Papel del supresor tumoral p19ARF en la respuesta inflamatoria

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 30-10-201

Modulation of the cooperativity in the assembly of multistranded supramolecular polymers

It is highly desirable that supramolecular polymers self-assemble following small changes in the environment. The degree of responsiveness depends on the degree of cooperativity at play during the assembly. Undertanding how to modulate and quantify cooperativity is therefore highly desirable for the study and design of responsive polymers. Here we show that the cooperative assembly of a porphyrin-based, double-stranded polymer is triggered by changes in building blocks and in salt concentration. We develop a model that accounts for this responsiveness by assuming the binding of the salt countercations to the double-stranded polymer. Using our assembly model we generate plots that show the increase in concentration of polymer versus the normalized concentration of monomer. These plots are ideally suited to appreciate changes in cooperativity, and show that, for our system, these changes are consistent with the increase in polymer length observed experimentally. Unexpectedly, we find that polymer stability increases when cooperativity decreases. We attribute this behaviour to the fact that increasing salt concentration stabilizes the overall polymer more than the nucleus. In other words, the cooperativity factor , defined as the ratio between the growth constant Kg and the nucleation constant Kn decreases as the overall stability of the polymer increases. Using our model to simulate the data, we generate cooperativity plots to explore changes in cooperativity for multistranded polymers. We find that, for the same pairwise association constants, the cooperativity sharply increases with the number of strands in the polymer. We attribute this dependence to the fact that the larger the number of strands, the larger is the nucleus necessary to trigger polymer growth. We show therefore that the cooperativty factor does not properly account for the cooperativity behaviour of multistranded polymers, or any supramolecular polymer with a nucleus composed of more than 2 building blocks, and propose the use of the corrected cooperativity factor m. Finally, we show that multistranded polymers display highly cooperative polymerisation with pairwise association constants as low as 10 M-1 between the building blocks, which should simplify the design of responsive supramolecular polymers

Ship in a bottle: confinement-promoted self-assembly

Understanding self-assembly in confined spaces is essential to fully understand molecular processes in confined cell compartments and will offer clues on the behaviour of simple confined systems, such as protocells and lipid-vesicle based devices. Using a model system composed of lipid vesicles, a membrane impermeable receptor and a membrane-permeable ligand, we have studied in detail how compartmentalization modulates the interaction between the confined receptor and its ligand. We demonstrate that confinement of one of the building blocks stabilizes complex self-assembled structures to the extent that dilution leads, counterintuitively, to the formation of long range assemblies. The behaviour of the system can be explained by considering a confinement factor that is analogous, although not identical, to the effective molarity for intramolecular binding events. The confinement effect renders complex self-assembled species robust and persistent under conditions where they do not form in bulk solution. Moreover, we show that the formation of stable complex assemblies in systems compartmentalized by semi-permeable membranes does not require the prior confinement of all components, but only that of key membrane impermeable building blocks. To use a macroscopic analogy, lipid vesicles are like ship-in-a bottle constructs that are capable of directing the assembly of the confined ship following the confinement of a few key wooden planks. Therefore, we believe that the confinement effect described here would have played an important role in shaping the increase of chemical complexity within protocells during the first stages of abiogenesis. Additionally, we argue that this effect can be exploited to design increasingly efficient functional devices based on comparatively simple vesicles for applications in biosensing, nanoreactors and drug delivery vehicles

Study of Calcium Phosphate Formation Driven by the Dissolution of a 45S5 Bioactive Glass

Although models have been proposed to explain the mechanisms of BG dissolution and subsequent CaP mineralisation, open questions remain. The processes in which phase transition occurs in aqueous solutions and their dynamics remain under-explored partly because traditional instruments/techniques do not allow for direct observations at the adequate time and length scales at which such phase transformations occur. For instance, given the crucial role of the silica gel in CaP formation during BG dissolution, uncertainty exists on how such silica gel forms on the BG surface. In the case of CaP formation driven by BG dissolution, questions can also be added, i.e., how CaP develops into an apatitic-like structure, how many transient phases there are, and, in general, the phenomena occurring in the solid-liquid interface during BG dissolution. Several approaches were taken to study CaP mineralisation driven by bioglass dissolution, mainly examining the solid-liquid interface and the BG after-reaction surface. Electron microscopy techniques were used, including liquid phase transmission electron microscopy (LP-TEM), scanning electron microscopy, and FIB cross-sections. LP-TEM was expected to give a fresh look to the BG dissolution and its adhered phenomena by enabling direct observation. Therefore, one of the aims was to establish whether this *in-situ* imaging technique was suitable for studying this material system. However, the original BG's (Novamin™) particle size was too big to be directly used in the liquid cell (LC). Therefore, the first aim was to develop a method to obtain sub-micron BG particles. *Ex-situ* experiments were also performed, and results were compared with *in-situ* experiments. Other analysis techniques, such as XPS, PXRD, FTIR, ICP-EOS, ToF-SIMS, etc., were utilised to help indirectly understand the studied material system. Ball-milled bioglass (BMBG) particles of suitable size were obtained and directly used in the liquid cell. Moreover, it was found that the grinding method utilised does not alter the BG particles’ chemical composition. Consequently, by judicially setting up LP-TEM experiments, novel observations of BG dissolution and CaP mineralisation processes occurred in their native liquid state. This study established the suitability of LP-TEM to study BG dissolution, determined its critical operational factors, the vast potential of *in-situ* imaging and how modern TEM microscopes could hugely benefit the investigation of material systems like bioglass. Furthermore, cross-sections of reacted BG blocks gave essential insights into the BG dissolution phenomena, particularly its strong dependency on experimental conditions, and tentative evidence has shown that soluble silica from BG dissolution may not reprecipitate/re-polymerise on BG particles’/BG blocks’ surface. On the other hand, *ex-situ* experiments of BMBG dissolution were also performed, giving meaningful insights into this material system (BMBG). However, differences were found when comparing *ex-situ* experiments and *in-situ* experiment results. Additionally, under the experimental conditions used in this study, complementary analysis techniques determined that CaP, during BG dissolution, transitions from ACP to a calcium-deficient nanocrystalline apatitic structure with minimal contents of Si4+ and Na+ ions that may be molecularly part of CaP. Some of the challenging topics of current and future BG research, i.e., controlled release of therapeutic ions or biomolecules, reliable BG coatings, and tunable mechanical properties, may be better tackled by improving the knowledge of BG dissolution. Moreover, for many years, the Hench model has been the core guidance of how bioglass dissolution and the subsequent CaP formation occur. However, this study shows tentative evidence that contributes to and somewhat differs from it.The main funder of this project was the Engineering and Physical Science Research Council (EPSRC). Also, Glaxo Smith Kline (GSK) helped with sponsorship