Biofisica de centromas y adn mediante manipulacion optica

Resumen

Recent years have witnessed the emergence of an entirely new field of science, often referred to as single-molecule biophysics. This interdisciplinary field is currently providing novel approaches to biochemical and biological problems. The capability of single-molecule methods of revealing the behavior of individual molecules having different conformation and properties is beyond the ability of their bulk counterparts, which describe the behavior of enormous ensembles of molecules, averaging the measured parameters over the entire molecular population. Optical tweezers are one of these new techniques which allow the real-time manipulation of single molecules and cells. They have been used in a variety of biological systems to lead to a new understanding of the mechanical properties of the fundamental building blocks of the cell, and of the mechanism by which molecular machines function. This thesis comprises the implementation of an optical tweezers instrument as one of its research projects. The technique, the basic ideas behind the operating principles and the explanation of the components of the built set-up are described in Chapter 2. The state-of-the-art optical tweezers instrument developed constitutes the main experimental tool of the present thesis. It is used in two different investigations. Firstly, we study the mechanical stability of single DNA molecules under low humidity conditions. We analyze the simultaneous effect of tension and ethanol on two DNAs differing in both sequence and secondary structure in buffer to analyze GC content dependencies. Our results support that condensation co-exists with the Aform and that such association reinforce the conformational B-A transition (Chapter 3). This study is complemented with the characterization by two other singlemolecule techniques, namely, magnetic tweezers and atomic force microscopy (Chapter 4). Secondly, we perform laser manipulation of individual Drosophila centrosomes which lead us to carry out single-organelle electrophoresis. We find that the centrosome is a net negatively charged organelle and, although we infer an effective charge significantly smaller than that of microtubules, we show that the charge of the centrosome has a remarkable influence over its own structure. Specifically, we investigate the hydrodynamic behavior of the centrosome by measuring its size by both Stokes law and thermal-fluctuation spectral analysis of force. We find that the hydrodynamic size of the centrosome is 60% larger than its electron microscopy diameter and that this physiological expansion is produced by the electric field that drains to the centrosome, a self-effect which modulates its structural behavior via environmental pH (Chapter 5).