Hyperpolarized (HP) magnetic resonance imaging has emerged as a promising method for probing the biological characteristics of living tissue. A sensor molecule (e.g. pyruvate, fumarate, acetate) is hyperpolarized and injected into a patient, and the resulting metabolic flux can be used to assess cell viability, tumour response to therapy, and perform pH mapping, to name a few examples. Two significant barriers stand in the way of the widespread implementation of HP imaging: (1) the current hyperpolarization method, dissolution dynamic nuclear polarization (dDNP) is cumbersome, expensive, and requires expertise to operate the equipment, and; (2) preclinical method development relies heavily on the use of animal models which inhibits rapid screening of experiments. To overcome these limitations, in this project I will: (1) implement parahydrogen-induced polarization as the hyperpolarization method, which is significantly cheaper, easier to use, and can produce the hyperpolarized metabolites at a higher rate than dDNP, and; (2) perform the hyperpolarized imaging in organ-on-a-chip devices, which closely mimic in vivo conditions but allow for experiments to be performed at a greater rate, and can use human tissue as opposed to animal tissue to more closely mimic real conditions. The specific focus in this project will be to investigate whether HP metabolic imaging can provide insight into the progression of muscular dystrophy in human muscle tissue.

Emergent collective behaviours such as flocks and waves are a hallmark of biological active matter, and occur prominently also in large groups of migrating epithelial cells, where they are involved in fundamental biological processes such as wound healing, morphogenesis and cancer cell invasion. Physical forces transduced between cells and arising between cells and the extracellular matrix (ECM) play an integral role in orchestrated multicellular phenomena. Another integral contribution is given by the actions of leader cells, that modulate and guide the migration of cohesive cell groups. How leader cells achieve this using intracellular and cell-ECM forces remains largely to be understood. Here we propose an experimental approach to generate leader cells using optogenetics and to study how leaders influence the collective behaviour of migratory cell groups. We will use epithelial cells expressing light-sensitive activators of RhoGTPases, which enable reversible and directional control of cell motility using blue light. Traction force microscopy and monolayer stress microscopy will be performed using these cells while we will create and control leaders. With functionalized substrates, we will study the mechanical role of leaders in different conditions, from confined two-cell systems to confluent monolayers exhibiting flocking. Finally, we will express the light-sensitive proteins in cancer cells, to study how their three-dimensional dissemination is affected by leaders. Our experimental approach combines physics-derived modelling and quantification of forces with advanced molecular biology tools. The latter will enable us to perform selected modifications on cells, targeting a wide array of proteins involved in cell-cell adhesion and force transduction. Our goal is to shed light on how leaders physically influence collective migration in physiologically relevant situations, paving the way for the in vivo applications of light-induced leader cells.

Our understanding of cell biology has reached the point in which cells can be exogenously engineered to carry out specific tasks. This is typically applied to generate gene circuits that respond to biochemical interactions between specific molecules. However, cells sense not only biochemical but also mechanical signals, in the process of mechanotransduction. Here, we propose to re-engineer cell mechanotransduction from scratch, in a manner that is not based on any endogenous cell signalling pathway. We will achieve this by harnessing our novel findings that force application to the cell nucleus regulates transport through nuclear pore complexes (NPCs), in such a way that proteins can be made to translocate to the cell nucleus with force by appropriately tuning their active and passive transport properties. First, we will implement a mechanosensing element, involving a precise understanding of the mechanical parameters regulating nucleocytoplasmic transport, and subsequent design of molecules with optimal mechanosensitivity (that is, force-dependent nuclear localization). Second, we will implement a control element, enabling a system to control to what extent, and for how long, force reaches the nucleus and triggers subsequent mechanosensing. Finally, we will implement a functional element, by which mechanosensitive molecules will be engineered to trigger the transcription of specific genes in the nucleus. As a proof-of-concept, we will apply this system to re-engineer three main properties of fibroblasts and mesenchymal cells (matrix remodelling, migration, and epithelial/mesenchymal plasticity), all involved in pathological responses to altered tissue mechanics. This project will deliver synthetic mechanotransduction, a novel tool that will be orthogonal and compatible with existing cell engineering approaches. Further, it will provide an answer to the fundamental question of how a functional, biological mechanotransduction system can be generated de novo.

Arthritis, a widespread inflammatory condition, affecting millions globally, necessitates urgent advancements in therapeutic approaches. Predominantly characterized by osteoarthritis (OA), this debilitating condition causes joint pain and stiffness, notably impacting the knee, hand, and hip joints. OA, a chronic degenerative disease, intensifies with age, imposing a significant economic burden on healthcare systems. The insufficiency of current treatments highlights the need for innovative therapies. Tissue engineering and regenerative medicine offer promising avenues, with platelet-rich plasma therapy (PRP) emerging as a forefront contender. PRP harnesses the regenerative potential of growth factors (GFs) to stimulate tissue repair processes, particularly in cartilage and bone cells. However, clinical application faces hurdles, notably the rapid degradation of GFs within the intricate synovial fluid (SF) environment, limiting their therapeutic efficacy and distribution. To overcome these challenges, scientists explore advanced drug delivery systems utilizing nanoparticles (NPs) as carriers. Although promising, passive NPs diffusion through viscous biological barriers, such as joint fluids, remains a significant obstacle. In response, OrthoBots introduces enzyme-powered NPs, termed nanobots, as active carriers of GFs within SF. By utilizing enzymatic propulsion, nanobots aim to enhance GF transport and distribution, facilitating targeted cartilage regeneration. This innovative approach holds transformative potential, potentially revolutionizing arthritis therapy by overcoming current limitations and offering more effective and personalized treatment strategies. Through systematic in vitro studies and in vivo proof-of-concept demonstrations, OrthoBots will pave the way for the next generation of arthritis therapeutics, addressing the unmet clinical needs and improving patient outcomes.