Innovative analytical strategies for the development of sensor devices and mass spectrometry methods

Il lavoro presentato in questa tesi di Dottorato è incentrato sullo sviluppo di strategie analitiche innovative basate sulla sensoristica e su tecniche di spettrometria di massa in ambito biologico e della sicurezza alimentare. Il primo capitolo tratta lo studio di aspetti metodologici ed applicativi di procedure sensoristiche per l’identificazione e la determinazione di biomarkers associati alla malattia celiaca. In tale ambito, sono stati sviluppati due immunosensori, uno a trasduzione piezoelettrica e uno a trasduzione amperometrica, per la rivelazione di anticorpi anti-transglutaminasi tissutale associati a questa malattia. L’innovazione di questi dispositivi riguarda l’immobilizzazione dell’enzima tTG nella conformazione aperta (Open-tTG), che è stato dimostrato essere quella principalmente coinvolta nella patogenesi. Sulla base dei risultati ottenuti, entrambi i sistemi sviluppati si sono dimostrati una valida alternativa ai test di screening attualmente in uso per la diagnosi della celiachia. Rimanendo sempre nel contesto della malattia celiaca, ulteriore ricerca oggetto di questa tesi di Dottorato, ha riguardato lo sviluppo di metodi affidabili per il controllo di prodotti “gluten-free”. Il secondo capitolo tratta lo sviluppo di un metodo di spettrometria di massa e di un immunosensore competitivo per la rivelazione di prolammine in alimenti “gluten-free”. E’ stato sviluppato un metodo LC-ESI-MS/MS basato su un’analisi target con modalità di acquisizione del segnale selected reaction monitoring per l’identificazione di glutine in diversi cereali potenzialmente tossici per i celiaci. Inoltre ci si è focalizzati su un immunosensore competitivo per la rivelazione di gliadina, come metodo di screening rapido di farine. Entrambi i sistemi sono stati ottimizzati impiegando miscele di farina di riso addizionata di gliadina, avenine, ordeine e secaline nel caso del sistema LC-MS/MS e con sola gliadina nel caso del sensore. Infine i sistemi analitici sono stati validati analizzando sia materie prime (farine) che alimenti (biscotti, pasta, pane, etc.). L’approccio sviluppato in spettrometria di massa apre la strada alla possibilità di sviluppare un test di screening multiplo per la valutazione della sicurezza di prodotti dichiarati “gluten-free”, mentre ulteriori studi dovranno essere svolti per ricercare condizioni di estrazione compatibili con l’immunosaggio competitivo, per ora applicabile solo all’analisi di farine estratte con etanolo. Terzo capitolo di questa tesi riguarda lo sviluppo di nuovi metodi per la rivelazione di HPV, Chlamydia e Gonorrhoeae in fluidi biologici. Si è scelto un substrato costituito da strips di carta in quanto possono costituire una valida piattaforma di rivelazione, offrendo vantaggi grazie al basso costo, alla possibilità di generare dispositivi portatili e di poter visualizzare il risultato visivamente senza la necessità di strumentazioni. La metodologia sviluppata è molto semplice, non prevede l’uso di strumentazione complessa e si basa sull’uso della isothermal rolling-circle amplification per l’amplificazione del target. Inoltre, di fondamentale importanza, è l’utilizzo di nanoparticelle colorate che, essendo state funzionalizzate con una sequenza di DNA complementare al target amplificato derivante dalla RCA, ne permettono la rivelazione a occhio nudo mediante l’uso di filtri di carta. Queste strips sono state testate su campioni reali permettendo una discriminazione tra campioni positivi e negativi in tempi rapidi (10-15 minuti), aprendo una nuova via verso nuovi test altamente competitivi con quelli attualmente sul mercato.

Semi-global techniques in image matching and change detection with applications to civil and environmental engineering.

This work of thesis wants to present a dissertation of the wide range of modern dense matching algorithms, which are spreading in different application and research fields, with a particular attention to the innovative “Semi-Global” matching techniques. The choice of develop a semi-global numerical code was justified by the need of getting insight on the variables and strategies that affect the algorithm performances with the primary objective of maximizing the method accuracy and efficiency, and the results level of completeness. The dissertation will consist in the metrological characterization of the proprietary implementation of the semi-global matching algorithm, evaluating the influence of several matching variables and functions implemented in the process and comparing the accuracy and completeness of different results (digital surface models, disparity maps and 2D displacement fields) obtained using our code and other commercial and open-source matching programs in a wide variety of application fields.

Valutazione del rischio ecologico e sviluppo di strategie innovative per il monitoraggio della qualita’ dei corpi idrici: applicazione di un nuovo approccio integrato in un bacino pilota.

Freshwater is extremely precious; but even more precious than freshwater is clean freshwater. From the time that 2/3 of our planet is covered in water, we have contaminated our globe with chemicals that have been used by industrial activities over the last century in a unprecedented way causing harm to humans and wildlife. We have to adopt a new scientific mindset in order to face this problem so to protect this important resource. The Water Framework Directive (European Parliament and the Council, 2000) is a milestone legislative document that transformed the way that water quality monitoring is undertaken across all Member States by introducing the Ecological and Chemical Status. A “good or higher” Ecological Status is expected to be achieved for all waterbodies in Europe by 2015. Yet, most of the European waterbodies, which are determined to be at risk, or of moderate to bad quality, further information will be required so that adequate remediation strategies can be implemented. To date, water quality evaluation is based on five biological components (phytoplankton, macrophytes and benthic algae, macroinvertebrates and fishes) and various hydromorphological and physicochemical elements. The evaluation of the chemical status is principally based on 33 priority substances and on 12 xenobiotics, considered as dangerous for the environment. This approach takes into account only a part of the numerous xenobiotics that can be present in surface waters and could not evidence all the possible causes of ecotoxicological stress that can act in a water section. The mixtures of toxic chemicals may constitute an ecological risk not predictable on the basis of the single component concentration. To improve water quality, sources of contamination and causes of ecological alterations need to be identified. On the other hand, the analysis of the community structure, which is the result of multiple processes, including hydrological constrains and physico-chemical stress, give back only a “photograph” of the actual status of a site without revealing causes and sources of the perturbation. A multidisciplinary approach, able to integrate the information obtained by different methods, such as community structure analysis and eco-genotoxicological studies, could help overcome some of the difficulties in properly identifying the different causes of stress in risk assessment. In synthesis, the river ecological status is the result of a combination of multiple pressures that, for management purposes and quality improvement, have to be disentangled from each other. To reduce actual uncertainty in risk assessment, methods that establish quantitative links between levels of contamination and community alterations are needed. The analysis of macrobenthic invertebrate community structure has been widely used to identify sites subjected to perturbation. Trait-based descriptors of community structure constitute a useful method in ecological risk assessment. The diagnostic capacity of freshwater biomonitoring could be improved by chronic sublethal toxicity testing of water and sediment samples. Requiring an exposure time that covers most of the species’ life cycle, chronic toxicity tests are able to reveal negative effects on life-history traits at contaminant concentrations well below the acute toxicity level. Furthermore, the responses of high-level endpoints (growth, fecundity, mortality) can be integrated in order to evaluate the impact on population’s dynamics, a highly relevant endpoint from the ecological point of view. To gain more accurate information about potential causes and consequences of environmental contamination, the evaluation of adverse effects at physiological, biochemical and genetic level is also needed. The use of different biomarkers and toxicity tests can give information about the sub-lethal and toxic load of environmental compartments. Biomarkers give essential information about the exposure to toxicants, such as endocrine disruptor compounds and genotoxic substances whose negative effects cannot be evidenced by using only high-level toxicological endpoints. The increasing presence of genotoxic pollutants in the environment has caused concern regarding the potential harmful effects of xenobiotics on human health, and interest on the development of new and more sensitive methods for the assessment of mutagenic and cancerogenic risk. Within the WFD, biomarkers and bioassays are regarded as important tools to gain lines of evidence for cause-effect relationship in ecological quality assessment. Despite the scientific community clearly addresses the advantages and necessity of an ecotoxicological approach within the ecological quality assessment, a recent review reports that, more than one decade after the publication of the WFD, only few studies have attempted to integrate ecological water status assessment and biological methods (namely biomarkers or bioassays). None of the fifteen reviewed studies included both biomarkers and bioassays. The integrated approach developed in this PhD Thesis comprises a set of laboratory bioassays (Daphnia magna acute and chronic toxicity tests, Comet Assay and FPG-Comet) newly-developed, modified tacking a cue from standardized existing protocols or applied for freshwater quality testing (ecotoxicological, genotoxicological and toxicogenomic assays), coupled with field investigations on macrobenthic community structures (SPEAR and EBI indexes). Together with the development of new bioassays with Daphnia magna, the feasibility of eco-genotoxicological testing of freshwater and sediment quality with Heterocypris incongruens was evaluated (Comet Assay and a protocol for chronic toxicity). However, the Comet Assay, although standardized, was not applied to freshwater samples due to the lack of sensitivity of this species observed after 24h of exposure to relatively high (and not environmentally relevant) concentrations of reference genotoxicants. Furthermore, this species demonstrated to be unsuitable also for chronic toxicity testing due to the difficult evaluation of fecundity as sub-lethal endpoint of exposure and complications due to its biology and behaviour. The study was applied to a pilot hydrographic sub-Basin, by selecting section subjected to different levels of anthropogenic pressure: this allowed us to establish the reference conditions, to select the most significant endpoints and to evaluate the coherence of the responses of the different lines of evidence (alteration of community structure, eco-genotoxicological responses, alteration of gene expression profiles) and, finally, the diagnostic capacity of the monitoring strategy. Significant correlations were found between the genotoxicological parameter Tail Intensity % (TI%) and macrobenthic community descriptors SPEAR (p<0.001) and EBI (p<0.05), between the genotoxicological parameter describing DNA oxidative stress (ΔTI%) and mean levels of nitrates (p<0.01) and between reproductive impairment (Failed Development % from D. magna chronic bioassays) and TI% (p<0.001) as well as EBI (p<0.001). While correlation among parameters demonstrates a general coherence in the response to increasing impacts, the concomitant ability of each single endpoint to be responsive to specific sources of stress is at the basis of the diagnostic capacity of the integrated approach as demonstrated by stations presenting a mismatch among the different lines of evidence. The chosen set of bioassays, as well as the selected endpoints, are not providing redundant indications on the water quality status but, on the contrary, are contributing with complementary pieces of information about the several stressors that insist simultaneously on a waterbody section providing this monitoring strategy with a solid diagnostic capacity. Our approach should provide opportunities for the integration of biological effects into monitoring programmes for surface water, especially in investigative monitoring. Moreover, it should provide a more realistic assessment of impact and exposure of aquatic organisms to contaminants. Finally this approach should provide an evaluation of drivers of change in biodiversity and its causalities on ecosystem function/services provision, that is the direct and indirect contributions to human well-being.