Embriogénesis somática de Pinus spp. bajo condiciones de estrés abióticomodelo para el estudio de los mecanismos que controlan la tolerancia

Resumen

Water, the most abundant molecule in our planet, is truly and empirically accepted as an essential component to sustain life on Earth. The extraordinary properties of this unique and universal "matrix of life" have served over this quarter century, as the driving force -for theoreticians and experimentalists- to perform novel research on the structural basis and molecular dynamics taking place in very different environments. Even today, scientists are still discovering new phenomena in water, especially at the nanoscale.When referred to bio-systems, water is usually seen as the liquid environment -or "matrix"- where physical and chemical processes take place. Here, the molecules of life are thought of being wet - this is, completely immersed in liquid water or, in other words, in fully hydrated conditions. While this is true for most living cells, a surprising number of processes require merely traces of water, which could be vapor or a layer as thin as nanometers. Indeed, this is observed in situations in which, because of extremes of either heat or cold, liquid water is scarce. Here, the hydration layer of the molecules - understood as an entity that differs from bulk water - should confer certain stability to keep the biomolecules still functional; and the processes taking place at the air interface should play a prominent role.In this direction, the transmission of many airborne viruses - through respiratory droplets (e.g. aerosols) - requires, actually, survival in dry conditions. Influenza A virus (IAV), is one of the best-known cases, for which the transmission and thus survival is tightly connected to seasonal factors. Seasonal epidemics generally occur during winter, when air temperature and humidity are low. Under these conditions, the respiratory droplets must confer some structural stability to the contained viruses; membrane collapse, for example, should be in principle avoided. However, this assumption is yet poorly understood and experimental data based on biophysical methods are in great demand to obtain a solid knowledge base for the restriction of virus transmission (in addition to vaccination strategies).This thesis represents the outcome of almost four years of intensive work in the field of applied biophysics in life sciences. Here, we explore the possibilities to mimic IAV by different model systems, by mostly focusing on the role of hydration water under various relative humidity (RH) conditions, namely hydration scenarios. To date, RH has been one of the less investigated and one of the most controversial environmental factors. Its effect on the transmission, survival and stability of the virus is still under debate, while the influence of air temperature, e.g., is quite well established.In this research, the structure of the IAV envelope is studied in a range of model systems and by a combination of very different techniques, providing new biophysical insights on the micro- and nanoscale, from dehydrated to fully hydrated conditions.To this aim, in Chapter 1, an overview on the scope of this investigation is provided. Here, we include an introduction to the science of hydration as well as information on the IAV structure and seasonality. Finally, a detailed description on the developed model systems is offered.In Chapter 2, all the experimental techniques used in this research are described and specific considerations for performing the experiments are noted.In Chapter 3, we introduce the first approximation for mimicking the IAV surface "spike" glycoproteins (e.g. hemagglutinin, HA), by the use of dimannoside-coated gold nanoparticles (Dimanno-AuNPs). In this research, unprecedented observations on the stability and hydration water of the particles are provided in solution - by Dynamic Light Scattering (DLS) and Zeta Potential (ZP) measurements - and after dehydration - by a combination of Vibrational Sum Frequency Generation Spectroscopy (VSFG), Fourier Transform Infrared Spectroscopy (FT-IR) and Electron Microscopy. The study culminates with the analysis on the water adsorption of the particles at various RH, by Atomic Force Microscopy (AFM). Here, the hydration properties of mannosides in air, from dehydrated states to high humidity are demonstrated.In Chapter 4, a supported lipid bilayer, with HA anchored at high surface density is proposed as a "flattened" and simplified model of the IAV envelope. The effect of RH is investigated, in complete cycles of hydration, dehydration and rehydration. The membrane performance is evaluated in terms of structure and dynamics, by combining Confocal Fluorescence Microscopy, Raster Image Correlation Spectroscopy (RICS), Line-Scan Fluorescence Spectroscopy (LSFCS) and AFM. The macro- and nanoscopic effects of HA under dehydration stress are studied in detail, revealing new information on the stabilization mechanisms operating in the IAV envelope at low RH.In Chapter 5, the use of a monovalent Live Attenuated Influenza Vaccine (LAIV) as a more realistic model of the IAV envelope is explored. Here, revealing the morphological features of the IAV envelope is attempted by performing AFM on solid surfaces in air. Although the detailed surface structure of the virus is not revealed, complete assembled viruses are identified after dehydration, suggesting that adsorbed LAIV can be used as an analogous model of the IAV envelope during airborne transmission.Finally, Chapter 6 contains concluding remarks for the studies presented in this thesis, and an outlook to further investigations.