This research proposal would create a novel instrument which would combine Physics and Biology, and which would make it possible to simultaneously record several types of information from biological cells. The Atomic Force Microscope is a nanometre-resolution microscope, which physically 'touches' the surface of the cell. It is sensitive to the shape and structure of the outer layer of the cell, and produces a 3-dimensional map of the cell surface. Confocal microscopy on the other hand is a powerful light microscope technique, in which the focal point of a laser beam can be controllably moved throughout the 3-dimensional body of the cell, and therefore we can build up a picture of the inside of the cell by moving this focal point around inside the cell. If we are able to record information from these 2 microscopes at the same time, we can correlate directly features on the cell surface with internal cellular properties. One example of this would be to record fluorescence from membranes whilst obtaining a surface map. Another application would be to purposefully indent the cell with the tip of the AFM probe, and record the response of the cell, as its internal skeleton adapts to the stimulus on the outside surface. We will also combine these two microscopes with a technique called electrophysiology which will give information on the electrical signals crossing the surface of the cell. Ultimately the information we obtain will give us more insights into cell processes and specialised functions, how cells communicate with one another, and also how they interact and adapt to their environment.

Pore-forming proteins are crucial armaments in the continuous battle between living organisms and the pathogens that threaten their fitness and survival. These proteins act on cells, which are the micrometre-scaled, basic units of all forms of life. Cells are separated and protected from their environment by a thin membrane. Pathogens such as bacteria can release pore-forming proteins ("toxins") that drill holes in the membranes of healthy cells in the host organism, to release nutrients for the bacteria, to invade these cells and/or kill them. Patients affected by bacterial pneumonia, for example, suffer from the devastating effects of such a toxin, pneumolysin, on lung tissue. The immune system, however, uses a similar mechanism to kill germs and infected or cancerous cells, thus preventing them from doing further damage to the organism. It secretes related, but somewhat different pore-forming proteins to perforate the membranes of such unwanted invaders. To perform these tasks, pore-forming proteins have developed sophisticated drilling mechanism. These proteins can convert from a soluble form in the aqueous, cellular environment into a very different form, in which 20-50 protein molecules assemble into a ring-shaped pore bound to the membrane. We can look at these forms with X-rays or electrons to deduce their three-dimensional structures. Thanks to such experiments, we now have a reasonably clear picture of the soluble proteins and their pore structure in the membrane. For some pore-forming proteins, scientists have even identified the changes inside the proteins which make this transition possible. However, if we wish to design drugs that prevent such pores from being formed, as in the example of bacterial pneumonia indicated above, it would be useful to know more about the steps in their formation. It is exactly this pore assembly that is still largely enigmatic. In this project, we will try to answer some specific questions about membrane pore formation. We would like to know how the proteins assemble on the membrane. Do they assemble one by one, or do they first form larger units that subsequently assemble in a pore? Do the proteins first need to assemble on the membrane, or can they dock in the membrane and assemble in pores afterwards? And at what point in this process will the membrane that is surrounded by the assembled protein be extruded to create a hole? To investigate the dynamics of this process, we rely on a technique called atomic force microscopy. Atomic force microscopy is the small-scale equivalent of reading Braille: With a tiny artificial finger, we feel the pore-forming proteins while they assemble on the membrane. Whereas X-ray crystallography and electron microscopy are limited to static samples, atomic force microscopy can probe active proteins while they are at work. We will thus apply atomic force microscopy to the membranes that are being exposed to attack by pore-forming proteins. Meanwhile, we will benefit from the more detailed views provided by electron microscopy to identify intermediate assemblies of pore-forming proteins, that are trapped by chemical bonds or by lowering the temperature. Electron microscopy will thus provide highly detailed pictures of pore forming proteins in different states of assembly and atomic force microscopy will enable us to see how the proteins transit between these different states.

BIOPOL is an interdisciplinary European training network at the interface of cell biology, physics and engineering. BIOPOL aims specifically at the understanding of fundamental mechanochemical principles guiding cellular behaviour and function and their relevance to human disease. A new supra-disciplinary research field is emerging bringing together the fields of molecular cell biology, physics and engineering aiming at an in depth understanding of fundamental cellular mechanochemical principles. BIOPOL combines exactly this required expertise in one joint training program for young researchers. BIOPOL has assembled a unique multidisciplinary consortium bringing together top scientists from the fields of molecular/developmental cell biology, membrane physics, engineering as well as specialists from the private sector. The scientific objectives focus on understanding of fundamental mechanisms of cellular mechanosensing in health and disease, the role of external forces in cell division and mechanochemical regulation of cell polarity including tissue formation. Finally, part of BIOPOL´s research program is the further development of cutting edge technologies like advanced atomic force microscopy, novel photonic tools like optical stretcher or innovative organ on a chip technology, exploiting physical cellular properties. BIOPOL´s collaborative cutting edge research program is integral part of its training program provided to early stage researcher and is further translated into seven state of the art experimental training stations representing the consortiums expertise. In addition, BIOPOL has developed a 3 years modular curriculum including workshops, summerschools, Business plan competitions and conferences with a specific agenda of transferable skill training elements highly relevant for scientific communication, translational research and in particular entrepreneurship.

NanoScience is the emerging research discipline of building designer materials or machines which do entirely new things, by combining thousands of atoms arranged in intricate assemblies and connections. Understanding and controlling this new science results in NanoTechnology estimated to be one of the massive opportunities in the 21st Century, for making devices that really do what we want cheaper, faster, cooler, smarter and more efficiently. The process of assembly is the key to fostering widespread implementation of nanoscience discoveries. This is an area in which the UK must be strong to reap the rewards of increased investment. Most emerging opportunities depend on radically improving such nano-organisation, needed to impact major societal themes of Energy, Healthcare and Nano. However despite all these claims, which are mostly well-founded conceptually, the difficult is in how to really build on this extreme scale. Bigger than molecules but smaller than machinery, we have only learnt in recent years how to grow a plethora of nano-components. But perfecting ways to bring together these nano-components into active devices is the new challenge. Traditional approaches that piece things together laboriously are completely unfeasible here. The aim of our Doctoral Training Centre in Assembly of Functional NanoMaterials and NanoDevices is to hothouse training of a high-calibre cadre of inter-disciplinary nano-researchers and spur them to develop entirely new ways to assemble nano-machinery for doing something useful. The academics involved in this Nano DTC have all had experience of helping to teach young researchers across a range of research fields such as Physics, Materials Science, Chemistry and Engineering, and have also shown a real interest in developing novel ideas into practical inventions and engaged with companies (many of them their own spin-offs). The University of Cambridge has a large number of scientific programmes in this area, so a large opportunity exists to join them up, with the PhD students all interacting very widely across these disciplines, as well as engaging with the nitty-gritty tools of how nano-innovation can make it out into the real world.The Nano DTC will operate as a distinct PhD nursery, with the entry co-housed and jointly mentored in the initial year of formal courses and project work. Students from a range of undergraduate disciplines will thus spend considerable time together while each postgraduate will have a selection of 1st year courses crafted on entry by the DTC management committee, depending on their specific skill set and aspirations. The initial year provides additional skills in disciplines outside their degree, understanding of the Enterprise landscape relating to Nano-Innovation, specific knowledge of the nanoscience and application of self-assembly to NanoDevices and NanoMaterials, and miniprojects spanning different disciplines to broaden students' experience and peer networks, aiding final PhD project selection. A range of joint activities are programmed in later years including Nano DTC cohort student-led conferences, and industry reviews.Although individual examples of nano-entrepreneurship can be found across the UK, graduate students are rarely exposed to this experience, and frequently it is seen as detrimental to their research progress. A repeated theme emerging from nano research-to-application projects is how early-stage nano-construction strategies benefit from being informed by eventual scale-up, implementation routes, market potential and societal awareness. In turn, this joined up approach feeds back into the basic science process, frequently stretching research programs beyond the well-trodden paths and stimulating high impact science as well as innovation. The aim of the Cambridge Nano DTC is to make this experience pervasive for a new brand of UK Nano PhD students.