Immunotherapy is a potential key weapon in the fight against cancer, in general, and Glioblastoma Multiforme (GBM), in particular. Still, a better understanding of how immunotherapy can be leveraged in GBM patients is direly needed. Disease complexity and blood-brain barrier impermeability mean that a standard vaccinology approach will not work. As part of a novel cancer management strategy, this project aims to create more efficacious vaccines using polymeric nanoparticles and self-assembled nanoplatforms in combination with modified oncolytic viruses. We will use biodegradable nanomaterials to protect the OV from body clearance and exploit the intranasal route for efficient delivery to the brain. We will incorporate the IL12 gene into a recombinant adenovirus vector backbone, which will stay under the nanoparticle protection until the drug reaches the tumour microenvironment and is released to reverse tumour-induced immunosuppression. This new strategy offers an efficient alternative to the current standard-of care - i.e. labour-intensive immunotherapy and possibly chemoradiation. The development of an off-the-shelf product, which provides additional anti-tumoral effects at different levels, would significantly prolong the life expectancy of GBM patients. This bioprocess would be scalable for production and compatible with Good Manufacturing Practices. The administration strategies, safety and efficacy of the anti-cancer agent will be validated in animal GBM models. The main training objective is to educate a strongly needed generation of interdisciplinary specialists in tight collaboration with large pharmacological industries, academia, and clinical centres. This will not only educate 14 outstanding early-stage researchers, but also greatly enhance their career perspectives through the unique opportunity to conduct transdisciplinary research and embark on a high-quality training in both academic and entrepreneurial industrial environments.

INSIDE-HEART brings together universities, companies and hospitals from countries (Italy, Finland, France, Israel, Netherlands, Spain, and Sweden) with the main scope of establishing a multi-disciplinary network to tackle the design and the early-phase validation of digital biomarkers, specifically targeting the diagnosis of supraventricular arrhythmias (SVAs) and their associated potential for adverse risk assessment, via the joint combination of signal processing, artificial intelligence and non-clinical devices. This will be achieved by performing excellent research through a unique doctoral training “without walls” among field-expert academic, industrial and clinical entities. The composite nature of the INSIDE-HEART network ensures a highly qualified training and research infrastructure for the specific goal, which aims to generate a new profile of researcher with multi-sectoral expertise able to fill the existing gap, i.e., the absence of digital biomarkers for SVAs reliably estimated with non-clinical devices, taking into account basic research, clinical needs and business interests. Research and training are designed to consider relevant aspects such as public concern of private data management, gender and ethics related to SVAs, all according to Responsible Research & Innovation principles and Open Science practices. All activities in INSIDE-HEART are designed to pursue innovation in three domains: i) Educational domain – by implementing a new multi-sectoral paradigm of PhD training to shape modern professional researchers with cross-competencies in the field, and able to accelerate the translation from basic science to market and clinics; ii) Basic science domain - by producing new knowledge about digital biomarkers by means of a multi-sectoral approach to explore the complex aspects related to SVAs; and iii) Technological domain – by developing new data-driven and model-based methodologies to compute digital biomarkers and support the clinical decision.

Single-molecule protein sensing and sequencing technologies are emerging on the horizon as new powerful tools, which ultimate goal is the characterization of the proteome, namely the total set of proteins made by a cell or organism. Despite recent advances, challenges in terms of full-length reads, high throughput and intrinsic complexity related to the larger set of building blocks (i.e. 20 amino acids) still remain. Furthermore, the unambiguous identification of proteins at low concentration is an open issue for conventional techniques, such as Mass Spectroscopy and ELISA. I aim to develop a plasmon-enhanced single-molecule sensing device to track and identify proteins, combining a dual-color amino-acid-specific technique to label the proteins and the enhancement to the fluorescence provided by properly-designed plasmonic nanostructures integrated within a nanochannel-based device. The plasmonic nanochannel is filled with a custom-made gel to slow down the motion of proteins and separate them by their molecular mass during the electrophoretic migration, meanwhile plasmonic nanostructures enhance the fluorescence signals acquired from each individual dual-color labelled protein. I will focus on the optimization of the device and testing its capabilities for the discrimination of clinically-relevant isoforms of Vascular endothelial growth factor (VEGFa) family. I will tackle the challenge of isoforms identification even further, by using the device to discriminate between VEGFa and VEGFb, which differ for only six amino acids at the C termini. Results from this work will provide a technique for the identification and quantification of protein isoforms with single-molecule resolution. Potentially, the use of this diagnostic tool will provide new insight into the role of VEGF isoforms and their up/down regulation into several diseases and in turns, also new avenues for targeted therapeutics.

One of the important shortcomings of modern anticancer therapies is their limited penetration depth of only a few cell layers into the tumor. Concentrated around the heterogeneous vasculature, these drugs produce only a local therapeutic effect. In this project we propose a method of overcoming this limitation by engineering a novel class of gas-filled nanostructures capable of homing to tumor tissues, and using their vibration in response to ultrasound energy to deliver drugs deeper into the tumor core. The proposed approach is based on ultrasonic cavitation, a phenomenon in which gas bubbles expand and collapse under the influence of ultrasound waves. This process produces fluid streaming that propels drugs deeper into the tumor mass. The use of ultrasound for drug delivery is attractive due to its availability and affordability. However, the use of this technology is currently limited by the properties of conventional microbubble-based cavitation nuclei: their large size prevents them from penetrating into the tumor and their short circulation times do not match the pharmacokinetic time constants of many drugs. To overcome these challenges, we will utilize gas vesicles (GVs), a unique class of genetically encoded, gas-filled protein nanostructures derived from buoyant photosynthetic microbes, as cavitation nuclei. Unlike microbubbles, GVs are physically stable and their nanoscale dimensions have the potential to enable them to extravasate into tumors and bind to specific cellular targets. We hypothesize that GVs can act as both imaging agents and cavitation nuclei. If so, this therapeutic approach could have vastly improved efficacy and selectivity and the potential to combine cavitation-enhanced drug delivery with emerging advancements in cell based therapeutics. This project will enable the applicant to diversify his capabilities and experience beyond ultrasound imaging and signal processing and re-inforce a position of professional maturity.

Membrane fusion is essential for many physiological and pathological processes: infection of enveloped viruses, fertilization, intracellular trafficking of vesicles and in some cases of organogenesis. These processes are mediated by specific proteins called fusogens. Fusexins are fusogens that are essential for sexual reproduction and exoplasmic merger of plasma membranes in protists, plants, invertebrates and class II enveloped viruses. The main goal of my project is to characterize the least understood class of membrane fusion: cell-cell fusion processes within the phylum Chordata mediated by proteins from the Fusexin superfamily. I will evaluate whether proteins from mouse gametes with predicted structural similarities to Fusexins fulfill the two criteria to be considered fusogens (i.e. necessity and sufficiency for membrane fusion). In parallel, I will elucidate the mechanisms of action of the amphioxus Br-FF-1 proteins, the first Fusexins described in Chordata. The project involves both high risk and feasible objectives, employing assays that are well established in the Podbilewicz lab as well as the development of new techniques (including in vitro fertilization assays, fusion in heterologous cells, pseudotyping of viruses and virus-cell infection). I expect that results obtained will shed light on the evolution of the Fusexin superfamily that maps to the beginning of the eukaryotic cells and enveloped viruses, and thoroughly characterize the mechanisms of action of cell-cell fusogens. My proposal has the potential to lead to breakthroughs in understanding and manipulating membrane fusion in sexual reproduction, organ development, diseases and tissue repair.