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.

In the diploma thesis the charge transfer reaction on thin layer N,N',N'',N'''-tetramethyl-tetra-3,4-pyridinoporphyrazinocobalt mediator is studied. The mediator is deposited on electrode surface formed by basal plane of highly ordered pyrolytic graphite. The modified electrode, which displays electrocatalytic activity to oxidation of propylene, has been characterized by cyclic voltammetry, backscattering spectroscopy and atomic force microscopy.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Author: Karolína Růžičková Supervisor: PharmDr. Barbora Švecová, Ph.D. Consultant: Mgr. Anna Nováčková Title of thesis: Evaluation of preparation of monolayer lipid skin barrier models Skin, the protective barrier of human body, consists of several layers. The uppermost one is the stratum corneum, part of epidermis, whose extracellular matrix is composed mainly of ceramides, cholesterol and free fatty acids. The composition and arrangement of skin lipids are essential for the proper skin barrier function. Various multilayer and monolayer models are used to study skin lipids at the molecular level. Some of the evaluation methods are Langmuir monolayers at the air interface. In this work I dealt with the behavior of monolayer lipid models at four different pH values of the liquid subphase. Lipids isolated from human skin, lipid mixture prepared from the individual components, and a mixture of fatty acids were compared as well. Langmuir isotherms and the Brewster angle microscopy at different compression rates were used for this purpose. The results showed that pH of the subphase has no major effect on lipids arrangement. Lipids were most likely to form a tight monolayer at neutral pH 7,0, at a...

Nanofibres are found in a broad variety of hierarchical biological systems as fundamental structural units, and nanofibrillar components are playing an increasing role in the development of advanced functional materials. Accurate determination of the mechanical properties of single nanofibres is thus of great interest, yet measurement of these properties is challenging due to the intricate specimen handling and the exceptional force and deformation resolution that is required. The atomic force microscope (AFM) has emerged as an effective, reliable tool in the investigation of nanofibrillar mechanics, with the three most popular approaches—AFM-based tensile testing, three-point deformation testing, and nanoindentation—proving preferable to conventional tensile testing in many (but not all) cases. Here, we review the capabilities and limitations of each of these methods and give a comprehensive overview of the recent advances in this field.

Selective ion and solute transport across cell membranes is a vital process occurring in all types of cells. Evolutionarily developed transport proteins work as membrane-embedded molecular machines, which alternately open a gate on each side of the membrane to bind and translocate specific ions. Sodium/proton exchange plays a crucial role in maintaining cytoplasmic pH and membrane potential, while, if not regulated, the process causes severe heart diseases in humans. Here I applied single-molecule force spectroscopy to investigate molecular interactions determining the structural stability of the sodium/proton antiporter NhaA of Escherichia coli, which serves as a model system for this class of proteins. Mechanical pulling of NhaA molecules embedded in the native lipid bilayer caused a step-wise unfolding of the protein and provided insights into its stability. Modified experiments allowed observing refolding of NhaA molecules and estimating folding kinetics for individual structural elements, as well as detecting eventual misfolded conformations of the protein. The activity of NhaA increases 2000fold upon switching pH from 6 to 8. Single-molecule force measurements revealed a reversible change in molecular interactions within the ligand-binding site of the transporter at pH 5.5. The effect was enhanced in the presence of sodium ions. The observation suggests an early activation stage of the protein and provides new insights into the functioning mechanism. When studying interactions of NhaA with the inhibitor 2-aminoperimidine, I exploited single-molecule force measurements to validate the binding mechanism and to describe quantitatively formation of the protein:inhibitor complex. The ability of single-molecule force measurements to probe structurally and functionally important interactions of membrane proteins opens new prospects for using the approach in protein science and applied research.

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.

Multiferroika, also Materialien, die gleichzeitig ferroelektrische und ferromagnetische Eigenschaften besitzen, sind sowohl für die Forschung um das Verständnis dieser Eigenschaften als auch für potentielle Anwendungen in neuartigen Speichern von großem Interesse. Die Rasterkraftmikroskopie spielt hierbei eine entscheidende Rolle, da mit ihrer Hilfe die Eigenschaften solcher Probensysteme auf der Nanometerlängenskala untersucht werden können. In der vorliegenden Arbeit werden drei unterschiedliche multiferroische Systeme auf ihre ferroelektrischen und ferromagnetischen Eigenschaften sowie auf deren Kopplung hin mit Hilfe verschiedener Methoden der Rasterkraftmikroskopie untersucht. Im Grundlagenteil dieser Arbeit wird dazu zunächst eine Methode vorgestellt, mit der magnetische Spitzen für die Rasterkraftmikroskopie charakterisiert werden können, so dass in experimentellen Untersuchungen die Wechselwirkung zwischen der untersuchenden Spitze und der untersuchten Probe besser abgeschätzt werden kann. Des Weiteren wird eine Möglichkeit vorgestellt, Kelvin-Sonden-Rasterkraftmikroskopie mit der magnetischen Rasterkraftmikroskopie zu kombinieren, um elektrostatische Artefakte bei den Untersuchungen der magnetischen Eigenschaften auszuschließen. Im experimentellen Teil der Arbeit werden zuerst die beiden einphasigen Multiferroika BiFeO3 und BiCrO3 untersucht. Es kann experimentell gezeigt werden, dass für die Untersuchung der ferromagnetischen Eigenschaften von Multiferroika die Kombination aus Kelvin-Sonden-Rasterkraftmikroskopie und magnetischer Rasterkraftmikroskopie notwendig ist und mit dieser Technik die magnetischen und elektrostatischen Kräfte ohne Übersprechen voneinander getrennt werden können. Mit Hilfe der Piezoantwort-Rasterkraftmikroskopie werden die ferroelektrischen Domänen dieser Systeme untersucht und lokal die Polarisationsrichtung in den einzelnen Domänen bestimmt. Weiterhin wird an einem Schichtsystem, bestehend aus einem Nickelfilm, der auf BaTiO3 aufgebracht ist, die magnetoelektrische Kopplung analysiert. Hierbei wird vor allem der Einfluss einer elektrischen Spannung auf die leichte magnetische Achse des Nickelfilms studiert, sowie die Veränderung der magnetischen Domänenstruktur in Abhängigkeit der angelegten elektrischen Spannung.:Abbildungsverzeichnis Abkürzungen 1. Einleitung I. Grundlagen 2. Ferroische Materialien 2.1. Ferromagnetika 2.2. Ferroelektrika 2.3. Kopplung ferroischer Eigenschaften: Multiferroika 3. Messmethoden 3.1. Grundlagen der Rasterkraftmikroskopie 3.1.1. Kontakt-Rasterkraftmikroskopie 3.1.2. Nichtkontakt-Rasterkraftmikroskopie 3.2. Piezoantwort-Rasterkraftmikroskopie 3.2.1. Aufbau und Grundlagen 3.2.2. Verwendete Messgeräte 3.3. Kelvin-Sonden-Rasterkraftmikroskopie 3.4. Magnet-Rasterkraftmikroskopie 3.4.1. Grundlagen 3.4.2. MFM in externen Magnetfeldern 3.4.3. Verwendete Messgeräte II Experimente 4. Experimente 4.1. Charakterisierung magnetischer Spitzen 4.2. Untersuchungen an BiFeO3 4.2.1. Eigenschaften von BiFeO3 4.2.2. Piezoantwort-Rasterkraftmikroskopie-Messungen 4.2.3. Magnetische-Rasterkraftmikroskopie-Messungen 4.3. Untersuchungen an BiCrO3 4.3.1 Eigenschaften von BiCrO3 4.3.2. Piezoantwort-Rasterkraftmikroskopie-Messungen 4.3.3. Magnetische-Rasterkraftmikroskopie-Messungen 4.4. Untersuchungen an Ni-Dünnfilmen auf BaTiO3 4.4.1. Magnetische Eigenschaften von Ni 4.4.2. Theoretische Beschreibung des Systems 4.4.3. Magnetische Rasterkraftmikroskopie-Messungen 5. Zusammenfassung und Ausblick Angang A A.1. Koordinatentransformation des piezoelektrischen Tensors A.2. Bestimmung der Polarisationsrichtung aus der Verzerrung Literaturverzeichnis Eigene Publikationen A.3. Vorträge A.4. Poster A.5. Veröffentlichungen Danksagung

A major distress mode in asphalt pavements is low temperature cracking, which results from the contraction and expansion of the asphalt pavement under extreme temperature changes. The potential for low temperature cracking is an interplay between the environment, the road structure and importantly the properties of the asphalt mixture. The thermal cracking performance of asphalt concrete mixtures can be evaluated by conducting thermal stress restrained specimen tests (TSRST) which is known to be correlated well with the fracture temperatures observed in the field. Although TSRST provides a good estimation of the field performance, it may be unrealistic to implement the obtained results in a design framework. On the other hand, recent studies showed Superpave indirect tension tests can be used to evaluate fracture performance (fatigue, moisture damage, low temperature cracking, etc.) of the asphalt concrete  mixtures. In addition, the obtained elastic and viscoelastic parameters from the Superpave IDT tests can be used as an input parameter to establish a design framework. The study presented in this thesis has a main objective to develop a framework using Superpave IDT test results as input parameters in order to evaluate the low temperature cracking performance of asphalt concrete mixtures. Moreover, the study aims to investigate micro-mechanically the low temperature cracking behavior of bitumen using atomic force microscopy (AFM) as a tool. The numerical model has been developed by integrating fracture energy threshold into an asphalt concrete thermal fracture model, considering non-linear thermal contraction coefficients. Based on the asphalt concrete mixture viscoelastic properties, this integrated model can predict thermally induced stresses and fracture temperatures. The elastic, viscoelastic and fracture energy input parameters of the model were measured by conducting indirect tension tests and the thermal contraction coefficients were measured experimentally. The proposed model has been validated by comparing the predicted fracture temperatures with the results obtained from TSRST tests. It was found that, while there is a quantitative discrepancy, the predicted ranking was correct. In the measurement of the thermal contraction coefficients it was observed that the thermal contraction coefficient in asphalt concrete is non-linear in the temperature range of interest for low temperature cracking. The implications of having non-linear thermal contraction coefficient were investigated numerically. In an effort to understand the effect of bitumen properties on low temperature fatigue cracking, AFM was used to characterize the morphology of bitumen. The AFM topographic and phase contrast image confirmed the existence of bee-shaped microstructure and different phases. The bitumen samples were subjected to both environmental and mechanical loading and after loading, micro-cracks appeared in the interfaces of the bitumen surface, confirming bitumen itself may also crack. It was also found that the presence of wax and wax crystallization plays a vital role in low temperature cracking performance of bitumen. / <p>QC 20120828</p>

Heteroepitaxial SiGeC layers have attracted immenseattention as a material for high frequency devices duringrecent years. The unique properties of integrating carbon inSiGe are the additional freedom for strain and bandgapengineering as well as allowing more aggressive device designdue to the potential for increased thermal budget duringprocessing. This work presents different issues on epitaxialgrowth, defect density, dopant incorporation and electricalproperties of SiGeC epitaxial layers, intended for variousdevice applications. Non-selective and selective epitaxial growth of Si1-x-yGexCy(0≤x≤30, ≤y≤0.02) layershave been optimized by using high-resolution x-ray reciprocallattice mapping. The incorporation of carbon into the SiGematrix was shown to be strongly sensitive to the growthparameters. As a consequence, a much smaller epitaxial processwindow compared to SiGe epitaxy was obtained. Differentsolutions to decrease the substrate pattern dependency (loadingeffect) of SiGeC growth have also been proposed. The key pointin these methods is based on reduction of surface migration ofthe adsorbed species on the oxide. In non-selective epitaxy,this was achieved by introducing a thin silicon polycrystallineseed layer on the oxide. The thickness of this seed layer had acrucial role on both the global and local loading effect, andon the epitaxial quality. Meanwhile, in selective epitaxy,polycrystalline stripes introduced around the oxide openingsact as migration barriers and reduce the loading effecteffectively. Chemical mechanical polishing (CMP) was performedto remove the polycrystalline stripes on the oxide. Incorporation and electrical properties of boron-doped Si1-x-yGexCylayers (x=0.23 and 0.28 with y=0 and 0.005) with aboron concentration in the range of 3x1018-1x1021atoms/cm3 have also been investigated. In SiGeClayers, the active boron concentration was obtained from thestrain compensation. It was also found that the boron atomshave a tendency to locate at substitutional sites morepreferentially compared to carbon. These findings led to anestimation of the Hall scattering factor of the SiGeC layers,which showed good agreement with theoretical calculations. Keywords:Silicon germanium carbon (SiGeC), Epitaxy,Chemical vapor deposition (CVD), Loading effect, Highresolution x-ray diffraction (HRXRD), Hall measurements, Atomicforce microscopy (AFM).

Since the 18th-century scientists are studying diatoms, fascinated by their beauty and diversity. Their nano- and micropatterned biosilica cell walls are outstanding examples of biologically controlled mineral formation. Although the knowledge about diatom cell wall formation increased over the last 60 years, the process is still far away from being completely understood. Diatom cell walls exhibit highly interesting material properties, making them appealing to material scientists. Due to those properties, diatom cell walls are on the brink of becoming powerful tools in nanotechnology. However, the production of tailored silica structures for nanotechnology requires a much better understanding of the processes and components involved in cell wall morphogenesis. Recent studies set the focus on insoluble organic matrices as important parts of this process, suggesting that they act as templates in silica morphogenesis. Therefore, in this study, the occurrence of insoluble organic matrices and their possible silica precipitation activity was analyzed in the three diatom species T. pseudonana, T. oceanica and C. cryptica. For all three species girdle band and valve derived insoluble organic matrices could be identified. The extracted insoluble organic matrices exhibited structural features present in the corresponding biosilica cell walls. The highest similarities were found in the valve derived matrices of C. cryptica. Accessibility studies showed that the biosilica associated insoluble organic matrices of T. pseudonana were only partially accessible, arguing for an entrapment of insoluble organic matrices in the silica, rather than an attachment to the surface of the cell wall. All examined insoluble organic matrices of the three species exhibited intrinsic silica precipitation activity. The most intriguing structures were formed by the insoluble organic matrices of C. cryptica, yielding a porous silica pattern. The addition of biosilica derived soluble components or long-chain polyamines promoted this process and moreover lead to the reconstitution of biosilica-like hierarchical silica pore patterns. The generated silica structures were templated by the underlying structure of the insoluble organic matrix. The result presented in this thesis make this the first study reporting the in vitro generation of diatom biosilica-like hierarchical silica pore patterns using all natural cell wall components. It supports the hypothesis of microplates acting as templates for biosilica morphogenesis and introduces an interesting experimental setup for silica-based in vitro studies on the mechanism of pore formation in diatoms.