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L’analisi scientifica è da un po’ di tempo entrata nel mondo dei beni culturali, non sono più solo gli archeologi e gli storici dell’arte a occuparsi dello studio dei reperti archeologici e storici, ma anche gli scienziati sono entrati a gamba tesa portando le loro conoscenze al servizio di questa vasta gamma di materiali. Lo studio di reperti botanici e zoologici, le competenze chimiche e mineralogiche, lo studio di fenomeni fisici, ha permesso di porre e rispondere a nuove domande, colmando così lacune sulla storia dell’umanità. Reti commerciali, migrazioni, tecniche produttive, molte sono state le scoperte avvenute grazie anche all’intervento scientifico, oltre a fornire strumenti utili al restauro e alla conservazione dei reperti. Nei miei tre anni di dottorato mi sono occupato dell’applicazione dell’analisi in Fluorescenza a Raggi X (XRF) per analizzare campioni metallici e ceramici. Questa tecnica ha il vantaggio di poter essere applicata in maniera non invasiva e non distruttiva su un reperto per ottenere informazioni sulla sua composizione elementare. In particolare, il nostro obiettivo è quello di ottenere informazioni sui diversi strati che compongono un campione. Spesso, infatti, i manufatti presentano una struttura stratificata causata dal passaggio degli anni o dalla natura stessa dell’oggetto, che presenza una serie di decorazioni superficiali. L’impiego di tecniche non invasive permetterebbe quindi di ottenere informazioni più dettagliate anche su campioni al momento inaccessibili, in quanto troppo fragili o troppo preziosi. Lo studio si è quindi avvalso di tecniche a scansione angolare, per cui il segnale di fluorescenza caratteristica dipende sia dalla posizione dell’analita all’interno del campione, sia dalla struttura e composizione dello stesso, sia dalla geometria di analisi. Per valutare l’applicabilità delle tecniche sono stati analizzati tre casi diversi: campioni metallici preparati ad hoc in laboratorio, un campione ceramico, e un campione di lustro. Per i primi due campioni è stata applicata l’XRF a Risoluzione Angolare (AR-XRF) per cui il campione è stato ruotato con uno step angolare inferiore a un grado, e misurato ad ogni step. Nel caso dei campioni metallici i profili misurati sono stati confrontati direttamente con i profili calcolati usando il metodo dei Parametri Fondamentali. Nel caso del campione ceramico invece, sono stati confrontati i rapporti dei profili, in quanto la geometria del campione era piuttosto complessa. Nello studio sui campioni metallici, composti da una doratura depositata su una lamina di rame, è stato possibile calcolare lo spessore dello strato superficiale. Nel caso del campione di Majolica sono invece state studiate le varie decorazioni, valutando così anche diversi limiti della tecnica, in particolar modo nel caso in cui la composizione della decorazione sia simile alla composizione dello strato sottostante, o nel caso in cui avvengano processi di diffusione a lunghe distanze. In casi in cui invece lo spessore della decorazione era limitato e ben separato dallo strato sottostante, è stato possibile ottenere informazioni sua sulla composizione della decorazione che sul suo spessore. Infine, lo studio delle ceramiche lustrate è stato effettuato presso la linea XRF del Sincrotrone di Elettra, impiegando tecniche di analisi in radenza. In questo caso stato possibile solo evidenziare come il nano-strato di nano-particelle di argento, caratteristico di questa tipologia di campioni, sia effettivamente distinguibile applicando queste tecniche, in quanto il profilo di fluorescenza di tale elemento è molto diverso dagli altri. Rimangono comunque dei problemi, legati soprattutto all’analisi dati e all’allineamento che vanno ancora risolti, rendendo necessari ulteriori studi a riguardo. In the last decades scientific analysis has been deeply employed in the world of cultural heritage, thus, archaeologists and art historians are no more the sole front line workers of this field. Scientists, and science, have joined the team, giving new inputs and tools for the study of historical and archaeological samples, allowing to explore new paths and receive new answers, collecting information otherwise inaccessible on human history and culture. New discoveries have been made on the trade networks, migrations and on the technologies employed; besides, science also gave precious inputs on conservation and restoring procedures, allowing to better preserve fragile and sensitive artifacts. In my three years as a PhD student, I worked on the application of X-Ray Fluorescence (XRF) analysis to analyze ceramic and metal samples. XRF is a non-invasive technique that retrieves the elemental composition of a sample. In particular the aim of my PhD project is to obtain information on the layered structure of an unknown sample, distinguishing and characterizing the different layers. Indeed, artifacts usually concerning the field of Cultural Heritage present a layered structure; sometimes it is due to the presence of alteration layers, other times, instead, the objects are made of different layers from the principle, for example in the case of a glazed ceramic or of a painting. The possibility to get this information in a non-invasive way will give the possibility to analyze objects that are nowadays unattainable, because they cannot be sampled. My project has, thus, focused on the analysis of three kinds of samples employing angular dependent techniques (Angle Resolved-XRF, Grazing Emission-XRF}, Grazing Incidence-XRF}); indeed, the fluorescence signal of an analyte depends on its position inside the sample, on the sample composition and on the geometry of analysis. The chosen specimens allowed to verify the feasibility of this analytical method in an increasing complexity: a gilded laboratory-made sample, a ceramic Majolica sherd, and an Italian renaissance lustered fragment. The first two samples have been analyzed through AR-XRF where the measure is performed while tilting the sample, one spectrum is collected for each tilting angle. In the case of the gilded sample the measured profiles have been compared directly with the calculated profiles employing the Fundamental Parameters method. For the ceramic Majolica sample, instead, we studied the ratio of the profiles, as the sample surface is not flat. In the study of the metallic samples, made of gilded copper plate, we could infer the thickness of the top-layer. While in the case of the Majolica sample, we studied the different decorations, evaluating the limits of the technique, in particular in the case that the top-layer composition is similar to the underling layer, or in the case of a long-range diffusion. Instead, in the case of two well-separate layers we could retrieve information on both the composition and the thickness of the layer. Finally, the study of the lustered ceramic has been carried out at the XRF beamline of the Elettra Synchrotron of Trieste, employing grazing techniques. In this case we could only highlight and distinguish the presence of the silver nanoparticles in the luster nanolayer, which is the peculiar feature of this kind of artifacts. However, there are still many questions left, especially concerning the data analysis and the alignment of the sample, which requires more investigations.

La fotosintesi è il processo chiave che sostiene la vita sulla Terra. L'efficienza della fotosintesi in condizioni ambientali variabili è mantenuta da meccanismi di regolazione che rispondono velocemente, ovvero l'emissione della fluorescenza della clorofilla a (F) e il quenching non fotochimico (NPQ). F è una radiazione elettromagnetica emessa a lunghezze d'onda più lunghe (640-850 nm) rispetto a quelle di eccitazione. NPQ è un processo complesso che comprende la de-epossidazione dei pigmenti del ciclo della xantofilla con conseguente dissipazione dell'energia in eccesso sotto forma di calore. L'obiettivo del mio progetto di dottorato era quello di sfruttare i metodi di telerilevamento di prossimità della vegetazione per chiarire il collegamento tra misurazioni iperspettrali in continuo di indici ottici relazionati alla fisiologia delle piante (F e PRI) e la funzionalità della vegetazione. Sono state utilizzate misure multiangolari per caratterizzare la risposta anisotropa della fluorescenza nel rosso lontano (F760), nel rosso (F687) e del PRI. Presento un ampio dataset sperimentale di misurazioni multiangolari di F, PRI e riflettanza (R) raccolte durante cicli giornalieri su quattro diversi target di vegetazione. Le risposte direzionali di F760, F687 e PRI in canopy orizzontalmente omogenee sono caratterizzate da valori maggiori nella direzione di retrodiffusione con un massimo nell'hotspot e da valori minori nella direzione di diffusione in avanti. La forma della risposta angolare di F e PRI è per lo più controllata dalla funzione di distribuzione dell'inclinazione delle foglie (LIDF), mentre la grandezza e la forma del picco dell'hotspot sono sensibili ad una combinazione di fattori - l'indice di area fogliare (LAI) e il rapporto tra la larghezza delle foglie e l'altezza della canopy. La valutazione quantitativa dell'impatto dell'anisotropia sui rendimenti apparenti della fluorescenza (Fy*760 e Fy*687) ha mostrato che, in media, le osservazioni off-nadir sono sovrastimate del 20-67% in direzione della retrodiffusione e sottovalutate in altre direzioni del 10-45%. I risultati ottenuti corroborano l'importanza di mantenere una geometria di osservazione nadirale per le misure in continuo di F e PRI. Un ulteriore elemento di complessità per l'interpretazione delle serie temporali di segnali ottici risiede nella dipendenza temporale delle dinamiche della vegetazione. In tale contesto, questo lavoro verifica l'applicabilità di una tecnica di decomposizione delle serie temporali altamente flessibile chiamata Singular Spectrum Analysis (SSA) per disaccoppiare le componenti lente e veloci delle dinamiche della vegetazione nelle serie temporali di F760, Fy*760 e PRI. In primo luogo, è stata sviluppato un proof of concept sulla base di serie temporali spettrali e di flussi a intervalli di mezz’ora simulate realisticamente con il modello SCOPE. Le simulazioni comprendevano due output - considerando ed escludendo l'effetto del ciclo di de-epossidazione delle xantofille e il fattore di amplificazione dell’efficienza di fluorescenza rispettivamente su PRI e F760. Le componenti SSA lente decomposte di PRI, F760 e Fy*760 hanno mostrato una buona correlazione con la variabilità costitutiva di riferimento simulata escludendo l'effetto della modulazione fisiologica. L'accuratezza delle componenti SSA veloci è stata validata con variabili fisiologicamente indotte di riferimento (∆PRI, ∆F), e con il NPQ e l'efficienza di utilizzo della luce (LUE). Quindi, questa metodologia è stata applicata su un set di serie temporali spettrali e di flussi acquisite in campo su grano invernale, permettendo di migliorare significativamente la correlazione delle componenti veloci di F e PRI con LUE rispetto alle serie temporali originali. l'approccio basato su SSA è uno strumento promettente per disaccoppiare le informazioni fisiologiche a partire da misurazioni in continuo del segnale ottico. Photosynthesis is the key process sustaining life on Earth. The efficiency of photosynthesis under varying environmental conditions is maintained by fast-responding regulatory mechanisms, namely chlorophyll a fluorescence emission (F) and non-photochemical quenching (NPQ). F is an electromagnetic radiation emitted at longer wavelengths (640-850 nm) than for excitation. NPQ is a complex process which includes the de-epoxidation of xanthophyll cycle pigments resulting in dissipation of excess energy as heat. This translates into changes in leaf absorbance at 531 nm, which can be detected with the photochemical reflectance index (PRI). Proximal sensing is a powerful tool for exploitation of subtle signals related to the downregulation of photosynthesis. However, F and PRI are also influenced by canopy structural and biochemical properties, illumination conditions and solar-view geometry, and its explicit interpretation is still challenging. The aim of my Ph.D. project was to exploit the methods of proximal sensing of vegetation to elucidate a link between continuous hyperspectral measurements of optical indicators related to plant physiology (F and PRI) and vegetation functioning. Multi-angular measurements were used to characterize the anisotropic response of far-red (F760), red (F687) fluorescence and PRI. I present an extensive experimental dataset of multi-angular measurements of F, PRI and R collected during a day over four different vegetation targets. Directional responses of F760, F687 and PRI in horizontally homogeneous canopies are characterized by increased values in the backward scattering direction with a maximum in the hotspot and decreased values in the forward scatter direction. The shape of F and PRI angular response is mostly controlled by leaf inclination distribution function (LIDF), while a magnitude and shape of the hotspot peak is sensitive to a combination of factors - leaf area index (LAI) and the ratio of leaf width to canopy height. Quantitative evaluation of the impact of anisotropy on fluorescence apparent yields (Fy*760 and Fy*687) showed, that, on average, off-nadir observations were overestimated by 20-67% in the backscatter direction and underestimated in other directions by 10-45%. The archived results reinforce the importance of maintaining nadir observation geometry for continuous measurements of F and PRI. Additional complexity for the interpretation of time series of optical signal lies in time-scale dependant vegetation dynamics. Here I test the applicability of highly-adaptive time series decomposition technique Singular Spectrum Analysis (SSA) for disentangling slow and fast varying components of vegetation dynamics in time series of F760, Fy*760 and PRI. First, the proof of concept was developed based on spectral and flux half-hourly time series realistically simulated with the SCOPE model. The simulations included two outputs - featuring and excluding the effect of the xanthophyll cycle de-epoxidation and fluorescence efficiency amplification factor on PRI and F760, respectively. The decomposed slow varying SSA-components of total PRI, F and Fy*760 showed a good correlation with reference constitutive variability simulated excluding the effect of physiological modulation (PRI0 , F0,760, Fy*0,760). The accuracy of disentangled fast varying SSA-components was validated with the reference physiologically induced variables (∆PRI, ∆F), and with non-photochemical quenching (NPQ) and light-use efficiency (LUE). Then, the application of this methodology on a field dataset of spectral and flux time series collected in winter wheat field allowed to significantly improve the correlation of fast components of F and PRI with the fast component of LUE in comparison with original time series. Therefore, SSA-based approach is a promising tool for decoupling physiological information from continuous measurements of optical signal which can foster the use of automated proximal sensing systems.

Negli ultimi decenni, il telerilevamento è stato utilizzato con successo per monitorare la vegetazione grazie alla sua capacità intrinseca di fornire informazioni quantitative ripetute e spazialmente distribuite circa le sue proprietà. Tuttavia, la ricerca si è concentrata prevalentemente sulla descrizione delle proprietà strutturali e biochimiche della vegetazione piuttosto che sulla comprensione del suo funzionamento. Negli ultimi decenni, il telerilevamento della fluorescenza della clorofilla indotta dal sole (F) è emerso come strumento innovativo utile allo studio del funzionamento delle piante. F è un debole segnale emesso nelle regioni spettrali del rosso e infrarosso vicino (~650-800 nm) dal cuore dell’apparato fotosintetico per dissipare l’eccesso di energia assorbita. Il suo potenziale risiede nella relazione esistente tra fotochimica e vie dissipative: poiché la fotochimica compete con la dissipazione sotto forma di F e calore, F può essere un indicatore diretto del funzionamento della fotosintesi. L’obiettivo principale di questo dottorato è stato l’utilizzo di dati ottici (i.e., riflettanza e fluorescenza) per migliorare la comprensione del funzionamento della vegetazione e della sua variabilità spaziale. In particolare, il lavoro si è focalizzato sullo studio del legame tra proprietà ottiche della vegetazione, plant traits (PT) e ecosystem functional properties (EFP) in una foresta di media latitudine. A tale scopo, sono state utilizzate tecniche di telerilevamento innovative per studiare il funzionamento della vegetazione a partire da dati ottici ad altissima risoluzione spettrale acquisiti tramite il sensore aviotrasportato ad immagine HyPlant. Il lavoro si è focalizzato su due obiettivi principali: i) l’analisi della relazione spaziale tra F e EFP per comprendere la variabilità della funzionalità a scala regionale; ii) l’analisi del potenziale di F come indice sintetico della diversità funzionale. I risultati dimostrano l’efficacia dell’utilizzo di F come proxy della funzionalità della vegetazione, sottolineando allo stesso tempo la complessità del legame tra F, PT e EFP e la necessità di integrare dati differenti per interpretare correttamente il segnale di F. In particolare, i risultati mostrano che: i) F è relazionata alla variabilità spaziale delle EFP, dimostrando che tale relazione tipicamente osservata nel dominio temporale si mantiene in quello spaziale; ii) F è uno strumento più promettente rispetto agli indici tradizionali basati sulla riflettanza per spiegare la diversità funzionale. Globalmente, i risultati mostrano il ruolo fondamentale dell’eterogeneità spaziale nel controllare l’uptake del carbonio, migliorando così la comprensione della complessa relazione tra F e funzionalità. Futuri studi in questa direzione sono una priorità per migliorare la comprensione del ruolo della vegetazione nel bilancio globale del carbonio. Remote Sensing (RS) data have been successfully exploited in the last decades to monitor vegetation due to their inherent capacity of providing repeated and spatially-distributed quantitative information about vegetation properties. However, most research focused on the description of the structural and biochemical properties of vegetation rather than on the understanding of its functioning. In the last decade, RS of sun-induced chlorophyll fluorescence (F) emerged as a novel and promising tool for assessing plant functional status. F is a weak signal emitted by the core of the photosynthetic machinery in the red and far-red spectral regions (~650-800 nm) as a side product of light absorption. The potential of F relies on the relationship existing between photochemistry and the energy dissipation pathways: since photochemistry competes with F emission and heat dissipation for the absorbed energy, F can be a direct indicator of plant actual functional state. The main aim of this Ph.D. research was to exploit optical data (i.e., reflectance and fluorescence) to advance the understanding of vegetation functioning and of its variability across space. In particular, the work aimed at better understanding the link between vegetation optical properties, plant traits (PTs) and ecosystem functional properties (EFPs) in a case study represented by a mid-latitude forest ecosystem. At this purpose, innovative RS techniques were exploited to infer information about the vegetation functioning from fine and ultra-fine spectral resolution optical measurements acquired with the HyPlant airborne imaging spectrometer. The analyses were focused on two main work streams: i) the investigation of the spatial relationship between F and EFPs to better understand the variability of the ecosystem functioning at regional scale; ii) the analysis of the potential of F as a synthetic descriptor of the ecosystem functional diversity. Results provided evidence of the effectiveness of F as a tool for assessing vegetation functioning, but also pointed out the complexity of the link existing between F, PTs and EFPs and the need to integrate different RS derived products to obtain an unambiguous interpretation of the F signal. In particular, results showed that: i) F can be related to the spatial variability of the EFPs, thus demonstrating that this link usually observed in the temporal domain holds in the spatial domain; ii) F is a more powerful tool compared to traditional reflectance-based indices for explaining the functional diversity. Overall, these results improved the understanding of the complex relationship between F and vegetation functioning by adding new insights into the critical role of the spatial heterogeneity in controlling the carbon uptake. Further research in this direction constitutes a high priority for advancing the understanding of the imprint of plants on the global carbon balance.

Intrinsically disordered proteins (IDPs) are proteins that do not fold into a stable globular structure but are biological active. These proteins are involved in regulation, recognition, and signaling process, where high-specificity/low affinity interactions with multiple partners are fundamental prerequisites. In the last thirty years, a class of small peptides, knowns as antimicrobial peptides (AMPs), similar to IDPs but involved in eukaryotic innate immune response against pathogen infections, have been the subjects of several studies to understand their mechanism of action and to discover and/or develop new antimicrobial substances, that can be used as antibiotic. In the following PhD thesis we elucidate the structure, mechanism and function of several peptides which are characteristic of AMPs and IDPs. In particular, we determined the structure and function of the AMP Chionodracine (CND). We demonstrated that CND can bind to membranes of different lipid compositions with a preference for membranes mimicking those of prokaryotic cells. We also observed that upon the binding with phospholipid vesicles, the peptide remains adsorbed at the lipid surface, only partially exposed to the aqueous environment. In aqueous solution, the peptide is unfolded but undergoes to disorder-to- order transition, upon binding with phospholipid membranes. Based on the promising results obtained for CND, we designed three different CND mutants, with increased net positive charge and hydrophobicity, and characterized their interaction with synthetic and natural membranes. We characterized the mutant’s activity and function in presence of different phospholipid membranes. These studies demonstrated that the mutations have increased antimicrobial properties of CND. The mutants showed a stronger ability to interact, bind and disrupt membranes of different lipid compositions that can be directly correlated to the number of positively charged amino acids present in the sequence. The heat-stable Protein Kinase Inhibitor (PKI) is a fully disordered 75 amino acid that regulate the activity and the intracellular localization of catalytic subunit of cAMP-dependent protein kinase A (PKA-C). To understand the molecular mechanism underlying recognition of PKA-C by PKI, we characterized the change in the conformational landscape that PKI undergoes upon binding to PKA-C (Apo to PKA-C bound). These studies were carried out using NMR spectroscopy, the only structural technique to provide atomic resolution detail on IDPs. Using chemical shift analysis and nuclear spin relaxation experiments, we demonstrated that PKI alone has minimal secondary structural elements with only two transient helixes. However, paramagnetic relaxation enhancement (PRE) experiments show that PKI is not fully disordered, but has transient tertiary intramolecular interaction between the C- and N-terminus. When PKI is bound to the kinase, in the presence of the non-hydrolysable ATP mimic, β,γ-Imidoadenosine 5′-triphosphate (ATPN), only the sequence that directly interacts with the PKA-C undergoes significant structural rearrangement while the majority of the peptide is still unfolded. Le proteine intrinsecamente disordinate (intrinsically disordered proteins – IDPs) sono catene proteiche che, diversamente dalle proteine globulari, sebbene non assumano una struttura tridimensionale, sono biologicamente attive. Le IDPs sono coinvolte in processi di regolazione, riconoscimento e segnalazione cellulare, dove interazioni ad alta affinità/ bassa specificità con partner multipli sono un prerequisito fondamentale. Negli ultimi trent’anni una classe di piccoli peptidi ad attività antimicrobica (antimicrobial peptides - AMPs), componenti fondamentali della risposta immunitaria eucariotica contro infezioni batteriche, e simili per alcune caratteristiche alle IDPs ma, sono diventati l’oggetto di studi finalizzati alla comprensione del loro meccanismo di azione e all’individuazione e/o implementazione di nuove sostanze and attività antimicrobica da utilizzare come alternativa ai comuni antibiotici. Nel seguente lavoro di tesi di dottorato, abbiamo elucidato la struttura, i meccanismi e la funzione di diversi peptidi appartenenti alle famiglie proteiche sopra citate. In particolare, abbiamo descritto e caratterizzato la struttura e l’attività del peptide antimicrobicoChionodracine (CND). In particolare abbiamo dimostrato che CND interagisce con membrane di diversa composizioni lipidica, prediligendo comunque le membrane la cui composizione fosfolipica mima quella delle membrane procariotiche. A seguito del legame con vescicole fosfolipidiche, il peptide si distribuisce sulla superficie del doppio strato lipidico. In soluzione acquosa, il peptide non ha una struttura tridimensionale definita mentre, in presenza di membrane fosfolipidiche, si organizza in una α-elica amfipatica. Sulla base degli studi effettuati su CND, abbiamo progettato tre diversi peptidi, aventi una maggiore carica positiva e idrofobicità rispetto al peptide wild-type. Come effettuato per CND, anche per i mutanti abbiamo caratterizzato l’attivitàdi interazione con le membrane, utilizzando organismi batterici modello, e vescicole lipidiche la cui composizione lipidica mimava quelle delle membrane procariotiche ed eucariotiche. Gli studi hanno dimostrato che le mutazioni introdotte hanno aumentato sia la capacità di interagire con le membrane modello dei mutanti. In particolare i peptidi mostrano una maggiore capacità di interazione, legame e rottura delle membrane di diversa composizione lipidica, PKI (heat-stable Protein Kinase Inhibitor) è un peptide costituito da 75 aminoacidi, non strutturato in soluzione, che regola l’attività e la localizzazione cellulare della sub-unità catalitica della proteina chinasi A (PKA-C). Al fine di comprendere il meccanismo molecolare alla base del riconoscimento di PKA-C da parte di PKI, abbiamo caratterizzato i cambiamenti conformazionali subiti da PKI a seguito del legame con l’enzima. Questi studi sono stati effettuati utilizzando la spettroscopia di risonanza magnetica nucleare (NMR), che è l’unica tecnica che fornisce informazioni dettagliate a livello atomico sugli IDPs. Utilizzando l’analisi dei chemical shifts ed esperimenti di nuclear spin relaxation, siamo stati in grado di dimostrare che PKI, da solo, presenta elementi minimi di strutture secondarie, con due eliche transitorie. Tuttavia, gli esperimenti di paramagnetic relaxation enhancement (PRE) hanno mostrato che PKI non è completamente disordinata in soluzione, bensì sono presenti interazioni terziare transienti tra la porzione N- e C-terminal. Una volta legata a PKA-C, in presenza di un analogo non idrolizzabile dell’ATP (ATPN), le porzioni del peptide che subiscono una arrangiamento strutturale sono quelle che direttamente interagiscono con la chinasi, mentre la maggior parte del peptide rimane non strutturato. Dottorato di ricerca in Scienze ambientali

Amongst all the properties that phthalocyanines possess, their photochemical behavior is extremely peculiar and useful for an extremely wide range of applications such as photodynamic therapy and photovoltaics. During the last years we have synthesized and tested a considerable number of new macrocyclic-based dyes for Dye-Sensitized Solar Cells bearing zinc in the central cavity. We have recently chosen to change the central ion to investigate the potentialities of other metals and we have focused our attention on copper derivatives because of their extremely high stability, fundamental for a long lifetime of the solar cell, and color intensity which is needed in order to maximize the light harvesting properties of the device. Even if Cu(II) is a well known fluorescence quencher because of its capability to induce an intersystem crossing in the molecular systems that interact with it, a consistent number of copper chlorines, porphyrins and corroles have been reported as photosensitizers1,2 showing interesting results in terms of efficiencies. Our contribution will deal with the fluorescence properties of some copper phthalocyanines with increasing degree of substitution synthesized as potential photochemical sensitizers, analyzed both with steady-state and time-resolved measurements. The results will be compared with those of the corresponding free-base and, when possible, zinc derivatives and in one specific case the evidence of a consistent fluorescence in a copper derivative has been highlighted.

Following previous studies on α and β polarizabilities of ketocyanines, a subgroup of D–π–A–π–D quadrupolar chromophores with moderately V-shaped structure, the present work analyses the effects of modifying the π-bridges connecting the D (NMe2) and A (CO) groups. This aim is pursued through a detailed comparison between the previously studied ketocyanines (KC2, KC3) and a Michler's ketone analogue (KM1) bearing styrenic (in the place of polyenic) π-bridges. First, we report a spectroscopic study, including absorption and fluorescence anisotropy spectra, aimed to probe the electronic peculiarities of KM1 as well as to derive consistent three-state model (TSM) parameters for the three compounds. The paper goes on with an extensive theoretical study, carried out in the framework of the density functional theory (DFT), encompassing the structure, the electronic spectrum, α and β polarizabilities and two-photon absorption (TPA) cross-sections (σTP). Calculations performed according to the sum-over-states (SOS) approach are discussed with reference to the performances of few-state descriptions, it is shown that such descriptions (including TSM), which have been proved to be quite reliable in the case of KC2 and KC3, lose their effectiveness with KM1 because of the electronic characteristics related to the styrenic π-bridges. As to the TPA cross-sections, the results of TSM and SOS approaches concerning the TSM g → c and g → e transitions are supplemented by those obtained using the quadratic response theory. A common qualitative conclusion, traceable to the degree of bending of the V-shaped structure, is that in the case of KM1 the allowed (g → e) and the “forbidden” (g → c) transitions both should be observable in the TPA spectrum, as confirmed by experiment.

Il presente lavoro di Tesi di Dottorato di Ricerca (Scuola di Dottorato in Scienza e Tecnica "Bernardino Telesio") dal titolo "Novel Organic Optoelectronic Materials" è stato svolto all'interno dei laboratori di "Organic Optoelectronic Materials" e "New Syntheses via Organometallic Catalysis" del Dipartimento di Chimica dell'Università della Calabria. Nuovi semiconduttori organici potenzialmente impiegabili in dispositivi optoelettronici quali OLEDs, celle solari, transitors ed elettrocromici sono state progettate, sintetizzate e caratterizzate. La prima parte del lavoro ha riguardato la sintesi e la caratterizzazione di derivati di triarilammine, molecole elettron-donatrici (anodiche), da impiegare in dispositivi elettrocromici ("smart windows") per l'attenuazione della radiazione solare nel vicino infrarosso (NIR). L'intensa ed estesa banda di assorbimento nel NIR prodotta delle specie mono-ossidate ha suggerito l'impiego di queste triarilammine in dispositivi "complementari" in cui vengono utilizzate insieme a molecole elettron-accetrici (catodiche) elettrocromiche nel visibile, per un'ampia modulazione dello spettro solare. Tali sistemi sono stati dispersi all'interno di matrici polimeriche al fine di realizzare dei gel elettrocromici le cui prestazioni sono state ampiamente studiate. La risposta elettrocromica del gel è stata inoltre provata in un dispositivo plastico, dimostrandone le potenziali applicazioni campo dell'elettronica flessibile. La seconda parte del lavoro invece, ha riguardato la sintesi e lo studio delle proprietà di cristalli liquidi semiconduttori di tipo "n" (elettron-accetori) da impiegare in celle solari organiche "bulk heterojunction". Tali molecole, appartenenti alla famiglia dei viologeni estesi, hanno mostrato interessantissime proprietà mesomorfiche fortemente influenzate dalla lunghezza delle catene alchiliche ( a 9, 10 e 11 atomi di carbonio) e dell'anione utilizzato, la bis(triflimmide). Le proprietà elettrochimiche di questi composti sono state investigate sia in soluzione che nelle mesofasi. Un'efficace elettrocromismo dovuta (i) al doppio strato elettrico creato all'elettrodo degli anioni presenti e (ii) all'elevata sovrapposizione degli orbitali di frontiera dell'esteso sistema π- coniugato è stato osservato nelle fasi colonnari e smettiche in cui le molecole si sono auto-assemblate. Infine, le proprietà elettrocromiche di questi composti sono state studiate all'interno di film plastici utilizzabili in dispositivi quali i displays. Dottorato di Ricerca in Mesophases and Molecular Materials, XXV Ciclo, a.a. 2011-2012 Università della Calabria

The field of nanoparticles is amazingly many-sided and consequently their applications range between many different areas from industry to bio-analysis and catalysis. In particular, luminescent nanoparticles attract close attention in the areas of biology, medical diagnosis and therapy, where they already find many applications. In this so fascinating and wide framework we have focussed our attention on luminescent silica nanoparticles able to act as sensing materials. We highlight here the importance, especially with the aim of sensing, of gaining precise knowledge and control of their structures; the performance of a chemosensor is, in fact, totally dependent on its design. We then briefly present the state of the art and the progress both in the synthetic protocols and in the application of luminescent silica nanoparticles as chemosensors. We present many recent examples, organized into two main sections, the first dealing with systems presenting the signalling units on the surface (dye coated silica nanoparticles, DCSNs) and the second with systems entrapping the dyes inside the silica matrix (dye doped silica nanoparticles, DDSNs).

The discovery of methods suitable for the conversion in vitro of native proteins into amyloid fibrils has shed light on the molecular basis of amyloidosis and has provided fundamental tools for drug discovery. We have studied the capacity of a small library of tetracycline analogues to modulate the formation or destructuration of β2-microglobulin fibrils. The inhibition of fibrillogenesis of the wild type protein was first established in the presence of 20% trifluoroethanol and confirmed under a more physiologic environment including heparin and collagen. The latter conditions were also used to study the highly amyloidogenic variant, P32G. The NMR analysis showed that doxycycline inhibits β2-microglobulin self-association and stabilizes the native-like species through fast exchange interactions involving specific regions of the protein. Cell viability assays demonstrated that the drug abolishes the natural cytotoxic activity of soluble β2-microglobulin, further strengthening a possible in vivo therapeutic exploitation of this drug. Doxycycline can disassemble preformed fibrils, but the IC(50) is 5-fold higher than that necessary for the inhibition of fibrillogenesis. Fibril destructuration is a dynamic and time-dependent process characterized by the early formation of cytotoxic protein aggregates that, in a few hours, convert into non-toxic insoluble material. The efficacy of doxycycline as a drug against dialysis-related amyloidosis would benefit from the ability of the drug to accumulate just in the skeletal system where amyloid is formed. In these tissues, the doxycycline concentration reaches values several folds higher than those resulting in inhibition of amyloidogenesis and amyloid destructuration in vitro.

In accordance with a first aspect of the present invention, it is provided a method for the preparation of an active nanoparticle, comprising a mixing step, during which at least one active compound is mixed with molecules of at least one surfactant in an organic solvent; an evaporation step, that is subsequent to the mixing step and during which the organic solvent is evaporated in order to obtain a residue; a reaction step, which is subsequent to the evaporation step and during which molecules of at least one alkoxysilane are added to the residue and silanized in presence of water; the alkoxysilane is chosen between a tetraalkoxysilane and a trialkoxysilane; the surfactant comprising the following structure: Hydro1-Lipo-Hydro2 wherein Lipo indicates a substantially hydrophobic chain, Hydro1 and Hydro2 each indicate a substantially hydrophilic chain. Advantageously, the active compound is substantially lipophilic.

The present invention relates to methods for the preparation of an active particle, active particles and uses of these active particles. The purpose of this invention is to provide active particles, uses of particles and methods for the preparation of particles, which allow overcoming, at least partially, the drawbacks of the state of the art and are, at the same time, easy and economical to implement. According to the present invention the following are provided: active particles, uses of particles and methods for the preparation of particles as specified in the independent claims which follow and, preferably, in any of the claims directly or indirectly dependent on the independent claims.

We present here the study of the photophysical properties of new dye-doped silica nanoparticles (DDNs) bearing dansyl fluorescent derivatives covalently linked to the silica matrix. The described experimental evidences show how the different location of the chromophores induces great changes in their photophysical behavior, suggesting that fluorophores located near the surface of the nanoparticles have a very different behavior with respect to the internal molecules. These latter ones, in fact, are shielded from the solvent and have a strong blue emission, while those at the periphery interact with the solvent and show a weaker red-shifted emission. As a consequence, the fluorescence properties of these nanoparticles are an average between the characteristics of the two different families of dyes. The relative amount of fluorophores located in the two compartments can be controlled simply by changing the size since, from our results, the thickness of the solvent permeable layer is not relevantly affected by the diameter of the nanoparticles. It is noteworthy that the fluorophores located in the outer shell exhibit very peculiar features: they are sensitive and interact with small molecules such as solvent molecules but, at the same time, they are not accessible to big receptor species such as beta-cyclodextrins. Such results indicate that most of the solvent-sensitive dansyl moieties are located within pores large enough to only accommodate solvent but not big molecules as cyclodextrins, giving precious insight on the morphology of the nanoparticles.

Despite the key role of magnesium in many fundamental biological processes, knowledge about its intracellular regulation is still scarce, due to the lack of appropriate detection methods. Here, we report the spectroscopic and photochemical characterization of two diaza-18-crown-6 hydroxyquinoline derivatives (DCHQ) and we propose their application in total Mg(2+) assessment and in confocal imaging as effective Mg(2+) indicators. DCHQ derivatives 1 and 2 bind Mg(2+) with much higher affinity than other available probes (K(d) = 44 and 73 microM, respectively) and show a strong fluorescence increase upon binding. Remarkably, fluorescence output is not significantly affected by other divalent cations, most importantly Ca(2+), or by pH changes within the physiological range. Evidence is provided on the use of fluorometric data to derive total cellular Mg(2+) content, which is consistent with atomic absorption data. Furthermore, we show that DCHQ compounds can be effectively employed to map intracellular ion distribution and movements in live cells by confocal microscopy. A clear staining pattern consistent with known affinities of Mg(2+) for biological ligands is shown; moreover, changes in the fluorescence signal could be tracked following stimuli known to modify intracellular Mg(2+) concentration. These findings suggest that DCHQ derivatives may serve as new tools for the study of Mg(2+) regulation, allowing sensitive and straightforward detection of both static and dynamic signals.

The steady-state and time-resolved fluorescence properties of the multitryptophan minimal subunit CaeSS2 from Carcinus aestuarii hemocyanin have been studied with the aim of probing the environment of the fluorophores within the protein matrix. Subunit a of Panulirus interruptus hemocyanin, whose X-ray structure is known, has been also studied. The results are compared with those collected with other two monomeric fractions (CaeSS1, CaeSS3) produced by dissociation of the native, oligomeric protein as well as with those of the hexameric aggregate. Three classes of tryptophan residues can be singled out by a combination of fluorescence quenching and lifetime measurements on the holo-Hc (the copper containing, oxygen binding form) and the apo-Hc (the copper-free derivative). One class of tryptophans is exposed to the protein surface. Some of these residues are proposed to be involved in the intersubunit interactions in CaeSS1 and CaeSS3 fractions whereas in CaeSS2 the protein matrix masks them. This suggests the occurrence of conformational rearrangements after detachment of the subunit from the native aggregate, which could explain the inability of CaeSS2 to reassociate. A second class of tryptophan has been correlatively assigned, by comparison with the results obtained with Panulirus interruptus hemocyanin, to residues in close proximity to the active site. The third class includes buried, active site-distant, residues.

Diel changes in photosynthetic oxygen evolution and several photochemical parameters measured by chlorophyll fluorescence quenching and induction were measured in outdoor dense cultures of the alga Monodus subterraneus (Eustigmatophyta). Cultures were maintained under two temperature regimes. In one, a rise in temperature was initiated in the morning by the increase in solar radiation up to the optimal temperature of 28 °C; in the other, a heating device was used to increase the rate of warming up in early morning. Although the two cultures were maintained at the same temperature and light intensity for most of the day, cultures exposed for only a short time to suboptimal temperature showed a larger decrease in allmost all the photosynthetic parameters. By comparing the diel changes in maximal photochemistry efficiency of photosystem II, the electron transfer rate and the photochemical and non photochemical chlorophyll fluorescence quenching of the cultures, we concluded that even a relatively short exposure to suboptimal morning temperatures induced photoinhibitory damage. The higher photochemical activity of the heated culture was also reflected in a significant increase in productivity, which was 60% higher in the morning heated cultures than in the non-heated cultures.

Autofluorescence of the cornea within specific wavelength region and dynamic light scattering measurements of ocular tissue have both been used for early-stage detection of the presence of diabetic retinopathy. In the present study, autofluorescence of the cornea and dynamic light scattering in the cornea and vitreous have been measured and compared using two innovative research instruments in twenty-two insulin-dependent diabetes mellitus patients, for the diagnosis of diabetic retinopathy. Corneal autofluorescence mean values in each diabetic retinopathy grade significantly correlated with dynamic light scattering measurements in the vitreous, and in the cornea, thus confirming that changes in the natural fluorescence is strictly correlated with molecular changes of ocular tissues. © 1998 Society of Photo-Optical Instrumentation Engineers. [S1083-3668(98)01103-4]

Time-resolved fluorescence spectroscopy shows promise as a high-sensitivity, high-discrimination tool for diagnostic purposes. The application of this technique to the study and the diagnosis of aging processes within ocular tissues is investigated. An age-related study of the pigments (melanin, lipofuscin, and melanolipofuscin) of the retinal pigment epithelium, performed using time-resolved fluorescence spectroscopy with picosecond gating, is presented. The study was performed on intact pigment granules, extracted with soft procedures, and the results have been correlated with the ultrastructure of the granules and with the results of continuous-wave absorption and fluorescence spectroscopy. Both classes of pigments exhibit age-related fluorescence properties. >

The measurement of fast fluorescence waveforms with a traditional sequential sampling technique results in an inaccurate determination of the fluorescence decay time(s) in the presence of fluorescence fading. This paper describes a random sampling technique that overcomes this difficulty. A suitable software for random sampling and averaging has been developed for a subnanosecond pulsed‐laser microfluorometer. Details of the random sampling routine are presented. The advantages of the random sampling technique as opposed to sequential sampling are discussed mathematically, and results of computer simulation for the two methods are shown.

A computer‐controlled system for time‐resolved fluorescence spectroscopy is described. The system provides the simultaneous acquisition of the fluorescence decay curve, of the time‐integrated emission spectrum, and of two time‐gated spectra. Gating within the fluorescence decay is provided by setting up windows in the output of the time‐to‐amplitude converter in single photon timing instrumentation. The gate parameters (position and width) are fully programmable. Depending on the laser source used, time resolutions between 45 and 150 ps are obtained. Therefore, in most situations no deconvolution procedures are required to elaborate the spectra. The system has been characterized with the aid of suitable test solutions. The results indicate that a correct choice of the gate parameters allows complementary information (in terms of both decay times and emission spectra) of the fluorescence parameters of molecular species in molecular systems.

The tetracycline dyes, particularly chlorotetracycline, have been employed as probes of membrane-associated calcium during the excitation process of nerve. Both squid giant axons, stained internally, and lobster nerves, stained externally, show a small increase in fluorescent light during the action potential. Increasing the calcium concentration bathing a lobster nerve leads to a larger optical signal. Adding fluoride ion to the inside of a squid axon, which might be expected to influence the internal calcium-ion concentration, also leads to a larger optical signal. Squid axons have been studied under conditions of voltage clamp and the hyperpolarizing response. Model studies were done with erythrocyte ghosts to clarify the influence of membranes and calcium on the fluores-cence of the tetracyclines. Chlorotetracycline may be monitoring calcium concentration associated with the inner surface of the nerve membrane.