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.

Synthesis and characterization of fluorescent atropoisomeric bis-arylboryl-carbazoles

This thesis project presents a work based on the study of bis-arylboryl-carbazoles a particular class of aminoboranes. The peculiarity of these compounds is the -B=N+ chemical moiety and their conformational behaviour coming from the combination of steric constrain and conjugation of the B-N bond. Our work is focused on three products: 9-(mesityl(naphthalen-1-yl)boraneyl)-9H-carbazole 1a, 9-(mesityl(2-methylnaphthalen-1-yl)boraneyl)-9H-carbazole 1b and 9-(anthracen-9-yl(mesityl)boraneyl)-9H-carbazole 1c. We firstly focused our attention on the synthesis optimizing conditions. Then the products were synthetized and characterized with NMR. The products were eventually analysed through conformational studies, by a theoretical approach with DFT calculations and by experimental techniques, such as standard kinetic and EXSY. In the end of this work the products were characterized through fluorescence studies both by DFT, TD-DFT calculations and experimentally by emission spectroscopy.