The study of metabolomics is a relatively new technique to identify metabolites that are produced by body processes and are involved in regulatory metabolic pathways. It is highly likely that certain metabolites will be altered in disease or during drug therapy and thus, being able to measure and identify these (biomarkers) will be critical for future health and disease diagnostics. For metabolomic profiling to be of value for clinicians in the diagnosis of disease, however, it is essential to establish accurate baseline data from healthy controls. Correct interpretation of metabolomic data will require a thorough knowledge of the impact of time of day as well as the effect of a person's internal biological (circadian) timing system on the metabolomic profile. Biological circadian rhythms and time of day variation occur in most physiological markers e.g. melatonin, cortisol, glucose; metabolites identified in plasma and urine will be no exception. However, to date there has been no systematic study of circadian variation in the human metabolome using established circadian protocols. In addition how a typical living environment (light/dark cycle, sleep/wake cycle, meals) affects metabolomic profiles needs to be determined. Using strictly controlled laboratory studies in healthy volunteers and cutting edge metabolomic technology we thus aim to characterise the effect of the circadian clock, the time of day, the light/dark environment, meals and sleep on rhythmic and non-rhythmic metabolites identified in plasma and urine. Metabolites that show rhythmic circadian and time-of-day variation (cycling) and those that do not (non-cycling metabolites), as well as metabolic processes affected by sleep and sleep deprivation, will be identified through the use of cutting edge, highly sensitive, liquid chromatography-mass spectrometric (LC-MS) techniques. At Surrey we have proven expertise in conducting circadian and sleep deprivation experiments. Using our recently established LC-MS methodology (Surrey and ICR) we have pilot data in healthy volunteers kept in controlled conditions similar to the proposed studies. Significant time of day variation has been observed in at least 20 plasma and 20 urine metabolites, based on Orthogonal Projections to Latent Structures (OPLS) analysis (Simca Software, Waters). Therefore in terms of expertise, clinical and analytical facilities and technical skills the proposal is feasible. Identification of metabolite rhythms and how these are affected by external factors (time of day, wakefulness, sleep, environmental lighting, regular meals) will provide reliable baseline data which will be crucial for the future use, and correct interpretation, of metabolomics in the detection and treatment of human disease. In addition, our Project Partner (Erasmus MC University Medical Center (EUMC), Rotterdam) will perform proteome and transcriptome analysis on selected samples across the 24 h day from both studies with a view to combining the data. The biological samples, metabolomic database and research findings will be shared and disseminated for the benefit of a wide range of professionals involved in disease diagnosis and treatment (e.g. clinicians, clinical biochemists) which will ultimately benefit society.

Caveolin-1 is emerging as a key molecule in breast cancer. Interestingly, previous studies have found that caveolin-1 can both help and hinder the formation and spread of breast cancer. Caveolin-1 also has an impact on how cancers respond to particular treatments. However, its exact role is not fully understood. In this study, a combination of molecular techniques will be used to assess the nature, expression and role of caveolin-1 in a large number of breast cancers of different types. Working within a team of experts at one of the world‘s leading cancer hospitals and research institutes, our project intends to find out if alterations in caveolin-1 and its associated processes can be used to predict the outcome of patients with breast cancer. We will undertake detailed experiments to investigate how caveolin-1 works inside breast cancer cells and the impact it has on other processes important for the development and progression of cancers. Improvements in our understanding may allow better selection of treatments for particular patients. This project will also determine if caveolin-1 can be directly targeted by newer treatments. Women who are particularly likely to benefit are those who do not respond to currently used hormonal or biological treatments.

Imaging technology is required to enable personalised adaptation of treatment for improved outcomes for patients with soft-tissue sarcoma, specifically techniques which can identify those patients who would benefit from neoadjuvant therapy prior to surgery, accurately assess tumour response, and enable expedient switching to a more efficacious agent/treatment regime as necessary. The objective of this project is to use advanced, clinicallytranslatable multi-parametric MRI strategies, coupled with computational pathology, to define imaging biomarkers associated with the heterogeneous phenotype that develops within patient-derived xenograft models of soft-tissue sarcoma, and for the assessment of tumour response to neoadjuvant therapy.

Mitochondria are specialized compartments found within cells that convert fuel into energy. During processes such as development, where energy demands are high, insulin activates multiple biological switches that ultimately generates new mitochondria. Decreases in mitochondrial number and function, and/or the accumulation of damaged mitochondria accelerate the ageing process. Moreover dysfunctional mitochondria can drive the onset of a chronic inflammatory state that underpins to the pathogenesis of type 2 diabetes, cancer, and neurodegenerative disorders. Insulin-resistance is a hallmark of such age-associated diseases strongly suggesting that suppression of insulin-mediated mitochondrial production is a common route to ageing and disease. However, the biochemical reactions that link insulin stimulation to mitochondrial health are largely unmapped. Gaining a systems-level view into the signaling networks that link insulin stimulation to mitochondrial production is essential. We aim to use a combination of genetic and phosphoproteomic technologies to map key biochemical reactions that are critical for insulin-mediated mitochondrial homeostasis. We are optimistic that a comprehensive understanding of these reactions will allow us to ultimately develop means to manipulate insulin signalling networks to improve well-being during the ageing process. Using a novel genetic screen that we have recently developed, we first aim to comprehensively identify all genes required for mitochondrial health. This cost-effective screen that use computational algorithms to automatically measure mitochondrial shape in millions of single cells following inhibition of each gene in the genome one at a time. If inhibition of a particular gene leads to abnormally shaped mitochondria this strongly suggests a role for this gene in promoting mitochondrial homeostasis. We have recently developed a cutting-edge mass-spectrometry based technology to monitor levels of protein phosphorylation on all proteins in a cell. Kinase proteins phosphorylate different proteins as a means to turn different proteins "on" on "off". By monitoring all phosphorylation events that occur in cells following stimulation of insulin, we can then determine which proteins are likely to be regulated by insulin. By completing these two aims we will thus identify sets of proteins that are both involved in mitochondrial health (through genetic screens completed in Aim 1), and that are regulated by insulin (through completion of Aim 2). In order to determine the kinases that are responsible for phosphorylating substrates identified in Aims 1 and 2, we will then combine genetics and mass spectrometry to monitor protein phosphorylation following systematic inhibition of all kinases in insulin-treated cells.

The genetic information embedded in our DNA is efficiently decoded into proteins via a RNA intermediate. In eukaryotes, the process of faithfully transcribing DNA into RNA is carried out by three distinct transcription machineries, RNA polymerase I, II and III. Each RNA polymerase is responsible for the transcription of a specific subset of genes. RNA polymerase III is the enzyme devoted to the transcription of short essential RNAs which are involved in fundamental cellular functions, such as the tRNAs and the 5S rRNA. To efficiently transcribe the eukaryotic genome, RNA Polymerase I, II and III rely on distinct sets of transcription factors, which selectively recognise a specific class of genes and accordingly recruit the cognate RNA polymerase. During the last twenty years, the eukaryotic transcription machineries have been extensively characterised: a detailed map of RNA polymerase II structure and the overall architecture of RNA polymerase I and III have been obtained. This structural information has been instrumental in understanding the function and the mechanisms of eukaryotic transcription machineries. Nevertheless, a very scarce amount of structural information is available regarding the mechanisms by which class-specific transcription factors recruit and assist their cognate RNA polymerase to form a transcriptionally competent pre-initiation complex. As a consequence, the process of transcription initiation remains obscure and, for this reason, we are aiming to obtain structural and functional information of Pol III pre-initiation complexes using an integrated structural biology approach. We are focussing on the Pol III system, since Pol III core pre-initiation complexes are particularly stable. Specific transcription factors required for the correct assembly of a pre-initiation complex are stably associated subunits of the Pol III enzyme, whereas in the Pol II system the analogous transcription factors are dissociable. To this end, we were able to isolate and purify crystallization-grade Pol III core pre-initiation complexes, using endogenous yeast RNA Polymerase III and transcription factors produced recombinantly. To study the structures of these large macromolecular complexes, we are integrating cryo-EM and crystallography, an approach that recently enabled us to structurally and functionally characterize the Pol III core enzyme. The structural information will be critical in order to understand the underlying mechanisms which govern the assembly of functional eukaryotic pre-initiation complexes which are able to accurately initiate transcription. As transcription initiation is a highly regulated process, our findings will have a profound impact on the field of gene expression regulation. Additionally, since the assembly of Pol III initiation complexes is a process often deregulated in cancer cells, our findings will provide an opportunity to develop and test new anti-cancer therapies based on normalization of Pol III transcription levels. Furthermore, Pol III products have been shown to act as essential effectors of the target-of-rapamycin (TOR) pathway to control cellular and organismal growth, hence the output of the proposed research can impact other important biological processes controlled by TOR, such as stress response and aging.