LINEAR AND NON-LINEAR PHOTOACTIVATION A TOOL FOR NEUROSCIENCE AND CANCER RESEARCH.

Photoactivation is a physical process that allows using light-matter interaction as a precursor to induce a specific Physico-chemical reaction. This reaction is induced after the absorption of a photon, having a suitable wavelength, by specific photoactivable molecules. Such molecules act as a molecular machine that performs functions useful both for therapeutic purposes but also for the study of biophysical processes. The ability to have high temporal and spatial control is one of the main advantages of photoactivation. Therefore combining the photoactivation with appropriate optical methods, manipulation of living specimens at molecular precision is possible. Nevertheless, a set of photoactivable molecules is the key player in realizing super-resolution microscopy, allowing the quantitative study of biological challenges at the subcellular scale. This work of thesis focuses the attention on two classes of photoactivable molecules, i.e., caged compounds and photosensitizers, with the attention at applications in the neuroscience and photodynamic therapy fields, respectively. Caged compounds are realized via covalent appendage of a light-sensitive protecting group “the cage” to a signaling molecule. In particular, it was used as a GABA caged molecule (RuBi-GABA). One photon (UV-VIS light) and two-photon (700-900nm) absorption were used to break the “cage” binding. The uncaged molecule (GABA) becomes active and can bind the GABAA receptor site generating a Cl- current across the neuronal membrane that can be recorded using the Patch Clamp Technique. This approach allows controlling the neurotransmitter release in time, space, and relative concentration. In particular, the uncaging method and fluorescence microscopy coupled to the patch-clamp technique provides a useful approach to detect a selected biological target in a temporally and spatially confined way. It was analyzed how the change of physical parameters such as uncaging distance, exposure time, laser power, linear and non-linear photoactivation influence the measurements, and it was determined how these parameters change concentration and volume of GABA release and consequently the GABAA response. Specifically, localization precision can be improved using advanced fluorescent optical methods such as super-resolved and non-linear fluorescence microscopy. This allows exploring the release of caged GABA topically applied in situ at defined concentration and in a specific region of neuronal cells for mapping the localization and the functional distribution of GABAA receptors in cerebellar granule cells in vitro. Finally, it was possible to explore the responses generated by specifics drugs in different regions of neurons. Photosensitizers are photoactivable molecules that, after absorption of light, can produce reactive species of oxygen, which induce cell damage and death. When those molecules are targeted to cancer cells the process could lead to the death of tumor cells. Such molecules are typically exploited in photodynamic therapy (PDT). This treatment modality is not invasive and explicates its function by the simultaneous presence of a PS, visible light, and tissue oxygen. Indeed, these specific molecules can target and localize in the neoplastic tissue and, upon photoactivation with visible light, they generate reactive oxygen species (ROS), causing confined damage. The reactive chemical species, and in particular singlet oxygen (1O¬2), are characterized by an active region of the order of 0.02 μm, where a series of reactions can produce oxidative damages and consequently cellular death for apoptosis or necrosis. The cellular death mechanism,such as apoptosis or necrosis,depends on(i)the cell line used,(ii)thequantity of light usedand(iii)and the cellarea in which the PSs accumulate. Besides, these cellular damages could produce an immune response, improving therapy efficiency. A particular class of PSs, such as hypericin, phthalocyanine, porphyrin, and curcumin, are an object of study for PDT. However, a general characteristic and drawback of PSs is the low solubility in water and the aggregate formation, which impair their photophysical properties, such as the quenching of productive, excited states. To overcome this limit, PSs carriers were exploited. Among them, proteins offer several potential advantages. It has been demonstrated that PS molecules spontaneously bind internal hydrophobic cavities of particular proteins, preserving their monomeric, photoactive (both photosensitizing and fluorescent) state. Proteins such as ApoMb and BSA were used as a carrier (ApoMb and BSA) for hypericin, phthalocyanine, porphyrin, and curcumin. These complexes were studied through advanced microscopical methods (spinning disk confocal, confocal, and STED microscopy) analyzing the difference in the use of PS or PS-protein complex, demonstrating how a protein can increase the PS efficacy. Therefore, were performed accumulation measurements, biocompatibility and bioavailability tests, colocalization measurements, and were studied the interactions of PSs with tumoral spheroids. In summary, the main goals of this work are: (i) the study of cellular tumor damages induced by photosensitizing molecules and its localization in cells and the increase of its efficacy using protein carriers exploit advanced microscopy technique (ii) the study of GABAA receptor with the use of GABA caged molecule developing a set-up that combines confocal and two-photon excitation fluorescence microscope with patch-clamp technique