Esta tese apresenta o estudo do efeito do solvente acetato de etila e acetona no comportamento em solução dos polímeros acetato butirato celulose (CAB) e acetato butirato carboximetil celulose (CMCAB) e nas características dos seus filmes finos obtidos pela técnica de revestimento rotacional ou por adsorção. As medidas de viscosidade e espalhamento de raio-X a baixo ângulo (SAXS) mostraram que o acetato de etila é um melhor solvente para CAB e CMCAB do que a acetona. A caracterização dos filmes foi feita através de medidas de elipsometria, microscopia de força atômica (AFM), espectrocospia vibracional por geração de soma de freqüências (SFG) e medidas de ângulo de contato. Os filmes de CMCAB obtidos por revestimento rotacional são mais espessos quando preparado em acetona do que em acetato de etila. Imagens de AFM mostraram que os filmes de CMCAB oriundos de soluções em acetato de etila são mais homogêneos e lisos do que aqueles preparados a partir de acetona. Medidas de SFG comprovaram a forte afinidade da acetona com SiO2/Si, mostrando que esse solvente cria uma nova camada para os filmes de CAB e CMCAB. Os valores de energia superficial calculados para CAB e CMCAB foram semelhantes ~ (49,0 ± 0,5) mJ/m², sendo a contribuição da componente dispersiva maior que a da componente polar. A adsorção das proteínas lisozima, albumina do soro bovino (BSA), concanavalina A e jacalina foram mais pronunciadas sobre os filmes de CMCAB do que sobre CAB. Indicando que a presença do grupo carboximetil (CM) contribui significativamente no processo de adsorção das biomoléculas. O efeito da rugosidade dos filmes de CAB e CMCAB sobre o processo de adsorção das proteínas foi estudado. No caso do CMCAB, a adsorção das proteínas foi mais pronunciada sobre o filme rugoso do que sobre o filme mais liso. Entretanto, para os filmes de CAB a rugosidade não teve um efeito significativo na adsorção das proteínas / The effect of ethyl acetate and acetone on the solution behavior of cellulose acetate butyrate (CAB) and carboxymehtylcellulose acetate butyrate (CMCAB) and on the characteristics of films obtained either by spin coating or adsorption was investigated. Viscosity and small angle X-ray scattering (SAXS) measurements showed that ethyl acetate is a better solvent than acetone for CAB e CMCAB. Films were characterized by means of ellipsometry, atomic force microscopy (AFM), sum frequency generation (SFG) and contact angle measurements. Spin-coated films of CMCAB from ethyl acetate solutions were thicker than those deposited from acetone solutions. AFM images revealed that CMCAB spin coated films from ethyl acetate solutions were homogeneous and flat. However, films obtained from solutions in acetone were very rough. SFG spectra showed that acetone binds strongly to SiO2/Si wafers, creating a new surface for CAB and CMCAB films. Surface energy values determined for spin-coated CAB and CMCAB were similar ~ (49,0 ± 0,5) mJ/m² with the dispersive component larger than the polar component. The adsorption of lysozyme, bovine serum albumin (BSA), concanavalin A and jacalin was more pronounced onto CMCAB films than that onto CAB films. Indicating that carboxymethyl group favored the adsorption process. The influence of surface roughness of CAB and CMCAB on protein adsorption has been investigated. In the case of CMCAB, protein adsorption was morepronounced onto rough films than that onto flat films. However, the roughness of CAB films exerted no significant influence on proteins adsorption

Esta tese aborda a aplicação de técnicas avançadas de processamento e análise de imagens em problemas originais envolvendo imagens de microscopia de força atômica. Para isso, foi desenvolvida uma série de algoritmos para a caracterização e o entendimento do processo de formação de novos materiais poliméricos com perspectivas de inúmeras aplicações tecnológicas. As análises envolveram a determinação da orientação da morfologia de substratos para alinhamento de cristais líquidos, contagem e estimativa dos raios de domínios em filmes automontados POMA/PVS, análise do aumento da fotoluminescência em filmes PPV e estudos da curvatura espontânea de macromoléculas de polímeros. Dentre os algoritmos principais podemos citar a determinação da inclinação dos autovalores da matriz de covariância das coordenadas dos pontos da forma, aplicação da técnica dos máximos regionais e diagramas de Voronoi, filtros passabanda 2D através da transformada de Fourier e extração da curvatura multiescala. A implementação destes algoritmos envolveu algoritmos básicos de análise de imagens tais como esqueletização, dilatações exatas e extração do contorno de formas. A principal contribuição deste trabalho foi a implementação do software denominado SPIA (Scanning Probe Image Analysis) que possui ferramentas para análise e processamento de imagens incluindo todas as que foram utilizadas no decorrer deste trabalho além de outras ferramentas. Este software foi desenvolvido em ambiente Delphi sob o paradigma da orientação a objetos para plataformas Windows NT/9X/2000/XP. Possui uma interface amigável e semelhante a outros softwares dedicados a processamento de imagens. Todas as técnicas aplicadas foram testadas extensivamente e os resultados que corroboram sua eficiência são mostrados ao longo da tese / This thesis address the application of advanced techniques of processing and analysisof images in original problems involving images of atomic force microscopy. For this, a series of algorithms for characterization and understanding of process of formation of new polymeric materials was developed and implemented, with perspectives of many technological applications. The analysis was applied to the determination of orientation of the morphology of substrates for alignment of liquid crystals, counting and estimative of radiuses of granules in layer-by-layer polymer films, analysis of enhancement of photoluminescence in PPV cast films, as well as the study of curvature spontaneous of macromolecules. The principal algorithms included are determination of inclination of eigenvectors of matrix of covariance of coordinates of points of shape,application of regional maxima technique and Voronoi diagrams, passband filters 2D through Fourier Transform and curvature multiscale. The implementation of these algorithms involved a series of image analysis algorithms such as squeletonization, exact dilations and extracting of contour of shapes. The principal contribution of this work was to develop a software called SPIA (Scanning Probe Image Analysis) that includes tools for analysis of processing of images including that were used in this work. This software was developed in Delphi under object orientation paradigm to Windows NT/9X/2000/XP. It has a friendly interface similar to other image processing softwares. All this techniques were tested extensively and the results that corroborate the robustness of the algorithms are included throughout the thesis

A Na, K-ATPase (NKA) é uma proteína de membrana encontrada em organismos eucariotos multicelulares cuja atividade e funções já são amplamente discutidas na literatura. Sua unidade funcional corresponde a um heterodímero formado por duas subunidades , com regiões transmembrana. Espécies multiméricas como dímeros e tetrâmeros dessa enzima também são conhecidos por exercer atividade enzimática. As interações lipídio-proteína são intrínsecas para a NKA, por tal motivo, proteolipossomos constituídos de DPPC e DPPC:DPPE foram preparados por co-solubilização. Como controle, lipossomos de mesma composição foram produzidos por extrusão e/ou sonicação. Para as imagens de AFM, as amostras foram fixadas com glutaraldeído, para proteção mecânica e contra desidratação das vesículas. Para lipossomos de DPPC as imagens topográficas de AFM das vesículas apresentaram formato oval, superfície perfeitamente lisa e diâmetro médio de 151 + 46 nm, enquanto as vesículas de composição DPPC:DPPE, apesar de lisas, tiveram cantos pontiagudos e diâmetro médio de 98 + 28 nm. Imagens de fase de ambas as composições não apresentaram qualquer indicativo de diferenças na composição química, provavelmente devido à natureza de carga neutra dos dois fosfolipídios. As imagens de fase por AFM para os proteolipossomos tanto de DPPC-NKA, quanto DPPC:DPPE-NKA, revelaram resultados inéditos na literatura, onde a inserção da NKA aparece como nítidas regiões transições de fase de composição química distinta quando comparadas com os lipossomos. No entanto, as mudanças de fase são diferentes entre as composições estudadas, aparecendo como manchas escuras circulares para DPPC-NKA e mais visíveis como interstícios brilhantes para composição de DPPC:DPPE-NKA. As vesículas de DPPC-NKA apresentaram diâmetro médio de 390 + 326 nm e, nas imagens de topografia tridimensionais, protusões de 38 a 115 nm correspondentes às regiões de mudanças de fase, que, indicaram o diâmetro dos microdomínios relacionados à proteína. Já nas imagens para DPPC:DPPE-NKA o diâmetro médio dos proteolipossomos foi de 189 + 156 nm, e as protusões apareceram entre os interstícios, variando de 20 a 66 nm. O estudo de DSC dos lipossomos revelou que a concentração de glutaraldeído nas condições das análises de AFM, em torno de 5% (v/v), afetam as características físico-químicas para as composições com DPPE. A AFM foi eficiente para confirmar a reinserção da NKA em proteolipossomos pelas imagens de fase, e, para medir o diâmetro dos microdomínios pelas imagens de topografia. / Na, K-ATPase (NKA) is a membrane protein present in eukaryotic multicellular organisms. Its functions and activity are already widely described in the literature. Its minimal functional structure is a heterodimer of two main subunits , with transmembrane domains. However, dimers and tetramers of the enzyme are also known to have enzymatic activity. Since there are intrinsic lipid-protein interactions, NKA proteoliposomes composed of DPPC and DPPC:DPPE (1:1 molar ratio) were prepared by the co-solubilization method and liposomes of the same compositions were obtained by extrusion and/or sonication to be used as control. The samples to the AFM study were prepared using glutaraldehyde to protect the vesicles from mechanical shocks and dehydration. Liposomes composed of DPPC and DPPC:DPPE (1:1 molar ratio) were prepared by extrusion and sonication, respectively, as control. The topographical images for DPPC liposomes showed vesicles with an oval shape and smoothed surfaces with a mean diameter of 151 + 46 nm. DPPC:DPPE vesicles also presented smoothed surfaces, but with pointed corners and mean diameter of 98 + 28 nm. Phase images for both lipid compositions showed no differences in chemical composition. For DPPC:DPPE samples, this can be explained by the neutral net charge of both lipids. The proteoliposomes observed in the AFM phase images showed darker and large circular spots in the vesicles. These spots represent delays in the phase oscillation of the AFM probe and are associated with different chemical composition. The phase changes showed the reconstitution of the NKA in the proteoliposomes. When compared with topographical images, this spots matched protrusions. The mean diameter of DPPC-NKA proteoliposomes determined by AFM was 390 + 326 nm. In the three-dimensional topographical images of composition, protrusions from 38 to 115 nm near the areas of different phases indicate the diameters of the NKA microdomains. The phase changes for DPPC:DPPE-NKA appeared as bright interstices with the protrusions of the topographical images in between them. The size of these protrusions ranged from 20 to 66 nm and the mean diameter of the proteoliposomes was 189 + 156 nm. The DSC liposomes data showed that the glutaraldehyde concentration used in the AFM analysis affect the physical chemistry properties of the samples with DPPE. AFM proved to be an efficient method to confirm the reconstitution of into proteoliposomes with phase images and to determine the diameter of the protein microdomains with the topographical images.

[pt] Neste trabalho foi estudada a deformação mecânica em semicondutores III-V resultante da nanolitografia por microscopia de força atômica (AFM). O AFM, equipado com uma ponta de diamante de raio de curvatura de 80 nm, foi usado para riscar a superfície do InP com forças da ordem de dezenas de mN ao longo de direções cristalográficas específicas. O padrão litografado na superfície foi caracterizado com o uso do AFM, enquanto uma análise da microestrutura do material foi feita com o uso da microscopia eletrônica de transmissão (MET). Foi realizado um estudo da deformação mecânica ao riscarmos o InP (100) com o uso do AFM utilizando forças normais variando de 7 uN a 120 uN e em direções cristalográficas das famílias <110> e <100>. Foi visto por MET, que é mais fácil produzir deformação plástica para riscos feitos na direção <110> do que na direção <100>, o que associamos à diferença na orientação dos vetores de Burgers ativados para os planos de escorregamento do InP para riscos ao longo das diferentes direções. Foi realizado também um estudo da influência da distância entre dois riscos consecutivos, feitos com o uso do AFM com força normal de 30 uN, no endurecimento por deformação plástica. Um significante endurecimento foi observado para distâncias entre riscos menores que 80 nm indicando que ocorre travamento entre discordâncias geradas por sucessivos riscos a distâncias menores que 80 nm. / [en] In this work, the mechanical deformation of III-V semiconductors resulting from atomic force microscopy (AFM) nanolithography was studied. The AFM, equipped with a diamond tip with 80nm radius, was used to scratch the InP surface with forces in the order of tens of mN along specific crystallographic directions. The pattern lithographed at the surface was characterized by AFM, while the material microstructure analyzes was performed by transmission electron microscopy (TEM). We studied the mechanical deformation of InP (100) produced by the AFM with forces in the range of 7uN to 120 uN along directions from the <110> and <100> families. It was observed by TEM that, it is easier to produce plastic deformation for scratches along the <110> than along the <100> directions, which was associated to the different orientations of the Burgers vectors activated for the InP slip planes for the scratches along the different directions. The influence of the distance between two scratches, performed with a normal force of 30 uN on the materials hardening process was performed as well. Significant hardening was observed at distances between scratches of 80nm or less suggesting that locking due to dislocation interaction is occurring at parallel scratches at distances smaller than 80nm.

Cell motility has been found to play a role in many important body functions as well as the embryogenenis of mulitcellular organisms like vertebrates. From a physics point of view the interesting questions behind every motion are: Why is it moving? Where do the forces come from? Today we know that the motility of many cells is dependent on an active polymer network. Actin, one of the most abundant proteins in the body, is constantly polymerized, being moved around and depolymerized in motile cells. Until now, only forces outside the cell like traction forces could be measured. The direct measurement of the force generated by polymerizing actin filaments has only been measured by our lab and the lab of M. Radmacher. In these measurements fish keratocytes were used. Whereas I did these experiments, for the first time, on mammalian cells. To measure forward forces on neuronal growth cones I stabilized the SFM, as measurement times went up from minutes to hours. Furthermore measurements had to be performed at 37°C instead of room temerature, this induced drifts of the substrate. I incorporated an optical trap into the microscope to track the motion of the substrate. A feedback loop moved the SFM cantilever to minimize relative motion of substrate and cantilever. For keratocytes I directly measured the forces produced by actin polymerization and, to my knowledge for the first time, the forces associated with the retrograde actin flow using a SFM. The result was that both actin and myosin play important but different roles in motility. For actin it turned out that considering just the polymerization was not enough. Actin depolymerization and the resulting entropic forces are a completely new physical effect in actin based cell motility. With this new force in the force balance I can explain all effects observed in my experiments without introducing any new biochemical feedback loops. Finally I showed that neuronal growth cones are very soft and weak structures. They are at least one order of magnitude softer and weaker as for example fibroblasts or cells forming the blood vessel walls. As neurons are usually located in soft environments this does not impede their normal outgrowth. It could even serve as a safety mechanism that prevents cell from growing into wrong areas like breaching the blood-brain-barrier, on a physical level. For a neuron the wall of a blood vessel feels like a brick wall for us.

The cellular membrane is a complex biological entity, far from being an inert assembly of protein and lipids which separates cells from the surrounding environment. A multitude of biological processes, ranging from active transport of ions into and out of the cell, to the immune response, are regulated at the level of the plasma membrane. The understanding of their molecular basis is among the central goals of modern biological research. In order to dissect the complexity of actual cell membranes, which involves a very complex network of intermolecular interactions, a “divide and conquer” strategy proves very useful. To this end, researchers try to isolate molecules from complex biological contexts to understand their function in simple model systems under controlled conditions. A variety of model membranes have thus been developed in order to gain insight into membrane processes. This approach has resulted in a deeper knowledge on how lipids and proteins interact and how these interactions govern the function of cellular membranes. In the recent past in fact, a connection has been established between the lateral structure of the plasma membrane and its biological function. Furthermore, a large range of biophysical techniques have been used to characterize protein-lipid microdomains. For example, atomic force microscopy (AFM) is a powerful technique which allows a highly detailed topographical characterization of lipid domains in physiological conditions. While AFM imaging offers an extremely high spatial resolution, up to the nanometer scale, the limited image acquisition speed (minutes) can pose a severe drawback in adequately studying fast dynamic processes. On the other hand, fluorescence based imaging techniques are much faster (10-3-100 s), but certainly lack the high spatial resolution that AFM offers. FCS in particular can also provide information about dynamic processes, like diffusion of fluorescent membrane components. For these reasons, implementing a combination of the above mentioned techniques on the same sample (e.g. cell membrane models) would prove extremely beneficial in the complete dynamic and structural characterization of molecular interactions. . The work described in this thesis can be summarized in two main points: i) the development of a novel combined approach of atomic force microscopy (AFM), laser scanning imaging (LSM), and fluorescence correlation spectroscopy (FCS) and ii) the study of the effects of ceramide in the lateral organization of model plasma membranes. We described one of the first simultaneous applications of AFM and FCS on biologically relevant systems. More specifically, model membranes showing complex phase separation were investigated with a combined approach of AFM, confocal fluorescence imaging, force measurements and FCS, based on commercially available instruments. AFM conveys information about the structural and mechanical properties of the different lipid phases. Different membrane domains can be distinguished based on height difference, elastic properties and line tension as measured by the AFM tip. Simultaneous optical measurements offer the correlation of these data in real time with the partition behavior and diffusion of fluorescent lipids and proteins. We established a clear link between the local membrane viscosity, probed by FCS, and the lipid-lipid interactions involved in line tension, probed by AFM force measurements. An example of a significant drawback circumvented by the AFM-FCS approach involves the use of AFM micromanipulation to eliminate unwanted interactions between lipid particles — similar to intra-cellular vesicles found in vivo experiments — and the membrane, which usually result in distorted FCS autocorrelation curves. Finally, the combined application of AFM and FCS on membrane-anchored proteins reconstituted in lipid bilayers has been instrumental in clarifying inconsistencies that arose in work that focused solely on either AFM or fluorescence techniques. We have shown that, in the case of proteins diffusing in the plane of the membrane, AFM can unambiguously detect only a small immobile fraction. Furthermore, since AFM detection of proteins might be facilitated by high local membrane viscosity (e.g. in ordered lipid phases), the measurement of protein partition between disordered and ordered membrane domains might be biased toward the latter. In this case, the use of FCS as a complementary technique allows a more thorough investigation and deeper understanding of the system of interest. The second part of this thesis dealt with the study of complex lipid mixtures which are used to model the putative lipid/proteins domains in cells, called “rafts”. Firstly, we proved how the combined fluorescence imaging/AFM approach is useful in general for studying supported lipid membranes and the role of lipid domains in biological contexts. We investigated the effect of environmental stress on biological membranes and the protective effects of several substances. Our experimental approach was shown to be a new valuable method to visualize the dehydration damage and its effects on the lateral organization of lipid domains. Our results demonstrated that disaccharides like trehalose or sucrose are effective in protecting lipid membranes, not only on a macroscopic scale — preserving the overall integrity of the bilayer — but also on a microscopic scale, preventing the clustering of microdomains. These phenomena are interesting in the context of biological damage to living cells which need to be stored for long time, like organs to be transplanted or blood platelets. Finally, a large section of this thesis focused on the effects of a specific lipid called “ceramide” on the lateral organization of proteins and lipids in the plasma membrane. Ceramide is produced by cells in several situations, like bacterial infections or apoptosis. As consequence of ceramide production in vivo, the local concentration and the dynamic behavior of lipids and membrane receptors are supposed to exhibit strong variations. In order to understand the molecular mechanisms responsible for these effects, we applied a combination of AFM, FCS and fluorescence imaging on simple model membranes containing ceramide. We could show for the first time that, in presence of raft-like Lo/Ld phase separation, physiological quantities of ceramide induced the formation of a highly ordered gel phase, constituted of ceramide and sphingomyelin. The enzymatic production of ceramide was monitored both in supported and in free-standing bilayers. In the second case, ceramide production was connected to selective vesicle budding from the raft-like phase. Since short-chain analogues are often used in both medical applications and biochemical research to mimic the effect of long-chain ceramides, we investigated the effect of chain-length on ceramide-induced membrane reorganization. We could show that only long-chain ceramides (C18 and C16) form highly ordered domains. Interestingly, FCS measurements indicated that the physical properties of the Lo raft-like domains are hardly affected by the presence of ceramide domains. Furthermore, the increased thickness of the Ld phase — as measured by AFM — and its higher viscosity — as measured by FCS — strongly support the hypothesis of ceramide-induced cholesterol displacement from rafts. On the other hand, short-chain ceramides showed completely different biophysical properties that lead to a destabilization of the raft domains, possibly acting as surfactants between the different lipid phases. Our findings contribute to the explanation of in vivo experiments where short-chain ceramides inhibit cell signaling by disrupting the lipid order in the plasma membrane. We have so far demonstrated that ceramide plays a fundamental role in lipid-lipid interactions. In a physiological context, it is also known to produce dramatic effects in living cells. Since a majority of the processes in vivo are thought to be governed by the activity of proteins, it is highly likely that ceramide not only affects lipid organization but also modifies protein-protein and protein-lipid interactions to produce its effects. To test this hypothesis, we reconstituted several membrane proteins in lipid bilayers containing Ld, Lo, and ceramide-rich domains. We were able to show that some membrane proteins are sorted into ceramide-rich domains. More specifically, the raft-associated proteins we tested were enriched in the highly ordered ceramide-rich domains, while the Ld-associated components were excluded from them. Furthermore, the inclusion of any membrane component in ceramide-rich domains is directly connected to a dramatic reduction of its in-plane diffusion. In an in vivo context, such a reorganization of membrane receptors might be used by the cell to alter the signaling process, for example, by i) separating raft receptors from inhibitors with lower raft affinity, ii) bringing both raft-associated receptors and raft-associated signaling molecules into contact, or iii) stabilizing the interactions between a receptor and its ligand by decreasing their diffusion coefficients. In conclusion, this thesis describes a novel combination of AFM, LSM, and FCS for the investigation of the lateral organization of biological membranes. Our results show that this approach applied on model membranes of increasing complexity is an effective tool for understanding the molecular mechanisms behind the organization of biological membranes. This report opens up new possibilities for further investigation in living cell membranes using the same methodology we have described.

La métastase des cellules cancéreuses est un processus caractérisé par un comportement de la cellule anormal. Les propriétés mécaniques particulières des cellules cancéreuses est l'une des caractéristiques pathologiques principales. Ces propriétés sont liées à leurs capacités à envahir les tissus avoisinants, à transmigrer et à proliférer vers de nouveaux sites. La progression du cancer est caractérisée par la perturbation et la réorganisation du cytosquelette d'actine ainsi que par des changements des propriétés mécaniques des cellules, probablement liés à la capacité dupliquée des cellules cancéreuses à migrer et à s'adapter à l'environnement.Les propriétés mécaniques sont essentielles pour la régulation des fonctions cellulaires comme la migration, l'adhésion, la prolifération, et la différentiation, et les anomalies sont associées aux pathologies, en particulier le cancer. Les propriétés mécaniques sont aussi dépendantes du micro-environnement de la cellule, et la rigidité des substrats modifie les propriétés mécaniques internes des cellules, ainsi que la structure du cytosquelette. Ainsi, comprendre les processus impliqués dans les variations des propriétés viscoélastiques est essentiel pour l'étude de la progression des tumeurs.La microscopie à force atomique (AFM) a prouvé être un outil fiable pour sonder les propriétés mécaniques statiques et dynamiques (sur de grande gamme de fréquence) de matériaux mous, comme les spécimens biologiques, à de petites échelles et grande résolution.Dans cette étude, nous proposons de nouveaux marqueurs du cancer basé sur une approche mécanobiologique. Les propriétés viscoélastiques de cellules cancéreuses de la vessie sont mesurées par des expériences d'indentation dynamiques par AFM. Cette méthode est validée en utilisant des gels de polyacrylamide et un modèle à fractions dérivées est proposé pour décrire le comportement mécanique de ces gels. Ensuite, le module de cisaillement complexe de trois lignées cellulaires à potentiel métastatique différent est mesuré à trois positions différentes de la cellule: le noyau, le périnoyau et la périphérie de la cellule. En utilisant des drogues d'inhibition de l'actine, les propriétés mécaniques sont corrélées à la microstructure de l'actine obtenue par microscopie confocale. Nous proposons un modèle simplifié pour décrire le comportement des modules élastiques G' et visqueux G''. Une relation entre l'invasivité des cellules cancéreuses et leur propriétés mécaniques est aussi mis en avant. En particulier, nous trouvons que le plateau élastique et la fréquence de transition (quand G'=G'') peuvent être utilisés comme marqueurs d'invasivité. Enfin, nous mesurons le module de cisaillement complexe de cellules cancéreuses adhérentes sur des environnements mécaniques et biologiques différents, et des propriétés intrigantes de la périphérie des cellules cancéreuses sont reportées. / Cancer cell metastasis is a multi-stage process characterized by cell malfunctional behavior. Some of the major pathological characteristics of cancer cells are their particular mechanical properties which are linked to their ability to invade surrounding tissues, transmigrate and proliferate at new sites. There are evidences that cancer progression is characterized by disruption and reorganization of the actin cytoskeleton as well as changes in the mechanical properties. This change is probably associated with the enhanced capability of cancer cells to migrate and adapt to changing environments.The mechanical properties are essential for the regulation of cell functions like migration, adhesion, proliferation and differentiation, and abnormalities are connected with pathologies, in particular cancer metastasis. The mechanical properties are also dependent on the micro-environment of the cell, as substrate stiffness changes cell internal mechanical properties, as well as the cytoskeleton structure. Thus, the understanding of the mechanics involved in the variation of the viscoelastic properties is crucial for the study of tumor progression.Atomic force microscopy (AFM) has proved to be a reliable tool to probe static and frequency–dependent mechanical properties of soft materials, like biological specimens, at small scale with high resolution.In this study, we propose new markers of cancer metastasis based on a cell mechanics approach. We report on the viscoelastic properties of human bladder cancer cells measured by dynamic indentation experiments using AFM. This method is first calibrated using polyacrylamide gels and a fractional model is proposed to describe the behavior of such gels. We then investigate the complex shear modulus of three different cell lines with different metastatic potential. We probe the elastic G' and viscous G'' modulus at three different locations across the cell: nucleus, perinucleus and the cell periphery. With the use of actin inhibitory drugs, we correlate mechanical properties and the actin microstructure obtained by confocal microscopy imaging. We propose a simplified power-law model to describe the behavior of the elastic and viscous moduli. We also report a relationship between the malignancy of cancer cells and their viscoelastic properties. In particular, we find that the elastic plateau modulus and the transition frequency (frequency at which G' = G'' ) can be used as markers of invasiveness. Then, we probe the complex shear modulus of cancer cells on different mechanical and biological environments and we report intriguing properties of the periphery of cancer cells.

La culture du blé est l’une des plus importantes au monde. Le grain de blé est un matériau composite naturel dont la majeure partie est constituée d’albumen amylacé formé d’un assemblage compact de granules d’amidons (glucides) enchâssés dans une matrice protéique (gluten). Pour obtenir des produits comme la farine, la structure de l'albumen doit être fragmentée en broyant les grains sous des fortes contraintes. La quantité et la qualité des produits obtenus dépendent du comportement de l’albumen à la fragmentation. En raison de sa nature composite, le comportement rhéologique du grain est tributaires des propriétés mécaniques des phases qui le composent (granules, gluten, pores), de leurs interactions, ainsi que de leur distribution spatiale. Les granules d’amidons sont de formes relativement sphériques et de tailles micrométriques, tandis que les protéines sont organisées en un réseau entourant les granules. L'interaction entre l'amidon et ce réseau protéique est influencée par certaines protéines, les puroindolines, dont la présence et le type d’allèle sont contrôlées génétiquement. Si les gènes codant pour les puroindolines sont présents sous forme sauvages, la dureté meunière, c’est à dire l’aptitude à la fragmentation du grain est faible. L’origine de ce comportement est liée à une adhérence limitée entre matrice protéique et amidon. L'absence totale de puroindolines chez le blé dur conduit au contraire à une dureté très élevée des grains et à une forte adhérence. L'objectif de cette thèse est d'étudier, à partir d’une approche multidisciplinaire, la biomécanique du fractionnement du grain de blé en mettant l'accent sur le rôle des granules d'amidon. Des échelles de taille différentes sont considérées : échelle micrométrique du granule et de la matrice protéique; agencement complexe de ces composants dans l'albumen et échelle millimétrique du grain. Ainsi, des expériences de broyage à l'échelle du grain ont été combinées avec des mesures nano-mécaniques par microscopie à force atomique (AFM) et des simulations numériques.Le comportement au broyage a été étudié en utilisant un micro-moulin instrumenté. Une comparaison a été effectuée entre des essais réalisés sur une variété de blé dur et sur la même variété dans laquelle ont été introduits les gènes codant pour les puroindolines. Un changement significatif du comportement mécanique des grains transformés, attribuable uniquement à la présence de puroindolines, a été observé - en termes d'énergie consommée, - de productivité en farine et - de taux d'amidon endommagé. Ces changements sont compatibles avec l'hypothèse d'une faible adhérence, entre granules d'amidon et matrice protéique, induite par la présence des puroindolines et montrent l'effet significatif de celles-ci sur le comportement à la fragmentation. Ces modifications de comportement mécanique peuvent être étudiées par des mesures AFM nano-mécaniques. Pour compléter des travaux antérieurs ayant permis la mesure des propriétés de l'amidon et du gluten, une méthode basée sur des mesures AFM en mode résonance de contact (CR-AFM) a été développée. Celle-ci permet de cartographier les propriétés directement à l'intérieur des granules d’amidon et prend en compte à travers un modèle théorique les variations importantes de topographie observées dans les sections de grains. Ces études CR-AFM de l'albumen ont ensuite porté sur les propriétés mécaniques des granules d'amidon d'origines botaniques différentes (céréales et légumineuses).Enfin, le rôle de la distribution bimodale en taille des granules d'amidon sur la fragmentation de l'albumen a été précisé à partir d’une étude numérique paramétrique détaillée. Les propriétés mécaniques élastiques et à la rupture ont été analysées en détail, ainsi que le rôle dominant de la ténacité des granules et de l'adhérence à l'interface sur l’endommagement de l’amidon. / The wheat grain is a natural composite material of worldwide importance. The major part of the grain is the starchy endosperm. To obtain food products, such as flour, the endosperm’s compact structure needs to be disintegrated, which is achieved by milling the grains under high forces. The quantity and quality of the milling products notably depend on the fragmentation behaviour of the endosperm.Due to the endosperm’s composite nature, this behaviour depends strongly on the mechanical properties of its components and their interaction. The main components of the endosperm are carbohydrates and proteins. The carbohydrates are deposited as starch in the form of granules of micro-meter size, whereas proteins form a network (gluten), which surrounds the starch granules. The interactions between starch and proteins is believed to be influenced by certain non-gluten proteins (puroindolines), whose presence and allelic state are genetically controlled. If puroindoline genes are present in the wild-type form, grain hardness is low, which have been related to low starch-protein adhesion. The complete absence of puroindolines in the durum wheat species leads to very high grain hardness and indicates a strong adhesion.The aim of this thesis was to investigate the biomechanics of wheat grain fractionation with a focus on the role of the starch granules therein, which was pursued with a multi-disciplinary approach. Different size scales were considered, from the micro meter-sized structures of starch and protein, the complexity of their arrangement in the endosperm, up to the millimeter-sized grains. In this work, grain-scale milling experiments were combined with nano-mechanical measurements by atomic force microscopy (AFM) and numerical simulations.The milling behaviour of a transgenic durum wheat line, which contained puroindoline genes, was determined by grain scale milling experiments and compared to the milling behavior of non-modified durum wheat. A significant change of milling behavior of the transformed durum wheat grains was observed in terms of milling energy, flour yield and starch damage, which was solely attributable to the presence of puroindolines. The observed changes were consistent with the hypothesis of a lower adhesion between starch granules and protein matrix due to the presence of puroindolines and confirmed the significant effect of puroindolines on the fragmentation behaviour, independent of the grain’s genetic background.The change of fragmentation behaviour is a result of modifications of the mechanical properties of the endosperm’s components and/ or their interaction. Such modifications can be investigated by AFM nano-mechanical measurements. Based on previous work illuminating the global nano-mechanical properties of starch and gluten, contact-resonance AFM (CR-AFM) was applied to obtain maps of the nano-mechanical properties inside the grains. Due to the high topography variations of grain section surfaces and the non-trivial correlation between surface slope and contact resonance-frequency, which hindered a straight-forward interpretation of CR-AFM measurements, a practical method based on existing analytical models of the cantilever vibration was developed to correct the measurements. CR-AFM studies of the endosperm were then focused specifically on the mechanical properties of starch granules and the link to starch structure, and applied to the study of starches from wheat in comparison to plants from different botanical origin (other cereals and legumes).Finally, the role of starch granules, their size distribution, and mechanical properties on endosperm fragmentation was analysed by parametric numerical studies. The influence of the bi-modal size distribution of granules on the mesoscale mechanical properties was shown, as well as the governing role of granule toughness and interface adhesion on the granule damage.

Les ELISA (Enzyme-Linked ImmunoSorbent Assay) sont une des technologies analytiques les plus utilisées dans la recherche et les applications biomédicales. Leur production nécessite la construction de films d’anticorps sur des surfaces constituées le plus souvent de polystyrène. La haute hydrophobie du polystyrène assure une adhésion forte et spontanée des anticorps permettant ainsi d’y construire facilement une monocouche d’anticorps. L’amélioration des ELISA passe certainement par l’amélioration et la compréhension des mécanismes physico-chimiques à l’œuvre lors de l’immobilisation des anticorps sur le polystyrène. Dans ce but, ce travail présente un essai de théorisation appuyé par des simulations numériques et des estimations expérimentales par microscopie à force atomique (AFM) et ELISA. En faisant référence à l’effet hydrophobe, la thermodynamique des processus irréversibles permet premièrement d’expliciter les raisons de l’adhésion des anticorps sur le polystyrène. Deuxièmement, des simulations numériques dans le cadre du modèle des additions séquentielles aléatoires (RSA) montrent la façon dont peuvent se saturer les surfaces en favorisant certaines orientations d’anticorps recherchées dans le cadre de l’ELISA. Finalement, l’amélioration du modèle RSA en un modèle RSA+R tenant compte des changements d’orientations par relaxation des anticorps illustre le lien entre les conditions de dépôt et la structure de la monocouche obtenue. Ces éléments semblent corroborés par l’expérience / ELISA (Enzyme-Linked ImmunoSorbent Assay) are widely used analytical technologies in research and biomedical fields. Their implementation require to build antibodies thin films onto predominantly composed polystyrene surfaces. The high hydrophobicity of polystyrene ensures spontaneous and strong antibodies adhesion allowing to easily build antibodies monolayers. ELISA improvements lie most probably throughout improvements and comprehension of physico-chemical mecanisms on which antibodies immobilization on polystyrene are relied. In this way, our work explains a therozation essay emphasized by numerical modelling and experimental estimations by atomic force microscopy (AFM) and ELISA. Keeping in mind the so-called hydrophobic effect, thermodynamics of irrversible processes allows in a first time explaining reasons of antibodies adhesion on polystyrene. In a second time, numerical modelling in the field of random sequential additions model (RSA) show a way of surfaces saturation involving a strong trend to favor some antibodies orientations expected for ELISA. Finally, a RSA improvement in a RSA+R model taking into account orientational changes by the way of relaxation shows a link between deposition conditions and obtained monolayer structure. Such results seem to be strongly correlated with experimental facts

Les arbres irriguent leurs organes par un système microfluidique complexe qui permet le transfert d’eau sous des pressions hydrostatiques négatives allant de -0.1 à -13 MPa. Dans ces conditions de métastabilité, les arbres vivent avec le risque d’une vaporisation soudaine de leur sève, qui conduit à l’embolie. Après cavitation, la poche de gaz créée croît ensuite par différence de pression et diffuse dans les vaisseaux du xylème à travers des membranes poreuses appelées “ponctuations”. Ces membranes permettent d’assurer le transfert hydrique vasculaire et de lutter contre la propagation de l’embolie gazeuse pouvant conduire à un état létal de la plante. Pour comprendre les mécanismes d’embolie d’air dans le xylème, nous avons pu étudier la structure des ponctuations du peuplier et caractériser par microscopie à force atomique (AFM) leurs propriétés mécaniques. Nous avons fait des expériences de nano-indentation et de flexion sur des échantillons secs et saturés en eau. Les premières expériences semblent montrer que les propriétés mécaniques sont peu affectées par la sorption d’eau (le module d’Young de la membrane primaire est E 0:40 GPa). Nous avons pu établir que le module des ponctuations était inférieur à celui de la paroi vaisseaux du xylème (E 8 GPa). Des expériences de micromoulages ont montré que les membranes des ponctuations se déforment sous l’effet d’une différence de pression. En tenant compte de la présence des parois secondaires, l’analyse par éléments finis de la déformation des ponctuations permet de calculer le module d’Young des membranes qui est identique à celui déterminé en AFM. Nous avons pu mettre en évidence que les mâchoires constituées par les parois secondaires limitent la déformation de la membrane au niveau de l’encastrement près du bord. Des expériences d’injection d’air ont permis de déterminer la pression critique (Pc = 1:8 MPa) et les diamètres critiques des pores présents sur les valves capillaires (dpore 160 nm). La taille des pores estimée est cohérente avec les données de la littérature. Nous avons proposé une première modélisation de la propagation d’une embolie sur la base de l’écoulement de Darcy dans la membrane. L’ensemble des résultats semble montrer que la diffusion de gaz est rendu possible par l’effet conjoint de la déformation de la membrane qui génère l’ouverture des pores diminuant ainsi la pression critique de passage du gaz et de la rupture des ponts capillaires présents dans les pores de la membrane. / The xylem vessels of trees constitute a model natural microfluidic system. In this work, we have studied the mechanism of air flow in the Populus xylem. The vessel microstructure was characterized by optical microscopy, transmission electronic microscopy (TEM), and atomic force microscopy (AFM) at different length scales. The xylem vessels have length ≈ 15 cm and diameter ≈ 20 μm. Flow from one vessel to the next occurs through ∼ 10 2 pits, which are grouped together at the ends of the vessels. The pits contain a thin, porous pit membrane with a thickness of 310 nm. We have measured the Young’s moduli of the vessel wall and of the pits (both water-saturated and after drying) by specific nanoindentation and nanoflexion experiments with AFM. We found that both the dried and water-saturated pit membranes have Young’s modulus around 0.4 MPa, in agreement with values obtained by micromolding of pits deformed by an applied pressure difference. Air injection experiments reveal that air flows through the xylem vessels when the differential pressure across a sample is larger than a critical value ∆P c ≈ 1.8 MPa. In order to model the air flow rate for ∆P ≥ ∆P c , we assumed the pit membrane to be a porous medium that is strained by the applied pressure difference. Water menisci in the pit pores play the role of capillary valves, which open at ∆P = ∆P c . From the point of view of the plant physiology, this work presents a basic understanding of the physics of bordered pits.