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This project focuses on translating an existing bioreactor design that can be used to grow a variety of tissues in the laboratory. In particular, we will be optimising the growth of cardiac muscle and neural tissue in a 3D perfusion bioreactor whilst incorporating electrical stimulation regimes. The bioreactor configuration is entirely novel and we will strengthen and finalise our existing IP and file a patent as a result of this grant. This project will engage with a variety of industrial collaborators in market steering capacity and tissue phenotype maintenance proof of concept data will be gained.

Recent advances in fluorescence microscopy have led to significant improvements in our ability to locate and discriminate small objects. Super-resolution is the term used for technologies that can resolve objects that are smaller or closer together than was previously thought possible according to the laws of Physics. It is now possible to see objects with nanometre resolution and precision as well as image at depth in living tissue and examine rapid biological events at high speed. Achieving all of these improvements at the same time is the "holy grail" of optical microscopy but this has proved practically difficult. At the University of Leicester, we have developed a new hybrid technology called SuperRAMP that allows high speed, multicolour, multiphoton, super-resolution imaging deep in living tissues. Super-resolution, Random Access MultiPhoton microscopy (SuperRAMP) is a unique technique that combines patterned illumination with mathematical methods to pinpoint discrete objects with nanometre precision at very high speeds. Devices that use sound waves passing across crystals are used to project patterns of infra-red light, which can pass through specimens better than visible wavelengths of light and produce better penetration into deep tissues. These patterns are processed mathematically to produce high-resolution images. SuperRAMP improves both the lateral (xy) and depth (z) resolution by 2-3-fold compared to standard microscopes to an absolute lateral resolution of 120 nm. It can collect two colour images at speeds of around 10 frames per second and from super-resolved points of interest at thousands of samples per second. No other microscope available today can operate at this resolution, at depth, and at this speed in living tissues. SuperRAMP will be combined with a second, complementary method called dSTORM to provide even higher resolution. dSTORM uses the same mathematical techniques used for SuperRAMP microscopy but without scanning. Although it takes longer to create an image, it has a higher resolution of 20 nm. We will develop a super-resolution, multi-user facility that will make these exciting technologies available to researchers at Leicester and to the wider community. This will help us to drive academic excellence amongst existing BBSRC funded researchers in projects that range from studies of the synthesis of new proteins, the processes of cell division, mechanisms of cell-cell communication in the central nervous system, the neurological bases for development and behaviour - including circadian clocks that control daily rhythms - in model animal systems including locusts, zebra fish, fruit flies, rats and mice. This facility will support research collaborations within the Midlands (Nottingham, Leicester and Warwick) and further afield (Cambridge) and allow us to strengthen our commercial impact by developing a show case facility for functional, super-resolution imaging. Having developed this revolutionary technology, we are uniquely placed to establish a world-leading centre of excellence for functional, super-resolution imaging.

Understanding evolutionary relationships and how characteristics of species (e.g. behaviours, genomes, morphological characteristics and proteins) evolve over time is a fundamental pursuit, either directly or indirectly, for all biologists. Computational tools to study how species characteristics change over time are called comparative methods. Among other things comparative methods are used to reconstruct ancestral forms, calculate how fast (or slow) characteristics change through time and to test if the evolution of species characteristics are correlated. Comparative methods are used thousands of time each year in scientific publications by biologists from all research areas. Recent advances in molecular sequencing technology and computer power have produced large and highly detailed maps of how species are related to each other. These maps are represented in a tree like form analogous to a family tree, they are known as phylogenies or phylogenetic trees. Phylogenetic trees are used in conjunction with species characteristics and comparative methods to help biologists infer historical processes of evolution. In 2013 two of the largest phylogenies were published, a near complete phylogeny of birds, comprising of almost 10,000 species and a large fish phylogeny of 8,000 species. These join a mammal phylogeny 5,000 species (2007), a 55,000 species tree of plants (2009) and a 6,000 species phylogeny of amphibians (2012). In contrast in the early 2000s a phylogeny of 100-200 species was considered very large. While the data and computing power have advanced inordinately over the last 20 years, the underlying statistics used in most comparative methods analysis has failed to keep pace. The statistical framework was laid down when a 30 species tree were considered large. This means that the vast majority of comparative methods assume that evolutionary processes are constant and homogeneous through time and through the tree. This assumption was not unreasonable when first introduced, as the available phylogenies consisted of a small number of closely related taxa which covered a narrow time period. Today the size of available phylogenies have grown enormously, they now cover more divergent groups and larger time frames and include a comprehensive sample of species. Using these trees we can now see that the homogenous assumption has been shown to produce incorrect results and hides important evolutionary information. Consider the evolution of body size in mammals, traditional comparative methods assume a homogeneous evolutionary process over hundreds of millions of years, affecting all species, at all time periods the same. But the evolutionary processes affecting some groups have been shown to be radically different, for example, flight in bats limit their body size, while being aquatic allows body size to increase. The assumption of a homogeneous process creates an averaging effect which is unable to detect important changes in evolutionary processes and produces results which are known to be wrong. This project will develop novel statistical methods which remove the assumption of a homogenous evolutionary process across the phylogenetic tree and through time. The methods will not only more accurately model heterogeneous evolutionary processes but of equal importance is their ability to automatically detect, without prior knowledge, the number and location of these shifts. The ability to automatically detect changes in evolutionary processes provides valuable biological insights allowing researchers to understand evolutionary processes on a finer scale than previously possible. These methods will directly benefit the thousands of researchers using comparative methods and bridge the gap between advances in data and the methods used to analyse them.

Ever large pieces of DNA such as genes and gene clusters are required for Synthetic Biology, and these are normally made by a combination of chemical and biochemical methods. The chemical methodology is required at the start of the process to generate very short pieces of DNA (oligonucleotides) by automated solid-phase methods. These are then used to build bigger pieces of DNA by biochemical methods that are based on the polymerase chain reaction (PCR amplification). The chemical synthesis of DNA can lead to damage which results in mistakes (mutations) in the final DNA product, and to avoid this the DNA has to be repaired by various enzymes. This is tedious and slows down the overall process, increasing costs and limiting the size of DNA that can be made. In this project we will analyse DNA made by modern ultra high throughput chemical methods and optimise the process to minimise mutations. We will also explore a different way to make large pieces of DNA; enzymatic ligation. In this process DNA constructs with modified bases can be made, which are very useful in gene expression and biomedical studies. These cannot be made by PCR amplification which erazes the modifications. Such modified DNA can only be properly made from highly pure oligonucleotides in very large numbers, placing stringent requiremenst on high-throughput oligonucleotide synthesis. Overall this project will greatly increase the capacity, quality and efficiency of DNA synthesis and is highly relevant to Synthetic Biology Centres in the UK and beyond.

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.

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 https://www.ukri.org/apply-for-funding/how-we-fund-studentships/. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.

Introduction. This PhD project examines how our internal clocks translate external timing (such as the light and dark environment) into complex physiological rhythms. It will specifically address the impact of clock disruption on mammalian physiology, and test whether pharmacological targeting of the clockwork may counteract environmental or pathological clock disruption. These studies closely align to the BBSRC's strategic initiatives on understanding biological rhythms and lifetime of health and build on a long-standing and successful collaboration between the University of Manchester and Pfizer. Background. Daily 24-hour rhythms are present in virtually all aspects of our behaviour and physiology, ranging from our sleep-wake cycle, to fluctuations in body temperature, blood pressure and circulating hormones. These rhythms are driven by internal timing systems (circadian clocks) that run throughout the body, and act within each tissue to orchestrate organ function and rhythmic activity. In mammals, circadian timing is headed by a 'master clock' located in a small area of the brain called the suprachiasmatic nucleus (SCN). Importantly, the SCN synchronises clocks in the rest of the brain and body, so that the activities of different organ systems are coordinated with each other, as well as with overriding behavioural cycles (i.e. when we eat, when we sleep, etc.). The SCN also keeps in time with the environment through direct relays of photic information from the retina. Understanding how the circadian system works, and how it directs diverse physiological pathways cross the body has become increasingly important because we now recognize that disruption of our clocks is commonly observed with many pathological conditions. For example, lifestyles that disturb these clocks, such as shift-work, increase the incidence of diseases including cancer and diabetes. Circadian disruption is also recognized as an important feature of many neurological disorders in including dementia and bipolar depression. Methodology. This PhD proposal will examine how internal timers align our physiology to the environment and what the consequences are when that alignment is disrupted. Importantly these studies will detail circadian clock function at both a molecular and physiological level. At a molecular level we will use clock-gene reporter systems and longitudinal measures of gene/protein expression to detail how the molecular clockwork in different tissues responds to changes in environmental inputs (such as light or temperature). These studies will be paralleled by comprehensive measures of behavioural and physiological rhythms (e.g. locomotor activity, thermogenesis, feeding behaviour, metabolic rate), using models of environmental challenge (e.g. repeated shift of the light/dark cycle; akin to chronic shift work) and genetic models of inappropriate entrainment (e.g. CK1etau and Aft mutant mice; which model human familial advanced sleep phase and delayed sleep phase). This will also allow us to test how clock re-setting stimuli and drugs (e.g. CK1e antagonists) impact on a broad-spectrum of physiological rhythms. These studies will benefit from an exciting new method to track oscillations in gene activity in free-moving mice. This is achieved by injecting a virus containing a light-emitting gene, which oscillates in response to activation of a specific clock-driven or metabolic pathway. Thus, we can track how the core clockwork of a given tissue responds to altered environment and/or to pharmacological targeting.

Summary: The transition from first-generation (1G) bioethanol to a more productive and sustainable bioeconomy based on plant biomass is essential for Brazil to remain at the forefront in clean energy production and to strengthen security of food and energy supplies, create jobs, and mitigate climate change. A key challenge associated with the transition from 1G to second-generation (2G) biofuels is the difficulty to release sugars from cell walls to produce bioethanol with economic viability. Understanding the factors that govern recalcitrance and engineering cell wall architectures and hydrolytic enzymes for enhanced sugar release is crucial for the commercial exploitation of lignocellulosic feedstocks and realization of the bio-economy concept. Another important aspect is the development of biomass crops that maintain biomass productivity and quality under water-scarce environments unsuited for growing food crops. This aspect is even more important in a scenario of predicted climate change, where we need to quickly adapt crops to challenging environmental conditions. Activities comprise a mix of networking, research and landscape-scoping activities, integrated between the partners, across three different work packages: WP1: Tailored enzyme cocktails matching feedstock & pretreatment. The aim within the framework of this 1-year award will be to provide preliminary data on the potential of cocktail-feedstock-pretreatment matching. Current commercial enzymes are based on the one-size-fits-all approach. Since enzymes are a major contributor to biomass processing costs, creating customized enzyme cocktails that are tailored to specific pretreatments and feedstocks, will reduce enzyme loading and therefore processing costs. This WP will deliver proof of concept for the enormous potential of matching enzyme cocktails to feedstock and pretreatment. WP2: Engineering the hydrolytic enzymes and feedstocks of the future. Synthetic biology offers exciting engineering opportunities to facilitate the release of sugars from plant cell wall biomass. One example is that of "biological pretreatment", a concept based on exploiting and redesigning some of the endogenous hydrolytic enzymes and processes already taking place in plants. Another attractive strategy to improve cell wall deconstruction is through the in planta expression of thermostable cell wall degrading enzymes. We will organize a workshop to effectively capture the potential of such synthetic biology approaches and produce a strategy document based around engineered ideotypes for both "in planta deconstruction" and "multifunctional hydrolytic enzymes". The outcomes of this work-package provides the foundation for further implementation of synthetic biology approaches in the area of plant cell walls and will position the partners at the forefront of this emerging research area. WP3: Effect of environmental and genetic factors on cell wall biomass quality and conversion. The composition and architecture of cell wall biomass can differ significantly depending on tissue, species, cultivar, and environmental conditions. Mapping variations in biomass quality is particularly important in the context of climate change and developing sugarcane varieties suitable for cultivation on marginal land. The integration of cell wall phenotyping data with those from saccharification assays will provide essential information on how differences in biomass tissue, varieties/genetics, and environmental conditions impact on cell wall quality and biomass deconstruction into its components sugars. The outcomes of this controlled environment experiment represent a platform for the translation and design of future field trial studies focussing on utilizing marginal land available in Brazil.

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 https://www.ukri.org/apply-for-funding/how-we-fund-studentships/. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.

Anaerobic ammonium oxidation or 'anammox' is the most recent addition to processes recognised in the biogeochemical nitrogen cycle (1,2). Originally discovered in a Dutch waste-water treatment plant, anammox bacteria combine nitrite and ammonium to make N2 in the absence of O2. It has since become clear that these bacteria are metabolically dominant in O2-minimum zones across the world's oceans and may produce one out of every two N2 molecules released annually into the atmosphere. Despite the environmental and biotechnological importance of anammox there is very little knowledge of the enzymology underpinning the process. This exciting and ambitious project builds on recent success in the purification of novel anammox enzymes and has the aim of resolving the structural, catalytic and redox properties of these enzymes (1-5). The successful applicant will be a talented biochemist or chemist with an enthusiasm for metalloproteins and enzymology who will develop advanced skills in the characterisation of redox enzymes by spectroscopic (EPR and MCD), spectro-electrochemical and protein film voltammetric methods. Research will be performed within the vibrant Centre for Molecular and Spectroscopic Biochemistry under the supervision of Professor Julea Butt and Dr Myles Cheesman in the School of Chemistry, University of East Anglia and in collaboration with Dr Jan Keltjens, Dr Boran Kartal and Prof Mike Jetten, Nijmegen University, NL.