Fluorescence imaging is a powerful technique that has transformed our understanding of biology. Recently, a number of techniques have been developed that overcome one of the fundamental limitations of fluorescence imaging, namely the diffraction limit. With these techniques, it is now possible to resolve sub-cellular architecture in multiple colors and 3D with unprecedented detail. However, the main limitation of these techniques has been the slow acquisition times making it difficult to study dynamic processes. Since biological samples are inherently highly dynamic, this limitation is a major hurdle that needs to be overcome. I will develop a correlative fluorescence imaging technique that combines the capabilities of super resolution and real-time imaging. With this correlative technique it will be possible to observe the dynamics of a biological sample in real-time and subsequently 'freeze' the dynamics (by fixation or low temperature) at a time of interest to obtain a super resolution image. The dynamics can therefore be correlated with ultrastructural information, combining the capabilities of real-time and super resolution imaging. I will apply this correlative imaging technique to study infection mechanism of Herpes Simplex Virus (HSV). HSV is a medically important virus that infects neurons and epithelial cells. Besides the health hazards that it poses, HSV also has important implications in gene therapy. However, the details of HSV infection mechanism remain poorly understood. Since HSV infection involves dynamic interactions between virus particles and sub-cellular components, both of which are tens of nanometers in length scale, this is an ideal model system in which the correlative super resolution and real-time imaging technique will lead to important insights that were previously unattainable.

Bacterial infection into host cells is an important and highly active field of research. Understanding the interactions and the distribution of the host-cell scaffolding protein network during bacterial entry poses a major challenge. The molecular architecture of actin comet tails, filamentous structures assembled by internalized bacteria to move inside the host-cell cytoplasm and from cell-to-cell, remains unknown. Cryo-electron tomography (cryo-ET) is the most advanced method for visualizing the architecture of hydrated cells at a resolution better than 5 nm. Cryo-ET will be used to visualize the three dimensional (3D) cytoskeleton reorganization directly in eukaryotic cells infected by Listeria. Measurements will be performed at cryo-temperatures, on vitrified cell samples preserved in a close-to-life state. Specimen thickness limitations will be overcome by the use of the focused ion beam (FIB) micro-machining method, to obtain 500 nm thick samples as required for the collection of good data. Cryo-ET will be combined with correlative cryo-fluorescence microscopy, to localize the scaffolding components recruited during Listeria uptake and motility: host-cell actin, septin and clathrin. We expect to achieve nanometer resolution maps of the cell area of interest. The distribution and the ultrastructure of the cytoskeletal scaffold at Listeria entry and of Listeria actin comet tails will be provided. The work will provide unprecedented insight into cytoskeleton architecture during bacterial pathogenesis. The applicant obtained her PhD at a structural biology institute in France. Joining the Baumeister laboratory in Germany, and collaborating with the Cossart group, will allow her to address new challenging questions on the structural organisation of the cell, at unprecedented resolution. The acquired combination of skills at a world-class level will contribute significantly to her professional maturity, and to increase the competitiveness of European science.

Lung cancer is the most common cancer in terms of both incidence and mortality, worldwide. With a median age at diagnosis of 71, lung cancer is mainly affecting the aging population. Airway stenosis is a key problem with significant morbidity and premature death. Endobronchial stenting is a proven therapy to keep the airways open. Nevertheless the currently used clinical stents have major disadvantages either by rapid re-occlusion due to tumour ingrowths (metal stents) or massive mucus retention due to the interrupted mucociliary function (coated stents). The aim of the project is to develop a viable endobronchial stent (syn. PulmoStent) for the treatment of broncho-tracheal cancer diseases. The concept is based on the combination of stent technologies with the principles of tissue engineering. The PulmoStent is a multi-layered structure providing (1) a functional respiratory epithelium on the luminal side, which allows the maintenance of the mucociliary function in the stented area, (2) an embedded micro- or nanosphere formulations, enabling the sustained, local release of tumour-specific therapeutics in combination with (3) a mechanical separating layer on the external side, enabling a local tumour suppression to avoid stent displacement and restenosis by a growing tumour. The PulmoStent is a step change beyond the state-of-the-art from a passive to a viable and functional active implant tailored to the patient. It focuses on a clearly identified clinical need for the treatment of lung cancer. The combination of different kinds of biomaterials to a co-scaffold system for the bio-functionalization of the stent will lead to an improved performance of endobronchial stents and thereby to longer durability. The novel PulmoStent will improve the quality of life and increase the life expectancy of lung cancer patients, because of the reduced mucus retention in the stented area, and herewith the reduced risk of life-threatening pneumonia and the local tumour suppression.

Malaria is a global health priority that has been targeted for elimination in recent years. Attaining the goals that define elimination of malaria in different countries depends critically on provision of effective antimalarials and further that these antimalarials are used appropriately in individual patients. Drug resistance is a major threat to malaria control and has important global public health implications. Over the past decades the genetic bases for resistance to most of the antimalarial classes currently in use has become defined. For some drugs and combinations, these mutations are the most important predictors of treatment failure. This proposal will innovate new technologies to confirm malaria diagnosis and detect drug resistance in malaria parasites by analysis of mutations in nucleic acids, using nanowire technology, and will result in the development of a simple, rapid and affordable point-of-care handheld diagnostic device. The device will be useful at many levels in malarial control by: 1. Optimising individual treatments for patients 2. Assessing the epidemiology of drug resistance in malaria endemic areas 3. Assessing population impacts of antimalarial interventions The development programme capitalises on highly original and proprietary advances made by QuantuMDx in the field of point-of-care diagnostics. This is complemented by academic expertise that has made major contributions to the understanding of antimalarial drug resistance mechanisms in laboratory models, as well as parasites obtained directly from patients. The impact of this proposal can be extended rapidly to other established and emerging infectious diseases.

At least 20% of human malignancies are caused by consequences of persistent infections. High-risk human papillomavirus (HPV) types cause over 500.000 cancer cases per year, rendering HPV the #2 human carcinogen after tobacco. Infection-related tumors are attractive targets for cancer vaccination, as they provide the opportunity to target antigens that are immunological non-self. Vaccination can be prophylactic, inducing immune responses preventing infection in the first place, or therapeutic, stimulating the immune system into eradicating established disease. Prophylactic immunization against HPV has become the paradigm for cancer immunoprevention. Unfortunately, current HPV vaccines have no therapeutic effect on existing infections. Studies on spontaneously regressing HPV-induced lesions show that cell-mediated immune responses are crucial in clearing established HPV infection. Cytotoxic T cells (CTL) kill infected cells after recognizing viral epitopes presented on HLA molecules on the cell surface. There are hundreds of different HLA types, and a given epitope is only applicable for the fraction of patients with the relevant HLA molecule. This project will define a set of T cell epitopes that elicit CTL-mediated HPV protection in the entire population, by including epitopes for all HLA supertypes. The applicant has established a methodology of determining which viral epitopes are presented on target cells during her past mobility period in the US. HPV-transformed cells of various HLA backgrounds are analyzed by nanospray mass spectrometry. Identified peptides are tested for immunogenicity and the ability to induce CTL. From these tests, a minimal set of functional epitopes providing >95% population protection coverage is selected for vaccine formulation. The technology is currently transferred to the DKFZ. If this epitope-specific, yet widely applicable therapeutic vaccination approach is successful, it can be used as a platform technology in other malignancies.

Infections associated with dental implants may cause peri-implantitis often resulting in implant loss and impaired function. Recent studies show an alarming increase in the incidence of the infections, while on the other hand the efficacy of the prevailing treatment method is decreasing due to the rising resistance of micro-organisms to antibacterial agents. The SMEs of the NanoTi consortium intend to bring a new titanium implant to the market that possesses the innate capability to resist bacterial infections without the addition of any antibacterial compound. In order to reach this goal the aim of the NanoTi project is to develop nanophase topography on the surface of titanium dental implants that will enable such an effect. This nanophase topography: • Reduces the susceptibility of titanium dental implants for infections; • Enables the surgical decontamination of implants if infection occurs; • Supports bone healing around the dental implant.

Bone has a remarkable capacity to heal. However, in some instances the amount of bone which is needed to heal exceeds its healing capacity. These cases arise following accidents, infection or surgery to remove cancerous tissue and they result in the need to perform approximately 600,000 surgical bone grafting procedures annually. These procedures have inherent disadvantages and so there is an urgent clinical need to develop a tissue engineering alternative to bone grafting. In this study an osteoconductive/osteoinductive nanoscaffold will be designed to retain growth factors with proven osteogenic potential within their structure. As such, relatively low doses of these expensive molecules can be retained at the bone defect site. The technology developed in this study has enormous potential to reduce the overall burden placed on patients and on European healthcare systems by reducing the costs involved in using Growth Factors in a variety of applications. To perform this work the Fellow will move from Athlone Institute of Technology, Ireland to join a leading orthopaedic research group at one of Harvard University's teaching hospitals where he will be trained in nanotoxicity testing, detection of growth factor release, cell loading and orthopaedic preclinical models. The Fellow has extensive knowledge in the field of biomaterials and orthopaedic research having trained at the AO Research Institute, Davos, Switzerland. However this fellowship will allow him to develop his knowledge in the field of biocompatibility testing. Knowledge developed in this area will be transferred back to Europe during the return phase of the fellowship. This knowledge will allow the Fellow to further refine the research carried out at Harvard. The goal of this research is to develop translational solutions to clinical problems. Indeed, the chance to work at Harvard would be hugely beneficial in developing direct links to clinicians at one of the world's most prestigious Universities.

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 project aims to develop a fully integrated, automated and user-friendly platform for infectious disease diagnosis. Malaria can be treated in just 48 hours, but delayed or false diagnosis or missing a relevant alternative cause of fever may be lethal. Therefore, other diseases with similar clinical symptoms will be investigated too. DiscoGnosis will integrate micro, nano, and bio components into a multi-functional point-of-care platform, performing simultaneously protein and genetic analysis to timely and accurately identify major pathogenic causes of fever, enabling proper treatment. A foil-based centrifugal microfluidic lab-on-a-chip cartridge, core of the platform, will integrate monolithically all necessary unit operations for raw sample treatment (blood-to-result regime), from sample collection and injection, to plasma separation, DNA extraction and purification. Low-cost production, scalable from prototype to batch fabrication (with proper quality control, calibration and standards specifications) will render the platform affordable to end users, even in developing countries; high sensitivity detection and multiplexity will rely on magnetic microparticles and quantum dot technologies, supported by dedicated optics development; rapid analysis (~30 min) will be achieved via isothermal DNA amplification protocols. The entire system will be validated in a controlled field test with standardized samples and by end-users in high-risk developing countries through partners' established contacts. Data management will be implemented to allow rational organization in the field and to reinforce the 'shield' of Europe against such diseases, as more than 30, 000 malaria cases are reported annually among returning European tourists. This generic point-of-care platform can be applied to many diseases (eg, cancer, cardiovascular, Alzheimer) by only changing its bio-components. The strong SME participation indicates the high commercialization potential of the project.

Antimicrobial agents, such as antibiotics, have dramatically reduced the number of deaths from infectious diseases over the last 70 years. However, through overuse and misuse of these agents, many micro-organisms have developed antimicrobial resistance. Oligonucleotide therapeutics have the potential to become the new class of antibacterials capable of treating a broad range of infections. By acting on novel targets, they circumvent current resistance mechanisms and with judicious use, can suppress the rise of future resistance. DNA-TRAP will build on a platform technology that uses proprietary nucleic acid-based Transcription Factor Decoys (TFDs) that act on novel genomic targets by capturing key regulatory proteins to block essential bacterial genes and defeat infection. Taking forward newly emerging insights and expertise that exists within each of the partners and through the mutual secondment of researchers, the project aims to develop a new class of nanoparticulate antibacterials capable of meeting the clinical challenge of drug-resistant infections such as Clostridium difficile and Pseudomonas aeruginosa. DNA-TRAP will establish a lasting, international partnership for transfer of knowledge between Industry and Academia in the field of nanomedicine. Exchange of knowledge and expertise between the partners is key to establishing the fundamental properties of nanostructured drug delivery systems to treat bacterial infections and through this, provide the basis for building a manufacturing platform to advance the experimental therapeutic into clinical trials. 17 researchers in the field of drug development and delivery from 2 commercial (SME) and 2 non-commercial partners across 2 member states, will have the opportunity to share and acquire new complementary and multidisciplinary knowledge, through inter-sectoral and interdisciplinary exchange, allowing for the development of new solutions and the establishment of further joint research projects.