Aim The aim of the project is to design and test novel high-throughput, high coverage strategies for quantitative mass spectrometry on complex biological samples, thereby bringing proteomics technology closer to the data speed and quality available in the genomics field through gene expression arrays. Techniques involving new labelling compounds with extreme multiplexing capabilities as well as highly efficient data collection will be designed, evaluated in silico and in a real experimental setting on in vitro models. The ultimate goal is to develop novel proteomics high-throughput analysis techniques that allow the deep investigation of complex proteomes to study their expression differences and to investigate the functional role of proteins in normal physiology. As a model for a very complex protein mixture the plasma proteome is used. Introduction In 2006 a state of the art proteomics facility in the Philips Research Lab in Eindhoven has started on the investigation of complex mammalian proteomes, using an ABI 4800 MALDI TOF/TOF and an ABI 4000 Q trap respectively. Extensive sample preparation and pre-fractionation techniques are used to reduce the protein content complexity before analysis by MS and MS/MS techniques. Chemical labelling approaches allow multiplexing of several samples to a single experimental procedure, thereby eliminating many systematic effects. A Philips research scientist has been placed part-time at the proteomics team at the European Bioinformatics Institute to prepare for data analysis. This institute is the European hotspot for biological data; work relevant to this project is on manual and automatic annotation of the human genome and proteome, repositories of proteomics data, protein interactions and text mining. Several methods have been proposed to improve throughput and / or coverage of proteomics experiments. Examples of these are pure software approaches such as building of inclusion and exclusion lists of precursor masses, based on previous experiments in the own lab, as well as on 'collective experience' from experiments performed and made available world-wide. Next, there are existing methods such as the isotopic or isobaric labelling techniques that allow some sample multiplexing. Furthermore, complexity reduction can be reached by the selection of one or at most a few peptides per protein. But to generate a breakthrough in throughput while maintaining or even increasing protein coverage, several innovative chemistries -currently in development- involving a combination of extreme multiplexing techniques and complexity reduction will be investigated. This will be done in three steps: in silico design and test, test on simple protein mixtures and finally application on plasma samples as a model for a complex proteome. Workplan Year 1: Literature survey, internship at the Philips lab in Eindhoven. In silico model of 'MudPit' experiments on plasma, through modelling of digestion, separation steps, post-translational modifications and /where known- protein abundances. Generation of expected survey spectra, including isotope distributions. In silico design and test of labelling and complexity reduction methods, test on simple protein mixtures. Year 2: Test one or more methods on plasma samples, evaluate results in terms of gain in throughput, dynamic range. Year 3: Application of the analysis result to high-throughput quantitative proteomics on 100 or more samples, either on a MALDI approach, or on a electrospray instrument (higher sensitivity, but requires a priori knowledge of fragmentation). Combination and statistical analysis of the experimental results. Year 4: Exploring the data; e.g. for correlations between different proteins due to co-regulation or interaction. Placing the results in the context of current biological understanding of plasma proteomics. Comparison to data from proteomics repositories, protein interaction databases, text mining results

Aim There is currently a great interest in understanding the molecular mechanism that underpin compartmentalisation and cross-talk of intracellular signalling processes in order to appreciate fundamental insights. In this, the cAMP signalling pathway has provided important paradigms. cAMP-phosphodiesterases (PDE's) underpin compartmentalised cAMP signalling in cells. This project focuses on understanding and exploiting the functional role of specific PDE's in male prostate epithelium. In particular defining interactions between the cAMP signalling system and that of the androgen receptor (AR), whose action is required for normal prostate development and function. Androgen Receptor signalling and its modulation by cAMP Prostate development and normal prostate function requires androgen signaling and action (1). The male androgens, in particular the testicular testosterone and its more active metabolite dihydrotestosterone (DHT), exert their action in the prostate through a nuclear hormone receptor, the Androgen Receptor (AR). The development and physiology of the male organism regarding sexual differentiation and maturation is strongly dependent on androgen and AR activity. The 110-kDa AR is a member of the super-family of nuclear receptors, and is activated upon binding of various lipophilic ligands (steroids, retinoids, etc). Ligand binding promotes the association of AR co-regulators, and the protein complex then translocates into the cell nucleus. DNA transcription is induced by binding of AR complexes to AREs (Androgen Responsive Elements) in the promotor region of target genes. The modulation of transcriptional activity of the AR appears to be influenced by a number of co-regulators and phosphorylation events. It has been suggested that AR activity can be induced in a ligand-independent manner by the elevation of cAMP levels. This has been presumed to be mediated via the activation of PKA. However, no analysis has yet been done to determine whether regulation can additionally occur through the recently discovered cAMP signalling system involving the cAMP GTP exchange factor, EPAC. Nevertheless, several potential PKA phosphorylation sites have been identified on the AR (Ser81/94/650) and phosphorylation by PKA has been speculated to provide a mechanism for ligand-independent activation of AR transcription. Thus work is needed to define the parameters that may control PKA phosphorylation at these sites, such as regulation by PDEs and the involvement of AKAPs and PKA isoforms as well as their functional significance. However, it has also been proposed that cAMP cross-talk with the AR may occur (additionally) at the level of CREB (cAMP responsive element-binding protein). This model suggests an indirect effect of PKA on AR stimulation by the phosphorylation of Ser133 of CREB. Such PKA phosphorylation promotes CREB binding to cAMP responsive elements in target candidate genes and the recruitment of additional co-activators, thereby enhancing target gene transcription. The presence of a putative cAMP responsive site (CRE) at the 5'-upstream regulatory region in the PSA gene makes CREB a conceivable candidate for mediating PKA effects. Thus PKA may regulate AR functioning at two localities, the AR itself and also CREB. Although it has been shown consistently over the years that the activation of cAMP-PKA pathway enhances AR transcriptional activity, the exact mechanism for this has not been shown. This effect could be mediated through either direct phosphorylation of the AR by cAMP-PKA, or mediated through indirect PKA phosphorylation effects. Workplan This study aims to identify the modulation of AR signalling by cAMP-PKA. These investigations will provide novel understanding about cAMP compartmentalisation and cross-talk in an important biological system. These multi-disciplinary studies using 'state-of-the-art' technologies will provide a first-rate training for the student involved.

Aim The aim of the project is to establish a 21st century approaches to the characterisation of protease activities in cellular extracts or body fluids. Proteolytic cleavage is now accepted as a key event in signal transduction (e.g., apoptotic cascade, NF-?B signalling) and in the governance of cellular metabolism. Furthermore the need for surrogate markers in clinical trials has heightened interest in protease, protein and peptide content of biological fluids. There is therefore increased interest in characterising protease activities in cellular function and extracellular fluids. However the available protease assays are still have a very low throughput. In this respect the low cost of drug assays in clinical laboratories, using multiple reaction monitoring (MRM) mass spectrometry (MS) may be a model that can be followed in protease assays. It is therefore our goal to develop a high-throughput protease assay at reasonable costs. Introduction Despite recent advances in the field, the substrates and in vivo roles for newly identified proteases are unknown and, even for proteases that have been well characterised, their biological functions are often not fully understood. New techniques are urgently required to identify the protease repertoire that is expressed and active in a cell, tissue or organism, as well as to identify as many natural substrates of each protease as possible by identifying substrate specificity in the primary sequence and then applying bioinformatics analysis to seek potential substrates. In the field of analytical chemistry, many small molecules (e.g., drug metabolites, hormones, protein degradation products) are routinely measured using this approach at high throughput with great precision (Coefficient of Variation or CV <5%). Most such assays employ electrospray ionisation followed by two stages of mass selection: a first stage (MS1) selecting the mass of the intact analyte (parent ion) and, after collision activated decomposition of the parent ion, a second stage (MS2) selecting a specific fragment of the parent, collectively generating a 'Selected Reaction Monitoring' (SRM) assay. In the Whetton lab a protocol for about 40 such transitions to be assessed on 40 different peptides has been used with great success in identifying specific post-translational modifications (Unwin et al 2005 Molecular and Cellular Proteomics 4: 1134-1144). This can be adapted to the project below. Workplan Year 0: On the award of the studentship a useful combination of synthetic peptide substrates, which provides potential substrates for all major classes of mammalian proteases will be synthesised. Year 1: Development of a sample assay workflow. Key elements include: definition of the components of the protease/substrate incubation suitable for downstream mass spectrometric assay. This will be followed by followed by peptide cleavage and product purification. Samples will be run on an AB QSTAR tandem mass spectrometer to define products. Information acquired on tandem MS profiles helps define specific fragment for MS2 in MRM assay. Years 2 & 3: Optimisation of the protease cleavage product purification procedure for a high-throughput approach. Determination of best MRM approach for each protease of interest using highly enriched proteases and defined peptide substrate library Translation of a selected sample assay workflow to an MRM based assay. Several hundred possible peptide products from protease reactions will be assessed with multiple MRM runs. Year 4: Testing of the developed high-throughput MRM assay for protease activity on mammalian cell culture extracts, cell culture supernatants as well as human body fluids like serum of healthy volunteers to validate the MRM protease multiplex assay and to characterize the protease activity content of these samples. As a standard reference to test for protease activity we will use array-based protease test systems.

The aim of the project is to establish a high-throughput/low-cost assay for the investigation and characterization of function role and the regulation of Androgen Receptor signaling in the normal prostate and the consequences of de-regulation for the physiological function of the prostate in the healthy organism. The prostate development and normal prostate function requires androgen signaling and action (1). The male androgens exert their action in the prostate through a nuclear hormone receptor, the Androgen Receptor (AR). It has been shown recently that androgen stimulation of a prostate cell lines in vitro changes the expression level of >600 genes (2). Recently, new potential AR genomic targets have been identified of which many show altered expression in prostate disease (3). The modulation of transcriptional activity of the AR appears to be influenced by a number of co-regulators and phosphorylation events. These involve for example signal transduction through e.g. TGF-?, IL-6, and IGF-I pathways (4). Despite all the progress in the field it is still unclear how exactly the key pathway (i.e., the Androgen pathway) in normal prostate development and growth is influenced and modulated by other cellular signaling pathways and - in particular - what type of de-function in the regulatory network of Androgen activity and regulation leads to the development of abnormal prostate behaviour (e.g., benign enlargements, malignancies etc). Therefore we aim to investigate the Androgen signaling network on a protein level in cellular models by systematically disturbing the members of the network (e.g., by siRNA) as well as known modulators of the network (e.g., EGF, TGF-?, IL-6, and IGF-I etc) followed by monitoring of the consequences on the protein levels of the members of the signaling network. By comparison of these types of experiments in different cellular models of the prostate epithelium we aim to unravel the most important members network, and/or the most important modulators of the of the Androgen signaling pathway for normal prostate function. As typically a high number of experimental conditions have to be tested to come to an understanding of the regulatory context of complex signaling networks, it is the goal of this project to establish a high-throughput multiplex MRM mass spectrometry assay that is able to measure a high number (>100) of the key proteins in the Androgen signaling network in a single experimental procedure. This requires the identification of the key members of the Androgen signaling network, the development and validation of an MRM assay for these proteins and the testing of multiplexing of many validated assays. Further, the establishment of cell culture of different prostate epithelial cell lines and testing/optimisation of conditions for 'network disturbance' experiments by e.g. siRNA. Year 1: Definition of the key members of the Androgen Receptor signaling pathway as well as main modulatory pathways (scientific literature). Cell culture conditions for prostate cell lines models. Years 2 & 3: Experimental conditions for siRNA investigations. MRM assay development of validation for key members of the Androgen Receptor signaling pathway. Multiplex assay optimisation for individually validated MRM assays. Validation of the developed multiplex MRM assay on cell line extracts. Years 4: 'Network disturbance' experiments by the introduction of commercially available and validated siRNA molecules to the identified key members of the Androgen Receptor signaling pathway as well as main modulatory pathways. Testing of the outcome by the developed MRM assay. Comment: the project will be supported by bioinformatics network modelling and analysis provided by Philips Research Key references (1) Roy, Vit Horm, 1999, 55, 309/352 (2) Valesco, Endocrinol, 2004, 145(8), 3913-3924 (3) Massie, EMBO Reports, 2007, AOP (4) Heinlein, 2002, Endocrine Reviews, 23(2), 175/200

We aim to develop air-stable high mobility (>0.1 cm^2/Vs) electron transporting (n-channel) organic field-effect transistors (OFETs) employing soluble fullerene derivatives. The main motivation for developing n-channel OFETs is that they enable complementary circuit design, a vital ingredient for the fabrication of the next generation large-scale, low-power, high-performance organic integrated circuits. As our material workhorse we choose the family of fullerenes due to their record-breaking electron mobility (~6 cm2/Vs). Emphasis is placed on soluble derivatives due to their processing advantage for large-area, low manufacturing cost applications. The novelty of the proposed work originates from our recent study where the first solution-processed, air-stable n-channel fullerene transistors have been demonstrated. To the best of our knowledge, this unique combination of solubility, ambient stability and electron transporting character has only been demonstrated previously in two organic molecules and can be considered as a significant breakthrough. The subject of the proposed work is very topical with huge technological importance in the area of organic electronics and it is anticipated to have significant impact both in academic research and industrial R&D worldwide.