Development of a fluorescent-based single liposome assay for studying calcium transporter LMCA1

The morphological and functional unit of all the living organisms is the cell. The transmembrane proteins, localized in the plasma membrane of cells, play a key role in the survival of the cells themselves. These proteins perform a variety of different tasks, for example the control of the homeostasis. In order to control the homeostasis, these proteins have to regulate the concentration of chemical elements, like ions, inside and outside the cell. These regulations are fundamental for the survival of the cell and to understand them we need to understand how transmembrane proteins work. Two of the most important categories of transmembrane proteins are ion channels and transporter proteins. The ion channels have been depth studied at the single molecule level since late 1970s with the development of patch-clamp technique. It is not possible to apply this technique to study the transporter proteins so a new technique is under development in order to investigate the behavior of transporter proteins at the single molecule level. This thesis describes the development of a nanoscale single liposome assay for functional studies of transporter proteins based on quantitative fluorescence microscopy in a highly-parallel manner and in real time. The transporter of interest is the prokaryotic transporter Listeria Monocytogenes Ca2+-ATPase1 (LMCA1), a structural analogue of the eukaryotic calcium pumps SERCA and PMCA. This technique will allow the characterization of LMCA1 functionality at the single molecule level. Three systematically characterized fluorescent sensors were tested at the single liposome scale in order to investigate if their properties are suitable to study the function of the transporter of interest. Further studies will be needed in order to characterize the selected calcium sensor and pH sensor both implemented together in single liposomes and in presence of the reconstituted protein LMCA1.

Noise mapping of highly trafficked roads of the province of Bologna according to D.Lgs. 194/2005

The European Community has stressed the importance of achieving a common understanding to deal with the environmental noise through community actions of the Member States. This implies the use of harmonized indicators and specific information regarding the values of indicators, the exceedance of limits and the number of people and dwellings exposed to noise. The D.Lgs. 149/2005 in compliance with the European Directive 2002/49/EC defines methodologies, noise indicators and types of outputs required. In this dissertation the work done for the noise mapping of highly trafficked roads of the Province of Bologna will be reported. The study accounts for the environmental noise generated by the road infrastructure outside the urban agglomeration of Bologna. Roads characterized by an annual traffic greater than three millions of vehicles will be considered. The process of data collection and validation will be reported, as long as the implementation of the calculation method in the software and the procedure to create and calibrate the calculation model. Results will be provided as required by the legislation, in forms of maps and tables. Moreover results regarding each road section accounted will be combined to gain a general understanding of the situation of the overall studied area. Although the understanding of the noise levels and the number of people exposed is paramount, it is not sufficient to develop strategies of noise abatement interventions. Thus a further step will be addressed: the creation of priority maps as the basis of action plans for organizing and prioritizing solutions for noise reduction and abatement. Noise reduction measures are reported in a qualitative way in the annex and constitute a preliminary research.