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Yadav, Rahul B. "Studies of the mTOR signalling pathway using advanced FRET-FLIM technique." Thesis, Oxford Brookes University, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.543796.
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West, Lucien. "Illuminating cAMP dynamics at the synapse with multiphoton FLIM-FRET Imaging." Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/43387.
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The study of signalling pathways within mammalian physiology has long been hindered by the size of the players involved, being far beyond the realms of the conventional light microscope. The advent of advanced fluorescent imaging techniques has revolutionised our capabilities to probe biological processes. The work in this thesis particularly utilised Förster resonance energy transfer (FRET), a fluorescence-based technique that can provide functional readouts of the processes underlying cellular function. Specifically I worked to develop and optimise a fluorescence imaging system for investigating the dynamics and function of cyclic adenosine monophosphate (cAMP), a ubiquitous second messenger. The neuroscientific study of how the brain can learn and recall memories is a rapidly advancing field. The current challenges of tackling dementias, such as Alzheimer's disease, and preventing memory loss can only be addressed through better understanding of how memories can be stored. It is now believed that neurons retain memories within their synapses, the femtolitre structures that determine the strength of these connections. cAMP has been shown to play a distinctive role in orchestrating the retention of long term memory at the synaptic level. However, its spatial and temporal activation profiles are still not fully understood. To address this, my PhD project combined FRET readouts with cutting edge imaging techniques applied to synapses in neuronal cultures that provide reasonably convenient optical access. By examining the structure of these synapses, along with the measurement of cAMP concentration in different neuronal regions, this project uncovered the highly compartmentalised nature of this signalling molecule, seen to act directly at the sites of strengthening synapses. Through the optimisation of a FRET imaging system for studying activity in neuronal tissues, this project establishes a method for the future investigation of a plethora of pathways underlying the healthy functioning of the mammalian brain.
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Doll, Franziska [Verfasser]. "Visualizing Protein-Specific Post-Translational Modifications with FLIM-FRET Microscopy / Franziska Doll." Konstanz : KOPS Universität Konstanz, 2018. http://d-nb.info/1223372219/34.
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Andrews, Natalie Julia. "Spatio-temporal mapping of protein activity in live zebrafish using FRET FLIM OPT." Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/59958.
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Optical projection tomography is a “mesoscopic” imaging technique, which fills a size gap in imaging, between techniques that provide subcellular resolution such as confocal laser scanning microscopy and cellular/whole body imaging techniques such as PET/MRI. OPT provides three dimensional (3D) whole body imaging utilising absorption or fluorescence contrast in mesoscopic (1-10 mm), transparent samples. This has the potential to be developed for use in biomedical research, for example in drug screening, developmental biology, and the study of disease mechanisms. To fully exploit the advantages of OPT, we image live zebrafish. These are the optimal size for imaging, and transparent mutants are readily available resulting in less scattering of optical radiation. They are also genetically tractable and many transgenic lines with fluorophore labels have been created. This thesis reports on the use of optical projection tomography (OPT) and fluorescence lifetime imaging (FLIM) to detect enzyme activity in whole live zebrafish. FRET biosensors for Caspase 1 and 3 activity were generated and the Caspase 3 FRET biosensor was validated in mammalian cell culture. Transgenic zebrafish expressing the biosensors ubiquitously under control of the ubiquitin (Ubi), macrophage (mpeg) and neutrophil (LysC) promoters were generated and used to validate the biosensors within live zebrafish. Caspase 3 is activated as part of the apoptotic pathway, in response to gamma irradiation. After exposure to 25 Gy irradiation, Caspase 3 activity is evident in the head region of 24 hpf zebrafish ubiquitously expressing the Caspase 3 biosensor, through both confocal and OPT FLIM imaging. FLIM OPT imaging allows visualisation of activity via production of 3D lifetime maps. Programs were generated to enable qualification and quantification of the data. Preliminary validation of the novel Caspase 1 biosensor indicates the biosensor is functional and it is possible to use FLIM to detect Caspase 1 activity in live zebrafish, in response to immune activation via tail transection. Overall, these findings demonstrate that FLIM OPT is a useful tool in mesoscopic imaging, able to identify enzyme activity in a whole live zebrafish, in 3D.
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Loukil, Abdelhalim. "Etude de la cycline A2 : interactions, dégradation et mise en évidence du rôle de l'autophagie." Thesis, Montpellier 2, 2012. http://www.theses.fr/2012MON20115.
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Le cycle cellulaire est finement régulé dans le temps et l'espace. Nous avons abordé les aspects dynamiques des interactions que la cycline A2 entretient avec ses partenaires Cdk1, Cdk2 et l'ubiquitine au cours du cycle cellulaire, dans des lignées cellulaires humaines. A cette fin, nous avons eu recours aux approches de FRET (Förster/fluorescence resonance energy transfer) et de FLIM (fluorescence lifetime imaging microscopy). Ceci nous a permis de montrer que les formes ubiquitinylées de la cycline A2 apparaissent principalement sous forme de foyers en prométaphase et se propagent ensuite à l'ensemble de la cellule. En outre, nous avons découvert que l'autophagie participe à la dégradation de cette cycline en mitose. Nous discutons les implications de ces observations quant à un rôle éventuel de la cycline A2 au moment de la formation de l'anneau de constriction, ainsi que de la participation de l'autophagie via cette cycline, dans la réponse aux dommages à l'ADN en mitoseThe cell cycle is finely regulated in time and space. We have studied the dynamical aspect of the interactions between cyclin A2 and its partners Cdk1, Cdk2 and ubiquitin during the cell cycle, in human cell lines. To this aim, we have used FRET (Förster/fluorescence resonance energy transfer) and FLIM (fluorescence lifetime imaging microscopy) techniques. We have thus shown that ubiquitylated forms of cyclin A2 are detected predominantly in foci in prometaphase, before spreading throughout the cell. Moreover, we have shown that autophagy contributes to cyclin A2 degradation in mitosis. We discuss the implications of these observations regarding a possible role of cyclin A2 when the cleavage furrow forms, and the participation of autophagy in DNA damage response in mitosis
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Nobis, Max. "In vivo FLIM-FRET imaging of pharmacodynamics and disease progression in mouse cancer models." Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7283/.
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Perrin, Aurélien. "Caenorhabditis elegans un modèle d’étude des différents compartiments du noyau : de l’étude d’un stress du nucléole par inhibition de la voie de neddylation à la mesure de la compaction de la chromatine in vivo." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT049/document.
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NEDD8, molécule de la famille de l’ubiquitine est essentielle au développement, à la croissance et à la viabilité d’un organisme, de plus c’est une cible prometteuse en thérapeutique. Nous avons découvert que l’inhibiteur spécifique de la NEDDylation, MLN4924 altère la morphologie sans fragmentation et augmente la surface du nucléole de cellules humaines et de noyaux de la lignée germinale de Caenorhabditis elegans. Une approche de protéomique quantitative (SILAC) combiné à l’analyse de la production des ARNr et des ribosomes montrent que MLN4924 change la composition protéique du nucléole sans affecter l’activité transcriptionnelle de l’ARN pol I. Notre analyse montre que MLN4924 active p53 par la voie RPL11/RPL5-Mdm2 caractéristique d’un stress du nucléole. Cette étude identifie le nucléole comme une cible intéressante dans l’utilisation d’inhibiteurs de la NEDDylation et apporte un nouveau mécanisme d’activation de p53 par inhibition de la voie NEDD8.Dans une seconde étude nous avons adapté la méthode de FLIM-FRET (« Fluorescence Lifetime Imaging Microscopy – Förster Resonance Energy Transfer ») à l’étude de la compaction de la chromatine à l’échelle du nanomètre dans un organisme vivant. Le nématode Caenorhabditis elegans s’est révélé être un modèle de choix. Au sein des chromosomes méiotiques, nous avons identifié différentes régions de compaction, de niveau variable par mesure du FRET entre histones fusionnées à des protéines fluorescentes. Par une approche originale d’ARN interférence et injection d’un « extra-chromosome » nous avons défini l’architecture à une nano-échelle de différents états de l’hétérochromatine et montré que cette organisation est contrôlée par les protéines HP1 « Heterochromatin Protein 1 » et SETDB1, une protéine « H3-Lysine 9 methyl transferase ». Nous avons également montré que la compaction de l’hétérochromatine est dépendante des condensines I et II et plus particulièrement la condensine I contrôle l’état faiblement compacté de la chromatine.Nos travaux ont confirmé que C. elegans est un modèle d’intérêt majeur pour l’étude des compartiments nucléaires et parfaitement adapté pour des études pré-cliniqueThe ubiquitin-like molecule NEDD8 is conserved and essential for viability, growth and development; its activation pathway is a promising target for therapeutic intervention. We found that the small molecule inhibitor of NEDDylation, MLN4924, alters the morphology and increases the surface size of the nucleolus in human cells and Caenorhabditis elegans germ cells in the absence of nucleolar fragmentation. Through SILAC proteomic analysis and rRNA production, processing and ribosome profiling, we show that MLN4924 changes the composition of the nucleolar proteome but does not inhibit RNA Pol I transcription. Further analysis demonstrates that MLN4924 activates the p53 tumour suppressor through the RPL11/RPL5-Mdm2 pathway, with characteristics of nucleolar stress. The study identifies the nucleolus as a target of the NEDDylation pathway and provides a mechanism for p53 activation upon NEDD8 inhibition.Then we adapted a quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) approach to assay the nano-scale chromatin compaction in a living organism, the nematode Caenorhabditis elegans. By measuring FRET between histone-tagged fluorescent proteins, we visualized distinct chromosomal regions and quantified the different levels of nanoscale compaction in meiotic cells. Using RNAi and repetitive extrachromosomal array approaches, we defined the heterochromatin state and showed that its architecture presents a nanoscale-compacted organization controlled by Heterochromatin Protein-1 (HP1) and SETDB1 H3-lysine-9 methyl-transferase homologs in vivo. Next, we functionally explored condensin complexes. We found that condensin I and condensin II are essential for heterochromatin compaction and that condensin I additionally controls lowly compacted regions. Our data show that, in living animals, nanoscale chromatin compaction is controlled not only by histone modifiers and readers but also by condensin complexes.We confirm that C. elegans is an interesting model to study nuclear signalling and perfectly adapt to be a platform for pre-clinical studies
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Engel, Stephanie Vanessa. "Assembly von Influenzaviren." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/15918.
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Es wird angenommen, dass das Influenzavirus-Glykoprotein Hämagglutinin (HA) für seine Funktion sowohl bei der Virusfreisetzung als auch bei der Fusion von viraler und zellulärer Membran mit Cholesterin- und Sphingolipidreichen Domänen, sogenannten Membran-Rafts, assoziiert sein muss. Aus diesem Grund sollte in dieser Arbeit die Membran-Raft-Affinität von HA in lebenden Zellen mittels FLIM-FRET gemessen werden. Dabei wurde mit Hilfe der Fluroreszenz-Lebenszeit-Messung (FLIM) der Förster-Resonanz-Energie-Transfer (FRET) von fluoreszenzmarkiertem HA auf einen etablierten Raft-Marker bestimmt. Diese Messungen zeigten, dass beide Proteine in gemeinsamen Klustern in der Plasmamembran vorkommen. Durch Cholesterinentzug und durch den Einsatz von Cytochalasin D, welches die Mikrofilamente zerstört, konnte diese Klusterbildung reduziert werden. Demnach tragen sowohl die Membran-Rafts als auch das Aktinnetzwerk zu dieser Klusterbildung bei. Mittels FLIM-FRET konnte zusätzlich bestätigt werden, dass die Signale für die Detergenslöslichkeit von HA in Triton-Extraktionsexperimenten, die Palmitylierung und die stark hydrophoben Aminosäuren zu Beginn der Transmembrandomäne (TMD), auch im lebenden System eine wichtige Rolle spielen. Zusätzlich konnten biochemische Experimente zeigen, dass die hydrophoben Aminosäuren zu Beginn der HA-TMD den intrazellulären Transport, nach der Trimerbildung, entscheidend verzögern. Diese Verzögerung ist vermutlich auf einer erschwerten Integration dieser Proteine in die Membran-Rafts begründet. Die virale Fusion mit der Wirtszellmembran wird durch eine pH5-Behandlung vermittelte Konformationsänderung von HA ausgelöst. FLIM-FRET-Messungen zeigten für die pH5-Konformation von HA eine verglichen mit der pH7-Konformation verringerte Klusterbildung mit dem Raft-Marker. Somit ist offensichtlich, dass die Membranfusion-vermittelnde HA-Konformation eine verringerte Raft-Affinität besitzt. Diese verringerte Raft-Affinität könnte eine wichtige Rolle bei der Störung der Lipide an der Fusionsstelle spielen und somit die Bildung und/oder Vergrößerung der Fusionspore erleichtern.It has been supposed that the hemagglutinin (HA) of influenza virus is recruited to cholesterol- and sphingolipid-enriched domains, also named membrane-rafts, to accomplish its function in virus budding and membrane fusion. This study aimed at verifying the affinity of HA for membrane-rafts in living cells using fluorescence-lifetime imaging microscopy to measure Förster’s resonance energy transfer (FLIM-FRET). FLIM-FRET revealed strong clustering between a fluorescence-tagged HA-protein and a well-established raft-marker in CHO cells. Clustering was significantly reduced when rafts were disintegrated by cholesterol depletion and when microfilaments were disrupted with cytochalasin D. Thus, membrane-rafts as well as the actin meshwork contribute synergistically to clustering. Clustering was also reduced by the removal of the known signals for the association of HA with detergent-resistant-membranes, the palmitoylation and the first amino acids in the transmembrane region (TMR). Since these mutations are obviously important for the raft-association of HA their function during the transport through the ER and the Golgi-complex was studied. These investigations showed that the exchange of the first three amino acids of the HA-TMR led to a decelerated transport after trimer-formation of the protein, probably due to retarded integration of these proteins into membrane-raft domains. Mediating viral fusion with the host cell membrane requires an irreversible conformational change of HA. FLIM-FRET studies of this low pH conformation unveiled that the clustering with the raft-marker is decisively reduced compared to the pre-fusion conformation of the protein. It might be assumed that the fusion-mediating conformation of HA reduces the proteins affinity for membrane-rafts. Therefore it is likely that this reduced affinity for rafts after the conformational change is relevant to cause perturbation of lipids at the fusion site and thereby facilitating the formation and/or enlargement of the fusion pore.
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Sizaire, Florian. "Développement d’un criblage automatisé de l’activité kinase d’un biosenseur Aurora A par FLIM." Thesis, Rennes 1, 2019. http://www.theses.fr/2019REN1B033.
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La surexpression d’Aurora A est un marquer majeur de certains cancers épithéliaux. Ce gène code pour la kinase multifonctionnelle Aurora A et son activation est requise pour l’entrée et la progression vers la mitose. Jusqu'à présent, aucun inhibiteur de cet oncogène n'a été approuvé par la FDA et il est donc primordial d'identifier de nouvelles molécules. Notre équipe a développé un biosenseur FRET (Forster Resonance Energy Transfer) pour l’activité kinase d’Aurora A, constitué de la kinase entière flanquée de deux fluorophores, une GFP et une mCherry. Le changement de conformation d’Aurora A lorsqu’elle est activée rapproche les fluorophores et augmente l’efficacité du FRET. Il est ainsi possible de suivre l’activation d’Aurora A dans les cellules vivantes exprimant le biosenseur à des niveaux endogènes. Nous pouvons mesurer le FRET en utilisant la technique de FLIM (Fluorescence Lifetime Imaging Microscopy) grâce à un microscope développé dans l’équipe et appelé fastFLIM. Mes travaux de thèse ont consisté à développer une stratégie de criblage robuste et automatisée en combinant les capacités du fastFLIM et le biosenseur d’activité d’Aurora A. Cette stratégie basée sur une automatisation des acquisitions et de l’analyse de données a permis de cribler une banque de molécules en plaque 96 puits afin de trouver de potentielles inhibiteurs de l’activité kinase d’Aurora A. De plus, j’ai participé à la validation du biosenseur pour un suivi de l’activité kinase dans des cellules vivantes en montrant que les variations de FRET mesurées correspondent bien à l’état de phosphorylation d’Aurora A sur le résidu Thréonine 288, marqueur de son activation. Enfin, j’ai participé à l’élaboration de nouvelles techniques de microscopie pour suivre l’activité du biosenseur. Pour cela, j’ai utilisé un biosenseur de type homoFRET avec l’enjeu de pouvoir utiliser plusieurs biosenseurs dans un contexte multiplex. J’ai aussi utilisé la technique de 2c-FCCS (2-colors Fluorescence Cross Correlation Spectroscopy) sur le biosenseur Aurora A afin de pouvoir mesurer le FRET dans des régions où celui-ci est faiblement exprimant et dont la mesure de durée de vie de fluorescence n’est pas possible par le FLIM. Ainsi, mes travaux de thèse s’inscrivent dans la tendance à développer une microscopie quantitative et autonome avec comme enjeu d’apporter un grande nombre de données phénotypiquesOverexpression of Aurora A is a major marker of some epithelial cancers. This gene encodes the multifunctional Aurora A kinase and its activation is required for entry and progression to mitosis. So far, no inhibitor of this oncogene has been approved by the FDA and it is therefore essential to identify new molecules. Our team developed a Forster Resonance Energy Transfer (FRET) biosensor for Aurora A kinase activity, consisting of the entire kinase flanked by two fluorophores, a GFP and a mCherry. The conformational change of Aurora A when it is activated brings the fluorophores closer and increases FRET efficiency. It is thus possible to follow the activation of Aurora A in living cells expressing the biosensor at endogenous levels. We can measure FRET using FLIM (Fluorescence Lifetime Imaging Microscopy) technique using a microscope developed in the team called fastFLIM. My thesis work consisted of developing a robust and automated screening strategy by combining the capabilities of fastFLIM and the Aurora A activity biosensor. This strategy based on automation of acquisitions and data analysis allowed to screen a library of 96-well plate molecules for potential inhibitors of Aurora A kinase activity. In addition, I participated in the validation of the biosensor for kinase activity monitoring in living cells, showing that the FRET variations measured correspond to the phosphorylation state of Aurora A on the Threonine 288 residue, a marker of its activation. Finally, I participated in the development of new microscopy techniques to monitor the activity of the biosensor. For that, I used a homoFRET biosensor with the challenge of being able to use several biosensors in a multiplex context. I also used the 2c-FCCS (2-color Fluorescence Cross Correlation Spectroscopy) technique on the Aurora A biosensor to measure FRET in regions where it is weakly expressing and whose lifetime measurement of Fluorescence is not possible by FLIM. Thus, my thesis work is part of the trend to develop a quantitative and autonomous microscopy with the challenge of providing a large number of phenotypic data
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Ziegler, Cornelia. "Imagerie quantitative de l'assemblage de la NADPH oxydase des phagocytes en cellules vivantes par des approches FRET-FLIM." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS048/document.
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La NADPH oxydase des phagocytes (NOX2) est responsable de la production d’anions superoxydes qui sont les précurseurs des autres formes réactives de l’oxygène. NOX2 est une enzyme majeure de la réponse immunitaire. Les dysfonctionnements de NOX2 sont associés à de nombreuses pathologies et donc il convient d’en comprendre les détails de la régulation. Cette oxydase est composée de cinq sous-unités : deux protéines membranaires, gp91phox et p22phox et 3 protéines cytosoliques p47phox, p67phox et p40phox. D’après les études in vitro avec des protéines purifiées, les protéines cytosoliques sont supposées former un complexe ternaire qui se déplace à la membrane avec une petite protéine G, Rac, au moment l’activation.L’objectif de ce projet est de caractériser les interactions spécifiques entre les sous-unités cytosoliques de NOX2 en cellules vivantes en utilisant le phénomène de transfert résonant d’énergie de type Förster (FRET) entre deux fluorophores, un donneur et un accepteur. Ici les fluorophores seront des protéines fluorescentes de la famille de la GFP. Elles sont fusionnées à deux sous-unités. L’efficacité du FRET dépend de la distance entre les fluorophores et permet ainsi de caractériser les interactions entre les protéines d’intérêt. Une méthode rapide d’identification des situations où le FRET est positif a été mise au point par cytométrie en flux. Des études détaillées et quantitatives ont ensuite été réalisées en utilisant l’imagerie de durée de fluorescence (FLIM) du donneur. Le FLIM, combiné à l’utilisation de donneurs présentant une durée de vie mono-exponentielle, permet de déterminer directement des efficacités de FRET apparentes et moléculaires, qui contiennent, toutes les deux, des informations qualitatives et quantitatives sur l’interaction et la structure des protéines impliquées. De ces données, il est possible d’extraire la fraction des donneurs interagissant avec un accepteur. Les informations obtenues à partir des données de FRET-FLIM permettent une meilleure compréhension de l’organisation et de la régulation de NOX2 tout en permettant une estimation des constantes de dissociation (Kd). Afin de confirmer ces résultats, des expériences de spectroscopie de corrélation de fluorescence à deux couleurs (FCCS) ont été réalisées. Cette méthode complétement indépendante n’est pas basée sur la distance entre fluorophores comme le FRET mais sur leur co-diffusion à travers un petit volume d’observation dans le cytoplasme cellulaire.L’approche FRET-FLIM nous a tout d’abord permis d’observer les interactions entre hétéro-dimères formés de deux sous-unites différentes en cellules vivantes et d’exclure la formation d’homo-dimères entre deux sous-unités identiques. Etant donné la bonne précision des mesures de FLIM, nous avons pu comparer les informations structurales obtenues en cellules avec les données structurales issues d’études sur les protéines purifiées in vitro et nous avons constaté qu’elles sont en bon accord. Nous avons ensuite aligné les structures disponibles pour proposer un premier modèle 3D du complexe cytosolique de la NADPH oxydase au repos dans le cytosol cellulaire.Les fractions de protéines en interaction sont pour tous les hétéro-dimères autour de 20% ce qui n’est pas en accord avec l’hypothèse courante qui propose que toutes les sous-unités cytosoliques soient sous forme de complexe. Toutefois nos premiers résultats de FCCS confirment ce résultat extrait des données de FRET-FLIM. Nous proposons donc que la complexation des sous-unités cytosolique pourrait être impliquée dans la régulation de la NADPH oxydase. Des études complémentaires seront nécessaires pour valider cette nouvelle hypothèse. Les constantes de dissociation Kd estimées à partir de nos résultats sont micromolaires et donc un ordre de grandeur plus élevé que les valeurs nanomolaires déterminées in vitro. Des mesures plus détaillées de FCCS pourront compléter et valider ces résultatsThe phagocyte NADPH oxidase (NOX2) is a key enzyme of the immune system generating superoxide anions, which are precursors for other reactive oxygen species. Any dysfunctions of NOX2 are associated with a plethora of diseases and thus detailed knowledge about its regulation is needed. This oxidase is composed of five subunits, the membrane-bound gp91phox and p22phox and the cytosolic p47phox, p67phox, and p40phox. The latter are assumed to be in a ternary complex that translocates together with the small GTPase Rac to the membranous subunits during activation.Our aim was to discover and to characterize specific interactions of the cytosolic subunits of NOX2 in live cells using a Förster Resonance Energy Transfer (FRET) based approach: Since FRET depends on the distance between two fluorophores, it can be used to reveal protein-protein interactions non-invasively by studying fluorescent protein tagged subunits. To have a rapid method on hand to reveal specific interactions, a flow cytometer based FRET approach was developed. For more detailed studies, FRET was measured by fluorescence lifetime imaging microscopy (FLIM), because it allows a direct determination of the apparent and molecular FRET efficiency, which contains both qualitative and quantitative information about the interaction and the structure of the interacting proteins. Furthermore, the FRET-FLIM approach enables an estimation of the fraction of bound donor. This information itself is important for a better understanding of the organisation and regulation of the NOX2, but it is also necessary for the calculation of the dissociation constant Kd from the FRET-FLIM data. To confirm the findings obtained by FRET-FLIM fluorescence cross correlation spectroscopy (FCCS) experiments were performed. This completely independent method is not based on distances like FRET but on the observation of the co diffusion of the fluorescently labelled samples when they move across a small observation volume inside the cells.The FRET-FLIM approach allowed us in a first step to discover heterodimeric interactions between all cytosolic subunits in live cells. Due to the good precision of the results, we were able to extract structural information about the interactions and to compare them with available structural data obtained from in vitro studies. The information from FRET-FLIM was coherent with in vitro data. We then aligned the available structures leading to the first 3D model of the cytosolic complex of the NADPH oxidase in the resting state in live cells.Additionally, the bound fraction for all heterodimeric interactions derived by FRET-FLIM is around 20 %, which is in contrast to the general belief that all cytosolic subunits are bound in complex. The first FCCS results support our findings. Therefore, we believe that the complexation of the cytosolic subunits could be involved in the regulation of the NADPH oxidase and should be investigated further. The estimated Kd derived from the FRET-FLIM approach is in the low micomolar range, which is an order of a magnitude higher than the nanomolar range of in vitro studies.In conclusion, we showed that our quantitative FRET-FLIM approach is not only able to distinguish between specific and unspecific protein-protein interactions, but gives also information about the structural organisation of the interacting proteins. The high precision of the FRET-FLIM data allow the determination of the bound fraction and an estimation of the Kd in live cells. FCCS is a complementary method, which can verify these quantitative findings. However, it cannot replace FRET-FLIM completely as it does not give any structural information.With respect to the biological outcome of this project, we can propose for the first time a 3D-model of the cytosolic complex of the NADPH oxidase covering the in vitro as well as the live cell situation. Additionally, the small bound fraction found here may raise new ideas on the regulation of this vital enzyme
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Betolngar, Dahdjim-Benoît. "Développement d’un nouveau couple de protéines fluorescentes pour le FRET : Validation et application à un biosenseur d’activité kinase A." Thesis, Paris 11, 2015. http://www.theses.fr/2015PA112068/document.
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Les protéines kinases A (PKA) sont des enzymes qui catalysent la phosphorylation de résidus sérine ou thréonine. L’activité des PKA peut être mesurée in cellulo grâce aux biosenseurs AKAR (A-Kinase Activity Reporter). AKAR est composé de 4 modules: la séquence substrat des PKA, un domaine de liaison aux acides aminés, et 2 protéines fluorescentes pouvant interagir par FRET (Förster Resonant Energy Transfer). La phosphorylation de la séquence consensus par la PKA et l’interaction de l’acide aminé phosphorylé avec le module senseur provoque une modification de la conformation d’AKAR et une augmentation du FRET entre les 2 protéines fluorescentes.L’objectif initial de ce travail était de produire un biosenseur AKAR basé sur un nouveau couple de protéines fluorescentes plus performantes, et insensibles au pH. Ce biosenseur devant à terme être utilisable en imagerie ratiométrique, et en FLIM (Fluorescence Lifetime Imaging Microscopy). Ainsi nous avons mis au point une nouvelle version d’AKAR: AqAKARCit. Cette version exploite la protéine Aquamarine, l'une des meilleures protéines cyan disponibles aujourd'hui, avec un rendement quantique de 89%, un déclin de fluorescence mono-exponentiel, et une insensibilité au pH dans tout le domaine physiologique. Les résultats obtenus en FLIM avec cet AqAKARCit ont permis des améliorations notables en stabilité et sensibilité.Une version baptisée AqAKARTagRFP a été construite, dans laquelle l’accepteur est une protéine fluorescente orange permettant une meilleure séparation spectrale entre donneur et accepteur et insensible aux variations de pH. Les étapes de caractérisations de AqAKARTagRFP ont été accomplies en FLIM, et en ratiométrie. AqAKARTagRFP permet d'obtenir de bonnes réponses en FRET par ratiométrie, mais reste difficilement utilisable en FLIM en raison d'une dynamique de réponse limitée. La sensibilité du biosenseur a été améliorée par une modification de l'ancrage de l'Aquamarine. Les modifications apportées à cette version nommée AqEAKARTagRFP la place au niveau des biosenseurs AKAR les plus performants actuellement disponibles.Les mesures de sensibilité au pH d’ AqEAKARTagRFP réalisées en FLIM ont révélé une insensibilité au pH du biosenseur sur une étendue de pH jamais atteinte jusqu’à présent. Cependant, en ratiométrie, on note malgré tout une sensibilité détectable aux pHs fortement acides (pH ≤ 6), ce qui ne permettra pas de l'utiliser pour l'imagerie ratiométrique de compartiments cellulaires acides. Un contrôle négatif non phosphorylable AqEAKARmutTagRFP a été étudié. Ce contrôle présente les mêmes variations de signal en réponse à des changements imposés de pH intracellulaire, révélant que ces variations sont indépendantes de l’activité PKA.L'étude de tandems CFP-Cit et Aq-Cit dépourvus de la partie senseur nous à permis d'analyser le comportement de FRET des couples cyan/jaune en fonction du pH. Un modèle décrivant ce comportement a été créé et appliqué à AKAR.Les expériences complémentaires faites sur CFPAKARCit sont en accord avec nos simulations mais la construction AqAKARCit révèle du FRET résiduel à pH acide que notre modèle numérique ne prévoit pas. Une sensibilité aux pH acides de la partie senseur d’AKAR qui provoquerait un changement de conformation du biosenseur et une augmentation de FRET pourrait expliquer ce phénomène.Ce travail de thèse a permis la mise au point d’un nouveau couple de protéines fluorescentes par le FRET insensibles au pH. Ce couple va permettre une meilleure caractérisation des sensibilités des biosenseurs existants comme nous l’avons montré avec AKAR. Ce couple de protéines fluorescentes pourra également être utilisé dans des compartiments cellulaires acides, par exemple pour étudier des interactions protéine/protéine. Enfin, grâce à une meilleure séparation spectrale en excitation et en émission, ce couple peut être utilisé dans des applications plus exigeantes comme la microscopie biphotoniqueProtein kinase A (PKA) are enzymes which catalyze the phosphorylation of serine or threonine residues. The activity of PKA can be measured in cellulo through AKAR biosensors (A-Kinase Activity Reporter). AKAR consists of 4 modules: the PKA substrate sequence, a phospho-amino binding domain, and two fluorescent proteins that can interact by FRET (Förster Resonant Energy Transfer). After action of the PKA, the interaction between the phophorylated amino acid and the phospho-amino binding domain causes a change in the conformation of AKAR and an increase in FRET between the two fluorescent proteins.The initial objective of this work was to produce an AKAR biosensor based on a new pair of improved fluorescent proteins, and insensitive to pH. This biosensor to eventually be used in ratiometric imaging and FLIM (Fluorescence Lifetime Imaging Microscopy). So we developed a new version of AKAR: AqAKARCit. This version uses the Aquamarine protein, one of the best cyan proteins available today, with a quantum yield of 89%, a near mono-exponential fluorescence decay, and insensitive to pH throughout the physiological range. The results obtained with this AqAKARCit allowed significant improvements in stability and sensitivity.A version called AqAKARTagRFP was built, in which the acceptor is an orange fluorescent protein allowing better spectral separation between donor and acceptor and insensitive to pH variations. The characterization of AqAKARTagRFP was performed in FLIM, and ratiometry. AqAKARTagRFP provides good answers in FRET by ratiometry but remains difficult to use in FLIM due to limited dynamic responses. The sensitivity of the biosensor has been improved by modification of the anchoring of Aquamarine. Changes to this version named AqEAKARTagRFP place it at the most efficient AKAR biosensors currently available.The pH-responsive measures of AqEAKARTagRFP made in FLIM showed insensitivity to pH on a range never reached so far. However, in ratiometry, there is still a detectable sensitivity to highly acidic pHs (pH ≤ 6), which will not allow to use it for ratiometric imaging of cellular acidic compartments. A negative unphosphorylatable control AqEAKARmutTagRFP was studied. This control presents the same signal variations in response to changes imposed on intracellular pH, revealing that these variations are independent of PKA activity.The study of the CFP-Cit and Aq-Cit tandems devoid of the sensor part allowed us to analyze the behavior of cyan / yellow FRET pairs regarding the pH. A model describing this behavior was created and applied to AKAR. Additional experiments on CFPAKARCit are in agreement with our simulations but AqAKARCit reveals residual FRET at acidic pHs that our numerical model does not predict. A sensitivity to acidic pH of the sensor module of AKAR which would cause a conformational change in the biosensor and an increase in FRET could explain this phenomenon.This thesis has allowed the development of a new pair of fluorescent proteins for FRET imaging insensitive to pH. This couple will allow better characterization of existing biosensors sensibilities as we have shown with AKAR. This pair of fluorescent proteins may also be used in acidic cellular compartments, for example to study protein / protein interactions. Finally, through improved spectral separation in excitation and in emission, this pair can be used in more demanding applications such as biphoton microscopy
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Janßen, Julia Annika. "In vivo FLIM-FRET as a novel technique to assess cAMP and cGMP in the intact zebrafish heart." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2018. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-232452.
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Introduction: 23 million patients worldwide suffer from heart failure. These patients depend on cardiac research, because cardiac research enables the development of new therapeutic strategies and –targets. In cardiomyocytes, the compartmentalization of cAMP and cGMP depends on many factors. T-tubuli and PDEs are responsible for the division of cells in microdomains in which localized and specific cAMP and cGMP-signaling occurs. The aim of this thesis was to develop a method to answer the open questions that remain about the physiological and pathophysiological significance of cAMP/cGMP compartmentalization. Methods: I used the zebrafish as a model, because the transparency of zebrafish larvae enabled non-invasive fluorescent imaging in cardiomyocytes in the living animal. I cloned the Fluorescence Resonance Energy Transfer (FRET) sensors EPAC1-camps for cAMP and cGi500 for cGMP and injected them into zebrafish fertilized embryos. Then I used the F0 generation for Fluorescence Lifetime Imaging (FLIM) -FRET-measurements of cAMP and cGMP. Ca2+ is an important downstream mediator of cAMP and cGMP, because Ca2+ regulates cardiac contraction. Therefore, I also cloned the Ca2+ sensor GCaMP6 and used the dye Fluo-4 AM to include intracellular Ca2+ in the imaging. Results: The cloned sensors for cAMP, cGMP and Ca2+ were successfully injected into the zebrafish and showed expression in individual cardiomyocytes. I developed a protocol to mount the living zebrafish embryos and to measure intracellular cAMP and cGMP with FLIM-FRET in vivo with high spatial resolution. I characterized the sensors in their functionality by showing that the sensors react to changes in intracellular concentrations of cAMP and cGMP. The results of this study include evidence that zebrafish have mechanisms that lead to cAMP/cGMP compartmentalization in the absence of T-tubuli, and these mechanisms keep compartmentalization constant even under extreme cAMP or cGMP increasing drug treatment. Furthermore, I imaged intracellular Ca2+ by confocal microscopy and developed a protocol to use Fluo-4 AM for Ca2+ imaging. Conclusion: The method used in this thesis should allow the investigation of subcellular cAMP/cGMP compartmentalization and Ca2+ and to subsequently answer open questions in the field, for example whether a change of cAMP compartmentalization leads to the pathological phenotypes of cardiac disease or if a changed compartmentalization of cAMP in cardiac disease influences Ca2+ concentrations and therefore contraction. Additionally, this method can be used to learn more about cAMP, cGMP und Ca2+ during regeneration in the heart, because the zebrafish cardiomyocytes can regenerateEinleitung: Weltweit sind mehr als 23 Millionen unter Herzinsuffizienz leidende Patienten auf die kardiologische Grundlagenforschung angewiesen, da diese die Voraussetzung für eine bessere Versorgung durch adaptierte und neue Behandlungswege schafft. In Kardiomyozyten hängt die Kompartimentierung von cAMP und cGMP von vielen Faktoren ab. T-Tubuli und PDEs werden unter anderem für die Aufteilung der Zellen in Mikrodomänen, in denen lokalisierte und spezifische cAMP- und cGMP-Signalgebung stattfinden kann, verantwortlich gemacht. Das Ziel dieser Arbeit war die Etablierung einer Methode, mithilfe derer offene Fragen bezüglich der physiologischen und insbesondere der pathophysiologischen Relevanz der cAMP- und cGMP Kompartimentierung beantwortet werden können. Methode: Als Modell diente der Zebrafisch, da die Transparenz von Zebrafisch Embryonen eine nicht-invasive Bildgebung von Fluoreszenz in Kardiomyozyten im lebenden Tier ermöglicht. Dafür klonierte ich die Förster Resonance Energy Transfer (FRET) -Sensoren EPAC1-camps als cAMP-Sensor und cGi500 als cGMP-Sensor und injizierte diese in befruchtete Zebrafisch Embryonen. Anschließend benutzte ich die F0-Generation für Fluorescence Lifetime Imaging (FLIM) -FRET-Messungen von cAMP und cGMP. Da Ca2+ als wichtiger downstream Mediator von cAMP und cGMP die kardiale Kontraktion reguliert, klonierte ich außerdem den Ca2+-Sensor GCaMP6 und benutzte den Farbstoff Fluo-4 AM, um intrazelluläres Ca2+ darzustellen. Ergebnisse: Die klonierten Sensoren für cAMP, cGMP und Ca2+ konnten erfolgreich in den Zebrafisch injiziert werden und zeigten alle Expression in einzelnen Kardiomyozyten. Ich entwickelte ein Protokoll, dass die Fixierung von lebenden Zebrafisch Embryonen und nachfolgender Bildgebung von cAMP und cGMP mit hoher zellulärer Auflösung mit FLIM-FRET in vivo erlaubte. Ich konnte eine funktionelle Charakterisierung der Sensoren durchführen, indem ich zeigte, dass sie auf Konzentrationsänderungen von intrazellulärem cAMP und cGMP reagieren sowie zeigen, dass Zebrafische trotz fehlender T-Tubuli eine signifikante cAMP- und cGMP Kompartimentierung aufweisen, auch unter extremen Bedingungen nach Gabe von cAMP/cGMP stimulierenden Substanzen in hoher Dosierung. Ich konnte zudem subzelluläres Ca2+ durch konfokale Mikroskopie bildgebend darstellen und entwickelte ein Protokoll, um mit Fluo-4 AM eine schnelle Möglichkeit zu haben, Ca2+ mit in die Messungen einzubeziehen. Ausblick: Die in dieser Arbeit benutzte Methode bietet eine gute Möglichkeit, subzelluläre cAMP- und cGMP-Kompartimentierung und Ca2+ zu untersuchen und damit zum Beispiel die Fragen zu beantworten, ob eine veränderte cAMP/cGMP Kompartimentierung zu Herzkrankheiten wie Hypertrophie führt oder ob eine veränderte cAMP Kompartimentierung den zellulären Ca2+ Haushalt und damit die kardiale Kontraktion beeinflusst. Darüber hinaus kann das von mir etablierte Protokoll dazu genutzt werden, mehr über cAMP, cGMP und Ca2+ während der Regeneration im Herzen zu lernen, da der Zebrafisch über ausgeprägte Regenerationsfähigkeiten verfügt
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Ellis, Jonathan. "FRET analysis of splicing factors involved in exon and intron definition in living cells." Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/4397.
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I have analyzed the interactions between SR proteins and splicing components that are bound at the 5’ or 3’ splice site using fluorescence resonance energy transfer (FRET) microscopy. The SR proteins interact with the U1 snRNP-associated 70 kDa protein (U170K) at the 5’splice site and with the small subunit of the U2 snRNP auxiliary factor (U2AF35) at the 3’ splice site. These interactions have been extensively characterized biochemically in the past, and are proposed to play roles in both intron and exon definition. We employed FRET acceptor photobleaching and fluorescence lifetime imaging microscopy (FLIM) to identify and spatially localise sites of direct interactions of SF2/ASF, and other SR proteins, with U2AF35 and U1-70K in live cell nuclei. These interactions were shown to occur more strongly in interchromatin granule clusters (IGCs). They also occur in the presence of the RNA polymerase II inhibitor, DRB, demonstrating that they are not exclusively co-transcriptional. FLIM data have also revealed a novel interaction between HCC1, a factor highly related to the large subunit of the U2AF splicing factor, with both subunits of U2AF that occur in discrete domains within the nucleoplasm but not within IGCs. These data demonstrate that the interactions defining intron and exon definition do occur in living cells in a transcription-independent manner.
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Devauges, Viviane. "Microscopie de fluorescence résolue en temps et en polarisation pour le suivi d’interactions protéiques en neurobiologie." Thesis, Paris 11, 2011. http://www.theses.fr/2011PA112315/document.
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Le suivi des interactions entre protéines, localisées à la membrane plasmique ou à l’intérieur de cellules, a été réalisé au cours de cette thèse par imagerie de fluorescence et par l’analyse de processus dits de FRET (Forster Resonance Energy Transfer). Pour quantifier le FRET entre nos protéines d’intérêt, nous avons choisi le contraste de durée de vie de fluorescence car cette méthode est indépendante de la concentration et de l’intensité de fluorescence. Afin d’obtenir une résolution suffisante pour des problématiques neurobiologiques, un microscope TIRFLIM (Total Internal Reflection Fluorescence Lifetime Imaging Microscopy) avait préalablement été développé. Celui-ci nous permet de faire de l’imagerie en plein champ avec une résolution axiale sub-longueur d’onde. Ce dispositif a été calibré et optimisé au cours de cette thèse pour répondre au mieux à des problématiques biologiques. Différentes approches ont ainsi été testées dans le but de calibrer la profondeur de pénétration de l’onde évanescente. Des surfaces plasmoniques ont entre autres été utilisées pour augmenter la sélectivité axiale du montage. Notre microscope a été dédié à l’étude de l’effet du cholestérol sur l’interaction entre la protéine précurseur de l’amyloïde APP, protéine transmembranaire impliquée dans la maladie d’Alzheimer et une de ses enzymes de clivage BACE1. Nous avons ainsi effectué un suivi dynamique de l’effet du cholestérol sur l’interaction entre APP et BACE1 dans des cellules HEK-293 et dans des cultures primaires de neurones d’hippocampe d’embryons de rat, de la membrane plasmique à l’intérieur des cellules grâce à notre dispositif TIRFLIM. La mesure d’anisotropie de fluorescence résolue en temps a également été implémentée sur notre montage. Ces mesures résolues en temps et en polarisation ont permis de mesurer le temps de corrélation rotationnelle de fluorophores et de mettre en évidence de manière qualitative différents niveaux d’homodimérisation de protéines impliquées dans la maladie d’AlzheimerIn the framework of this thesis, we have used FRET (Forster Resonance Energy Transfer) as a mechanism to follow the interaction of proteins from the plasma membrane to the cytoplasm of cells. To quantify FRET, we have chosen Fluorescence Lifetime Imaging Microscopy (FLIM) since this method is independent of the concentration and intensity of the fluorophores. To have a good axial resolution, a TIRFLIM set-up (Total Internal Reflection Fluorescence Lifetime Imaging Microscopy) was developed and this allowed us to perform wide-field imaging with sub-wavelength axial resolution. This set-up was calibrated and optimized in order to answer biological questions. Different approaches were tested in order to measure the penetration depth of the evanescent field and especially plasmonic surfaces were used to further enhance the axial resolution. Our set-up was dedicated to the study of the effect of cholesterol on the interaction between the Amyloid Precursor Protein (APP), a transmembrane protein involved in Alzheimer Disease, and one of its cleaving enzyme (BACE1). We performed a dynamic tracking of APP and BACE1 proximity under the effect of cholesterol, in HEK-293 cells and primary cultures of embryonic rat hippocampal neurons, thanks to our TIRFLIM set-up.Time-resolved fluorescence anisotropy has been implemented on our set-up. This has enabled us to measure the rotational correlation time of fluorophores and to investigate quantitatively different states of homodimerization of proteins involved in Alzheimer’s disease
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Déméautis, Claire. "Développement de nouvelles méthodes pour dépasser les limitations rencontrées dans le suivi de biosenseur FRET par microscopie de fluorescence quantitative." Thesis, Rennes 1, 2016. http://www.theses.fr/2016REN1B035/document.
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La microscopie de fluorescence est devenue un outil incontournable en biologie. En particulier, il est possible de suivre dans le temps et dans l’espace en cellules vivantes l’activité de protéines en utilisant des biosenseurs FRET génétiquement encodés. Mon travail de thèse a consisté à développer de nouvelles méthodes de microscopie de fluorescence quantitative pour dépasser les limitations rencontrées dans le suivi de biosenseurs FRET. En premier lieu, j’ai eu à développer une méthodologie pour le suivi de deux biosenseurs FRET génétiquement encodés par mesure de durée de vie (FLIM) en simultané avec une seule longueur d’onde d’excitation. En effet, il n’est pas facile de suivre deux activités biochimiques par biosenseurs FRET dans un même échantillon vivant par microscopie de fluorescence à cause de l’existence de fuites spectrales dans les canaux de détection des différentes protéines fluorescentes et l’utilisation de deux longueurs d’onde d’excitation pour chacun des deux donneurs. En combinant les deux couples de protéines fluorescentes mTFP1/ShadowG et LSSmOrange/mKate2, les artefacts de fuite spectrale ont pu être négligés. Il a été possible de suivre les activités kinases des protéines ERK et PKA en simultané par FLIM. À l’aide de cette méthodologie, nous avons pu mettre en évidence une activation de la voie PKA lors d’une stimulation à l’EGF. En second lieu, j’ai développé une méthode pour le suivi de biosenseur FRET par la technique de spectroscopie à corrélation croisée de fluorescence (FCCS). Le suivi d’activité de certaines protéines peut s’avérer difficile du fait de leur faible expression au sein de l’échantillon vivant et de leur localisation. La méthode de FCCS nécessite une faible concentration de fluorophores et peut donc s’adapter à ces échantillons. Le FRET a un effet direct sur l’amplitude de corrélation croisée lorsque celle-ci est mesurée en suivant la co-diffusion de deux protéines fluorescentes attachées à un même biosenseur. Une diminution de ratio d’amplitude des courbes d’autocorrélation (verte ou rouge) sur l’amplitude de la courbe de corrélation croisée correspond à la présence de FRET. Nous avons pu mesurer cette diminution dans des cellules exprimant le biosenseur FRET Aurora A WT (FRET) en comparaison avec un mutant K162M (non FRET). Ces premiers résultats sont très prometteurs pour l’utilisation de cette approche au suivi d’un biosenseur faiblement exprimé en cellules vivantes. L’amélioration du suivi de biosenseur FRET, par les méthodes de microscopie de fluorescence quantitative présentées dans ce travail, doit pouvoir permettre la réponse à des questions d’intérêt biologiques nécessitant le suivi multiplex de FRET ou la mesure de biosenseurs à faible niveau d’expression, là où les techniques conventionnelles se heurtaient à leur limitationFluorescence microscopy has become an invaluable tool in biology. In particular, it allows to follow in time and space the activity of proteins, using genetically encoded FRET biosensors, in live cell imaging. In my thesis work, I have developed new quantitative fluorescence microscopy methods to overcome the limitations encountered in monitoring FRET biosensors. First, I developed a methodology to monitor simultaneously two genetically encoded FRET biosensors by lifetime measures (FLIM) with a single excitation wavelength. Previously, it was not easy to follow two biochemical activities by FRET biosensors in the same live sample by fluorescence microscopy. Two reasons for that: the existence of spectral bleed through in the detection channel of each fluorescent proteins and the use of two excitation wavelengths for the two donors. By combining two fluorescent proteins pairs: mTFP1 / ShadowG and LSSmOrange / mKate2, the “spectral bleed through” artifact became negligible. It became then possible to follow the kinase activity of PKA and ERK proteins simultaneously by FLIM. Using this methodology, we were able to show an activation of the PKA pathway upon stimulation with EGF. Second, I developed a method to monitor FRET biosensor by fluorescence cross-correlation spectroscopy technique (FCCS). Monitoring the activity of certain proteins may be difficult due to their low expression in living sample and their sub-cellular localization. The FCCS methods requires a low concentration of fluorophores and can therefore be adapted to these samples. FRET has a direct effect on the cross-correlation amplitude when it is measured by following the co-diffusion of two fluorescent proteins attached to a same biosensor. An amplitude ratio decrease, of the autocorrelation curves (green or red) on the amplitude of the cross-correlation curve, corresponds to the presence of FRET. We were able to measure this ratio decreases in cells expressing the FRET biosensor Aurora A wild type (FRET) compared to the K162M mutant one (non-FRET). These first results are very promising to monitor the activity of a weakly expressed protein in living cells biosensor using this approach. The improvement of FRET biosensor monitoring, by quantitative fluorescence microscopy methods presented in this work, will help to answer biological questions of interest requiring the measurement of multiplex FRET monitoring or biosensors at low level expression, where conventional techniques are facing these limitations
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Scolari, Silvia. "Lateral organization of the transmembrane domain and cytoplasmic tail of influenza virus hemagglutinin revealed by time resolved imaging." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/16007.
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Der Viruspartikelzusammenbau hängt von der Anreicherung viraler Untereinheiten in spezifischen Domänen der PM ab. Es wird vorgeschlagen, dass Membran-Rafts – geordnete, sphingomyelin- und cholesterinreiche Mikrodomänen in der PM – als lokale Rekrutierungsstellen dienen. Hämagglutinin (HA) ist ein homotrimeres Glykoprotein in der Hülle des Influenzavirus. Es dient der Bindung an die Wirtszelle und der Fusion mit dem Endosom. Es wird angenommen, dass HA bei der Abschnürung der Viruspartikel von der Zelle mitwirkt. Zwei Hauptbeobachtungen führten zu der Hypothese, dass sich HA in Lipid-Mikrodomänen einlagert: HA wurde biochemisch in Detergens-resistenten Membranen nachgewiesen und die Virushülle ist mit raftbildenden Lipiden angereichert. Um die Rolle der HA-Transmembrandomäne für die Lipid-Raft-Inkorporation aufzuklären, wurde ein Konstrukt entwickelt, das den C-Terminus von HA mit dem gelb fluoreszierenden Protein YFP fusioniert, und die Transmembrandomäne, nicht aber die N-terminale Ektodomäne von HA enthält. In transfizierten Säugetierzellen wurde der Förster-Resonanz-Energie-Transfer (FRET) zwischen diesem Konstrukt und einem GPI-verankerten cyan fluoreszierenden Protein CFP (Raft-Marker) durch Fluoreszenz-Lebenszeit-Mikroskopie (FLIM) gemessen. Die Ergebnisse zeigen, dass sich HA-Konstrukte in Cholesterin-abhängigen Lipiddomänen anreichern, was durch eine erhöhte FRET-Effizienz nachgewiesen wurde. Zudem führen ein Cholesterinentzug aus der PM und die Deletion hochkonservierter Palmitylierungsstellen zu einer signifikanten Verringerung selbiger; sehr gering war diese zwischen dem HA-Konstrukt und einem Nicht-Raft-Marker. Darüberhinaus konnte durch ortsspezifische Mutagenese gezeigt werden, dass die verwendeten Konstrukte disulfidbrückenverbundene Oligomere bilden, welche Voraussetzung für den Transport der Konstrukte an die PM sind. Zeitaufgelöste Anisotropiemessungen ergaben für diese ein starkes Homo-FRET-Signal, welches die Oligomerisierungshypothese bestätigt.Numerous enveloped viruses bud from the host cell plasma membrane (PM). Assembly of the new viral particles depends on the accumulation of the viral subunits at specific sites of the cell membrane. Lipid domains or rafts enriched of sphingomyelin and cholesterol were suggested as sites for local recruitment of viral components. Hemagglutinin (HA), a homotrimeric glycoprotein embedded in the envelope of influenza virus, mediates binding of the virus to the host cell and fusion between the viral envelope and the endosomal membrane. HA might play an important role in budding of the viral particles from the host cell. Two observations led to the suggestion that HA entraps in lipid microdomains. First, HA was rescued in DRM fractions, second the viral envelope was found to be enriched in lipids generally forming rafts. To elucidate the role of the HA transmembrane domain in lipid raft localization we expressed constructs harboring the transmembrane domain and the cytoplasmic tail but lacking the N-terminal ectodomain of HA in the PM of mammalian cells. We studied energy transfer (FRET) between these constructs and a GPI anchored CFP as a raft marker by fluorescence lifetime imaging microscopy (FLIM). Our results suggest that HA constructs are indeed sorted into cholesterol-dependent lipid domains since cholesterol depletion of the PM caused a significant decrease of FRET efficiency. Likewise, deletion of the three highly conserved palmitoylation sites of HA is also accompanied by a reduction of FRET efficiency. Site directed mutagenesis demonstrated that TMD-HA constructs form disulfide linked oligomers and that oligomerization is fundamental for the transport to the PM. This result was corroborated by time resolved anisotropy measurements that revealed strong homoFRET between TMD-HA-YFP molecules, thus indicating protein clustering. Accordingly, trimerization of full length HA is fundamental for stability and the subsequent delivery of the protein to the cell surface.
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Altenbach, Kirsten. "Development and analysis of recombinant fluorescent probes for use in live cell imaging of filamentous fungi." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4739.
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The molecular cloning and subsequent engineering of the green fluorescent protein (GFP) of the jellyfish Aequoria victoria allowed a novel approach to the investigation of cell signalling. GFP and its mutants can now not only be used to target specific organelles in living cells but also function as a basis for a variety of sensors for biologically important ions and molecular interactions. GFP-based Ca2+- sensors have been successfully used for studies in mammalian and plant cells. In filamentous fungi, however, they have not yet been reported to work. Since only little is known about calcium signalling in filamentous fungi, this project aimed to improve existing GFP-based Ca2+- sensors by exchanging the original fluorophores for improved versions and expressing those in the filamentous fungus Aspergillus niger. During this project, the donor and acceptor fluorophores of 3 existing Ca2+-FRETprobes based on cameleons and troponin C-sensors, have been changed, 2 novel positive FRET controls have been designed and these , as well as donor and acceptor fluorophores alone, have been expressed in the filamentous fungus Aspergillus niger. The probes were assessed using different imaging techniques, such as conventional confocal laser scanning microscopy (CLSM), fluorescence lifetime imaging microscopy (FLIM) and spectral imaging using a Leica TSC SP5 confocal and IRIS, a novel spectral imaging device designed at Heriot Watt University. Problems were encountered that prevented FRET analysis using CLSM and IRIS. These were due mainly to the difference in expression level of the constructs and the distribution of the emission bandpasses of the IRIS system. Analysis of the spectral data obtained on the Leica confocal system and analysis of the FLIM results, however, revealed significant differences between the donor only and the positive FRET controls. Spectra of the positive FRET controls and the Ca2+-sensitive probes showed emission peaks of both the donor and the acceptor fluorophores upon excitation of the donor fluorophore alone while analysis of the FLIM results revealed an additional decay component in the positive FRET controls. Both results are very strong indicators that we can detect FRET in living hyphae of Aspergillus niger transformed with the probes designed during this project.
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Fries, Maximilian Werner. "Multiplexed biochemical imaging reveals the extent and complexity of non-genetic heterogeneity in DNA damage-induced caspase dynamics." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/273868.
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Genetically identical cells show a heterogeneous response to a multitude of signals such as growth factors and DNA damage. While this heterogeneity has been shown to be a major determinant of treatment success in several diseases including cancer, little is known about how differences in biochemical signalling networks underlie such heterogeneity. State-of-the-art methodologies to study biochemical networks are often invasive and enable to quantify biochemical events only on cell populations or at a single point in time for a single cell, and therefore, cannot adequately quantify the fast, asynchronous and heterogeneous responses. In order to address these limitations, we have developed a unique sensing platform based on fluorescence lifetime imaging microscopy (FLIM) capable to multiplex at least three biosensors by utilizing Förster Resonance Energy Transfer (FRET) efficiently. After an overall introduction in Chapter 1, I describe the rational design and characterization of novel FRET pairs aiming to utilize the visible spectrum efficiently in combination with FLIM in Chapter 2. We combined blue, green and red donor fluorescent proteins that are excited at the same wavelength (840 nm for two-photon excitation) with genetically encoded quenchers, i.e. non-fluorescent chromoproteins as acceptors. This sensing platform enables the simultaneous detection of three biochemical reactions within single living cells providing new opportunities to characterize and understand non-genetic heterogeneity. In Chapter 3, I will demonstrate the first application of this novel platform by studying the activity of three key enzymes in DNA damage-induced cell death, caspase-2, -3, and -9. We confirm the heterogeneous nature of Cisplatin-induced cell death in genetically identical cells but reveal the existence of at least three subpopulations of cells characterized by distinct caspase dynamics. By combining biochemical and morphological information we infer the existence of different biochemical network topologies that are associated with alternative death phenotypes each cell adopts, such as apoptosis and programmed necrosis. Finally, deconvolution of cellular populations and direct measurement of a three-node caspase network - formerly impossible - permitted us to design perturbations of cell fate choices utilizing clinically relevant inhibitors. These perturbations resulted in changes in cell fate in response to Cisplatin, a clinically desirable outcome that suggests new avenues for combinatorial drugging and a new strategy to reveal cancer vulnerabilities that may be otherwise confounded by typical genetic and non-genetic heterogeneity.
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Balloi, Eleonora. "Investigating conformational changes of proteins using Förster Resonance Energy Transfer." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/investigating-conformational-changes-of-proteins-using-frster-resonance-energy-transfer(fd5f53e7-9464-40bf-9c87-6fbd0a3d8804).html.
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Förster Resonance Energy Transfer (FRET)-based techniques are gaining an increasing importance in cell biology and cell-matrix adhesion studies because they allow both the detection of conformational changes of target proteins and their localisation in cells. Frequency Domain-Fluorescence Lifetime Microscopy (FD-FLIM) is currently considered one of the most reliable methods to measure FRET in live cells. However, due to its dependence on many technical prerequisites, its use is not yet widespread. The purpose of this work was to first establish FD-FLIM measurements of FRET on a new FD-FLIM microscope module. Then we aimed to apply FD-FLIM-FRET measurements to the study of conformational changes of the cell matrix-adhesion proteins vinculin and integrin and of the growth factor receptor Tie-2. In the first part of the work, published FRET probes including distance-sensors and two sets of vinculin-based probes were extensively tested with FD-FLIM, sensitised emission and ratiometric FRET. FD-FLIM was shown to be the most accurate method in approximating molecular distances between fluorophores. Moreover this study unveiled specific caveats associated with both existing vinculin FRET probes. FD-FLIM was then used to study conformational changes of the extracellular matrix receptor alphavβ3 integrin and of the angiopoietin receptor Tie-2 using specific FRET probes designed by us. While data showed that the alphav-integrin-FRET probe localised to adhesion sites, more experiments will be required to evaluate its full functionality. The Tie-2-FRET probe was fully functional and, upon ligand binding, allowed the detection of a bending movement of the extracellular domain towards the cell membrane. Finally, a combination of FRET, immunofluorescence and tension release experiments were used to show that intracellular tension is not required to maintain integrins in their activated conformation. However, intracellular tension is required to recruit other key proteins such as vinculin, talin and tensin to adhesions sites. Overall this work demonstrates the importance of FD-FLIM-FRET as a tool to investigate conformational changes of adhesion proteins and transmembrane receptors within the cell environment.
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Ashraf, Waseem. "Mécanismes d'interaction de l'intégrateur épigénétique UHRF1 avec l'acétyltransférase TIP60." Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAJ078/document.
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UHRF1 est une protéine nucléaire responsable du maintien et de la régulation de l'épigénome des cellules. Elle favorise la prolifération cellulaire et est surexprimée dans la plupart des cancers. TIP60, l'un des partenaires le plus important d’UHRF1, est impliqué dans le remodelage de la chromatine et la régulation transcriptionnelle grâce à son activité acétyltransférase. Ensemble, les deux protéines régulent la stabilité et l'activité d'autres protéines telles que la DNMT1 et la p53. Le but de cette étude était d'explorer le mécanisme d'interaction entre UHRF1 et TIP60 en visualisant cette interaction dans les cellules. La microscopie par imagerie à temps de vie de fluorescence et d'autres techniques de biologie moléculaire ont été utilisées. Les résultats ont montré que UHRF1 interagit directement avec le domaine MYST de TIP60 et cette interaction se produit dans la phase S du cycle cellulaire. Les deux protéines ont également montré une réponse similaire aux dommages à l'ADN, ce qui prédit une cohérence dans leur fonction dans le mécanisme de réparation de l'ADN. La surexpression de TIP60 a également induit la baisse du niveau d’UHRF1 et de DNMT1 ainsi qu’une induction d'apoptose dans les cellules ce qui suggère un rôle de TIP60 dans la régulation des fonctions oncogéniques d’UHRF1UHRF1 is a nuclear protein maintaining and regulating the epigenome of cells. Its promotes proliferation and is found upregulated in most of cancers. TIP60 is one of the important interacting partner of UHRF1 and is involved in chromatin remodeling and transcriptional regulation through its acetyltransferase activity. Together they regulate the stability and activity of other proteins such as DNMT1 and p53. The aim of this thesis was to explore the mechanism of interaction between UHRF1 and TIP60 by visualizing this interaction in cells. Fluorescent lifetime imaging microscopy and other molecular biology techniques were employed for this purpose. Results of this study showed that UHRF1 interacts directly to the MYST domain of TIP60 and this interaction prevails in the S-phase of cell cycle. Both proteins also showed a similar response to DNA damage predicting a coherence in their function in DNA repair mechanism. Overexpression of TIP60 also downregulated UHRF1 and DNMT1 and induced apoptosis in cells suggesting a role of TIP60 in regulation of oncogenic functions of UHRF1
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Siche, Stefanie. "Die Proteine HA und M2 von Influenzaviren." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2016. http://dx.doi.org/10.18452/17499.
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Die Assemblierung von Influenzaviren erfolgt an Rafts der apikalen Wirtszellplasmamembran mit denen das Hämagglutinin (HA) über Acylierungen im C-Terminus und hydrophobe Aminosäuren seiner Transmembrandomäne (TMD) interagiert. M2 besitzt eine cytoplasmatische amphiphile Helix (AH), die ebenso potenzielle Raft-Motive aufweist: Eine Acylierung und Cholesterol-Bindemotive. In dieser Arbeit wurde per Konfokalmikroskopie an polarisierten Zellen, die fluoreszenzmarkierte M2-Varianten exprimierten, gezeigt, dass diese M2-Motive nicht für den apikalen Transport, der vermutlich durch Raft-ähnliche Vesikel erfolgt, benötigt werden. Messungen des Förster-Resonanzenergietransfers über Fluoreszenz-Lebenszeit-Mikroskopie (FLIM-FRET) in der Plasmamembran lebender Zellen, die fluoreszenzmarkiertes HA und M2 koexprimierten, ergaben, dass diese Motive auch nicht für die Interaktion mit den durch HA, in Abhängigkeit von dessen Raft-Motiven, stabilisierten Raft-Domänen notwendig sind. Mittels reverser Genetik konnten infektiöse WSN-Viren mit fehlender Acylierung am Ende der HA-TMD, nicht jedoch Viren ohne die zwei cytoplasmatischen Acylierungen hergestellt werden. Weiterhin ergaben Wachstumsanalysen, dass die Acylierung von HA und M2 für den gleichen Schritt des viralen Replikationszyklus von Bedeutung sind. Für die M2-AH wurde postuliert, dass sie die Membrankrümmung detektiert und durch Insertion in die Wirtszellmembran die Virusabschnürung bewirkt. Infektiöse Viren ohne M2 oder ohne die AH konnten ebenso wie Viren mit M2 mit einer Helix mit reduzierter Amphiphilität in dieser Arbeit nicht hergestellt werden. Allerdings führte die Substitution der AH durch typische krümmungsdetektierende oder modulierende Helices zu Viren, deren Wachstum um zwei bis vier Titerstufen im Vergleich zum Wildtyp reduziert war. Die Helix-Amphiphilität scheint wichtig zu sein, aber auch die Sequenz oder bestimmte Aminosäuren sind offenbar für eine effiziente Virusreplikation notwendig.The assembly of influenza virus particles occurs at the apical plasma membrane of the host cell at membrane rafts which the hemagglutinin (HA) interacts with via acylations in its C-terminal region and via hydrophobic amino acids in the transmembrane domain (TMD). M2 possesses a cytoplasmic amphiphilic helix (AH) that also contains potential raft motifs: an acylation and cholesterol-binding motifs. In this work, confocal microscopy of polarised cells, which were expressing fluorescently labelled M2-variants, demonstrated that these motifs of M2 are not required for apical transport, which is assumed to be mediated by raft-like vesicles. Furthermore, FLIM-FRET (Förster resonance energy transfer measured via fluorescence lifetime imaging microscopy) analyses, performed in the plasma membrane of living cells coexpressing fluorescently labelled HA and M2, revealed that these M2-motifs are not required for association with the large coalesced raft phase organised by HA. In contrast, deleting HA’s raft-targeting features clearly reduced clustering with M2. While the removal of the two cytoplasmic acylations prevented the rescue of infectious virus by reverse genetics, a mutant virus without acylation in the HA-TMD could be rescued. Moreover, growth analyses revealed that the acylations of HA and M2 are important for the same step in the viral replication cycle. It has been postulated that the M2-AH detects membrane curvature and accomplishes membrane scission by inserting into the host cell membrane. Viruses without M2, without the M2-AH or with M2 containing a helix with reduced amphiphilicity could not be produced in this work. However, substituting the AH by typical curvature-sensing or -generating helices led to viruses with two to four orders of magnitude reduced growth as compared to wildtype virus. The amphiphilicity of the helix seems to be important, but also the sequence or specific amino acids appear to be necessary for an efficient virus replication.
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Ramirez-Garces, Diana. "Analyses of CRN effectors (crinkler and necrosis) of the oomycete aphanomyces euteiches." Toulouse 3, 2014. http://thesesups.ups-tlse.fr/2489/.
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L'oomycète Aphanomyces euteiches est un pathogène racinaire de légumineuses cultivées (pois, luzerne. . . ) et de la plante modèle Medicago truncatula. Les oomycètes, comme d'autres microorganismes pathogènes eucaryotes, secrètent et transloquent des molécules à l'intérieur des cellules de l'hôte (effecteurs intracellulaires/cytoplasmiques) dans le but de manipuler les fonctions de la plante et de faciliter l'infection. Les protéines CRN (Crinkling and Necrosis) constituent une famille d'effecteurs nucléaires largement répandue chez les oomycètes et récemment décrites chez des espèces fongiques. Leurs cibles et rôle dans la virulence ainsi que leurs mécanismes de sécrétion et de translocation sont encore mal compris. Nous avons entrepris la caractérisation fonctionnelle des protéines AeCRN5 et AeCRN13 d'A. Euteiches ainsi que de l'homologue d'AeCRN13 du champignon pathogène d'amphibien Batrachochytrium dendrobatidis, BdCRN13. Les analyses d'expression génique et protéique ont permis de montrer que AeCRN5 et AeCRN13 sont exprimés durant l'infection des racines de M. Truncatula. Des résultats préliminaires d'immuno-localisation d'AeCRN13 ont révélé, pour la première fois, la sécrétion et translocation d'un CRN durant l'infection. Leur expression hétérologue, à la fois dans les cellules de plantes et d'amphibiens, a montré que ces protéines se localisent dans les noyaux où leurs activités conduisent à la perturbation de la physiologie de l'hôte. En développant un système in vivo basé sur la technique de FRET-FLIM, nous avons démontré que ces CRN ciblent les acides nucléiques: AeCRN5 cible l'ARN des plantes tandis qu'AeCRN13 et BdCRN13 lient l'ADN. Ces deux effecteurs CRN13 exhibent un motif de type HNH, lequel est typiquement retrouvé dans des endonucleases. Nous avons démontré que les CRN13 présentent une activité nuclease in vivo conduisant à la génération de coupures double brin de l'ADN. Ce travail a permis de mettre en évidence un nouvel mécanisme d'action des effecteurs de microorganismes eucaryotes et apporte des nouveaux aspects pour la compréhension de l'activité des protéines CRN d'oomycète mais aussi, pour la première fois, de champignonThe oomycete Aphanomyces euteiches is an important pathogen infecting roots of legumes (pea, alfalfa. . . ) and the model legume Medicago truncatula. Oomycetes and other microbial eukaryotic pathogens secrete and deliver effector molecules into host intracellular compartments (intracellular/cytoplasmic effectors) to manipulate plant functions and promote infection. CRN (Crinkling and Necrosis) proteins are a wide class of intracellular, nuclear-localized effectors commonly found in oomycetes and recently described in true fungi whose host targets, virulence roles, secretion and host-delivery mechanisms are poorly understood. We addressed the functional characterization of CRN proteins AeCRN5 and AeCRN13 of A. Euteiches and AeCRN13's homolog of the chytrid fungal pathogen of amphibians Batrachochytrium dendrobatidis, BdCRN13. Gene and protein expression studies showed that AeCRN5 and AeCRN13 are expressed during infection of M. Truncatula's roots. Preliminary immunolocalization studies on AeCRN13 in infected roots indicated that the protein is secreted and translocated into root cells, depicting for the first time CRN secretion and translocation into the host during infection. The heterologous ectopic expression of AeCRNs and BdCRN13 in plant and amphibian cells indicated that these proteins target host nuclei and lead to the perturbation of host physiology. By developing an in vivo FRET-FLIM-based assay, we revealed that these CRNs target host nucleic acids: AeCRN5 targets plant RNA while AeCRN13 and BdCRN13 target DNA. Both CRN13 exhibit a HNH-like motif commonly found in endonucleases and we further demonstrated that both CRN13 display a nuclease activity in vivo inducing double-stranded DNA cleavage. This work reveals a new mode of action of intracellular eukaryotic effectors and brings new aspects for the comprehension of CRN's activities not only in oomycetes but, for the first time, also in true fungi
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Crosby, Kevin C. "Macromolecular Organization and Cell Function: A Multi-System Analysis." Diss., Virginia Tech, 2008. http://hdl.handle.net/10919/30259.
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The interior of the cell is a densely crowded and complex arena, full of a vast and diverse array of molecules and macromolecules. A fundamental understanding of cellular physiology will depend not only upon a reductionist analysis of the chemistry, structure, and function of individual components and subsystems, but also on a sagacious exegesis of the dynamic and emergent properties that characterize the higher-level system of living cells. Here, we present work on two aspects of the supramolecular organization of the cell: the controlled assembly of the mitotic spindle during cell division and the regulation of cellular metabolism through the formation of multienzyme complexes.
During division, the cell undergoes a profound morphological and molecular reorganization that includes the creation of the mitotic spindle, a process that must be highly controlled in order to ensure that accurate segregation of hereditary material. Chapter 2 describes results that implicate the kinase, Zeste-white3/Shaggy (Zw3/Sgg), as having a role in regulating spindle morphology.
The congregation of metabolic enzymes into macromolecular complexes is a key feature of cellular physiology. Given the apparent pervasiveness of these assemblies, it seems likely that some of the mechanisms involved in their organization and regulation might be conserved across a range of biosynthetic pathways in diverse organisms. The Winkel laboratory makes use of the flavonoid biosynthetic pathway in Arabidopsis as an experimental model for studying the architecture, dynamics, and functional roles of metabolic complexes. Over the past several years, we have accumulated substantive and compelling evidence indicating that a number of these enzymes directly interact, perhaps as part of a dynamic globular complex involving multiple points of contact between proteins. Chapter 3 describes the functional analysis of a predicted flavonol synthase gene family in Arabidopsis. The first evidence for the interaction of flavonoid enzymes in living cells, using fluorescent lifetime imaging microscopy fluorescent resonance energy transfer analysis (FLIM-FRET), is presented in Chapter 4.Ph. D.
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Valenta, Hana. "Live-cell investigation of the NADPH oxidase active state using fluorescent proteins and quantitative spectro-microscopies." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASF010.
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En cellules vivantes, les interactions dynamiques entre les protéines jouent un rôle clé dans la régulation de nombreuses voies de signalisation et événements biochimiques. C’est également le cas de la NADPH oxydase (NOX) des phagocytes, qui est une enzyme majeure du système immunitaire inné. Elle génère des anions superoxyde (O₂•⁻), précurseurs d’espèces réactives de l’oxygène (ROS), qui sont essentielles dans la lutte contre les infections microbiennes. La NADPH oxydase est un complexe protéique composé de six sous-unités ; deux protéines membranaires (NOX2 et p22phox) formant le centre catalytique, trois protéines cytosoliques (p67phox, p47phox et p40phox) et une petite GTPase Rac. Le mécanisme d’activation de la NADPH oxydase est basé sur l’assemblage de toutes les sous-unités cytosoliques avec les sousunités membranaire, où les interactions protéineprotéine jouent un rôle important. Un défaut d’activité de la NADPH oxydase cause une maladie granulomateuse septique chronique (CGD) caractérisée par des infections sévères et récurrentes. En revanche, des niveaux élevés de ROS contribuent aux maladies cardiovasculaires et neurodégénératives. Ainsi, l’activité de la NADPH oxydase doit être strictement régulée. Une meilleure compréhension de la machinerie de la NADPH oxydase au niveau moléculaire aidera à identifier les aspects clés de l’activité enzymatique et donc les potentielles cibles thérapeutiques. Le but de ma thèse était d’étudier l’état actif de la NADPH oxydase en utilisant des stratégies de microscopie à fluorescence en cellules vivantes. Pour détecter les interactions protéine-protéine, nous avons exploité le phénomène de transfert résonant d’énergie de type Förster (FRET) mesuré par imagerie de durée de fluorescence (FLIM). Étant donné que le phénomène de FRET a lieu uniquement entre des fluorophores proches spatialement (< 10 nm), il est approprié pour révéler les interactions à l’échelle nanométrique entre les sous-unités de la NOX étiquetées par des protéines fluorescentes (PFs) et il fournit également des informations sur la topologie du complexe enzymatique. Les approches de FRET-FLIM ont été réalisées soit avec des sous-unités séparées, soit avec une protéine de fusion chimérique appelée "Trimère". Le Trimère est composé des domaines essentiels des protéines cytosoliques p47phox, p67phox et Rac1, permettant une activité constitutive de la NADPH oxydase dans les cellules, sans avoir besoin d’un stimulant. Dans une première étape, nous avons travaillé avec les sous-unités individuelles étiquetés par les PFs dans les cellules de type fibroblastes ou dans des modèles de phagocytes. Nous avons comparé le PMA et l’acide arachidonique en tant qu’activateurs de la NADPH oxydase en termes de cinétique d’activation et de production de ROS. Les conditions expérimentales les plus convenables ont été explorées par la microscopie TIRF, qui permet d’exciter sélectivement des fluorophores situés près de la membrane plasmique et ainsi rend possible de suivre la formation du complexe actif de la NADPH oxydase en temps réel. Dans la deuxième partie de ma thèse, nous avons utilisé majoritairement le Trimère. Les expériences FRET-FLIM ont révélé que le Trimère forme des clusters dans la membrane plasmique. L’activité continue de la NADPH oxydase incité par le Trimère a été également examinée en termes de conséquences sur la physiologie des cellules vivantes. Nous avons montré que la production continue de ROS à long terme conduit à l’acidification du pH intracellulaire et déclenche l’apoptoseIn living cells, dynamic interactions between proteins play a key role in regulating many signaling pathways and biochemical events. It is also the case of the phagocyte NADPH oxidase (NOX), a key enzyme of the innate immune system. It generates superoxide anions (O₂•⁻), precursors of reactive oxygen species (ROS), such as hydrogen peroxide or hydroxyl radical that are critical for host responses to microbial infections. The NADPH oxidase is a protein complex composed of six subunits; two membrane proteins (NOX2 and p22phox) forming the catalytic core, three cytosolic proteins (p67phox, p47phox and p40phox) and a small GTPase Rac. The sophisticated activation mechanism of the NADPH oxidase relies on the assembly of all cytosolic subunits with the membrane-bound components, whereby proteinprotein interactions play an important role. Lack of the NADPH oxidase activity leads to chronic granulomatous disease (CGD) characterized by severe and recurrent infections. On the other hand, enhanced levels of ROS contribute to cardiovascular and neurodegenerative diseases. Thus, the NADPH oxidase activity needs to be tightly regulated in order to maintain physiological levels of ROS. Understanding the NADPH oxidase machinery at the molecular level will help to identify the key aspects of its enzyme activity and thereby potential therapeutic targets. The aim of my PhD project was to investigate the active state of the NADPH oxidase in living cells using state of the art fluorescence microscopy strategies. To detect the protein-protein interactions Förster Resonance Energy Transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) was a method of choice. As the FRET phenomenon occurs only between fluorophores in close proximity (< 10 nm), it is well-suited to reveal interactions of the NADPH oxidase subunits labeled by fluorescent proteins at nanoscale level, but also it provides information about the topology of the enzyme complex. FRET-FLIM was performed either with separated NOX subunits or with a chimeric fusion protein called “Trimera”. The Trimera is composed of the essential domains of the cytosolic proteins p47phox, p67phox and Rac1, enabling constitutive, robust NADPH oxidase activity in cells without the need of a stimulant. First, we worked with the individual FP-labeled cytosolic subunits in COSNOX cells (stably expressing NOX2/p22phox subunits) or macrophages and compared PMA and arachidonic acid as activators of the NADPH oxidase in terms of the activation kinetics and the total ROS production. By introducing mutations into the p47phox and p67phox subunits we were able to modulate the oxidase activity. The final validated working conditions were explored by TIRF microscopy, an imaging method allowing selective excitation of the fluorophores situated in the vicinity of the plasma membrane, and thus enabling to monitor the realtime formation of the active NADPH oxidase complex. We also focused on NOX2, the catalytic center of the NADPH oxidase that we labeled by FPs and prepared for further FRET-FLIM experiments aiming the investigation of NOX2/cytosolic subunits interactions. Second, we employed the Trimera that acts as a single activating protein of the NADPH oxidase. FRET-FLIM experiments revealed that theFP-Trimera forms clusters in the plasma membrane. The continuous long-term NOX activity elicited by the Trimera was also examined in terms of consequences on the physiology of living cells. We showed that the sustained ROS production leads to acidification of the intracellular pH and triggers apoptosis
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Muhammad, Sharif Ossai. "Characterizing the interaction between VE-PTP, Tie2 and VE-Cadherin." VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/2869.
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Many signaling pathways have been shown to be involved in the formation of the vascular system. Among them are the endothelial specific receptor families such as VEGF, Ang/Tie, as well as other signaling pathways such as semaphorins, which are also involved, in axonal guidance. It is known that the interaction between receptor tyrosine kinase, Tie2, VE-Cadherin, and VE-PTP mediate endothelial cell quiescence and adhesion. However, the structural basis of these interactions is not well understood. The aim of our study is to characterize the binding interactions between these players. Another important part of our study is describing the cross-talk between vasculature and nervous system by characterizing the Neuropilin/Plexin/Semaphorin system. VE-Cadherin along with neuropilins plays an essential role by directing VEGF signals to the appropriate location and coordinating the activation of downstream molecules. We characterize the interaction between Tie2, VE-PTP and VE-Cadherin by (FRET)-based proximity assay, fluorescence lifetime imaging, and co-immunoprecipitation assays. Our data showed a consistent localization of the protein and FRET signal for Tie2 and VE-PTP prior to ligand recognition. We showed the association between Tie2 and VE-Cadherin complex by co-immunoprecipatation. However, our FRET data was not consistent. The examination of VE-PTP and VE-Cadherin for association and localization of the protein showed a very unique, mutually exclusive localization of the protein. Our study of Neuropilin/Plexin/Semaphorin system showed changes in the protein localization, FRET signal and morphology upon stimulation of HEK293 cells expressing Nrp/plexin with Sema3D. In this system VE-Cadherin along with neuropilins plays an essential role by directing VEGF signals to the appropriate location and coordinating the activation of downstream molecules. The characterization of extracellular binding between Tie2, VE-PTP, and VE-Cadherin, will help to better understand the molecular mechanisms of normal and tumor angiogenesis to develop new anti-angiogeneic therapies.
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Ganesan, Sundar. "Local protein turnover as a regulatory mechanism of growth and collapse of neuronal growth cones." Doctoral thesis, [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=976327376.
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Ahmad, Tanveer. "Inhibition de la protéine UHRF1 par les partenaires épigénétiques et les épi-drogues." Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ089.
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UHRF1 est protéine nucléaire sur-exprimée dans les cellules cancéreuses. Elle joue un rôle essentiel dans la méthylation de l'ADN, favorise la prolifération cellulaire et inhibe l’expression des gènes suppresseurs de tumeurs. TIP60 est un partenaire importantd’UHRF1 qui participe au remodelage de la chromatine, à la régulation transcriptionnelle des gènes et à d'autres activités cellulaires grâce à son activité acétyltransférase. Les deux protéines sont impliquées dans la régulation de l'activité et la stabilité d’importantes protéines telles que la DNMT1 et la p53. Le but de cette thèse était d'étudier le mécanisme d'interaction de TIP60 avec UHRF1 et d'explorer l'effet de la surexpression de TIP60 dans la régulation d’UHRF1. Un autre objectif était de dépister et de développer des inhibiteurs de l’UHRF1 qui puisse cibler son activité. Pour atteindre ces objectifs, nous avons utilisé diverses approches, y compris des techniques biologiques et biophysiques. Les résultats ont révélé qu’UHRF1 interagit avec le domaine MYST de TIP60 durant la phase S du cycle cellulaire. La surexpression de TIP60 régule la dégradation d’UHRF1 (un oncogène) de manière dépendent d’une ubiquitination, expliquant ainsi son rôle suppresseur de tumeur.De plus, un inhibiteur d’UHRF1 appartenant à la famille des anthraquinones a été identifié.Cette molécule inhibe l'activité de basculement de la cytosine méthylée réalisée par le domaine SRA d’UHRF1. Elle a également altéré l'interaction UHRF1/DNMT1 et réduit les niveaux de méthylation globauxUHRF1 is a nuclear protein that is in cancer cells. It plays an essential role in DNA methylation, promotes cell proliferation and inhibits the expression of tumor suppressor genes. TIP60 is an important partner of UHRF1 which participates in chromatin remodeling,transcriptional gene regulation and other cellular activities through its acetyltransferase activity. Both proteins are involved in regulating the activity and stability of important proteins such as DNMT1 and p53. The purpose of this thesis was to study the interaction mechanism of TIP60 with UHRF1 and to explore the effect of over expression of TIP60 inthe regulation of UHRF1 expression. Another objective was to identify and develop UHRF1inhibitors that could target its activity. To achieve these objectives, we used different approaches, including biological and biophysical techniques. The results revealed thatUHRF1 interacts with the MYST domain of TIP60 during the S phase of the cell cycle. The over expression of TIP60 induces the degradation of UHRF1 (an oncogene), in a poly-ubiquitination dependent way, explaining in parts its tumor suppressing role. In addition, an anthraquinone UHRF1 inhibitor was found. This molecule inhibits the flipping activity of methylated cytosine produced by SRA domain of UHRF1. It also altered theUHRF1/DNMT1 interaction and reduced global methylation levels
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Janßen, Julia Annika [Verfasser], Ali [Akademischer Betreuer] El-Armouche, Christopher [Akademischer Betreuer] Antos, Henning [Gutachter] Morawietz, and Ali [Gutachter] El-Armouche. "In vivo FLIM-FRET as a novel technique to assess cAMP and cGMP in the intact zebrafish heart / Julia Annika Janßen ; Gutachter: Henning Morawietz, Ali El-Armouche ; Ali El-Armouche, Christopher Antos." Dresden : Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2018. http://d-nb.info/1151816477/34.
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Mauritz, Jakob Martin Andreas. "Homeostasis and volume regulation in the Plasmodium falciparum infected red blood cell." Thesis, University of Cambridge, 2011. https://www.repository.cam.ac.uk/handle/1810/240497.
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The thesis reports on the application of advanced microanalytical techniques to answer a fundamental open question on the homeostasis of Plasmodium falciparum infected red blood cells, namely how infected cells retain their integrity for the duration of the parasite asexual reproduction cycle. The volume and shape changes of infected cells were measured and characterized at femtolitre resolution throughout the intraerythrocytic cycle using confocal microscopy. Fluorescence lifetime imaging and electron probe X-ray microanalysis were applied for the quantification of intracellular haemoglobin and electrolyte concentrations. The cytomechanical properties of uninfected and infected red cells were studied using a novel optical stretcher device, which enabled individual cells to be trapped and manipulated optomechanically in microfluidic channels. Combined, these methods offered a unique insight into the homeostatic and rheological behaviour of malaria-infected red cells. The results were analysed by comparison with predictions from a detailed physiological model of the homeostasis and volume regulation of infected cells, providing broad support to the view that excess haemoglobin consumptions by the parasite was necessary for the integrity of infected cells (the colloidosmotic hypothesis). The dissertation is introduced with an overview of malaria, red blood cells homeostasis and the changes induced by Plasmodium falciparum infection. In the following, this description is extended to an in-depth theoretical analysis of the infected red blood cell homeostasis, from which the need to characterise certain parameters arises. The subsequent chapters address sequentially the assessment of the haemoglobin and electrolyte concentration, cell shape and volume changes and ultimately alterations in cell elasticity. The experimental part is complemented with a comparison of the resulting data to the predictions from the theoretical analysis and an outlook on future work.
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Tramier, Marc. "Imagerie des déclins de fluorescence pour l'étude de la dynamique et des interactions de macromolécules en cellules vivantes." Phd thesis, Université Pierre et Marie Curie - Paris VI, 2001. http://tel.archives-ouvertes.fr/tel-00003477.
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Le but de notre travail est de développer une imagerie des déclins de fluorescence et d'en démontrer les potentialités pour l'étude de la dynamique macromoléculaire et des interactions entre macromolécules en cellules vivantes. Notre approche repose sur la mesure de la corrélation temporelle de photons uniques de fluorescence (TCSPC) simultanément à la détermination de la localisation spatiale (le long d'une ligne) de la région d'émission. Des images monodirectionnelles de déclins de fluorescence ont ainsi été obtenues représentant la cinétique de fluorescence en différentes régions subcellulaires. Nous avons également mis au point la mesure de déclins d'anisotropie de fluorescence provenant d'un petit volume subcellulaire (1 µm3) sous microscope en mode confocal. Les résultats présentés dans ce mémoire démontrent l'intérêt de cette approche technologique pour des problématiques de biologie cellulaire. Le marquage fluorescent endogène de protéines a été réalisé en les fusionant à la GFP ou un de ses variants spectraux. Les interactions protéine-protéine ont été étudiées soit par hétéroFRET en mesurant la diminution de la durée de vie de fluorescence du chromophore donneur, soit par homoFRET en mesurant la cinétique de dépolarisation de la fluorescence. Nous avons mis en évidence (i) la formation d'hétérodimères de p45 du facteur de transcription NF-E2 avec deux partenaires dans différents compartiments subcellulaires par hétéroFRET, et (ii) l'homodimérisation de la thymidine kinase du virus de l'herpès simplex type 1 de façon plus concluante par homoFRET que par hétéroFRET. Par ailleurs, la mesure des déclins d'anisotropie de fluorescence de l'éthidium comme sonde de la dynamique torsionnelle de l'ADN a révélé l'existence d'une très forte restriction de cette dynamique dans la chromatine non perturbée. Les développements supplémentaires de notre système pour une imagerie bi-dimensionnelle puis tri-dimensionnelle sont prometteurs.
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Devauges, Viviane. "Microscopie de fluorescence résolue en temps et en polarisation pour le suivi d'interactions protéiques en neurobiologie." Phd thesis, Université Paris Sud - Paris XI, 2011. http://tel.archives-ouvertes.fr/tel-00816666.
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Le suivi des interactions entre protéines, localisées à la membrane plasmique ou à l'intérieur de cellules, a été réalisé au cours de cette thèse par imagerie de fluorescence et par l'analyse de processus dits de FRET (Forster Resonance Energy Transfer). Pour quantifier le FRET entre nos protéines d'intérêt, nous avons choisi le contraste de durée de vie de fluorescence car cette méthode est indépendante de la concentration et de l'intensité de fluorescence. Afin d'obtenir une résolution suffisante pour des problématiques neurobiologiques, un microscope TIRFLIM (Total Internal Reflection Fluorescence Lifetime Imaging Microscopy) avait préalablement été développé. Celui-ci nous permet de faire de l'imagerie en plein champ avec une résolution axiale sub-longueur d'onde. Ce dispositif a été calibré et optimisé au cours de cette thèse pour répondre au mieux à des problématiques biologiques. Différentes approches ont ainsi été testées dans le but de calibrer la profondeur de pénétration de l'onde évanescente. Des surfaces plasmoniques ont entre autres été utilisées pour augmenter la sélectivité axiale du montage. Notre microscope a été dédié à l'étude de l'effet du cholestérol sur l'interaction entre la protéine précurseur de l'amyloïde APP, protéine transmembranaire impliquée dans la maladie d'Alzheimer et une de ses enzymes de clivage BACE1. Nous avons ainsi effectué un suivi dynamique de l'effet du cholestérol sur l'interaction entre APP et BACE1 dans des cellules HEK-293 et dans des cultures primaires de neurones d'hippocampe d'embryons de rat, de la membrane plasmique à l'intérieur des cellules grâce à notre dispositif TIRFLIM. La mesure d'anisotropie de fluorescence résolue en temps a également été implémentée sur notre montage. Ces mesures résolues en temps et en polarisation ont permis de mesurer le temps de corrélation rotationnelle de fluorophores et de mettre en évidence de manière qualitative différents niveaux d'homodimérisation de protéines impliquées dans la maladie d'Alzheimer.
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Blandin, Pierre. "Développement instrumental pour la microscopie de fluorescence résolue en temps : applications biomédicales." Phd thesis, Université Paris Sud - Paris XI, 2008. http://tel.archives-ouvertes.fr/tel-00376116.
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Pour certaines problématiques biomédicales, notamment en neurobiologie, l'imagerie des échantillons exige des résolutions spatiale et temporelle élevées, et seules des techniques de microscopie optique innovantes vont permettre d'imager la cellule dans des conditions physiologiques. Pour répondre à ces contraintes nous avons développé en intégralité un dispositif de microscopie de fluorescence en réflexion totale interne et résolue en temps. En effet, l'imagerie de fluorescence résolue en temps (FLIM) permet, en complément des informations de localisation et de topologie apportées par la microscopie conventionnelle, une analyse dynamique de processus moléculaires et métaboliques au sein des cellules. Associée à la technique de microscopie en réflexion totale interne, qui confère au système d'imagerie une résolution axiale sub-longueur d'onde tout en acquérant des images en champ large, la technique FLIM permet d'analyser l'activité membranaire des cellules en s'affranchissant de la fluorescence des autres entités de la cellule.Ce travail s'est articulé autour de trois axes principaux : l'étude et le développement d'un dispositif d'excitation de la fluorescence basé sur un oscillateur laser solide picoseconde dont le spectre est élargi par effets non linéaires dans des fibres optiques microstructurées ; le développement et la caractérisation d'un dispositif de microscopie de fluorescence par onde évanescente (TIRF) couplé à une détection résolue en temps en plein champ ; et l'application de ce dispositif à l'étude d'un précurseur membranaire du peptide amyloïde impliqué dans la maladie d'Alzheimer.
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Lee, Jiun-De, and 李俊德. "Researching of SNAP25-Rabphilin FRET Pair Within PC12 Cells by FLIM╱FRET." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/66797649204078167675.
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碩士國立陽明大學
生醫光電工程研究所
95
Exocytosis, one kind of processes in membrane traffic, is proposed to contain four sequential steps: docking, priming, fusion, and recycling. While exocytosis is occurring, each step was regulated by the interaction between proteins and between protein and lipid. In terms of the technology of observing interaction between proteins, using Fluorescence Resonance Energy Transfer (FRET) into fluorescence microscopy is able to obtain approximate 200nm spatial resolution beyond the optics limit. Since the lifetime of fluorescent molecule will change during FRET, we used Fluorescence Lifetime Imaging Microscopy (FLIM) to observe both donor only (pEGFP-SNAP25 transfection) and coexistent of donor-acceptor (co-transfection of pEGFP-SNAP25 and mRFP-Rabphilin) ,and evaluated the difference in between to assure the existence of FRET phenomenon. Synaptosomal associated protein of 25 kDa (SNAP25), located in membrane protein, belongs to one type of Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE). According to the localization, SNAP25 is also termed to be the target-membrane associated protein (t-SNAREs). Rabphilin, the GTP-Rab3A specific binding protein, is also related to neurotransmitter and hormone secretion. It is first time that the interaction between SNAP25 and Rabphilin was found using biochemical methods by Fukuda in 2005. In this study, we examined the interaction between SNAP25-EGFP and mRFP-Rabphilin during the stimulated exocytosis within neuroendocrine PC12 cells through getting the FLIM images by the FLIM/FRET technique. The PC12 cells have P2X and P2Y receptors. If the receptors detected ATP, the calcium concentration in cells would be increased, and it would occur exocytosis. We found that during the usage of ATP for less than 10 seconds, the fluorescent lifetime distribution of samples which contain both GFP and RFP have shift about 170 ps. That is, during the short time when cells are stimulated and exocytosis occurs, FRET of SNAP25 and Rabphilin happens. Therefore, we believe that when exocytosis occurs, SNAP25 and Rabphilin would have interaction.
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Mooney, Chance. "Quantitative FLIM-FRET Measurement of Voltage Dependent Prestin Conformational Changes." Thesis, 2013. http://hdl.handle.net/1911/72010.
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The transmembrane protein prestin forms an integral part of the mammalian sense of hearing by providing the driving force for the electromotility of the outer hair cell, a specialized cell that resides within the cochlea. This provides the cochlea with an ability to amplify mechanical vibrations, allowing for a high degree of sensitivity and selectivity in auditory transduction. The phenomenon, driven by changes in the transmembrane potential, is thought to be the result of conformational changes in self-associating prestin oligomers. We have previously utilized Forster resonance energy transfer (FRET), by both sensitized emission and acceptor photobleach methods, to detect prestin self -association. While these methods can qualitatively confirm prestin-prestin association, determining nanoscale changes in prestin organization requires greater accuracy than either technique provides. In this thesis, a FRET methodology based on fluorescence lifetime imaging (FLIM), detected by time correlated single photon counting (TCSPC), is implemented and utilized to quantitatively measure conformational changes within prestin-prestin oligomers in response to voltage stimulus.
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Ho, Pei-Yun, and 何佩芸. "Monitoring interactions among integrinαIIbβ3、NHE1、NCX1 that trigger calcium oscillations by FLIM-FRET technology." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/85697334784946103616.
Full textAbstract:
碩士國立陽明大學
生醫光電工程研究所
95
Chinese hamster ovary cells expressing exogenous human integrinαIIbβ3 on their plasma membrane (CHOαIIbβ3) exhibited active calcium oscillations when plated on substrates coated with fibrinogens or disintegrins. In these cells, profound targeting of sodium-proton exchanger NHE1 and sodium-calcium exchanger NCX1 from intracellular vesicles to the plasma membrane was noticed, where they co-localized with integrins. Such molecular interactions could be clearly demonstrated using FRET-FLIM technique based on fluorescence lifetime measurements. Further more, targeting and interaction of the ion exchangers with integrinαIIbβ3, and their functional coupling to initiate calcium influx, were dependent on lipid microdomain, or lipid rafts.
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Veľas, Lukáš. "Vliv lipidového složení a modelových peptidů na laterální organizaci lipidových vrstev." Master's thesis, 2017. http://www.nusl.cz/ntk/nusl-355698.
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Oxidized phospholipids (OxPLs) are known to be present in living organisms due to oxidative stress. However, the physiological function of OxPLs is still not fully understood. They have been shown to be present in many inflammatory diseases such as atherosclerosis and neurodegenerative diseases like Parkinson's and Alzheimer's disease. In this work we present the influence of two truncated OxPLs on the lateral heterogeneity of a model lipid membrane. Specifically, we studied the effect of 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3- phosphocholine (POVPC) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) on the formation of nanodomains present in giant unilamellar vesicles containing 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol and sphingomyelin. Only few techniques are capable of detecting nanometer-sized domains in the membrane with high resolution. Time resolved Förster resonance energy transfer (TR-FRET) combined with Monte Carlo (MC) simulations provide a strong tool not only to detect lateral heterogeneities but also characterize them with the resolution of 2 nm. Profound effects on the nanodomain size were observed in the presence of both studied OxPLs and differences were detected, as PGPC with a carboxylic group drives formation of larger nanodomains than POVPC...
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Kumaran, Nair Deepak. "Insights into molecular mechanisms regulating the activity of multidomain proteins in living cells using FRET-FLIM = Einblicke in die molekularen Mechanismen zur Regulierung der Aktivität von Multidomain-Proteinen in lebenden Zellen mit Hilfe von FRET-FLIM Untersuchungen /." 2008. http://diglib.uni-magdeburg.de/Dissertationen/2008/deekumarannair.htm.
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Jose, Mini [Verfasser]. "Einsatz von Fret-Flim zur Beobachtung von Protein-Protein_Wechselwirkungen in lebenden Zellen: Einblicke in Interaktionen des präsynaptischen Proteins Bassoon = Investigating protein-protein interactions by fret-flim in living cells: mediated by the presynaptic proteins bassoon / von Mini Jose." 2007. http://d-nb.info/986796913/34.
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Kumaran, Nair Deepak [Verfasser]. "Einblicke in die molekularen Mechanismen zur Regulierung der Aktivität von Multidomain-Proteinen in lebenden Zellen mit Hilfe von FRET-FLIM-Untersuchungen = Insights into molecular mechanisms regulating the activity of multidomain proteins in living cells using FRET-FLIM / von Deepak Kumaran Nair." 2008. http://d-nb.info/98885290X/34.
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Janßen, Julia Annika. "In vivo FLIM-FRET as a novel technique to assess cAMP and cGMP in the intact zebrafish heart." Doctoral thesis, 2017. https://tud.qucosa.de/id/qucosa%3A30747.
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Introduction: 23 million patients worldwide suffer from heart failure. These patients depend on cardiac research, because cardiac research enables the development of new therapeutic strategies and –targets. In cardiomyocytes, the compartmentalization of cAMP and cGMP depends on many factors. T-tubuli and PDEs are responsible for the division of cells in microdomains in which localized and specific cAMP and cGMP-signaling occurs. The aim of this thesis was to develop a method to answer the open questions that remain about the physiological and pathophysiological significance of cAMP/cGMP compartmentalization.
Methods: I used the zebrafish as a model, because the transparency of zebrafish larvae enabled non-invasive fluorescent imaging in cardiomyocytes in the living animal. I cloned the Fluorescence Resonance Energy Transfer (FRET) sensors EPAC1-camps for cAMP and cGi500 for cGMP and injected them into zebrafish fertilized embryos. Then I used the F0 generation for Fluorescence Lifetime Imaging (FLIM) -FRET-measurements of cAMP and cGMP. Ca2+ is an important downstream mediator of cAMP and cGMP, because Ca2+ regulates cardiac contraction. Therefore, I also cloned the Ca2+ sensor GCaMP6 and used the dye Fluo-4 AM to include intracellular Ca2+ in the imaging.
Results: The cloned sensors for cAMP, cGMP and Ca2+ were successfully injected into the zebrafish and showed expression in individual cardiomyocytes. I developed a protocol to mount the living zebrafish embryos and to measure intracellular cAMP and cGMP with FLIM-FRET in vivo with high spatial resolution. I characterized the sensors in their functionality by showing that the sensors react to changes in intracellular concentrations of cAMP and cGMP. The results of this study include evidence that zebrafish have mechanisms that lead to cAMP/cGMP compartmentalization in the absence of T-tubuli, and these mechanisms keep compartmentalization constant even under extreme cAMP or cGMP increasing drug treatment. Furthermore, I imaged intracellular Ca2+ by confocal microscopy and developed a protocol to use Fluo-4 AM for Ca2+ imaging.
Conclusion: The method used in this thesis should allow the investigation of subcellular cAMP/cGMP compartmentalization and Ca2+ and to subsequently answer open questions in the field, for example whether a change of cAMP compartmentalization leads to the pathological phenotypes of cardiac disease or if a changed compartmentalization of cAMP in cardiac disease influences Ca2+ concentrations and therefore contraction. Additionally, this method can be used to learn more about cAMP, cGMP und Ca2+ during regeneration in the heart, because the zebrafish cardiomyocytes can regenerate.Einleitung: Weltweit sind mehr als 23 Millionen unter Herzinsuffizienz leidende Patienten auf die kardiologische Grundlagenforschung angewiesen, da diese die Voraussetzung für eine bessere Versorgung durch adaptierte und neue Behandlungswege schafft. In Kardiomyozyten hängt die Kompartimentierung von cAMP und cGMP von vielen Faktoren ab. T-Tubuli und PDEs werden unter anderem für die Aufteilung der Zellen in Mikrodomänen, in denen lokalisierte und spezifische cAMP- und cGMP-Signalgebung stattfinden kann, verantwortlich gemacht. Das Ziel dieser Arbeit war die Etablierung einer Methode, mithilfe derer offene Fragen bezüglich der physiologischen und insbesondere der pathophysiologischen Relevanz der cAMP- und cGMP Kompartimentierung beantwortet werden können. Methode: Als Modell diente der Zebrafisch, da die Transparenz von Zebrafisch Embryonen eine nicht-invasive Bildgebung von Fluoreszenz in Kardiomyozyten im lebenden Tier ermöglicht. Dafür klonierte ich die Förster Resonance Energy Transfer (FRET) -Sensoren EPAC1-camps als cAMP-Sensor und cGi500 als cGMP-Sensor und injizierte diese in befruchtete Zebrafisch Embryonen. Anschließend benutzte ich die F0-Generation für Fluorescence Lifetime Imaging (FLIM) -FRET-Messungen von cAMP und cGMP. Da Ca2+ als wichtiger downstream Mediator von cAMP und cGMP die kardiale Kontraktion reguliert, klonierte ich außerdem den Ca2+-Sensor GCaMP6 und benutzte den Farbstoff Fluo-4 AM, um intrazelluläres Ca2+ darzustellen. Ergebnisse: Die klonierten Sensoren für cAMP, cGMP und Ca2+ konnten erfolgreich in den Zebrafisch injiziert werden und zeigten alle Expression in einzelnen Kardiomyozyten. Ich entwickelte ein Protokoll, dass die Fixierung von lebenden Zebrafisch Embryonen und nachfolgender Bildgebung von cAMP und cGMP mit hoher zellulärer Auflösung mit FLIM-FRET in vivo erlaubte. Ich konnte eine funktionelle Charakterisierung der Sensoren durchführen, indem ich zeigte, dass sie auf Konzentrationsänderungen von intrazellulärem cAMP und cGMP reagieren sowie zeigen, dass Zebrafische trotz fehlender T-Tubuli eine signifikante cAMP- und cGMP Kompartimentierung aufweisen, auch unter extremen Bedingungen nach Gabe von cAMP/cGMP stimulierenden Substanzen in hoher Dosierung. Ich konnte zudem subzelluläres Ca2+ durch konfokale Mikroskopie bildgebend darstellen und entwickelte ein Protokoll, um mit Fluo-4 AM eine schnelle Möglichkeit zu haben, Ca2+ mit in die Messungen einzubeziehen. Ausblick: Die in dieser Arbeit benutzte Methode bietet eine gute Möglichkeit, subzelluläre cAMP- und cGMP-Kompartimentierung und Ca2+ zu untersuchen und damit zum Beispiel die Fragen zu beantworten, ob eine veränderte cAMP/cGMP Kompartimentierung zu Herzkrankheiten wie Hypertrophie führt oder ob eine veränderte cAMP Kompartimentierung den zellulären Ca2+ Haushalt und damit die kardiale Kontraktion beeinflusst. Darüber hinaus kann das von mir etablierte Protokoll dazu genutzt werden, mehr über cAMP, cGMP und Ca2+ während der Regeneration im Herzen zu lernen, da der Zebrafisch über ausgeprägte Regenerationsfähigkeiten verfügt.
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Błasiak, Ewa. "Fluorescencyjne badania oddziaływań receptorów dopaminowych w układzie modelowym in vitro : rola polimorfizmów pojedynczego nukleotydu w obrębie receptorów D_{2}." Praca doktorska, 2015. http://ruj.uj.edu.pl/xmlui/handle/item/42458.
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Chakraborty, Sandeep, and 夏柏杉. "Fluorescence based methods (FLIM and FRET) to study the metabolic state of Parkinson’s disease in cell model and in vitro protein intractions." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/41738728403186503249.
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博士國立陽明大學
生醫光電研究所
104
Fluorescence based spectroscopic and microscopic techniques have been widely used in the field of scientific research and medical diagnostics for their unique advantages, such as specificity, sensitivity, simplicity, and speed. Over the years, two of the most frequently used techniques based on fluorescence are Förster/fluorescence resonance energy transfer (FRET) spectroscopy (and microscopy) and fluorescence lifetime imaging microscopy (FLIM). Fluorescence lifetime imaging technique quantifies the average time a fluorophore remains in the excited state before descending to the ground state. This technique can easily distinguish two molecules with similar fluorescence emission bands, or the same molecule with different structural conformations based on their fluorescence lifetime. In this work, we applied two-photon fluorescence lifetime imaging microscopy (2P-FLIM) to observe two endogenous fluorescent molecules viz. reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD). NADH have longer lifetime when it binds to protein and free NADH has shorter lifetime. On the other hand, FAD has shorter lifetime when it binds to protein, while the free FAD has longer time. In this thesis, we exploited these properties of NADH and FAD to monitor the cellular metabolic state in Parkinson’s disease (PD) cellular model in terms of the cellular redox ratios NADH/NAD+ and FADH2/FAD via 2P-FLIM. NADH and FAD are two co-enzymes which take part in the ATP production of cells in the inner-mitochondrial membrane; we monitored via 2P-FLIM to map the cellular metabolism in PD. The cellular redox state can be interpreted in terms of the fluorescence lifetime components values of NADH and FAD, and also the relative contributions of free to protein-bound NADH (and FAD). Two-photon excitation was used for lifetime imaging of NADH and FAD, as it causes less photobleaching and yields higher cell viability. PD is a progressive neurological disorder due to the loss of dopaminergic neurons in substantia nigra of mid-brain region and several lines of evidence suggest that mitochondrial dysfunction is responsible for the disease pathology. In this work, PC12 cells were first treated with nerve growth factor (NGF) to differentiate it into neuronal cells which were further treated with 1-methyl-4-phenylpyridinium (MPP+) to establish the PD cellular model. A systematic FLIM data analysis showed a statistically significant (p < 0.001) decrease in the fluorescence lifetime of both free and protein-bound NADH, as well as free and protein-bound FAD in MPP+ treated cells. On the relative contributions of the free and protein-bound NADH and FAD to the life time, however, both the free NADH contribution and the corresponding protein-bound FAD contribution increased significantly (p < 0.001) in MPP+ treated cells, compared to control cells. These results, which indicate a shift in energy production in the MPP+ treated cells from oxidative phosphorylation towards anaerobic glycolysis, can potentially be used as cellular metabolic metrics to assess the condition of PD at the cellular level. In this thesis, we also developed an organic fluorophore based steady-state quantitative FRET assay with a new and modified algorithm to extract FRET emission signal. This method was further applied to quantify the interaction between leukocyte function-associated antigen-1(LFA-1) and intercellular adhesion molecule-1 (ICAM-1) in terms of the dissociation constant (Kd). The interaction between these two transmembrane proteins plays a significant role in cellular adhesion including the extravasation and inflammatory response of leukocytes, and also in the formation of immunological synapse. Moreover, the LFA-1/ICAM-1 interaction may serve as a potential therapeutic target for the treatment of several diseases as irregular expressions of LFA-1 or ICAM-1 or both may lead to autoimmune diseases, metastasis cancer, etc. In addition, we also developed the FRET assay into a screening platform to identify peptides and small molecules that inhibit the LFA-1/ICAM-1 interaction. For the FRET pair, we used Alexa Fluor 488-LFA-1 conjugate as the donor and Alexa Fluor 555-human recombinant ICAM-1 (D1-D2-Fc) as the acceptor. From our quantitative FRET analysis, the Kd between LFA-1 and D1-D2-Fc was determined to be 17.93 ± 1.34 nM. Both the Kd determination and screening assay were performed in a 96-well plate platform, providing the opportunity to develop it into a high-throughput assay. In future, these approaches can be further developed/optimized for the study of in vivo Parkinson’s disease pathogenesis and for small molecules based drug screening.
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Chang, Jui-Yun. "Calcium/Calmodulin-Dependent Protein Kinase II Serves as a Biochemical Integrator of Calcium Signals for the Induction of Synaptic Plasticity." Diss., 2016. http://hdl.handle.net/10161/12854.
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Repetitive Ca2+ transients in dendritic spines induce various forms of synaptic plasticity by transmitting information encoded in their frequency and amplitude. CaMKII plays a critical role in decoding these Ca2+ signals to initiate long-lasting synaptic plasticity. However, the properties of CaMKII that mediate Ca2+ decoding in spines remain elusive. Here, I measured CaMKII activity in spines using fast-framing two-photon fluorescence lifetime imaging. Following each repetitive Ca2+ elevations, CaMKII activity increased in a stepwise manner. This signal integration, at the time scale of seconds, critically depended on Thr286 phosphorylation. In the absence of Thr286 phosphorylation, only by increasing the frequency of repetitive Ca2+ elevations could high peak CaMKII activity or plasticity be induced. In addition, I measured the association between CaMKII and Ca2+/CaM during spine plasticity induction. Unlike CaMKII activity, association of Ca2+/CaM to CaMKII plateaued at the first Ca2+ elevation event. This result indicated that integration of Ca2+ signals was initiated by the binding of Ca2+/CaM and amplified by the subsequent increases in Thr286-phosphorylated form of CaMKII. Together, these findings demonstrate that CaMKII functions as a leaky integrator of repetitive Ca2+ signals during the induction of synaptic plasticity, and that Thr286 phosphorylation is critical for defining the frequencies of such integration.
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Lee, Seok-Jin. "Spatiotemporal Dynamics of Calcium/calmodulin-dependent Kinase II in Single Dendritic Spines During Synaptic Plasticity." Diss., 2011. http://hdl.handle.net/10161/3818.
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Synaptic plasticity is the leading candidate for the cellular/molecular basis of learning and memory. One of the key molecules involved in synaptic plasticity is Calcium/calmodulin-dependent Kinase II (CaMKII). Synaptic plasticity can be expressed at a single dendritic spine independent of its neighboring dendritic spines. Here, we investigated how long the activity of CaMKII lasts during synaptic plasticity of single dendritic spines. We found that CaMKII activity lasted ~2 minutes during synaptic plasticity and was restricted to the dendritic spines undergoing synaptic plasticity while nearby dendritic spines did not show any change in the level of CaMKII activity. Our experimental data argue against the persistent activation of CaMKII in dendritic spines undergoing synaptic plasticity and suggest that the activity of CaMKII is a spine-specific biochemical signal necessary for synapse-specificity of synaptic plasticity. We provide a biophysical explanation of how spine-specific CaMKII activation can be achieved during synaptic plasticity. We also found that CaMKII is activated by highly localized calcium influx in the proximity of Voltage-dependent Calcium Channels (VDCCs) and a different set of VDCCs and their respective Ca2+ nanodomains are responsible for the differential activation of CaMKII between dendritic spines and dendritic shafts.
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Chen, Yu-Ling, and 陳郁伶. "Characterization of L0038 Encoded in the Pathogenicity Island of Enterohemorrhagic Escherichia coli O157:H7 and Researching of Protein-Protein Interaction in Type III Secretion System by FLIM/FRET." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/pgd792.
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碩士國立陽明大學
微生物及免疫學研究所
97
Enterohemorrhagic Escherichia coli (EHEC) O157:H7, a pathogen causes hemorrhagic diarrhea and hemolytic-uremic syndrome, employs the type III secretion system to deliver proteins into host cells. Mechanistically, a pedestal-like structure is formed between the bacterium and the host cell, then a tight bacterial attachment is achieved. These characteristics have been attributed to a pathogenic island on the bacterial chromosome known as the locus for enterocyte effacement(LEE), a cluster of 41 predicted open reading frame(ORFs), of which some have been characterized. Among the uncharacterized ORFs, l0038 encoded a protein of 152 amino acid residue with a predicted pI of 5.5. An l0038–deleted strain created by one-step inactivation of chromosomal gene gave decrease of EspA synthesis and lost most of secrected proteins, suggested that L0038 may act as one of the TTSS components.Unfortunately, there is no way to complement the secretion phenotype of the deleted strain byexogenously expressing L0038, so the hypothesis might need further investigation. In this study, the interaction between L0038 and EscU, and also that between EscU and L0050 were demonstrated by bacterial two-hybrid assay. The sequential interactions between EspA, L0050, EscU and L0038 may imply the decrease of EspA in l0038 mutant may be due to the fail in recruitment of EspA to TTSS machinery. The protein interactions indeed play an essential role in building the TTSS and in secreting the TTS proteins. Accordingly, we apply the technology of FRET/FLIM in in vivo analyzing the interactions between bacterial proteins. Co-expression of both fluorescence-tags fused CesT and Tir in JM109 was used as positive control to set up the FLIM assay in bacteria. Although the possible interference of non-intact fusion protein ruined the analysis result, the shorter life time of donor fluorescence as comparing with that with donor fluorescence protein only demonstrated that there is an in vivo protein-protein interaction between CesT and Tir. Results clearly showed the success in applying FLIM assay in analysis of bacterial samples.
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Ebrecht, René. "The signal transduction of synapse formation and it's failure in Rett syndrome." Doctoral thesis, 2016. http://hdl.handle.net/11858/00-1735-0000-0023-3E2B-2.
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Jayachandran, Christina. "Molecular DNA Sensors to Measure Distribution of Cytoskeletal Forces." Doctoral thesis, 2019. http://hdl.handle.net/21.11130/00-1735-0000-0005-1463-5.
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Ghelani, Tina. "Orientation and organization of the presynaptic active zone protein Bassoon: from the Golgi to the synapse." Doctoral thesis, 2016. http://hdl.handle.net/11858/00-1735-0000-0028-881D-E.
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