living cells using state of the art fluorescence microscopy strategies. To detect the protein-protein interactions Förster Resonance Energy Transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) was a method of choice. As the FRET phenomenon occurs only between fluorophores in close proximity (< 10 nm), it is well-suited to reveal interactions of the NADPH oxidase subunits labeled by fluorescent proteins at nanoscale level, but also it provides information about the topology of the enzyme complex. FRET-FLIM was performed either with separated NOX subunits or with a chimeric fusion protein called “Trimera”. The Trimera is composed of the essential domains of the cytosolic proteins p47phox, p67phox and Rac1, enabling constitutive, robust NADPH oxidase activity in cells without the need of a stimulant. First, we worked with the individual FP-labeled cytosolic subunits in COSNOX cells (stably expressing NOX2/p22phox subunits) or macrophages and compared PMA and arachidonic acid as activators of the NADPH oxidase in terms of the activation kinetics and the total ROS production. By introducing mutations into the p47phox and p67phox subunits we were able to modulate the oxidase activity. The final validated working conditions were explored by TIRF microscopy, an imaging method allowing selective excitation of the fluorophores situated in the vicinity of the plasma membrane, and thus enabling to monitor the realtime formation of the active NADPH oxidase complex. We also focused on NOX2, the catalytic center of the NADPH oxidase that we labeled by FPs and prepared for further FRET-FLIM experiments aiming the investigation of NOX2/cytosolic subunits interactions. Second, we employed the Trimera that acts as a single activating protein of the NADPH oxidase. FRET-FLIM experiments revealed that theFP-Trimera forms clusters in the plasma membrane. The continuous long-term NOX activity elicited by the Trimera was also examined in terms of consequences on the physiology of living cells. We showed that the sustained ROS production leads to acidification of the intracellular pH and triggers apoptosis
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A, S. Jijumon. "Systematic characterization of a large number of Microtubule-Associated Proteins using purification-free TIRF-reconstitution assays Purification of tubulin with controlled post-translational modifications by polymerization–depolymerization cycles Microtubule-Associated Proteins: Structuring the Cytoskeleton Purification of custom modified tubulin from cell lines and mouse brains by polymerization-depolymerization cycles." Thesis, université Paris-Saclay, 2021. http://www.theses.fr/2021UPASL007.
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Le cytosquelette des microtubules (MTs) est constitué de filaments dynamiques impliqués dans une multitude de fonctions telles que la division cellulaire, le maintien de forme des cellules, les battements ciliaires ou encore la différenciation neuronale. Une régulation stricte des fonctions des MTs est donc d'une grande importance pour l'homéostasie cellulaire, et toute perturbation pourrait potentiellement conduire à des maladies comme le cancer, les ciliopathies ou la neurodégénérescence. Dans un contexte cellulaire, les propriétés des MTs peuvent être contrôlées par leurs interactions avec une grande variété de protéines associées (MT-associated proteins ; MAPs). Notre connaissance de ces interacteurs s'est continuellement enrichie au cours des dernières décennies, mais il n'existe à ce jour aucune étude systématique visant à décrire et à classer ces protéines en fonction de leurs mécanismes de liaison et de leurs effets structuraux sur les MTs. Dans mon projet de thèse, j’ai mis au point un essai permettant une analyse rapide et systématique à la base des lysats clarifiés de cellules humaines surexprimant une multitude des différents MAPs. Le comportement dynamique des MT en présence d'environ 50 MAPs différentes a été imagé à l'aide de la microscopie TIRF. Cela nous permet d'étudier le comportement des MAP dans une situation proche de leur environnement naturel, mais en éliminant la complexité de l'espace intracellulaire, telle que l'encombrement par des organelles et des filaments du cytosquelette à l'intérieur de l'espace intracellulaire confiné. En effet, la plupart des MAPs étaient bien solubles dans notre approche d'extraction, tandis que les approches de purification pour plusieurs d'entre elles ont conduit à leur précipitation, rendent les expériences de reconstitution in vitro classique impossible. Ma nouvelle approche m’a permis de définir plusieurs nouvelles protéines comme de véritables MAP. J’ai montré que des MAPs non-caractérisées auparavant ont des effets étonnamment différents sur la polymérisation et la structure des MTs, créant ainsi une variété de réseaux de MT distincts. J’ai également démontré que mon approche permet d'étudier les structures des MAPs associées aux MTs par cryo-microscopie électronique, ou d'étudier le dynamique des MTs porteuses de mutations trouvées dans les pathologies humaines. J’ai également démontré que mon approche permet à tester la sensibilité des MAPs aux modifications post-traductionnelles de la tubuline, ou d'étudier le rôle des MAPs dans les interactions entre l'actine et les MTs. Mon approche expérimentale permet donc de mieux comprendre comment les MAP et les MT contrôlent ensemble le fonctionnement du cytosqueletteMicrotubules (MTs) are dynamic filaments involved in a plethora of functions such as cell division, cell shape, ciliary beating, neuronal differentiation. Strict regulation of MT functions is therefore of high importance for the cellular homeostasis, and any perturbations could potentially lead to diseases like cancer, ciliopathies and neurodegeneration. At the protein level, there are accumulating studies showing that MT properties can be controlled via interaction with a large variety of MT-associated proteins (MAPs). Our knowledge of MAPs has been enriched over time, but up to this date no systematic studies exist that aim to describe and categorize these proteins according to their binding mechanisms and structural effects on MTs. In my PhD project, I have developed an assay for rapid and systematic analysis of MAPs using cleared lysates of cultured human cells in which I overexpress a variety of different MAPs. The dynamic behaviour of growing MTs in the presence of those MAPs were imaged using TIRF microscopy. This allows me to study the behaviour of around 50 MAP candidates in a situation close to their natural environment, but eliminating complexity coming from different organelles and crammed cytoskeleton filaments inside the confined intracellular space. Indeed, most MAPs were nicely soluble in the extract approach, while purification attempts of several of them led to protein precipitation, thus making classical invitro reconstitution approaches impossible. This novel approach allowed me to compare many MAPs under similar experimental conditions, and helped to define several novel proteins as bona-fide MAPs. I demonstrate that previously uncharacterized MAPs have strikingly different effects on MT polymerization and MT structure, thus creating a variety of distinct MT arrays. I further extended this cell-free pipeline to study structures of MAPs bound to MTs by cryo-electron microscopy, or to study the MT interactions of MAPs carrying patient mutations. Finally, I demonstrated that my approach can be used to test the sensitivity of MAPs to tubulin PTMs, as well as to study the role of MAPs in actin-MT crosstalk. In the future, this novel approach will allow for a better mechanistic understanding of how MAPs and MTs together control cytoskeleton functions
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Juillot, Dimitri. "Etude du mécanisme de l'arrêt de division pendant la transformation génétique naturelle chez streptococcus pneumoniae." Electronic Thesis or Diss., université Paris-Saclay, 2023. http://www.theses.fr/2023UPASL153.
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Streptococcus pneumoniae (le pneumocoque) est une bactérie commensale de l'homme pouvant provoquer dans certaines conditions des pneumonies, des méningites ou des septicémies. Les stratégies de lutte utilisées contre le pneumocoque reposent sur l'emploi d'antibiotiques ou de vaccins. Ces deux approches se heurtent cependant à son remarquable potentiel de plasticité génétique qui dépend de sa capacité à transformer. La transformation est un mécanisme d'échange d'ADN très conservé chez les bactéries, qui contribue à leur diversification, leur évolution et leur adaptation. La capacité des bactéries à transformer est liée à un état physiologique dit de ‘compétence'. Chez le pneumocoque, la compétence se développe de façon transitoire dans toutes les cellules de la population lors de leur phase de multiplication. Elle s'accompagne d'un blocage du processus de division qui permet aux cellules de finaliser les étapes terminales de la transformation sans compromettre l'intégrité de leur génome. ComM, une protéine synthétisée pendant la compétence, apparait nécessaire et suffisante pour induire ce blocage. Le but de mon projet de thèse était de comprendre le mécanisme d'action de ComM. Pour mesurer l'impact de ComM, et plus généralement du développement de la compétence, sur la dynamique des protéines de la division, j'ai utilisé la technique de microscopie de fluorescence par réflexion totale interne (TIRF), Une autre stratégie a consisté à mettre en évidence les connexions potentielles entre les protéines de la transformation et celles de la division, par une approche double hybride. Mes résultats ont montré que le développement de la compétence ralentie la mobilité de deux protéines clés de la synthèse du septum. Par ailleurs, les expériences de double hybride ont révélé plusieurs interactions entre ComM et les protéines de la division, parmi lesquelles la protéine MurA1, impliquée dans la synthèse du précurseur du PG. Sur la base de mes résultats, je propose un modèle dans lequel ComM pourrait moduler l'activité de MurA1 pour diminuer la synthèse de PG septal. A terme, ce projet pourrait inspirer de nouvelles pistes pour bloquer la croissance du pneumocoque et donc lutter contre ce pathogène humain majeurStreptococcus pneumoniae (the pneumococcus) is a commensal bacterium that can cause pneumonia, meningitis or septicemia under certain conditions. Control of pneumococcal infections is based on the use of antibiotics or vaccines. However, both of these approaches come up against the pneumococcus remarkable potential for genetic plasticity, which depends on its ability to uptake exogenous DNA through transformation. Transformation is a highly conserved DNA exchange mechanism in bacteria that contributes to their diversification, evolution and adaptation. The ability of bacteria to transform requires the development of a specific physiological state called 'competence'. In the pneumococcus, competence develops transiently in all cells of the population during their multiplication phase. It is accompanied by an arrest of the division process, which allows the cells to complete the final stages of transformation without compromising the integrity of their genome. ComM, a protein of unknown function synthesized during competence, appears necessary and sufficient to induce this inhibition of cell division. The goal of this PhD project was to decipher the mechanism of action of ComM. To measure the impact of ComM, and more generally the development of competence, on the dynamics of cell division proteins, I used total internal reflection fluorescence (TIRF) microscopy. Another strategy was to identify potential interactions between transformation and division proteins, using a yeast two-hybrid approach. My results show that the development of competence interferes with the mobility of the two key proteins of the septal peptidoglycan synthesizing machinery FtsW and PBP2x. Furthermore, two-hybrid experiments revealed several interactions between ComM and division proteins, in particular the MurA1 protein, involved in the synthesis of the precursor of the PG. On the basis of my results, I propose a model in which ComM could modulate MurA1 activity to decrease the septal PG synthesis. Ultimately, this project could inspire new strategies to block the growth of the pneumococcus and thus fight against this major human pathogen
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Myklatun, Ahne. "Production and Application of Micronsized Polysaccharide Particles - Studying Perturbation of a Model Mucus Barrier with Total Internal Reflection Fluorescence (TIRF) Microscopy and Atomic Force Microscopy (AFM) Indentation." Thesis, Norges teknisk-naturvitenskapelige universitet, Institutt for fysikk, 2011. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-13742.
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The overall aim of this project was to produce homogeneously sized polysaccharide microparticles and apply these and similar sized particles as probes for investigation of mucin layers as a model for a biological barrier. Small polysaccharide particles have many applications, e.g. within the medical field of drug delivery. In this study a microfluidic system was developed to produce alginate beads, which can be used in drug delivery systems. Different designs, continuous phases and concentrations were tested in order to find an optimal system. Beads in the size range of 10 µm were produced using a device with T-shaped design and three inlets. An electrostatic bead generator was also used to make alginate beads, however the beads produced were too large to be used in the experiments with the mucin layers.One of the many challenges when working with drug delivery systems is the mucus barrier protecting the epithelial cells. In this study a model mucus barrier was made by immobilizing mucins, the glycoprotein responsible for the physical properties of the barrier. A procedure for fluorescence labelling of polystyrene beads with quantum dots was developed, and penetration of these beads into the model barrier was measured with total internal reflection fluorescence (TIRF) microscopy. In TIRF the excitation field intensity decays exponentially, and the emitted fluorescence intensity from the beads gives an indication of the distance between the beads and the surface. Measurements performed on mucin layers of different concentrations indicate that mucin concentrations above 0,5 mg/ml will result in a layer too thick or too dense to give a intensity signal. At mucin concentration 0,05 mg/ml fluorescence was observable in TIRF, and it was clearly weaker than for the control with a bead directly on a glass surface. This indicates that the beads hover over the surface due to the mucin layers, and show that it is in principle possible to measure the penetration depth of beads into mucin layer using TIRF. To simulate the condition in the lungs of cystic fibrosis (CF) patients, the mucin layers were incubated with alginate. Measurements were performed to see how this affected the penetration of the beads into the layer. A weaker fluorescent signal was obtained for these samples in TIRF, which suggests that there has been interaction between mucin and alginate. It was in addition investigated how different concentrations of G-blocks in the solution affected the penetration into the mucin-alginate layer. These testes were carried out using both TIRF and atomic force microscopy (AFM) nanoindentation experiments. The TIRF measurements were inconclusive, while the nanoindentation experiments showed decreased interaction between mucin-alginate layer and a bead.
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Brüning, Dennis [Verfasser], Ingo [Akademischer Betreuer] Rustenbeck, and Simone [Akademischer Betreuer] Baltrusch. "TIRF-Mikroskopische Untersuchungen der Insulin-Granula und des Aktin-Zytoskeletts in primären Beta-Zellen und Untersuchungen des zellulären Energiestoffwechsels mit PercevalHR / Dennis Brüning ; Ingo Rustenbeck, Simone Baltrusch." Braunschweig : Technische Universität Braunschweig, 2019. http://d-nb.info/1201818826/34.
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Luo, Yi. "Nucleosome Regulation of Transcription Factor Binding Dynamics: a Single-molecule Study." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1449093157.
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Loe, Ashley M. "TOWARDS AN UNDERSTANDING OF PHARMACOLOGICALLY INDUCED INTRACELLULAR CHANGES IN NICOTINIC ACETYLCHOLINE RECEPTORS: A FLUORESCENCE MICROSCOPY APPROACH." UKnowledge, 2016. http://uknowledge.uky.edu/chemistry_etds/69.
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Upregulation of nicotinic acetylcholine receptors (nAChRs) is a well-documented response to chronic nicotine exposure. Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels consisting of alpha (α2-10) and beta (β2-4) subunits. Nicotine, an agonist of nAChRs, alters trafficking and assembly of some subtypes of nAChRs, leading to an increase in expression of high sensitivity receptors on the plasma membrane. These physiological changes in nAChRs are believed to contribute to nicotine addiction, although the mechanism of these processes has not been resolved. Recently, many studies have converged on the idea that nicotine induces upregulation by an intracellular mechanism. In this dissertation, expression levels of nAChRs were quantified upon exposure to nicotine and its primary metabolite, cotinine. A pH sensitive variant of GFP, super ecliptic pHluorin (SEP), was integrated with a nAChR subunit to study expression and trafficking of nAChRs by differentiating intracellular and plasma membrane inserted receptors. In this work, cotinine is shown to increase the number of α4β2 nAChRs within a cell. Cotinine also affects trafficking of α4β2, evident by a redistribution of intracellular receptors and an increase in single vesicle insertion events on the plasma membrane. This work shows both nicotine and cotinine alter the overall assembly of α4β2 to favor the high sensitivity (α4)2(β2)3 version. Since cotinine and nicotine induce similar physiological changes in nAChRs, the metabolite potentially plays a role in the mechanism of nicotine addiction.
Although an intracellular mechanism for upregulation has been supported, a shift in assembly to the high sensitivity (α4)2(β2)3 version exclusively in the endoplasmic reticulum has not previously been detected. In order to study organelle specific changes in stoichiometry, a novel method was developed to isolate single nAChRs in nanovesicles derived from native cell membranes. Separation of nanovesicles originating from the endoplasmic reticulum and plasma membrane, encompassing isolated nAChRs, allows precise changes in stoichiometry to be monitored in subcellular regions. In this work, single molecule bleaching steps of green fluorescent protein (GFP) encoded in each alpha subunit of the pentamer are detected. The number of bleaching steps, or transitions to a nonfluorescent state upon continuous excitation, corresponds to the number of GFP-labeled alpha subunits present. Therefore, the stoichiometry can be deduced by detection of two bleaching steps, as in (α4)2(β2)3, or three bleaching steps, seen in (α4)3(β2)2. Using this method on isolated nAChRs, a shift to assembly of high sensitivity (α4)2(β2)3 receptors is detected definitively within the endoplasmic reticulum. In addition, an increase in (α4)2(β2)3 receptors located on the plasma membrane is shown when nicotine is present. This work provides convincing evidence that nicotine acts intracellularly, within the endoplasmic reticulum, to alter stoichiometry of nAChRs.
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Zahedi, Shadi. "Are Mitochondria a Potential Target for Anti-Cancer Therapy in Carcinoid Tumors?" University of Toledo Health Science Campus / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=mco1280427079.
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Zhang, Zhihui. "Assembly and Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator and Associated Proteins." UKnowledge, 2018. https://uknowledge.uky.edu/chemistry_etds/101.
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Cystic Fibrosis (CF) is an autosomal recessive genetic disease that leads to severe malfunction in many organs, but particularly the lungs. The primary cause of this malfunction is the decrease of the airway surface liquid layer on the lung epithelium. The lack of hydration leads to mucus build up on the epithelial lining, leading to blockage of airways. The underlying cause of CF is the dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), which results from mutations in the protein. Almost 90% of CF patients are caused by the deletion of the phenylalanine at position 508 of CFTR, which is believed to affect the folding and stability of CFTR. The misfolded ΔF508-CFTR undergoes ER associated degradation (ERAD), causing the failure of ΔF508-CFTR trafficking to the cell surface. Small molecule correctors yield moderate improvements in the trafficking of ΔF508-CFTR to the plasma membrane. It is currently not known if correctors increase trafficking through improved cargo loading of transport vesicles or through direct binding to CFTR. In this dissertation, real-time measurements of trafficking were utilized to identify the mechanistic details of chemical, biochemical, and thermal factors that impact CFTR correction, using the corrector molecule VX-809, a secondary mutation (I539T), and low temperature conditions. Each individually improved trafficking of ΔF508-CFTR to approximately 10% of wild-type levels. The combination of VX-809 with either low temperature or the I539T mutation increased the amount of CFTR on the plasma membrane to nearly 40%, indicating synergistic activity. The number of vesicles reaching the surface was significantly altered; however the amount of channel in each vesicle remained the same. Therefore, a 2 step therapeutic approach might be an ideal treatment for CF. The first step would be composed of a compound that mimics the mechanism of stabilization provided by low temperature or the I539T mutation, while the second step would be VX-809 or a similar corrector compound. These studies suggest that understanding how low temperature and second site suppressors alter ΔF508-CFTR could be key to the development of future therapeutics for the effective treatment of CF.
The precise pathophysiology of cystic fibrosis is not well studied. The involvement of another transport protein, epithelial sodium channel (ENaC), makes the situation more complicated. ENaC and CFTR are colocalized on the apical surface of epithelia cells. With our fluorescence microscopy techniques, we explored the effects of CFTR on the residence time of ENaC on the cell membrane. A reliable approach measuring the half-life of protein on the cell membrane is required for this study. We present a new approach to quantify the half-life of membrane proteins on the cell surface, through tagging the protein with the photoconvertible fluorescent protein, Dendra2. Total internal reflection fluorescence microscopy (TIRF) is applied to limit visualization of fluorescence to proteins located on the plasma membrane. Photoconversion of Dendra2 works as a pulse chase experiment by monitoring only the population of protein that has been photoconverted. As the protein is endocytosed the red emission decreases due to the protein leaving the TIRF field of view. The half-life of the protein on the plasma membrane was calculated upon imaging over time and quantifying the change in red fluorescence. Our method provides a unique opportunity to observe real-time protein turnover at the single cell level without addition of protein synthesis inhibitors. This technique will be valuable for the future protein half-life study.
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Xu, Zhen. "Auto-inhibition mechanism of the guanine nucleotide exchange factor Tiam1." Diss., University of Iowa, 2016. https://ir.uiowa.edu/etd/5880.
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The Rho family of guanosine triphosphatases (GTPases) function as binary molecular switches, which play an important role in the regulation of actin cytoskeleton rearrangement and are involved in several critical cellular processes including cell adhesion, division and migration. Rho GTPases are specifically activated by their associated guanine nucleotide exchange factors (RhoGEFs). Dysregulation of RhoGEFs function through mutation or overexpression has been implicated in oncogenic transformation of cells and linked to several kinds of invasive and metastatic forms of cancer. T-cell lymphoma invasion and metastasis 1 (Tiam1) is a multi-domain Dbl family GEF protein and specifically activates Rho GTPase Rac1 through the catalytic Dbl homology and Pleckstrin homology (DH-PH) bi-domain. Previous works have shown that the nucleotide exchange function of the full-length Tiam1 is auto-inhibited and can be activated by N-terminal truncation, phosphorylation and protein-protein interactions. However, the molecular mechanisms of Tiam1 GEF auto-inhibition and activation have not yet been determined. In this study, the N-terminal PH-CC-Ex domain of Tiam1 is shown to directly inhibit the GEF function of the catalytic DH-PH domain in vitro. Using fluorescencebased kinetics experiments, we demonstrate that the auto-inhibition of Tiam1 GEF function occurs by a competitive inhibition model. In this model, the maximum velocity of catalytic activity remains unchanged, but the Michaelis-Menten constant of the auto-inhibited Tiam1 (the PH-PH fragment) on the substrate Rac1 is increased compared to the activated Tiam1 (the catalytic DH-PH domain alone). Through small angle X-ray scattering (SAXS), the structure of auto-inhibited Tiam1 (the PH-PH fragment) is shown to form a closed conformation in which the catalytic DH-PH domain is blocked by the N-terminal PH-CC-Ex domain. Taken together, these findings demonstrate the molecular mechanism of Tiam1 GEF autoinhibition in which the PH-CC-Ex domain of Tiam1 inhibits its GEF function by preventing the substrate Rho GTPase Rac1 from accessing the catalytic DH-PH bi-domain.
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Brehove, Matthew Steven. "Access to the Genome: A Study of Transcription Factor Binding Within Nucleosomes." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1480603783786784.
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Schneider, René. "A novel parabolic prism-type TIR microscope to study gold nanoparticle-loaded kinesin-1 motors with nanometer precision." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-110212.
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Movement of motor proteins along cytoskeletal filaments is fundamental for various cellular processes ranging from muscle contraction over cell division and flagellar movement to intracellular transport. Not surprisingly, the impairment of motility was shown to cause severe diseases. For example, a link between impaired intracellular transport and neurodegenerative diseases, such as Alzheimer’s, has been established. There, the movement of kinesin-1, a neuronal motor protein transporting vesicles along microtubules toward the axonal terminal, is thought to be strongly affected by roadblocks leading to malfunction and death of the nerve cell. Detailed information on how the motility of kinesin-1 deteriorates in the presence of roadblocks and whether the motor has a mechanism to circumvent such obstructions is scarce. In this thesis, kinesin-1 motility was studied in vitro in the presence of rigor kinesin-1 mutants, which served as permanent roadblocks, under controlled single-molecule conditions.
The 25 nm wide microtubule track, consisting of 13 individual protofilaments, resembles a multi-lane environment for transport by processive kinesin-1 motors. The existence of multiple traffic-lanes, allows kinesin-1 to utilize different paths for cargo transport and potentially also for the circumvention of roadblocks. However, direct observation of motor encounters with roadblocks has been intricate in the past, mainly due to limitations in both, spatial and temporal resolution. These limitations, intrinsic to fluorescent probes commonly utilized to report on the motor positions, originate from a low rate of photon generation (low brightness) and a limited photostability (short observation time). Thus, studying kinesin-1 encounters with microtubule-associated roadblocks requires alternative labels, which explicitly avoid the shortcomings of fluorescence and consequently allow for a higher localization precision.
Promising candidates for replacing fluorescent dyes are gold nanoparticles (AuNPs), which offer an enormous scattering cross-section due to plasmon resonance in the visible part of the optical spectrum.
Problematic, however, is their incorporation into conventionally used (fluorescence) microscopes, because illumination and scattered light have the same wavelength and cannot be separated spectrally. Therefore, an approach based on total internal reflection (TIR) utilizing a novel parabolically shaped quartz prism for illumination was developed within this thesis. This approach provided homogenous and spatially invariant illumination profiles in combination with a convenient control over a wide range of illumination angles. Moreover, single-molecule fluorescence as well as single-particle scattering were detectable with high signal-to-noise ratios. Importantly, AuNPs with a diameter of 40 nm provided sub-nanometer localization accuracies within millisecond integration times and reliably reported on the characteristic 8 nm stepping of individual kinesin-1 motors moving along microtubules. These results highlight the potential of AuNPs to replace fluorescent probes in future single-molecule experiments. The newly developed parabolic prism-type TIR microscope is expected to strongly facilitate such approaches in the future.
To study how the motility of kinesin-1 is affected by permanent roadblocks on the microtubule lattice, first, conventional objective-type TIRF microscopy was applied to GFP-labeled motors. An increasing density of roadblocks caused the mean velocity, run length, and dwell time to decrease exponentially. This is explained by (i) the kinesin-1 motors showing extended pausing phases when confronted with a roadblock and (ii) the roadblocks causing a reduction in the free path of the motors. Furthermore, kinesin-1 was found to be highly sensitive to the crowdedness of microtubules as a roadblock decoration as low as 1 % sufficed to significantly reduce the landing rate.
To study events, where kinesin-1 molecules continued their runs after having paused in front of a roadblock, AuNPs were loaded onto the tails of the motors. When observing the kinesin-1 motors with nanometer-precision, it was interestingly found that about 60 % of the runs continued by movements to the side, with the left and right direction being equally likely. This finding suggests that kinesin-1 is able to reach to a neighboring protofilament in order to ensure ongoing transportation. In the absence of roadblocks, individual kinesin-1 motors stepped sideward with a much lower, but non-vanishing probability (0.2 % per step). These findings suggest that processive motor proteins may possess an intrinsic side stepping mechanism, potentially optimized by evolution for their specific intracellular tasks.
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Köster, Darius Vasco. "Role of Caveolae in Membrane Tension." Doctoral thesis, Universitätsbibliothek Leipzig, 2010. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-63103.
Full textAbstract:
Caveolae sind charakteristische Plasmamembraneinstülpungen, die in vielen Zelltypen vorkommen und deren biologische Funktion umstritten ist. Ihre besondere Form und ihre Häu gkeit in Zellen, die stets mechanischen Belastungen ausgesetzt sind, führten zu der Annahme, dass Caveolae die Plasmamembran vor mechanischen Belastungen schützen und als Membranreservoir dienen. Dies sollte mit dieser Dissertation experimentell geprüft werden. Zunächst wurde der Ein uss der Caveolae auf die Membranspannung von Zellen im Normalzustand untersucht. Dann wurden die Zellen mechanisch belastet. Mit Fluoreszensmikroskopie wurde das Verschwinden von Caveolae nach Strecken der Zellen oder nach einem hypo-osmotischen Schock beobachtet. Messungen der Membranspannung vor und unmittelbar nach dem hypo-osmotischem Schock zeigten, dass Caveolae einen Anstieg der Membranspannung verhindern, unabhängig von ATP und dem Cytoskelett. Die Erzeugung von Membranvesikel mit Caveolae erlaubte es, diesen Effekt der Caveolae in einem vereinfachten Membransystem zu beobachten. Schliesslich wurden Muskelzellen untersucht. Zellen, die genetisch bedingt weniger Caveolae haben und mit Muskelschwundkrankheiten in Verbingung stehen, waren mechanisch weniger belastbar als gesunde Zellen. Zusammenfassend wird mit dieser Dissertation die These bestärkt, dass Caveolae einem Anstieg der Membranspannungen entgegenwirken. Dass dies in Zellen und in Vesikeln unabhängig von Energie und Cytoskelett geschieht, lässt auf einen passiven, mechanisch getriebenen Prozess schliessen. Diese Erkenntnis trägt zum Verständnis der Rolle von Caveolae in Zellen bei und kann dem besseren Verständnis von Krankheiten bedingt durch Caveolin-Mutationen, wie z.B. Muskelschwundkrankheiten, dienenCaveolae, the characteristic plasma membrane invaginations present in many cells, have been associated with numerous functions that still remain debated. Taking into account the particular abundance of caveolae in cells experiencing mechanical stress, it was proposed that caveolae constitute a membrane reservoir and bu er the membrane tension upon mechanical stress. The present work aimed to check this proposition experimentally. First, the in uence of caveolae on the membrane tension was studied on mouse lung endothelial cells in resting conditions using tether extraction with optically trapped beads. Second, experiments on cells upon acute mechanical stress showed that caveolae serve as a membrane reservoir bu ering surges in membrane tension in their immediate, ATP- and cytoskeleton-independent attening and disassembly. Third, caveolae incorporated in membrane vesicles also showed the tension bu ering. Finally, in a physiologically more relevant case, human muscle cells were studied, and it was shown that mutations with impaired caveolae which are described in muscular dystrophies render muscle cells less resistant to mechanical stress. In Summary the present work provides experimental evidence for the hypothesis that caveolae bu er the membrane tension upon mechanical stress. The fact that this was observed in cells and membrane vesicles in an ATP and cytoskeleton independent manner reveals a passive, mechanically driven process. This could be a leap forward in the comprehension of the role of caveolae in the cell, and in the understanding of genetic diseases like muscular dystrophies
Cavéoles sont des invaginations caractéristiques de la membrane plas- mique présents dans beaucoup de types cellulaires. Ils sont liées à plusieurs fonctions cellulaires, ce qui sont encore débattues. Prenant compte de l importance des cavéoles dans les cellules soumises au stress mécanique, les cavéoles sont proposées de constituer un réservoir membranaire et de tamponner la tension membranaire pendant des stresses mécaniques. Cette étude a eu le but de tester cette hypothèse expérimentalement. En premier, l in uence des cavéoles sur la tension membranaire au repos a été étudiée sur des cellules endothéliales du poumon de la souris. Puis, on a montré que les cavéoles tamponnent l augmentation de la tension membranaire après l application d un stress mécanique. En suite, la réalisation des vésicules membranaires contenant des cavéoles a permit de montrer leur rôle comme réservoir membranaire dans un système simpli é. Finalement, dans un contexte physiologiquement plus relevant, l étude des cellules musculaires a montrée que les mutations du cavéolin associées aux dystrophies musculaires rendent les cellules moins résistante aux stresses mécaniques. En conclusion, cette étude supporte l\'hypothèse que les cavéoles tamponnent la tension membranaire pendant des stresses mécaniques. Le fait que cela se passe dans les cellules et les vésicules indépendamment d ATP et du cytosquelette révèlent un processus passif et mécanique. Cela pourrait servir à une meilleure compréhension du rôle des cavéoles dans la cellule et les maladies génétiques comme les dystrophies musculaires
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Blandin, Pierre. "Développement instrumental pour la microscopie de fluorescence résolue en temps : applications biomédicales." Phd thesis, Université Paris Sud - Paris XI, 2008. http://tel.archives-ouvertes.fr/tel-00376116.
Full textAbstract:
Pour certaines problématiques biomédicales, notamment en neurobiologie, l'imagerie des échantillons exige des résolutions spatiale et temporelle élevées, et seules des techniques de microscopie optique innovantes vont permettre d'imager la cellule dans des conditions physiologiques. Pour répondre à ces contraintes nous avons développé en intégralité un dispositif de microscopie de fluorescence en réflexion totale interne et résolue en temps. En effet, l'imagerie de fluorescence résolue en temps (FLIM) permet, en complément des informations de localisation et de topologie apportées par la microscopie conventionnelle, une analyse dynamique de processus moléculaires et métaboliques au sein des cellules. Associée à la technique de microscopie en réflexion totale interne, qui confère au système d'imagerie une résolution axiale sub-longueur d'onde tout en acquérant des images en champ large, la technique FLIM permet d'analyser l'activité membranaire des cellules en s'affranchissant de la fluorescence des autres entités de la cellule.Ce travail s'est articulé autour de trois axes principaux : l'étude et le développement d'un dispositif d'excitation de la fluorescence basé sur un oscillateur laser solide picoseconde dont le spectre est élargi par effets non linéaires dans des fibres optiques microstructurées ; le développement et la caractérisation d'un dispositif de microscopie de fluorescence par onde évanescente (TIRF) couplé à une détection résolue en temps en plein champ ; et l'application de ce dispositif à l'étude d'un précurseur membranaire du peptide amyloïde impliqué dans la maladie d'Alzheimer.
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Denoyelle, Quentin. "Theoretical and Numerical Analysis of Super-Resolution Without Grid." Thesis, Paris Sciences et Lettres (ComUE), 2018. http://www.theses.fr/2018PSLED030/document.
Full textAbstract:
Cette thèse porte sur l'utilisation du BLASSO, un problème d'optimisation convexe en dimension infinie généralisant le LASSO aux mesures, pour la super-résolution de sources ponctuelles. Nous montrons d'abord que la stabilité du support des solutions, pour N sources se regroupant, est contrôlée par un objet appelé pré-certificat aux 2N-1 dérivées nulles. Quand ce pré-certificat est non dégénéré, dans un régime de petit bruit dont la taille est contrôlée par la distance minimale séparant les sources, le BLASSO reconstruit exactement le support de la mesure initiale. Nous proposons ensuite l'algorithme Sliding Frank-Wolfe, une variante de l'algorithme de Frank-Wolfe avec déplacement continu des amplitudes et des positions, qui résout le BLASSO. Sous de faibles hypothèses, cet algorithme converge en un nombre fini d'itérations. Nous utilisons cet algorithme pour un problème 3D de microscopie par fluorescence en comparant trois modèles construits à partir des techniques PALM/STORMThis thesis studies the noisy sparse spikes super-resolution problem for positive measures using the BLASSO, an infinite dimensional convex optimization problem generalizing the LASSO to measures. First, we show that the support stability of the BLASSO for N clustered spikes is governed by an object called the (2N-1)-vanishing derivatives pre-certificate. When it is non-degenerate, solving the BLASSO leads to exact support recovery of the initial measure, in a low noise regime whose size is controlled by the minimal separation distance of the spikes. In a second part, we propose the Sliding Frank-Wolfe algorithm, based on the Frank-Wolfe algorithm with an added step moving continuously the amplitudes and positions of the spikes, that solves the BLASSO. We show that, under mild assumptions, it converges in a finite number of iterations. We apply this algorithm to the 3D fluorescent microscopy problem by comparing three models based on the PALM/STORM technics
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Ramírez, Espinoza Jesica Ivonne. "Evaluación económica para el establecimiento de una engorda de bovinos en corral en el sur del Estado de México." Tesis de Licenciatura, Universidad Autónoma del Estado de México, 2018. http://hdl.handle.net/20.500.11799/94304.
Full textAbstract:
La evaluación de proyectos es una alternativa viable que reduce el riesgo de una inversión. El objetivo de este trabajo fue evaluar una propuesta de inversión, sin endeudamiento con terceros, para una engorda de bovinos en corral, en una región del sur del Estado de México en 2017, a través de la metodología de formulación y evaluación de proyectos bajo certidumbre. Los resultados indicaron la existencia de viabilidad comercial, técnica y financiera. Durante un horizonte de cinco años y una tasa de rentabilidad mínima aceptable de 12%, el proyecto se paga y genera un VAN de $279,398, una TIR de 18.20% y una TIRM (Tasa Interna de Retorno Modificada) de 16.60%. La inversión se recupera en 4.70 años. Se concluye que con el valor de los indicadores de rentabilidad, el proyecto es rentable, tiene cierto riesgo de incurrir en pérdidas, sin embargo se sugiere tomar la decisión de inversión.
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Morten, Michael J. "Developing novel single molecule analyses of the single-stranded DNA binding protein from Sulfolobus solfataricus." Thesis, University of St Andrews, 2015. http://hdl.handle.net/10023/7568.
Full textAbstract:
Single-stranded DNA binding proteins (SSB) bind to single-stranded DNA (ssDNA) that is generated by molecular machines such as helicases and polymerases. SSBs play crucial roles in DNA translation, replication and repair and their importance is demonstrated by their inclusion across all domains of life. The homotetrameric E. coli SSB and the heterotrimeric human RPA demonstrate how SSBs can vary structurally, but all fulfil their roles by employing oligonucleotide/oligosaccharide binding (OB) folds. Nucleofilaments of SSB proteins bound to ssDNA sequester the ssDNA strands, and in doing so protect exposed bases, keep the ssDNA in conformations favoured by other proteins that metabolise DNA and also recruit other proteins to bind to ssDNA. This thesis focuses on the SSB from the archaeon S. solfataricus (SsoSSB), and has found SsoSSB to be a monomer that binds cooperatively to ssDNA with a binding site size of 4-5 nucleotides. Tagging ssDNA and SsoSSB with fluorescent labels allowed the real time observation of single molecule interactions during the initial nucleation event and subsequent binding of an adjacent SsoSSB monomer. This was achieved by interpreting fluorescent traces that have recorded combinations of FRET, protein induced fluorescent enhancement (PIFE) and quenching events. This novel analysis gave precise measurements of the dynamics of the first and second monomers binding to ssDNA, which allowed affinity and cooperativity constants to be quantified for this important molecular process. SsoSSB was also found to have a similar affinity for RNA, demonstrating a promiscuity not found in other SSBs and suggesting further roles for SsoSSB in the cell - possibly exploiting its capacity to protect nucleic acids from degradation. The extreme temperatures that S. solfataricus experiences and the strength of the interaction with ssDNA and RNA make exploring the application of SsoSSB for industrial uses an interesting prospect; and its rare monomeric structure provides an opportunity to investigate the action of OB folds in a more isolated environment than in higher order structures.
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Pérez, González Daniel Cibrán. "Single-molecule studies of nucleic acid folding and nucleic acid-protein interactions." Thesis, University of St Andrews, 2017. http://hdl.handle.net/10023/12039.
Full textAbstract:
Nucleic acids and proteins, some of the building blocks of life, are not static structures but highly dynamic entities that need to interact with one another to meet cellular demands. The work presented in this thesis focuses on the application of highly sensitive fluorescence methods, both at ensemble and single-molecule level, to determine the dynamics and structure of specific biomolecular interactions with nanometer resolution and in temporal scales from nanoseconds to minutes, which includes most biologically relevant processes. The main aims of my PhD can be classified in three areas: i) exploring new fluorescent sensors with increased specificity for certain nucleic acid structures; ii) understanding how some of these nucleic acids sense the presence of small molecules in the cellular environment and trigger gene regulation by altering their structure; and iii) understanding how certain molecular machines, such as helicase proteins, are able to unwind the DNA double helix by using chemical energy in the form of ATP hydrolysis.