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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.

Life-End summary Sustainable production of safe chicken is an international priority and preserving bird welfare is a key component of this. Current intensive (broiler) production can compromise bird health and welfare and food safety and there are strong links between poor bird welfare and the Campylobacter public health threat. Campylobacter is the most common cause of bacterial diarrhoea in the EU and despite millions of pounds of research funding it is estimated that contaminated chicken caused ~700000 human campylobacteriosis cases in the UK in 2013 with around 100 deaths. Infection is characterised by severe abdominal pain and acute (sometimes bloody) diarrhoea and costs the UK an estimated £1 billion per year. Campylobacter contamination of chicken takes two forms. First, surface contamination of carcasses leads to cross-contamination in the kitchen. Second, and perhaps of greater importance than currently thought, contamination within muscle and liver tissues, increasing the health risk by facilitating bacterial survival during cooking. Chickens in poor production environments or exposed to stress are more susceptible to Campylobacter and in such birds the bacteria show greater extra-intestinal spread to edible tissues, possibly as a consequence of disturbance to the gut environment. Therefore, improvements in broiler welfare have great potential to improve public health but there is an urgent need for information on the effect of stress to inform targeted interventions to reduce Campylobacter in broiler chickens. One acutely stressful event in the life of broilers, in any production stream, is harvest when birds are removed from the farm for slaughter. We define the process as comprising: food withdrawal, catching, transport and stunning by either gas or electricity. Although there is a growing body of evidence that these stressors can increase Campylobacter growth rates as well as extra-intestinal spread, there is a paucity of data on their relative importance or how they may select for particular types of Campylobacter. By examining the harvest processes using large scale industry-relevant experimental conditions, state-of-the-art genomics, molecular microbiology and mathematical modelling techniques, we will determine the impact of harvest on gut health in broilers. We will combine this with a study to identify bacterial genetic determinants involved in extra-intestinal spread of Campylobacter to edible tissues. We will quantify the relative impact of each stage of harvest on the gut bacterial population and the physiology and immunity of the birds, and investigate the role these play in controlling extra-intestinal spread of Campylobacter. This multidisciplinary research programme will enhance understanding of the influence of the harvest process on bird gut health and Campylobacter. The quantitative information and modelling will be used to provide direct advice to industry about the elements of the harvest processes that provide the best opportunity for interventions that will mitigate the ongoing challenge of Campylobacter contamination in chicken meat.

Understanding the regulation of root branching is of vital agronomic importance as it determines the efficiency of water uptake and nutrient acquisition. By using microCT imaging, we recently observed that the distribution of water in soil profoundly influences root branching. Plants achieve this via a novel mechanism termed 'hydropatterning' by our US collaborator Jose Dinneny (Bao et al, PNAS, 2014), where lateral roots (LR) form on the side of the main root in contact with water, but rarely on the dry side. LR hydropatterning occurs in both dicot and monocot roots and therefore appears to be a highly conserved adaptive trait. Mutant studies in Arabidopsis revealed that LR hydropatterning is dependent on the auxin response (transcription) factor ARF7. This project investigates whether LR hydropatterning is dependent on ARF7 (and its target genes) and whether this is a highly conserved mechanism in land plants using genetic and microCT imaging approaches, respectively.

The fine structural detail within cells has traditionally been examined using either scanning EM (SEM) or transmission EM (TEM). TEM is performed through a single thin section of the structure of interest and therefore often just gives a 'snapshot' 2D image. SEM gives a 3D-like image but only images the surface of the structure and not the detail therein. With TEM images, researchers often question what is happening above and below the single section and, to answer this, serial-sectioning is required. This is very time-consuming and technically difficult - the orientation of each section cannot be controlled and, therefore, 3-D-reconstruction of the digital images from these multiple serial sections requires frequent re-alignment that takes many hours. Even the most skilled technician will lose some sections resulting in incomplete series. Remarkably, these problems can be overcome by the 3View system, an automated sectioning and image capture system incorporated into a high resolution SEM. Computer-controlled, multiple sections and images can be made with one sample and 3-D-reconstructions performed without the need for realignment. This results in the best features of TEM and SEM being combined to show how cells are organized - both internally and in relation to their neighbouring cells and environment - at unprecedented levels of spatial information. Our immediate research projects using this equipment will cover a wide range of subjects - how do cells of the heart develop structures to beat every second? how do mitochondria - the 'batteries of the cell' - re-model themselves under different conditions? how can stem cells transform themselves into different cell types? how does the brain makes split-second judgments and more. Completely new understanding of these subjects will arise from being able to produce high resolution 3-D images of the structures of interest with this technology.

About 60% of the population have fasting blood cholesterol concentrations high enough to be a risk factor for coronary heart disease, which is responsible for the deaths of >60,000 people annually in the UK, many of which are potentially preventable with appropriate changes to diet and lifestyle. Water soluble types of dietary fibre (SDF), confer many health benefits; in particular, oat and barley beta-glucan (BG) is effective at reducing blood cholesterol and lipid concentrations and this has been recognised by European Food Safety Authority (EFSA). However, further processing of BG in foods can reduce its efficacy, and so a recommended intake of BG of at least 3 g/day requires large quantities (>100g) of unprocessed cereals such as oats and barley to be consumed daily. Most consumers would find this difficult or unpalatable, so approaches are required to incorporate BG into palatable, commonly consumed foods with demonstrable blood cholesterol and lipid lowering properties. Despite the clear health benefits of BG, its mechanisms of action are still not well understood. It has been suggested that the ability of BG to lower cholesterol is triggered by the interference of this polymer and other types of SDF with various stages of lipid digestion, thus reducing or delaying lipid uptake. This then disturbs the recycling of bile salts (bio-surfactants produced by the liver which aid lipid digestion) from the gut back into the liver. Bile salts are cholesterol derivatives, so increased production of bile salts in the liver could reduce plasma cholesterol concentrations. It is not clear which aspects of lipid digestion are affected by BG, and therefore which molecular properties are crucial for its functionality. The aim of this proposal is to determine the mechanisms of action of SDF and in particular BG on lipid digestion. Various mechanisms have been proposed, and they mostly involve the lipid digestion and transport processes. Therefore in vitro lipid digestion studies will allow us to study in detail all the different stages of lipid digestion including lipase activity and adsorption, bile salt adsorption and transport, and micelle formation and transport. The molecular and biochemical properties of BG and other other types of SDF will be determined using a range of state of the art techniques, including analytical centrifugation, and will allow us to determine which properties are important for functionality. Based on these findings, the effect of processing on BG will be studied to determine exactly why processing attenuates its ability to reduce blood cholesterol and lipids. This knowledge will then allow us to propose strategies by which SDF can be incorporated into palatable, manufactured foods and still retain their ability to reduce blood cholesterol and lipid concentrations.

Phoma stem canker, caused by the fungal pathogen Leptosphaeria maculans, is a damaging disease on oilseed rape in the UK, causing annual yield losses > £100M despite use of fungicides costing £20M. With recent loss of the most effective fungicides (e.g. Punch C) through EU legislation and predicted global warming, potential yield losses will increase. Use of host resistance to control this disease is becoming ever more important. However, new sources of resistance are often rendered ineffective due to pathogen population changes. The aims of this project are to monitor the emergence of new virulent races of L. maculans and to develop new control strategies to increase durability of host resistance. To maintain effectiveness of cultivar resistance against L. maculans, this project will use molecular technology to investigate the differences between different regions in distribution of virulent races of L. maculans in populations assessed from spore samples and from crop plant samples. Results will be used to guide deployment of cultivars with suitable resistance for the region where the corresponding avirulent pathogen races are predominant. Resistance against L. maculans relies on major resistance (R) gene-mediated qualitative resistance and minor gene-mediated quantitative resistance. The fungus L. maculans has high evolutionary potential to overcome host resistance. Resistance can be rendered ineffective in 2-3 years due to L. maculans population changes from avirulent to virulent. This project will investigate the molecular events leading to virulent mutations in L. maculans by exploiting new genomic information about L. maculans. Modelling work shows that the range and severity of phoma stem canker will increase under predicted global warming. To investigate the effects of environmental factors (e.g. temperature) on operation of different R genes for resistance against L. maculans, severity of phoma stem canker on cultivars with different R genes will be assessed in field experiments in different regions (i.e. different environments) and in controlled environment experiments at different temperatures. Recent work showed that R gene-mediated resistance against L. maculans (an apoplastic pathogen) operates by recognition of pathogen effectors through receptor-like proteins (RLPs). The sequences of R genes stable at increased temperatures and the genome sequences of host Brassica napus and its related species B. rapa and B. oleracea will be used to identify candidate R genes coding for RLPs. New knowledge obtained from this project will be used to develop strategies to improve control of phoma stem canker by using effective cultivar resistance. Improved control of this disease will benefit growers by reducing yield losses. It will also address the challenge of food security. The environment will also benefit from reduced use of fungicides.

The aim of this ICASE PhD project is to investigate the role of adipose tissue in hair follicle growth and cycling as a basis for novel therapeutic interventions. The research is in partnership with Unilever R&D. Adipose tissue is a key constituent of human skin whose functions extend far beyond energy storage and thermoregulation. Several mutant mouse models with defects in adipocytes have elucidated some of these previously unknown roles. For example, a genetic mouse model lacking early B-cell factor 1(Ebf1) exhibited reduced intradermal adipocytes. Hair follicles in these mice failed to re-enter the anagen phase of the hair growth cycle and remained in the resting telogen stage (Hesslein 2009). This demonstrates the importance of pre-adipocytes in the promotion of telogen to anagen transition during the hair follicle cycle in mice, and has raised increasing interest in the role of the adipocyte-hair follicle communication in the regulation of hair growth. However, the adipocyte-hair follicle communication in human skin, and how it may be manipulated in a clinically or cosmetically desirable manner, is entirely unknown. There is increasing insight into the importance of this bidirectional communication for hair follicle cycling and (murine) wave pattern formation, as well as adipocyte differentiation and function (Plikus 2008, Schmidt 2012, Donati 2014). As such, improved characterisation of this communication could provide novel targets and strategies for therapeutic intervention. The mechanism behind the potential interaction of adipocyte signals with the hair follicle is not well understood but platelet derived growth factor (PDGF) signalling may play a significant role. PDGFA mRNA is significantly elevated in adipocyte precursor cells (Festa 2011) and mice lacking PDGFA show a delay in hair follicle stem cell activation (Tomita 2006). There is strong possibility that understanding and influencing signalling of intradermal adipocytes in the hair follicle microenvironment could impact the hair follicle with potential benefits such as promoting hair growth & prolonging the follicle growing phase. This study will span the Centre for Dermatology Research and Unilever R&D facility, Colworth. Extensive training will be provided in histology, immunohistochemistry and state-of-the-art double-immunostaining and image analysis techniques. Carina will also become familiar with light, fluorescent and confocal microscopy, qRT-PCR and gene silencing in intact human skin/hair follicles.

United States

During the last twenty years, there has been an explosion in new microscopy techniques which exploit the high peak intensities from laser sources for excitation of fluorescent dyes used as markers in live cells. These methods, which are based on nonlinear optics, offer several advantages for the biologist over more traditional imaging techniques. These include imaging of deeper tissue thanks to longer excitation wavelengths, avoidance of damaging short-wavelengths, and an overall reduction in photo-bleaching. However, it has been generally accepted that these nonlinear microscopy methods must use a laser focused to a tiny spot which is then scanned around the specimen. This limits the capture rate of information to around 1 frame/second. This is a major limitation to the method for studying live cells, since rapid and important changes in the intra-cellular biochemistry are often missed. A few methods for increasing the imaging speed of nonlinear microscopy have been demonstrated, but only one is commercially available (which is essential when the technology is to be used in a biology research laboratory). This technique involves splitting a single high-intensity laser beam into up to 64 lower intensity 'beamlets' which are then scanned around the specimen, but this unfortunately can result in a 'patchwork quilt' effect which introduces unwanted artifacts into the images and can render interpretation and analysis difficult. To provide the advantages of nonlinear microscopy but at fast capture speeds, we propose to capitalize on innovations in sensor technology and use a less well-focused laser beam, which will illuminate the full image field. This 'wide-field' method is known to biologists, but in a linear (single-photon) rather than nonlinear (two-photon) approach, and therefore is a simple adaptation to existing instrumentation that is familiar to the end-user. The key difference in our technology over a conventional fluorescence microscope will be the light source, which we will change from a light-emitting diode to a high peak intensity laser (which we already have in our laboratory). We will also use small modifications to the microscope and add a sensitive scientific camera detector. Our calculations show that nonlinear excitation of fluorescence is possible at capture speeds of up to 100 frames/second. We will test this new technology with non-biological specimens initially, and then apply the method to two different cell types to study both fast and slow calcium signalling events. If we are successful, this technology is almost certain to change how cell biologists obtain images of their specimens which, in turn, will likely have a long-term impact on pharmacology and the development of new medicines.