Oxidative stress on biomaterials

Reactive Oxygen Species (ROS) is a family of oxygen-based active molecules, which are deeply involved in numerous crucial processes of cellular metabolism, where the redox equilibrium is very important. An imbalance between ROS and the cell ability to neutralize them with antioxidants results in an Oxidative Stress (OS). This leads to cell alterations and impaired physiological functions. OS is involved in a growing list of human pathologies, from brain disorder, like Alzheimer and Parkinson's disease, to various forms of cancer (skin melanoma) and eye pathologies (cataract, macular disease), as well as in the aging process. Sometimes, inducing OS selectively in diseased cells can also act as a powerful therapy. This is the basis of PhotoDynamic Therapy (PDT), a newly developed medical application to cure cancer. It is based on ROS generation through photo-sensitive molecules (also referred to as PhotoSensitizers, or PS) under specific illumination. The goal of this work was to develop an efficient way to tune and characterize OS and related damage to biological systems. For this purpose, we synthesized a water-soluble derivative of fullerene-based PS, since the pristine molecule is known to generate ROS under visible illumination at very high yield. The efficiency of our custom-made PS in generating ROS was characterized by Electron Spin Resonance (ESR). Parallel studies were performed on a porphyrin-based PS and on TiO2 nanoparticles. The effects of oxidative stress provoked via PS were investigated for selected proteins and for a large variety of cells. The extent of OS was finely tuned by adjusting the experimental conditions such as PS concentration in the buffer. The changes upon OS were studied by three modern experimental techniques: ESR, Synchrotron Infrared MicroSpectroscopy (SIRMS) and Atomic Force Microscopy (AFM). We have obtained a promising set of data on the consequences of OS detected by these local techniques. During the OS certain chemical bonds are interrupted and others are created which manifest in the change of the lattice vibrations detected for entire cells using SIRMS. These stresses affect, first of all, the constituents of the cells. For example, OS on a protein induces conformational changes as measured by ESR using spin labeled proteins. We have shown that larger organizations of proteins like cytoskeletal filaments must also change under OS, since the cell stiffness decreases considerably. AFM measurements on various cell types (neurons, fibroblast, bladder cell, glioblastoma…) and on healthy and cancerous cells have clearly shown the progressive decrease of the Young's modulus as OS was switched on.

Statistical Study of the Unfolding of Multimodular Proteins and their Energy Landscape by Atomic Force Microscopy

Fabrizio Benedetti

The aim of the present thesis is to investigate several aspects of: the proteins mechanics, interprotein interactions and to study also new techniques, theoretical and technical, to obtain and analyze the force spectroscopy experiments. The first section is dedicated to the statistical properties of the unfolding forces in a chain of homomeric multimodular proteins. The basic idea of this kind of statistic is to divide the peaks observed in a force extension curve in separate groups and then analyze these groups considering their position in the force curves. In fact in a multimodular homomeric protein the unfolding force is related to the number of not yet unfolded modules (we call it "N"). Such effect yields to a linear dependence of the most probable unfolding force of a peak on ln(N). We demonstrate how such dependence can be used to extract the kinetic parameters and how, ignoring it, could lead to significant errors. Following this topic we continue with non kinetic methods that, using the resampling from the rupture forces of any peak, could reconstruct the rupture forces for all the other peaks in a chain. Then a discussion about the Monte Carlo simulation for protein pulling is present. In fact a theoretical framework for such methodology has to be introduced to understand the various simulations done. In this chapter we also introduce a methodology to study the ligand receptor interactions when we directly functionalize the AFM tip and the substrate. In fact, in many of our experiments, we see a "cloud of points" in the force vs loading rate graph. We have modeled a system composed by "N" parallel springs, and studying the distribution of forces obtained in the force vs loading rate graph we have establish a procedure to restore the kinetic parameters used. Such procedure has then been used to discuss real experiments similar to biotin-avidin interaction. In the following chapter we discuss a first order approximation of the Bell-Evans model where a more explicit form of the potential is considered. In particular the dependence of the curvature of the potential on the applied force at the minimum and at the metastable state is considered. In the well known Bell-Evans model the prefactors of the transition rate are fixed at any force, however this is not what happen in nature, where the prefactors (that are the second local derivative of the interacting energy with respect to the reaction coordinate in its minimum and maximum) depend on the force applied. The results obtained with the force spectroscopy of the Laminin-binding-protein are discussed, in particular this protein showed a phase transition when the pH was changed. The behavior of this protein changes, from a normal WLC behavior to a plateau behavior. The analysis of the force spectroscopy curves shows a distribution of length where the maximum of the first prominent peak correspond to the full length of the protein. However, length that could be associated with dimers and trymers are also present in this distribution. Later a new approach to study the lock and key mechanism, using "handles" with a specific force extension pattern, is introduced. In particular handles of (I27)3 and (I27–SNase)3 were biochemically attached to: strept-actin molecules, biotin molecules, RNase and Angiogenin. The main idea is to have a system composed by "handle-(molecule A)-(molecule B)-handle" where the handles are covalently attached to the respective molecules and the two molecules "A and B" are attached by secondary bonds. This approach allows a better recognition of the protein-protein interaction enabling us to filter out spurious events. Doing a statistic on the rupture forces and comparing this with the statistic of the detachments of the system of the bare handles, we are able to extract the information of the interaction between the molecule A and B. The two last chapters are of more preliminary character that the previous part of the thesis. A section is dedicated to the estimation of effective mass and viscous drag of the cantilevers studied by autocorrelation and noise power spectrum. Usually the noise power spectrum method is the most used, however the autocorrelation should give approximately the same information. The parameters obtained are important in high frequency modulation techniques. In fact, they are needed to interpret the results. The results of these two methods show a good agreement in the estimation of the mass and the viscous drag of the various cantilever used. Afterwards a chapter is dedicated to the discussion of the force spectroscopy experiments using a low frequency modulation of the cantilever base. Such experiments allow us to record the phase and the amplitude shift of the modulation signal used. Using the amplitude channel we managed to restore the static force signal with a lower level of noise. Moreover these signals give us direct information about the dynamic stiffness and the lose of energy in the system, information that, using the standard technique would be difficult (or even impossible) to obtain.

EPFL2012

Characterization of Solid-Liquid Interfaces with High-Resolution Atomic Force Microscop

Maria Ricci

Solid-liquid interfaces are ubiquitous. Despite the unquestioned relevance, many scientific research fields rely on simplified or macroscopic descriptions, discarding the molecular nature of the constituents. However, it is the specific molecular interactions and the complex chemistry in the interfacial region that are fundamental in phenomena such as heterogeneous catalysis, electrochemistry, crystal growth, membrane transport and body-antibody recognition. In particular, the models used to describe the so-called electrical double layer of ionic distributions at charged surfaces in liquid, are based on continuum assumptions. For example, the Gouy-Chapman-Stern model assumes that a dense, homogenously distributed layer with a thickness of a hydrated ion (referred to as the "Stern layer"), is adsorbed onto the charged surface, proceeded by an exponentially decaying diffuse ionic cloud, in which ions are point charges in a continuous dielectric media. In this thesis, I will first illustrate how the assumption of a homogeneous ion lateral density in the Stern Layer fails to describe the actual solid-water interface of several relevant surfaces. Then I will show how the lateral organization of the ions indeed plays a role as important as the vertical modulation of the charge distribution. This is because the molecular nature of the ions, the surface, and the water molecules themselves each contribute to the precise lateral organization of the constituents. As result, the ions can reside on discrete surface sites, defined both laterally and vertically in space. Moreover, the water-mediated interaction can impart a correlation in the reciprocal lateral distribution of the ions. To achieve this level of description, high-resolution atomic force microscopy (AFM) is used. Unlike most of other experimental techniques, AFM is able to characterize interfaces locally at the atomic/molecular level. In fact, in the AFM small-amplitude regime, detection of even single ions adsorbed at the surface of the solid is possible by measuring the perturbation they induce on the local solvation environment. This capability represents an invaluable tool for the exploration of phenomena occurring at the Stern layer of solid-liquid interfaces. Following, I will investigate the properties of several solid-aqueous interfaces in the presence of different ionic concentrations and species, ranging from hard (mica) to soft systems such as organic self-assembled monolayers, lipid bilayers, and the dynamic surface restructuring of calcite when covered with fatty acid molecules. The experimental study, combined with molecular dynamic simulations, reveals a water-induced ion-ion attractive interaction for some ionic species. These results show that water alone is the driving force to induce order within the Stern layer, creating hydration-correlation effects on mica, self-assembled monolayers and possibly lipid bilayers. The local modulation of the hydration properties of the surface is fundamental to highly dynamic systems such as calcite, where steps act as nucleating points for the processes of dissolution and growth. The specific interaction of foreign ions and fatty acids modify these processes as well as the equilibrium between the crystal and the solution. This thesis provides clear experimental evidence of new mechanisms developing at solid-liquid interfaces paving the way for a deeper and more conscious understanding of the colloidal properties of materials.

EPFL2015

Statistical Study of the Unfolding of Multimodular Proteins and their Energy Landscape by Atomic Force Microscopy

Fabrizio Benedetti

EPFL2012

Characterization of Solid-Liquid Interfaces with High-Resolution Atomic Force Microscop

Maria Ricci

EPFL2015