ERC FUNDED PROJECTS

The main objective of 4D-PET is to develop an innovative whole-body PET scanner based in a new detector concept that stores 3D position and time of every single gamma interaction with unprecedented resolution. The combination of scanner geometrical design and high timing resolution will enable developing a full sequence of all gamma-ray interactions inside the scanner, including Compton interactions, like in a 3D movie. 4D-PET fully exploits Time Of Flight (TOF) information to obtain a better image quality and to increase scanner sensitivity, through the inclusion in the image formation of all Compton events occurring inside the detector, which are always rejected in state-of-the-art PET scanners. The new PET design will radically improve state-of-the-art PET performance features, overcoming limitations of current PET technology and opening up new diagnostic venues and very valuable physiological information

2 048 386 €

Start date: 2017-01-01, End date: 2021-12-31

Although we have extensive knowledge of many important processes in cell biology, including information on many of the molecules involved and the physical interactions among them, we still do not understand most of the dynamical features that are the essence of living systems. This is particularly true for the actin cytoskeleton, a major component of the internal architecture of eukaryotic cells. In living cells, actin networks constantly assemble and disassemble filaments while maintaining an apparent stable structure, suggesting a perfect balance between the two processes. Such behaviors are called “dynamic steady states”. They confer upon actin networks a high degree of plasticity allowing them to adapt in response to external changes and enable cells to adjust to their environments. Despite their fundamental importance in the regulation of cell physiology, the basic mechanisms that control the coordinated dynamics of co-existing actin networks are poorly understood. In the AAA project, first, we will characterize the parameters that allow the coupling among co-existing actin networks at steady state. In vitro reconstituted systems will be used to control the actin nucleation patterns, the closed volume of the reaction chamber and the physical interaction of the networks. We hope to unravel the mechanism allowing the global coherence of a dynamic actin cytoskeleton. Second, we will use our unique capacity to perform dynamic micropatterning, to add or remove actin nucleation sites in real time, in order to investigate the ability of dynamic networks to adapt to changes and the role of coupled network dynamics in this emergent property. In this part, in vitro experiments will be complemented by the analysis of actin network remodeling in living cells. In the end, our project will provide a comprehensive understanding of how the adaptive response of the cytoskeleton derives from the complex interplay between its biochemical, structural and mechanical properties.

2 349 898 €

Start date: 2017-09-01, End date: 2022-08-31

Air pollution is the main urban-related environmental hazard. It appears to affect brain development, although current evidence is inadequate given the lack of studies during the most vulnerable stages of brain development and the lack of brain anatomical structure and regional connectivity data underlying these effects. Of particular interest is the prenatal period, when brain structures are forming and growing, and when the effect of in utero exposure to environmental factors may cause permanent brain injury. I and others have conducted studies focused on effects during school age which could be less profound. I postulate that: pre-natal exposure to urban air pollution during pregnancy impairs foetal and postnatal brain development, mainly by affecting myelination; these effects are at least partially mediated by translocation of airborne particulate matter to the placenta and by placental dysfunction; and prenatal exposure to air pollution impairs post-natal brain development independently of urban context and post-natal exposure to air pollution. I aim to evaluate the effect of pre-natal exposure to urban air pollution on pre- and post-natal brain structure and function by following 900 pregnant women and their neonates with contrasting levels of pre-natal exposure to air pollutants by: i) establishing a new pregnancy cohort and evaluating brain imaging (pre-natal and neo-natal brain structure, connectivity and function), and post-natal motor and cognitive development; ii) measuring total personal exposure and inhaled dose of air pollutants during specific time-windows of gestation, noise, paternal stress and other stressors, using personal samplers and sensors; iii) detecting nanoparticles in placenta and its vascular function; iv) modelling mathematical causality and mediation, including a replication study in an external cohort. The expected results will create an impulse to implement policy interventions that genuinely protect the health of urban citizens.

2 499 992 €

Start date: 2018-09-01, End date: 2023-08-31

T cell engineering with chimeric antigen receptors has opened the door to effective immunotherapy. CARs are fusion genes encoding receptors whose extracellular domain comprises a single chain variable fragment (scFv) antibody that binds to a tumour surface epitope, while the intracellular domain comprises the signalling module of CD3ζ along with powerful costimulatory domains (e.g. CD28 and/or 4-1BB). CARs are a major breakthrough, since they allow bypassing HLA restrictions or loss, and they can incorporate potent costimulatory signals tailored to optimize T cell function. However, solid tumours present challenges, since they are often genetically unstable, and the tumour microenvironment impedes T cell function. The tumour vasculature is a much more stable and accessible target, and its disruption has catastrophic consequences for tumours. Nevertheless, the lack of affinity reagents has impeded progress in this area. The objectives of this proposal are to develop the first potent and safe tumour vascular-disrupting tumour immunotherapy using scFv’s and CARs uniquely available in my laboratory. I propose to use these innovative CARs to understand for the first time the molecular mechanisms underlying the interactions between anti-vascular CAR-T cells and tumour endothelium, and exploit them to maximize tumour vascular destruction. I also intend to employ innovative engineering approaches to minimize the chance of reactivity against normal vasculature. Lastly, I propose to manipulate the tumour damage mechanisms ensuing anti-vascular therapy, to maximize tumour rejection through immunomodulation. We are poised to elucidate critical interactions between tumour endothelium and anti-vascular T cells, and bring to bear cancer therapy of unparalleled power. The impact of this work could be transforming, given the applicability of tumour-vascular disruption across most common tumour types.

2 500 000 €

Start date: 2013-08-01, End date: 2018-07-31

Imagine being able to design into living cells and organisms de novo vesicle transport mechanisms that do not naturally exist? At one level this is a wild-eyed notion of synthetic biology. But we contend that this vision can be approached even today, focusing first on the process of exocytosis, a fundamental process that impacts almost every area of physiology. Enough has now been learned about the natural core machinery (as recognized by the award of the 2013 Nobel Prize in Physiology or Medicine to the PI and others) to take highly innovative physics/engineering- and DNA-based approaches to design synthetic versions of the secretory apparatus that could someday open new avenues in genetic medicine. The central idea is to introduce DNA-based functional equivalents of the core protein machinery that naturally form (coats), target (tethers), and fuse (SNAREs) vesicles. We have already taken first steps by using DNA origami-based templates to produce synthetic phospholipid vesicles and complementary DNA-based tethers to specifically capture these DNA-templated vesicles on targeted bilayers. Others have linked DNA oligonucleotides to trigger vesicle fusion. The next and much more challenging step is to introduce such processes into living cells. We hope to break this barrier, and in the process start a new field of research into “synthetic exocytosis”, by introducing Peptide-Nucleic Acids (PNAs) of tethers and SNAREs to re-direct naturally-produced secretory vesicles to artificially-programmed targets and provide artificially-programmed regulation. PNAs are chosen mainly because they lack the negatively charged phosphate backbones of DNA, and therefore are more readily delivered into the cell across the plasma membrane. Future steps, would include producing the transport vesicles synthetically within the cell by externally supplied origami-based PNA or similar cages, and - much more speculatively - ultimately using encoded DNA and RNAs to provide these functions.

3 000 000 €

Start date: 2015-09-01, End date: 2021-08-31

Fifteen years ago it was widely believed that asthma was an allergic/atopic disease caused by allergen exposure in infancy; this produced atopic sensitization and continued exposure resulted in eosinophilic airways inflammation, bronchial hyper-responsiveness and reversible airflow obstruction. It is now clear that this model is at best incomplete. Less than one-half of asthma cases involve allergic (atopic) mechanisms, and most asthma in low-and-middle income countries is non-atopic. Westernization may be contributing to the global increases in asthma prevalence, but this process appears to involve changes in asthma susceptibility rather than increased exposure to “established” asthma risk factors. Understanding why these changes are occurring is essential in order to halt the growing global asthma epidemic.This will require a combination of epidemiological, clinical and basic science studies in a variety of environments. A key task is to reclassify asthma phenotypes. These are important to: (i) better understand the aetiological mechanisms of asthma; (ii) identify new causes; and (iii) identify new therapeutic measures. There are major opportunities to address these issues using new techniques for sample collection from the airways (sputum induction, nasal lavage), new methods of analysis (microbiome, epigenetics), and new bioinformatics methods for integrating data from multiple sources and levels. There is an unprecedented potential to go beyond the old atopic/non-atopic categorization of phenotypes. I will therefore conduct analyses to re-examine and reclassify asthma phenotypes. The key features are the inclusion of: (i) both high and low prevalence centres from both high income countries and low-and-middle income countries; (ii) much more detailed biomarker information than has been used for previous studies of asthma phenotypes; and (iii) new bioinformatics methods for integrating data from multiple sources and levels.

2 348 803 €

Start date: 2016-01-01, End date: 2020-12-31

"Prostate cancer is the commonest solid cancer in men in the European Community. There is evidence for genetic predisposition to the development of prostate cancer and our group has found the largest number of such genetic variants described to date worldwide. The next challenge is to harness these discoveries to advance the clinical care of populations and prostate cancer patients to improve screening and target treatments. This proposal, BARCODE, aims to be ground-breaking in this area. BARCODE has two components (1) to profile a population in England using the current 77 genetic variant profile and compare screening outcomes with those from population based screening studies to determine if genetics can target screening more effectively in this disease by identifying prostate cancer that more often needs treatment and (2) genetically profiling men with prostate cancer in the uro-oncology clinic for a panel of genes which predict for worse outcome so that these men can be offered more intensive staging and treatment within clinical trials. This will use next generation sequencing technology using a barcoding system which we have developed to speed up throughput and reduce costs. The PI will spend 35% of her time on this project and she will not charge for her time spent on this grant as she is funded by The University of London UK. The research team at The Institute Of Cancer Research, London, UK is a multidisciplinary team which leads the field of genetic predisposition to prostate cancer and its clinical application and so is well placed to deliver on this research. This application will have a dramatic impact on other researchers as it is ground –breaking and state of the art in its application of genetic findings to public health and cancer care. It will therefore influence the work being undertaken in both these areas to integrate genetic profiling and gene panel analysis into population screening and cancer care respectively."

2 499 123 €

Start date: 2014-10-01, End date: 2019-09-30

This proposal seeks to develop a novel technique for fluid and particle manipulations, based upon exploiting the mechanical interactions between acoustic waves and phononic. The new platform involves generating surface acoustic waves (SAWs) on piezoelectric chips, but, unlike previous work, the ultrasonic waves are first coupled into a phononic lattice, which is placed in the path of the ultrasonic wave. The phononic lattice comprises a miniaturised array of mechanical elements which modulates the sound in a manner analogous to how light is “patterned” using a hologram. However, whilst in an optical hologram, the pattern is created by exploiting the differences in refractive indices of the elements of the structure, here the ultrasonic field is modulated both by the elastic contrast between the elements in the array, as well as by the dimensions of the array and its surrounding matrix (including the size and pitch of the features within the array). The result of passing the acoustic wave through a phononic crystal is the formation of new and complex ultrasonic landscapes. As part of the proposed work we aim to understand the physics of this technology and to exploit its development in a range of medical devices. We will show that by using phononic crystals it is possible to create highly controllable patterns of acoustic field intensities, which propagate into the fluid, creating pressure differences that result in unique flow patterns to enable a new platform for including biological sample processing, medical diagnostics, drug delivery and blood clotting devices – all on low cost disposable devices. Different frequencies of ultrasound will interact with different phononic structures to give different functions, providing a toolbox of different functions. Just as in electronics, where discrete components are combined to create circuits, so we propose to combine different phononic lattices to create fluidic microcircuits with important new applications.

2 208 594 €

Start date: 2014-04-01, End date: 2019-03-31

The aetiology of many musculoskeletal (MS) diseases is related to biomechanical factors. However, the tools to assess the biomechanical condition of patients used by clinicians and researchers are often crude and subjective leading to non-optimal patient analyses and care. In this project innovations related to imaging, sensor technology and biomechanical modelling are utilized to generate versatile, accurate and objective methods to quantify the (pathological) MS condition of the lower extremity of patients in a unique manner. The project will produce advanced diagnostic, pre-planning and outcome tools which allow clinicians and researchers for detailed biomechanical analysis about abnormal tissue deformations, pathological loading of the joints, abnormal stresses in the hard and soft tissues, and aberrant joint kinematics. The key objectives of this proposal are: 1) Develop and validate image-based 3-D volumetric elastographic diagnostic methods that can quantify normal and pathological conditions under dynamic loading and which can be linked to biomechanical modelling tools. 2) Create an ultrasound (US)-based system to assess internal joint kinematics which can be used as a diagnostic tool for clinicians and researchers and is a validation tool for biomechanical modelling. 3) Generate and validate an ambulant functional (force and kinematic) diagnostic system which is easy to use and which can be used to provide input data for biomechanical models. 4) Create and validate a new modelling approach that integrates muscle-models with finite element models at a highly personalized level. 5) Generate biomechanical models which have personalized mechanical properties of the hard and soft tissues. 6) Demonstrate the applicability of the personalized diagnostic and pre-planning platform by application to healthy individuals and patient subjects. Support from the ERC will open new research fields related to biomechanical patient assessment and modeling of MS pathologies.

2 456 400 €

Start date: 2013-05-01, End date: 2018-04-30

In 2010, 800 billion Euros was spent on brain diseases in Europe and the cost is expected to increase due to the aging population. – Here I propose to exploit our new discovery for research to alleviate this disease burden. In work selected by Nature Medicine among the top 10 ”Notable Advances” and by Science as one of the 10 ”Breakthroughs of the year” 2015, we discovered a meningeal lymphatic vascular system that serves brain homeostasis. We want to reassess current concepts about cerebrovascular dynamics, fluid drainage and cellular trafficking in physiological conditions, in Alzheimer’s disease mouse models and in human postmortem tissues. First, we will study the development and properties of meningeal lymphatics and how they are sustained during aging. We then want to analyse the clearance of macromolecules and protein aggregates in Alzheimer’s disease in mice that lack the newly discovered meningeal lymphatic drainage system. We will study if growth factor-mediated expansion of lymphatic vessels alleviates the parenchymal accumulation of neurotoxic amyloid beta and pathogenesis of Alzheimer’s disease and brain damage after traumatic brain injury. We will further analyse the role of lymphangiogenic growth factors and lymphatic vessels in brain solute clearance, immune cell trafficking and in a mouse model of multiple sclerosis. The meningeal lymphatics could be involved in a number of neurodegenerative and neuroinflammatory diseases of considerable human and socioeconomic burden. Several of our previous concepts have already been translated to clinical development and we aim to develop proof-of-principle therapeutic concepts in this project. I feel that we are just now in a unique position to advance frontline European translational biomedical research in this suddenly emerging field, which has received great attention worldwide.

2 420 429 €

Start date: 2017-08-01, End date: 2022-07-31

Recent evidence demonstrates that cancer is overtaking cardiovascular disease as the number one cause of mortality in Europe. This is largely due to the lack of preventative measures for common (e.g. breast) or highly fatal (e.g. ovarian) human cancers. Most cancers are multifactorial in origin. The core hypothesis of this research programme is that the extremely high risk of BRCA1/2 germline mutation carriers to develop breast and ovarian cancer is a net consequence of cell-autonomous (direct effect of BRCA mutation in cells at risk) and cell non-autonomous (produced in distant organs and affecting organs at risk) factors which both trigger epigenetic, cancer-initiating effects. The project’s aims are centered around the principles of systems medicine and built on a large cohort of BRCA mutation carriers and controls who will be offered newly established cancer screening programmes. We will uncover how ‘cell non-autonomous’ factors work, provide detail on the epigenetic changes in at-risk tissues and investigate whether these changes are mechanistically linked to cancer, study whether we can neutralise this process and measure success in the organs at risk, and ideally in easy to access samples such as blood, buccal and cervical cells. In my Department for Women’s Cancer we have assembled a powerful interdisciplinary team including computational biologists, functionalists, immunologists and clinician scientists linked to leading patient advocacy groups which is extremely well placed to lead this pioneering project to develop the fundamental understanding of cancer development in women with BRCA mutations. To reset the epigenome, re-establishing normal cell identity and consequently reducing cancer risk without the need for surgery and being able to monitor the efficacy using multicellular epigenetic outcome predictors will be a major scientific and medical breakthrough and possibly applicable to other chronic diseases.

2 497 841 €

Start date: 2017-09-01, End date: 2022-08-31

The frontier in cancer therapy of orchestrating the immune system to attack tumors is producing unprecedented survival benefit in some patients. The corollary is lack of efficacy both in ostensibly responsive tumor types as well as others that are mostly non-responsive. The basis lies in pre-existing and adaptive resistance mechanisms that circumvent induction of tumor-reactive cytotoxic T cells (CTLs) capable of infiltrating solid tumors and eliminating cancer cells. A priori, cancers induced by expression of human papillomavirus oncogenes should be responsive to immunotherapy: these cancers encode immunogenic neo-antigens – the oncoproteins E6/7 – necessary for their manifestation. Rather, such tumors are poorly responsive to immunotherapies. Results from my lab and others using mouse models of HPV-induced cancer have established an actionable hypothesis: during tumorigenesis, such tumors erect multiple barriers to the induction, infiltration, and killing of cancer cells by tumor antigen-reactive CTLs. These include overarching systemic antigen-nonspecific immunosuppression mediated by expanded populations of myeloid cells in spleen and lymph nodes, complemented by immune response-impairing barriers operative in the tumor microenvironment. A spectrum of models will probe these barriers, genetically and pharmacologically, establishing their functional importance, alone and in concert. A major focus will be on how oncogene-expressing keratinocytes elicit a marked expansion of immunosuppressive myeloid cells in spleen and lymph nodes, and how these myeloid cells in turn inhibit development and activation of CD8 T cells and antigen-presenting dendritic cells. Then we’ll assess the therapeutic potential of barrier-breaking strategies combined with immuno-stimulatory modalities. This project will deliver new knowledge about multi-faceted barriers to immunotherapy in these refractory cancers, helping lay the groundwork for efficacious immunotherapy.

2 500 000 €

Start date: 2020-01-01, End date: 2024-12-31

Breast cancer is a major cause of death in the United States and the Western World. Advanced medical technologies and therapeutic strategies are necessary for the successful detection, diagnosis, and treatment of breast cancer. Here, we propose to use novel technologies (tissue microarrays (TMA) and automated quantivative bioimaging (AQUA)) to identify new therapeutic and prognostic markers for human breast cancer. More specifically, we will study the activation status of a new signaling pathway which we have implicated in breast cancer pathogenesis, using both mouse animal models and cells in culture. For this purpose, we will study the association of CAPER expression with pre-malignant lesions and progression from pre-malignancy to full-blown breast cancer. We expect that this new molecular marker will allow us to improve diagnostic accuracy for individual patients, enhancing both the prognostic predictions as well as the prediction of drug responsiveness for a given patient.

1 500 000 €

Start date: 2010-01-01, End date: 2014-12-31

Mechanisms governing interaction between multicellular organisms and microbes are central for understanding pathogenesis, symbiosis and the function of ecosystems. We propose to address these mechanisms by pioneering an interdisciplinary approach for understanding cellular signalling, response processes and organ development. The challenge is to determine factors synchronising three processes, organogenesis, infection thread formation and bacterial infection, running in parallel to build a root nodule hosting symbiotic bacteria. We aim to exploit the unique possibilities for analysing endocytosis of bacteria in model legumes and to develop genomic, genetic and biological chemistry tools to break new ground in our understanding of carbohydrates in plant development and plant-microbe interaction. Surface exposed rhizobial polysaccharides play a crucial but poorly understood role in infection thread formation and rhizobial invasion resulting in endocytosis. We will undertake an integrated functional characterisation of receptor-ligand mechanisms mediating recognition of secreted polysaccharides and subsequent signal amplification. So far progress in this field has been limited by the complex nature of carbohydrate polymers, lack of a suitable experimental model system where both partners in an interaction could be manipulated and lack of corresponding methods for carbohydrate synthesis, analysis and interaction studies. In this context our legume model system and the discovery that the legume Nod-factor receptors recognise bacterial lipochitin-oligosaccharide signals at their LysM domains provides a new opportunity. Combined with advanced bioorganic chemistry and nanobioscience approaches this proposal will engage the above mentioned limitations.

2 399 127 €

Start date: 2011-05-01, End date: 2016-04-30

The regulation of cell migration is central in pattern formation, homeostasis and disease. The proposed research is aimed at investigating the molecular basis for cell motility and the associated polarization of the cell. In view of the dynamic nature of these processes, we have chosen to utilize the migration of Primoridal Germ Cells (PGCs) in zebrafish - a model that offers unique experimental advantages for imaging and experimental manipulations. The fact that molecules facilitating the motility of zebrafish PGCs are evolutionary conserved and the finding that the cells are directed by chemokines, molecules that control a wide range of cell trafficking events in vertebrates, make this in vivo study of particular importance. The proposed work involves both the functional analysis of previously identified candidates and the identification of molecules, which have a presently unknown effect on the migration process. For both objectives, we will employ novel experimental schemes. We will examine the role of proteins in achieving functional cell polarity compatible with efficient motility and response to directional cues, using unique techniques and analysis tools in the context of the living organism. The precise function of the identified proteins will be determined by combining mathematical tools aimed at quantitatively gauging the role of the molecules in conferring proper cell shape, biophysical methods aimed at measuring forces, rigidity and cytoplasm flow and determination of the effect on the organization of relevant structures using cryo electron tomography. Together, this approach would provide a non-conventional understanding of cell migration by correlating structural, morphological and dynamic cellular properties with the ability of cells to effectively migrate towards their target.

1 960 600 €

Start date: 2011-06-01, End date: 2017-05-31

"Centrioles are critical for the formation of cilia, flagella and centrosomes, as well as for human health. The mechanisms governing centriole formation constitute a long-standing question in cell biology. We will pursue an innovative multidisciplinary research program to gain further insight into these mechanisms, using human cells, C. elegans and Trichonympha as model systems. This program is expected to also have a major impact by contributing a novel cell free assay to the field, thus paving the way towards making synthetic centrioles. Six specific aims will be pursued: 1) Deciphering HsSAS-6/STIL distribution and dynamics. We will use super-resolution microscopy, molecular counting, photoconversion and FCS to further characterize these two key components required for centriole formation in human cells. 2) The SAS-6 ring model as a tool to redirect centriole organization. Utilizing predictions from the SAS-6 ring model, we will assay the consequences for centrioles and cilia of altering the diameter and symmetry of the structure. 3) Determining the architecture of C. elegans centrioles. We will conduct molecular counting and cryo-ET of purified C. elegans centrioles to determine if they contain a spiral or a cartwheel, as well as identify SAS-6-interacting components. 4) Comprehensive 3D map and proteomics of Trichonympha centriole. We will obtain a ~35 Å 3D map of the complete T. agilis centriole, perform proteomic analysis to identify its constituents and test their function using RNAi. 5) Regulation of cartwheel height and centriole length. We will explore whether cartwheel height is set by SAS-6 proteins and perform screens in human cells to identify novel components regulating cartwheel height and centriole length. 6) Novel cell free assay for cartwheel assembly and centriole formation. Using SAS-6 proteins on a lipid monolayer as starting point, we will develop and utilize a cell-free assay to reconstitute cartwheel assembly and centriole format"

2 499 270 €

Start date: 2014-04-01, End date: 2019-03-31

Despite decades of research, developing ways to overcome drug resistance in cancer is the most challenging bottleneck for curative therapies. This is because, in some forms of cancer, the cancer stem cells from which the diseases arise are constantly evolving, particularly under the selective pressures of drug therapies, in order to survive. The events leading to drug resistance can occur within one or more individual cancer stem cell(s) – and the features of each of these cells need to be studied in detail in order to develop drugs or drug combinations that can eradicate all of them. The BCR-ABL+ and BCR-ABL- myeloproliferative neoplasms (MPN) are a group of proliferative blood diseases that can be considered both exemplars of precision medicine and of the drug resistance bottleneck. While significant advances in the management of MPN have been made using life-long and expensive tyrosine kinase inhibitors (TKI), patients are rarely cured of their disease. This is because TKI fail to eradicate the leukaemia stem cells (LSC) from which MPN arise and which persist in patients on treatment, often leading to pervasive drug resistance, loss of response to therapy and progression to fatal forms of acute leukaemia. My goal is to change the way we study the LSC that persist in MPN patients as a means of delivering more effective precision medicine in MPN that is a “game-changer” leading to therapy-free remission (TFR) and cure. Here, I will apply an innovative strategy, ChAMPioN, to study the response of the MPN LSC to TKI in innovative pre-clinical laboratory models and directly in patients with MPN - up to the resolution of individual LSC. This work will reveal, for the first time, the molecular and clonal evolution of LSC during TKI therapies, thus enabling the development of more accurate predictions of TKI efficacy and resistance and rational approaches for curative drug therapies.

3 005 818 €

Start date: 2018-01-01, End date: 2022-12-31

Eukaryotic cells inherit much of their genomic information in the form of chromosomes during cell division. Centimetre-long DNA molecules are packed into micrometer-sized chromosomes to enable this process. How DNA is organised within mitotic chromosomes is still largely unknown. A key structural protein component of mitotic chromosomes, implicated in their compaction, is the condensin complex. In this proposal, we aim to elucidate the molecular architecture of mitotic chromosomes, taking advantage of new genomic techniques and the relatively simple genome organisation of yeast model systems. We will place particular emphasis on elucidating the contribution of the condensin complex, and the cell cycle regulation of its activities, in promoting chromosome condensation. Our previous work has provided genome-wide maps of condensin binding to budding and fission yeast chromosomes. We will continue to decipher the molecular determinants for condensin binding. To investigate how condensin mediates DNA compaction, we propose to generate chromosome-wide DNA/DNA proximity maps. Our approach will be an extension of the chromosome conformation capture (3C) technique. High throughput sequencing of interaction points has provided a first glimpse of the interactions that govern chromosome condensation. The role that condensin plays in promoting these interactions will be investigated. The contribution of condensin s ATP-dependent activities, and cell cycle-dependent post-translational modifications, will be studied. This will be complemented by mathematical modelling of the condensation process. In addition to chromosome condensation, condensin is required for resolution of sister chromatids in anaphase. We will develop an assay to study the catenation status of sister chromatids and how condensin may contribute to their topological resolution.

2 076 126 €

Start date: 2010-04-01, End date: 2015-03-31

Background: Therapeutic angiogenesis with vascular endothelial growth factors (VEGFs) has great potential to become a novel, minimally invasive new treatment for a large number of patients with severe myocardial ischemia. However, this requires development of new technology. Advancing state-of-the-art: We propose a paradigm shift in gene therapy for chronic ischemia by activating endogenous VEGF-A,-B and -C genes and angiogenic transcription programs from the native promoters instead of gene transfer of exogenous cDNA to target tissues. We will develop a new platform technology (endogenetherapy) based on our novel concept of the release of promoter pausing and new promoter-targeted upregulating short hairpinRNAs, tissue-specific superenhancerRNAs activating specific transcription centers involving gene clusters in different chromosomal regions, small circularRNAs formed from self-splicing exons-introns that can be regulated with oligonucleotides and small molecules such as metabolites, nuclear RNAi vectors and specific CRISPR/gRNAmutatedCas9-VP16 technology with an ability to target integration into genomic safe harbor sites. After preclinical studies in mice and in a newly developed chronic cardiac ischemia model in pigs with special emphasis on the analysis of clinically relevant blood flow, metabolic and functional outcomes based on MRI, ultrasound, photoacoustic and PET imaging, the best construct will be taken to a phase I clinical study in patients with severe myocardial ischemia. Since endogenetherapy also involves epigenetic changes, which are reversible and long-lasting, we expect to efficiently activate natural angiogenic programs. Significance: If successful, this approach will begin a new era in gene therapy. Since there is a clear lack of technology capable of targeted upregulation of endogenous genes, the novel endogenetherapy approach may become widely applicable beyond cardiovascular diseases also in other areas of medicine and biomedical research.

2 437 500 €

Start date: 2015-11-01, End date: 2020-10-31

CODIR proposes a new configuration of fundamental and applied biomedical and engineering multidisciplinary research to explore and characterise colon behavior necessary for the project and wider objectives. Scope and focus is on novel robotic hydro-colonoscopy (RHC), which stems from two considerations: (i) replacement of the flexible colonosocope with a patient-friendly system for inspection of the mucosal surface colon and (ii) the very recent concept of hydro-colonoscopy whereby water is used instead of traditional air insufflation. RHC can enable a breakthrough in patient-compliant complete endoscopic examination and biopsy of the colon for the further study of life threatening disorders of the colon commonly categorized as inflmmatory bowel disease, all of unknown aetilogy despite intensive research. CODIR will provide new insights for biomedical investigation and research applicable to many biomedical fields: biologic [absorption of water and electrolyte from the colon, characterisation of surface topograpgy of the colon, mechanical properties of colonic wall], imaging, mechatronics robot functionality and a novel colonic irrigation and filling system. The ambition is to develop a one-stop holistic system which cleans the colon of faecal debris and then introduces a tethered swimming/ submerging robot for inspection of the mucosal aspect of colon under the control of a clinician operating the endoluminal mini-robot from a control console. A secondary, very important outcome of CODIR is to increase patient compliance (currently 50%) for screening colonoscopy in early diagnosis of colorectal cancer, the worlds second commonest cancer. RHC can overcome major disadvantages of existing colonoscopy examination: discomfort, sedation, thus increasing compliance and enabling future research.

2 999 948 €

Start date: 2011-08-01, End date: 2016-07-31

How cells retain, lose, and regain developmental plasticity is poorly understood due to ignorance of the molecular mechanisms regulating gene expression. Each gene is regulated by a unique set of factors and as a consequence the trans-acting factors and cis-acting chromatin modification states regulating a given gene are extremely rare. Transcription is affected by events taking place many thousands of base pairs away from the start, a property enabling developmental and evolutionary plasticity, presumably made possible by DNA looping or translocation of factors along chromatin. Most factors regulating a given gene function at many other genes, complicating interpretation of the consequences of altering the activity of such factors. It is difficult to exclude the possibility that phenotypes are knock-on effects. This could be surmounted if it were possible to observe individual genes in real time in three-dimensional space and to analyse the immediate consequences of altering the activity of regulatory factors. Of these, those capable of inter-connecting DNAs or of translocating large distances along chromatin are of interest. Cohesin is such a factor, composed of three core subunits, a pair of Smc proteins and a kleisin subunit, that interact with each other to form a huge tripartite ring, within which it is thought chromatin fibres are entrapped. In proliferating cells, cohesin’s primary function is to connect sister chromatids during DNA replication until the onset of anaphase, possibly by virtue of co-entrapment within a single ring. However, cohesin is present in most quiescent cells and it is becoming clear that it also regulates gene expression and recombination. This proposal has two goals: To image gene expression on polytene chromosomes and to investigate cohesin’s role during ecdysone-induced transcription. The advantage of this system is that we can use micro-injection of TEV protease to inactivate cohesin. A second goal is to develop the TEV system to

2 421 212 €

Start date: 2012-05-01, End date: 2018-04-30

The clinical phenotype and the outcome of Crohn's disease (CD) and ulcerative colitis (UC), the opposite ends of chronic inflammatory bowel diseases (IBD), are heterogeneous and represent the result of a complex interplay of the gut microbiome with the immune system in genetically predisposed individuals. Disease management is much less heterogeneous as all patients are treated using non-specific anti-inflammatory agents, and only 30-50% achieve clinical and mucosal remission -the goal of therapy nowadays- therefore leaving large margins for improvement. The advances in knowledge about the factors triggering disease onset should be translated to approach the disease from a molecular angle. Key cellular pathways have emerged including bacterial recognition, autophagy, endoplasmic reticulum stress and intestinal barrier function. Functional/molecular characterization of these pathways in a given patient, correlation with meaningful clinical outcomes, and tailoring an individual therapeutic approach has never been attempted and will represent a breakthrough in the current paradigm of treating multifactorial inflammatory conditions. This project aims to functionally characterize patients with CD/UC for the major pathways by using integrated (epi)genetic, transcriptomic, immunologic, barrier integrity and metagenomic studies. From these readouts we will construct an index [the Crohn’s and Ulcerative Colitis Characterization and Intervention trial (CrUCCial) index], reflecting the proportional contribution of each of the pathogenic mechanisms in a given patient. We will next study the correlation of this index and its components to meaningful clinical outcomes and finally, the index will be tested in a pilot study of newly diagnosed patients in whom the disease will be targeted individually based on the components of the CrUCCial index. Our approach, from diagnosis over prognosis to therapy, will revolutionize the paradigm of disease management.

2 494 500 €

Start date: 2016-09-01, End date: 2021-08-31

In physiological situations, the extracellular matrix (ECM) sequesters cytokines, localizes them, and modulates their signaling. Thus, physiological signaling from cytokines occurs primarily when the cytokines are interacting with the ECM. In therapeutic use of cytokines, however, this interaction and balance have not been respected; rather the growth factors are merely injected or applied as soluble molecules, perhaps in controlled release forms. This has led to modest efficacy and substantial concerns on safety. Here, we will develop a protein engineering design for second-generation cytokines to lead to their super-affinity binding to ECM molecules in the targeted tissues; this would allow application to a tissue site to yield a tight association with ECM molecules there, turning the tissue itself into a reservoir for cytokine sequestration and presentation. To accomplish this, we have undertaken preliminary work screening a library of cytokines for extraordinarily high affinity binding to a library of ECM molecules. We have thereby identified a small peptide domain within placental growth factor-2 (PlGF-2), namely PlGF-2123-144, that displays super-affinity for a number of ECM proteins. Also in preliminary work, we have demonstrated that recombinant fusion of this domain to low-affinity binding cytokines, namely VEGF-A, PDGF-BB and BMP-2, confers super-affinity binding to ECM molecules and accentuates their functionality in vivo in regenerative medicine models. In the proposed project, based on this preliminary data, we will push forward this protein engineering design, pursuing super-affinity variants of VEGF-A and PDGF-BB in chronic wounds, TGF-beta3 and CXCL11 in skin scar reduction, FGF-18 in osteoarthritic cartilage repair and CXCL12 in stem cell recruitment to ischemic cardiac muscle. Thus, we seek to demonstrate a fundamentally new concept and platform for second-generation growth factor protein engineering.

2 368 170 €

Start date: 2014-05-01, End date: 2019-04-30

A cell’s decision to die is governed by multiple input signals received from a complex network of programmed cell death (PCD) pathways, including apoptosis and programmed necrosis. Additionally, under some conditions, autophagy, whose function is mainly pro-survival, may act as a back-up death pathway. We propose to apply new approaches to study the molecular basis of two important questions that await resolution in the field: a) how the cell switches from a pro-survival autophagic response to an apoptotic response and b) whether and how pro-survival autophagy is converted to a death mechanism when apoptosis is blocked. To address the first issue, we will screen for direct physical interactions between autophagic and apoptotic proteins, using the protein fragment complementation assay. Validated pairs will be studied in depth to identify built-in molecular switches that activate apoptosis when autophagy fails to restore homeostasis. As a pilot case to address the concept of molecular ‘sensors’ and ‘switches’, we will focus on the previously identified Atg12/Bcl-2 interaction. In the second line of research we will categorize autophagy-dependent cell death triggers into those that directly result from autophagy-dependent degradation, either by excessive self-digestion or by selective protein degradation, and those that utilize the autophagy machinery to activate programmed necrosis. We will identify the genes regulating these scenarios by whole genome RNAi screens for increased cell survival. In parallel, we will use a cell library of annotated fluorescent-tagged proteins for measuring selective protein degradation. These will be the starting point for identification of the molecular pathways that convert survival autophagy to a death program. Finally, we will explore the physiological relevance of back-up death mechanisms and the newly identified molecular mechanisms to developmental PCD during the cavitation process in early stages of embryogenesis.

2 500 000 €

Start date: 2013-03-01, End date: 2018-02-28

Hematopoietic stem cells (HSC) are used in clinical therapies for leukemia and blood-related genetic disorders. Whereas the number of patients requiring treatment continues to increase, HSC transplantations are limited due to insufficient patient-matched donor HSCs. The current challenge is to create more matched HSCs. As evidenced by the Nobel Prize award this year, reprogramming of somatic cells to pluripotent stem cells (iPS) is one of the most important breakthroughs of recent times. This innovative advance contributes to our ability to reprogram patient-specific cells not only to pluripotency, but also to directly program them to other desired cell lineages. The study of healthy and diseased patient cells in this context will have huge impact on the development of new drug and cell-based treatments. My research is uniquely positioned at the interface of fundamental and translational research at the University of Edinburgh Centre for Inflammation Research and Centre for Regenerative Medicine. Through more than a decade of HSC developmental research, my group has shown that HSCs arise from endothelial cells in a natural reprogramming event. We are one of the few groups worldwide that can isolate these special endothelial cells and show that they yield robust transplantable HSCs (the gold-standard for clinically relevant HSCs). Using our unique expertise I aim to foster new translational strategies to de novo generate human HSCs from patient somatic cells. My aims are to 1) mark and manipulate the program for HSC generation during the endothelial to HSC transition (EHT); 2) define extrinsic molecules affecting EHT and engineer novel niches; 3) reprogram human somatic cells or endothelial derived iPS cells directly to HSCs. These aims will be realized through novel multi-color reporter mouse and ES/iPS lines indicating EHT in real-time, allowing for the isolation and functional validation of de novo HSC generation. These novel models and cultures will significantly advance research and technology, to have major impact on the field.

2 500 000 €

Start date: 2015-01-01, End date: 2019-12-31

Cells in multicellular organisms organise along body and tissue axes. Cellular processes, such as division plane orientation, must be aligned with these polarity axes to generate functional 3-dimensional morphology, particularly in plants, where cell walls prevent cell migration. While some polarly localized plant proteins are known, molecular mechanisms of polarity establishment or its translation to division orientation are elusive, in part because regulators in animals and fungi appear to be missing from plant genomes. Cell polarity is first established in the embryo, but this has long been an intractable experimental model. My team has developed the genetic, cell biological and biochemical tools that now render the early Arabidopsis embryo an exquisite model for studying cell polarity and oriented division. Recent efforts already led to the unexpected identification of a novel family of deeply conserved polar plant proteins that share a structural domain with key animal polarity regulators. In the DIRNDL project, we will capitalize upon our unique position and foundational results, and use complementary approaches to discover the plant cell polarity and division orientation system. Firstly, we will address the function of the newly identified conserved polarity proteins, and determine mechanistic convergence of polarity regulators across multicellular kingdoms. Furthermore, we will use proteomic approaches to systematically identify polar proteins, and a genetic approach to identify regulators of polarity and division orientation, essential for embryogenesis. We will functionally analyse polar proteins and regulators both in Arabidopsis and the liverwort Marchantia to help prioritize conserved components, and to facilitate genetic analysis of protein function. Finally, we will use a cell-based system for engineering polarity de novo using the regulators identified in the project, and thus reveal the mechanisms that provide direction in plant development.

2 500 000 €

Start date: 2019-09-01, End date: 2024-08-31

Developmental biology seeks not only to learn more about the fundamental processes of growth and pattern per se, but to understand how they synergize to enable the morphogenesis of multicellular organisms. Our goal is to perform real-time analyses of these developmental processes in an intact developing organ. By applying a vital imaging approach, we can circumvent the normal limitations of inferring cellular dynamics from static images or molecular data, and obtain the real dynamic view of growth and patterning. The wing imaginal disc of Drosophila, which starts out as a simple epithelial structure and gives rise to a precisely structured adult limb, will serve as an ideal model system. This system has the combined advantages of relative simplicity and genetic tractability. We will create several innovations that expand the current toolkit and thus facilitate the detailed dissection of growth and patterning. A key early step will be to develop novel reporters to dynamically and faithfully monitor signaling cascades involved in growth and patterning, such as the Dpp and Hippo pathways. We will also implement quantification techniques that are currently being set up in collaboration with an experimental physicist, to deduce, and alter, the mechanical forces that develop in the cells of a growing tissue. The large amount of quantitative data that will be generated allow us derive computational models of the individual pathways and their interaction. The focus of the study will be to answer the following questions: 1) Is the Hippo pathway regulated spatially and temporally, and by what signaling pathways? 2) Do mechanical forces play a pivotal controlling role in organ morphogenesis? 3) What are the global effects on growth, when pathways controlling patterning, cell competition or compensatory proliferation are perturbed? The proposed project will bring the approaches taken to define the mechanisms underlying and controlling growth and patterning to the next level.

2 310 000 €

Start date: 2009-02-01, End date: 2014-01-31

Different morphologies evolve in different organisms in response to changing environments. As land plants evolved, developmental mechanisms were either generated de novo, or were recruited from existing toolkits and adapted to facilitate changes in form. Some of these changes occurred once, others on multiple occasions, and others were gained and then subsequently lost in a subset of lineages. Why have certain forms survived and others not? Why does a fern look different from a flowering plant, and why should developmental biologists care? By determining how many different ways there are to generate a particular morphology, we gain an understanding of whether a particular transition is constrained. This basic information allows an assessment of the extent to which genetic variation can modify developmental mechanisms and an indication of the degree of developmental plasticity that is possible and/or tolerated both within and between species. This proposal aims to characterize the developmental mechanisms that underpin the diverse shoot forms seen in extant plant species. The main goal is to compare developmental mechanisms that operate in vegetative shoots of bryophytes, lycophytes, ferns and angiosperms, with a view to understanding the constraints that limit morphological variation. Specifically, we will investigate the developmental basis of three major innovations that altered the morphology of vegetative shoots during land plant evolution: 1) formation of a multi-cellular embryo; 2) organization of apical growth centres and 3) patterning of leaves in distinct spatial arrangements along the shoot. To facilitate progress we also aim to develop transgenic methods, create mutant populations and generate digital transcriptomes for model species at key phylogenetic nodes. The proposed work will generate scenarios to explain how land plant form evolved and perhaps more importantly, how it could change in the future.

2 230 732 €

Start date: 2009-07-01, End date: 2015-06-30

The majority of animals show an external bilateral symmetry, precluding the observation of multiple internal left-right (L/R) asymmetries which are fundamental for organ packaging and function. A prominent molecular pathway converging on and downstream of the Pitx2 transcription factor confers left-handed information in the left side of the embryo, with players expressed on the right ensuring that the left determinants are excluded. Therefore, conferring or excluding left identity in left and right hand sides, respectively, drives L/R asymmetry. Some indications suggest that a program actively specifying right–handed information could exist on the right. Our recent findings support this view. In a screening for novel regulators of the epithelial to mesenchymal transition (EMT), we have identified a transcription factor, EMT2, which similarly to well known factor Snail, it is an EMT inducer. The EMT is crucial for the development of tissues during embryonic development and for the progression of carcinomas to the invasive state. Strikingly, again as Snail, in addition to promote EMT, the EMT2 factor is predominantly expressed on the right side and may operate instructing L/R identity on the right-hand side of the embryo. With this background, our knowledge of the EMT and a series of genome-wide high-throughput approaches and a comprehensive functional analysis using the chick, the fish and the mouse as model systems we propose to reveal the putative molecular pathways conveying right-handed information and to reveal commonalities between L/R pathways and the EMT. In the long run, we aim at better understanding human pathologies that involve these morphogenetic and cellular processes, including pathological situs conditions (i.e. altered organ positioning) and cancer progression.

2 460 000 €

Start date: 2013-05-01, End date: 2018-12-31

Immunotherapy with checkpoints blockers is transforming the treatment of advanced cancers. Colorectal cancer (CRC), a cancer with 1.4 million new cases diagnosed annually worldwide, is refractory to immunotherapy (with the exception of a minority of tumors with microsatellite instability). This is somehow paradoxical as CRC is a cancer for which we have shown that it is under immunological control and that tumor infiltrating lymphocytes represent a strong independent predictor of survival. Thus, there is an urgent need to broaden the clinical benefits of immune checkpoint blockers to CRC by combining agents with synergistic mechanisms of action. An attractive approach to sensitize tumors to immunotherapy is to harness immunogenic effects induced by approved conventional or targeted agents. Here I propose a new paradigm to identify molecular determinants of resistance to immunotherapy and develop personalized in silico and in vitro models for predicting response to combination therapy in CRC. The EPIC concept is based on three pillars: 1) emphasis on antitumor T cell activity; 2) systematic interrogation of tumor-immune cell interactions using data-driven modeling and knowledge-based mechanistic modeling, and 3) generation of key quantitative data to train and validate algorithms using perturbation experiments with patient-derived tumor organoids and cutting-edge technologies for multidimensional profiling. We will investigate three immunomodulatory processes: 1) immunostimulatory effects of chemotherapeutics, 2) rewiring of signaling networks induced by targeted drugs and their interference with immunity, and 3) metabolic reprogramming of T cells to enhance antitumor immunity. The anticipated outcome of EPIC is a precision immuno-oncology platform that integrates tumor organoids with high-throughput and high-content data for testing drug combinations, and machine learning for making therapeutic recommendations for individual patients.

2 460 500 €

Start date: 2018-10-01, End date: 2023-09-30

Embryonic development progresses through successive cell fate decisions and intricate three-dimensional morphogenetic transformations. Implantation is the defining event in mammalian pregnancy during which a fundamental morphogenetic transformation is initiated: the body axes are established and the embryonic germ layers created. Despite its importance, a comprehensive understanding of the molecular mechanisms, transcriptional pathways, cellular interactions, as well as the spatio-temporal development of the embryo at implantation stages is at present lacking, due to the embryo’s inaccessibility. To overcome these limitations, we generated a culture system that allows the development of mouse implanting embryos outside of the mother. This system provides the opportunity to address how architectural features and signaling events integrate to induce the emergence of the body plan. Combining this new technology with the analysis of genetically engineered mouse embryos, the aim of this research proposal is to fill the knowledge gap between pre and post-implantation development. Single cell sequencing, two-photon microscopy, high-content forward genetic screening, and modeling will be merged with a functional assessment of embryo development in vivo to reveal the determinants of implantation and early post-implantation development. This global understanding will be employed to explore the extent to which stem cells can recapitulate embryonic development, with tremendous potential for regenerative medicine. Knowledge of the cellular and molecular mechanisms that intertwine lineage specification, developmental potential, and tissue morphogenesis will offer novel insight on the pathological causes of embryo lethality and congenital disorders. The proposed studies will shed light on this crucial yet mysterious stage of development in the mouse and, by extrapolation, offer outstanding potential to advance our understanding of human development.

2 477 951 €

Start date: 2016-01-01, End date: 2021-12-31

Heart failure (HF) affects 15 million Europeans and entails higher mortality and health care costs than cancer. EPLORE addresses this issue by prospective epidemiological research in 4 European countries and by a proof-of-concept clinical trial. WP1 will for the first time at the population level document the incidence and progression of subclinical LV dysfunction and clarify whether asymptomatic LV dysfunction, as picked up by the newest echocardiographic techniques, predicts cardiovascular (CV) outcomes, including HF. WP2 will investigate the contribution of ventricular-arterial coupling disease and mechanical LV dyssynchrony to subclinical LV dysfunction. WP3 will identify a set of urinary polypeptides that signify early LV dysfunction and validate these biomarkers by showing that they predict deterioration of LV function, progression to HF and the incidence of CV complications over and beyond established risk factors. WP3 will also search for novel panels of circulating biomarkers, of which combined measurement will add information (accuracy, sensitivity and specificity) to established biomarkers (e.g., NT-proBNP) and identify genetic variants involved in the progression of LV dysfunction, either causally or as biomarker. WP4 consists of a randomised clinical trial to translate in a high-risk high-gain setting the results of WPs 1-3 into clinical practice and to identify a new treatment modality that potentially slows progression of diastolic LV dysfunction. Dissemination in WP5 will contribute to new guidelines for the prevention and treatment of HF. WP6 includes governance, monitoring research strategies and output, and protection of IPR. In conclusion, EPLORE will advance risk stratification and the early diagnosis of subclinical HF. The project will potentially result into specific treatments for diastolic LV dysfunction and inform guidelines for prevention and treatment of HF. It will benefit 20% of Europeans who currently have subclinical LV dysfunction.

2 391 440 €

Start date: 2012-07-01, End date: 2017-06-30

Multicellularity in plants evolved independently from other eukaryotes and presents a unique, alternative way how to deal with challenges of life. A major plant developmental module is the directional transport for the plant hormone auxin. The crucial components are PIN auxin transporters, whose polar, subcellular localization determines directionality of auxin flow through tissues. PIN-dependent auxin transport represents a unique model for studying the functional link between basic cellular processes, such as vesicle trafficking and cell polarity, and their developmental outcome at the level of the multicellular organism. Despite decades of intensive research, the classical approaches in the established models are approaching their limits and many crucial questions remain unsolved, in particular related to PIN structure, regulatory motifs and evolutionary origin I propose to start a new direction in my research using an evolutionary perspective. This promises to overcome current limitations and provides not only (i) interesting insights into PIN evolution and diversification, but also (ii) a unique opportunity to study how evolutionary conserved cellular mechanisms of e.g. endocytic trafficking evolved specific plug-ins to make them subject to plant-specific regulations. The characterization of (iii) prokaryotic PIN origin will provide a so urgently needed (iv) entry into PIN structural studies. To achieve these goals, we will also establish novel (v) genetic and cell biological models in the ancestral lineage of the land plants that will be of a great use for any plant evolutionary studies. The intellectual and methodological challenges of such interdisciplinary strategy combining several lower and higher plant models are obvious, but our preliminary results at several fronts promise its feasibility and success to gain deeper understanding of exciting questions on evolution and mechanisms behind the coordination and specification of developmental programs.

2 410 292 €

Start date: 2018-01-01, End date: 2022-12-31

The evolution of the first rooting systems approximately 470 million years ago was a critical event in the history of life on Earth because it allowed the growth of complex multicellular eukaryotic photosynthetic organisms – plants - on the surface of the land. Rooting systems are important because they facilitate the uptake of every chemical element in the plant body with the exception of carbon. The root systems of the first land plants (liverworts) comprised a mass of unicellular tip-growing filaments (rhizoids) that grew from the plant surface into the soil. All root systems that evolved since then similarly comprise a system of tipgrowing filamentous cells located at the interface between the plant and the soil, indicating that the differentiation of filamentous root cells has been critical for root function for the past 470 million years. This proposal aims to characterize the origin and evolution of this essential cellular differentiation process. The proposed research is in three parts: First we propose to define the mechanism that controlled the development of the first land plant root system by identifying genes that control liverwort rooting system (rhizoids) development and characterizing their regulatory interactions. Second we propose to determine if the mechanism that controlled the development of the first land plant root system was inherited from algal ancestors. Third we propose to characterize the mechanism that controls filamentous root hair growth in Arabidopsis in response to environmental factors, and determine if it is conserved among land plants. In combination, these experiments will define the genetic mechanisms underpinning the development and evolution of one of the fundamental developmental processes in land plants.

2 463 835 €

Start date: 2010-10-01, End date: 2015-09-30

Pancreatic islet transplantation is essential for diabetes treatment. Outcome varies due to transplantation site, quality of islets and the fact that transplanted islets are affected by the same challenges as in situ islets. Tailor-making islets for transplantation by tissue engineering combined with a more favorable transplantation site that allows for both monitoring and local modulation of islet cells is thus instrumental. We have established the anterior chamber of the eye (ACE) as a favorable environment for long term survival of islet grafts and the cornea as a natural body window for non-invasive, longitudinal optical monitoring of islet function. ACE engrafted islets are able to maintain blood glucose homeostasis in diabetic animals. In addition to studies in non-human primates we are performing human clinical trials, the first patient already being transplanted. Tissue engineering of native islets is technically difficult. We will therefore apply genetically engineered islet organoids. This allows us to generate i) standardized material optimized for transplantation, function and survival, as well as ii) islet organoids suitable for monitoring (sensor islet organoids) and treating (metabolic islet organoids) insulin-dependent diabetes. We hypothesize that genetically engineered islet organoids transplanted to the ACE are superior to native pancreatic islets to monitor and treat insulin-dependent diabetes. Our overall aim is to create a platform allowing monitoring and treatment of insulin-dependent diabetes in mice that can be transferred to large animals for validation. The objective is to combine tissue engineering of islet cell organoids, transplantation to the ACE, synthetic biology, local pharmacological treatment strategies and the development of novel micro electronic/micro optical readout systems for islet cells. This regenerative medicine approach will follow our clinical trial programs and be transferred into the clinic to combat diabetes.

2 500 000 €

Start date: 2020-01-01, End date: 2024-12-31

Since a few decades, human patients who suffer from severe illnesses or multiple trauma, conditions that were previously lethal, are being treated in intensive care units (ICUs). Modern intensive care medicine bridges patients from life-threatening conditions to recovery with use of mechanical devices, vasoactive drugs and powerful anti-microbial agents. By postponing death, a new unnatural condition, intensive-care-dependent prolonged (>1 week) critical illness, has been created. About 25% of ICU patients today require prolonged intensive care, sometimes for weeks or months, and these patients are at high risk of death while consuming 75% of resources. Although the primary insult was adequately dealt with, many long-stay patients typically suffer from hypercatabolism, ICU-acquired brain dysfunction and polyneuropathy/myopathy leading to severe muscle weakness, further increasing the risk of late death. As hypercatabolism was considered the culprit, several anabolic interventions were tested, but these showed harm instead of benefit. We previously showed that fasting early during illness is superior to forceful feeding, pointing to certain benefits of catabolic responses. In healthy humans, fasting activates catabolism to provide substrates essential to protect and maintain brain and muscle function. This proposal aims to investigate whether evolutionary conserved catabolic fasting pathways, specifically lipolysis and ketogenesis, can be exploited in the search for prevention of brain dysfunction and muscle weakness in long-stay ICU patients, with the goal to identify a new metabolic intervention to enhance their recovery. The project builds further on our experience with bi-directional translational research - using human material whenever possible and a validated mouse model of sepsis-induced critical illness for objectives that cannot be addressed in patients - and aims to close the loop, from a novel concept to a large randomized controlled trial in patients.

2 500 000 €

Start date: 2018-10-01, End date: 2023-09-30

The Mexican influenza A virus (H1N1) reminds us that the threat of an influenza pandemic is real. The 1918 Spanish flu virus, also started as a low pathogenic virus that mutated into a highly pathogenic virus within months, causing more than 50 million deaths. The Mexican influenza A virus (H1N1) may follow the same path. FLUPLAN will expand our knowledge of the packaging signals that govern reassortment events between influenza A viruses in general and between the Mexican influenza A virus (H1N1) and circulating human, porcine and highly pathogenic avian influenza A viruses in particular. FLUPLAN will thus lead to fundamental insights in the mechanisms that govern reassortment phenomena, providing a risk assessment concerning the pandemic potential of circulating avian and mammalian influenza A viruses. This will provide us with a panel of possible reassortant viruses of potentially pandemic nature. The MVA vaccine vector system that efficiently induced broad protective immunity against HPAI-H5N1 viruses in macaques, will be used for the preparation of a repository of MVA-H based pandemic vaccine seed viruses.The selection will be based on the reassortant viruses mentioned above, and on a repository of avian influenza viruses of the 16HA subtypes including the Mexican influenza A virus (H1N1) of avian/swine origin. The added value of including a relevant MVA-NP in the immunization schedule to obtain broader and longer protection will be determined in a macaque infection model. Collectively these studies will provide us with a highly versatile system that anticipates on future pandemic events by having seed viruses for vaccine development ready to go on the shelf, for the rapid production of broadly protective pandemic vaccines, which will save time and thus lives.

2 187 758 €

Start date: 2010-06-01, End date: 2015-05-31

A foremost health problem stems from the burden of degenerative diseases, including heart failure, neurodegeneration, retinal degeneration and diabetes, essentially linked to the aging of the human population and the incapacity of post-mitotic tissues to undergo efficient repair. This is an ambitious, highly innovative project aimed at developing an in vivo selection procedure, based on gene transfer of two genetic libraries cloned into Adeno-Associated Virus (AAV)-based vectors, for the identification of novel secreted factors or microRNAs providing benefit against various degenerative diseases. Two arrayed libraries will be generated, one coding for ~1,300 cDNAs from the mouse secretome, the other for all known microRNAs (~800 genes). Pools of vectors from each library will be obtained with serotypes suitable for in vivo transduction of different organs. The vectors will be injected in a series of mouse models of degenerative disorders involving damage to cardiomyocytes,, neurodegeneration, retinal degeneration and loss of beta-cells in the pancreas. The degenerative conditions will drive the selection for secreted factors or miRNA putatively preventing cell apoptosis, enhancing residual cell function or, in the best possible scenario, promoting tissue regeneration. This in vivo selection approach, which is supported by very encouraging preliminary results, has never been attempted before and is rendered possible by the property of AAV vectors to be produced at high titers, infect tissues at high multiplicity, persist in the transduced cells for prolonged period of times and efficiently express their transgenes in vivo. In addition to its final goal of identifying novel biotherapeutics, the project entails the successful achievement of several intermediate objectives and is expected to extend both technology and knowledge beyond the state-of-the art.

1 824 000 €

Start date: 2010-04-01, End date: 2015-03-31

Background: Poor angiogenesis and collateral vessel formation lead to coronary heart disease, claudication, infarctions and amputations while malignant glioma is one of the most aggressive proangiogenic tumors leading to death in a few months. For these diseases either stimulation or blocking, respectively, of angiogenesis may provide novel treatment options. Advancing State-of-the-Art: Our hypothesis is that in ischemia it will be possible to support natural growth of blood vessels with Therapeutic angiogenesis and lymphangiogenesis by using local gene transfer of the new members of vascular endothelial growth factor (VEGF) family and their receptors. New co-receptors, designer mutants and PCR suffling products of VEGFs will be used. New vector technology will be used to achieve long-lasting effects of VEGFs. We aim to develop novel site-specifically integrating, targeted, regulated vectors to precisely express the new VEGFs, their soluble decoy receptors and single-chain therapeutic antibodies (scFv) for pro- and anti-angiogenic purposes. As novel approaches, we have developed metabolically biotinylated lenti- and adenoviruses suitable for targeting and Epigenetherapy where siRNA/miRNAs and short nuclear RNAs regulate endogenous gene expression at the VEGF promoter level via modification of histone code. scFv library for endothelial cells and lentivirus-siRNA library directed to all human and mouse kinases will be screened to identify new mediators of angiogenesis in order to develop next generation pro- and antiangiogenic therapies. Based on our strong track record in Clinical applications, the best new pro- and antiangiogenic approaches will be taken to phase I clinical studies in myocardial ischemia and malignant glioma. Significance: This work should lead to significant advances and new therapies for severe ischemia and malignant glioma. Epigenetherapy and new site-specifically integrating, regulated vectors should be widely applicable in medicine.

2 200 000 €

Start date: 2010-06-01, End date: 2015-05-31

Much of what we know about how embryos determine their axes of symmetry comes from research in invertebrates (mainly Drosophila) and cold-blooded vertebrates (mainly Xenopus). In both cases, polarity is set up by the localisation of maternal determinants in the cytoplasm of the fertilised egg. These determinants are inherited differentially by daughter cells, leading them to acquire different fates, which effectively fixes the axes of the embryo by the 8 cell stage. In contrast, in amniotes (reptiles, birds and mammals) embryonic polarity remains plastic until much later, just before gastrulation, when the embryo may contain as many as 50,000 cells. If an embryo at this stage is cut into fragments, each fragment can generate a complete embryo. This property, called "embryonic regulation", is thought to be responsible for the generation of monozygotic (identical) and conjoined ( Siamese ) twins in humans and other amniotes. We know almost nothing about how polarity is determined in higher vertebrates or about the mechanisms of embryonic regulation and twinning. This project uses a multi-disciplinary systems approach to reveal the gene interaction network controlling polarity, regulation and twinning. The project will also generate a mathematical model of early development or "virtual embryo", allowing prediction of experimental outcomes and clinical scenarios.

1 997 899 €

Start date: 2010-06-01, End date: 2016-02-29

Although several therapies target cellular pathways, current small molecules drug discovery is based on identification of inhibitors to single proteins, without knowledge of whether they are the most advantageous target. The objective of this proposal is to develop a novel method for drug discovery, combining phenotypic cell based screens with functional genetic networks to determine the molecular mechanisms of numerous small molecule inhibitors. This method will enable identification of numerous distinct inhibitors of a particular pathway, as well as providing their molecular mechanism. Cancer cells harbour gene mutations that make them more reliant on other cellular pathways for survival. Such cellular pathways can be targeted to selectively kill the cancer cells using the concept of synthetic lethality. In this project we want to identify inhibitors of homologous recombination to target cancer using synthetic lethality. To establish a functional genetic network for homologous recombination, we will first identify all recombination proteins using multiple genome-wide RNAi screens. Then the synthetic sick or lethal interaction map between all recombination proteins is determined by co-depletion of these. Such synthetic sick or lethal network will identify numerous putative targets for anti-cancer treatment. Importantly, using this network for chemical-genetic functional interactions will assist in determinating of the molecular mechanisms of inhibitors. Chemical-genetic networks based on synthetic sickness or lethality can potentially change future drug discovery methods as well as providing new mechanistic insights into the field of toxicology.

2 500 000 €

Start date: 2011-03-01, End date: 2016-02-29

Animal growth is a complex process that is intimately linked to the developmental program in order to form fit adults with proper size and proportions. Genetics is an important determinant of growth, exemplified by the role of local diffusible molecules in setting organ proportions for a given species. In addition to this genetic control, organisms use adaptation mechanisms allowing modulating the size of individuals according to environmental cues, among which nutrition. Therefore, sophisticated cross-talks between local and global cues are at play for the determination of the final size of an individual. The major objective of this project is to tackle the mechanisms involved in coupling growth control with environmental cues, as well as the mechanisms participating in growth arrest and the determination of final size. Our project proposes a blend of physiological and genetic approaches on the Drosophila model, with the use of tissue-targeted loss-of-function to unravel some of the important cross-talks existing between organs for the control of growth at the global level. We will develop these approaches to (i) unravel the molecular nature of tissue cross-talks involved in nutrient sensing and the control of insulin/IGF secretion; (ii) tackle the feed-back mechanisms linking the developmental clock to the growing state of tissues and organs. These projects should bring new contributions in two separate fields related to growth control, Developmental Biology and Physiology, in an attempt to merge these complementary approaches into a broader vision of this fascinating biological question.

2 500 000 €

Start date: 2011-07-01, End date: 2016-06-30

Targeting therapeutic genes selectively into the central nervous system (CNS) is a crucial precondition for translation of gene therapy strategies into human trials. The current multidisciplinary proposal integrates expertise identified as essential in the effective acceleration of research to overcome bottlenecks in the field including: 1) Inefficiency of therapy delivery to the CNS because of factors like the blood-brain barrier (BBB); 2) Poor understanding of disease mechanisms at the molecular and cellular levels. These problems must be overcome to develop fully effective treatments for neurological disorders. Currently the adeno-associated (AAV)-based system is one of the most refined and effective gene delivery systems for neuronal cells. In contrast to all other systems, it has been possible to engineer AAV9 to deliver genes through the BBB to the CNS by intravascular (IV) administration. However, following IV delivery, these vectors also target liver and other tissues, with significant potential for untoward effects. This has prompted us to adopt two major strategies: i) targeting of AAV9 vectors at the level of transcription by insertion of hybrid motor neuron specific promoters into the vector genome; ii) development of a CNS-targeted delivery approach based on state-of-the art nanoparticle-mediated encapsulation of AAV9 vectors. We anticipate that engineering strategies with the ability to restrict transgene expression to CNS tissue will significantly overcome various existing hurdles in CNS gene therapy development. Our objectives are to: 1) explore mechanisms leading to penetration of scAAV9 vectors through BBB since the exact mechanism of AAV9 diffusion through BBB is unknown; 2) design novel targeted strategies with enhanced tropism to CNS; 3) use CNS targeted vectors to investigate mechanisms of motor neuron death linked to mutations in RNA processing genes; 4) utilise CNS-targeted systems to test therapeutic strategies for motor neuron diseases.

2 499 959 €

Start date: 2012-03-01, End date: 2017-10-31

The liver is a vital organ for synthesis and detoxification. The most significant liver diseases are hepatitis, non alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver steatohepatitis (NASH), carcinoma and cirrhosis. An additional and important cause of liver injury is adverse drug reactions (ADRs). In particular NAFLD is the most common liver disease affecting between 20% and 44% of European adults and 43-70% of patients with type 2 diabetes, and is one prime cause for chronic and end-stage liver disease, such as cirrhosis and primary hepatocellular carcinoma. This proposal is based on recent findings in the laboratory: The development of novel 3D spheroid system with chemically defined media allowing studies of chronic drug toxicity, relevant liver disease and liver function for 5 weeks in vitro, the finding of the role of miRNA in hepatocyte dedifferentiation and that hepatocytes during spheroid formation first de-differentiate but later in spheroids re-differentiate to an in vivo relevant phenotype. This forms the basis for the main objectives: i) to study diseased liver in vitro with identification of mechanisms, biomarkers and novel drug candidates for treatment of NAFLD and fibrosis, ii) evaluate drug toxicity sensitivity and mechanisms in diseased liver systems and iii) further develop methods for hepatocyte proliferation and regeneration in vitro for transplantation purposes, including genetic editing in cases of hepatocytes obtained from patients with genetically inherited liver diseases. This work is carried out in close contact with the Hepatology unit at the Karolinska Hospital partly using resources at the Science for Life Laboratory at Karolinska. It is anticipated that the project can provide with novel mechanisms, biomarkers and new targets for treatment of liver disease as well as novel methods for clinically applicable liver regeneration without the use of stem cells or transformed cells.

2 413 449 €

Start date: 2017-09-01, End date: 2022-08-31

Chronic liver diseases such as liver cirrhosis and hepatocellular carcinoma (HCC) are major challenges for global health. HCC is the second leading and fastest rising cause of cancer death worldwide. The limited availability of therapeutic options reflects our poor understanding of the molecular and clinical mechanisms involved in progression of liver disease. Chronic hepatitis C virus (HCV) infection is a main risk factor for HCC. Although HCC may be avoided by addressing the underlying cause in early stage disease, strategies to prevent HCC in patients with established cirrhosis and advanced fibrosis, in which the risk of HCC persists despite treatment of the underlying cause are lacking. Indeed, even HCV cure does not eliminate the risk of HCC development when advanced fibrosis is already present. Since fibrosis/cirrhosis-driven carcinogenesis is the mechanism of HCC development common to all major etiologies, we propose to use HCV-induced liver disease as a model to decipher the pan-etiology sequence of molecular events underlying disease progression and HCC. Our own data provide solid evidence that HCV infection alters pathways implicated in liver disease progression, including cirrhosis deterioration, HCC development, and overall and liver-specific death. Thus, the molecular investigation of these pathways will identify key cell circuits for the understanding of the pathogenesis of liver disease and HCC in general, and as broadly applicable pan-etiology diagnostic and therapeutic targets. Using a novel patient-derived cell culture model system for liver disease biology combined with advanced functional genomics, novel animal models and clinical investigation, we aim to uncover the cell circuits that are of clinical relevance for liver disease progression and cancer. By providing novel targets and biomarkers for liver disease and HCC prevention, this proposal will have a marked impact on the management and prognosis of patients with liver disease and HCC.

2 305 000 €

Start date: 2016-01-01, End date: 2020-12-31

Sensory cilia are organelles extending like antennas from many eukaryotic cells, with crucial functions in sensing and signalling. Cilia consist of an axoneme built of microtubules, enveloped by a specialized membrane. Ciliary development and maintenance depend critically on a specific, microtubule-based intracellular transport mechanism, intraflagellar transport (IFT). In my laboratory, we study the chemosensory cilia of C. elegans, which sense water-soluble molecules in the animal’s environment for chemotaxis. Over the past years, we have developed a unique set of quantitative, single-molecule fluorescence microscopy tools that allow us to visualize and quantify IFT dynamics with unprecedented detail in living animals. So far, our focus has been on the cooperation of the motor proteins driving IFT. The overall objective of my current proposal is to zoom out and shed light on the connection between ciliary structure, chemosensory function and IFT, from a systems perspective. Recent work has indicated that axoneme length is controlled by IFT. Preliminary results from my laboratory show that axoneme length changes dynamically in response to perturbations of IFT or cilia. Furthermore, we have shown that IFT is substantially affected upon exposure of animals to known repellent solutions. The four major aims in my proposal are to: • determine how directional changes in IFT are regulated and are affected by external disturbances, • understand the dynamics of the axonemal microtubules and how IFT affects these dynamics and vice versa, • study how sensory ciliary function affects IFT and ciliary structure, • further develop our (single-molecule) fluorescence microscopy toolbox by improving instrumentation and using better fluorescent probes and sensors. These experiments will place my lab in a unique position to push forward our understanding of the relationship between structure, function and dynamics of transport of this fascinating and fundamental organelle.

2 499 580 €

Start date: 2018-09-01, End date: 2023-08-31

"Because of its biological complexity, cancer is still poorly understood. Chronic inflammation has been shown, both experimentally and epidemiologically, to be a predisposition to, and also an inseparable aspect of clinically prevalent cancer entities. Therefore, a detailed understanding of both tumour and immune cell functions in cancer progression is a prerequisite for more successful therapeutic startegies. My team was the first to reveal the lymphocyte-intrinsic PKC/NR2F6 axis as an essential signalling node at the crossroads between inflammation and cancer. It is the mission of this project to identify molecular signatures that influence the risk of developing tumours employing established research tools and state-of-the-art genetic, biochemical, proteomic and transcriptomic as well as large scale CRISPR/Cas9 perturbation screening-based functional genomic technologies. Defining this as yet poorly elucidated effector pathway with its profoundly relevant role would enable development of preventive and immune-therapeutic strategies against NSCLC lung cancer and potentially also against other entities. Our three-pronged approach to achieve this goal is to: (i) delineate biological and clinical properties of the immunological PKC/NR2F6 network, (ii) validate NR2F6 as an immune-oncology combination target needed to overcome limitations to ""first generation anti-PD-1 checkpoint inhibitors"" rendering T cells capable of rejecting tumours and their metastases at distal organs and (iii) exploit human combinatorial T cell therapy concepts for prevention of immune-related adverse events as well as of tumour recurrence by reducing opportunities for the tumour to develop resistance in the clinic. Insight into the functions of NR2F6 pathway and involved mechanisms is a prerequisite for understanding how the microenvironment at the tumour site either supports tumour growth and spread or prevents tumour initiation and progression, the latter by host-protective cancer immunity."

2 484 325 €

Start date: 2018-10-01, End date: 2023-09-30

Lung cancer is the most common cause of death from cancer worldwide with 1.59 million deaths annually and it places the highest economic burden of all cancers on the EU at €18.8billion. These figures call for a major transformation of the therapeutic management of lung cancer. The recent developments in immunotherapy give new hope. At present a significant minority of patients benefit from immune therapy, which remains not curative when delivered alone. It is thus clear that improving the efficacy of immune therapy is needed as well as the identification of predictive biomarkers. Combining immunotherapy with radiotherapy improves effectiveness, but resistance is still common, most probably because of hypoxia. In HYPOXIMMUNO, we will open up a new paradigm in treating metastatic NSCLC. I will provide for the first time (pre)clinical proof of concept of (i) highly innovative CT-based radiomics and PET-hypoxia imaging patient stratification tools and (ii) a tri-modal curative hypoxia-targeting treatment strategy that complements current immunotherapy and radiotherapy approaches. This tri-modal curative treatment strategy is based on (1) high precision Stereotactic Radiotherapy (SABR) to kill cancer cells and trigger the immune system, combined with (2) a hypoxia-activated prodrug to target the hypoxic tumour cells resistant to immunotherapy and (3) a promising and innovative tumour specific antibody (Ab)-based immunotherapy (immunocytokine L19-IL2 to “push the accelerator” with or without a checkpoint inhibitor to “release the break”) to form a powerful synergistic immuno-oncology strategy. In HYPOXIMMUNO, I envision to lay the foundation for the next breakthrough in oncology, by improving patient selection and developing highly innovative cancer therapies that target hypoxia, to improve the quality of life and prolong survival of cancer patients, in particular patients with metastatic NSCLC (watch the animation summarising the project: https://youtu.be/oP9Gp4a0b_Q).

2 499 399 €

Start date: 2016-12-01, End date: 2021-11-30

Non-invasive preclinical and clinical imaging is a powerful tool and has a huge potential, specifically in the realm of oncology. Recently, our laboratory developed a novel multimodality imaging system, which combines positron emission tomography (PET) and magnetic resonance imaging (MRI), yielding temporally and spatially matched data. However, the molecular PET and functional MRI signals are very complex and are often not fully understood. Thus, we will cross-validate the complementary PET/MRI information with proteomics and metabolomics data to gain a better understanding of the in vivo image data and yield finally an accurate holistic tumor profile. The cross-validation will be supported by image-guided accurately dissected tumor substructures. Tumor metabolism, receptor status, hypoxia, perfusion, apoptosis and angiogenesis will be investigated by established PET tracers. In the same imaging session, functional parameters of the tumor, such as perfusion, oxygenation and morphology will be assessed by MRI. Beyond this, novel imaging ligands for senescence, tumor stroma, and fatty acid synthase, which have been recently recognized as emerging key-players in tumor progression and therapy resistance, will be developed. The individual in vivo and in vitro parameters will be fed into a data mining utilizing a computer learning approach with regression and classification methods to detect common patterns and the related pharmacokinetics behind the in vivo imaging parameters. Analysis of the dynamic PET data will be performed by compartment analysis and kinetic modelling. Overall aim is to gain a better understanding of imaging data, provide an accurate holistic in vivo tumor profile to support prognostic parameters for tumor progression and therapy response. Finally, the revealed information will lead to a more accurate selection of imaging biomarkers for diagnosis and therapy control and will provide input for new strategies in tumor-specific tracer development.

2 494 800 €

Start date: 2013-06-01, End date: 2018-05-31