Membrane elastic heterogeneity studied at nanometrical scale on living cells

The aim of this thesis was to explore the cell mechanical properties using the Atomic Force Microscope (AFM). The cell membrane contains lipids microdomains, called rafts, enriched in cholesterol and sphingolipids. The rafts are believed to play an important role in signal processing by acting as a "signaling platform". Indeed, membrane proteins involved in signal transduction concentrates into these rafts and are coupled with signaling pathways inside the cell. The mechanical properties of these rafts were characterized by targeting one of its component, the glycosylphosphatidylinositol-anchored protein (GPI-AP). During this work, we found these domains to be stiffer than the surrounding membrane. Several control experiments were performed to consolidate this finding. The extraction of cholesterol, one of the major component of raft, was shown to dramatically reduce the stiffness of the raft to reach the surrounding membrane value. The stiffness specificity of rafts may be related to the lower diffusion rate of proteins and can be, therefore, an important property for its role as a signaling platform. During this thesis, we also introduced a new AFM imaging mode, which we called "stiffness tomography". With this imaging mode, we were able to distinguish stiff materials inclusion located into the sample. Different control experiments were done to validate this imaging mode. A virtual experiment was performed with the help of the finite element modeling. This permitted us to validate our methodology, but also pointed us its limitations. The stiffness tomography was also used on living cells and showed significant differences between native and cytoskeleton depolimerized cells. Since no postprocessing tool was available at the beginning of this work, the software development was a very significant part of the project. Its development resulted in a toolbox (a collection of function), that is available for future software development. A non negligible part of the development consisted in the toolbox documentation that is reported in the appendix C. This software permitted to process force volume AFM les and to characterize the elastic properties of the cell membrane with a high precision and reliability

Atomic Force Microscopy Imaging of Living Cells

Over the last two decades, Atomic Force Microscopy (AFM) has emerged as the tool of choice to image living organisms in a near-physiological environment. Whereas fluorescence microscopy techniques allow labeling and tracking of components inside cells and the observation of dynamic processes, AFM is mainly a surface technique that can be operated on a wide range of substrates including biological samples. AFM enables extraction of topographical, mechanical and chemical information from these sample

Continuous monitoring of shelf lives of materials by application of data loggers with implemented kinetic parameters

The evaluation of the shelf life of, for example, food, pharmaceutical materials, polymers, and energetic materials at room or daily climate fluctuation temperatures requires kinetic analysis in temperature ranges which are as similar as possible to those at which the products will be stored or transported in. A comparison of the results of the evaluation of the shelf life of a propellant and a vaccine calculated by advanced kinetics and simplified 0th and 1st order kinetic models is presented. The obtained simulations show that the application of simplified kinetics or the commonly used mean kinetic temperature approach may result in an imprecise estimation of the shelf life. The implementation of the kinetic parameters obtained fromadvanced kinetic analyses into programmable data loggers allows the continuous online evaluation and display on a smartphone of the current extent of the deterioration of materials. The proposed approach is universal and can be used for any goods, any methods of shelf life determination, and any type of data loggers. Presented in this study, the continuous evaluation of the shelf life of perishable goods based on the Internet of Things (IoT) paradigm helps in the optimal storage/shipment and results in a significant decrease of waste

Latent class analysis reveals clinically relevant atopy phenotypes in 2 birth cohorts

Phenotypes of childhood-onset asthma are characterized by distinct trajectories and functional features. For atopy, definition of phenotypes during childhood is less clear.; We sought to define phenotypes of atopic sensitization over the first 6 years of life using a latent class analysis (LCA) integrating 3 dimensions of atopy: allergen specificity, time course, and levels of specific IgE (sIgE).; Phenotypes were defined by means of LCA in 680 children of the Multizentrische Allergiestudie (MAS) and 766 children of the Protection against allergy: Study in Rural Environments (PASTURE) birth cohorts and compared with classical nondisjunctive definitions of seasonal, perennial, and food sensitization with respect to atopic diseases and lung function. Cytokine levels were measured in the PASTURE cohort.; The LCA classified predominantly by type and multiplicity of sensitization (food vs inhalant), allergen combinations, and sIgE levels. Latent classes were related to atopic disease manifestations with higher sensitivity and specificity than the classical definitions. LCA detected consistently in both cohorts a distinct group of children with severe atopy characterized by high seasonal sIgE levels and a strong propensity for asthma; hay fever; eczema; and impaired lung function, also in children without an established asthma diagnosis. Severe atopy was associated with an increased IL-5/IFN-γ ratio. A path analysis among sensitized children revealed that among all features of severe atopy, only excessive sIgE production early in life affected asthma risk.; LCA revealed a set of benign, symptomatic, and severe atopy phenotypes. The severe phenotype emerged as a latent condition with signs of a dysbalanced immune response. It determined high asthma risk through excessive sIgE production and directly affected impaired lung function

Maturation of the gut microbiome during the first year of life contributes to the protective farm effect on childhood asthma

Peer reviewedPostprin

Latent class analysis reveals clinically relevant atopy phenotypes in 2 birth cohorts

Background: Phenotypes of childhood-onset asthma are characterized by distinct trajectories and functional features. For atopy, definition of phenotypes during childhood is less clear. Objective: We sought to define phenotypes of atopic sensitization over the first 6 years of life using a latent class analysis (LCA) integrating 3 dimensions of atopy: allergen specificity, time course, and levels of specific IgE (sIgE). Methods: Phenotypes were defined by means of LCA in 680 children of the Multizentrische Allergiestudie (MAS) and 766 children of the Protection against allergy: Study in Rural Environments (PASTURE) birth cohorts and compared with classical nondisjunctive definitions of seasonal, perennial, and food sensitization with respect to atopic diseases and lung function. Cytokine levels were measured in the PASTURE cohort. Results: The LCA classified predominantly by type and multiplicity of sensitization (food vs inhalant), allergen combinations, and sIgE levels. Latent classes were related to atopic disease manifestations with higher sensitivity and specificity than the classical definitions. LCA detected consistently in both cohorts a distinct group of children with severe atopy characterized by high seasonal sIgE levels and a strong propensity for asthma; hay fever; eczema; and impaired lung function, also in children without an established asthma diagnosis. Severe atopy was associated with an increased IL-5/IFN-gamma ratio. A path analysis among sensitized children revealed that among all features of severe atopy, only excessive sIgE production early in life affected asthma risk. Conclusions: LCA revealed a set of benign, symptomatic, and severe atopy phenotypes. The severe phenotype emerged as a latent condition with signs of a dysbalanced immune response. It determined high asthma risk through excessive sIgE production and directly affected impaired lung function.Peer reviewe

Stiffness tomography by atomic force microscopy

A method of analyzing a sample with a probe based measurement instrument using stiffness tomography. Stiffness or elastic modulus data obtained through contact measurement with the sample as a function of depth is used in the detection of features, particularly subsurface features, based on stiffness or elastic modulus differential. In an AFM, a probe tip is indented, preferably until it reaches a desired depth, and force-deflection data is measured as a function of depth. The FD data is broken into subsets or segments, each of which is independently analyzed to determine a stiffness or elastic modulus. The stiffness or elastic modulus data is used in determining whether there is great enough difference or contrast in any portion of the data to detect presence of a feature with a stiffness different than its surroundings. Such data can be imaged and/or manipulated to provide an image of subsurface features

Thermal process safety based on reaction kinetics and reactor dynamics

The specific problem of non-converted reactant accumulation encountered in fed-batch reactor is a challenging task of process safety. In order to avoid such situation that may lead to a runaway reaction, the reaction kinetics and reactor dynamic must be known. In this work, an innovative approach to determine an optimal feed profile, based on a reaction kinetic and reactor dynamic, is presented. This approach was applied to the reaction system involved in the Morton International Inc. incident. Great improvements were observed in the control of the accumulation, decreasing the number of necessary experiments needed for the scale-up studies