Дисертації з теми "Single Particle Tracking (SPT"
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1
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.
Анотація:
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.
2
Mawoussi, Kodjo. "Effet de l'encombrement des protéines sur la diffusion des lipides et des protéines membranaires." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066541/document.
Анотація:
La diffusion latérale des lipides et des protéines transmembranaires est essentielle pour les fonctions biologiques. Dans le contexte cellulaire, la fraction surfacique des protéines membranaires est élevée, atteignant environ de 50 à 70% selon le type de membrane. La diffusion se fait donc dans un milieu très encombré. Le but de ce travail est d'étudier in vitro l'effet de l'encombrement des protéines sur la diffusion des protéines et des lipides. Jusqu'à présent, les mesures de diffusion latérale ont généralement été réalisées à faible densité de protéines, et l'effet de l'encombrement des inclusions membranaires ou des protéines membranaires a été peu étudié expérimentalement. Nous avons utilisé une méthode de suivi de particules uniques (SPT) pour suivre les trajectoires de la pompe à protons Bactériorhodopsine (BR) et de lipides marqués avec des quantum dots au bas de vésicules unilamellaires géantes (GUVs) en fonction de la fraction de surface totale (Ф) de BR reconstituée dans la membrane constituée par ailleurs de 1,2-Dioleoyl-sn-glycéro-3-phosphocholine (DOPC)Lateral diffusion of lipids and transmembrane proteins is essential for biological functions. In the cellular context, the surface fraction of membrane proteins is high, reaching approximately 50 to 70% depending on the membrane type. Therefore, diffusion occurs in a very crowded environment. The aim of this work is to study in vitro the effect of protein crowding on their own diffusion and on those of the surrounding lipids. So far, lateral diffusion measurements generally have been carried out at low protein density, and the effect of proteins crowding has not been much studied experimentally. We used a single particle tracking (SPT) method to track the trajectories of the Bacterorhodopsin (BR) proton pump and of lipids labeled with quantum dots at the bottom of giant unilamellar vesicles (GUVs) as a function of the total surface fraction (Ф) of BR reconstituted in 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane
3
Mereghetti, Alessio. "Performance evaluation of the SPS scraping system in view of the high luminosity LHC." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/performance-evaluation-of-the-sps-scraping-system-in-view-of-the-high-luminosity-lhc(600579c0-0877-415d-bf8d-32896497b5ff).html.
Анотація:
Injection in the LHC is a delicate moment, since the LHC collimation system cannot offer adequate protection during beam transfer. For this reason, a complex chain of injection protection devices has been put in place. Among them, the SPS scrapers are the multi-turn cleaning system installed in the SPS aimed at halo removal immediately before injection in the LHC. The upgrade in luminosity of the LHC foresees beams brighter than those currently available in machine, posing serious problems to the performance of the existing injection protection systems. In particular, the integrity of beam-intercepting devices is challenged by unprecedented beam parameters, leading to interactions potentially destructive. In this context, a new design of scrapers has been proposed, aimed at improved robustness and performance. This thesis compares the two scraping systems, i.e. the existing one and the one proposed for upgrade. Unlike any other collimation system for regular halo cleaning, both are "fast" systems, characterised by the variation of the relative distance between the beam and the absorbing medium during cleaning, which enhances the challenge on energy deposition values. Assets/liabilities of the two systems are highlighted by means of numerical simulations and discussed, with particular emphasis on energy deposition in the absorbing medium, time evolution of the beam current during scraping and losses in the machine. Advantages of the system proposed for upgrade over the existing one are highlighted. The analysis of the existing system takes into account present operational conditions and addresses the sensitivity to settings previously not considered, updating and extending past studies. The work carried out on the upgraded system represents the first extensive characterisation of a multi-turn cleaning system based on a magnetic bump. Results have been obtained with the Fluka-SixTrack coupling, developed during this PhD activity from its initial version to being a state-of-art tracking tool for cleaning studies in circular machines. Relevant contributions to the development involve the handling of time-varying impact conditions. An extensive benchmark against a test of the scraper blades with beam has been carried out, to verify the reliability of results. Effcts induced in the tested blades confirm the high values of energy deposition predicted by the simulation. Moreover, the comparison with the time profile of the beam intensity measured during scraping allowed the reconstruction of the actual settings of the blades during the test. Finally, the good agreement of the quantitative benchmark against readouts of beam loss monitors finally proves the quality of the analyses and the maturity of the coupling.
4
Relich, Peter Kristopher II. "Single Particle Tracking| Analysis Techniques for Live Cell Nanoscopy." Thesis, The University of New Mexico, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10251887.
Анотація:
Single molecule experiments are a set of experiments designed specifically to study the properties of individual molecules. It has only been in the last three decades where single molecule experiments have been applied to the life sciences; where they have been successfully implemented in systems biology for probing the behaviors of sub-cellular mechanisms. The advent and growth of super-resolution techniques in single molecule experiments has made the fundamental behaviors of light and the associated nano-probes a necessary concern amongst life scientists wishing to advance the state of human knowledge in biology. This dissertation disseminates some of the practices learned in experimental live cell microscopy. The topic of single particle tracking is addressed here in a format that is designed for the physicist who embarks upon single molecule studies. Specifically, the focus is on the necessary procedures to generate single particle tracking analysis techniques that can be implemented to answer biological questions. These analysis techniques range from designing and testing a particle tracking algorithm to inferring model parameters once an image has been processed. The intellectual contributions of the author include the techniques in diffusion estimation, localization filtering, and trajectory associations for tracking which will all be discussed in detail in later chapters. The author of this thesis has also contributed to the software development of automated gain calibration, live cell particle simulations, and various single particle tracking packages. Future work includes further evaluation of this laboratory's single particle tracking software, entropy based approaches towards hypothesis validations, and the uncertainty quantification of gain calibration.
5
Sanamrad, Arash. "Biological Insights from Single-Particle Tracking in Living Cells." Doctoral thesis, Uppsala universitet, Beräknings- och systembiologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-229342.
Анотація:
Single-particle tracking is a technique that allows for quantitative analysis of the localization and movement of particles. In this technique, trajectories are constructed by determining and connecting the positions of individual particles from consecutive images. Recent advances have made it possible to track hundreds of particles in an individual cell by labeling the particles of interest with photoactivatable or photoconvertible fluorescent proteins and tracking one or a few at a time. Single-particle tracking can be used to study the diffusion of particles. Here, we use intracellular single-particle tracking and trajectory simulations to study the diffusion of the fluorescent protein mEos2 in living Escherichia coli cells. Our data are consistent with a simple model in which mEos2 diffuses normally at 13 µm2 s−1 in the E. coli cytoplasm. Our approach can be used to study the diffusion of intracellular particles that can be labeled with mEos2 and are present at high copy numbers. Single-particle tracking can also be used to determine whether an individual particle is bound or free if the free particle diffuses significantly faster than its binding targets and remains bound or free for a long time. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that, unlike bound subunits, free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with co-transcriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.
6
Guerrier, Mark Paul. "The development and evaluation of phosphorescent particle tracking." Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.324887.
7
Woringer, Maxime. "Tools to analyze single-particle tracking data in mammalian cells." Electronic Thesis or Diss., Sorbonne université, 2019. http://www.theses.fr/2019SORUS419.
Анотація:
Ce travail présente des outils pour analyser la régulation de la transcription dans les cellules eucaryotes, en particulier pour le suivi de facteurs de transcription (TF) individuels dans les cellules de mammifères. Un noyau de cellule eucaryote est complexe et contient de nombreuses molécules (ADN, ARN, protéines, ATP, etc). Ces molécules interagissent avec des TF et influencent la transcription. Certaines de ces interactions peuvent être étudiées par des techniques de biochimie. La plupart, en particulier les interactions faibles, non covalentes, sont invisibles par ces méthodes. La microscopie de cellules vivantes et le suivi de molécules uniques (SPT en anglais) sont de plus en plus utilisées pour étudier ces phénomènes. L'inférence des paramètres biophysiques d'un facteur de transcription, par exemple son coefficient de diffusion, son nombre de sous-populations ou son temps de résidence sur l'ADN sont cruciaux pour comprendre sa dynamique et son influence sur la transcription. Des outils validés et précis sont donc nécessaires pour analyser les données de SPT. Pour être utile, un outil de SPT doit être non seulement validé, mais aussi accessible à des non-programmeurs. Ils doivent aussi tenir compte des biais expérimentaux présents dans les données. Nous proposons un outil d'analyse de SPT, qui se fonde sur l'estimation du propagateur de la diffusion. Ce outil a été validé et est accessible par une interface web. Nous avons montré qu'il donne des résultats proches de l'état de l'art. Il a été testé dans deux cadres : (1) l'étude de la diffusion augmentée par la catalyse enzymatique in vitro et (2) l'analyse de la dynamique du TF c-Myc dans des cellules de mammifèresThis work aims at providing tools to dissect the regulation of transcription in eukaryotic cells, with a focus on single-particle tracking of transcription factors in mammalian cells. The nucleus of an eukeryotic cell is an extremely complex medium, that contains a high concentration of macromolecules (DNA, RNA, proteins) and other small molecules (ATP, etc). How these molecules interact with transcription factors, and thus influence transcription rates is an area of intense investigations. Although some of these interactions can be captured by regular biochemistry, many of them, including weak, non-covalent interactions remain undetected by these methods. Live-cell imaging and single-particle tracking (SPT) techniques are increasingly used to characterize such effects. The inference of biophysical parameters of a given transcription factor (TF), such as its diffusion constant, the number of subpopulations or its residence time on DNA, are crucial to understanding how TF dynamics and transcription intertwine. Accurate and validated SPT analysis tools are needed. To be used by the community, SPT tools should not only be carefully validated, but also be easily accessible to non-programmers. They should also be designed to take into account known biases of the imaging techniques. In this work, we first propose a tool, accessible through a web interface, based on the modeling of the diffusion propagator. We validate it extensively and show that it exhibits state-of-the art performance. We apply this tool to two experimental settings: (1) the study of catalysis-enhanced diffusion in-vitro and (2) the analysis of the dynamics of the c-Myc transcription factor in mammalian cells
8
Piette, Nathalie. "Micropatterning subcellulaire pour étudier la connectivité neuronale." Electronic Thesis or Diss., Bordeaux, 2024. http://www.theses.fr/2024BORD0034.
Анотація:
L'impression protéique a initialement été utilisée pour reproduire et comprendre l’influence de la matrice extracellulaire sur les cellules et certains de leurs composants. Au cours de la dernière décennie, l'impression subcellulaire s’est développée, permettant d’étudier les interactions protéiques et leur rôle dans les voies de signalisation ainsi que dans la formation de synapses, immunologiques ou neuronales.La connexion synaptique est médiée par les protéines d’adhésion synaptique présentes de chaque côté de la synapse. En raison de la complexité de l’environnement synaptique mais également du manque de modèle in vitro permettant d’étudier la connexion synaptique dans un environnement biomimétique et contrôlé, les rôles exacts de ces protéines dans la synaptogénèse restent encore incertains. L’impression protéique subcellulaire est une solution potentielle pour combler ce manque. Pour cela, nous avons développé deux modèles biomimétiques basés sur l’impression protéique : un premier, utilisant des cellules hétérologues, permettant d’obtenir des informations sur la cinétique d’interaction des couples protéiques et ainsi de lier cela à leur fonction potentielle. Et un deuxième, utilisant des neurones primaires hippocampique, permettant de former des synapses artificielles pour étudier la nano-organisation de la synapse au cours du développement.Le système d’impression protéique PRIMO, commercialisé par Alvéole, qui co-finance cette thèse, est peu utilisé par les neuroscientifiques. En plus des objectifs biologiques, l'objectif industriel de cette thèse est de développer des méthodologies et des preuves de concept afin de démontrer les avantages et la faisabilité de la technologie PRIMO en neuroscience.En couplant notre premier modèle avec des techniques d’imagerie sur cellules vivantes (sptPALM et FRAP), nous avons pu différencier des cinétiques d’interaction entre différents couples de protéines d’adhésion synaptique mais également pour des interactions avec des protéines d’échafaudage. Une interaction labile pour SynCAM1, qui est connue pour son rôle dans la morphologie synaptique. Une forte et stable interaction pour Neuroligine1- Neurexine1β, due à la dimérisation de Neuroligine1, qui est indispensable pour la fonctionnalité de la synapse.Avec le second modèle, nous avons démontré, en présence de LRRTM2, la formation spécifique de synapses artificielles. Ces hémi-synapses présentent des caractéristiques morphologiques et fonctionnelles proches de synapses natives, avec la présence de vésicules et d’une activité calcique spontanée. Cependant, nous n’avons pas réussi à former de postsynapses artificielles avec Neurexine1β. Basés sur nos observations et une analyse bibliographique, nous avons formulé l’hypothèse que la postsynapse pourrait être le compartiment initiateur de la synaptogenèse.En conclusion, cette étude démontre : (1) que l’impression subcellulaire est un excellent modèle pour étudier la connectivité synaptique et l’adhésion de manière générale, aussi bien d’un point de vue fonctionnel qu’organisationnel. (2) Que les modèles d’hémi-synapses utilisant l’impression protéique sont plus spécifiques que les anciens modèles. (3) Que le système PRIMO ouvre de nombreuses perspectives en neurosciences via ses capacités d’impressions quantitativesMicropatterning was initially employed to replicate and understand the influence of the extracellular matrix on cells and some of their components. Over the past decade, subcellular printing has emerged, enabling the study of protein interactions and their role in signaling pathways as well as in the formation of synaptic, immunological, or neuronal pathways.The synaptic connection is mediated by synaptic adhesion proteins present on each side of the synapse. Due to the complexity of the synaptic environment and the lack of in vitro models to study synaptic connection in a biomimetic and controlled environment, the exact roles of these proteins in synaptogenesis remain uncertain. Subcellular protein printing presents a potential solution to address this gap. For this purpose, we have developed two biomimetic models based on protein printing: a first one using heterologous cells, providing insights into the interaction kinetics of protein pairs and linking them to their potential function. And a second one using primary neurons, allowing the formation of artificial synapses to study synaptic nano-organization during development.The protein printing system PRIMO, commercialized by Alvéole, which is co-funding this thesis, is underutilized by neuroscientists. Besides these biological objectives, the industrial aim of this thesis is to develop methodologies and proofs of concept to demonstrate the advantages and feasibility of the PRIMO technology in neuroscience.By coupling our first model, based on heterologous cells, with live-cell imaging techniques (sptPALM and FRAP), we differentiated interaction kinetics among various synaptic adhesion protein pairs and also for interactions with scaffold proteins. A labile interaction was observed for SynCAM1, known for its role in synaptic morphology. A strong and stable interaction was evident for Neuroligin1/Neurexine1β due to Neuroligin1's dimerization, which is essential for synaptic functionality.With the second model using primary hippocampal neurons, we demonstrated, in the presence of LRRTM2, the specific formation of artificial synapses. These hemi-synapses exhibited morphological and functional characteristics close to native synapses, including the presence of vesicles and spontaneous calcium activity. However, we were unable to form artificial postsynapses with Neurexine1β. Based on our observations and bibliographic analysis, we hypothesize that the postsynapse could be the initiating compartment for synaptogenesis.In conclusion, this study demonstrates: (1) that subcellular printing is an excellent model to study synaptic connectivity and adhesion from both a functional and organizational perspective. (2) That models of hemi-synapses using micropatterning are more specific than previous models. (3) That the PRIMO system opens numerous perspectives in neuroscience through its quantitative printing capabilities
9
Naeem, Asad. "Single and multiple target tracking via hybrid mean shift/particle filter algorithms." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/12699/.
Анотація:
This thesis is concerned with single and multiple target visual tracking algorithms and their application in the real world. While they are both powerful and general, one of the main challenges of tracking using particle filter-based algorithms is to manage the particle spread. Too wide a spread leads to dispersal of particles onto clutter, but limited spread may lead to difficulty when fast-moving objects and/or high-speed camera motion throw trackers away from their target(s). This thesis addresses the particle spread management problem. Three novel tracking algorithms are presented, each of which combines particle filtering and Kernel Mean Shift methods to produce more robust and accurate tracking. The first single target tracking algorithm, the Structured Octal Kernel Filter (SOK), combines Mean Shift (Comaniciu et al 2003) and Condensation (Isard and Blake 1998a). The spread of the particle set is handled by structurally placing the particles around the object, using eight particles arranged to cover the maximum area. Mean Shift is then applied to each particle to seek the global maxima. In effect, SOK uses intelligent switching between Mean Shift and particle filtering based on a confidence level. Though effective, it requires a threshold to be set and performs a somewhat inflexible search. The second single target tracking algorithm, the Kernel Annealed Mean Shift tracker (KAMS), uses an annealed particle filter (Deutscher et al 2000), but introduces a Mean Shift step to control particle spread. As a result, higher accuracy and robustness are achieved using fewer particles and annealing levels. Finally, KAMS is extended to create a multi-object tracking algorithm (MKAMS) by introducing an interaction filter to handle object collisions and occlusions. All three algorithms are compared experimentally with existing single/multiple object tracking algorithms. The evaluation procedure compares competing algorithms' robustness, accuracy and computational cost using both numerical measures and a novel application of McNemar's statistic. Results are presented on a wide variety of artificial and real image sequences.
10
Zelman-Femiak, Monika [Verfasser], and Gregory [Akademischer Betreuer] Harms. "Single Particle Tracking ; Membrane Receptor Dynamics / Monika Zelman-Femiak. Betreuer: Gregory Harms." Würzburg : Universitätsbibliothek der Universität Würzburg, 2012. http://d-nb.info/1026414768/34.
11
Spille, Jan-Hendrik [Verfasser]. "Three-dimensional single particle tracking in a light sheet microscope / Jan-Hendrik Spille." Bonn : Universitäts- und Landesbibliothek Bonn, 2014. http://d-nb.info/1052581986/34.
12
Ernst, Dominique [Verfasser], and Jürgen [Akademischer Betreuer] Köhler. "Single-Particle Orbit Tracking - Setup, Characterisation and Application / Dominique Ernst. Betreuer: Jürgen Köhler." Bayreuth : Universität Bayreuth, 2013. http://d-nb.info/1059353709/34.
13
Liu, Honghui Liu. "Using Tunable Lens to Extend the Range of the Single-Particle Tracking Microscop." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1525655236634443.
14
Hävermark, Tora. "Single-particle tracking for direct measurements of Trigger Factor ribosome binding in live cells." Thesis, Uppsala universitet, Molekylärbiologi, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-445886.
Анотація:
Trigger Factor (TF) is a prokaryotic chaperone protein that exerts its major chaperone activity while associated with translating ribosomes, assisting de novo folding of the emerging nascent chain. Although much is known about the kinetics behind TF-ribosome binding, most results are based on in vitro experiments which fail to mimic the cellular environment. Single-particle approaches have gained increasing power for studying binding kinetics of biomolecules in living cells. One such method is single-particle tracking by super-resolution fluorescence microscopy, where the position of a fluorescently labelled particle is recorded over time, giving information about the movement of the particle inside the cell. Changes in diffusion behaviour is then used as an indicator of changes in biological activities. In this work, a diffusion model that qualitatively and quantitatively describes TF’s binding to ribosomes is presented. The model was obtained by single-particle tracking of TF labelled with HaloTag. Particle movements were analysed with a Hidden Markov Model-based algorithm that fit the trajectories to a defined set of different diffusion states, where fast diffusion could be related to free TF and slow diffusion to a ribosome-bound state. Moreover, the model could distinguish between two types of ribosome interactions: TF’s stable binding to ribosomes and a faster sampling behaviour. The average time spent stably bound to ribosomes is 670 ms and these interactions account for 53% of TF’s activity. TF is one of many processing proteins that interact with the emerging peptide chain during translation. By using the same approach on more of these factors, the interplay between them and the growing nascent chain can be characterized, giving an increased understanding of the highly complex translation machinery.
15
Ali, Rehan. "An Automated Analysis Of Single Particle Tracking Data For Proteins That Exhibit Multi Component Motion." ScholarWorks @ UVM, 2018. https://scholarworks.uvm.edu/graddis/870.
Анотація:
Neurons are polarized cells with dendrites and an axon projecting from their cell body. Due to this polarized structure a major challenge for neurons is the transport of material to and from the cell body. The transport that occurs between the cell body and axons is called Axonal transport. Axonal transport has three major components: molecular motors which act as vehicles, microtubules which serve as tracks on which these motors move and microtubule associated proteins which regulate the transport of material. Axonal transport maintains the integrity of a neuron and its dysfunction is linked to neurodegenerative diseases such as, Alzheimer’s disease, Frontotemporal dementia linked to chromosome 17 and Pick’s disease. Therefore, understanding the process of axonal transport is extremely important.
Single particle tracking is one method in which axonal transport is studied. This involves fluorescent labelling of molecular motors and microtubule associated proteins and tracking their position in time. Single particle tracking has shown that both, molecular motors and microtubule associated proteins exhibit motion with multiple components. These components are directed, where motion is in one direction, diffusive, where motion is random, and static, where there is no motion. Moreover, molecular motors and microtubule associated proteins also switch between these different components in a single instance of motion.
We have developed a MATLAB program, called MixMAs, which specializes in analyzing the data provided by single particle tracking. MixMAs uses a sliding window approach to analyze trajectories of motion. It is capable of distinguishing between different components of motion that are exhibited by molecular motors and microtubule associated proteins. It also identifies transitions that take place between different components of motion. Most importantly, it is not limited by the number of transitions and the number of components present in a single trajectory. The analysis results provided by MixMAs include all the necessary parameters required for a complete characterization of a particle’s motion. These parameters are the number of different transitions that take place between different components of motion, the dwell times of different components of motion, velocity for directed component of motion and diffusion coefficient for diffusive component of motion.
We have validated the working of MixMAs by simulating motion of particles which show all three components of motion with all the possible transitions that can take place between them. The simulations are performed for different values of error in localizing the position of a particle. The simulations confirm that MixMAs accurately calculates parameters of motion for a range of localization errors. Finally, we show an application of MixMAs on experimentally obtained single particle data of Kinesin-3 motor.
16
Trenkmann, Ines, Daniela Täuber, Michael Bauer, Jörg Schuster, Sangho Bok, Shubhra Gangopadhyay, and Christian von Borczyskowski. "Investigations of solid liquid interfaces in ultra-thin liquid films via single particle tracking of silica particles." Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-191734.
Анотація:
Single particle tracking with a wide field microscope is used to study the solid liquid interface between the viscous liquid tetrakis(2 ethylhexoxy)-silane and a silicon dioxide surface. Silicon dioxide nanoparticles (5 nm diameter) marked with the fluorescent dye rhodamine 6G are used as probes. The distributions of diffusion coefficients, obtained by mean squared displacements, reveal heterogeneities with at least two underlying diffusion components. Measurements on films with varying film thicknesses show that the slower component is independent of the film thickness, while the faster one increases with the film thickness. Additionally, we could show that the diffusion behavior of the particles cannot be sufficiently described by only two diffusion coefficients.
17
Trenkmann, Ines, Jörg Schuster, Shubhra Gangopadhyay, and Christian von Borczyskowski. "Investigation of solid liquid interface in ultra-thin liquid films via single particle tracking of colloidal particles." Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-191812.
18
Trenkmann, Ines, Daniela Täuber, Michael Bauer, Jörg Schuster, Sangho Bok, Shubhra Gangopadhyay, and Christian von Borczyskowski. "Investigations of solid liquid interfaces in ultra-thin liquid films via single particle tracking of silica particles." Diffusion fundamentals 11 (2009) 108, S. 1-12, 2009. https://ul.qucosa.de/id/qucosa%3A14082.
Анотація:
Single particle tracking with a wide field microscope is used to study the solid liquid interface between the viscous liquid tetrakis(2 ethylhexoxy)-silane and a silicon dioxide surface. Silicon dioxide nanoparticles (5 nm diameter) marked with the fluorescent dye rhodamine 6G are used as probes. The distributions of diffusion coefficients, obtained by mean squared displacements, reveal heterogeneities with at least two underlying diffusion components. Measurements on films with varying film thicknesses show that the slower component is independent of the film thickness, while the faster one increases with the film thickness. Additionally, we could show that the diffusion behavior of the particles cannot be sufficiently described by only two diffusion coefficients.
19
Trenkmann, Ines, Jörg Schuster, Shubhra Gangopadhyay, and Christian von Borczyskowski. "Investigation of solid liquid interface in ultra-thin liquid films via single particle tracking of colloidal particles." Diffusion fundamentals 11 (2009) 115, S. 1-2, 2009. https://ul.qucosa.de/id/qucosa%3A14089.
20
Barry, Zachary Thomas. "Single-particle tracking and fluorescence correlation spectroscopy for systems-level analysis of molecular dynamics in diverse biological systems." Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/112494.
Анотація:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017.Cataloged from PDF version of thesis.
Includes bibliographical references.
Fluorescence microscopy has proven to be immensely powerful for the study of biological systems at both the cellular and systems biological levels. The ability to specifically label a single molecular species fluorescently has enabled the study of complex cellular structures through the visualization of their constituent components both individually as well as in context of the overall structure. Since the advent of engineered fluorescent proteins (such as GFP) and other proteins capable of being genetically encoded as fusion constructs, the utility of fluorescence microscopy has increased exponentially in terms of the ability to efficiently, specifically label desired molecules while limiting perturbations to the biology under study. With this enhanced ability of visualization came a hand-in-hand evolution of computational techniques to extract quantitative information from microscopy images. In this thesis, I focus on the application of fluorescence imaging at the biophysical level in living cells: analyzing the motion/dynamics of single molecules and complexes, which are small relative to the structures of the cell, in order to elucidate their molecular function and mechanism. The motion of these "particles" within living cells is necessarily related to their functions as well as their interacting partners, which can vary dynamically during their lifetimes. Observation and analysis of this motion using a combination of fluorescence microscopy and robust quantitative analysis allows one to infer these characteristics. Here, I study three diverse biological systems in the context of live-cell fluorescence microscopy and biophysical analysis: 1) the transport of 0-actin mRNA particles in primary mouse neurons, 2) kinetochore motion during cell division, specifically focusing on anaphase dynamics, and 3) the motion of cell-growth-implicated membrane proteins in Bacillus subtilis.
Funded by the NSF Physics of Living Systems PHY 1305537.
by Zachary Thomas Barry.
Ph. D.
21
Takeyama, Mao. "Convective heat transfer of saturation nucleate boiling induced by single and multi-bubble dynamics." Kyoto University, 2021. http://hdl.handle.net/2433/261621.
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Sharma, Gaurav. "Direct numerical simulation of particle-laden turbulence in a straight square duct." Thesis, Texas A&M University, 2003. http://hdl.handle.net/1969.1/155.
Анотація:
Particle-laden turbulent flow through a straight square duct at Reτ = 300 is studied using direct numerical simulation (DNS) and Lagrangian particle tracking. A parallelized 3-D particle tracking direct numerical simulation code has been developed to perform the large-scale turbulent particle transport computations reported in this thesis. The DNS code is validated after demonstrating good agreement with the published DNS results for the same flow and Reynolds number. Lagrangian particle transport computations are carried out using a large ensemble of passive tracers and finite-inertia particles and the assumption of one-way fluid-particle coupling. Using four different types of initial particle distributions, Lagrangian particle dispersion, concentration and deposition are studied in the turbulent straight square duct. Particles are released in a uniform distribution on a cross-sectional plane at the duct inlet, released as particle pairs in the core region of the duct, distributed randomly in the domain or distributed uniformly in planes at certain heights above the walls. One- and two-particle dispersion statistics are computed and discussed for the low Reynolds number inhomogeneous turbulence present in a straight square duct. New detailed statistics on particle number concentration and deposition are also obtained and discussed.
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Leung, Siu Ling. "Fabrication of Multimodal Organic-Inorganic Hybrid Nanovesicles and Study on their Intracellular Fates in Cancer Cells by Single Particle Tracking." Diss., The University of Arizona, 2013. http://hdl.handle.net/10150/312657.
Анотація:
Creation of magic bullets might be the dream of all scientists working on anticancer therapeutics. In reality, there are always pros and cons. Chemotherapeutics such as doxorubicin and paclitaxel can effectively inhibit growth of cancer cells; however, the drugs at a high dosage are not selective and can cause severe damage to normal tissues and/or organs. To minimize its side effects, anticancer therapeutics are often encapsulated using nanovesicles (NVs). Antibodies that target specific cancer cells may be conjugated with drug-carrying NVs to further improve the selectivity of drug delivery. In the design and fabrication of NV-based drug carriers, many structural and micro-environmental factors affect cellular uptake and internalization of drug-carrying NVs, drug release and distribution in tissues, and therapeutic efficiency. Two goals of my dissertation study is (1) to fabricate multimodal NVs for bio-imaging, selective targeting and drug delivery and (2) to unfold interplays between different factors and intracellular fates of these NVs in prostate cancer cells. This proposed work separates into four phases: (1) Novel organic-inorganic liposomal cerasomes will be used as drug carriers for delivery of potent anticancer doxorcubicin (DOX). Liposomes will be stabilized via an addition of inorganic polyorganosiloxane networks on their surfaces, creating liposomal cerasomes, and its potential to store and release DOX will be investigated. (2) Prostate cancer cells will be used as a model system to study endocytic pathway of cerasomes. Among various types of human cancer, prostate cancer is the second leading cause of death for man. It is anticipated that the proposed study will shed new insights into endocytosis drug-carrying NVs. Results from the study may facilitate the development of cerasome-based therapeutics for treating prostate cancer patients. (3) Since diagnostic, imaging and therapy are three major biomedical applications in nanotechnology, multifunctional NVs with combined bio-imaging, therapeutic efficiency and selective targeting modalities are essential as next-generation nano-carriers. Here, we proposed to incooperate fluorophores and prostate cancer targeted antibodies into cerasomes to achieve our multimodal NVs. (4) Single particle tracking (SPT) analysis will be used to study the intracellular fates of cerasome uptake in single cell level. SPT is an advanced imaging technique permits the real-time monitoring of cellular entry, intracellular transport and internalization of multiple NVs. Our main focus is to identify the different in cellular transportation mechanism between targeted and non-targeted vesicles. Moreover, SPT will be integrated into a microfluidic system, which not only minimizes the consumption of reagents/ materials but also allows us to precisely control biochemical environments around cells.
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Subburaj, Yamunadevi [Verfasser], and Ana J. [Akademischer Betreuer] García-Sáez. "Single Particle Tracking to Characterize the Mechanism of Pore Formation by Pore Forming Proteins / Yamunadevi Subburaj ; Betreuer: Ana J. García-Sáez." Tübingen : Universitätsbibliothek Tübingen, 2014. http://d-nb.info/1162897333/34.
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Zanetti, Domingues Laura Carolina. "Single-particle tracking of the human epidermal growth factor receptors (HER) 1-3 in a breast cancer model : the effect of tyrosine kinase inhibitors." Thesis, King's College London (University of London), 2014. http://kclpure.kcl.ac.uk/portal/en/theses/singleparticle-tracking-of-the-human-epidermal-growth-factor-receptors-her-13-in-a-breast-cancer-model-the-effect-of-tyrosine-kinase-inhibitors(929e12cf-48bc-484f-b304-3f09d74203c3).html.
Анотація:
Over-expression and mutational activation of the Human Epidermal Growth Factor Receptor (HER) 1-4 receptor family are implicated in tumorigenesis and cancer progression. Perturbations of their associated signalling cascades and feed-back loops can amplify aberrant signalling. Inhibition of these signals is a central objective of anti-cancer targeted therapeutics, but to date most drugs have limited success. The reasons are not understood and underscore a lack of understanding of the molecular basis of receptor signalling and drug action. At the heart of these processes are combinatorial interactions leading to receptor homo- and hetero-dimerization To characterise the interactions between HER family homo- and hetero-dimers in basal and activated states, receptors were labelled in their non-active configuration with Affibodies conjugated to fluorophores or activated by fluorescently-tagged ligands. This will enable future application of the method to primary samples. Cell culture substrates and fluorescent dyes were optimised to reduce the incidence of non-specific probe adhesion and a set of parameters to guide the choice of fluorescent dyes for Single-Molecule applications was determined. Alexa Fluor 488- and CF640R- tagged probes were used for pairwise live-cell tracking of HER1-3 receptors in presence or absence of clinical-grade tyrosine kinase inhibitors. These experiments highlighted the role of tyrosine kinase activity in determining HER1 diffusion at the plasma membrane and likelihood of interaction. Activated HER1 homo-interactions and hetero-interactions with HER2 were the predominant colocalisation pairs identified. Precise determination of the dwell-time distribution of all pairs is pending and will require more precise assessment of photobleaching lifetime contaminations and denoising. Analysis of the diffusion of HER1 with a novel, globally-optimised Bayesian tracking algorithm developed by Dr Wareham (University of Cambridge) revealed the presence of two populations of receptors characterised by their directional motion components. The most directional population depends on the presence of an intact actin cytoskeleton and on tyrosine kinase activity. The methods presented in this thesis, will be extended to study the dynamics and kinetics of the whole HER family, laying the foundations for the construction of a quantitative model of the system.
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El, Beheiry Mohamed Hossam. "Towards whole-cell mapping of single-molecule dynamics." Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066618/document.
Анотація:
La compréhension fondamentale de fonctions biologiques est accordée par l’imagerie de molécules uniques dans les cellules vivantes. Cet environnement nanoscopique est compliqué et largement mal compris. La microscopie de localisation permet cet environnement d’être sondé avec le suivi de molécules uniques. Depuis longtemps, l’obstacle principal qui empêcher la compréhension de processus physiques à cette échelle était la pénurie d’information accessible; de fortes hypothèses ne peuvent pas être établies à partir de quelques dizaines de trajectoires de molécules uniques. L’introduction des techniques de suivi de molécules à haute densité a recadré les possibilités. Dans cette thèse, les outils d’inférence Bayesienne ont été développé pour élucider le comportement de molécules uniques à partir la cartographie de leurs paramètres physiques de mouvement. Ces cartes décrivent, entre autre, les hétérogénéités aux échelles locales mais aussi à l’échelle de la cellule entière. Notamment elles dévoilent les détails quantitatifs sur les processus cellulaires de base. En utilisant cette approche cartographique, les interactions entre récepteurs et protéines d’échafaudage chez les neurones et les cellules non-neuronales sont étudiés. Une étude sur les interactions imprimées dans la cellule est également effectuée grace à un système prometteur d’impression de protéine. Les différents types d’interactions entre constructions chimériques du récepteur de glycine et de protéines d’échafaudage de géphyrine sont décrits et distingué in situ. Enfin, les perspectives vis-à-vis l’obtention de cartes tridimensionnelles de dynamiques de molécules uniques sont également commentéesImaging of single molecules inside living cells confers insight to biological function at its most granular level. Single molecules experience a nanoscopic environment that is complicated, and in general, poorly understood. The modality of choice for probing this environment is live-cell localisation microscopy, where trajectories of single molecules can be captured. For many years, the great stumbling block in comprehension of physical processes at this scale was the lack of information accessible; statistical significance and robust assertions are hardly possible from a few dozen trajectories. It is the onset of high-density single-particle tracking that has dramatically reframed the possibilities of such studies. Importantly, the consequential amounts of data it provides invites the use of powerful statistical tools that assign probabilistic descriptions to experimental observations. In this thesis, Bayesian inference tools have been developed to elucidate the behaviour of single molecules via the mapping of motion parameters. As a readout, maps describe heterogeneities at local and whole-cell scales. Importantly, they grant quantitative details into basic cellular processes. This thesis uses the mapping approach to study receptor-scaffold interactions inside neurons and non-neuronal cells. A promising system in which interactions are patterned is also examined. It is shown that interactions of different types of chimeric glycine receptors to the gephyrin scaffold protein may be described and distinguished in situ. Finally, the prospects of whole-cell mapping in three-dimensions are evaluated based on a discussion of state-of-the-art volumetric microscopy techniques
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Bhat, Siddharth. "Design and characterization in depleted CMOS technology for particle physics pixel detector." Thesis, Aix-Marseille, 2019. http://www.theses.fr/2019AIXM0267.
Анотація:
L’expérience ATLAS commencera à fonctionner avec l’accélérateur LHC à hauteluminosité (HL-LHC) en 2026 pour augmenter la probabilité de nouvelles découvertes. La technologie du détecteur de pixels monolithiques en "depleted" CMOS a été une des options envisagées pour la couche externe du détecteur pixel d’ATLAS mis a niveau et est une technologie à fort potentiel pour les futurs détecteurs a pixels. Dans cette thèse, plusieurs prototypes ont été développés utilisant différentes technologies CMOS appauvries, par exemple, LFoundry (LF) 150 nm, TowerJazz (TJ) à 180 nm et Austriamicrosystems AG (AMS) à 180 nm. Dans un environnement à haute énergie, tel que HL-LHC, les événements uniques (SEU), qui deviennent un sujet de préoccupation pour le bon fonctionnement des circuits. Plusieurs puces de test dans les technologies AMS, TowerJazz et LFoundry avec différentes structures tolérantes au SEU ont été prototypées et testées. Un schéma d’alimentation alternatif appelé schéma d’alimentation série est prévu pour le futur détecteur Inner Tracker (ITk) de l’expérience ATLAS. Pour répondre aux exigences de l’expérience ATLAS concernant l’environnement d’une couche pixélisée dans un environnement de collisionneur à rayonnement élevé, de nouveaux développements avec des capteurs "depleted" CMOS ont été développés dans le régulateur Shunt-LDO et la polarisation de capteur conçus dans la technologie d’imagerie CMOS TowerJazz 0,18 um modifiée. Dans le processus TowerJazz modifié, deux niveaux de tension différents sont utilisés pour l'épuisement du capteur. Polarisation les tensions sont générées à l'aide d'un circuit de pompe à charge négativeThe ATLAS experiment will start operating at the High Luminosity LHC accelerator (HL-LHC) in 2026 to increase the probability of new discoveries. Depleted CMOS monolithic pixel detector technology has been one of the options considered for the outer layer of an upgraded ATLAS pixel detector and is a high potential technology for future pixel detectors. In this thesis, several prototypes have been developed using different depleted CMOS technologies, for instance, LFoundry (LF) 150 nm, TowerJazz (TJ) 180 nm and austriamicrosystems AG (AMS) 180 nm. In a high-energy environment like HL-LHC, Single Event Upsets (SEU), which become of concern for reliable circuit operation. Several test-chips in AMS, TowerJazz and LFoundry technologies with different SEU tolerant structures have been prototyped and tested. The SEU tolerant structures were designed with appropriate electronics simulations using Computer Aided Design (CAD) tools in order to study the sensitivity of injected charge to upset a memory state. An alternative powering scheme named Serial Powering scheme is foreseen for the future Inner Tracker (ITk) detector of the ATLAS experiment. To meet the requirements ofthe ATLAS experiment to the environment of a pixelated layer in a high radiation collider environment, new developments with depleted CMOS sensors have been made in Shunt-LDO regulator and sensor biasing which are designed in modified TowerJazz 180 nm CMOS imaging technology. In the TowerJazz modified process, two different voltage levels are used for the purpose of sensor depletion. The bias voltages are generated by using a negative charge pump circuit
28
Butler, Corey. "Quantitative single molecule imaging deep in biological samples using adaptive optics." Thesis, Bordeaux, 2017. http://www.theses.fr/2017BORD0632/document.
Анотація:
La microscopie optique est un outil indispensable pour la recherche de la neurobiologie et médecine qui permet l’étude des cellules dans leur environnement natif. Les processus sous-cellulaires restent néanmoins cachés derrière les limites de la résolution optique, ce qui rend la résolution des structures plus petites que ~300nm impossible. Récemment, les techniques de la localisation des molécules individuelles (SML) ont permis le suivi des protéines de l’échelle nanométrique grâce à l’ajustement des molécules uniques à la réponse impulsionnelle du système optique. Ce processus dépend de la quantité de lumière recueilli et rend ces techniques très sensibles aux imperfections de la voie d’imagerie, nommé des aberrations, qui limitent l’application de SML aux cultures cellulaires sur les lamelles de verre. Un système commercial d’optiques adaptatives est implémenté pour compenser les aberrations du microscope, et un flux de travail est défini pour corriger les aberrations dépendant de la profondeur qui rend la 3D SML possible dans les milieux biologiques complexes. Une nouvelle méthode de SML est présentée qui utilise deux objectifs pour détecter le spectre d’émission des molécules individuelles pour des applications du suivi des particules uniques dans 5 dimensions (x,y,z,t,λ) sans compromis ni de la résolution spatiotemporelle ni du champ de vue. Pour faciliter les analyses de manière quantitative des Go de données générés, le développement des outils biochimiques, numériques et optiques est présenté. Ensemble, ces approches ont le but d’amener l’imagerie quantitative des molécules uniques dans les échantillons biologiques complexesOptical microscopy is an indispensable tool for research in neurobiology and medicine, enabling studies of cells in their native environment. However, subcellular processes remain hidden behind the resolution limits of diffraction-limited optics which makes structures smaller than ~300nm impossible to resolve. Recently, single molecule localization (SML) and tracking has revolutionized the field, giving nanometer-scale insight into protein organization and dynamics by fitting individual fluorescent molecules to the known point spread function of the optical imaging system. This fitting process depends critically on the amount of collected light and renders SML techniques extremely sensitive to imperfections in the imaging path, called aberrations, that have limited SML to cell cultures on glass coverslips. A commercially available adaptive optics system is implemented to compensate for aberrations inherent to the microscope, and a workflow is defined for depth-dependent aberration correction that enables 3D SML in complex biological environments. A new SML technique is presented that employs a dual-objective approach to detect the emission spectrum of single molecules, enabling 5-dimensional single particle imaging and tracking (x,y,z,t,λ) without compromising spatiotemporal resolution or field of view. These acquisitions generate ~GBs of data, containing a wealth of information about the localization and environment of individual proteins. To facilitate quantitative acquisition and data analysis, the development of biochemical, software and hardware tools are presented. Together, these approaches aim to enable quantitative SML in complex biological samples
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Lanoiselée, Yann. "Revealing the transport mechanisms from a single trajectory in living cells." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLX081/document.
Анотація:
Cette thèse est dédiée à l’analyse et la modélisation d'expériences où la position d'un traceur dans le milieu cellulaire est enregistrée au cours du temps. Il s’agit de pouvoir de retirer le maximum d’information à partir d’une seule trajectoire observée expérimentalement. L’enjeu principal consiste à identifier les mécanismes de transport sous-jacents au mouvement observé. La difficulté de cette tâche réside dans l’analyse de trajectoires individuelles, qui requiert de développer de nouveaux outils d’analyse statistique. Dans le premier chapitre, un aperçu est donné de la grande variété des dynamiques observables dans le milieu cellulaire. Notamment, une revue de différents modèles de diffusion anormale et non-Gaussienne est réalisée. Dans le second chapitre, un test est proposé afin de révéler la rupture d'ergodicité faible à partir d’une trajectoire unique. C’est une généralisation de l’approche de M. Magdziarz et A. Weron basée sur la fonction caractéristique du processus moyennée au cours du temps. Ce nouvel estimateur est capable d’identifier la rupture d’ergodicité de la marche aléatoire à temps continu où les temps d'attente sont distribués selon une loi puissance. Par le calcul de la moyenne de l’estimateur pour plusieurs modèles typiques de sous diffusion, l’applicabilité de la méthode est démontrée. Dans le troisième chapitre, un algorithme est proposé afin reconnaître à partir d’une seule trajectoire les différentes phases d'un processus intermittent (e.g. le transport actif/passif à l'intérieur des cellules, etc.). Ce test suppose que le processus alterne entre deux phases distinctes mais ne nécessite aucune hypothèse sur la dynamique propre dans chacune des phases. Les changements de phase sont capturés par le calcul de quantités associées à l’enveloppe convexe locale (volume, diamètre) évaluées au long de la trajectoire. Il est montré que cet algorithme est efficace pour distinguer les états d’une large classe de processus intermittents (6 modèles testés). De plus, cet algorithme est robuste à de forts niveaux de bruit en raison de la nature intégrale de l’enveloppe convexe. Dans le quatrième chapitre, un modèle de diffusion dans un milieu hétérogène où le coefficient de diffusion évolue aléatoirement est introduit et résolu analytiquement. La densité de probabilité des déplacements présente des queues exponentielles et converge vers une Gaussienne au temps long. Ce modèle généralise les approches précédentes et permet ainsi d’étudier en détail les hétérogénéités dynamiques. En particulier, il est montré que ces hétérogénéités peuvent affecter de manière drastique la précision de mesures effectuées sur une trajectoire par des moyennes temporelles. Dans le dernier chapitre, les méthodes d’analyses de trajectoires individuelles sont utilisées pour étudier deux expériences. La première analyse effectuée révèle que les traceurs explorant le cytoplasme montrent que la densité de probabilité des déplacements présente des queues exponentielles sur des temps plus longs que la seconde. Ce comportement est indépendant de la présence de microtubules ou du réseau d’actine dans la cellule. Les trajectoires observées présentent donc des fluctuations de diffusivité témoignant pour la première fois de la présence d’hétérogénéités dynamiques au sein du cytoplasme. La seconde analyse traite une expérience dans laquelle un ensemble de disques de 4mm de diamètre a été vibré verticalement sur une plaque, induisant un mouvement aléatoire des disques. Par une analyse statistique approfondie, il est démontré que cette expérience est proche d'une réalisation macroscopique d'un mouvement Brownien. Cependant les densités de probabilité des déplacements des disques présentent des déviations par rapport à la Gaussienne qui sont interprétées comme le résultat des chocs inter-disque. Dans la conclusion, les limites des approches adoptées ainsi que les futures pistes de recherches ouvertes par ces travaux sont discutées en détailThis thesis is dedicated to the analysis and modeling of experiments where the position of a tracer in the cellular medium is recorded over time. The goal is to be able to extract as much information as possible from a single experimentally observed trajectory. The main challenge is to identify the transport mechanisms underlying the observed movement. The difficulty of this task lies in the analysis of individual trajectories, which requires the development of new statistical analysis tools. In the first chapter, an overview is given of the wide variety of dynamics that can be observed in the cellular medium. In particular, a review of different models of anomalous and non-Gaussian diffusion is carried out. In the second chapter, a test is proposed to reveal weak ergodicity breaking from a single trajectory. This is a generalization of the approach of M. Magdziarz and A. Weron based on the time-averaged characteristic function of the process. This new estimator is able to identify the ergodicity breaking of continuous random walking where waiting times are power law distributed. By calculating the average of the estimator for several subdiffusion models, the applicability of the method is demonstrated. In the third chapter, an algorithm is proposed to recognize the different phases of an intermittent process from a single trajectory (e.g. active/passive transport within cells, etc.).This test assumes that the process alternates between two distinct phases but does not require any hypothesis on the dynamics of each phase. Phase changes are captured by calculating quantities associated with the local convex hull (volume, diameter) evaluated along the trajectory. It is shown that this algorithm is effective in distinguishing states from a large class of intermittent processes (6 models tested). In addition, this algorithm is robust at high noise levels due to the integral nature of the convex hull. In the fourth chapter, a diffusion model in a heterogeneous medium where the diffusion coefficient evolves randomly is introduced and solved analytically. The probability density function of the displacements presents exponential tails and converges towards a Gaussian one at long time. This model generalizes previous approaches and thus makes it possible to study dynamic heterogeneities in detail. In particular, it is shown that these heterogeneities can drastically affect the accuracy of measurements made by time averages along a trajectory. In the last chapter, single-trajectory based methods are used for the analysis of two experiments. The first analysis carried out shows that the tracers exploring the cytoplasm show that the probability density of displacements has exponential tails over periods of time longer than the second. This behavior is independent of the presence of both microtubules and the actin network in the cell. The trajectories observed therefore show fluctuations in diffusivity, indicating for the first time the presence of dynamic heterogeneities within the cytoplasm. The second analysis deals with an experiment in which a set of 4mm diameter discs was vibrated vertically on a plate, inducing random motion of the disks. Through an in-depth statistical analysis, it is demonstrated that this experiment is close to a macroscopic realization of a Brownian movement. However, the probability densities of disks’ displacements show deviations from Gaussian which are interpreted as the result of inter-disk shocks. In the conclusion, the limits of the approaches adopted as well as the future research orientation opened by this thesis are discussed in detail
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Stirnnagel, Kristin. "Herstellung autofluoreszierender retroviraler Partikel zur Analyse der zellulären Aufnahmemechanismen von Foamyviren." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-84198.
Анотація:
Foamyviren (FV) gehören zur Familie der Retroviridae, werden aber aufgrund besonderer Eigenschaften in eine eigene Unterfamilie, die Spumaretrovirinae, eingeordnet. FV besitzen vor allem in vitro einen sehr breiten Tropismus, so dass bisher keine Zelllinie bekannt war, die nicht durch FV infiziert werden konnte. Obwohl diese Besonderheit darauf schließen lässt, dass ein sehr ubiquitäres Molekül auf der Wirtszelloberfläche für die FV-Bindung verwendet wird, ist der Rezeptor für die Virus-Aufnahme noch nicht bekannt. Dass FV einen pH-abhängigen Aufnahmemechanismus verwenden, lässt eine endozytotische Aufnahme vermuten. Dennoch sind die frühen Replikationsschritte, die zur Fusion der viralen und Wirtszellmembran führen, nur unzureichend charakterisiert. Deswegen wurden in der vorliegenden Arbeit funktionelle autofluoreszierende FV hergestellt, um die Bindung und Aufnahmemechanismen foamyviraler Partikel in Wirtszellen mit fluoreszenzmikroskopischen Analysen zu untersuchen.
Mit diesen Methoden konnten erstmalig vier Zelllinien identifiziert werden, die nicht mit FV infizierbar sind, und damit mögliche Kandidaten für die Identifizierung des unbekannten FV Rezeptors darstellen. Des Weiteren wurden die fluoreszierenden FV erfolgreich eingesetzt, um die Fusionsereignisse zwischen viraler und zellulärer Membran in Echtzeit in lebenden Zellen zu untersuchen. Die durchgeführte „Single Virus Tracking“-Analyse zeigte, dass PFV (Prototype FV) Env-tragende Partikel sowohl an der Plasmamembran als auch in vermeintlichen Endosomen fusionieren können, wohingegen SFV (Simian FV) Env-tragende Partikel die Fusion wahrscheinlich nur in Endosomen auslösen können.
Hinweis zur Nutzung der Filmdateien: Öffnen mit QuickTimePlayer bzw. ImageJ
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Haas, Kalina. "Nanoscale co-organization of AMPAR and Neuroligin probed with single-molecule based microscopy." Thesis, Bordeaux 2, 2013. http://www.theses.fr/2013BOR22134/document.
Анотація:
Il est bien admis que la compréhension de la structuration moléculaire à l’intérieur des cellules neuronales est essentielle pour appréhender le fonctionnement du cerveau. Pour cette raison, l’étude de l’organisation des molécules clés neuronales et synaptiques contribue grandement à comprendre le mystère du cerveau. AMPA sont des récepteurs ionotropiques du glutamate jouent le rôle central dans la plasticité synaptique et la transmission synaptique basale dans le système nerveux central. Distribution des récepteurs AMPA sur la membrane neuronale est remarquablement hétérogène. Ils sont organisés en agrégats fonctionnels distincts, appelés nanodomaines. Des travaux antérieurs ont montré que Neuroligin, la molécule d’adhésion post-synaptique, ancres récepteurs AMPA par PSD95 dans la membrane post-synaptique et constitue en même temps un complexe d’adhésion trans-synaptique avec présynaptique Neurexin, impliqué dans le recrutement de machines de libération vésiculaire sur le site présynaptique. De cette façon, NRLG fonctionnellement organise synapses par la poste de recrutement de molécules présynaptiques essentielles pour réponse synaptique. Ici, nous avons étudié l’effet de la modulation de NRLG1 (modification du niveau d’expression ou de l’activité) sur le dynamique et nano-organisation des récepteurs AMPA au niveau des synapses individuelles. Notre hypothèse est que le complexe NRX-NRG pourrait être impliquée dans la localisation précise des récepteurs post-synaptiques et son apposition avec zone active présynaptique, jouant ainsi le rôle important dans la transduction du signal approprié. Taille de la densité post-synaptique (PSD) est de l’ordre de 500 nm, alors que diamètre moyen des nanodomaines AMPAR 100 nm. Une telle petite dimension nécessitait l’application de techniques de microscopie de super-résolution, dont la résolution de l’ordre de 20-40 nm est presque un ordre de grandeur mieux que microscopie fluorescence limitée par la diffraction. Nanoscopie fluorescence permettent visualiser des cellules jusqu’au niveau presque moléculaire. Pour atteindre mes objectifs, j’ai mis en place différents nanoscopies de localisation d’une seule molécule, qui s’appuient sur séparés dans l’espace et le temps de détection de population choisi de sondes de fluorescence. Il a été proposé que le trafic membranaire des récepteurs de neurotransmetteurs peut contribuer à la modulation de l’efficacité synaptique. J’ai sondé propriétés diffusionnelles des récepteurs AMPA avec suivi de particules unique, qui a été pendant longtemps appliqué pour sonder l’hétérogénéité de la membrane cellulaire. Localisation relative des biomolécules à la base de la compréhension de leur relation fonctionnelle. Il est bien admis que la juxtaposition de deux objets, ainsi que leur colocalisation, peuvent témoigner de leur association. Avec les récents développements dans l’acquisition multi couleur de la molécule unique et images de super-résolution à base d’ensemble, il est maintenant possible d’explorer la colocalisation à l’échelle nanométrique entre biomolécules dans des cellules vivantes et fixe. Malgré l’ la popularité et l’application très répandue, il n’existe que quelques paradigmes d’analyse quantitative pour la colocalisation des images multicolores super-résolution. Ici, avec l’aide de paradigmes conventionnels de mesure de la colocalisation et statistiques multivariées, nous analysons et présentons en isolement l’échelle du détail et proximité des macromolécules au sein de zones fonctionnelles de synapses. En outre, nous utilisons ces paradigmes pour évaluer marqueurs fluorescents impliqués dans la production de routine de la molécule unique fondée images super-résolution. Nous étendons notre analyse élucider en profondeur le co-agrégation des molécules clés synaptiques, PSD95 et récepteurs AMPA, qui sont impliqués dans l’organisation synaptique et transmission basaleThe brain is made of complex networks of interconnected neuronal cells. All our mental activities are underlain by electrochemical signals passing through dedicated neuronal circuits. Climbing further up on the complexity ladder, information processing by neurons is performed by multiple molecules assembling and interacting together. It is well accepted that the understanding of the molecular structuring inside neuronal cells is essential to apprehend functioning of the brain. For this reason, study of the organization of the key neuronal and synaptic molecules greatly contributes to understand the mystery of the brain. AMPA receptors (AMPARs) are ionotropic glutamate receptors that play a central role in synaptic plasticity and basal synaptic transmission in the central nervous system. The distribution of AMPARs on the neuronal membrane is remarkably heterogeneous. They are organized in distinct functional aggregates, called nanodomains. Previous work demonstrate that the postsynaptic adhesion molecule Neuroligin (NRLG) anchors AMPARs through PSD-95 in the postsynaptic membrane while simultaneously forming a trans-synaptic adhesion complex with presynaptic Neurexin (NRX), and recruiting vesicular release machinery at the presynaptic site. In this way, NRLG functionally organizes synapses by recruiting post and pre-synaptic molecules essential for regulation of synaptic responses. Here we studied the effect of NRLG modulation (modification of expression level or activity) on AMPAR nano-dynamics and nano-organization at individual synapses. Our hypothesis is that the NRX-NRLG complex could be involved in the precise localization of postsynaptic receptors and their apposition with the neurotransmitter release sites in the presynaptic active zone, thus playing important role in proper signal transduction. The size of the postsynaptic density (PSD) is in the order of 500 nm, whereas the average diameter of AMPAR nanodomains 100 nm. Such small dimension necessitated the application of super-resolution microscopy techniques, whose resolution in the range of 20-40 nm is almost an order of magnitude better than diffraction limited fluorescence microscopy. Probe-based far-field fluorescence nanoscopies allow visualizing cells down to almost molecular level. To achieve my goals, I implemented different single-molecule localization nanoscopies which rely on the detection of selected populations of fluorescence probes that are separated in space and time. It was proposed that membrane trafficking of neurotransmitter receptors may contribute to modulation of synaptic efficacy. I have probed diffusional properties of AMPARs with single particle tracking, which has long been applied to probe heterogeneity of the cell membrane. Relative localization of biomolecules provides the basis for understanding their functional relationship. It is well accepted that the juxtaposition of two objects, as well as their colocalization, may give evidence of their association. With the recent developments in multi-color acquisition of single molecule and ensemble based super resolution images, it is now possible to explore the colocalization at the nanoscale between biomolecules in live and fixed cells. Despite the popularity and wide spread application of super resolution imaging, there exist only a few quantitative analysis paradigms for the colocalization of multicolor super-resolution images. Here, with the aid of conventional colocalization measurement paradigms and multivariate statistics, we analyze and report in detail the scale segregation and proximity of macromolecules within functional zones of synapses. Furthermore, we use these paradigms to evaluate fluorescent tags involved in the routine generation of single molecule based super-resolution images. We extend our analysis to elucidate in depth the co-aggregation and clustering of two key synaptic molecules, PSD95 and AMPARs, which are involved in basal synaptic organization and transmission
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Gross, Linda C. M. "Applications of droplet interface bilayers : specific capacitance measurements and membrane protein corralling." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:0b7ffba6-b86d-499c-a93f-3b2fc46a427b.
Анотація:
Droplet Interface Bilayers (DIBs) have a number of attributes that distinguish them from conventional artificial lipid bilayers. In particular, the ability to manipulate bilayers mechanically is explored in this thesis. Directed bilayer area changes are used to make precise measurements of the specific capacitance of DIBs and to control the two dimensional concentration of a membrane protein reconstituted in the bilayer. Chapter 1 provides a general introduction to the role of the lipid membrane en- vironment in the function of biological membranes and their integral proteins. An overview of model lipid bilayer systems is given. Chapter 2 introduces work carried out in this laboratory previously and illustrates the experimental setup of DIBs. Some important bilayer biophysical concepts are covered to provide the theoretical background to experiments in this and in later chapters. Results from the characterisation of DIBs are reported, and an account of the development of methods to manipulate the bilayer by mechanical means is given. Chapter 3 describes experiments that apply bilayer area manipulation in DIBs to achieve precise measurement of specific capacitance in a range of lipid systems. Chapter 4 reports results from experiments investigating the response of bilayer specific capacitance to an applied potential. Chapter 5 covers the background and experimental setup for total internal fluo- rescence microscopy experiments in DIBs and describes the expression, purification and characterisation of the bacterial β-barrel membrane protein pore α-Hemolysin. Chapter 6 describes experiments that apply the mechanical manipulation of bilayer area in DIBs to the corralling and control of the surface density of α-Hemolysin.
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Gennerich, Arne. "Fluoreszenzkorrelationsspektroskopie und Rasterkorrelationsmikroskopie molekularer Prozesse in Nervenzellen." Doctoral thesis, [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=971029008.
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Haziza, Simon. "Quantification du transport intraneuronal par suivi de nanodiamants fluorescents. Application à l’étude de l’impact fonctionnel de facteurs de risque génétiques associés aux maladies neuropsychiatriques." Thesis, Université Paris-Saclay (ComUE), 2015. http://www.theses.fr/2015SACLN013/document.
Анотація:
L’identification de biomarqueurs des maladies mentales telles que l’autisme, la schizophrénie ou la maladie d’Alzheimer, est d’une importance capitale non seulement pour établir un diagnostic objectif, mais aussi pour suivre l’effet des traitements. La création et le maintien de fonctions neuronales sub-cellulaires, telle que la plasticité synaptique, sont fortement dépendants du transport intraneuronal, essentiel pour acheminer d’importants composants à des positions spécifiques. Un transport actif défaillant semble être partiellement responsable d’anomalies de la plasticité synaptique et de la morphologie neuronale présentes dans de nombreuses maladies neuropsychiatriques. Cette thèse décrit (i) la mise au point d’une méthode de quantification du transport intraneuronal reposant sur le suivi de nanoparticules de diamants fluorescents (fNDs); (ii) l’application de cette technique simple et faiblement invasive à l’analyse fonctionnelle de variants génétiques associés à des maladies neuropsychiatriques. Ce manuscrit comporte quatre chapitres. Le premier détaille l’architecture polygénique complexe des maladies mentales et démontre la pertinence d’étudier le transport intraneuronal. Les deuxième et troisième chapitres sont dédiés à la méthode et détaillent les stratégies d’internalisation des fNDs, les outils de quantification du transport intraneuronal et la validation de la technique. La forte brillance, la photo-stabilité parfaite et l’absence de toxicité cellulaire font des fNDs un outil de choix pour étudier la dynamique du transport intraneuronal sur une durée d’observation de plusieurs heures avec une haute résolution spatiotemporelle et une bonne puissance statistique. Enfin, dans le quatrième chapitre, nous appliquons cette nouvelle méthode d’analyse fonctionnelle pour étudier l’effet de variants génétiques associés à l’autisme et à la schizophrénie. Pour cela, nous utilisons des lignées de souris transgéniques ayant une faible surexpression des gènes MARK1 et SLC25A12, ainsi que des AAV-shRNA pour induire une haplo-insuffisance du gène AUTS2. Notre méthode de diagnostic moléculaire s’avère suffisamment sensible pour déceler des variations fines de la dynamique du transport intraneuronal, ouvrant la voie à de futurs développements en nanomédecine translationnelleThe identification of molecular biomarkers of brain diseases as diverse as autism, schizophrenia and Alzheimer’s disease, is of crucial importance not only for an objective diagnosis but also to monitor response to treatments. The establishment and maintenance of sub-cellular neuronal functions, such as synaptic plasticity, are highly dependent on intracellular transport, which is essential to deliver important materials to specific locations. Abnormalities in such active transport are thought to be partly responsible for synaptic plasticity and neuronal morphology impairment found in many neuropsychiatric and neurodegenerative diseases. This thesis reports (i) the development of a quantification technic of intraneuronal transport based on fluorescent nanodiamonds (fNDs) tracking; (ii) the application of this simple and minimally invasive approach to the functional analysis of neuropsychiatric disease-related genetic variants.This manuscript falls into four chapters. The first one details the complex polygenic architecture of mental disorders and demonstrates the disease relevance of monitoring the intraneuronal transport. The second and the third chapters are dedicated to the nanodiamond-tracking assay and describe the fNDs internalisation strategies, the spatiotemporal quantitative readouts and the validation of the technic. The high brightness, the perfect photostability and the absence of cytotoxicity make fNDs a tool of choice to perform high throughput long-term bioimaging at high spatiotemporal resolution. Finally, in the fourth chapter, we apply this new functional analysis method to study the effect of genetic variants associated to autism and schizophrenia. We established transgenic mouse lines in which MARK1 and SLC25A12 genes were slightly overexpressed, and AAV-shRNA to induce AUTS2 gene haploinsufficiency. Our molecular diagnosis assay proves sufficiently sensitive to detect fine changes in intraneuronal transport dynamic, paving the way for future development in translational nanomedicine
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Kohram, Maryam. "A Combined Microscopy and Spectroscopy Approach to Study Membrane Biophysics." University of Akron / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=akron1436530389.
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Constals, Audrey. "Etude de la régulation glutamate dépendante de la mobilité des récepteurs AMPA et de son rôle physiologique." Thesis, Bordeaux 2, 2013. http://www.theses.fr/2013BOR22058.
Анотація:
Les récepteurs AMPA (rAMPA) sont les récepteurs ionotropiques du glutamate responsables de la majeure partie des courants excitateurs rapides dans la transmission synaptique rapide. Lors de la libération de glutamate, le rAMPA passe par 3 états conformationnels majoritaires : pore fermé/agoniste non lié, pore ouvert/agoniste lié et pore fermé/agoniste lié. Le contrôle du nombre et de l’organisation dans la synapse des rAMPA, via une combinaison de diffusion latérale et d’endo/exocytose, est essentiel à la régulation de l’intensité de la transmission synaptique. Les interactions existant entre les protéines de la densité post-synaptique et les protéines partenaires des récepteurs régulent la diffusion des récepteurs, contrôlant leur nombre et leur organisation à la post-synapse. Mon travail de thèse a consisté à étudier l’impact de l’activation des rAMPA sur leur mobilité et leur organisation à la post-synapse. En effet, la fixation de glutamate sur les récepteurs ainsi que leur désensibilisation entraînent des modifications structurales majeures affectant leurs interactions avec les protéines d’échafaudage et les protéines accessoires. L’impact de telles modifications sur les propriétés de diffusion et sur l’organisation sub-synaptique de ces rAMPA était jusqu’à présent inconnu. Mes travaux démontrent une mobilisation des rAMPA synaptiques consécutivement à leur activation par le glutamate. A l’échelle moléculaire, je propose que le passage de l’état activé à l’état désensibilisé des rAMPA entraîne un changement d’affinité de ces derniers pour une de leur protéine partenaire : la Stargazin. Cette régulation glutamate dépendante de la diffusion des rAMPA participe au maintien de la fidélité de la transmission synaptique rapideAMPA receptors (AMPAR) are ionotropic glutamate receptors which are responsible for the vast majority of fast excitatory synaptic currents in fast transmission. Upon release of glutamate, AMPAR undergo three main conformational states: pore closed/agonist unbound, pore open/agonist bound and pore closed/agonist bound. Controlling the number of AMPAR and their organization in the synapse, through a combination of lateral diffusion and endo/exocytosis, is essential to regulate the intensity of synaptic transmission. The interactions between proteins of the post-synaptic density and accessory receptor proteins regulate the distribution of receptors, controlling their number and organization in the post-synapse. During my PhD, I studied the impact of AMPAR activation on their mobility and organization in the post-synapse. Indeed, the binding of glutamate to AMPAR and their following desensitization lead to major structural changes on the receptor which impacts on their interactions with scaffolding proteins and accessory proteins. The impact of such modifications on the lateral diffusion and sub-synaptic organization of AMPAR was not known yet. My findings show a mobilization of synaptic AMPAR following their activation by glutamate. At the molecular level, I suggest that the transition from the activated state to the desensitized state of AMPAR leads to a change in affinity of the receptor for their partner protein: Stargazin. This glutamate dependent regulation of AMPAR diffusion participates in maintaining the fidelity of fast synaptic transmission
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Orré, Thomas. "Mécanismes moléculaires d’activation des intégrines par la kindline-2 lors de l’adhésion cellulaire." Thesis, Bordeaux, 2017. http://www.theses.fr/2017BORD0824/document.
Анотація:
Les adhérences focales (AF), structures adhésives reliant la cellule à la matrice extra-cellulaire (MEC), constituent de véritables plateformes de signalisation biochimique et mécanique qui contrôlent l'adhérence, la migration, la différenciation et la survie cellulaire. Les récepteurs transmembranaires intégrines sont au coeur des AF, où elles connectent la MEC au cytosquelette d'actine. Au début des années 2000, la protéine intracellulaire taline, qui se lie aux parties cytoplasmiques bêta des intégrines, était considérée comme le principal activateur des intégrines. Néanmoins, il a depuis été montré que la kindline, autre protéine intracellulaire se liant aux parties bêta cytoplasmiques, jouait également un rôle essentiel dans l'activation des intégrines. Ainsi,plusieurs études ont mis en évidence que la kindline et la taline étaient complémentaires et avaient une action synergique durant l'activation des intégrines. Les bases moléculaires de ces phénomènes restent à déterminer. De plus, la plupart des données sur lerôle de la kindline dans l'adhérence et l'activation des intégrines provient d'expériences menées sur des cellules en suspension et/ou avec l'intégrine plaquettaire αIIbβ3. Ainsi, la régulation de ces processus par la kindline dans les cellules adhérentes est encore peu comprise. Dans cette étude, nous combinons la microscopie PALM et le suivi de protéines individuelles pour révéler le rôle et le comportement de la kindline à l'intérieur et à l'extérieur des AF au cours des événements moléculaires clés se déroulant au niveau de la membrane plasmique, et qui mènent à l'activation des intégrines. Nous avons observé que les intégrines bêta1 etbêta3 portant une mutation ponctuelle inhibant l'interaction avec la kindline montrent un défaut d'immobilisation dans les AF. Nous avons également observé que la kindline-2, qui est enrichie dans les AF, diffusait librement au niveau de la membrane plasmique,à l'intérieur et à l'extérieur des AF. Ceci constitue une distinction majeure par rapport à la taline, qui, au niveau de la membrane plasmique, est essentiellement observée dans les AF où elle est immobile, montrant qu'elle est recrutée dans les AF directement depuis le cytosol sans diffusion latérale membranaire (Rossier et al. 2012). Afin d'identifier les bases moléculaires du recrutement et de la diffusion membranaire de la kindline, nous avons utilisé différents variants mutés de kindline précédemment décrits. Le mutant kindline-2-QW614/615AA (liaison aux intégrines inhibée) montre une diffusion membranaire accrue, ce qui suggère que la kindline peut diffuser au niveau de la membrane plasmique sans être associée aux intégrines. Par ailleurs, la baisse d'immobilisation au niveau des AF observée avec ce mutant montre qu'une partie de l'immobilisation de la kindline est due aux intégrines, suggérant l'existence d'un complexe intégrine-kindline immobile dans les AF. La délétion du domaine PleckstrinHomology (PH) de la kindline diminue considérablement son recrutement et sa diffusion membranaire. Nous avons évalué le rôle fonctionnel du recrutement et de la diffusion membranaire de la kindline en réexprimant ces mutants dans des cellules déplétéesen kindline-1 et -2 (cellules KO kindline-1 -/-, kindline-2 -/-). Ces expériences montrent que le recrutement et la diffusion membranaire de la kindline sont cruciaux pour l'activation des intégrines durant l'étalement cellulaire et favorisent la formation d’adhérences. Cela suggère que la kindline utilise un chemin différent de celui de la taline pour atteindre et activer les intégrines,ce qui pourrait expliquer au niveau moléculaire comment la kindline complémente la taline durant l'activation des intégrinesFocal adhesions (FAs) are adhesive structures linking the cell to the extracellular matrix (ECM) and constitute molecular platforms for biochemical and mechanical signals controlling cell adhesion, migration, differentiation and survival. Integrin transmembrane receptors are core components of FAs, connecting the ECM to the actin cytoskeleton. During the early 2000s, the intracellular protein talin, which directly binds to the cytoplasmic tail of β-integrins, was considered as the main integrin activator. Nevertheless, it has been shown that kindlin, another intracellular protein that bind to β-integrin, is also a critical integrin activator. In fact, several studies have shown that kindlin and talin play complementary and synergistic roles during integrin activation. The molecular basis of these phenomena remains to determine. Moreover, most studies focusing on the role of kindlin during integrin activation and cell adhesion have been performed with suspended cells and/or with the platelet integrin αIIbβ3. Here we combined PALM microscopy with single protein tracking to decipher the role and behavior of kindlin during key molecular events occurring outside and inside FAs at the plasma membrane and leading to integrin activation, as we have done previously for talin (Rossier et al., 2012). We found that beta1 and beta3-integrins with a point mutation inhibiting binding to kindlin show reduced immobilization inside FAs. We also found that kindlin-2, which is enriched inside FAs, displayed free diffusion at the plasma membrane outside and inside FAs. This constitutes a major difference with talin, which, at the plasma membrane level, is observed almost exclusively in FAs, where it is immobile, which shows that talin is recruited into FAs directly from the cytosol without lateral diffusion along the plasma membrane (Rossier et al. 2012). To determine the molecular basis of kindlin membrane recruitment and diffusion, we used a kindlin variant known to decrease binding to integrins (kindlin-2- QW614/615AA). This mutant displayed increased membrane diffusion, suggesting that kindlin-2 can freely diffuse at the plasma membrane without interacting with integrins. Moreover, the kindlin-2-QW mutant showed decreased immobilization inside FA, showing that part of kindlin immobilization depends on interaction with integrins. This suggests that kindlin can form an immobile complex with integrins inside focal adhesions. Deletion of the kindlin pleckstrin homology (PH) domain strongly reduced the membrane recruitment and diffusion of kindlin. We assessed the functional role of kindlin membrane recruitment and diffusion by re-expressing different kindlin-2 mutants in kindlin-1/kindlin-2 double KO cells. Those experiments demonstrated that kindlin-2 membrane recruitment and diffusion are crucial for integrin activation during cell spreading and favor adhesion formation. This suggests that kindlin uses a different route from talin to reach integrins and trigger their activation, providing a possible molecular basis for their complementarity during integrin activation
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Linarès-Loyez, Jeanne. "Développement de la microscopie par auto-interférences pour l'imagerie super-résolue tridimensionnelle au sein de tissus biologiques épais." Thesis, Bordeaux, 2019. http://www.theses.fr/2019BORD0167/document.
Анотація:
Le travail de cette thèse a été consacré au développement d’un nouvelle technique SELFI (pour self-interferences, auto-interférences en anglais). Cette méthode permet d’obtenir une localisation tridimensionnelle d’émetteurs fluorescents individuels. Nous avons démontré que cela permet l'imagerie super-résolue en 3D et le suivie 3D de molécules uniques en profondeur dans des échantillons biologiques denses et complexes. La technique SELFI se base sur l'utilisation des interférences auto-référencées (également appelées « auto-interférences ») pour remonter à la localisation 3D d’un émetteur en une seule mesure. Ces interférences sont générées via l’utilisation d'un réseau de diffraction placé en sortie du microscope de fluorescence : le signal de fluorescence diffracte sur le réseau et les ordres interfèrent, après une courte propagation, sur le détecteur. Les interférences ainsi formées sont décodées numériquement pour remonter à la localisation 3D d'une molécule fluorescente au sein de l'échantillon. Une molécule unique peut ainsi être localisée avec une précision d'une dizaine de nanomètre, et cela jusqu'à une profondeur d'au moins 50µm au sein d'un échantillon biologique vivant épais (par exemple un tissu biologique).En combinant la méthode SELFI à différentes techniques de super-résolution (PALM, dSTORM et uPAINT), nous montrons que cette méthode de localisation tridimensionnelle permet de retrouver la hiérarchie et l'organisation de protéines dans des objets biologiques. En effectuant du SELFI-PALM, nous avons pu observer différentes protéines des points focaux d’adhésion (talin-C terminale et paxiline) et retrouver les différences de hauteur attendues, et ceux sur des échantillons de cellules vivantes. Ces résultats confirment la résolution accessible avec la technique SELFI (environ 25nm) même pour un faible nombre de photons collectés (environ 500 photons par molécule).Nous mettons en évidence la robustesse de la technique SELFI en reconstruisant des images de super-résolution 3D de structures denses en profondeur dans des échantillons tissulaires complexes. En effectuant du SELFI-dSTORM, nous avons observé le réseau d’actine sur des cellules cultivées en surface de la lamelle dans un premier temps, et à différentes profondeurs (25 et 50 microns) au sein de tissus artificiels dans un second temps.Du suivi 3D de particule unique a aussi été effectué sein de tissus biologiques vivants. Nous avons observé la diffusion libre de quantum dots à différentes profondeurs (jusqu’à 50 microns, limité par l’objectif utilisé) dans des tranches vivantes de cerveau.Nous avons appliqué la technique SELFI à la détection de récepteurs postsynaptiques NMDA. Cela nous a permis d'observer, sur des échantillons de neurones en culture primaire mais aussi au sein de tranches de cerveaux de rats, une différence d'organisation entre les deux sous-unités GluN2A et GluN2B de ce récepteur au glutamate.Enfin, nous avons démontré l'importance de suivre l'évolution de l'environnement des échantillons biologiques vivants lors des acquisitions permettant la détection de molécules individuelles. Grâce à l'utilisation additionnelle et simultanée de l'imagerie de phase quantitative, nous avons pu étudier la dynamique de la membrane cellulaire durant l’activation par un facteur de croissance. L'analyse corrélative entre les images de phase quantitative en lumière blanche et les détections de molécules fluorescentes uniques permet d'obtenir de nouvelles informations pertinentes sur l'échantillon étudiéThe work of this thesis was devoted to the development of a new technique SELFI (for self-interferences). This method unlocks the three-dimensional localization of individual fluorescent emitters. We have demonstrated that this allows 3D super-resolved imaging and 3D tracking of single molecules deep into dense and complex biological samples. The SELFI technique is based on the use of self-referenced interference to go back to the 3D location of a emitter in a single measurement. These interferences are generated using a diffraction grating placed at the exit of the fluorescence microscope: the fluorescence signal diffracts on the grating and, after a short propagation, the orders interfere on the detector. The formed interferences are digitally decoded to extract the 3D location of a fluorescent molecule within the sample. A single molecule can thus be localized with a precision of approximatively ten nanometers up to a depth of at least 50 µm in a thick living biological sample (for example a biological tissue).By combining the SELFI method with different super-resolution techniques (PALM, dSTORM and uPAINT), we show that this three-dimensional localization method grants the access to the hierarchy and organization of proteins in biological objects. By performing SELFI-PALM, we observed different proteins of the adhesion focal points (talin C-terminal and paxilin) and found the expected elevation differences, and those within living cell samples. These results confirm the resolution capability of the SELFI technique (about 25 nm) even for a small number of photons collected (about 500photons per molecule).We highlight the robustness of the SELFI technique by reconstructing 3D super-resolution images of dense structures at depth in complex tissue samples. By performing SELFI-dSTORM, we observed the actin network in cells grown on the surface of the coverslip at first, and at different depths (25 and 50 microns) within artificial tissues in a second time.3D single particle tracking has also been performed in living biological tissues. We observed the free diffusion of quantum dots at different depths (up to 50 microns) in living brain slices.We applied the SELFI technique to the detection of NMDA postsynaptic receptors. We observed, in primary culture of neurons but also within slices of rat brains, a difference in organization between the two subunits GluN2A and GluN2B of this glutamate receptor.Finally, we show the importance of following the evolution of the living biological sample environment during the acquisition of images leading to detections of single molecules. Thanks to the additional and simultaneous use of quantitative phase imaging, we were able to study cell membrane dynamics during the activation by a growth factor. The correlative analysis between white light quantitative phase images and single fluorescent molecule detections provides new relevant information on the sample under study
39
Renancio, Cédric. "Étude du trafic vésiculaire des récepteurs glutamatergiques de type AMPA : caractérisation d’une nouvelle protéine auxiliaire." Thesis, Bordeaux 2, 2013. http://www.theses.fr/2013BOR22139/document.
Анотація:
Les récepteurs du glutamate de type AMPA (rAMPA) sont les acteurs principaux de la transmission synaptique excitatrice rapide. Leur abondance au niveau de la densité postsynaptique est essentielle pour l'établissement et le maintien de la fonction synaptique, et est le résultat d'un trafic hautement dynamique. De nombreuses études ont permis de caractériser les mécanismes de diffusion membranaire impliqués dans l’adressage des rAMPA jusqu’à la synapse. Le rôle majeur des protéines auxiliaires des rAMPA dans la modulation de cette étape de trafic a été démontré. Par ailleurs, il est suggéré que la localisation synaptique des rAMPA est aussi régulée lors des phases plus précoces du trafic intracellulaire, c’est-à-dire de l'appareil de Golgi vers la membrane plasmique via les vésicules post-Golgiennes. Cependant le trafic vésiculaire post-Golgien des rAMPA n'a jamais été visualisé et reste donc encore très mal compris. En collaboration avec l'équipe de Guus Smit (Amsterdam), j’ai participé à la caractérisation d’une nouvelle protéine auxiliaire des rAMPA, appelée Shisa6. Dans le cadre de ce projet, j’ai pu étudier le rôle de cette protéine sur la diffusion membranaire des rAMPA en utilisant une technique de suivi de particule unique (Quantum dot) développée au laboratoire. Mon projet de thèse principal a consisté à étudier le trafic vésiculaire post-Golgien des rAMPA par le développement d’une nouvelle méthode d’étude. En effet, l'échec dans la visualisation dynamique du trafic vésiculaire des récepteurs pourrait être expliqué par un faible rapport signal/bruit, conséquence d'une faible concentration vésiculaire en rAMPA combinée à un bruit de fond important dû aux marquages provenant du réticulum endoplasmique (RE) et de la membrane plasmique. Dans le but de surpasser cette difficulté, nous avons mis au point un outil ingénieux (système ARIAD) afin de bloquer les rAMPA dans le RE et contrôler, par l'ajout d'un ligand, leur sécrétion du RE jusqu'à la membrane plasmique. Grâce à cet outil, nous avons non seulement augmenté considérablement la concentration des rAMPA dans les vésicules post-Golgiennes, mais aussi éliminé le bruit de fond membranaire. Par la technique de FRAP nous avons pu éliminer le bruit de fond provenant du RE. Une telle approche, combinée à des techniques d'imagerie sur neurones vivants, nous a permis de visualiser pour la première fois le trafic vésiculaire post-Golgien des rAMPA et de l’étudierAMPA-type glutamate receptors (AMPAR) are the main actors of the fast excitatory synaptic transmission. Their abundance at the postsynaptic density is essential for the establishment and maintenance of synaptic function, and is the result of a highly dynamic trafficking. Many studies have characterized the membrane diffusion mechanisms involved in the AMPAR synaptic localization, and revealed the critical role of the AMPAR auxiliary proteins in the modulation of this trafficking. Furthermore, it is suggested that AMPAR synaptic localization is also regulated during the early steps of the intracellular trafficking, from the Golgi apparatus to the plasma membrane via the post-Golgi vesicles. However, the post-Golgi vesicular trafficking of AMPAR has never been visualized and therefore remains poorly understood. In collaboration with the Guus Smit team (Amsterdam), I participated in the caracterization of a novel AMPAR auxiliary protein called Shisa6. As part of this project, I studied the role of this protein on the AMPAR membrane diffusion, using a method of single particle tracking (Quantum dot) developed in the laboratory. My main thesis project was to study the post-Golgi vesicular trafficking of AMPAR through the development of a new experimental protocol. Indeed, the failure in the dynamic visualization of the receptor vesicular trafficking could be explained by a low signal/noise ratio resulting of a poor AMPAR vesicular concentration, combined with a high background noise due to receptors localized both in the endoplasmic reticulum (ER) and at the plasma membrane. In order to overcome this difficulty, we have used an ingenious tool (ARIAD system) so as to block AMPAR into the ER and, by adding a ligand, control their trafficking from the ER to the plasma membrane. Thanks to this tool we have not only significantly increased the AMPAR concentration in the post-Golgi vesicles, but also eliminated the plasma membrane background noise. The FRAP imaging technique was used in order to remove the ER background noise. Such methodological approach combined with imaging techniques in living neurons, allowed us to clearly visualize for the first time the post-Golgi vesicular trafficking of AMPAR, and to study the mechanisms involved in this trafficking
40
Octeau, Vivien. "Microscopie de nano-objets individuels : étude de la diffusion des intégrines dans les sites d'adhésion focales de cellules vivantes." Thesis, Bordeaux 1, 2010. http://www.theses.fr/2010BOR14047/document.
Анотація:
L’effet photothermique permet de détecter efficacement des nanoparticules d’or avec un microscope en champ lointain grâce à leur forte absorption de la lumière. L’absence de problème photophysique fait des nanoparticules d’or une alternative au marquage de biomolécules par des sondes fluorescentes. La méthode PhACS (Photothermal Absorption Correlation Spectroscopy) utilise les fluctuations de signal photothermique dues au passage de nanoparticules dans le volume de détection pour étudier leur diffusion. Cette méthode permet également la mesure précise de diamètres hydrodynamiques de nanoparticules fonctionnalisées. La méthode SNaPT (Single Nano-Particle Tracking) réalise le suivi bidimensionnel de nanoparticules individuelles grâce à une localisation effectuée par triangulation. Nous avons appliqué cette méthode pour étudier la diffusion des intégrines alphaV-beta3 marquées par des nanoparticules d’or de 5 nm dans les adhérences focales, points d’ancrage entre le cytosquelette de la cellule et la matrice extracellulaire. Nous observons que ces intégrines ont tendance à former des agrégats qui alternent entre un mouvement diffusif et un mouvement confiné. Ce résultat appelle maintenant à un nouveau modèle où nous aurions une redistribution continue des intégrines au sein des adhérences focalesGold nanoparticles may be detected with optical far-field microscopy by use of the photothermal effect due to their strong light absorbance. With no photophysic issues, gold nanoparticles are an alternative to fluorescent probes for use in biological systems. The PhACS method (Photothermal Absorption Correlation Spectroscopy) is used to study diffusion by measuring the autocorrelation of photothermal signal fluctuations due to nanoparticles passing through the detection volume. This method is sensitive enough to mesure the precise hydrodynamic diameter of functionalised nanoparticles. The SnaPT method (Single Nano-Particle Tracking) can track 2-dimensional motion of individual nanoparticles by pinpointing the localization with a triangulation method. The SNaPT method was used to study motion of alphaV-beta3 integrins that were bound to a 5 nm gold nanoparticle inside focal adhesion, where the cell cytoskeleton is linked to the extracullular matrix. The integrin was found to organize into clusters oscillating between the bound and diffuse states. These observations require new working models where integrins would be constantly redistributed
41
Salomon, Antoine. "Modélisations statistiques pour l'analyse de la diffusion des molécules et du trafic intracellulaire en microscopie de fluorescence." Electronic Thesis or Diss., Université de Rennes (2023-....), 2023. http://www.theses.fr/2023URENS125.
Анотація:
En permettant de visualiser de manière sélective les dynamiques des particules dans les cellules, la microscopie de fluorescence est un outil de premier plan pour la recherche biomédicale. Il existe par conséquent une forte demande d'outils algorithmiques capables d'analyser automatiquement des données microscopiques brutes. Après une présentation de la théorie et des techniques entourant les dynamiques des particules, la microscopie de fluorescence, les méthodes de suivi et la classification de mouvements, nous présentons dans cette thèse une nouvelle méthode de cartographie basée sur des estimateurs à noyaux spatio-temporels permettant d'estimer de manière robuste la diffusion et la dérive intracellulaires à partir des données de suivi. Nous l'évaluons dans un ensemble étendu d'expériences utilisant des données simulées, réelles, 2D et 3D et montrons que notre méthode fournit des cartes de diffusion et de dérive précises et exactes tout en étant plus performante que les méthodes existantes. En tant que telle, elle permet aux biologistes d'étudier les dynamiques intracellulaires de particules spécifiquement marquées avec une plus large gamme d'acquisitions et de techniques de microscopie de fluorescence. En outre, nous présentons une méthode de détection de domaines de confinement utilisant également le suivi de particules et la classification de mouvementsFluorescence microscopy is a tool of primary interest in biomedical research as it allows to selectively visualize particle dynamics within the cell. Hence, there is a high demand for algorithmic tools capable of automatically analyzing raw microscopic data. After a presentation of the theory and techniques surrounding particle dynamics, fluorescence microscopy, tracking methods and motion classification, we present in this thesis a new mapping method based on spatiotemporal kernel estimators that robustly estimate intracellular diffusion and drift from tracking data. We evaluate it in an extensive set of experiments using simulated, real, 2D and 3D data and show that our method provides precise and accurate diffusion and drift maps while outperforming existing methods. As such, it allows biologists to study intracellular dynamics of specifically tagged particles with a wider range of acquisitions and fluorescence microscopy techniques. In addition, we present a confinement domain detection method, using particle tracks and motion classification as well
42
Simon, Apolline A. "Décryptage des paramètres physico-chimiques critiques favorisant la diffusion efficace des nanoparticules dans des modèles tumoraux." Electronic Thesis or Diss., Bordeaux, 2024. http://www.theses.fr/2024BORD0046.
Анотація:
La bio-imagerie d’environnements biologiques, complexes et hétérogènes utilisant des nanoparticules est uniquement pertinente si leurs propriétés intrinsèques et de surface sont contrôlées pour favoriser une diffusion en profondeur. En effet, la forme (rapports de forme, nanotubes, nanosphères), les dimensions (quelques nanomètres à quelques dizaines de nanomètres), les charges de surface et les interactions de surface avec le milieu environnant sont des paramètres décisifs. Ils régulent par exemple la mobilité et le devenir des nanoparticules à l’intérieur des milieux biologiques, dont par exemple les microenvironnements tumoraux. Dans cette thèse, nous nous sommes principalement concentrés sur les nanotubes de carbone, semi-conducteurs et à mono-parois dans le but d’adapter leur diffusion dans des environnements biologiques en contrôlant leurs propriétés de surface. Ce choix a été motivé car ces nanotubes présentent des atouts majeurs pour des applications en bio-imagerie. Leur longueur d’onde d’émission se trouve dans l’infrarouge court (SWIR), ce qui correspond à la deuxième fenêtre de transparence biologique. Ils sont également photostables et il a été prouvé qu’ils présentent une pénétration importante dans le tissu de par leur morphologie 1D. Pour étudier la mobilité des nanotubes dans des environnements complexes, nous avons suivi leurs trajectoires à l’échelle de la particule unique et appliqué les approches de microscopie de fluorescence à super-résolution. Nous avons tout d’abord détecté des modifications morphologiques associées à une fibrose à un stade précoce sur des tranches de foie de souris. Pour cela nous avons employé une stratégie de microscopie corrélative pour identifier l’environnement biologique in situ (noyaux et membranes des cellules) autour des nanotubes en plus de l’étude de leurs mobilités. Ce premier projet nous a motivé à explorer une deuxième stratégie pour disperser des nanotubes en augmentant leur brillance tout en conservant leur furtivité. Nous avons étudié de quelle manière le changement du coating autour du nanotubes (taille moléculaire du PEG ou présence d’une insaturation) impactait leur brillance et leur diffusivité. Cette diffusion a été testée sur différents modèles avec une hétérogénéité croissante allant du gel d’agarose à des extraits de matrice extracellulaire. Nous avons distingué deux tailles moléculaires de PEG découlant sur des dispersions compatibles à nos études. Enfin, dans le but d’étendre la bibliothèque de nanoparticules émettant dans le SWIR pour des applications de bio-imagerie, nous avons étudié un autre type de nanoobjets luminescents : des clusters d’or et des nanoparticules polymères chargées avec des clusters. L’analyse de leur luminescence ainsi que leur potentiel pour des études de suivi de particules uniques ont été évalués. L’analyse de clusters d’or uniques a été réalisée montrant une excellente brillance, mais uniquement dans un environnement sec. Par ailleurs, nous avons prouvé que les nanoparticules polymériques étaient détectables au niveau de la particule unique lorsqu’elles diffusaient dans un environnement aqueux ce qui fait d’elles d’excellentes candidates pour des applications de bio-imagerieBioimaging of complex and heterogeneous biological environments using nanoparticles is only relevant if one controls their intrinsic and surface properties to promote their diffusion in depth. Indeed, the shape (i.e. aspect ratios, nanotubes, nanospheres), the dimension (from a few nanometers up to a few tens of nanometers), the surface charges and the surface interactions with the surrounding environment are key parameters. They regulate for instance the mobility and the future of nanoparticles inside the biological milieu, such as tumoral microenvironments. In this PhD thesis, we mainly focused on semiconducting single-walled carbon nanotubes with the aim to tailor and apply their diffusion in bio-environments by controlling their surface properties. This choice was motivated by their exceptional advantages for bio-imaging applications. Their emission wavelength is in the short-wave infrared region (SWIR), which corresponds to the second window of biological transparency. In addition, they are photostable and it has been proved that they show a high tissue penetration ability due to their nanoscale 1D morphology. To study the mobility of nanotubes in complex environments, we tracked their trajectories at the single particle level and applied super-resolution fluorescence microscopy approaches. We first detected morphological modifications associated with early-stage fibrosis on murine liver slices. To that end, we employed a correlative microscopy strategy to identify the in situ biological environment (cell membranes and nuclei) surrounding the nanotubes in addition to the study of their mobilities. This first work motivated us to explore a second strategy to suspend the nanotubes to enhance their brightness while maintaining their stealth behaviours. We investigated how changing the coating around the nanotubes (PEG molecular size or presence of an insaturation) impacted their brightness and diffusivity. Diffusion has been tested within various models with growing complexity from an agarose gel to extracts of the extracellular matrix. We distinguished two molecular sizes of PEG rising to suspensions of nanotubes suitable for our studies. Finally, with the aim of expanding the library of SWIR-emitting nanoparticles for biological imaging, we investigated another type of luminescent nanoobjects: gold nanoclusters and polymeric nanoparticles loaded with such clusters. The analysis of their luminescence as well as their potential for single particle tracking were evaluated. Single gold cluster analysis has been conducted showing excellent brightness, but only in a dried environment. In addition, the polymeric nanoparticles were shown to be detectable at the single particle level diffusing within an aqueous media constituting promising candidates for bioimaging applications
43
Höfer, Chris Tina. "Influenza virus assembly." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2015. http://dx.doi.org/10.18452/17251.
Анотація:
Influenza A Viren besitzen ein segmentiertes, einzelsträngiges RNA-Genom, welches in Form viraler Ribonukleoprotein (vRNP)-Komplexe verpackt ist. Während das virale Genom im Zellkern repliziert wird, finden Assemblierung und Knospung reifer Viruspartikel an der apikalen Plasmamembran statt. Für die Virusbildung müssen die einzelnen viralen Komponenten hierher gebracht werden. Während intrinsische apikale Signale der viralen Transmembranproteine bekannt sind, sind der zielgerichtete Transport und der Einbau des viralen Genoms in neuentstehende Virionen noch wenig verstanden. In dieser Arbeit wurden potentielle Mechanismen des vRNP-Transportes untersucht, wie die Fähigkeit der vRNPs mit Lipidmembranen zu assoziieren und die intrinsische subzellulären Lokalisation des viralen Nukleoproteins (NP), eines Hauptbestandteils der vRNPs. Es konnte gezeigt werden, dass vRNPs nicht mit Lipidmembranen assoziieren, was mittels Flotation aufgereinigter vRNPs mit Liposomen unterschiedlicher Zusammensetzung untersucht wurde. Die Ergebnisse deuten jedoch darauf hin, dass das virale M1 in der Lage ist, Bindung von vRNPs an negativ-geladene Lipidmembranen zu vermitteln. Subzelluläre Lokalisation von NP wurde des Weiteren durch Expression fluoreszierender NP-Fusionsproteine und Fluoreszenzphotoaktivierung untersucht. Es konnte gezeigt werden, dass NP allein nicht mit zytoplasmatischen Strukturen assoziiert, stattdessen aber umfangreiche Interaktionen im Zellkern eingeht und mit hoher Affinität mit bestimmten Kerndomänen assoziiert, und zwar den Nukleoli sowie kleinen Kerndomänen, welche häufig in der Nähe von Cajal-Körperchen und PML-Körperchen zu finden waren. Schließlich wurde ein experimenteller Ansatz etabliert, welcher erlaubt, den Transport vRNP-ähnlicher Komplexe mittels Fluoreszenzdetektion aufzuzeichnen und Einzelpartikelverfolgungsanalysen durchzuführen. Unterschiedliche Phasen des vRNP-Transportes konnten beobachtet werden und ein 3-Phasen-Transportmodell wird skizziert.Influenza A viruses have a segmented single-stranded RNA genome, which is packed in form of viral ribonucleoprotein (vRNP) complexes. While the viral genome is replicated and transcribed in the host cell nucleus, assembly and budding of mature virus particles take place at the apical plasma membrane. Efficient virus formation requires delivery of all viral components to this site. While intrinsic apical targeting signals of the viral transmembrane proteins have been identified, it still remains poorly understood how the viral genome is transported and targeted into progeny virus particles. In this study, potential targeting mechanisms were investigated like the ability of vRNPs to associate with lipid membranes and the intrinsic ability of the viral nucleoprotein (NP) – which is the major protein component of vRNPs – for subcellular targeting. It could be shown that vRNPs are not able to associate with model membranes in vitro, which was demonstrated by flotation of purified vRNPs with liposomes of different lipid compositions. Results indicated, however, that the matrix protein M1 can mediate binding of vRNPs to negatively charged lipid bilayers. Intrinsic subcellular targeting of NP was further investigated by expression of fluorescent NP fusion protein and fluorescence photoactivation, revealing that NP by itself does not target cytoplasmic structures. It was found to interact extensively with the nuclear compartment instead and to target specific nuclear domains with high affinity, in particular nucleoli and small interchromatin domains that frequently localized in close proximity to Cajal bodies and PML bodies. An experimental approach was finally established that allowed monitoring the transport of vRNP-like complexes in living infected cells by fluorescence detection. It was possible to perform single particle tracking and to describe different stages of vRNP transport between the nucleus and the plasma membrane. A model of three-stage transport is suggested.
44
Glushonkov, Oleksandr. "Imagerie de fluorescence à haute résolution : étude de la localisation nucléolaire de la protéine de la nucléocapside du VIH." Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAJ028/document.
Анотація:
Au cours de ce travail de thèse expérimental, nous nous sommes intéressés à l’étude de la localisation nucléaire et nucléolaire de la protéine de la nucléocapside (NC) du VIH-1. Des études antérieures menées au laboratoire avaient mis en évidence une très forte accumulation de la NC dans les nucléoles. Ce compartiment nucléaire est connu pour être ciblé par de nombreux virus afin de promouvoir leur réplication. Des expériences de microscopie électronique avaient révélé la structure complexe du nucléole et montré qu’il est composé de trois sous-compartiments : les centres fibrillaires, le compartiment fibrillaire dense et le compartiment granulaire dans lesquels se déroule la synthèse des ribosomes. Afin de caractériser la localisation de la NC dans ces trois sous-compartiments, nous avons développé une approche de microscopie optique à haute résolution permettant d’obtenir des images à deux couleurs avec une résolution spatiale améliorée. Pour cela, nous avons mis au point un protocole qui permet d’utiliser simultanément une protéine fluorescente photocommutable et un fluorophore organique introduit par immunomarquage. Après avoir minimisé les aberrations optiques et corrigé les dérives mécaniques inhérentes au montage, nous avons visualisé simultanément la localisation de la NC surexprimée dans des cellules HeLa avec des marqueurs spécifiques des trois sous-compartiments nucléolaires (immunomarquage). La microscopie de fluorescence à haute résolution a permis de résoudre pour la première fois les différents compartiments et de montrer que la NC se localise préférentiellement dans le compartiment granulaire. Finalement, des expériences préliminaires avec des cellules vivantes ont permis de mettre en évidence que la NC est transportée de manière active dans le noyau et qu’elle pourrait interagir directement avec des protéines nucléolairesDuring this experimental thesis work, we investigated the nuclear and nucleolar localization of the nucleocapsid protein (NC) of HIV-1. Previous studies performed in our laboratory evidenced a strong accumulation of NC in a subnuclear structure called nucleolus. Playing role in multiple cellular processes, nucleolus is often targeted by viruses to promote their replication. Electron microscopy revealed three nucleolar components (fibrillar centers, dense fibrillar component and granular component) associated to specific steps of the ribosome biogenesis. To characterize the distribution of the NC in these three sub-compartments and therefore shed light on the nucleolar localization of NC during the replication cycle, we developed a high-resolution optical microscopy approach. After having minimized the optical aberrations and corrected the mechanical drifts inherent to the imaging setup, the NC-mEos2 fusion protein overexpressed in HeLa cells was visualized simultaneously with immunolabeled nucleolar markers. The use of high-resolution fluorescence microscopy enabled us to resolve for the first time the three nucleolar compartments and to demonstrate the preferential localization of NC in the granular compartment of nucleolus. Finally, preliminary experiments performed with living cells showed that NC is actively transported in the nucleus and therefore may interact directly with nucleolar proteins
45
FANTACCI, CLAUDIO. "Distributed multi-object tracking over sensor networks: a random finite set approach." Doctoral thesis, 2015. http://hdl.handle.net/2158/1003256.
Анотація:
The aim of the present dissertation is to address distributed tracking over a network of heterogeneous and geographically dispersed nodes (or agents) with sensing, communication and processing capabilities. Tracking is carried out in the Bayesian framework and its extension to a distributed context is made possible via an information-theoretic approach to data fusion which exploits consensus algorithms and the notion of Kullback–Leibler Average (KLA) of the Probability Density Functions (PDFs) to be fused.
The first step toward distributed tracking considers a single moving object. Consensus takes place in each agent for spreading information over the network so that each node can track the object. To achieve such a goal, consensus is carried out on the local single-object posterior distribution, which is the result of local data processing, in the Bayesian setting, exploiting the last available measurement about the object. Such an approach is called Consensus on Posteriors (CP). The first contribution of the present work is an improvement to the CP algorithm, namely Parallel Consensus on Likelihoods and Priors (CLCP). The idea is to carry out, in parallel, a separate consensus for the novel information (likelihoods) and one for the prior information (priors). This parallel procedure is conceived to avoid underweighting the novel information during the fusion steps. The outcomes of the two consensuses are then combined to provide the fused posterior density. Furthermore, the case of a single highly-maneuvering object is addressed. To this end, the object is modeled as a jump Markovian system and the multiple model (MM) filtering approach is adopted for local estimation. Thus, the consensus algorithms needs to be re-designed to cope with this new scenario. The second contribution has been to devise two novel consensus MM filters to be used for tracking a maneuvering object. The novel consensus-based MM filters are based on the First Order Generalized Pseudo-Bayesian (GPB1) and Interacting Multiple Model (IMM) filters.
The next step is in the direction of distributed estimation of multiple moving objects. In order to model, in a rigorous and elegant way, a possibly time-varying number of objects present in a given area of interest, the Random Finite Set (RFS) formulation is adopted since it provides the notion of probability density for multi-object states that allows to directly extend existing tools in distributed estimation to multi-object tracking. The multi-object Bayes filter proposed by Mahler is a theoretically grounded solution to recursive Bayesian tracking based on RFSs. However, the multi-object Bayes recursion, unlike the single-object counterpart, is affected by combinatorial complexity and is, therefore, computationally infeasible except for very small-scale problems involving few objects and/or measurements. For this reason, the computationally tractable Probability Hypothesis Density (PHD) and Cardinalized PHD (CPHD) filtering approaches will be used as a first endeavour to distributed multiobject filtering. The third contribution is the generalisation of the single-object KLA to the RFS framework, which is the theoretical fundamental step for developing a novel consensus algorithm based on CPHD filtering, namely the Consensus CPHD (CCPHD). Each tracking agent locally updates multi-object CPHD, i.e. the cardinality distribution and the PHD, exploiting the multi-object dynamics and the available local measurements, exchanges such information with communicating agents and then carries out a fusion step to combine the information from all neighboring agents.
The last theoretical step of the present dissertation is toward distributed filtering with the further requirement of unique object identities. To this end the labeled RFS framework is adopted as it provides a tractable approach to the multi-object Bayesian recursion. The δ- GLMB filter is an exact closed-form solution to the multi-object Bayes recursion which jointly yields state and label (or trajectory) estimates in the presence of clutter, misdetections and association uncertainty. Due to the presence of explicit data associations in the δ-GLMB filter, the number of components in the posterior grows without bound in time. The fourth contribution of this thesis is an efficient approximation of the δ-GLMB filter, namely Marginalized δ-GLMB (Mδ-GLMB), which preserves key summary statistics (i.e. both the PHD and cardinality distribution) of the full labeled posterior. This approximation also facilitates efficient multi-sensor tracking with detection-based measurements. Simulation results are presented to verify the proposed approach. Finally, distributed labeled multi-object tracking over sensor networks is taken into account. The last contribution is a further generalization of the KLA to the labeled RFS framework, which enables the development of two novel consensus tracking filters, namely the Consensus Marginalized δ-Generalized Labeled Multi-Bernoulli (CM-δGLMB) and the Consensus Labeled Multi-Bernoulli (CLMB) tracking filters. The proposed algorithms provide a fully distributed, scalable and computationally efficient solution for multi-object tracking.
Simulation experiments on challenging single-object or multi-object tracking scenarios confirm the effectiveness of the proposed contributions.
46
Zelman-Femiak, Monika. "Single Particle Tracking ; Membrane Receptor Dynamics." Doctoral thesis, 2011. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-65420.
Анотація:
Single-molecule microscopy is one of the decisive methodologies that allows one to clarify cellular signaling in both spatial and temporal dimentions by tracking with nanometer precision the diffusion of individual microscopic particles coupled to relevant biological molecules. Trajectory analysis not only enables determination of the mechanisms that drive and constrain the particles motion but also to reveal crucial information about the molecule interaction, mobility, stoichiometry, all existing subpopulations and unique functions of particular molecules. Efficacy of this technique depends on two problematic issues the usage of the proper fluorophore and the type of biochemical attachment of the fluorophore to a biomolecule. The goal of this study was to evolve a highly specific labeling method suitable for single molecule tracking, internalization and trafficking studies that would attain a calculable 1:1 fluorophore-to-receptor stoichiometry. A covalent attachment of quantum dots to transmembrane receptors was successfully achieved with a techinque that amalgamates acyl carrier protein (ACP) system as a comparatively small linker and coenzyme A (CoA)-functionalized quantum dots. The necessity of optimization of the quantum dot usage for more precise calculation of the membrane protein stoichiometries in larger assemblies led to the further study in which methods maximizing the number of signals and the tracking times of diverse QD types were examined. Next, the optimized techniques were applied to analyze behavior of interleukin-5 β-common chain receptor (IL-5Rβc) receptors that are endogenously expressed at low level on living differentiated eosinophil-like HL-60 cells. Obtained data disclosed that perused receptors form stable and higher order oligomers. Additionally, the mobility analysis based on increased in number (>10%) uninterrupted 1000-step trajectories revealed two patterns of confined motion. Thereupon methods were developed that allow both, determination of stoichiometries of cell surface protein complexes and the acquisition of long trajectories for mobility analysis. Sequentially, the aforementioned methods were used to scrutinize on the mobility, internalization and recycling dynamics characterization of a G protein-coupled receptor (GPCRs), the parathyroid hormone receptor (PTHR1) and several bone morphogenetic proteins (BMPs), a member of the TGF-beta superfamily of receptors. These receptors are two important representatives of two varied membrane receptor classes. BMPs activate SMAD- and non-SMAD pathways and as members of the transforming growth factor β (TGF-β) superfamily are entailed in the regulation of proliferation, differentiation, chemotaxis, and apoptosis. For effective ligand induced and ligand independent signaling, two types of transmembrane serine/threonine kinases, BMP type I and type II receptors (BMPRI and BMPRII, respectively) are engaged. Apparently, the lateral mobility profiles of BMPRI and BMPRII receptors differ markedly, which determinate specificity of the signal. Non-SMAD signaling and subsequent osteoblastic differentiation of precursor cells particularly necessitate the confinement of the BMP type I receptor, resulting in the conclusion that receptor lateral mobility is a dominative mechanism to modulate SMAD versus non-SMAD signaling during differentiation. Confined motion was also predominantly observed in the studies devoted to, entailed in the regulation of calcium homeostasis and in bone remodeling, the parathyroid hormone receptor (PTHR1), in which stimulation with five peptide ligands, specific fragments of PTH: hPTH(1–34), hPTHrP(107–111)NH2; PTH(1–14); PTH(1–28) G1R19, bPTH(3–34), first four belonging to PTH agonist group and the last to the antagonist one, were tested in the wide concentration range on living COS-1 and AD293 cells. Next to the mobility, defining the internalization and recycling rates of the PTHR1 receptor maintained in this investigation one of the crucial questions. Internalization, in general, allows to diminish the magnitude of the receptor-mediated G protein signals (desensitization), receptor resensitization via recycling, degradation (down-regulation), and coupling to other signaling pathways (e.g. MAP kinases). Determinants of the internalization process are one of the most addressed in recent studies as key factors for clearer understanding of the process and linking it with biological responses evoked by the signal transduction. The internalization of the PTH-receptor complex occurs via the clathrin-coated pit pathway involving β-arrestin2 and is initiated through the agonist occupancy of the PTHR1 leading to activation of adenylyl cyclase (via Gs), and phosphatidylinositol-specific phospholipase Cβ (via Gq). Taken together, this work embodies complex study of the interleukin-5 β-common chain receptor (IL-5Rβc) receptors, bone morphogenetic proteins (BMPs) and the parathyroid hormone receptor with the application of single-molecule microscopy with the newly attained ACP-quantum dot labeling method and standard techniquesDie Einzelmolekül-Mikroskopie, das Verfolgen der Diffusion einzelner, mikroskopischer Partikel, welche an relevanten biologischen Molekülen gekoppelt sind, ist eine der entscheidenden Verfahren zur räumlichen und zeitlichen Quantifizierung der Zellsignalisierung und hat eine Genauigkeit im Nanometerbereich. Die so gewonnene Trajektorienanalyse ermöglicht nicht nur die Bestimmung der Mechanismen, die der Bewegung der Partikel zugrunde liegen, sondern liefert auch wichtige Informationen über die molekulare Wechselwirkungen, Bewegungsfreiheit und Stöchiometrie sowie über alle existierenden Subpopulationen und besondere Funktionen der einzelnen Moleküle. Die Wirksamkeit dieser Technik hängt von der Verwendung des geeigneten Flurophors und der Art seiner biochemischen Anhaftung ab. Das Ziel dieser Arbeit war die Entwicklung eines hochspezifischen Markierungsverfahrens, das zur Verwendung der Einzelmolekül-Mikroskopie für Studien im Bereich Endozytose geeignet ist und gleichzeitig eine Fluorophore-Rezeptor Stöchiometrie von 1:1 erreicht. Eine kovalente Anhaftung von Quantenpunkten an Membranrezeptoren wurde erfolgreich in einer Methode realisiert, die ACP-Systeme (Engl. Acyl-Carrier-Protein) mit Koenzym A (CoA-) funktionalisierten Quantenpunkten amalgamiert. Die notwendige Optimierung der Verwendung von Quantenpunkten mit dem Ziel einer genaueren Berechnung der Stöchiometrie von Membranproteinen sehr großer Anzahl führte zu weiteren Studien. In diesem Zusammenhang wurden Methoden zur Maximierung der Signalanzahl und Beobachtungszeiten diverser Quantenpunktentypen untersucht. Im nächsten Schritt wurden die optimierten Verfahren angewendet, um das Verhalten von IL-5Rßc (Engl. Interleukin-5 ß-common chain receptor) Rezeptoren, die endogen auf niedriger Stufe auf lebende differenzierte eosinophile-ähnlichen HL-60 Zellen existieren, zu analysieren. Die gewonnenen Daten haben gezeigt, dass die Rezeptoren sich in stabilen Oligomeren hoher Ordnung bilden, was zusätzlich mit den Ergebnissen der Analyse der Mobilität, die auf einer hohen Anzahl unterbrochener 1000-Schritt Trajektorien basiert, zwei abgegrenzte Bewegungsmuster ergab. Daraufhin wurden Methoden entwickelt, die eine Bestimmung der Stöchiometrie von Zelloberflächen-Proteinkomplexen und die Erfassung umfangreicher Trajektorien zur Bewegungsanalyse ermöglichen. Im Weiteren wurden die zuvor genannten Methoden zur genauen Überprüfung der Mobilität, Endozytose und der Charakterisierung der rückläufigen Dynamik der repräsentativen Rezeptoren von zwei verschiedenen Membranrezeptoren Klassen, des Parathormon-Rezeptors (Engl. the parathyroid hormone receptor), der zu der G-Protein-gekoppelter Rezeptor Gruppe (GPCRs) gehört und der Rezeptoren der knochenmorphogenetischen Proteine (BMPs) verwendet. BMPs aktivieren SMAD- und non-SMAD Signalkaskaden und als ein Bestandteil des TGF-β-Signalszstem sind sie in die Proliferation, die Differenyiation, die Chemotaxis und die Apoptose involviert. Zwei BMP Rezeptor Typen, BMP Typ I und BMP Typ II (BMPRI und BMPRII) sind nötig für die effektive Signalwirkung. Offenbar sind die Bewegungsmuster für BMPRI und BMPRII sehr unterschiedlich, was hier die Genauigkeit des Signals festlegt. Non-SMAD Kaskade und die nachfolgende Differenzierung von den Osteoblastenzellen benötigt das abgegrenzte Bewegungsmuster von BMPRI. Daraus folgert, dass die laterale Mobilität ein Hauptmechanismus in der SMAD gegen non-SMAD Signalwirkung während der Differenziation ist. Das abgegrenzte Bewegungsmuster war auch für den Parathormon Rezeptor (Engl. the parathyroid hormone receptor) (PTHR1), der in die Calcium Homeostase und den Knochenumbau involviert ist, in den Studien zu beobachten. In diesen Studien wurden fünf Peptide Ligande, spezifische Teile von dem PTH: hPTH(1–34), hPTHrP(107–111)NH2; PTH(1–14); PTH(1–28) G1R19, bPTH(3–34), von denen die ersten vier zu der Agonistengruppe und der Letzte zu der Antagonistengruppe gehören, in verschiedenen Konzentrationen mit lebenden COS-1 und AD293 Zellen verwendet. (oder aufgebracht) Eine der Hauptfragen war die Festlegung der Rate der PTHR1 Internalisierung und des Recycling in dieser Forschung. Im Allgemeinen reduziert Internalisierung die Stärke der Signale, die von den G Proteinen kommen und durch die Rezeptoren übermittelt (die Desensibilisierung) werden. Durch den Rücklauf werden die Rezeptoren wieder sensibilisiert, degradiert und können somit an anderen Signalkaskaden ankoppeln (zB. MAP-Kinase ). Die Determinanten der Internalisierung sind das Hauptthema in den aktuellen Studien, da sie der Schlüssel zum besseren Verständnis der Internalisierung und zu den nachfolgenden biologischen Antworten sind. Die Internalisierung von dem PTH Rezeptor verläuft entsprechend des Clathrin-coated Pit Weges mit der Teilnahme von β-arrestin2 und ist durch den Ligand eingeleitet, der zur Aktivierung von adenylyl cyclase (via Gs), und phosphatidylinositol-specific phospholipase Cβ (via Gq) führt. Zusammenfassend ist diese Arbeit unter Verwendung von Einzelmolekül-Mikroskopie mit der neuen ACP-Quantumpunktmethoden sowie standard Markierungsmethoden ein komplexes Studium über die IL-5Rßc Rezeptoren, die BMP Rezeptoren und den PTH Rezeptor
47
Li, Zhenning. "Single View Human Pose Tracking." Thesis, 2013. http://hdl.handle.net/10012/7543.
Анотація:
Recovery of human pose from videos has become a highly active research area in the last decade because of many attractive potential applications, such as surveillance, non-intrusive motion analysis and natural human machine interaction. Video based full body pose estimation is a very challenging task, because of the high degree of articulation of the human body, the large variety of possible human motions, and the diversity of human appearances.
Methods for tackling this problem can be roughly categorized as either discriminative or generative. Discriminative methods can work on single images, and are able to recover the human poses efficiently. However, the accuracy and generality largely depend on the training data. Generative approaches usually formulate the problem as a tracking problem and adopt an explicit human model. Although arbitrary motions can be tracked, such systems usually have difficulties in adapting to different subjects and in dealing with tracking failures.
In this thesis, an accurate, efficient and robust human pose tracking system from a single view camera is developed, mainly following a generative approach. A novel discriminative feature is also proposed and integrated into the tracking framework to improve the tracking performance.
The human pose tracking system is proposed within a particle filtering framework. A reconfigurable skeleton model is constructed based on the Acclaim Skeleton File convention. A basic particle filter is first implemented for upper body tracking, which fuses time efficient cues from monocular sequences and achieves real-time tracking for constrained motions. Next, a 3D surface model is added to the skeleton model, and a full body tracking system is developed for more general and complex motions, assuming a stereo camera input. Partitioned sampling is adopted to deal with the high dimensionality problem, and the system is capable of running in near real-time. Multiple visual cues are investigated and compared, including a newly developed explicit depth cue.
Based on the comparative analysis of cues, which reveals the importance of depth and good bottom-up features, a novel algorithm for detecting and identifying endpoint body parts from depth images is proposed. Inspired by the shape context concept, this thesis proposes a novel Local Shape Context (LSC) descriptor specifically for describing the shape features of body parts in depth images. This descriptor describes the local shape of different body parts with respect to a given reference point on a human silhouette, and is shown to be effective at detecting and classifying endpoint body parts. A new type of interest point is defined based on the LSC descriptor, and a hierarchical interest point selection algorithm is designed to further conserve computational resources. The detected endpoint body parts are then classified according to learned models based on the LSC feature. The algorithm is tested using a public dataset and achieves good accuracy with a 100Hz processing speed on a standard PC.
Finally, the LSC descriptor is improved to be more generalized. Both the endpoint body parts and the limbs are detected simultaneously. The generalized algorithm is integrated into the tracking framework, which provides a very strong cue and enables tracking failure recovery. The skeleton model is also simplified to further increase the system efficiency. To evaluate the system on arbitrary motions quantitatively, a new dataset is designed and collected using a synchronized Kinect sensor and a marker based motion capture system, including 22 different motions from 5 human subjects. The system is capable of tracking full body motions accurately using a simple skeleton-only model in near real-time on a laptop PC before optimization.
48
Chu, Jia-Yin, and 朱佳音. "Ratiometric Fluorescence pH Sensing and Single-particle Tracking in Cancer Cell." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/25319646567471306974.
Анотація:
碩士國立臺灣大學
化學研究所
100
The intracellular pH is an important modulator of cell functions. Since the activities of most proteins are affected by very small changes of the proton concentration, there are a number of cellular mechanisms that finely regulate the intracellular pH values (pHi). Therefore, monitoring pHi with high spatial resolution could help us to elucidate many physiological or pathogenic processes taking place within cells. We synthesized 100 nm mesoporous silica nanoparticles with a pH-sensitive dye, FITC and a pH-insensitive dye, RITC. This nanoparticle was employed to estimate the pHi by the fluorescence intensity ratio of FITC to RITC. We conjugated high amount of dyes to improve the photostability of the dyed nanoparticles. Besides, we modified the surface by post-modification to form two different nanoparticles, positively charged TA-hMSN and negatively charged THPMP-hMSN. The nanoparticles were characterized with various physical methods. Then, we used the single-particle tracking (SPT) system to examine the interaction between the nanoparticles and HeLa cells. We obtained the trajectories of nanoparticles during the uptake process and were able to estimate the fluorescence ratio in the local environment simultaneously.
49
Epperla, Chandra Prakash, and 艾培仁. "Single Particle Tracking and Nanothermometry of Fluorescent Nanodiamonds in Membrane Nanotubes." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/t7992w.
Анотація:
博士國立清華大學
化學系
105
Fluorescent nanodiamond (FND) is a novel carbon based material that has drawn much attention in recent years due to its uniquely embedded defect centers named nitrogen-vacancy (N-V) centers. Most notable is the negatively charged nitrogen-vacancy (NV ̶ ) color center which emits a highly photostable far-red fluorescence emission. Since it does not photobleach or photoblink, it can be used to track for longer times. The FND also exhibits a very good biocompatibility and its surface can be easily functionalized through covalent or non-covalent interactions with biomolecules. The NV ̶ center has been used to sense environmental variables such as temperature and electric or magnetic fields by studying the shifts in their electronic transitions or spin transitions at electronic ground state. All these characteristics make FND a promising fluorescent probe for biological applications. Cell-to-cell communication is essential for the development and maintenance of multicellular organisms. Recently discovered membrane nanotubes (MNTs) are capable of creating intercellular communication pathways through which transport of proteins and other cytoplasmic components occurs. These cellular connections are very heterogeneous in both structure, function, and have been found to be formed in numerous cell types. MNTs are also known to participate in pathogenesis of many diseases such as Alzheimer’s, Parkinson’s and HIV. Hence, it is important to understand the dynamics of transport along these nanotubes and to explore the potential of MNTs as drug delivery channels. This doctoral thesis presents several applications of variously functionalized FNDs in membrane nanotubes. We applied protein functionalized FNDs as a photostable tracker, as well as a protein carrier, to illustrate the transport events in MNTs of human cells. Proteins, including bovine serum albumin and green fluorescent protein, were coated on 100-nm FNDs by physical adsorption. Then single-particle tracking of the bio-conjugates in the transient membrane connections was carried out by fluorescence microscopy. We observed different types of motions and velocity distribution of cargos that took takes place inside the MNTs. Our results demonstrate the promising applications of this novel carbon-based nanomaterial for intercellular delivery of biomolecular cargo down to the single-particle level. Further, we have studied the thermostability of both MNTs and cell membrane. We have developed gold nanorods (GNR) functionalized FNDs as a two-in-one optical nanodevice that can heat and sense the temperature simultaneously. We used all-optical method to study the nanothermometry of GNR-FNDs. We also demonstrated the photoporation on MNTs using GNR-FND nanohybrids to selectively deliver drugs to cytoplasm. Finally, we performed hyperlocalized hyperthermia on cell membrane for the treatment of cancer cell. During such a treatment, cancer cells can be killed selectively, while healthy cells remain unaffected. Our results demonstrate promising applications of this novel carbon-based nanomaterial for intercellular delivery of biomolecular cargo down to the single-particle level and a new paradigm for hyperthermia research and application.
50
Shih, Hsien-Kang, and 石憲剛. "3D Human Motion Tracking Based on Single Video Input and Particle Filtering." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/50924743055631440780.
Анотація:
碩士國立中正大學
電機工程所
97
This study uses monocular video sequence as input for the tracking of humans’ 3D motion pose, enabling accurate estimation of 3D motion parameters. Our method adopts silhouette, edge, and color features for matching the input images with the projected 3D human model. We also adopts an improved annealed particle filter to accurately estimate the system states (i.e., the human 3D pose).To have the particles search in solution space more efficient, we estimate motion vectors information between consecutive frames to control the search range of the particles in every degree of freedom of the humans 3D model and the search range of the particles at every time instance. Besides that, this study also proposes several algorithms to reduce the number of particles, but still maintains the tracking accuracy. Compared with the traditional particle filter and the annealed particle filter, experiments show that our method makes particles more clustering around true solutions with higher weights, thus having better tracking results than the other two methods. Compared with the annealed particle filter, experiments also show that our method can reduce the required number of particles by about 43%, without sacrificing the tracking accuracy.