Intracellular chloride and pH are fundamental regulators of neuronal excitability and they are often co-modulated during excitation-inhibition activity. The study of their homeostasis requires simultaneous measurements in vivo in multiple neurons. Combining random mutagenesis screening, protein engineering and two-photon-imaging this thesis work led to the discovery of new chloride-sensitive GFP mutants and to the establishment of ratiometric imaging procedures for the quantitative combined imaging of intraneuronal pH and chloride. These achievements have been demonstrated in vivo in the mouse cortex, in real-time monitoring the dynamic changes of ions concentrations during epileptic-like discharges, and in glioblastoma primary cells, measuring osmotic swelling responses to various drugs treatment.

MicroRNAs are a class of small non-coding RNAs involved in post-transcriptional regulation. Their role in almost all processes of the cell, make microRNAs ubiquitary players of cell development, growth, differentiation, cell to cell communication and cell death. Thus, cells’ physiological or pathological conditions are reflected by variations in the levels of expression of microRNAs, enabling them to be used as biomarkers of such states. In the past decade, there has been an exponential increase of studies using microRNAs as potential biomarkers for cancer, neurodegenerative diseases, inflammation and cardiac diseases, from tissues and liquid biopsies. However, none of them has reached the clinics yet, due to inconsistency of results through the literature and lack of assay standardization and reproducibility. Technological limitations of microRNAs detection have been, to date, the biggest challenge for using these molecules in clinical settings. In fact, although microarrays, RT-qPCR and RNA-seq are well-established technologies, they all require complex procedures and trained personnel, for performing RNA extraction, labelling of the target and PCR amplification. All these steps introduce variability and, in addition, since no universally standardized protocol – from sample extraction to analyte detection - has been produced yet, methodological procedures are difficult to reproduce. For this reason, we developed a new platform for the rapid detection of microRNAs in biofluids composed of an innovative silicon-photomultiplier (SiPM) based detector and a new chemistry for nucleic acid testing (Chem-NAT). Chem-NAT exploits a dynamic labelling chemistry which allows the sensitive detection of nucleic acids till single base level. On the other hand, SiPM-based device, compared to normal vacuum photomultipliers, grants miniaturization and higher capacity of fitting in a bench-top solution for clinical settings, among other advantages. The new platform – ODG – has been validated for the direct detection – neither RNA extraction nor PCR amplification needed - of microRNA-21 in plasma of lung cancer patients. In this work, we also explored the use of microRNAs as biomarkers in metastatic castration resistant prostate cancer (mCRPC). We collected plasma samples from mCRPC patients before and after abiraterone acetate treatment – androgen deprivation type of drug – and performed a miRnome analysis for discovering microRNAs predicting the efficacy of the drug. We chose miR-103a-3p and miR-378a-5p and we validated them via TaqMan RT-qPCR. We discovered that the ratio between the two microRNAs is able to predict the efficacy of abiraterone acetate and follow the responsiveness in time. In liquid biopsies, extracellular vesicles are getting increasing importance for diagnostic and prognostic purposes. Therefore, in this work we also explored the expression of some microRNAs in extracellular vesicles from plasma, isolated via nickel-based method. We discovered that microRNA-21 and microRNA-223 are not enriched in vesicles from healthy individuals.