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Biology is complex; cells are made up of 1000s of proteins, a similar number of metabolites and tens of thousands of genes. A goal of biological research is to understand how this complexity brings about the functions of life. One way to achieve this goal is to understanding the connections between the 1000s of components that make up cells. Measuring the connections between all the components is challenging, particularly because cells are dynamical systems that are constantly changing. Accurate descriptions of the dynamical network interactions that take place in a cell are required to make the advances required for improved crops for food security and new medicines. We have adapted a new tool set from Engineering to describe biological networks in a mathematical form. We make models of each of the connections which are used to predict how the system will change over time, which is very useful in discovering how cells respond to signals such as changes in temperature, hormones or drugs. Our new mathematical tool set allows researchers to identify and quantify the changes in a biological network, which can lead to the discovery of the gene(s) or pathways that are involved in responses to stresses or drugs and might underlie disease. Our new mathematical tool set will have wide utility in understanding a wide range of cellular systems, from the effects of drugs in humans to the response of a crop plant to environmental changes or attack by pests. Our development of a tool that measures how biological networks change is important for understanding biology, curing disease and improving crop plants to provide enhanced food security. We propose to develop this so called Nu gap analysis as a practical tool for biologists. In our implementation, we identify and describe connections in biological systems using simple liner models. The Nu gap measures the difference between the mathematical descriptions of the connections obtained in different conditions, such as following a response to a drug, or an environmental stress. To develop the Nu gap as a practical tool we will undertake a research programme that increases with complexity over time. This will permit rigorous testing, development and deployment of Nu gap analyses. First, we will perform theoretical analyses of the Nu gap on models derived from fabricated datasets designed specifically to assess the strengths and limitations of the Nu gap. This will inform as to where application of the toolset would be best, and conversely the situations where the Nu gap might be less informative. Having developed good theoretical understanding of the system, we will apply the Nu gap to real world data obtained by our laboratories. We will begin using data describing the circadian regulation of gene expression in the model plant Arabidopsis. A major goal will be to investigate the effect of a pharmacological and a genetic perturbation to the circadian system. Both profoundly affect the functioning of the circadian clock, but the mechanisms by which these affect the circadian clock is uncertain. We will move from investigating the fundamental properties of the circadian clock in the model plant Arabidopsis to using linear modelling and Nu gap analyses to describe the circadian clock in a major crop, barley. The circadian clock regulates many important agronomic traits such as flowering time, seed set and cold tolerance. Our studies have the potential to inform breeders of useful gene targets. Recognising that biological systems are more than a series of interactions between genetic components we will extend our analysis to incorporate the physiology of the cell, such as changes in the concentration of calcium in the cytosol, which act as key regulators of signalling in stressful conditions.

Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at www.rcuk.ac.uk/StudentshipTerminology. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.

The epidermis is the outer covering of the skin and plays an essential role in protecting our bodies from bacteria and other pathogens. It is made up of multiple layers of cells that are stacked on top of one another. The deepest cell layer, furthest from the skin surface, contains stem cells. Their role is to divide throughout our lives to make more cells that subsequently mature as they move through the upper cell layers. The most mature cells are specialised to form a protective barrier on the skin surface. If a tiny piece of skin is removed from the body and taken to the laboratory, it is possible to grow sheets of epidermis that have similar properties to normal epidermis and indeed these sheets can be used to repair burns and other types of skin wound. One interesting feature of human epidermis is that the stem cells are clustered in specific positions in the basal cell layer, which correlate with natural undulations in the boundary between the epidermis and the underlying connective tissue. As we age the undulations become much less pronounced, but in contrast in some common skin diseases such as psoriasis the undulations become more prominent. We would like to understand why the stem cells are clustered in this way and whether the size of the undulations affects their behaviour. To investigate this we will grow human epidermal cells on special surfaces made out of artificial materials, such as the rubbery substance used as bath sealant, that recreate the undulations. We will measure whether cells in different positions on these surfaces are more likely to remain as stem cells or to leave the basal layer and mature. We will investigate whether on substrates that resemble aged skin the cells show an altered ability to divide and mature. We will discover how the substrates direct the stem cells to behave by identifying the signalling events that take place inside the stem cells. We will also find out whether we can over-ride the effects of the substrates by artificially stimulating changes in gene expression inside the cells. The outcome of the project will be to explain, for the first time, why stem cells lie in specific locations in human epidermis and whether the information provided by their location contributes to the changes in the epidermis that are linked to skin ageing.

The twin objectives of RegulaTomE are to determine the importance of transcriptional regulation of the metabolic pathways defining quality traits in tomato and to identify such transcriptional regulators at the molecular level. The selected quality traits include antioxidant capacity which impacts shelf life and nutritional value as well as traits determining fruit flavor and over-ripening which influence organoleptic properties and shelf-life. RegulaTomE will use the natural variation available in introgression lines (ILs) resulting from wild species crosses to tomato to assess the importance of transcriptional regulation, identify additional regulatory genes and assess underlying genetic and epigenetic variation. RegulaTomE will assess the potential for direct or indirect use of natural variation from an untapped wild species resource for crop improvement. To identify genes regulating metabolic pathways using the Solanum lycopersicoides ILs, and to capture genetic and epigenetic variation for application to gene discovery and tomato improvement, resources need to be developed, including a genome reference sequence for S. lycopersicoides and metabolite, DNA methylation and transcriptome profiles of IL fruit. RegulaTomE will lead to regulatory gene identification and new tools for metabolic engineering of fruit quality. The natural variation in fruit quality revealed by the S.lycopersicoides ILs could be applied to tomato improvement either directly through introgression into cultivated varieties or indirectly through the identification of target loci and corresponding allelic variation making positive contributions to quality traits within S. lycopersicum breeding germplasm. The outputs of RegulaTomE will provide a framework of understanding as well as tools, in the form of genes, target loci and molecular markers, to support development of longer shelf-life, more nutritious and more flavorsome fleshy fruits in other horticultural crops.

The use of antibodies as catalysts has long been the topic of research and discussion but initial attempts to utilise the concept in useful therapeutic or industrial settings has so far proved disappointing [1]. Significantly reduced catalytic rates in comparison to traditional enzyme catalysts remains the key reason for this lack of progress but recent technological developments that have launched a new generation of antibody formats and their use in "imprinting" enzyme catalytic sites [2] suggest that this topic is now worth revisiting. The VHH antibody format, for example, is capable of maintaining functional activity within a cell and can be expressed at high concentrations, which highlights its potential for overcoming any intrinsic low catalytic rate under specific circumstances. In particular, they may find use in catalysing useful synthetic process within engineered microorganisms when it is impractical or impossible to do this with an enzyme. It is also possible to envisage the use of libraries of intracellular antibody catalysts to generate libraries of natural product-like chemical compounds for use in inhibitor screening against pharmaceutically-relevant targets. Catalytic antibodies may also find therapeutic utility in degrading aggregated or misfolded protein in diseases such as Alzheimer's Disease [3]. This BBSRC-sponsored PhD study proposal will utilise the synthetic biology expertise at SYNBIOCHEM (Manchester Institute of Biotechnology) and the antibody format expertise and libraries at UCB to investigate this novel area of research and lay the groundwork for the future synthetic biology uses of catalytic antibodies. The work will be divided into three main areas of research: 1. The use of non-proprietary UCB antibody phage libraries to identify catalytic antibodies in reactions of particular interest to SYNBIOCHEM and where they already possess protein and crystal structures that could be used in the imprinting process. Early targets will be centred on conformational catalysis in driving the synthesis of different monoterpenoid scaffolds from geranyl diphosphate. In the second phase of the work we will target enzyme activities from enzymes that are known not to express functionally in E. coli. A good example is progesterone 5B-reductase, which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily, and has been considered a key enzyme in cardenolide biosynthesis since it is the first stereospecific enzyme of the pathway leading to 5B-configured intermediates. Both these enzyme classes are major targets for metabolic engineering. Importantly, the second phase study will establish alternative routes to catalytic modules/parts based on antibody technology, for which expression of the natural enzyme is not possible in standard expression hosts such as E. coli. 2. The intracellular expression in microorganisms of VHH catalytic antibodies to test if they can be used for synthetic biology purposes in the production of novel chemical products 3. A detailed analysis of the potential to use catalytic antibodies to generate libraries of useful small molecules based on the chemically diverse monoterpenoid scaffold for use in generating "hits" against a therapeutically relevant target (to be defined by UCB). We envisage that a 6 month placement at UCB will be essential for the student to gain knowledge in the use and application of our antibody libraries and their intracellular expression.

Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at www.rcuk.ac.uk/StudentshipTerminology. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.

One of the greatest threats to public health in the 21st century is the rise of multi-drug resistant bacterial infections, which has been caused by a shortage in new types of antibiotics, as well as the improper use of antibiotics in medicine and agriculture. This has prompted the World Health Organisation to warn that "the need for action to avert a developing global crisis in health care is increasingly urgent" and the UK's Chief Medical Officer, Prof. Dame Sally Davies, to declare that "we are also not developing new drugs fast enough". One incredibly rich resource for antibiotics are microorganisms that live in soil, and the majority of clinically used antibiotics come from these bacteria. These bacteria have evolved the ability to produce natural products with excellent antibacterial activities, as the ability to kill surrounding bacteria is a big advantage when competing for nutrients. One compound I am currently researching is an antibiotic called bottromycin. The molecule has some highly unusual structural features and is a very active antibiotic towards dangerous infections like the "superbug" MRSA (methicillin-resistant Staphylococcus aureus), which kills tens of thousands of people worldwide each year. Bottromycin has actually been known for many years but problems with its stability and availability have prevented it from being used clinically. However, its entirely novel structure and mode of action make it highly promising antibiotic of the future. An understanding of bottromycin biosynthesis would provide the information necessary for the pathway to be modified to alter the structure of this antibiotic and increase the amount that can be made. Natural products are produced by the action of a series of enzymes (proteins), which are encoded by genes (DNA) in the bacterial genome. I have previously identified the genes required for bottromycin production and will now focus on the details of each biochemical step. I will also develop methods to make new bottromycin-like compounds. From a purely scientific perspective, the biochemical steps in this pathway are highly unusual, and an understanding of the enzyme mechanisms will increase our understanding of enzyme function. The experimental techniques involved in this work include the fermentation of bacterial cultures, the purification of enzymes and the analysis of biochemical reactions using mass spectrometry, which determines the mass of a compound and can provide important structural information.

Cell migration is important for normal development of animal embryos and tissue repair and regeneration in adults. It is controlled by environmental cues provided by diffusible factors and interactions of cells with the structural components of the three-dimensional extracellular matrix (ECM) through which they move. Two important and interacting classes of adhesion receptors in animal cells are the integrins and the syndecans. Integrins are a large family of heterodimeric molecules, the nature of the aB dimers determining their specificity for ECM ligands. Syndecans are a family of four heparan sulphate proteoglycans which bind growth factors and also act themselves as ECM-binding receptors. These two classes of molecules work together to control cell adhesion and its signalling consequences, and in particular integrins a5B1 and aVB3 and syndecan-4 are required for cell adhesion and migration on fibronectin-rich matrices via the formation and turnover of focal adhesions. Another recently discovered class of molecules known as the ADAMTS metalloproteinases (a disintegrin and metalloproteinase with thrombospondin-like motifs) also influence cell migration. The project will address the hypothesis that ADAMTS metalloproteinases regulate cell movement by affecting cell surface syndecan-4 presentation or function, leading to changes in integrin a5B1 and aVB3 trafficking and focal adhesion dynamics. This project will provide training in advanced cell and molecular techniques including immunolocalization, fluorescence activated cell sorting (FACS) and timelapse videomicroscopy to study the adhesion and migration of mouse embryo fibroblasts (MEFs) that are deficient in either integrins a5, B3, syndecan-4 or ADAMTS-1 or -15.

Priority area: Basic Bioscience Underpinning Heath Keywords: LC-MS, pulmonary circulation, oestrogen metabolism Abstract: Gender exerts profound influences on vascular health and 'healthy ageing'. Women are more at risk of developing cardio-pulmonary dysfunction and this may increase post-menopause. Few studies have, however, directly examined the possibility that gender and age may induce changes on the normal pulmonary vascular function and oestrogen metabolism that might pre-dispose women to vascular risk factors. Here we will determine gender differences in the normal function of the pulmonary vasculature, in particular the role & influence of oestrogen, oestrogen metabolism & oestrogen metabolites. Whilst there are several papers and reviews concerning the influence of oestrogen on the vasculature, the influence of oestrogen metabolites on the normal ageing vasculature is very under-researched. Likewise, the influence of gender on normal proliferative signalling pathways is largely under-investigated. Our preliminary data on human pulmonary artery smooth muscle cells (hPASMCs) suggests there are gender differences in signalling pathways & that oestrogen may be the reason for the gender differences. We have recently demonstrated that oestrogen itself can decrease signalling in hPASMCs through the BMPR2 pathway increase MAPK signalling; hence proliferation of female hPASMCs is greater than in male cells. We have shown that microRNA expression in hPASMCs can be influenced by gender and oestrogens. For example, microRNA96 is decreased in hPASMCs from female lung & this causes an increase in serotonin-induced proliferation via the 5-HT1B receptor. It is emerging that oestrogen metabolites may play a more influential role on normal vasculature function than oestrogen itself. One limitation to these investigations is our ability to actually measure oestrogen metabolism and metabolites in vascular tissue. Over the last two year we have developed a novel HPLC/LC-MS 'steroidomic' method for assessing oestrogen metabolism in hPASMCs. We can now apply this technology to understand the role of oestrogen & oestrogen metabolism in the normal function and ageing of pulmonary arteries. Year 1-2. The student would assist the development of LC-MS techniques to analyse oestrogen metabolites in hPASMCs & plasma We have already identified some metabolites that accumulate in PASMCs & that have either pro- or anti-proliferative effects & at first we will examine these (e.g. 16-OHE1/2, 4-OHE1/2, 2-OHE1/2, 2 and 4-MeOHE1/2). Following this, measurements will be made in plasma at days 7, 14, 21 and 28 of the menstrual cycle from normal healthy volunteers. Similar analysis will be made in samples from post-menopausal women and age-matched men. The student will also examine the expression of key microRNAs in these samples, especially those that may interact with oestrogen metabolism or action (e.g. miRNA-22, miRNA-206, miRNA-27b). Year 2-4. hPASMCs will be derived from healthy men & women & these will be grouped according to age. This will be in collaboration with Nick Morrell (Cambridge). The effects of normal ageing and gender on basal and stimulated oestrogen metabolism will be determined. The influence of oestrogen synthesis & metabolising enzymes on this will be determined by applying aromatase inhibitors such as anastrozole and CYP1B1 inhibitors such as TMS and /or by siRNA techniques to silence these enzymes. In addition, the activity and expression of key signalling pathways will be determined (BMPR2, pERK, pAkt, reactive oxygen species etc). The synthesis of oestrogen will be determined by examining aromatase expression & via aromatase activity assay. The student will also repeat key experiments on other pulmonary cell types such as fibroblasts & also vascular smooth muscle cells from human resistance arteries (from gluteal biopsy material). High fidelity training in in vivo skills is also available if the the student wishes this.

Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at www.rcuk.ac.uk/StudentshipTerminology. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.