Contraction speed of the actomyosin cytoskeleton in the absence of the cell membrane

The contraction of the actomyosin cytoskeleton, which is produced by the sliding of myosin II along actin filaments, drives important cellular activities such as cytokinesis and cell migration. To explain the contraction velocities observed in such physiological processes, we have studied the contraction of intact cytoskeletons of Dictyostelium discoideum cells after removing the plasma membrane using Triton X-100. The technique developed in this work allows for the quantitative measurement of contraction rates of individual cytoskeletons. The relationship of the contraction rates with forces was analyzed using three different myosins with different in vitro sliding velocities. The cytoskeletons containing these myosins were always contractile and the contraction rate was correlated with the sliding velocity of the myosins. However, the values of the contraction rate were two to three orders of magnitude slower than expected from the in vitro sliding velocities of the myosins, presumably due to internal and external resistive forces. The contraction process also depended on actin cross-linking proteins. The lack of α-actinin increased the contraction rate 2-fold and reduced the capacity of the cytoskeleton to retain internal materials, while the lack of filamin resulted in the ATP-dependent disruption of the cytoskeleton. Interestingly, the myosin-dependent contraction rate of intact contractile rings is also reportedly much slower than the in vitro sliding velocity of myosin, and is similar to the contraction rates of cytoskeletons (different by only 2–3 fold), suggesting that the contraction of intact cells and cytoskeletons is limited by common mechanisms.

New steps in the development of the second generation TEC GESMEY

The paper shows the results of the new steps that have been done in the development of the tidal energy converter GESMEY. These are the design, construction and trials into the sea of a 1/10 scale prototype and also the construction with the same scale of the buoy BOSCEM, that anchors the device and lets it in the correct work position and depth, along the two directions of the flow that the daily tidal cycle have. Inside the paper is described the objectives and the methodology of the experimental trials that were ca rry out the last summer with the scale prototype. GESMEY is a new type of tidal energy converter (TEC) that has the capability to exploit currents in waters over forty meters by itself and it gets only using its internal ballast system the necessary equilibrium between hy drostatics and hydrodynamics forces to make the emersion and the immersion procedures without any other help. Finally the paper shows the description of the results obtained over the performance of the devices along the immersion, emersion and floating transport manoeuvres and afterwards the results, that were obtained along the generation power tests that were carried out, are shown.

Calculation of the spatial distribution of photovoltaic field by arbitrary 2D ilumination patterns en LiNbO3; application to photovoltaic particle trapping.

Patterns of evanescent photovoltaic field induced by illumination on a surface of lithium niobate (LN) have been calculated and compared with the experimental patterns of nano- and microparticles trapped by dielectrophoretic forces. A tool for this calculation has been developed. Calculo de distribución espacial de campo por efecto fotovoltaico con patrones arbitrarios de iluminación, en LiNbO3

Mathematical Analysis of the Effect of Rotor Geometry on Cup Anemometer Response

The calibration coefficients of two commercial anemometers equipped with different rotors were studied. The rotor cups had the same conical shape, while the size and distance to the rotation axis varied.The analysis was based on the 2-cup positions analytical model, derived using perturbation methods to include second-order effects such as pressure distribution along the rotating cups and friction.Thecomparison with the experimental data indicates a nonuniformdistribution of aerodynamic forces on the rotating cups, with higher forces closer to the rotating axis. The 2-cup analytical model is proven to be accurate enough to study the effect of complex forces on cup anemometer performance.

Fourier analysis of the aerodynamic behavior of cup anemometers

The calibration results (the transfer function) of an anemometer equipped with several cup rotors were analyzed and correlated with the aerodynamic forces measured on the isolated cups in a wind tunnel. The correlation was based on a Fourier analysis of the normal-to-the-cup aerodynamic force. Three different cup shapes were studied: typical conical cups, elliptical cups and porous cups (conical-truncated shape). Results indicated a good correlation between the anemometer factor, K, and the ratio between the first two coefficients in the Fourier series decomposition of the normal-to-the-cup aerodynamic force

Numerical Simulation of the Aerodynamic Behavior of High Velocity Trains under Synthetic Crosswinds of Different Shear and Turbulence Characteristics

A numerical simulation of the aerodynamic behavior of high-speed trains under synthetic crosswinds at a 90º yaw angle is presented. The train geometry is the aerodynamic train model (ATM). Flow description based on numerical simulations is obtained using large eddy simulation (LES) and the commercial code ANSYSFluent V14.5. A crosswind whose averaged velocity and turbulence characteristics change with distance to the ground is imposed. Turbulent fluctuations that vary temporally and spatially are simulated with TurbSim code. The crosswind boundary condition is calculated for the distance the train runs during a simulation period. The inlet streamwise velocity boundary condition is generated using Taylor?s frozen turbulence hypothesis. The model gives a time history of the force and moments acting on the train; this includes averaged values, standard deviations and extreme values. Of particular interest are the spectra of the forces and moments, and the admittance spectra. For comparison, results obtained with LES and a uniform wind velocity fluctuating in time, and results obtained with Reynolds averaged Navier Stokes equations (RANS), and the averaged wind conditions, are also presented.

Methods for the analysis of multi-scale cell dynamics from fluorescence microscopy images

La embriogénesis es el proceso mediante el cual una célula se convierte en un ser un vivo. A lo largo de diferentes etapas de desarrollo, la población de células va proliferando a la vez que el embrión va tomando forma y se configura. Esto es posible gracias a la acción de varios procesos genéticos, bioquímicos y mecánicos que interaccionan y se regulan entre ellos formando un sistema complejo que se organiza a diferentes escalas espaciales y temporales. Este proceso ocurre de manera robusta y reproducible, pero también con cierta variabilidad que permite la diversidad de individuos de una misma especie. La aparición de la microscopía de fluorescencia, posible gracias a proteínas fluorescentes que pueden ser adheridas a las cadenas de expresión de las células, y los avances en la física óptica de los microscopios han permitido observar este proceso de embriogénesis in-vivo y generar secuencias de imágenes tridimensionales de alta resolución espacio-temporal. Estas imágenes permiten el estudio de los procesos de desarrollo embrionario con técnicas de análisis de imagen y de datos, reconstruyendo dichos procesos para crear la representación de un embrión digital. Una de las más actuales problemáticas en este campo es entender los procesos mecánicos, de manera aislada y en interacción con otros factores como la expresión genética, para que el embrión se desarrolle. Debido a la complejidad de estos procesos, estos problemas se afrontan mediante diferentes técnicas y escalas específicas donde, a través de experimentos, pueden hacerse y confrontarse hipótesis, obteniendo conclusiones sobre el funcionamiento de los mecanismos estudiados. Esta tesis doctoral se ha enfocado sobre esta problemática intentando mejorar las metodologías del estado del arte y con un objetivo específico: estudiar patrones de deformación que emergen del movimiento organizado de las células durante diferentes estados del desarrollo del embrión, de manera global o en tejidos concretos. Estudios se han centrado en la mecánica en relación con procesos de señalización o interacciones a nivel celular o de tejido. En este trabajo, se propone un esquema para generalizar el estudio del movimiento y las interacciones mecánicas que se desprenden del mismo a diferentes escalas espaciales y temporales. Esto permitiría no sólo estudios locales, si no estudios sistemáticos de las escalas de interacción mecánica dentro de un embrión. Por tanto, el esquema propuesto obvia las causas de generación de movimiento (fuerzas) y se centra en la cuantificación de la cinemática (deformación y esfuerzos) a partir de imágenes de forma no invasiva. Hoy en día las dificultades experimentales y metodológicas y la complejidad de los sistemas biológicos impiden una descripción mecánica completa de manera sistemática. Sin embargo, patrones de deformación muestran el resultado de diferentes factores mecánicos en interacción con otros elementos dando lugar a una organización mecánica, necesaria para el desarrollo, que puede ser cuantificado a partir de la metodología propuesta en esta tesis. La metodología asume un medio continuo descrito de forma Lagrangiana (en función de las trayectorias de puntos materiales que se mueven en el sistema en lugar de puntos espaciales) de la dinámica del movimiento, estimado a partir de las imágenes mediante métodos de seguimiento de células o de técnicas de registro de imagen. Gracias a este esquema es posible describir la deformación instantánea y acumulada respecto a un estado inicial para cualquier dominio del embrión. La aplicación de esta metodología a imágenes 3D + t del pez zebra sirvió para desvelar estructuras mecánicas que tienden a estabilizarse a lo largo del tiempo en dicho embrión, y que se organizan a una escala semejante al del mapa de diferenciación celular y con indicios de correlación con patrones de expresión genética. También se aplicó la metodología al estudio del tejido amnioserosa de la Drosophila (mosca de la fruta) durante el cierre dorsal, obteniendo indicios de un acoplamiento entre escalas subcelulares, celulares y supracelulares, que genera patrones complejos en respuesta a la fuerza generada por los esqueletos de acto-myosina. En definitiva, esta tesis doctoral propone una estrategia novedosa de análisis de la dinámica celular multi-escala que permite cuantificar patrones de manera inmediata y que además ofrece una representación que reconstruye la evolución de los procesos como los ven las células, en lugar de como son observados desde el microscopio. Esta metodología por tanto permite nuevas formas de análisis y comparación de embriones y tejidos durante la embriogénesis a partir de imágenes in-vivo. ABSTRACT The embryogenesis is the process from which a single cell turns into a living organism. Through several stages of development, the cell population proliferates at the same time the embryo shapes and the organs develop gaining their functionality. This is possible through genetic, biochemical and mechanical factors that are involved in a complex interaction of processes organized in different levels and in different spatio-temporal scales. The embryogenesis, through this complexity, develops in a robust and reproducible way, but allowing variability that makes possible the diversity of living specimens. The advances in physics of microscopes and the appearance of fluorescent proteins that can be attached to expression chains, reporting about structural and functional elements of the cell, have enabled for the in-vivo observation of embryogenesis. The imaging process results in sequences of high spatio-temporal resolution 3D+time data of the embryogenesis as a digital representation of the embryos that can be further analyzed, provided new image processing and data analysis techniques are developed. One of the most relevant and challenging lines of research in the field is the quantification of the mechanical factors and processes involved in the shaping process of the embryo and their interactions with other embryogenesis factors such as genetics. Due to the complexity of the processes, studies have focused on specific problems and scales controlled in the experiments, posing and testing hypothesis to gain new biological insight. However, methodologies are often difficult to be exported to study other biological phenomena or specimens. This PhD Thesis is framed within this paradigm of research and tries to propose a systematic methodology to quantify the emergent deformation patterns from the motion estimated in in-vivo images of embryogenesis. Thanks to this strategy it would be possible to quantify not only local mechanisms, but to discover and characterize the scales of mechanical organization within the embryo. The framework focuses on the quantification of the motion kinematics (deformation and strains), neglecting the causes of the motion (forces), from images in a non-invasive way. Experimental and methodological challenges hamper the quantification of exerted forces and the mechanical properties of tissues. However, a descriptive framework of deformation patterns provides valuable insight about the organization and scales of the mechanical interactions, along the embryo development. Such a characterization would help to improve mechanical models and progressively understand the complexity of embryogenesis. This framework relies on a Lagrangian representation of the cell dynamics system based on the trajectories of points moving along the deformation. This approach of analysis enables the reconstruction of the mechanical patterning as experienced by the cells and tissues. Thus, we can build temporal profiles of deformation along stages of development, comprising both the instantaneous events and the cumulative deformation history. The application of this framework to 3D + time data of zebrafish embryogenesis allowed us to discover mechanical profiles that stabilized through time forming structures that organize in a scale comparable to the map of cell differentiation (fate map), and also suggesting correlation with genetic patterns. The framework was also applied to the analysis of the amnioserosa tissue in the drosophila’s dorsal closure, revealing that the oscillatory contraction triggered by the acto-myosin network organized complexly coupling different scales: local force generation foci, cellular morphology control mechanisms and tissue geometrical constraints. In summary, this PhD Thesis proposes a theoretical framework for the analysis of multi-scale cell dynamics that enables to quantify automatically mechanical patterns and also offers a new representation of the embryo dynamics as experienced by cells instead of how the microscope captures instantaneously the processes. Therefore, this framework enables for new strategies of quantitative analysis and comparison between embryos and tissues during embryogenesis from in-vivo images.

Hysteresis in transverse galloping: the role of the inflection points

Transverse galloping is here considered as a one-degree-of-freedom oscillator subjected to aerodynamic forces, which are described by using the quasi-steady hypothesis. The hysteresis of transverse galloping is also analyzed. Approximate solutions of the model are obtained by assuming that the aerodynamic and damping forces are much smaller than the inertial and stiffness ones. The analysis of the approximate solution, which is obtained by means of the method of Krylov–Bogoliubov, reveals the existing link between the hysteresis phenomenon and the number of inflection points at the aerodynamic force coefficient curve, Cy(α)Cy(α); CyCy and αα being, respectively, the force coefficient normal to the incident flow and the angle of attack. The influence of the position of these inflection points on the range of flow velocities in which hysteresis takes place is also analyzed.

On the circulation and the position of the forward stagnation point on airfoils

Lift and velocity circulation around airfoils are two aspects of the same phenomenon when airfoils are not stalled and the Kutta—Joukowski theorem applies. This theorem establishes a linear dependence between lift and circulation, which breaks when stalling occurs. As the angle of attack increases beyond this point, the circulation vanishes. Since the circulation determines to a great extent the position of the forward stagnation point on an airfoil, the measurement of this position is an easy and simple way to determine the circulation, which is of help in understanding the role of the latter in the generation of aerodynamic forces on airfoils.

Design of a generalized tool for the performance assessment under sail based on analytical, numerical and empirical results : a global sailing yacht meta-model

La evaluación de las prestaciones de las embarcaciones a vela ha constituido un objetivo para ingenieros navales y marinos desde los principios de la historia de la navegación. El conocimiento acerca de estas prestaciones, ha crecido desde la identificación de los factores clave relacionados con ellas(eslora, estabilidad, desplazamiento y superficie vélica), a una comprensión más completa de las complejas fuerzas y acoplamientos involucrados en el equilibrio. Junto con este conocimiento, la aparición de los ordenadores ha hecho posible llevar a cabo estas tareas de una forma sistemática. Esto incluye el cálculo detallado de fuerzas, pero también, el uso de estas fuerzas junto con la descripción de una embarcación a vela para la predicción de su comportamiento y, finalmente, sus prestaciones. Esta investigación tiene como objetivo proporcionar una definición global y abierta de un conjunto de modelos y reglas para describir y analizar este comportamiento. Esto se lleva a cabo sin aplicar restricciones en cuanto al tipo de barco o cálculo, sino de una forma generalizada, de modo que sea posible resolver cualquier situación, tanto estacionaria como en el dominio del tiempo. Para ello se comienza con una definición básica de los factores que condicionan el comportamiento de una embarcación a vela. A continuación se proporciona una metodología para gestionar el uso de datos de diferentes orígenes para el cálculo de fuerzas, siempre con el la solución del problema como objetivo. Esta última parte se plasma en un programa de ordenador, PASim, cuyo propósito es evaluar las prestaciones de diferentes ti pos de embarcaciones a vela en un amplio rango de condiciones. Varios ejemplos presentan diferentes usos de PASim con el objetivo de ilustrar algunos de los aspectos discutidos a lo largo de la definición del problema y su solución . Finalmente, se presenta una estructura global de cara a proporcionar una representación virtual de la embarcación real, en la cual, no solo e l comportamiento sino también su manejo, son cercanos a la experiencia de los navegantes en el mundo real. Esta estructura global se propone como el núcleo (un motor de software) de un simulador físico para el que se proporciona una especificación básica. ABSTRACT The assessment of the performance of sailing yachts, and ships in general, has been an objective for naval architects and sailors since the beginning of the history of navigation. The knowledge has grown from identifying the key factors that influence performance(length, stability, displacement and sail area), to a much more complete understanding of the complex forces and couplings involved in the equilibrium. Along with this knowledge, the advent of computers has made it possible to perform the associated tasks in a systematic way. This includes the detailed calculation of forces, but also the use of those forces, along with the description of a sailing yacht, to predict its behavior, and ultimately, its performance. The aim of this investigation is to provide a global and open definition of a set of models and rules to describe and analyze the behavior of a sailing yacht. This is done without applying any restriction to the type of yacht or calculation, but rather in a generalized way, capable of solving any possible situation, whether it is in a steady state or in the time domain. First, the basic definition of the factors that condition the behavior of a sailing yacht is given. Then, a methodology is provided to assist with the use of data from different origins for the calculation of forces, always aiming towards the solution of the problem. This last part is implemented as a computational tool, PASim, intended to assess the performance of different types of sailing yachts in a wide range of conditions. Several examples then present different uses of PASim, as a way to illustrate some of the aspects discussed throughout the definition of the problem and its solution. Finally, a global structure is presented to provide a general virtual representation of the real yacht, in which not only the behavior, but also its handling is close to the experience of the sailors in the real world. This global structure is proposed as the core (a software engine) of a physical yacht simulator, for which a basic specification is provided.

Application of the Boundary Method to the determination of the properties of the beam cross-sections

Using the 3-D equations of linear elasticity and the asylllptotic expansion methods in terms of powers of the beam cross-section area as small parameter different beam theories can be obtained, according to the last term kept in the expansion. If it is used only the first two terms of the asymptotic expansion the classical beam theories can be recovered without resort to any "a priori" additional hypotheses. Moreover, some small corrections and extensions of the classical beam theories can be found and also there exists the possibility to use the asymptotic general beam theory as a basis procedure for a straightforward derivation of the stiffness matrix and the equivalent nodal forces of the beam. In order to obtain the above results a set of functions and constants only dependent on the cross-section of the beam it has to be computed them as solutions of different 2-D laplacian boundary value problems over the beam cross section domain. In this paper two main numerical procedures to solve these boundary value pf'oblems have been discussed, namely the Boundary Element Method (BEM) and the Finite Element Method (FEM). Results for some regular and geometrically simple cross-sections are presented and compared with ones computed analytically. Extensions to other arbitrary cross-sections are illustrated.

Broken symmetry and strangeness of the semiconductor impurity band metal-insulator transition

The filamentary model of the metal-insulator transition in randomly doped semiconductor impurity bands is geometrically equivalent to similar models for continuous transitions in dilute antiferromagnets and even to the λ transition in liquid He, but the critical behaviors are different. The origin of these differences lies in two factors: quantum statistics and the presence of long range Coulomb forces on both sides of the transition in the electrical case. In the latter case, in addition to the main transition, there are two satellite transitions associated with disappearance of the filamentary structure in both insulating and metallic phases. These two satellite transitions were first identified by Fritzsche in 1958, and their physical origin is explained here in geometrical and topological terms that facilitate calculation of critical exponents.

Analysis of the role of the Spitzenkörper in fungal morphogenesis by computer simulation of apical branching in Aspergillus niger

High-resolution video microscopy, image analysis, and computer simulation were used to study the role of the Spitzenkörper (Spk) in apical branching of ramosa-1, a temperature-sensitive mutant of Aspergillus niger. A shift to the restrictive temperature led to a cytoplasmic contraction that destabilized the Spk, causing its disappearance. After a short transition period, new Spk appeared where the two incipient apical branches emerged. Changes in cell shape, growth rate, and Spk position were recorded and transferred to the fungus simulator program to test the hypothesis that the Spk functions as a vesicle supply center (VSC). The simulation faithfully duplicated the elongation of the main hypha and the two apical branches. Elongating hyphae exhibited the growth pattern described by the hyphoid equation. During the transition phase, when no Spk was visible, the growth pattern was nonhyphoid, with consecutive periods of isometric and asymmetric expansion; the apex became enlarged and blunt before the apical branches emerged. Video microscopy images suggested that the branch Spk were formed anew by gradual condensation of vesicle clouds. Simulation exercises where the VSC was split into two new VSCs failed to produce realistic shapes, thus supporting the notion that the branch Spk did not originate by division of the original Spk. The best computer simulation of apical branching morphogenesis included simulations of the ontogeny of branch Spk via condensation of vesicle clouds. This study supports the hypothesis that the Spk plays a major role in hyphal morphogenesis by operating as a VSC—i.e., by regulating the traffic of wall-building vesicles in the manner predicted by the hyphoid model.

Mechanical separation of the complementary strands of DNA

We describe the mechanical separation of the two complementary strands of a single molecule of bacteriophage λ DNA. The 3′ and 5′ extremities on one end of the molecule are pulled progressively apart, and this leads to the opening of the double helix. The typical forces along the opening are in the range of 10–15 pN. The separation force signal is shown to be related to the local GC vs. AT content along the molecule. Variations of this content on a typical scale of 100–500 bases are presently detected.

Regulation of the Actin Cytoskeleton by Thrombin in Human Endothelial Cells: Role of Rho Proteins in Endothelial Barrier Function

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.