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Analyzing Interactions Between Cells And Extracellular Matrix By Atomic Force MicroscopyFriedrichs, Jens 10 December 2009 (has links) (PDF)
Interactions of cells with the extracellular matrix (ECM) have important roles in various physiological and pathological processes, including tissue morphogenesis during embryonic development, wound healing and tumor invasion. Although most of the proteins involved in cell-ECM interactions have been identified, the underlying mechanisms and involved signaling pathways are incompletely understood. Here, atomic force microscope-based imaging and single-cell force measurements were used to characterize the interactions of different cell types with ECM proteins.
The interplay between cells and ECM is complex. However, two interaction types, protein-protein and protein-carbohydrate, predominate. Integrins, adhesion receptors for ECM, mediate the former, galectins, a family of animal lectins, the latter. In the second chapter of this thesis, the contributions of both receptor families to the interactions of epithelial MDCK cells with ECM proteins are presented. It was found that galectins-3 and 9 are highly expressed in MDCK cells and required for optimal long-term adhesion (90 minutes) to ECM proteins collagen-I and laminin-111. Interestingly, early adhesion (< 2 minutes) to laminin-111, was integrin-independent and instead mediated by carbohydrate interactions and galectins. In contrast, early adhesion to collagen-I was exclusively mediated by integrins. Moreover, cells frequently entered an enhanced adhesion state, marked by a significant increase in the force required for cell detachment. Although adhesion was mediated by integrins, adhesion enhancement was especially observed in cells depleted for galectin-3. It was proposed that galectin-3 influences integrin-mediated adhesion complex formation by altering receptor clustering.
To control their attachment to ECM proteins, cells regulate integrin receptors. One regulatory process is integrin crosstalk, where the binding of one type of integrin influences the activity of another type. In the third chapter, the implementation of a single-cell force spectroscopy assay to identify such crosstalks and gain insight into their mechanisms is described. In this assay the interactions of integrin receptors being specifically attached to one ligand are characterized in dependence of another ligand-bond receptor pair. With this assay a crosstalk between collagen-binding integrin α1β1 and fibronectin-binding integrin α5β1 was identified in HeLa cells. This crosstalk was directional from integrin α1β1 to integrin α5β1 and appeared to regulate integrin α5β1 by inducing its endocytosis.
In the fourth and final chapter, mechanisms of matrix-induced cell alignment were studied by imaging cells on two-dimensional matrices assembled of highly aligned collagen fibrils. Integrin α2β1 was identified as the predominant receptor mediating cell polarization. Time-lapse AFM demonstrated that during alignment cells deform the matrix by reorienting individual collagen fibrils. Cells deformed the collagen matrix asymmetrically, revealing an anisotropy in matrix rigidity. When matrix rigidity was rendered uniform by chemical cross-linking or when the matrix was formed from collagen fibrils of reduced tensile strength, cell polarization did not occur. This suggested that both the high tensile strength and pliability of collagen fibrils contribute to the anisotropic rigidity of the matrix and lead to directional cellular traction and cell polarization. During alignment, cellular protrusions contacted the collagen matrix from below and above. This complex entanglement of cellular protrusions and collagen fibrils may further promote cell alignment by maximizing cellular traction.
The work presented here adds to the understanding of cell-ECM interactions. Atomic force microscopy imaging allowed characterizing the behavior of cells on nanopatterned collagen matrices whereas single-cell force spectroscopy revealed insights into the regulation of cell adhesion by galectins. Furthermore, methodological advances in the single-cell force spectroscopy assay allowed the intracellular regulation of receptor molecules to be studied. The work demonstrates that atomic force microscopy is a versatile tool to study cell-ECM interactions.
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Mechanical properties of pore-spanning membranes prepared from giant vesicles / Mechanische Eigenschaften von Poren-Spanning Membranen aus Riesenvesikeln vorbereitetKocun, Marta 23 May 2011 (has links)
No description available.
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Mechanical compression of coiled carbon nanotubesBarber, Jabulani Randall Timothy 26 February 2009 (has links)
Carbon nanotubes are molecular-scale tubes of graphitic carbon that possess many unique properties. They have high tensile strength and elastic modulus, are thermally and electrically conductive, and can be structurally modified using well established carbon chemistries. There is global interest in taking advantage of their unique combination of properties and using these interesting materials as components in nanoscale devices and composite materials.
The goal of this research was the correlation of the mechanical properties of coiled carbon nanotubes with their chemical structure. Individual nanocoils, grown by chemical vapor deposition, were attached to scanning probe tip using the arc discharge method. Using a scanning probe microscope the nanocoils are repeatedly brought into and out of contact with a chemically-modified substrate. Precise control over the length (or area) of contact with the substrate is achievable through simultaneous monitoring the cantilever deflection resonance, and correlating these with scanner movement. The mechanical response of nanocoils depended upon the extent of their compression. Nonlinear response of the nanocoil was observed consistent with compression, buckling, and slip-stick motion of the nanocoil. The chemical structure of the nanocoil and its orientation on the tip was determined using scanning and transmission electron microscopy.
The mechanical stiffness of eighteen different nanocoils was determined in three ways. In the first, the spring constant of each nanocoil was computed from the slope of the linear response region of the force-distance curve. The assumptions upon which this calculation is based are: 1) under compression, the cantilever-nanocoil system can be modeled as two-springs in series, and 2) the nanocoil behaves as an ideal spring as the load from the cantilever is applied. Nanocoil spring constants determined in this fashion ranged from 6.5x10-3 to 5.16 TPa for the CCNTs understudy. In the second, the spring constant of the nanocoil was computed from measuring the critical force required to buckle the nanocoil. The critical force method measured the force at the point where the nanocoil-cantilever system diverges from a linear region in the force curve. Nanocoil spring constants determined in this fashion ranged from 1.3x10-5 to 10.4 TPa for the CCNTs understudy. In the third, the spring constant of each nanocoil was computed from the thermal resonance of the cantilever-nanocoil system. Prior to contact of the nanocoil with the substrate, the effective spring constant of the system is essentially that of the cantilever. At the point of contact and prior to buckling or slip-stick motion, the effective spring constant of the system is modeled as two springs in parallel. Nanocoil spring constants determined in this fashion ranged from 2.7x10-3 to 0.03 TPa for the CCNTs understudy.
Using the thermal resonance of the cantilever system a trend was observed relating nanocoil structure to the calculated modulus. Hollow, tube-like nanostructures had a higher measured modulus than solid or fibrous structures by several orders of magnitude. One can conclude that the structure of carbon nanocoils can be determined from using their mechanical properties. This correlation should significantly contribute to the knowledge of the scientific and engineering community. It will enable the integration of carbon nanocoils in microelectromechanical (MEMS) or nanoelectromechanical systems (NEMS) as resonators, vibration dampers, or any other application in which springs are used within complex devices.
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Studium interakce membránových proteinů na molekulární úrovni pomocí silové spektroskopie, optické spektroskopie a metod výpočetní biochemie / Membrane protein interactions studied on single molecular level by force spectroscopy, optical spectroscopy and methods of computational biochemistryMATĚNOVÁ, Martina January 2011 (has links)
I have set for a challenging study that combined experimental and theoretical approaches in an attempt to resolve a role of small aminoacids in intermolecular interactions. First, I have proposed a hypothesis that described the interaction among individual aminoacids forming D helices of D1 and D2 proteins based on molecular dynamic simulations of a simplified model representing the reaction centre of photosystem II. Stability of the putative interhelical hydrogen bond network connecting D1 and D2 proteins was investigated experimentally with dynamic force spectroscopy using atomic force microscope. The results of both methods are in a full agreement with each other and reveal the key role of D1-Gly208 aminoacid in stability and functionality of photosystem II by providing milieu for weak interactions among three contact points at the cross of D helices: D1-Gly208 (O) and D2-Cys211 (O?), D1-Ser209 (O?) and D2-Ile204 (O), D1-Ser212 (O?) and D2-Gly207 (O). Mutation of the D1-Gly208 led to the increase in probability of the binding among the aforementioned aminoacids, undesirably strengthening the overall interactions among the proteins compromising photosynthetic capacity (D1-Ser208) or disabling of autotrophic growth (D1-Val208).
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Studium interakce membránových proteinů na molekulární úrovni pomocí silové spektroskopie, optické spektroskopie a metod výpočetní biochemie / Membrane protein interactions studied on single molecular level by force spectroscopy, optical spectroscopy and methods of computational biochemistryMATĚNOVÁ, Martina January 2011 (has links)
I have set for a challenging study that combined experimental and theoretical approaches in an attempt to resolve a role of small aminoacids in intermolecular interactions. First, I have proposed a hypothesis that described the interaction among individual aminoacids forming D helices of D1 and D2 proteins based on molecular dynamic simulations of a simplified model representing the reaction centre of photosystem II. Stability of the putative interhelical hydrogen bond network connecting D1 and D2 proteins was investigated experimentally with dynamic force spectroscopy using atomic force microscope. The results of both methods are in a full agreement with each other and reveal the key role of D1-Gly208 aminoacid in stability and functionality of photosystem II by providing milieu for weak interactions among three contact points at the cross of D helices: D1-Gly208 (O) and D2-Cys211 (O?), D1-Ser209 (O?) and D2-Ile204 (O), D1-Ser212 (O?) and D2-Gly207 (O). Mutation of the D1-Gly208 led to the increase in probability of the binding among the aforementioned aminoacids, undesirably strengthening the overall interactions among the proteins compromising photosynthetic capacity (D1-Ser208) or disabling of autotrophic growth (D1-Val208).
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Étude de l'influence du récepteur LRP-1 sur le potentiel invasif de cellules tumorales : mesures nanomécaniques et d'adhérence par microscopie à force atomique / Study of the influence of the LRP-1 receptor on the invasive potential of cancer cells : nanomechanical and adhesion measurements by atomic force microscopyLe cigne, Anthony 01 July 2016 (has links)
Le récepteur low-density lipoprotein receptor-related protein 1 (LRP-1) est capable d’internaliser des protéases impliquées dans la progression du cancer, et constitue donc une cible thérapeutique prometteuse. Cependant, LRP-1 peut également réguler certaines protéines membranaires. Son ciblage dans une stratégie de modulation de la protéolyse pourrait donc affecter l’adhésion et la dynamique du cytosquelette. Dans ce travail, nous avons étudié l’influence de l’invalidation de LRP-1 sur des paramètres originaux corrélés au potentiel invasif de cellules cancéreuses par microscopie à force atomique (AFM). Cette invalidation induit des changements dans la dynamique d’adhérence des cellules et dans la morphologie, tels qu’un renforcement des fibres de stress et un étalement plus prononcé, causant une augmentation de la surface et de la circularité cellulaires. L’analyse des propriétés mécaniques par AFM a montré que ces différences sont acccompagnées par une augmentation du module d’Young. De plus, les mesures montrent une diminution globale de la motilité cellulaire et une perturbation de la persistance directionnelle. Une augmentation de la force d’adhésion entre cellules invalidées pour LRP-1 et une bille fonctionnalisée à la gélatine a également été observée. Enfin, nos données de spectroscopie de force enregistrées à l’aide d’une pointe fonctionnalisée par un anticorps anti-sous-unité d’intégrine β1 montrent que l’invalidation de LRP-1 modifie la dynamique des intégrines. Dans leur ensemble, nos résultats montrent que des techniques classiquement utilisées dans l’investigation de cellules cancéreuses peuvent être couplées à l’AFM pour ouvrir l’accès à des paramètres complémentaires, pouvant faciliter la discrimination entre différents degrés de potentiel invasif. / The low-density lipoprotein receptor-related protein 1 (LRP-1) can internalize proteases involved in cancer progression and is thus considered a promising therapeutic target. However, it has been demonstrated that LRP-1 is also able to regulate membrane-anchored proteins. Thus, strategies that target LRP-1 to modulate proteolysis could also affect adhesion and cytoskeleton dynamics. Here, we investigated the effect of LRP-1 silencing on parameters reflecting cancer cells’ invasiveness by atomic force microscopy (AFM). The results show that LRP-1 silencing induces changes in the cells’ adhesion behavior, particularly the dynamics of cell attachment. Clear alterations in morphology, such as more pronounced stress fibers and increased spreading, leading to increased area and circularity, were also observed. The determination of the cells’ mechanical properties by AFM showed that these differences are correlated with an increase in Young’s modulus. Moreover, the measurements show an overall decrease in cell motility and modifications of directional persistence. An overall increase in the adhesion force between the LRP-1-silenced cells and a gelatin-coated bead was also observed. Ultimately, our AFM-based force spectroscopy data, recorded using an antibody directed against the β1 integrin subunit, provide evidence that LRP-1 silencing modifies integrin dynamics. Together, our results show that techniques traditionally used for the investigation of cancer cells can be coupled with AFM to gain access to complementary phenotypic parameters that can help discriminate between specific phenotypes associated with different degrees of invasiveness.
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Single-Molecule Force Manipulation and Nanoscopic Imaging of Protein Structure-Dynamics-Function RelationshipRoy Chowdhury, Susovan 01 September 2021 (has links)
No description available.
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Propriétés biophysiques des cardiomyocytes vivants en condition physio/physiopathologique et architecture des récepteurs couplés aux protéines G explorées par microscopie à force atomique / Biophysical properties of cardiomyocytes in physio / physiopathological conditions and of G protein coupled receptors architecture explored by atomic force microscopyLachaize, Véronique 11 October 2016 (has links)
L'insuffisance cardiaque est un réel problème de santé publique avec 1 millions de patients souffrant de cette pathologie cette année en France. Elle est définie incapacité de fournir un débit sanguin suffisant à l'organisme. Cette diminution de débit est traduite par la perte de fonction contractile du coeur provoqué par la nécrose des cellules responsable de cette fonction : les cardiomyocytes. Dans cette étude j'ai pu étudier les modifications topographiques et biomécaniques de la membrane du cardiomyocyte vivant en amont de sa rupture lors de la nécrose, par une technologie issue des nanosciences : la microscopie à force atomique (AFM). Mes travaux ont fait apparaitre une membrane très structurée chez le cardiomyocyte sain et une perte de cette architecture dans un temps précoce de l'installation de l'insuffisance cardiaque. L'utilisation de la microscopie électronique à transmission à montrer que les anomalies mises en évidences par AFM ont pour origine un réarrangement mitochondriale. Dans une seconde étude je me suis intéressée à l'organisation oligomérique d'une famille particulière de récepteur transmembranaire, les récepteurs couplés aux protéines G. Ces protéines sont une des cibles privilégiées pour les traitements pharmacologiques de l'insuffisance cardiaque tel que le bêta-bloquants et les vasodilatateurs. Ce mécanisme d'oligomérisation pourrait être la clef des effets secondaires liés à ces traitements. Afin d'étudier la conformation oligomérique, j'ai utilisé la spectroscopie de force à l'échelle de la molécule unique pour mettre en évidence différentes populations oligomérique de ces récepteurs sur la surface membranaire. Les résultats ont montré une distribution des populations oligomériques en fonction des conditions (densité de plasmide codants pour les récepteurs/stimulation avec agoniste synthétique ou naturel). Il est possible qu'il y ait une régulation des voies de signalisations par l'oligomérisation des récepteurs activés. La différence d'activité possible de chaque population oligomérique (monomère/dimère/tétramère/hexamère) semble être une explication plausible aux effets secondaire des agents pharmacologique. Mes travaux de thèse ont permis la mise en évidence de nouvelle piste par une technologie innovante, la microscopie à force atomique, dans le traitement de l'insuffisance cardiaqu / Heart failure is a public health problem with 1 million patients this year in France. This pathology is defined inability to heart pump sufficiently to maintain blood flow to meet the body's needs. This decrease is explicated by the loss of contractile function of the heart, caused by the necrosis of the contractile cells: cardiomyocytes. In this study, I was able to study the topographic and biomechanical modification of the cardiomyocyte membrane upstream of its rupture during necrosis, by technology derived from nanosciences : atomic force microscopy (AFM). My work reveals a highly structured membrane in healthy cardiomyocytes and a loss of this architecture in an early stage of the heart failure installation. In a second study I was interested in the oligomeric organization of a transmembrane receptors family , G protein-coupled receptors. These proteins are a privileged target for the pharmacological treatments on heart failure such as beta- Blockers and vasodilators. This oligomerization mechanism could be the key to the side effects associated with treatments. In order to study the oligomeric conformation, I used single molecule force spectroscopy and I reveal different oligomeric populations of these receptors on the membrane. The results showed a oligomeric populations distribution according the conditions (plasmid density coding for receptors / stimulation with synthetic or natural agonist). It is possible that there is a regulation of the signaling pathways, using the oligomerization for specific activation receptors. The possible difference in activity of each oligomeric population (monomer / dimer / tetramer / hexamer) appears to be a plausible explanation for the side effects of pharmacological agents. My thesis work allowed the discovery of a new track by an innovative technology, atomic force microscopy, in the treatment of heart failure.
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Einzelmolekül-Kraftspektroskopie zur Untersuchung der Wechselwirkungen zwischen Tau-Peptiden und monoklonalen AntikörpernStangner, Tim 11 March 2015 (has links)
In dieser Dissertation werden die Bindungseigenschaften von Rezeptor-Ligand-Komplexen mit Hilfe von Optischen Pinzetten untersucht. Aufgrund ihrer außerordentlichen Orts- (2nm) und Kraftauflösung (0,2pN) ist es möglich, diese spezifischen Interaktionen anhand einzelner Bindungsereignisse zu charakterisieren. Als Modellsysteme dienen die Wechselwirkungen zwischen den phosphorylierungsspezifischen, monoklonalen Antikörpern HPT-101 und HPT-104 und dem Morbus Alzheimer relevanten Tau-Peptid. Dieses pathogen veränderte Peptid wird krankheitsspezifisch an den Aminosäuren Threonin231 und Serin235 phosphoryliert, sodass die Detektion dieses Phosphorylierungsmusters mit Hilfe von monoklonalen Antikörpern eine mögliche Früherkennung der Alzheimer-Krankheit darstellt. Eine notwendige Voraussetzung dafür ist jedoch die exakte Kenntnis der Bindungsstellen des Liganden am Rezeptor. Ziel des ersten Teils dieser Arbeit ist es, das Epitop des monoklonalen Antikörpers HPT-101 zu bestimmen. Dazu werden mögliche bindungsrelevante Aminosäuren durch ein Alanin ausgetauscht (Alanin-Scan) und so insgesamt sieben neue Tau-Isoformen aus dem ursprünglichen doppelt-phosphorylierten Peptid Tau[pThr231/pSer235] hergestellt. Die jeweiligen Interaktionen zwischen den modifizierten Peptiden und dem Antikörper werden mit der dynamischen Kraftspektroskopie untersucht und mit Hilfe eines literaturbekannten Modells analysiert. Die sich daraus ergebenden Bindungsparameter (Lebensdauer der Bindung, charakteristische Bindungslänge, freie Aktivierungsenergie und Affinitätskonstante) werden zusammen mit den relativen Bindungshäufigkeiten erstmals genutzt, um Kriterien für essentielle, sekundäre und nicht-essentielle Aminosäuren im Tau-Peptid zu definieren. Bemerkenswerterweise existieren für insgesamt drei dieser Parameter (Bindungslebensdauer, Bindungslänge und Affinitätskonstante) scharfe Klassengrenzen, mit denen eine objektive Einteilung des Epitops von Antikörper HPT-101 möglich ist. Die erhaltenen Ergebnisse sind in überzeugender Weise im Einklang mit ELISA-Messungen zu diesem Antikörper-Peptid-Komplexen, sie liefern jedoch einen tieferen Einblick in die Natur einer spezifischen Bindung, da den kraftspektroskopischen Messungen auch die Bindungskinetik zugänglich ist. Das zweite Projekt der vorliegenden Dissertation etabliert eine Methodik, um die Datenvarianz in der Bestimmung der relativen Bindungshäufigkeit zu reduzieren. Anhand einer Kombination aus Fluoreszenz- und kraftspektroskopischen Messungen werden die Wechselwirkungen zwischen dem monoklonalen Antikörper HPT-104 und dem fluoreszenzmarkierten Peptid Tau[Fl-pThr231] untersucht. Es wird gezeigt, dass durch Vorsortieren der Peptid-beschichteten Kolloide, entsprechend ihrer Oberflächenbeladung, die Datenvarianz in den Bindungshäufigkeitsmessungen signifikant reduziert wird.
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Kinetics and dynamics of single biomoleculesSturm, Sebastian 11 August 2016 (has links)
This thesis contains several contributions to the theoretical description and interpretation of biophysical single-molecule measurements: (i) For semiflexible polymers, we derive an efficient formulation of their local transverse dynamics in terms of a Generalized Langevin Equation.
The elastic and frictional properties of the polymer are condensed into a memory kernel that is a function of the polymer\''s length and stiffness, the level of backbone tension, the position of the force probe along the polymer backbone and the boundary conditions at the polymer ends.
At short times, the memory kernel attains a universal limiting form that depends neither on the polymer length nor on the boundary conditions; we obtain analytical results that accurately describe this regime.
We discuss how to quickly and reliably evaluate the memory kernel for arbitrary times using a spectral decomposition method, and use an extensive body of numerical data to obtain analytical approximations to the memory kernel that cover the complementary long-time limit wherein polymer friction can be subsumed under a renormalized drag coefficient.
(ii) Based on a systematic nonequilibrium treatment of an overdamped, one-dimensional stochastic escape process driven by external force, we develop a theory of Dynamic Force Spectroscopy (DFS) that generalizes previously available DFS theories to the high loading rates realized in novel experimental assays and in computer simulations.
(iii) Extrapolating to future DFS experiments that may operate at far higher time resolution than presently achievable, we discuss the fast nonequilibrium relaxation of a semiflexible linker after bond rupture.
Based on a rigorous theory of tension propagation in semiflexible polymers, we predict the relaxation of force within the force actuator, show that this relaxation is dominated by linker contraction, and demonstrate quantitative agreement of our predictions with experimental data obtained by a collaborating experimentalist group.
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