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.

Optimal pose selection for the calibration of an overconstrained Cable-Driven Parallel Robot

In this project an optimal pose selection method for the calibration of an overconstrained Cable-Driven Parallel robot is presented. This manipulator belongs to a subcategory of parallel robots, where the classic rigid "legs" are replaced by cables. Cables are flexible elements that bring advantages and disadvantages to the robot modeling. For this reason, there are many open research issues, and the calibration of geometric parameters is one of them. The identification of the geometry of a robot, in particular, is usually called Kinematic Calibration. Many methods have been proposed in the past years for the solution of the latter problem. Although these methods are based on calibration using different kinematic models, when the robot’s geometry becomes more complex, their robustness and reliability decrease. This fact makes the selection of the calibration poses more complicated. The position and the orientation of the endeffector in the workspace become important in terms of selection. Thus, in general, it is necessary to evaluate the robustness of the chosen calibration method, by means, for example, of a parameter such as the observability index. In fact, it is known from the theory, that the maximization of the above mentioned index identifies the best choice of calibration poses, and consequently, using this pose set may improve the calibration process. The objective of this thesis is to analyze optimization algorithms which aim to calculate an optimal choice of poses both in quantitative and qualitative terms. Quantitatively, because it is of fundamental importance to understand how many poses are needed. Not necessarily a greater number of poses leads to a better result. Qualitatively, because it is useful to understand if the selected combination of poses actually gives additional information in the process of the identification of the parameters.