Viruses and bacteria utilize proteins to attach and infect cells and tissues. Viruses have envoloped proteins that especifically recongnize receptors in the surface of the target cells. HIV-1 recognices receptor CD4 in the surface of T cell throghout its envolope glycoprotein gp120. In the case of bacteria, they attach to tissues using long filament called pilus. Bacterial pilus type 1 is composed of several protein subunit arranged in chain, FimA-FimG-FimG-FimH. The more external domain, FimH, is the adhesin binding domain that establishes the mechanical anchoring to tissues. Both, bacterial and viral proteins withstand mechanical forces than can go from few piconewtons to hundreds. However, very little is known about how force modify the structure and features of these proteins and ultimately how its affects infection. In this project we aim to investigate the effect of mechanical forces in the anchoring proteins and its role during attachment. We will concentrate in viral receptors CD4 and bacterial fimbriae proteins (Fim). We will use a novel atomic force spectometer that allows us to apply calibrated forces to a single protein molecule. This technique allows also monitoring chemical reaction under force such as the reduction of disulfide bonds or the binding of peptides and antibodies. These processes are known to be implicated in the infection of viruses and bacteria and they may have a mechanical origin. We will use bioinformatics and high-throughout screening techniques to identify molecules that alter the nanomecanichs of these anchoring proteins and that can potentially be used to prevent infections.

Conformational changes in bio-molecules are closely linked to their function and thus of major interest for an understanding of many biological processes. For a number of biologically active RNA molecules there are indications from biochemical assays that they are able to regulate their function by switching between different conformations of similar energetic stability. An important example is the RNA genome of the Human Immunodeficiency Virus (HIV), which is responsible for the AIDS disease, one of the most serious pandemics ever caused by a virus. Despite an intensive research effort the ultimate vaccine or drug against this disease has not yet been successfully developed. The difficulty related to this can be explained by the extraordinary high rate of genetic evolution of the RNA genome, which is related to a high mutation rate and a tendency of the RNA molecules to form dimers. The mechanism by which dimerization is initiated and regulated in the viral life cycle is not very well understood but is believed to be mediated by conformational changes in the RNA. The objective of the present project is to address in detail this issue with a new experimental technique, single-molecule fluorescence- resonance-energy-transfer (FRET) microscopy, which promises important new knowledge that cannot be obtained with traditional, ensemble-averaging methods. The activity proposed here is very much in line with the objectives of the Marie Curie Actions, implying the transfer of knowledge and promotion of European scientific excellence, and depending on interdisciplinary collaborations at the national and European levels. Furthermore, it can be classified as nanoscience as well as life science and biotechnology, all of which are declared priority levels of the European Commission.

Molecular diagnostics of microbial pathogens is an integral part of modern medicine. The growing need for direct genotyping and/or the screening of the transcriptome calls for the development of alternative technologies. The STREP consortium plans to develop a cost-effective platform for the identification bacterial species based on the SLIC-Nanobiosystem. Using tmRNA transcripts of the bacterial ssrA gene, we will be able to detect, quantify and identify bacterial species in a single homogenous assay format. The SLIC-Nanobiosystem consists of a self-assembled lipid bilayer membrane that integrates a synthetic ligand-gated ion channel (SLIC). The SLIC comprises a capture molecule that can specifically bind a given analyte, a process that is monitored via electrical impedance spectroscopy. With this system the effect from even a few channels can be resolved thus providing an ultra-sensitive, highly stable and versatile biosensor platform. We intend to employ transcripts (tmRNA) of the ssrA gene in order to identify bacterial species present in clinical samples. These transcripts occur in high abundance and contain a core sequence that is species specific, a feature which will be exploited to identify infectious disease pathogens. Identification of the different bacterial tmRNA transcripts will be accomplished by displaying a library of nucleic acid capture probes on the SLIC. This will enable species identification and discrimination between one or more species present in the sample if mixed species infection is present. Since the detection equipment will be based on electronics, the realization of miniaturized/compact and cost-effective instruments will be possible. Our approach will lay the foundation for a new generation of multiparametric molecular testing systems, that will open novel opportunities within the area of point-of-care applications in the clinical diagnostics market.

The aim of the RTN is to explore the potential of gold Glyco-Nano-Particles (GNPs), to deepen the understanding ofbiomolecular interactions and to solve biomedical problems. The innovative combination of a variable metallic core with aselected set of ligands can turn GNPs into vaccines or anti-adhesion agents or dedicated vehicles to deliver compounds safely at their target location.GlycoGold will operate as a virtual research training centre at the interface of physics, chemistry, immunology, molecularbiology, and bioinformatics. It integrates and expands the knowledge base and infrastructures of the partners. It has workpackages (WPs) that deal with materials, tools and insight into biomolecular interactions, and WPs geared towards theapplication of GNPs as vaccines, and control of interactions. The Training&Mobility and Design&Innovation WPs and activenetworking ensure a smooth project workflow. The tasks are assigned to international task forces.The research is timely, because the required techniques have evolved to a point that an integrated multinational approach isviable. Secondly, interest of industries in obtaining this information is high. Thirdly, the necessary overall knowledge to perform the research in a single laboratory does not exist in Europe, and very few graduates have the integrated knowledge to operate in this area, despite the large demand for them.The network provides an interdisciplinary training environment for these specialists, where training is offered through (i) handson experience, (ii) summer schools aimed to bridge the knowledge gap between the physical and life sciences, (iii) short courses, lectures, demonstrations and practical training sessions, and (iv) activities for individual career development.Dedicated international and multilingual science promoting activities are scheduled annually aimed at young science students and in particular at high-school pupils.

The development of new polymeric biomaterials designed to stimulate specific cellular responses at the molecular level such as activation of signalling pathways that control gene activity involved in maintenance, growth, and functional regeneration of liver tissue in vitro could be an important step in tissue engineering. The project is aimed to the development of polymeric synthetic and biodegradable biomaterials to control liver cell responses in vitro and in vivo systems. Isolated hepatocytes are able to continue the full range of known in vivo liver specific functions for only a short time. The in vitro maintenance of competent hepatocytes is decidable so that the liver functions can be studied in a controlled environment. Engineered liver tissue constructs may provide an inexpensive and reliable in vitro physiological model with great control of variables for studying disease, drug, infection and molecular therapeutics. New modified polyetheretherketone PEEK-WC membranes will be prepared in hollow fiber configurations. Membranes will be prepared by phase inversion technique, which permits to obtain membranes with various structural properties by means of kinetic and thermodynamic parameter control. In addition, the development of synthetic polymeric materials consisting of nanofiber network scaffolds represents an entirely new approach to tissue engineering that has relied in the past on materials that where either of unknown composition (i.e. Matrigel) or not possible to design (i.e. Collagens). Thus, the design and preparation of synthetic three-dimensional nanofiber network scaffolds that highly mimic the extracellular matrix will be a valuable tool in the field. The surface of membranes/scaffolds to be utilized in the project will be modified by non equilibrium plasma-chemical processes such as Plasma Deposition of thin films (PE-CVD) and Plasma Treatments to adapt their properties to the best compatibility with cells.

The recently invented fluorescence lifetime imaging nanoscopy (FLIN) provides a groundbreaking tool for the study of single molecules (SM) and single molecular motors (SMM) as well as a broad array of phenomena in the NanoWorld. Previous limitations for SMM studies, resolution, short observation times, and photo-dynamic reactions, are now overcome by minimal-invasive picosecond FLIN. FLIN is the extension of the extremely successful fluorescence lifetime imaging microscopy (FLIM) into the nano-domain, with 10 to 100 nm space resolution. FLIN results from the combination of 4pi-microscopy with novel ultrasensitive, nonscanning imaging detectors, based on time- and space-correlated single photon counting (TSCSPC) that allows ultra-low excitation levels. This results in long-period (#gt; 1 hour), minimal-invasive observation of living cells and SM/SMM, without any cell damage or irreversible bleaching. Minimal-invasive FLIN (MI-FLIN) with global point spread function modelling allows observation of SMM movement at 1-nm accuracy and 10-nm resolution. Parallel to (i) MI-FLIN/FLIM implementation, the consortium will (ii) improve sensitivity, time- and space-resolution as well as throughput of the TSCSPC detectors, (iii) explore an array of novel applications provided by MI-FLIM/FLIN, such as nanometer SMM-tracking, (iv) develop a super-background-free TIRF microscope to improve detectability of SM/SMM, and (v) examine the behaviour of four different types of SMM and their dependence on energy-input. Enhanced basic understanding of biological and artificial machines and motors will lead to improved model systems and proceed one day to the design of artificial systems, improving the interface of biological and non-biological worlds. Furthermore, biological SMM are involved in many disease states such as Alzheimers, Werner syndrome and infectious diseases. Our studies aim to improve understanding of how these motors operate and how they break down in disease.

The emergence of new pathogens or variations has created recently severe threats to human health (E.coli O157:H7, SARS, the avian-flu disease). The gravity of the problem resides on the fact that their impact and spreading is growing dramatically due to the ongoing increase in worldwide human mobility in combination with trade in livestock, and food products. However, detecting the source of infection through conventional analytical methods requires complicated and time-consuming protocols. The project aims the development of a quick and low-cost diagnostic device (Lab on a Card) that develops and integrates technology advances in optoelectronics, microfluidic and microbiology, capable to detect, in-situ, DNA pathogens in 15 minutes. The device consists of a hand held base unit and a cartridge or labcard that will carry out a Real Time Polymerase Chain Reaction automatically, from sample preparation to an optical detection. The labcard, made of a photoresist called SU-8, on a plastic film, contains all the disposable components, whereas the base unit includes all the standard electronics and optics. The range of applications is limitless (infectious diseases, flu, tuberculosis, hepatitis, AIDS, cancer, etc). and it will be validated on Salmonellosis and Campylobacter detection. The scenario where this diagnostic device is used covers hospitals, food factories and private homes. Its impact will be enormous, reducing the incidence of infectious illness, and providing EU governments with a certificated tool to quickly monitor and survey the sources of pathogen contamination. An especial emphasis will be applied to develop and use microfabrication processes that are compatible with mass production and low cost of the devices offering a protected and disruptive technology to European enterprises. The successful achievement of this project will open the door for many other analytical miniaturisation to be developed.

'As the average age of us ''European nationals'' increases, the demand for major joint replacements is expected to rise in accordance. Although implants are considered an excellent solution to many health problems, any time a medical device is implanted into one's body there is a high risk for infection not only in the short but in the medium term as well. Additionally, the long term wear behavior of the surfaces in contact significantly affects the life of implants. In this manner, there is a need for new multifunctional coatings. In comparison with other implant materials, tetrahedral amorphous carbon (ta-C) have excellent blood and tissue biocompatibilities and therefore many artificial joints and cardiovascular implants are being coated today with ta-C materials. Recent studies have shown the possibility of incorporating certain toxic elements (e.g. Cu, Ag, or V) into hard carbon coatings with the idea of providing the implants with necessary infection resistance. However, until now, these coatings could only be deposited either by hybrid pulsed laser deposition (PLD) techniques or by multi-step processes, making their manufacturing expensive and unrealistic.We propose to investigate the development, the mechanical properties and wear behavior of heterophase ta-C:Me nanocomposite coatings deposited from two pulsed cathodic arc (PCA) plasma sources. One of the advantages of this novel deposition method over hybrid PLD techniques is its potential application to large surface areas and a comparatively less expensive technology while producing an intense highly ionized plasma plume which is necessary for the deposition of the hard ta-C phase. After the deposition, glow discharge optical emission spectroscopy (GDOES) will be used to obtain a quick analysis of the homogeneity of the metallic phase vs. depth allowing us to adjust the process parameters and improve the manufacture and design of novel ta-C:Me coatings.'

The heart of this proposal is to give access for European fellows to interdisciplinary knowledge and training focused on the elaboration and properties of nano-objects (metal nanoparticles, dendrimers and surfactant aggregates). The training site localized at the University Paul Sabatier in Toulouse is composed of three internationally recognized research teams working with a multidisciplinary approach. It will provide to the fellows the common technical and methodological background on three complementary aspects of those nano-objects: 1) the chemical synthesis leading to size and shape controlled nano-objects, 2) the control of their organization and mesostructure in volume or along a surface 3) their properties in catalysis, biology and material science. The training will combine laboratory work focused on a 'cutting edge' research project and lectures. Those lectures (selected academic courses, weekly seminars, talks given by invited world famous scientists and lectures dedicated to the EST fellows) will broaden and deepen the fellow's knowledge in the field, promote innovation and interdisciplinarity and help the trainee's career development. Through the multidisciplinary research project, the fellows will be trained in various chemical syntheses (organochemistry, chemistry of heteroelements, surfactant chemistry), in the characterization of the nano-object mesostructures (they will have access to the most advanced characterization techniques) and in applications of the nanoobjects in connection with societal issues (catalysts for green chemistry, environnemental sensors, drug carriers, transfection, anti-HIV agents for health care issue…).This training will ensure the development of a pool of young scientists having a large multidisciplinary background and able to deal with the challenges of the nanomaterial field. It will increase synergies and collaborations between European research centers and favor the leadership of the European activity in this field.

COCHISE is the first step of an activity aimed at the development of enabling micro-technologies to monitor physiological cellular interactions at the single cell level with a high throughput. It will be applied first to the immunological monitoring of anti-tumor vaccinations, singling out the rare effector cells (in the order of 10-3) that are actually active against tumor cells. The sensor that we are developing consists of an orderly matrix of about 4,000 living cells deposited in microwells created in a biocompatible substrate that also serves as a high-density circuit board. The microwells are monitored by an external microscope and have an embedded addressable impedance sensor. The key point is that each microwell can force contact between individual cells, and detect consequences of these contacts. The project integrates on the same platform several technologies such as electronic sensing, microfluidic interfaces for cell dispensing, control of osmotic balance of nutrients, management of evaporation, surface nano-modifications for management of fluid flows (e.g. hydrophilic and/or hydrophobic surface tend to drive or repel droplets) and avoidance or induction of surface cell adhesion. At the system level, cell delivery will leverage recent results that allow to delivery single cells in an effective way. An important side of the research is the definition of new therapeutic and diagnostic protocols for the immunotherapy of cancer. As a first step, we will apply our technology to the analysis of anti-tumor lytic effector cells, for a precise quantification of how many lytic events happen in the array, their locations and timings. A major advantage is that the cells are kept alive and can be retrieved individually for further analysis, such as gene expression profiling.