DERIVATI IMIDAZOPIRIDINICI COME INIBITORI DI PROTEINE CHINASI: SINTESI E VALUTAZIONE FUNZIONALE

Nel mio lavoro di tesi mi sono occupato della sintesi di una serie di derivati che mi conducesse ad ottenere molecole in grado di agire da inibitori della proteina chinasi RET (REarranged during Transfection) per il trattamento del tumore tiroideo. La tiroide è una ghiandola endocrina che produce ormoni detti ormoni tiroidei, che entrano nel circolo sanguigno e hanno la funzione di regolare il metabolismo. La tiroide rilascia il suo secreto dalle cellule follicolari sotto stimolazione di un altro ormone, il TSH (od ormone tireostimolante) prodotto dalla ghiandola pituitaria. Gli ormoni tiroidei sono prodotti dai tireociti e sono: la tiroxina (T4) e la triiodotironina (T3) contenenti rispettivamente 4 e 3 atomi di iodio. Tutte le forme di cancro a carico di ghiandole prendono il nome di adenocarcinomi: nel caso della tiroide si può avere un adenocarcinoma papillare (80%) oppure follicolare (11%) od a cellule di Hürtle (3%); il restante 6% di tumori maligni è costituito dal 5% da tumori midollari (di origine neuroendocrina), una forma particolarmente aggressiva, ma per fortuna rara (circa l'1%), è il cosiddetto carcinoma anaplastico della tiroide che da’ precocemente metastasi a distanza. Il cancro papillare tiroideo è il tumore maligno alla tiroide più comune, ha una incidenza media di 0,5-10 casi ogni 100.000 persone. L’eziologia dell’insorgenza del carcinoma papillare tiroideo non è stata ancora ben chiarita ma fra i fattori di rischio accertati c'è il cosiddetto gozzo, caratterizzato da numerosi noduli benigni della ghiandola dovuti a carenza di iodio, che può in alcuni casi predisporre alla trasformazione maligna delle cellule. Un altro fattore di rischio accertato è l'esposizione a radiazioni: il tumore della tiroide è più comune in persone che sono state trattate per altre forme tumorali con radioterapia sul collo oppure che sono state esposte a ricadute di materiale radioattivo. Le donne sono più colpite degli uomini nella proporzione di quattro a uno. Gli ultimi anni sono stati caratterizzati da significativi passi avanti nella comprensione delle basi molecolari che scatenano l’insorgenza del cancro alla tiroide. Le alterazioni principali responsabili della trasformazione neoplastica sono: la deregolazione genica e l’attivazione di vie di crescita tumorali ubiquitarie, come la segnalazione MAPK (Mitogen Activated Protein Kinase) e PI3K/AKT (phosphatidylinositol 3-kinase/AKT). Altre specifiche mutazioni possono avvenire nelle vie di trasduzione del segnale e nelle proteine mediatrici di queste vie, come le proteine RAS. In particolare il recettore RET appartiene alla famiglia delle proteine tirosin-chinasi e l’oncogene RET/PTC, che codifica per un recettore RET mutato, gioca un ruolo importante nello sviluppo del carcinoma papillare della Tiroide. Il trattamento terapeutico attualmente raccomandato per il cancro papillare tiroideo è la tiroidectomia totale seguita da una radioterapia con 131I. Tuttavia nel 20% dei casi il trattamento non ha successo e i pazienti vedono l’aspettativa di vita media calare drasticamente rispetto al resto della popolazione. La ricerca si è quindi diretta verso lo studio di composti in grado di inibire la cascata di segnali mediata da RET; un’opzione terapeutica attraente. Lo scopo del mio progetto di tesi è stato quindi quello di sintetizzare molecole a nucleo imidazopiridinico, correlate al composto hit di partenza 2,6-difenil-H-imidazo[1,2-α]piridina individuato come possibile nuovo inibitore di RET

Unmanned Aerial Vehicles for Debris Survey in Coastal Areas: Long-Term Monitoring Programme to Study Spatial and Temporal Accumulation of the Dynamics of Beached Marine Litter

Unmanned aerial vehicles (UAVs) are becoming increasingly accessible tools with widespread use as environmental monitoring systems. They can be used for anthropogenic marine debris survey, a recently growing research field. In fact, while the increasing efforts for offshore investigations lead to a considerable collection of data on this type of pollution in the open sea, there is still little knowledge of the materials deposited along the coasts and the mechanism that leads to their accumulation pattern. UAVs can be effective in bridging this gap by increasing the amount of data acquired to study coastal deposits, while also limiting the anthropogenic impact in protected areas. In this study, UAVs have been used to acquire geo-referenced RGB images in a selected zone of a protected marine area (the Migliarino, Massacciuccoli, and San Rossore park near Pisa, Italy), during a long-term (ten months) monitoring programme. A post processing system based on visual interpretation of the images allows the localization and identification of the anthropogenic marine debris within the scanned area, and the estimation of their spatial and temporal distribution in different zones of the beach. These results provide an opportunity to investigate the dynamics of accumulation over time, suggesting that our approach might be appropriate for monitoring and collecting such data in isolated, and especially in protected, areas with significant benefits for different types of stakeholders

Rat pial microvascular responses to melatonin during bilateral common carotid artery occlusion and reperfusion

The present study assessed the in vivo rat pial microvascular responses induced by melatonin during brain hypoperfusion and reperfusion (RE) injury. Pial microcirculation of male Wistar rats was visualized by fluorescence microscopy through a closed cranial window. Hypoperfusion was induced by bilateral common carotid artery occlusion (BCCAO, 30 min); thereafter, pial microcirculation was observed for 60 min. Arteriolar diameter, permeability increase, leukocyte adhesion to venular walls, perfused capillary length (PCL), and capillary red blood cell velocity (V(RBC) ) were investigated by computerized methods. Melatonin (0.5, 1, 2 mg/kg b.w.) was intravenously administered 10 min before BCCAO and at the beginning of RE. Pial arterioles were classified in five orders according to diameter, length, and branchings. In control group, BCCAO caused decrease in order 2 arteriole diameter (by 17.5 ± 3.0% of baseline) that was reduced by 11.8 ± 1.2% of baseline at the end of RE, accompanied by marked leakage and leukocyte adhesion. PCL and capillary V(RBC) decreased. At the end of BCCAO, melatonin highest dosage caused order 2 arteriole diameter reduction by 4.6 ± 2.0% of baseline. At RE, melatonin at the lower dosages caused different arteriolar responses. The highest dosage caused dilation in order 2 arteriole by 8.0 ± 1.5% of baseline, preventing leakage and leukocyte adhesion, while PCL and V(RBC) increased. Luzindole (4 mg/kg b.w.) prior to melatonin caused order 2 arteriole constriction by 12.0 ± 1.5% of baseline at RE, while leakage, leukocyte adhesion, PCL and V(RBC) were not affected. Prazosin (1 mg/kg b.w.) prior to melatonin did not significantly change melatonin's effects. In conclusion, melatonin caused different responses during hypoperfusion and RE, modulating pial arteriolar tone likely by MT1 and MT2 melatonin receptors while preventing blood-brain barrier changes through its free radical scavenging action

A new beach topography-based method for shoreline identification

The definition of shoreline is not the same for all contexts, and it is often a subjective matter. Various methods exist that are based on the use of different instruments that can determine and highlight a shoreline. In recent years, numerous studies have employed photogrammetric methods, based on different colours, to map the boundary between water and land. These works use images acquired by satellites, drones, or cameras, and differ mainly in terms of resolution. Such methods can identify a shoreline by means of automatic, semi-automatic, or manual procedures. The aim of this work is to find and promote a new and valid beach topography-based algorithm, able to identify the shoreline. We apply the Structure from Motion (SfM) techniques to reconstruct a high-resolution Digital Elevation Model by means of a drone for image acquisition. The algorithm is based on the variation of the topographic beach profile caused by the transition from water to sand. The SfM technique is not efficient when applied to reflecting surfaces like sea water resulting in a very irregular and unnatural profile over the sea. Taking advantage of this fact, the algorithm searches for the point in the space where a beach profile changes from irregular to regular, causing a transition from water to land. The algorithm is promoted by the release of a QGIS v3.x plugin, which allows the easy application and extraction of other shorelines

Segmentation of lung fields in digital chest radiographs by artificial neural networks

Lung field segmentation is a basic step for virtually any quantitative procedure. In this view, due to the imaging process and the complexity of the imaged district, an efficient use of prior anatomical knowledge is crucial. In this report we describe a new approach to lung field segmentation which is based on fuzzy boundary modeling and a neural network architecture including supervised multilayer networks and topology preserving maps

Rat Pial Microvascular Responses to Transient Bilateral Common Carotid Artery Occlusion and Reperfusion: Quercetin’s Mechanism of Action

The aim of the present study was to assess quercetin’s mechanism of action in rat pial microvessels during transient bilateral common carotid artery occlusion (BCCAO) and reperfusion. Rat pial microcirculation was visualized using fluorescence microscopy through a closed cranial window. Pial arterioles were classified in five orders of branchings. In ischemic rats, 30 min BCCAO and 60 min reperfusion caused arteriolar diameter decrease, microvascular leakage, leukocyte adhesion in venules, and reduction of capillary perfusion. Quercetin highest dose determined dilation in all arteriolar orders, by 40 ± 4% of baseline in order 2 vessels, and prevented microvascular permeability [0.15 ± 0.02 normalized gray levels (NGL)], leukocyte adhesion, and capillary failure. Protein kinase C (PKC) inhibition exerted by chelerythrine prior to quercetin attenuated quercetin-induced effects: order 2 arterioles dilated by 19.0 ± 2.4% baseline, while there was an increase in permeability (0.40 ± 0.05 NGL) and leukocyte adhesion with a marked decrease in capillary perfusion. Tyrosine kinase (TK) inhibition by tyrphostin 47 prior to quercetin lessened smaller pial arterioles responses, dilating by 20.7 ± 2.5% of baseline, while leakage increased (0.39 ± 0.04 NGL) sustained by slight leukocyte adhesion and ameliorated capillary perfusion. Inhibition of endothelium nitric oxide synthase (eNOS) by NG-nitro-L-arginine-methyl ester (L-NAME) prior to PKC or TK reduced the quercetin’s effects on pial arteriolar diameter and leakage. eNOS inhibition by L-NAME reduced quercetin effects on pial arteriolar diameter and leakage. Finally, combined inhibition of PKC and TK prior to quercetin abolished quercetin-induced effects, decreasing eNOS expression, while blocking ATP-sensitive potassium (KATP) channels by glibenclamide suppressed arteriolar dilation. In conclusion, the protective effects of quercetin could be due to different mechanisms resulting in NO release throughout PKC and TK intracellular signaling pathway activation

Protective Effects of Quercetin on Rat Pial Microvascular Changes during Transient Bilateral Common Carotid Artery Occlusion and Reperfusion

The aim of this study was to assess the in vivo effects of quercetin on pial microvascular responses during transient bilateral common carotid artery occlusion (BCCAO) and reperfusion. Rat pial microcirculation was visualized by fluorescence microscopy through a closed cranial window. Pial arterioles were classified in five orders of branchings. Capillaries were assigned order 0, the smallest arterioles order 1, and the largest ones order 5. In ischemic rats, 30 min BCCAO and 60 min reperfusion caused arteriolar diameter decrease (by 14.5 ± 3.3% of baseline in order 2), microvascular leakage [0.47 ± 0.04, normalized gray levels (NGL)], leukocyte adhesion in venules (9 ± 2/100 μm venular length, v.l./30 s), and reduction of capillary perfusion (by 40 ± 7% of baseline). Moreover, at the end of BCCAO and reperfusion there was a significant increase in reactive oxygen species (ROS) formation when compared with baseline. Quercetin highest dose determined dilation in all arteriolar orders (by 40 ± 4% of baseline in order 2) and prevented microvascular permeability (0.15 ± 0.02 NGL), leukocyte adhesion (3 ± 1/100 μm v.l./30 s) as well as ROS formation, while capillary perfusion was protected. Inhibition of endothelial nitric oxide synthase (NOS) prior to quercetin reduced arteriolar dilation (order 2 diameter increase by 10.3 ± 2.5% of baseline) and caused permeability increase (0.29 ± 0.03 NGL); inhibition of neuronal NOS or inducible NOS did not affect quercetin-induced effects. Inhibition of guanylyl cyclase prior to quercetin reversed the quercetin’s effects on pial arteriolar diameter and leakage. In conclusion, quercetin was able to protect pial microcirculation from ischemia–reperfusion damage inducing arteriolar dilation likely by nitric oxide release. Moreover, quercetin scavenger activity blunted ROS formation preserving the blood–brain barrier integrity

Long term remodeling of rat pial microcirculation after transient middle cerebral artery occlusion and reperfusion.

Objective: The aim of this study was to assess the in vivo structural and functional remodeling of pial arteriolar networks in the ischemic area of rats submitted to transient middle cerebral artery occlusion (MCAO) and different time intervals of reperfusion. Methods and results: Two closed cranial windows were implanted above the left and right parietal cortex to observe pial microcirculation by fluorescence microscopy. The geometric characteristics of pial arteriolar networks, permeability increase, leukocyte adhesion and capillary density were analyzed after 1 h or 1, 7, 14 or 28 days of reperfusion. MCAO and 1-hour reperfusion caused marked microvascular changes in pial networks. The necrotic core was devoid of vessels, while the penumbra area presented a few arterioles, capillaries and venules with severe neuronal damage. Penumbra microvascular permeability and leukocyte adhesion were pronounced. At 7 days of reperfusion, new pial arterioles were organized in anastomotic vessels, overlapping the ischemic core and in penetrating pial arterioles. Vascular remodeling caused different arteriolar rearrangement up to 28 days of reperfusion and animals gradually regained their motor and sensory functions. Conclusions: Transient MCAO-induced pial-network remodeling is characterized by arteriolar anastomotic arcades. Remodeling mechanisms appear to be accompanied by an increased expression of nitric oxide synthases