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Proteins are polymers that are crucial to all aspects of life. Proteins are biologically produced polymers that are synthesised by polymerisation of monomeric amino acids. The template for the polymerisation process is messenger ribonucleic acid (mRNA), which in turn is encoded for by DNA, which is used for relatively long-term storage of information in the cells of all living organisms. However, once they have been synthesised, proteins can be further modified in processes that are often crucial for their physiological function. One such process is reaction with atmospheric oxygen, a small and high diffusible molecule. We are interested in defining how and why proteins react with oxygen from the atmosphere. In pioneering work it was found that atmospheric oxygen reacts with collagen, a material which helps cells to stick together in animals, in a reaction catalysed by oxygenases. Oxygenases are types of enzymes (or biological catalysts), that incorporate atmospheric oxygen into their reaction products. Many oxygenases use a metal, such as iron, to help capture oxygen. Subsequent to the discovery of its role in collagen biosynthesis, it was found that oxygenases play key roles in the production of antibiotics, such as the penicillins. More recently, we have found that oxygenases also catalyse the hydroxylation of proteins. Some of the protein targets of oxygenases are important from biological and medicinal perspectives. A breakthrough was the discovery that the physiological mechanism by which cells in animals respond to limiting oxygen is actually regulated by oxygenase catalysed hydroxylation of proteins, involved in regulating the conversion of DNA to mRNA. Following this discovery we, and others, have found other protein-hydroxylases, acting on a range of protein-residues. We are now in an exceptionally good position to work out how these enzymes work, including developing an understanding of how they bind their protein substrates. We will use crystallographic and other techniques, that will provide detailed information on how the enzymes work as machines. The structural and mechanistic studies will lay the groundwork in order to exploit the basic science to artificially alter the activity of the oxygenases, using them for the production of high-value modified proteins, and to provide knowledge that will be useful for the pharmaceutical industry in targeting them for diseases. Overall the work will enable the United Kingdom to remain at the forefront of basic science research on oxygenases and the exploitation of this research for the development of new medicines and catalysts for high value chemical production.

In recent years it has become clear that there are many more lymphocytes resident in non-lymphoid tissues than previously thought and that tissue resident cells may be crucial for protection against pathogens, but many questions remain to be resolved. For example: 1. What is the importance of local versus systemic immunity in different infections and species? 2. Does protection of a host animal also prevent transmission? 3. How a correct balance of local and systemic protective immunity best be induced and maintained in different diseases? 4. What is the role of the innate immune system and signals from pathogen associated molecular patterns in initiating protection? 5. How does the microbiota of the host influence development of systemic and local immunity? We shall ask these questions initially in pigs using influenza virus as a model. We shall use different routes of infection and methods of immunisation to establish the hallmarks of protective local and systemic immune responses. Using different vaccine vectors, adjuvants and formulations, we shall dissect the essential innate and adaptive components of a protective response. Furthermore we shall determine whether these hallmarks are reflected in prevention of transmission. Using antibiotic treatment, diet or administration of defined commensal organisms, we shall manipulate the microbiota and define their effect on protective immune responses. The mechanisms will be further analysed in mouse transgenic or homologous recombinant (knockout) models. These studies will help us understand the role of local and systemic immunity in protection against pathogens and to identify in which circumstances it will be essential to recruit local immunity to achieve successful protective immunity by vaccination

Delivering sustainable food production in the face of climate change with reduced fertilizer and pesticide input requires a revolution in crop improvement. Meanwhile, modern crops are becoming increasingly depleted in gene biodiversity. Extending crop biodiversity and supporting future crop improvements can be achieved by 'mining' allelic variants of genes from ancestral wild germplasm. Novel strategies to utilize multi-parental breeding populations and apply the genomics revolution offer a promising route towards exploitation of exotic germplasm for breeding. We will test both approaches using wild barley (Hordeum vulgare ssp. spontaneum) as a model to improve agronomic performance of cultivated barley under abiotic and biotic stress conditions. For this, we will explore a nested association mapping (NAM) approach using the first cereal NAM population HEB-25 ('Halle Exotic Barley') to simultaneously test 25 wild barley accessions for beneficial gene effects. HEB-25 consists of 1,420 BC1S3 lines, each of which carrying ca. 25% of wild barley genome from one of 25 exotic donors on a 75% genetic background of the recipient spring barley cultivar Barke. First, the HEB-25 lines will be assessed for allele content, employing state-of-the-art Exome Capture with Next Generation Sequencing (EC-Seq) to discover single nucleotide polymorphisms (SNP) for 21,643 genes in each of the 1,420 HEB-25 lines. We expect to map roughly 400,000 SNPs, giving several SNPs per gene with the goal to distinguish any wild barley allele from the recipient Barke allele. Second, the HEB-25 lines will be cultivated in field trials in Germany, Scotland and Israel, to assess phenotypic stress performance under either nitrogen deficiency, drought or pathogen attack. Morphologic, agronomic, and nutrient content traits will be scored, as well as resistances against the important barley diseases leaf rust, yellow rust and net blotch. In addition, agronomic performance will be modeled in Israel by up-to-date remote sensing technology, to establish non-invasive phenotype prediction models. Third, the collected data sets will be archived and further processed in a central data warehouse at Halle, built around a custom web-accessible relational database, enabling universal access to the project outputs. Fourth, the resulting genotypic and phenotypic data sets will be combined in a Genome-Wide Association Scan (GWAS) to identify wild barley alleles that improve plant performance under stress. Because the gene resolution is extremely high this study will yield individual high confidence candidate alleles that putatively regulate the studied traits. Fifth, to validate the identified trait-improving exotic alleles in follow up studies, high-resolution offspring populations derived from the originally studied HEB-25 lines will be developed in Halle by backcrossing. The expected outcome of the BARLEY-NAM project will be beneficial in two directions. On one hand, the molecular regulation of new HEB properties will be studied in detail using the developed backcross lines. On the other hand, trait improving exotic alleles will be used in future breeding programs. This will ultimately lead to new elite barley cultivars with improved properties and, simultaneously, extend the biodiversity of our modern elite barley gene pool due to the incorporation of wild barley germplasm.

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

Medical advances have led to a welcome increase in life expectancy. However, increased aging populations pose new challenges and emphasize the need for novel approaches to aid and repair tissue lost through damage or disease. By 2020 approximately 20% of the UK population will be over 65 and the numbers of hip fractures worldwide will increase from 1.7 million in 1990 to 6.3 million in 2050. Thus, there is now an urgent need to understand how to maintain stem cells and then how to direct them into cell types we want such as bone and cartilage to enhance bone repair and improve quality of life for the patient. However, despite intensive research interest there is limited information on how to reproducibly maintain the bone stem cells (known as skeletal or mesenchymal stem cells); or indeed how to tell a bone stem cell to make bone or cartilage. Fortunately, human bone marrow contains these special skeletal stem cells. These stem cells can easily be obtained from these tissues and have the potential to form a variety of tissue types such as cartilage, bone, muscle, tendon, ligament and fat (for this reason, skeletal stem cells are currently one of the most exciting and promising areas for tissue engineering and reparative medicine and in the future this will allow stem cell-based therapies to be developed to treat or cure diseases). We are particularly interested in understanding how to maintain stem cells and to switch (differentiate) these bone stem cells to new bone and cartilage fat for regenerative medicine. For this approach to be successful, it is crucial to understand the way in which these skeletal stem cells change to become mature bone or retain their stem characteristics. Unlocking the molecular signals is the key to developing understanding and being able to undertake these studies in the absence of chemical cues is critical (to avoid confusing signals due to the chemicals used); we have powerful early data showing small nucleotides called microRNAs are key. MicroRNAs (miRNAs), are very small (only 18-25 nucleotides) non-protein-coding single-stranded RNAs that have the ability to regulate gene transcription. They have important and varied roles in many biological and disease related processes. There is new and exciting data to suggest i) a number of miRNAs are specifically expressed in stem cells, ii) they can control stem cell self-renewal, and, iii) they can control the ability of stem cells to form different tissue types. In addition we have data on nanotopographical surfaces (surfaces that can change or maintain our stem cells without any chemical cues) indicating the key role of microRNAs in keeping or changing bone stem cells. We will look at how these microRNAs affect cell behaviour and cytoskeleton (proteins involved in cell adhesion, spreading, metabolism and signalling), cell growth and differentiation. The results of this proposed project will open the way to modulate bone stem cells and thus drive the stems cells towards the desired cell type and provides exciting healthcare opportunities that will benefit many.

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

Trypanosoma theileri is a ubiquitous single celled organism present in cattle herds worldwide. Using knowledge of gene expression and protein trafficking in other trypanosomatid organisms, we have successfully developed T. theileri as a delivery system for vaccine antigens and therapeutics. Following recommendations from several Animal Health companies, we now aim to develop and demonstrate the potential of T. theileri as an effective and practical vaccine-vehicle, providing a clear route to commercialisation. In a two-step development path, Phase 1 will focus on technical refinements and analysis of the viability of the vaccine-vehicle during storage and distribution. The flexibility of the system to express antigens from a range of pathogens will also be established. Phase 2 will entail confirmation of the in vivo efficacy of the refinements developed in Phase 1. Combined the experiments will address key hurdles to the commercial development of this patented vaccine system.

Stomata are pores that provide for gaseous exchange across the impermeable cuticle of plant leaves. They open and close to balance the requirement for CO2 entry for photosynthesis against the need to reduce the transpiration of water vapour and prevent leaf drying. Stomatal transpiration is at the centre of a crisis in water availability and crop production that is expected to unfold over the next 20-30 years: globally, agricultural water usage has increased 6-fold in the past 100 years, twice as fast as the human population, and is projected to double again before 2030. Thus stomata represent an important target for breeders interested in manipulating crop performance. Stomatal movements are driven by solute transport - and consequent uptake/loss of water - across the cell membrane of the guard cells which surround the stomatal pore. Significantly, stomatal responses are slow compared to photosynthesis in the face of environmental fluctuations, especially of light. Improving water use efficiency (=amount of carbon fixed in photosynthesis/amount of water transpired) should be possible, without a cost to carbon assimilated in photosynthesis, if the speed of stomatal responses, especially to light, can be enhanced. However, the complexity of guard cell transport and its coupling to gas exchange and transpiration has presented a formidable barrier to systematic reverse-engineering aimed at enhancing stomatal responses through genetic manipulation and other means. Quantitative systems analysis offers an effective approach in silico to exploring the link between microscopic gene function and the macroscopic characteristics of assimilation and transpiration. As a first step to bridging this gap in understanding, we developed previously the OnGuard software for quantitative dynamic modelling of the guard cell. OnGuard models build explicitly on the wealth of molecular, biophysical and kinetic knowledge for guard cell transport and metabolism that drive stomatal movement; they accommodate stomata of different plant species, over the full range of conditions studied in the laboratory to date; and they have been shown to incorporate the real predictive power needed to guide experiments at the cellular and physiological levels that start with molecular manipulations in silico. The next major step towards establishing in silico strategies for crop design, based on our deep knowledge of stomatal guard cells, will be to establish and validate this computational link to incorporate carbon assimilation and water use efficiency at leaf and whole-plant levels. We propose now to develop such a strategy in models of the leaf, and scaling to the crop in the field, that capture CO2 uptake and transpiration. We will build the next-generation OnGuard models that incorporate CO2 uptake and transpiration, and we will incorporate computational statistical methods to accelerate model construction. Most important, the models will provide the essential micro-macro link to connect molecular function with physiological traits of the whole plant in water use and photosynthetic carbon assimilation and will enable scaling to the crop in the field. We will test this second generation of OnGuard models and validate their outputs to examine the longstanding hypothesis that significant erosion in the efficiency of water use by plants arises because of the mismatch in dynamic environmental responses between stomata and photosynthesis. Additionally, we will explore the connection of these traits with oscillations known to occur in stomatal aperture and in the signalling events (e.g. cytosolic-free [Ca2+]) previously documented at the cellular level in single guard cells. All studies will focus on the crop plant Vicia for which there is much data at the single-cell and whole-leaf levels, and on Arabidopsis for which we have mutants with well-defined effects on stomatal kinetics.

UK products composed of >51% whole grains can claim, "People with a healthy heart tend to eat more whole grains foods as part of a healthy lifestyle". A number of components present in wholegrain have been hypothesised to contribute to the beneficial cardiovascular effects associated with wholegrain consumption, including phenolic acids. Recent dietary interventions in humans using phenolic acid-rich foods (blueberry, coffee, champagne) are capable of improving the function of the circulatory system. Oats are a rich source of phenolic acids, although the degree to which they contribute to the human health benefits associated with wholegrain intake has not been investigated in humans. We hypothesise that phenolic acid-rich oats will be effective at inducing acute, beneficial improvements in circulatory function in humans thus contributing to a healthy cardiovascular function. Building on existing human work conducted in our laboratory, this study is designed to determine how phenolic acids may enhance human vascular function by focussing on how they modulate the function of the human circulatory system. The proposal will provide crucial information in three areas. Firstly, it will provide information regarding the variation in phenolic content that exists in both commercial varieties of oat products and newly developed ones. Secondly, we will test the ability of phenolic acid-rich oat intake to produce improvements in circulatory function in healthy humans using established, gold standard, clinical measures of blood flow around the body. Thirdly, we will strive to determine the processes in the body by which these beneficial effects on blood vessel function are mediated. The outcomes of this proposal have implications for exploiting the potential beneficial health effects of phenolic acids of the diet, particularly with relation to oat and other wholegrain intake. Oats are a sustainable, relatively cheap, UK product that offers an alternative to more expensive and less sustainable foods/beverages such as cocoa and berries, also shown to exert such biological effects on the circulation. The concept of healthy ageing is rooted at the core of this proposal through the application of sound dietary recommendations. Data emanating from the proposal are necessary before academia/industry may work to exploit such public health properties of foods in the future. There has been a large growth in the desire for such functional foods (21% market growth in the USA) and cause-and-effect data are essential for the development of EFSA/FDA health claims on specific oat products. The impact of bad diet/lifestyle and obesity on blood vessel health/disease is well reported and whilst wholegrain oat intake is recommended for the maintenance of a healthy circulation and heart, no studies to date have provided evidence for the health effects of the polyphenols in oats. Over three million Britons currently suffer from cardiovascular-related problems in the UK, with annual costs for the health system exceeding £30billion. Yet it has been estimated that 80%-90% of premature cardio-vascular events are preventable, and a healthy diet is paramount in this. We aim to work with a major food company, PepsiCo, to design and test a novel oat food/beverage with the potential to counteract the loss of correct blood vessel function. Understanding more about how specific diets may promote healthy cardiovascular aging will help us to define more effective public health advice and reduce the cost of services, by encouraging the population to make more informed healthy food choices.

Valorisation of low-value waste glycerol could transform the economics of biodiesel production.Key challenges for achieving this with biocatalysis are the rate of glycerol conversion to products, integrating bioprocess steps and tolerance of crude feedstock contaminants by host strains. We are industrial and academic bioprocess, cell-engineering, omics and bio-systems specialists. We will port pathways into Saccharomyces cerevisiae and Pichia pastoris for production of two valuable compounds from glycerol: chiral amino-alcohols (CAA) and 1,2-propanediol (PDO). Cells will be characterised with high throughput micro-scale process techniques and at scale. Transcriptomic and metabolomic data will be combined with process insights to dictate refactoring of both pathways and host 'chassis'. These steps will be repeated for second-iteration cells, driving better performance. New strains, processes and integrated methods will be of use to biodiesel and Industrial Biotechnology sectors.