Open Access mandate for Publications and Research data
Funder: EC Project Code: 101067016
Funder Contribution: 215,938 EUR
Quantifying the multiscale functionality of light-triggered hierarchically-structured assemblies represents a challenge in materials science underpinning the design of efficient next-generation optoelectronics, photovoltaics, and energy storage nanodevices. Critical to this challenge is the availability of new metrology and inspection tools which allow to probe the out-of-equilibrium dynamics of these materials, while interacting with light pulses, with quantitative contrast to all its components. DECIPHER proposes to combine phase retrieval image reconstruction methods with the advances in pulsed electron source technology, to build a break-through microscope capable of directly visualizing the nanoscale dynamics of functionalized materials with fs-ps temporal resolution and Å-nm spatial resolution. The proposal has three main objectives: (i) Construct a next-generation ultrafast electron diffraction imaging system. (ii) Implement cutting-edge phase retrieval methods to enable full-field quantitative imaging across length scales with sensitivity to heavy and light elements. (iii) Leverage these new methods to directly visualize light-activated functioning NP supracrystals, with unique sensitivity to their quantitative chemical/elemental composition and, simultaneously, to their 2D/3D topography. This approach will enable the study of nanoscale dynamical behavior with unprecedented detail and provide vital feedback toward the design of energy-efficient, high-performance devices.
To meet medical needs worldwide, tissue engineering must move from successful pre/clinical products towards an effective process to meet Worldwide medical needs, but this is challenging since a quantitative design framework has not emerged, yet. Synthetic biology (SYNBIO) was the solution that genetic engineers found to the same problem: “Despite tremendous individual successes in genetic engineering and biotechnology […], why is the engineering of useful synthetic biological systems still an expensive, unreliable and ad hoc research process?” asked Dr. Endy in a 2005 letter to Nature. The SYNBIO solution included: i) libraries of DNA parts with well-characterized effect on cells; ii) tools to computationally design system-level assemblies, or designer-DNA; and, iii) bottom-up engineering of cell functions using progressively more complex designer-DNA. Effectively, SYNBIO introduced a computer-aided design and manufacturing (CAD/M) platform that transformed the process of engineering cells. However, since inputs from the extracellular matrix (ECM) have largely been ignored, progress towards programmable tissue-level behavior have been more modest. Here, we will build on my experience with computational and experimental models in cardiac tissue engineering to develop a CAD/M framework for engineering cardiac tissues with computationally predictable properties, or designer-ECM. To characterize ECM-cell interactions, we will use traction force and super-resolution microscopy with fluorescence in-situ sequencing. To model multiscale ECM-cell interactions, we will use ordinary differential equations and subcellular element models. Finally, we will leverage ECM parts and human induced pluripotent stem cells to bioprint designer-ECM that recapitulate three phases of heart development: trabeculation, compaction, and maturation. With synthetic matrix biology (SYNBIO.ECM), we will develop a CAD/M-based process and a new class of products for cardiac tissue engineering.
Collagen maturation (through post-translational modifications, quaternary assembly and transport) is critical for tissues and organ development and homeostasis. This complex process involves numerous actors in different sub-cellular compartments, which in turn depend on highly regulated trafficking systems. Although the mechanisms of vesicular trafficking, membrane tethering and fusion are conserved throughout eukaryotic evolution, highly specialized tethering complexes have evolved to regulate specific fusion events. Recently, a novel pathway regulating collagen trafficking has been identified. Central to this pathway are two trafficking proteins, homologous to, but not part of, classical vesicle tethering components. At present, very little is known about their specific functions. Genetic mutations in both proteins induce rare but highly invalidating and often lethal phenotypes, characterized by abnormal collagen distribution, disorders of the extracellular matrix organization and impairment in endosomal trafficking. In this proposal, I will develop a multidisciplinary approach, merging cutting edge methodologies of affinity purification coupled to mass spectrometry with structural biology and biophysics to decipher the molecular mechanisms driving membrane tethering and, more broadly, endosomal trafficking, through this new complex. These results will unveil unprecedented mechanisms of vesicular trafficking, offering a potential for translational research on collagen-related diseases and also on disorders related to vesicular trafficking, possibly paving the way to future therapeutic strategies.
Colorectal cancer (CRC) results from the accumulation of genetic and epigenetic changes in colonic epithelial cells. Epigenome studies revealed that virtually all CRCs contain aberrantly methylated genes and perturbed methylation patterns. Ten-Eleven Translocation (TET) protein family dioxygenases oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further to other oxidized 5mCs, supporting active DNA demethylation and helping maintain epigenomic stability. Loss of TET1 is an oncogenic driver in some CRCs. My preliminary analysis indicates that human CRCs have low TET2 mRNA levels compared to normal colorectal tissue, and suggests that low TET2 expression predicts increased mutational load and reduced overall survival. However, whether TET2 deficiency contributes to CRC pathogenesis, or represents a bystander event, remains to be established. In this proposal, I will elucidate the role of TET2 in CRC pathogenesis by testing whether TET2 knockdown induces methylome and transcriptome reprogramming, ultimately promoting (epi)genomic instability and tumor growth. I will also investigate correlations between TET2 defects and molecular/clinico-pathological parameters, and probe TET2 expression as predictive biomarker of response to CRC therapies. With these aims, I will use a multi-disciplinary approach, combining cell biology, cancer epigenetics, bioinformatics, human and mouse studies with cutting-edge techniques such as 3D cell culture and RNA-seq. This study should establish a clear causal link between TET2 loss and CRC pathogenesis, providing new insight into the mechanism of TET2-mediated tumor suppression and leading to the development of innovative therapies that exploit vulnerabilities of TET2-deficient CRC cells. Overall, this project has both basic and translational significance, and the potential to advance our understanding of CRC carcinogenesis and therapeutic response.