BB/E021409/1 A multicellular 3D stem cell model to define the role of stroma in epithelial differentiation Closed Research Grant Biology In aging men the disorders of prostate are a major medical problem. Benign prostatic hyperplasia and cancer are increasingly prevalent. To find cures for these diseases it is essential to understand how the prostate grows and functions normally. All organs have their own population of stem cells which grow and develop into a variety of cells which communicate to form correct organ architecture and function. This occurs as a result of signals from the stem cell's own genes but also from signals provided by neighbouring cells, known as stroma. In the prostate, how this occurs is unknown. We propose to develop a model to grow gland-like structures from adult stem cells in the laboratory. The model will be employed to understand how stromal cells influence prostate cellular architecture. We aim to identify proteins which act as signals from the stroma to change epithelial shape. The shape of a cell has important effects on cell function. These experiments will increase our knowledge of how tissues develop and function. Development of tissue-like models based on human cells will provide a valuable gap between results from animal models and human clinical studies, to help understand the basic mechanisms of human physiology and disease. Such model systems will reduce the need for animal experimentation, which is currently the best way to investigate complex cell interactions in tissues. We anticipate the model will aid university directed research into human differentiation and disease mechanisms, but also for the pharmaceutical industry to screen new drugs for efficacy and safety in humans before trial. Recent advances in our lab have resulted in the isolation of human adult prostate stem cells and the development of 3D models of prostatic acini from basal cells. Results from 3D modelling indicate that stroma is important for epithelial morphogenesis and differentiation. Importantly, stromal cultures increase epithelial cell polarity and columnar cell shape. Using electron microscopy and RT-PCR our preliminary data has found that these morphological effects are accompanied by increased desmosomal expression. We now wish to develop our tissue engineering to produce a 3D model of prostatic acini using a homogeneous population of stem cells. A stem cell model will allow the study of full epithelial differentiation and the stem cell niche. It is important to model the prostate with human cells because the mouse prostate has a different anatomy, cell structure and protein function, and does not develop equivalent diseases to humans. The model will be used to investigate our hypothesis that 'stroma signals to control epithelial cell shape and polarity'. We will confirm which desmosomal isoforms are present in prostate epithelial acini and which are upregulated by stromal cultures, using Western Blotting and real time PCR. Upregulated desmosomal isoforms will be used as markers for epithelial cell polarity and shape. A differential gene expression profile will be generated from stroma grown with epithelial acini in 3D culture and stroma grown in 3D culture without acini, using microarray analysis. Candidate stromal genes will be identified that signal to upregulate epithelial polarity (desmosomal expression) and their function will be confirmed using siRNA knockdown studies. This is a novel pathway for epithelial cell differentiation which has not been studied before. NE/K001906/1 Biogeochemistry, macronutrient and carbon cycling in the benthic layer Active Research Grant School of Ocean and Earth Science The coasts and shelf seas that surround us have been the focal point of human prosperity and well-being throughout our history and, consequently, have had a disproportionate effect on our culture. The societal importance of the shelf seas extends beyond food production to include biodiversity, carbon cycling and storage, waste disposal, nutrient cycling, recreation and renewable energy. Yet, as increasing proportions of the global population move closer to the coast, our seas have become progressively eroded by human activities, including overfishing, pollution, habitat disturbance and climate change. This is worrying because the condition of the seabed, biodiversity and human society are inextricably linked. Hence, there is an urgent need to understand the relative sensitivities of a range of shelf habitats so that human pressures can be managed more effectively to ensure the long-term sustainability of our seas and provision of societal benefits. Achieving these aims is not straightforward, as the capacity of the seabed to provide the goods and services we rely upon depends on the type of substrate (rock, gravel, sand, mud) and local conditions; some habitats are naturally dynamic and relatively insensitive to disturbance, while others are comparatively stable and vulnerable to change. This makes it very difficult to assess habitat sensitivities or make general statements about what benefits we can expect from our seas in the future. Recently, NERC and DEFRA have initiated a major new research programme on Shelf Sea Biogeochemistry that will improve knowledge about these issues. In response to this call, we have assembled a consortium of leading scientists that includes microbiologists, ecologists, physical oceanographers, biogeochemists, mathematical modellers and policy advisors. With assistance from organisations like CEFAS, Marine Scotland and AFBI, they will carry out a series of research cruises around the UK that will map the sensitivity and status of seabed habitats based on their physical condition, the microbial and faunal communities that inhabit them, and the size and dynamics of the nitrogen and carbon pools found there. The latest marine technologies will measure the amount of mixing and flow rates just above the seabed, as well as detailed seabed topography. These measurements will allow better understanding of the physical processes responsible for movement and mixing of sediment, nutrient, and carbon. At the same time, cores will be retrieved containing the microbial and faunal communities and their activity and behaviour will be linked to specific biogeochemical responses. Highly specialised autonomous vehicles, called landers, will also measure nutrient concentrations and fluxes at the seabed. Components of the system can then be experimentally manipulated to mimic scenarios of change, such as changing hydrodynamics, disturbance or components of climate change. This will be achieved in the field by generating different flow regimes using a submerged flume or, in the laboratory, using intact sediment communities exposed to different levels of CO2, temperature and oxygen. By measuring the biogeochemical response and behaviour of the microbial and faunal communities to these changes, we will generate an understanding of what may happen if such changes did occur across our shelf seas. We will use all of this information to assess the relative vulnerability of areas of the UK seabed by overlaying the observation and experimental results over maps of various human pressures, which will be of value to planners and policymakers. Mathematical models will test future scenarios of change, such as opening or closing vulnerable areas to fishing or anticipated changes in the factors that control nutrient and carbon stocks. This will be valuable in exploring different responses to external pressures and for deciding which management measures should be put in place to preserve our shelf seas for future generations Commercial private sector and the knowledge economy: new and innovative methodologies, equipment and techniques, and combined state-of-the-art technologies (>2.3 million in-kind, see JeS) will assess what the primary physical and biogeochemical controls of shelf productivity are up to shelf sea scales. Since many interests rely on the marine environment, beneficiaries will be varied. By sharing expertise and knowledge, a world-leading manufacturer of microsensors and microscale instrumentation and an internationally recognized marine environmental data acquisition company will benefit from exploitable opportunities, e.g. new visualisation tools that enable holistic understanding of large-scale ecosystem processes. Policy professionals, governmental and devolved governmental organisations: The importance of shelf seas to society extends beyond fisheries to wider issues, such as biodiversity, carbon cycling and storage, waste disposal, nutrient cycling, and renewable energy resources. Consortium expertise will contribute to these UK priority challenges. The UK Marine & Coastal Access Act (MCAA), UK Climate Change Act, EU Habitats Directive and EU Marine Strategy Framework Directive (MSFD) support sustainable use of the marine environment. They also support the UK vision for achieving 'clean, healthy, safe productive and biologically diverse ocean and seas' (UK Marine Science Strategy). We will provide a coherent framework for sound evidence based-science in support of these policy instruments and statutory requirements. For example, the MSFD aims to achieve Good Environmental Status in EU marine waters by 2020, but we lack understanding of the magnitude and synchronicity of change in SSEs. Our research will directly inform Descriptor 1 (biological diversity) and 6 (seabed integrity) for a wide range of sediment habitats over time, which is important because the determination of good environmental status may have to be adapted over time (addressed in Task 2) "in vie of the dynamic nature of marine ecosystems and their natural variability, and given that the pressures and impacts on them may vary with the evolvement of different patterns of human activity and the impact of climate change" (MSFD). Our work will also inform environmental monitoring programmes: OSPARs Joint Assessment and Monitoring programme, the Eutrophication Monitoring Programme and The Clean Seas Environment Monitoring Programme (CSEMP, led by consortium member CEFAS). Task 1-3 complement the outcomes of CESEMP and provide scientific evidence to OSPAR. Similarly, experimental scenarios and modelling approaches will provide needed information for (i) the EU Water Framework Directive (the requirement for 'good chemical and ecological status' by 2015 does not account for climate change) and, (ii) the UK White Paper for MCAA (it is unclear how commitments to "look ahead at the predicted impacts of climate change on the marine environment, how marine activities will contribute towards it, and how they are affected by it" will be achieved). Finally, other EU instruments, such as the Habitats Directive (introduced in 1992), the EU Common Fisheries Policy (revised in 2002) and national legislation such as the UK MCAA and Scottish Marine Act, assume that removal (or control) of direct pressures will result in ecosystem recovery and/or species persistence. Our programme includes experimental scenarios and modelling approaches to provide further information on the vulnerability of SSEs in environmental futures under multiple pressures (Task 3). Our outputs will also help NERC meet its science theme challenges. Public, wider community: active engagement with a variety of organisations is detailed in Pathways to Impact (PtI). Skills& training: In addition to academic progression, early career researchers will gain experience and receive mentoring in running a large interdisciplinary programme, as well as training in communication skills and scientific methodology 138395 Marine environments 75 46902 Geosciences 15 13097 Ecol, biodivers.& systematics 5 33851 Microbial sciences 5 21005 Sediment/Sedimentary Processes 15 143045 Ecosystem Scale Processes 15 63200 Biogeochemical Cycles 60 108367 Community Ecology 5 80410 Responses to environment 5