Gelation, characterization and enhancement of Amyloid networks and modelling of Fibronectin binding towards Growth Factors

Abstract

Nature is a constant inspiration for us. The mechanisms on which life is based are, at the same time, incredibly complex and surprisingly harmonious; individual macromolecules interact in a seemingly chaotic dance, but which has an order and a melody. Amyloid fibrils are a class of bio-macromolecules that is being increasingly brought under the spotlight. In vivo, amyloids can be both pathological and functional. In fact, they are responsible of severely debilitating diseases, such as Alzheimer’s and Parkinson’s, but they also assume pivotal roles in key biological functions. In vitro, amyloid fibrils synthesized from food sources are more and more used as building blocks of innovative materials, the applications of which range from filtration of water pollutants to drug delivery. Consequently, extensive studies on how amyloid fibrils interact and how they form gels are pivotal, both for finding novel therapeutic approaches for the above-mentioned diseases, and for optimizing the synthesis of materials that meet human needs, while preserving the resilience of our Planet. Fibronectin is another important biomolecule, which is largely present in the extra-cellular matrix. The heparin-mediated conformational dance of fibronectin tunes its interactions with other key molecules, such as growth factors and, among these, the Vascular Endothelial Growth Factor (VEGF, the role of which is crucial for angiogenesis). A deeper understanding of how fibronectin and VEGF interact would lead to the possibility of synthesizing more effective drugs, the target of which is angiogenesis in pathological conditions (like cancer). This doctoral thesis contains studies on both the biomolecules mentioned above. Regarding amyloid fibrils, the aim of the studies we present is to characterize amyloid fibril gels (both over the course of their formation and structurally, when already formed), and to strengthen them through inclusion of polysaccharides. More in details, we show that in amyloid gels, the preparation of which is based on the passage of ions through a semipermeable membrane, the dynamic of the ions is non-diffusive but, instead, advective. This suggests the presence of a force that acts on the ions, that we hypothesize being arising from the Donnan effect. We also show that the ionic strength of the gels has a key role in tuning the entity and the frequency of restructuring phenomena, the role of which is to release the stresses accumulated by the fibrils over the gelation process. From a structural characterisation perspective, we show that the mesh size of amyloid networks can be measured through Dynamic Light Scattering (DLS) experiments, combining the theory of scattering from semi-flexible polymers together with a rigorous approach that takes the nonergodicity of the studied materials into account. Finally, with a more applied focus, we show an approach to strengthen amyloid fibril hydrogels and aerogels through inclusion of polysaccharides. The sugar chains improve the compression resistance of the materials, without interfering with the surface properties of the amyloid fibrils. In the case of fibronectin, we show how this molecule is able to bind not only with VEGF, but also with an important receptor of this growth factor (VEGFR2). More in detail, heparin has an important role in extending fibronectin molecules, exposing binding sites that can interact with both VEGF and VEGFR2. The interactions are favoured at acidic pH, due to modifications in the protonation state of the binding sites of the ligands: this piece of information deepens the understanding of how hypoxia and low extracellular pH are related to angiogenesis. Moreover, thanks to Surface Plasmon Resonance studies and their modeling, we show that not all the fibronectin molecules interact equally with VEGF and VEGFR2, and can instead be divided into two classes, with different kinetics and affinities. To resume, the presented studies broaden the actual knowledge on amyloid fibrils and fibronectin, and pave the way for synthesizing new functional materials and for facing severe diseases with novel therapeutic approaches.