Cette thèse interdisciplinaire a été dédiée à la caractérisation des propriétés mécaniques de myoblastes (murins et humains) et de myotubes (murins) à l'aide de la microscopie à force atomique (AFM). En modifiant ou en inhibant la dynamique du cytosquelette (CSK) d’actine de ces cellules, nous avons pu montrer que ces propriétés mécaniques variaient. L’enregistrement de courbes de force indentation nous a permis de montrer que la présence de cellules adhérentes introduisait sur les leviers d’AFM un amortissement visqueux supplémentaire à celui d’une paroi solide, et que cet amortissement visqueux dépendait de sa vitesse d’approche et que celui-ci restait non négligeable pour les plus faibles vitesses (1μm/s). Nous avons observé que les propriétés mécaniques des précurseurs de muscles devenaient non linéaires (comportement plastiques) pour des grandes déformations (>1μm) et qu’elles dépendaient de l’état, du type de cellule et de leur environnement. En combinant des expériences d’AFM, des modèles visco-élastiques et des méthodes d'analyse multi-échelle basées sur la transformation en ondelettes, nous avons illustré la variabilité des réponses mécaniques de ces cellules (de visco-élastiques à visco-plastiques). À l'aide de courbes de force-indentation, de l’imagerie morpho-structurale (DIC, microscopie à fluorescence) et de traitements pharmacologiques, nous avons éclairé le rôle essentiel des processus actifs (dépendants de l’ATP) dans la mécanique de myoblastes, en discutant tout particulièrement ceux des moteurs moléculaires (myosine II) couplés aux filaments d’actine. En particulier, nous avons montré que les fibres de stress du cytosquelette d’actine situées autour du noyau pouvaient présenter des évènements de remodelage soudains (ruptures) et que ces ruptures étaient une mesure indirecte de l’aptitude de ces cellules à tendre leur CSK. Nous avons enfin montré qu’il était possible de généraliser cette approche à des cas cliniques humains, en l’occurrence des myoblastes primaires de porteurs sains et de patients atteints de dystrophie musculaire de Duchenne, ouvrant la voie à des études plus larges sur d’autres types cellulaires et pathologies. / This interdisciplinary thesis was dedicated to the atomic force microscopy (AFM) characterization of the mechanical properties of myoblasts (murine and human) and myotubes (murine). We reported that the mechanical properties of these cells were modified when their actin cytoskeleton (CSK) dynamics was inhibited or altered. Recording single AFM force indentation curves, we showed that adherent layers of myoblasts and myotubes introduced on the AFM cantilever an extra hydrodynamic drag as compared to a solid wall. This phenomenon was dependent on the cantilever scan speed and not negligible even at low scan velocities (1μm/s). We observed that the mechanical properties of the muscle precursor cells became non-linear (plastic behaviour) for large local deformations (>1μm) and that they varied depending on the state, type and environment of the cells. Combining AFM experiments, viscoelastic modeling and multi-scale analyzing methods based on the wavelet transform, we illustrated the variability of the mechanical responses of these cells (from viscoelastic to viscoplastic). Through AFM force indentation curves analysis, morpho-structural imaging (DIC, fluorescence microscopy) and pharmacological treatments, we enlightened the important role of active (ATP-dependent) processes in myoblast mechanics, focusing especially on those related to the molecular motors (myosin II) coupled to the actin filaments. In particular, we showed that the perinuclear actin stress fibers could exhibit some abrupt remodelling events (ruptures), which are characteristic of the ability of these cells to tense their CSK. Finally, we showed that this approach can be generalized to some human clinical cases, namely primary human myoblasts from healthy donors and patients affected by Duchenne muscular dystrophy, paving the way for broader studies on different cell types and diseases.

II existe de nombreuses études mettant en relation le vieillissement et le stress oxydant, mais peu d'entre elles ont caractérisé l'évolution des marqueurs des défenses antioxydantes, notamment au niveau cutané, au cours du vieillissement dit extrinsèque ou environnemental.Nous avons cherché à mimer in vitro le vieillissement extrinsèque cutané, à partir d'un modèle de cellules épidermiques en culture, la lignée de kératinocytes humains HaCaT, soumises à un stress chimique, l'exposition au glyphosate, un principe actif entrant dans la composition de nombreuses formulations pesticides. Ainsi, nous avons exploré notre modèle par une approche multi-échelle originale et innovante: tout d'abord, à l'échelle moléculaire, par une étude protéomique quantitative différentielle des taux d'expression protéique intracytosolique suite à l'induction du stress ; puis, à l'échelle cellulaire en cytomètrie en flux, par la caractérisation fonctionnelle du stress oxydant et de la mort cellulaire qui en découle ; enfin, à l'échelle micro- et nanométrique, en microscopie confocale et à force atomique, par le suivi de l'évolution des propriétés morphologiques et viscoélastiques des kératinocytes stressés.Ce travail de thèse a démontré que le glyphosate altérait signifïcativement l'état et la fonction barrière de l'épiderme humain, ciblant notamment les kératinocytes, dont l'équipement moléculaire et les fonctions vitales d'adhérence et de dynamique membranaire se retrouvent fondamentalement dégradées. L'ensemble de ces résultats constitue un « tableau » qui évoque parfaitement les événements, les signaux et les comportements cellulaires s'installant au cours du vieillissement cutané. / Numerous studies relate ageing to oxidative stress. Few of them described thé évolution of markers of antioxidant défenses, in particular at thé cutaneous level, during extrinsic or environmental ageing. We tried to mimic in vitro cutaneous extrinsic ageing, with a model of epidermic cells in culture, thé human kératinocytes cell line HaCaT, subjected to a chemical stress, Glyphosate, an active ingrédient présent in thé composition of numerous pesticide formulations. Indeed, we examined thé effects of induced ageing in thé loss of kératinocytes integrity in culture, by an original and innovative multi-scale approach: Firstly, at thé molecular scale, we assessed thé stress induced modifications of intracytosolic protein expression by a differential quantitative proteomic study; then, at thé .cellular scale, with flow cytometry, by thé functional characterization of thé oxidative stress and resulting cell death; fmally, at thé micro- and nanoscale, with confocal and atomic force microscopy by thé following thé évolution of morphological and viscoelastic properties of stressed kératinocytes. This PhD work demonstrated that glyphosate impaired thé state and thé barrier function of thé human skin, in particular by fundamentally impairing kératinocytes through thé molecular equipment, thé vital adhésion fonctions and membrane dynamics. Ail thèse results give us a global image of events, signais and cell behavior occurring in skin aging.

Abstract Carbon nanotubes (CNTs) have extraordinary mechanical and electrical properties. Together with their small dimensions and low density, they are attractive candidates for building blocks in future nanoelectromechanical systems and for many other applications. The extraordinary properties are however only attained by perfectly crystalline CNTs and quickly deteriorate when defects are introduced to the structure. The growth technique affects the crystallinity where in general CNTs grown by arc-discharge are close to perfectly crystalline, while CVD-grown CNTs have large defect densities. Mechanical deformation also affects these properties, even without introducing defects. When CNTs are bent they behave similarly to drinking straws, i.e. they buckle or ripple and their bending stiffness drops abruptly. In this thesis, the mechanical behaviour of individual CNTs and vertically aligned carbon nanofibers (VACNFs) has been studied by performing force measurements inside electron microscopes. Cantilevered CNTs, and VACNFs, were bent using a force sensor, yielding force-deflection curves while their structure was imaged simultaneously. We have found that CNTs grown by arc-discharge have a high enough crystallinity to possess a Young’s modulus close to the ideal value of 1 TPa. CVD-grown CNTs possess a Young’s modulus that is about one order of magnitude smaller, due to their large defect density. The VACNFs are yet another order of magnitude softer as a result of their cup-stacked internal structure.  We also found that a high defect density will increase the critical strain for the rippling onset and the relative post-rippling stiffness. Multi-walled CNTs with a small inner diameter are less prone to ripple and have a larger relative post-rippling stiffness. Our findings show large variations in the onset of rippling and the bending stiffness before and after rippling. These variations open up possibilities of tailoring the mechanical properties for specific applications. / Baksidetext Carbon nanotubes (CNTs) have extraordinary mechanical and electrical properties. Together with their small dimensions and low density, they are attractive candidates for building blocks in nanoelectromechanical systems (NEMS), and many other applications.  In this thesis the mechanical behaviour of individual CNTs and vertically aligned carbon nanofibers has been studied by performing force measurements inside electron microscopes. We have found that the mechanical behaviour is very sensitive to the defect density and the internal structure of the CNTs. The extraordinary properties are only attained by defect free CNTs and quickly deteriorate if defects are introduced to the structure. Mechanical deformations also alter these properties. Single-walled CNTs behave similarly to drinking straws when bent, i.e. they buckle, while the inner tubes of multi-walled CNTs prevent buckling. Instead a more distributed rippling pattern is created for multi-walled CNTs. Both these deformation behaviours will cause an abrupt drop in the bending stiffness, which is detrimental for many applications. The findings in this work will have implications for the design of future NEMS. / <p>Artikel 2 Image formation mechanisms tidigare som manuskript, nu publicerad: urn:nbn:se:kau:diva-16425 (MÅ 150924)</p>

The rise in the world population can be correlated with an increase in energy need. Fossil fuels are not going to able to cover this need in energy because not only are they limited, they also have a negative effect on the environment. A reason the more to switch renewable energy. One of the most popular renewable energy source is solar energy. The organic solar cell could be a low-cost, light-weight and flexible option for photovoltaics. This thesis will discuss the morphology of the active layer of an organic solar cell. The polymer poly(9,9-dioctylfluorenyl-2,7-diyl) and the fullerene derivate [6,6]-phenyl C61-butyric acid methyl ester were used as model components for the active layer. These two components were processed in different solvents, different ratios, different total concentrations and were either dip- or spin-coated on glass substrates. These samples were analyzed with atomic force microscopy, steady state and time resolved fluorescence and UV/Vis spectroscopy. The analysis show that the morphology of the films processed in chloroform and tetrahydrofuran would react very similar in α-phase and β-phase by dip- and spin-coated samples. Xylene would react the opposite as tetrahydrofuran and chloroform while ethylbenzene would react little with different samples. / De stijging in wereldpopulatie kan gelinkt worden met een stijging in energieverbruik. Het is niet aan te raden om fossiele brandstoffen te gebruiken voor deze energiestijging want niet alleen zijn ze beperkt aanwezig op aarde ook zijn ze niet goed voor het milieu. Een reden te meer om naar duurzame energie over te schakelen. Één van de meeste populaire energiebronnen is zonne-energie. Hierbij zou de organische zonnecel een goedkope, lichte en flexibele optie zijn. Deze thesis zal de morfologie van de actieve laag van een zonnecel bespreken. Het polymeer poly(9,9-dioctylfluorenyl-2,7-diyl) en het fullereen derivaat [6,6]-fenyl C61-butylzuur waren de twee model componenten voor de actieve laag. Deze twee componenten werden in verschillende oplosmiddelen, verschillende verhoudingen en verschillende totaal concentraties bereidt en werden vervolgens gedipcoated of gespincoated op glazen substraten. De stalen werden vervolgens geanalyseerd door atomic force microscopy, steady state en time resolved fluorescence en UV/Vis spectroscopy. De analyse toont dat de morfologie van de films bereidt in chloroform en tetrahydrofuraan gelijkaardig reageren in α- fase en β-fase bij gedipt- en gespincoaten stalen. Terwijl xyleen net omgekeerd reageert als chloroform en tetrahydrofuraan. Bij ethylbenzeen zou de fases maar heel weinig veranderen bij de verschillende stalen.

La bactérie Pseudomonas aeruginosa (PA) est un pathogène responsable de 20%-30% des infections nosocomiales en milieu hospitalier. Pour les individus sains, elle ne présente pas de réel danger, mais pour les personnes atteintes par la mucoviscidose et les patients immunodéprimés, elle est la cause principale de mortalité et des infections pulmonaires. PA a développé des souches multi-résistantes aux antibiotiques et des nouvelles approches thérapeutiques plus efficaces sont donc nécessaires. Elle se fixe à la surface des cellules-hôtes par une interaction entre des protéines (lectines) présentes sur sa membrane et des sucres présents sur la membrane cellulaire. L’interaction lectine-sucre joue un rôle important dans l’adhésion de la bactérie puis dans la fabrication d’un biofilm pathogène.Une nouvelle approche thérapeutique consiste à créer des molécules synthétiques (glycomimes) de plus grande affinité que les sucres présents sur les cellules. Pour cela, plus de 150 glycomimes ont été synthétisés et examinés afin de trouver le meilleur candidat pour empêche le processus d'infection de bactéries. Certains d'entre eux ont été choisis et étudiés par la Microscopie à Force Atomique (AFM). Cette thèse est consacrée à l’étude des interactions lectine-glycomime et aussi cellule-bactérie par AFM. L’imagerie combinée avec la modélisation permet de comprendre le rôle du glycomime sur la géométrie des complexes créés et la spectroscopie permet de mesurer les forces d’interaction présentes lors de l’adhésion, au niveau moléculaire et cellulaire. Une réduction de l’adhésion bactérienne a été observée après l’introduction du glycomime, confirmant son rôle d’inhibiteur et la validité de toute la démarche. L’objectif ultime est l’identification des meilleurs glycomimes à introduire afin de développer de nouveaux médicaments. / Pseudomonas aeruginosa (PA) is a human opportunistic pathogen responsible for 20% -30% of nosocomial infections in French hospitals. For healthy people, it presents no real danger, but for people with cystic fibrosis disease and immune-compromised patients, it is the leading cause of mortality and lung infections. PA has developed antibiotic multi-resistant strains and new and more effective therapeutic approaches are needed. It binds to the surface of the host cells by an interaction between proteins (lectins) present on the membrane and sugars of the host-cell membrane. The lectin-sugar interaction plays an important role in adherence of the bacteria and in the manufacture of a pathogenic biofilm.A new therapeutic approach is to create synthetic molecules (glycoclusters) of greater affinity than the natural sugars present on the cells. To this aim, more than 150 glycoclusters have been synthetized and screened to find the best candidate to inhibit the bacteria infection process. Some of them have been selected and studied by Atomic Force Microscopy (AFM). In particular, this thesis is devoted to study the lectin-glycocluster and cell-bacteria interactions by AFM. The combination of AFM imaging with molecular dynamic simulations let understanding the role of the geometry of the glycoclusters on the complex formation, while AFM spectroscopy accesses the lectin-glycocluster interaction forces at the molecular and cellular levels. The reduction of bacterial adhesion has been observed upon the addition of the glycocluster. This confirms the anti-adhesive properties of the glycocluster and validates the procedure. The ultimate goal is the identification of the best glycoclusters in order to develop new drugs.

Pore forming toxins are known for their ability to efficiently form transmembrane pores which eventually leads to cell lysis. PFTs have potential applications in devel-oping novel drug and gene delivery strategies. Although structural aspects of many pore forming toxins have been studied, very little is known about the dynamics and subsequent rupture mechanisms. In the first part of the thesis, a combined experimental and modeling study to understand the lytic action of Cytolysin A (ClyA) toxins on red blood cells (RBCs) has been presented. Lysis experiments are carried out on a 1% suspension of RBCs for different initial toxin concentrations ranging from 100 – 500 ng/ml and the extent of lysis is monitored spectrophotometrically. Using a mean field approach, we propose a non – equilibrium adsorption-reaction model to quantify the rate of pore formation on the cell surface. By analysing the model in a pre-lysis regime, the number of pores per RBC to initiate rupture was found to lie between 400 and 800. The time constants for pore formation are estimated to lie between 1-25 s and monomer conformation time scales were found to be 2-4 times greater than the oligomerization times. Using this model, we are able to predict the extent of cell lysis as a function of the initial toxin concentration. Various kinetic models for oligomerization mechanism have been explored. Irreversible sequential kinetic model has the best agreement with the available experimental data. Subsequent to the mean field approach, a population balance model was also formulated. The mechanics of cell rupture due to pore formation is poorly understood. Efforts to address this issue are concerned with understanding the changes in the membrane mechanical properties such as the modulus and tension in the presence of pores. The second part of the thesis is concerned with using atomic force microscopy to measure the mechanical properties of cells. We explore the possibility of employing tapping mode AFM (TM-AFM) to obtain the elastic modulus of soft samples. The dynamics of TM-AFM is modelled to predict the elastic modulus of soft samples, and predict optimal cantilever stiffness for soft biological samples. From experiments using TM-AFM on Nylon-6,6 the elastic modulus is predicted to lie between 2 and 5 GPa. For materials having elastic moduli in the range of 1– 20 GPa, the cantilever stiffness from simulations is found to lie in the range of 1 – 50 N/m. For soft biological samples, whose elastic moduli are in the range of 10-1000 kPa, a narrower range of cantilever stiffness (0.1 – 0.6 N/m), should be used.

L’épithélium digestif est recouvert d’une couche protectrice de mucus, qui est un hydrogel perméable et viscoélastique. La couche de mucus est formée d'un réseau de fibres de mucines. Ces dernières sont des glycoprotéines de haut poids moléculaire avec un squelette protéique riche en sérine et thréonine, lié à une grande variété de O-glycanes qui représentent une source nutritionnelle pour les bactéries et/ou des ligands potentiels pour les adhésines bactériennes, contribuant ainsi probablement à la sélection et l'implantation d'un microbiote régio-spécifique. Nous nous sommes intéressés aux capacités muco-adhésives de L. lactis TIL448 par le couplage de (i) la microscopie à force atomique (AFM), à l'échelle de la cellule unique et en mode statique et (ii) la méthode hydrodynamique en chambre à écoulement cisaillé, à l'échelle de l’ensemble de la population bactérienne. Dans l'optique d'identifier la nature et le rôle fonctionnel des déterminants de surface mis en jeu, nous avons testé, outre la souche sauvage, la souche curée de plasmides TIL1230 et deux mutants TIL1289 et TIL1290, altérés dans la synthèse de pili et d'une protéine "mucus-binding", respectivement. Pour relier les propriétés muco-adhésives et diffusives de L. lactis, les capacités de migration de la souche TIL448 et de ses dérivés ont ensuite été évaluées dans des suspensions de PGM à concentration variable (0,5% et 5% (m/v)), en mettant en œuvre une nouvelle méthode "Diffusion Front Tracking" (DFT). Cette méthode consiste à suivre le front de diffusion de la suspension bactérienne au cours du temps au sein du réseau de PGM, dans une chambre de Hele-Shaw, couplée à une caméra CCD. Les bactéries L. lactis sont préalablement marquées avec la fuschine pour mieux visualiser le front de diffusion. Par ailleurs, nous avons démontré que les bactéries L. lactis ont tendance à être plus diffusives dans PGM 0,5% (m/v) que dans PGM 5% (m/v). La microstructure du réseau de mucines a donc été caractérisée par des approches de microrhéométrie 1 point (1P) et 2 points (2P) et de suivi de particules fluorescentes / The digestive epithelium is covered with a protective mucus layer, regarded as a viscoelastic and permeable hydrogel. Mucins are large glycoproteins with a serine and threonine-rich protein backbone, linked to a wide variety of O-linked oligosaccharide side chains arranged in a bottle-brush configuration. Such O-glycans are nutritive sources for bacteria and/or potential ligands for bacterial adhesins, probably contributing in this way to the selection of the species-specific microbiota. In this thesis, we focused on unraveling multi-scale interactions between a vegetal L. lactis subsp. lactis isolate, TIL448 and a model mucin, Pig Gastric Mucin (PGM). Our study, based on the combination of different biophysical approaches and tools, has allowed dissecting the muco-adhesive and diffusive phenotype of L. lactis TIL448, in relation with the nature of the bacterial surface determinants and the structural, mechanical and rheological properties of the PGM network. Firstly, the muco-adhesion of TIL448 were examined using the single-cell scale AFM measurements with dedicated lacto-probes and shear stress flow chamber experiments at the bacterial population level, under laminar flow conditions. We also tested the plasmid-cured strain and two mutants, obtained by disruption of the genes encoding the major pilin and the mucus-binding protein. Then, the diffusion ability of L. lactis was determined by implementing a novel method, named Diffusion Front Tracking (DFT). It consists of tracking the diffusion front of stained cell suspensions over time within the PGM network. In a second part, in order to have a more thorough understanding of the L. lactis muco-adhesive and diffusive ability, the microstructure and mechanical properties of PGM were determined. Gel microstructure for varying PGM concentration was probed by the analysis of diffusivities of 200-nm and 500-nm fluorescent nanoparticles with different surface properties (carboxyl-terminated, amine-terminated and neutral charged tracers), using fluorescence Multiple-Particle Tracking

Les pathologies inflammatoires vasculaires (PV) et articulaires (PA) représentent aujourd'hui la cause principale de la mortalité et d’invalidité dans les pays industrialisés. Comme les causes exactes favorisant leur apparition restent inconnues, le présent travail a proposé de nouvelles méthodes physiques susceptibles de détecter les premiers stades inflammatoires en utilisant des marqueurs spécifiques et d'étudier les changements mécaniques et structuraux subis par les tissus vasculaires et le liquide synovial (LS). Les PV peuvent être détectées en utilisant les examens IRM. Afin d’améliorer l’efficacité des agents de contraste IRM ceux-ci peuvent être greffés avec des anticorps. En utilisant la Spectroscopie de Force (SF), un mode de la Microscopie à Force Atomique, l’affinité établie entre un nouvel anticorps, le Fucoidan, et le marqueur spécifique P-Selectine a été analysé. L’étude sur PV a été finalisée en utilisant les mêmes techniques SF en mesure d’indentation afin de connaitre les changements de propriétés mécaniques entre les tissus vasculaires sains et pathologiques. Les modifications dans la dynamique du LS déclenchées par l'une des molécules incapables de réagir selon leur fonctionnalité peuvent conduire aux PA. Aussi la technique SF a été utilisée pour étudier le comportement de chaque composant moléculaire du LS. Il a été prouvé l’affinité de ces composants pour les bicouches lipidiques (BL), fréquemment rencontrées dans le corps humain. L’étude a été complétée par l’analyse des changements intervenant dans la dynamique des BL en présence/absence des composants principaux de LS. Les investigations ont été réalisées par un test de Récupération de Fluorescence Après Photoblanchiment. Enfin un test tribologique a été conduit pour étudier la variation du coefficient de frottement entre les BL et les composants du LS / As vascular (VP) and articular (AP) inflammatory pathologies represent nowadays the principal cause of mortality and disability in industrialized countries, the exact causes favoring their occurrence remain still unknown. The present work aimed at proposing new physical methods to detect the early inflammatory stages through recognition of specific markers and to study the structural and mechanical changes undergone by pathological vascular tissues and synovial fluid (SF). Vascular pathologies can be detected through contrasted MRI pictures. In order to improve the capacity of contrast agents to target specific markers they can be antibody-grafted. Atomic Force Microscopy’s mode Force Spectroscopy (AFM-FS) was used to evaluate the affinity between the Fucoidan as a new antibody, and the P-Selectin vascular inflammatory marker, for capacity to target that marker. Further study of VP used the FS techniques for nanoindentation to study changes in mechanical properties between healthy and pathological vascular tissues. Modifications in SF’s dynamics triggered by one of the molecular component not fulfilling its role may lead to AP. To investigate this issue, each of the main SF’s molecular components had their affinity tested versus the ever-present lipid bilayers using AFM-FS techniques. Furthermore changes in lipid bilayers’ dynamics in the presence/absence of the main SF components were analyzed by Fluorescence Recovery After Photobleaching technique. Finally a tribological test was performed to study the variation of the friction coefficient between the lipid bilayers and SF’s main components

Orientador: Mônica Alonso Cotta / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin / Made available in DSpace on 2018-08-18T17:58:13Z (GMT). No. of bitstreams: 1 Lorite_GabrielaSimone_D.pdf: 14464970 bytes, checksum: f2238701a88fbbba91e337eda7db2965 (MD5) Previous issue date: 2011 / Resumo: Apresentamos nesta tese uma investigação dos processos físico-químicos da adesão e desenvolvimento de biofilmes da bactéria fitopatogênica Xylella fastidiosa (Xf) em diferentes superfícies. Este estudo visa corroborar ou complementar diferentes aspectos dos modelos atualmente em discussão para biofilmes bacterianos, além de, num caráter tecnológico, fornecer subsídios para um eventual controle da formação dos biofilmes de Xf. Para isso utilizamos uma abordagem diferenciada à comumente empregada em biologia - e mais próxima à ciência dos materiais - visando isolar e quantificar a relevância dos parâmetros que contribuem na formação de um biofilme em diferentes superfícies, tentando aproximá-las do xilema da planta. Nossos resultados mostram uma semelhança de desenvolvimento (forma, tamanho e quantidade) nos biofilmes de Xf cultivados em superfícies de vidro e Si, e um melhor desenvolvimento dos biofilmes em Si do que nas superfícies de etil celulose, EC, e acetato de celulose, AC. De um ponto de vista fenomenológico, os biofilmes apresentam diferentes estruturas, taxa de desenvolvimento e tendências na expressão gênica entre superfícies com e sem presença de celulose derivatizada. Na caracterização das superfícies utilizadas consideramos o efeito do meio de cultura em suas propriedades. Nas superfícies de vidro e Si, constatamos a formação de um filme condicionante devido à adsorção dos constituintes deste meio. O grau de hidrofobicidade das superfícies de vidro e Si diminui significativamente após contato com o meio de cultura enquanto as superfícies de celulose derivatizada apresentam pouca (EC) ou nenhuma alteração (AC); observamos também um aumento do potencial de superfície (PS) para Si e EC e, ainda, uma diminuição de PS em AC. Estas evidências sugerem uma correlação entre PS mais altos e grau de hidrofobicidade baixos com a presença de biofilmes de Xf em maior número e tamanho. Além disso, medidas de espectroscopia de força utilizando ponta funcionalizada com a proteína de adesão XadA1 evidenciam também um comportamento distinto na superfície de AC. Estes resultados apontam para a importância da interação eletrostática no processo inicial de adesão da bactérias às superfícies estudadas. Por fim, a presença de material extracelular ao redor dos biofilmes e células de Xf em superfícies de vidro e Si foi observada indicando a presença de uma matriz exopolimérica protetora. Espectroscopia de infravermelho mostra a presença de polissacarídeos ¿ constituinte da matriz polimérica ¿ desde o estágio inicial de formação do biofilme, indicando uma possível contribuição dessa matriz para o processo de adesão que precede o desenvolvimento do biofilme / Abstract: In this work, we report an investigation on relevant physicochemical processes of bacterial surface adhesion and biofilm development for the phytopathogen Xylella fastidiosa (Xf) on different surfaces. This study aims to corroborate or supplement different aspects of the bacterial biofilms models currently under discussion and, in a technological point of view, provides experimental input for an eventual control of Xf biofilm formation. For this purpose, we have used an approach not commonly found in biology ¿ and more similar to materials science ¿ in order to isolate and quantify the relevance of the parameters that contribute to the formation of a biofilm on different surfaces, as well as trying to make these surfaces closer to the plant xylem. Our results show a similar development (shape, size and quantity) for Xf biofilms grown on Si and glass surfaces, and an improved development of biofilms grown on Si than on ethyl cellulose, EC, and cellulose acetate, AC. Under a phenomenological point of view, biofilms present different structures, rate of development and trends in gene expression between surfaces with and without the presence of derivatized cellulose. In order to characterize the surfaces, we considered the effect of culture medium on their properties. In Si and glass surfaces, we observe the formation of a conditioning film due to adsorption of the constituents of the culture medium. The degree of hydrophobicity of glass and Si surfaces decreases significantly after contact with this medium. On the other hand, cellulose derivatized surfaces present lower (EC) or no (AC) modifications of this property. In addition, we observed an increase in the surface potential (SP) for Si and EC and also a SP decrease for AC. These evidences suggest a correlation between higher SP values and lower degree of hydrophobicity with the presence of biofilms of Xf in larger numbers and size. Furthermore, force spectroscopy measurements using a functionalized tip with the adhesion protein XadA1 also show a different behavior on the AC surface. These results reveal the importance of electrostatic interaction in the initial bacterial adhesion for the surfaces studied here. Finally, the presence of extracellular material around the Xf cells and biofilms on glass and Si surfaces was observed indicating the presence of a protective exopolymeric matrix. Infrared spectroscopy shows the presence of polysaccharides - a constituent of the polymeric matrix - from the very initial stages of biofilm formation, indicating a possible contribution of this matrix for the adhesion process which precedes biofilm development / Doutorado / Biofísica / Doutora em Ciências

Die Fortschritte bei synthetischen Polymeren konzentrieren sich in erster Linie auf die Bemühungen, Kunststoffe auf molekularer Ebene zu entwickeln, um vielversprechende Eigenschaften und Funktionen auf makroskopischer Ebene zu erreichen. Daher sind die Verbesserung der Materialsynthese sowie die Zunahme der Komplexität des makromolekularen Designs zu einem wichtigen Forschungsschwerpunkt geworden. Es wurden bemerkenswerte synthetische Strategien zur Entwicklung von sequenzgesteuerten Oligomeren entwickelt, die durch eine präzise Mikrostruktur eine anschließende Faltung zu kontrollierten multizyklischen Origamis ermöglichten. Allerdings bleiben die derzeitigen synthetischen Methoden für die Herstellung von Präzisionspolymeren mit hohem Molekulargewicht bis zu einem gewissen Grad statistisch. Daher ist es nach wie vor schwierig, große synthetische Makromoleküle zu entwerfen, die sich zu präzisen und einheitlichen zyklischen Strukturen zusammenfügen können. Sind parallele Fortschritte bei der Charakterisierung von großen multizyklischen makromolekularen Strukturen sehr gefragt, da die meisten der derzeitigen Techniken nur in der Lage sind, Indizien für die Strukturorganisation zu liefern. Die vorliegende Arbeit untersucht dabei die Synthese und die Morphologie von dynamischen und kontrollierten zyklischen Polymeren. Das synthetische Konzept basiert auf der Herstellung von sequenzgesteuerten Makromolekülen mittels regulierten Einbaus von reaktiven Selenol- oder Thiolgruppen an gewünschten Positionen innerhalb einer Polymerkette. Die kontrollierte oxidative Dimerisierung der funktionellen Gruppen führt zu Diselenid- bzw. Disulfidbrücken und bewirkt eine intramolekulare Vernetzung zur Erzeugung einer dynamischen einkettigen Zyklisierung. Um Einblicke auf molekularer Ebene zu gewinnen und den Grad an struktureller Kontrolle aufzuzeigen, wird eine synthetische Strategie entwickelt, die eine direkte Visualisierung der erhaltenen Polymerkonformation ermöglicht. / The field of material science has evolved drastically in the last decades. Progress in synthetic polymers primarily focuses on efforts to design materials at a molecular level to reach promising properties and functions. Improving material synthesis and increasing the complexity of macromolecular design have become a major research focus. Remarkable synthetic strategies have been developed toward the elaboration of sequence-defined oligomers, in which the precise microstructure can allow subsequent folding into controlled and precise multi-cyclic origamis. However, current synthetic routes toward precision polymers with high molecular weight remain statistical to some degree, which reflects a loss of structural control. Thus, designing large synthetic macromolecules that can fold into precise and uniform cyclic-shape structures remains difficult to reach. Parallel progress in characterization of large multi-cyclic macromolecular designs are highly demanded since most of the current techniques are only capable of providing circumstantial evidence of structural organization. Macromolecules with dynamic intramolecular crosslinks have become relevant due to their ability to potentially reach equilibrium structures in response to external stimuli. In this study, controlled synthetic route and morphology characterization of dynamic cyclic polymers are investigated. The synthetic concept is based on the preparation of sequence-controlled macromolecules to guide the insertion of reactive selenol or thiol groups at desired positions within a polymer chain. Controlled oxidative dimerization of the functional groups leads to diselenide or disulfide bridges respectively and induces intramolecular crosslinking to generate dynamic cyclization. To gain insight into the molecular level to reveal the degree of structural control, a synthetic strategy is developed to access an additional analytic tool and enable direct visualization of the obtained polymer conformations.