Calcium Signaling in Live Cells on Elastic Gels under Mechanical Vibration at Subcellular Levels

A new device was designed to generate a localized mechanical vibration of flexible gels where human umbilical vein endothelial cells (HUVECs) were cultured to mechanically stimulate these cells at subcellular locations. A Fluorescence Resonance Energy Transfer (FRET)-based calcium biosensor (an improved Cameleon) was used to monitor the spatiotemporal distribution of intracellular calcium concentrations in the cells upon this mechanical stimulation. A clear increase in intracellular calcium concentrations over the whole cell body (global) can be observed in the majority of cells under mechanical stimulation. The chelation of extracellular calcium with EGTA or the blockage of stretch-activated calcium channels on the plasma membrane with streptomycin or gadolinium chloride significantly inhibited the calcium responses upon mechanical stimulation. Thapsigargin, an endoplasmic reticulum (ER) calcium pump inhibitor, or U73122, a phospholipase C (PLC) inhibitor, resulted in mainly local calcium responses occurring at regions close to the stimulation site. The disruption of actin filaments with cytochalasin D or inhibition of actomyosin contractility with ML-7 also inhibited the global calcium responses. Therefore, the global calcium response in HUVEC depends on the influx of calcium through membrane stretch-activated channels, followed by the release of inositol trisphosphate (IP3) via PLC activation to trigger the ER calcium release. Our newly developed mechanical stimulation device can also provide a powerful tool for the study of molecular mechanism by which cells perceive the mechanical cues at subcellular levels

Mechanical Alterations in airway smooth muscle after interaction with indoor pollutant – A mathematical model.

The viscoelasticity of mammalian lung is determined by the mechanical properties and structural regulation of the airway smooth muscle (ASM). The exposure to polluted air may deteriorate these properties with harmful consequences to individual health. Formaldehyde (FA) is an important indoor pollutant found among volatile organic compounds. This pollutant permeates through the smooth muscle tissue forming covalent bonds between proteins in the extracellular matrix and intracellular protein structure changing mechanical properties of ASM and inducing asthma symptoms, such as airway hyperresponsiveness, even at low concentrations. In the experimental scenario, the mechanical effect of FA is the stiffening of the tissue, but the mechanism behind this effect is not fully understood. Thus, the aim of this study is to reproduce the mechanical behavior of the ASM, such as contraction and stretching, under FA action or not. For this, it was created a two-dimensional viscoelastic network model based on Voronoi tessellation solved using Runge-Kutta method of fourth order. The equilibrium configuration was reached when the forces in different parts of the network were equal. This model simulates the mechanical behavior of ASM through of a network of dashpots and springs. This dashpot-spring mechanical coupling mimics the composition of the actomyosin machinery of ASM through the contraction of springs to a minimum length. We hypothesized that formation of covalent bonds, due to the FA action, can be represented in the model by a simple change in the elastic constant of the springs, while the action of methacholine (MCh) reduce the equilibrium length of the spring. A sigmoid curve of tension as a function of MCh doses was obtained, showing increased tension when the muscle strip was exposed to FA. Our simulations suggest that FA, at a concentration of 0.1 ppm, can affect the elastic properties of the smooth muscle ¯bers by a factor of 120%. We also analyze the dynamic mechanical properties, observing the viscous and elastic behavior of the network. Finally, the proposed model, although simple, incorporates the phenomenology of both MCh and FA and reproduces experimental results observed with in vitro exposure of smooth muscle to FA. Thus, this new mechanical approach incorporates several well know features of the contractile system of the cells in a tissue level model. The model can also be used in different biological scales.CNPqFAPES

Mechanical alterations in airway smooth muscle after interaction with indoor pollutant - A\ud mathematical model

The viscoelasticity of mammalian lung is determined by the mechanical properties and structural regulation of the airway smooth muscle (ASM). The exposure to polluted air may deteriorate these properties with harmful consequences to individual health. Formaldehyde (FA) is an important indoor pollutant found among volatile organic compounds. This pollutant permeates through the smooth muscle tissue forming covalent bonds between proteins in the extracellular matrix and intracellular protein structure changing mechanical properties of ASM and inducing asthma symptoms, such as airway hyperresponsiveness, even at low concentrations. In the experimental scenario, the mechanical effect of FA is the stiffening of the tissue, but the mechanism behind this effect is not fully w1derstood. Thus, the aim of this study is to reproduce the mechanical behavior of the ASM, such as contraction and stretching, under FA action or not. For this, it was created a two-dimensional viscoelastic network model based on Voronoi tessellation solved using Runge-Kutta method of fourth order. The equilibrium configuration was reached when the forces in different parts of the network were equal. This model simulates the mechanical behavior of ASM through of a network of dashpots and springs. This dashpot-spring mechanical coupling mimics the composition of the actomyosin machinery of ASM through the contraction of springs to a minimum length. We hypothesized that formation of covalent bonds, due to the FA action, can be represented in the model by a simple change in the elastic constant of the springs, while the action of methacholinc (MCh) reduce the equilibrium length of the spring. A sigmoid curve of tension as a function of MCh doses was obtained, showing increased tension when the muscle strip was exposed to FA. Our simulations suggest that FA, at a concentration of 0.1 ppm, can affect the elastic properties of the smooth muscle fibers by a factor of 120%. We also analyze the dynamic mechanical properties, observing the viscous and elastic behavior of the network. Finally, the proposed model, although simple, ir1corporates the phenomenology of both MCh and FA and reproduces experirnental results observed with ir1 vitro exposure of smooth muscle to .FA. Thus, this new mechanical approach incorporates several well know features of the contractile system of the cells ir1 a tissue level model. The model can also be used in different biological scales.CNPqFAPES

Numeric reconstruction of cytoskeleton with finite element method and topology optimization method

The importance of mechanical aspects related to cell activity and its environment is becoming more evident due to their influence in stem cell differentiation and in the development of diseases such as atherosclerosis. The mechanical tension homeostasis is related to normal tissue behavior and its lack may be related to the formation of cancer, which shows a higher mechanical tension. Due to the complexity of cellular activity, the application of simplified models may elucidate which factors are really essential and which have a marginal effect. The development of a systematic method to reconstruct the elements involved in the perception of mechanical aspects by the cell may accelerate substantially the validation of these models. This work proposes the development of a routine capable of reconstructing the topology of focal adhesions and the actomyosin portion of the cytoskeleton from the displacement field generated by the cell on a flexible substrate. Another way to think of this problem is to develop an algorithm to reconstruct the forces applied by the cell from the measurements of the substrate displacement, which would be characterized as an inverse problem. For these kind of problems, the Topology Optimization Method (TOM) is suitable to find a solution. TOM is consisted of an iterative application of an optimization method and an analysis method to obtain an optimal distribution of material in a fixed domain. One way to experimentally obtain the substrate displacement is through Traction Force Microscopy (TFM), which also provides the forces applied by the cell. Along with systematically generating the distributions of focal adhesion and actin-myosin for the validation of simplified models, the algorithm also represents a complementary and more phenomenological approach to TFM. As a first approximation, actin fibers and flexible substrate are represented through two-dimensional linear Finite Element Method. Actin contraction is modeled as an initial stress of the FEM elements. Focal adhesions connecting actin and substrate are represented by springs. The algorithm was applied to data obtained from experiments regarding cytoskeletal prestress and micropatterning, comparing the numerical results to the experimental onesCNPqFAPES

Comparison of physical properties of two exogenous surfactants: new parameter.

The pulmonary surfactant has essential physical properties for normal lung function. The most important property is the surface tension. In this work, it was evaluated the surface tension of two commercial exogenous surfactants used in surfactant replacement therapy, poractant alfa (Curosurf, Chiesi Farmaceuticals, Italy) and beractant (Survanta, Abbott Laboratories, USA) using new parameters. A Langmuir film balance (Minitrough, KSV Instruments, Finland) was used to measure surface tension of poractant alfa and beractant samples. For both samples, we prepared a solution of 1 mg/m dissolved in chloroform (100π`), which was applied over a subphase of milli-Q water (175 ml) in the chamber of the balance. The chamber has two moving barriers that can change its surface area between a maximal value of 112.5 cm 2 , and a minimal value of 22.5 cm 2, defining a balance cycle. Each surfactant had its surface tension evaluated during 20 balance cycles for three times. Four quantities were calculated from the experiment: Minimum Surface Tension (MTS), defined as the surface tension at minimal surface area during the first cycle; Mean Work Cycle (MWC), defined as the mean hysteresis area of the measured surface tension curve of the last 16 balance cycles; Critical Active Surface Area in Compression (CASAC) or in Expansion (CASAE), defined as the maximal chamber area where the surfactant is active on the surface in compression or expansion. The t-test was applied to verify for statistical significance of the results. Comproved with the MST is the same reported in literature, the differences between MWC, CASAC, and CASAE were statistically significant (p<0.001). The MWC, CASAC and CASAE were higher for poractant alfa than for beractant. A higher MWC for poractant alfa means higher elastic recoil of the lung in comparison with beractant. Using a different methodology, our results showed that poractant alfa is probably more effective in a surfactant replacement therapy than beractant due the use of poractant alfa in relation to the use of beractant in preterm infants with Respiratory Distress Syndrome (RDS).CNPqFAPES

Anomalous diffusion evidenced by particle tracking at high frame rates.

Diffusion is a common phenomenon in nature and generally is associated with a system trying to reach a local or a global equilibrium state, as a result of highly irregular individual particle motion. Therefore it is of fundamental importance in physics, chemistry and biology. Particle tracking in complex fluids can reveal important characteristics of its properties. In living cells, we coat the microbead with a peptide (RGD) that binds to integrin receptors at the plasma membrane, which connects to the CSK. This procedure is based on the hypothesis that the microsphere can move only if the structure where it is attached move as well. Then, the observed trajectory of microbeads is a probe of the cytoskeleton (CSK), which is governed by several factors, including thermal diffusion, pressure gradients, and molecular motors. The possibility of separating the trajectories into passive and active diffusion may give information about the viscoelasticity of the cell structure and molecular motors activity. And also we could analyze the motion via generalized Stokes-Einstein relation, avoiding the use of any active techniques. Usually a 12 to 16 Frames Per Second (FPS) system is used to track the microbeads in cell for about 5 minutes. Several factors make this FPS limitation: camera computer communication, light, computer speed for online analysis among others. Here we used a high quality camera and our own software, developed in C++ and Linux, to reach high FPS. Measurements were conducted with samples for 10£ and 20£ objectives. We performed sequentially images with different intervals, all with 2 ¹s exposure. The sequences of intervals are in milliseconds: 4 5 ms (maximum speed) 14, 25, 50 and 100 FPS. Our preliminary results highlight the difference between passive and active diffusion, since the passive diffusion is represented by a Gaussian in the distribution of displacements of the center of mass of individual beads between consecutive frames. However, the active process, or anomalous diffusion, shows as long tails in the distribution of displacements.CNPqFAPES

Design of multi-phase micromechanisms using the topology optimization method.

Um micromecanismo é, essencialmente, um dispositivo de dimensões milimétricas ou até micrométricas que executa uma tarefa específica como atuar como garra, pinça, grampo, etc. Quando acoplados a um sistema eletrônico, são chamados de sistemas microeletromecânicos ou \"Micro-Electro-Mechanical Systems\" (MEMS). Esses dispositivos são quase todos constituídos por mecanismos flexíveis, onde o movimento é dado pela flexibilidade de sua estrutura, sem juntas e pinos. Uma das formas de atuação de micromecanismos é a eletrotermomecânica, onde uma atuação elétrica sobre o próprio mecanismo é convertida em calor, por efeito Joule, que gera tensões térmicas responsáveis pela deformação estrutural desejada. Recentemente, vários grupos de pesquisa no mundo estão desenvolvendo micromecanismos fabricados com dois (ou até mais) materiais, o que permite obter maiores deformações sem que seja excedido o limite de resistência do material e mais flexibilidade no projeto de micromecanismos que realizem diferentes tarefas quando sujeito a diversas atuações (multiflexíveis). As técnicas de processo de fabricação de micromecanismos atingiram um alto nível de maturidade. No entanto, a modelagem e, em particular, o desenvolvimento de métodos computacionais sistemáticos para o projeto estão ainda no seu estágio inicial. Atualmente, o projeto de micromecanismos com vários materiais vem sendo realizado por métodos de tentativa e erro, dependendo da intuição e experiência do projetista. Além disso, o projeto genérico de um MEMS eletrotermomecânico é uma tarefa complexa, que leva em conta conhecimentos multidisciplinares. Dessa forma, o objetivo desse trabalho de mestrado foi desenvolver um software para o projeto de MEMS multifásicos, atuados eletrotermicamente, usando um método de projeto genérico e sistemático, como o Método de Otimização Topológica (MOT). Utilizando um modelo de interpolação de material de função de pico, qualquer número de materiais pode ser considerado sem que haja aumento na quantidade de variáveis de projeto se comparado à otimização com apenas um material e vazio. Visando maximizar o deslocamento de saída contra uma peça de rigidez conhecida, foram projetados mecanismos atuados por tensão elétrica, alguns considerando multiflexibilidade. Um estudo da influência dos parâmetros da otimização foi realizado. Como uma alternativa à atuação eletrotermomecânica, foram projetados mecanismos atuados por fluxo de calor.A micromechanism is essentially a device of milimetric, or even micrometric, dimensions that can actuate as a gripper, tweezers, clamp, etc. When coupled to an electronic system, they are called \"Micro-Electro-Mechanical Systems\" (MEMS). Almost all of these devices are constituted by compliant mechanisms, where the motion is allowed by the compliance of its own structure, rather than the presence of joint and pins. One of the forms of micromechanisms actuation is the electrothermomechanical, where an electric actuation applied to the mechanism is converted in heat, by Joule effect, that generates the thermal stress responsible for the desired structural deformation. Recently, many research groups around the world are developing micromechanisms manufactured with two (or even more) materials, what allows larger displacements without exceeding the materials ultimate tensile strength, and gives more flexibility in the design of micromechanisms that accomplish different tasks when under different actuations (multiflexible mechanisms). The manufacturing process techniques of micromechanisms reached a high level of maturity, however, the modelling and, particularly, the development of systematic computational methods for design are still in early stages. Nowadays, micromechanism design with many materials is being carried on by \"try and error\" methods, depending on designer intuition and experience. Also, a generic design of an electrothermomechanical MEMS is a complex task that needs multidisciplinary knowledge. Thus, the objective of this work is to develop a software for the design of multi-phase MEMS, electrothermomechanically actuated, using a method for systematic and generic design, such as Topology Optimization Method (TOM). Using a peak function material interpolation model, any number of materials can be considered without increasing the amount of design variables if compared to an optimization with only one material and void. Mechanisms actuated by electric tension were designed considering the maximization of output displacement against a work piece with known stiffness. The design of microactuators considering multiflexibility was also performed. A study of optimization parameters influence is presented. As an alternative to electrothermomechanical actuation, some mechanisms actuated by heat flow were designed

Rapid and localized mechanical stimulation and adhesion assay: TRPM7 involvement in calcium signaling and cell adhesion

A cell mechanical stimulation equipment, based on cell substrate deformation, and a more sensitive method for measuring adhesion of cells were developed. A probe, precisely positioned close to the cell, was capable of a vertical localized mechanical stimulation with a temporal frequency of 207 Hz, and strain magnitude of 50%. This setup was characterized and used to probe the response of Human Umbilical Endothelial Vein Cells (HUVECs) in terms of calcium signaling. The intracellular calcium ion concentration was measured by the genetically encoded Cameleon biosensor, with the Transient Receptor Potential cation channel, subfamily M, member 7 (TRPM7) expression inhibited. As TRPM7 expression also regulates adhesion, a relatively simple method for measuring adhesion of cells was also developed, tested and used to study the effect of adhesion alone. Three adhesion conditions of HUVECs on polyacrylamide gel dishes were compared. In the first condition, the substrate is fully treated with Sulfo-SANPAH crosslinking and fibronectin. The other two conditions had increasingly reduced adhesion: partially treated (only coated with fibronectin, with no use of Sulfo-SANPAH, at 5% of the normal amount) and non-treated polyacrylamide gels. The cells showed adhesion and calcium response to the mechanical stimulation correlated to the degree of gel treatment: highest for fully treated gels and lowest for non-treated ones. TRPM7 inhibition by siRNA on HUVECs caused an increase in adhesion relative to control (no siRNA treatment) and non-targeting siRNA, but a decrease to 80% of calcium response relative to non-targeting siRNA which confirms the important role of TRPM7 in mechanotransduction despite the increase in adhesion

Numeric reconstruction of 2D cellular actomyosin network from substrate displacement

Introduction: One of the fundamental structural elements of the cell is the cytoskeleton. Along with myosin, actin microfilaments are responsible for cellular contractions, and their organization may be related to pathological changes in myocardial tissue. Due to the complexity of factors involved, numerical modeling of the cytoskeleton has the potential to contribute to a better understanding of mechanical cues in cellular activities. In this work, a systematic method was developed for the reconstruction of an actomyosin topology based on the displacement exerted by the cell on a flexible substrate. It is an inverse problem which could be considered a phenomenological approach to traction force microscopy (TFM). Methods An actomyosin distribution was found with a topology optimization method (TOM), varying the material density and angle of contraction of each element of the actomyosin domain. The routine was implemented with a linear material model for the bidimensional actomyosin elements and tridimensional substrate. The topology generated minimizes the nodal displacement squared differences between the generated topology and experimental displacement fields obtained by TFM. The structure resulting from TOM was compared to the actin structures observed experimentally with a GFP-attached actin marker. Results The optimized topology reproduced the main features of the experimental actin and its squared displacement differences were 11.24 µm2, 27.5% of the sum of experimental squared nodal displacements (40.87 µm2). Conclusion This approach extends the literature with a model for the actomyosin structure capable of distributing anisotropic material freely, allowing heterogeneous contraction over the cell extension