L’attuale rilevanza rappresentata dalla stretta relazione tra cambiamenti climatici e influenza antropogenica ha da tempo posto l’attenzione sull’effetto serra e sul surriscaldamento planetario così come sull’aumento delle concentrazioni atmosferiche dei gas climaticamente attivi, in primo luogo la CO2. Il radiocarbonio è attualmente il tracciante ambientale per eccellenza in grado di fornire mediante un approccio “top-down” un valido strumento di controllo per discriminare e quantificare il diossido di carbonio presente in atmosfera di provenienza fossile o biogenica. Ecco allora che ai settori applicativi tradizionali del 14C, quali le datazioni archeometriche, si affiancano nuovi ambiti legati da un lato al settore energetico per quanto riguarda le problematiche associate alle emissioni di impianti, ai combustibili, allo stoccaggio geologico della CO2, dall’altro al mercato in forte crescita dei cosiddetti prodotti biobased costituiti da materie prime rinnovabili. Nell’ambito del presente lavoro di tesi è stato quindi esplorato il mondo del radiocarbonio sia dal punto di vista strettamente tecnico e metodologico che dal punto di vista applicativo relativamente ai molteplici e diversificati campi d’indagine. E’ stato realizzato e validato un impianto di analisi basato sul metodo radiometrico mediante assorbimento diretto della CO2 ed analisi in scintillazione liquida apportando miglioramenti tecnologici ed accorgimenti procedurali volti a migliorare le performance del metodo in termini di semplicità, sensibilità e riproducibilità. Il metodo, pur rappresentando generalmente un buon compromesso rispetto alle metodologie tradizionalmente usate per l’analisi del 14C, risulta allo stato attuale ancora inadeguato a quei settori applicativi laddove è richiesta una precisione molto puntuale, ma competitivo per l’analisi di campioni moderni ad elevata concentrazione di 14C. La sperimentazione condotta su alcuni liquidi ionici, seppur preliminare e non conclusiva, apre infine nuove linee di ricerca sulla possibilità di utilizzare questa nuova classe di composti come mezzi per la cattura della CO2 e l’analisi del 14C in LSC. / The ongoing concern raised by the interconnected relationship between climate changes and anthropogenic influence has long focused the attention on the greenhouse effect and related global warming as well as on atmospheric concentrations of climatically active gases, first of all the carbon dioxide. Radiocarbon is currently the environmental tracer for excellence providing through a "top -down" determination a reliable monitoring tool able to discriminate and quantify the origin of the carbon dioxide present in the atmosphere from fossil or biogenic sources. Traditional 14C fields of investigation, like archaeometric dating, are so joined by new application areas concerning both the energy sector as regards power plant emissions, fuels, carbon capture and storage technologies and the growing so called biobased products market deriving from renewable raw materials. In this PhD thesis the “radiocarbon world” was therefore explored from both the point of view of methodological and technical aspects and applications relating to numerous and different fields of investigation. A system for radiocarbon analysis based on radiometric method using direct CO2 absorption and Liquid Scintillation Counting was developed and validated providing technological improvements and procedural measures aimed at improving the performance of the method in terms of simplicity, sensitivity, and reproducibility. The method generally represents a good compromise with respect to the traditionally used methods for 14C analysis although it is currently still inadequate for those applications where a high precision is necessary, but competitive for modern samples with a high concentration of 14C. The investigations performed on several Ionic Liquids, while preliminary and inconclusive, finally opens new research lines about the possible use of this new class of compounds as analytical means for CO2 capture and 14C analysis in LSC.

Recent advances in the fast growing area of therapeutic/diagnostic proteins and antibodies - novel and highly specific drugs - as well as the progress in the field of functional proteomics regarding the correlation between the aggregation of damaged proteins and (immuno) senescence or aging-related pathologies, underline the need for adequate analytical methods for the detection, separation, characterization and quantification of protein aggregates, regardless of the their origin or formation mechanism. Hollow fiber flow field-flow fractionation (HF5), the miniaturized version of FlowFFF and integral part of the Eclipse DUALTEC FFF separation system, was the focus of this research; this flow-based separation technique proved to be uniquely suited for the hydrodynamic size-based separation of proteins and protein aggregates in a very broad size and molecular weight (MW) range, often present at trace levels. HF5 has shown to be (a) highly selective in terms of protein diffusion coefficients, (b) versatile in terms of bio-compatible carrier solution choice, (c) able to preserve the biophysical properties/molecular conformation of the proteins/protein aggregates and (d) able to discriminate between different types of protein aggregates. Thanks to the miniaturization advantages and the online coupling with highly sensitive detection techniques (UV/Vis, intrinsic fluorescence and multi-angle light scattering), HF5 had very low detection/quantification limits for protein aggregates. Compared to size-exclusion chromatography (SEC), HF5 demonstrated superior selectivity and potential as orthogonal analytical method in the extended characterization assays, often required by therapeutic protein formulations. In addition, the developed HF5 methods have proven to be rapid, highly selective, sensitive and repeatable. HF5 was ideally suitable as first dimension of separation of aging-related protein aggregates from whole cell lysates (proteome pre-fractionation method) and, by HF5-(UV)-MALS online coupling, important biophysical information on the fractionated proteins and protein aggregates was gathered: size (rms radius and hydrodynamic radius), absolute MW and conformation.

Volatile organic compounds (VOCs) are the key aroma producers in fruits and sensory quality of fruits is widely determined by qualitative and quantitative composition of VOCs. The aroma of grape is a complex of hundreds of VOCs belonging to different chemical classes like alcohols, esters, acids, terpenes, aldehydes, furanones, pyrazines, isoprenoids and many more. VOCs play important role as they determine the flavor of grapes and wine made from it. The objective of this thesis is to study of VOCs through development of different mass spectrometry based analytical methodologies and its applications for the comprehensive investigation and construction of database of the VOCs in grapes. First part of the study was dedicated to generation of the comprehensive database of grape VOCs through the screening of multiple grape varieties (n=124) representing different species, color and origin. The experiment was carried out using headspace solid-phase microextraction (HS-SPME) and gas chromatography mass spectrometry (GC-MS) based approach and according to metabolomics protocols. A customized dataset of reference standards (>350) was generated and, an automated pipeline for data analysis was created in collaboration with data management group of the institute. The results showed annotation of “level 1†of 117 VOCs in grape. The established database in this experiment will represent the significant portion of the future Grape Metabolome database. The second part of the study was dedicated to study the differential behavior of volatile organic compounds and their glycosylated precursors qualitatively and semi quantitatively. Volatile secondary metabolites also exist in the form of nonvolatile and odorless glycosylated precursors in grape and studies have confirmed that concentration of these precursors can be much higher than its free counterparts. The elevated concentrations of volatiles in glycosylated forms can significantly affect the wine aroma because of possible chemical modifications throughout the process of fermentation and wine ageing. In addition, the investigation of the biosynthesis and accumulation of VOCs in the fruit tissues requires the consideration of both the free and bound forms. To study the phenomenon an experiment was carried using solid phase extraction (SPE) of the free and glycosylated precursors; with enzymatic hydrolysis aglycone part of the precursors was released followed by subsequent GC-MS analysis. Over 10 different selected grape varieties were analyzed. Sixty-six significant different aroma compounds in grapes (pre and post hydrolysis) were identified. Identification was done based on several parameters like retention time, retention index and MS spectral database. The multivariate statistical analysis by two-way hierarchical clustering with heat map visualization showed distribution of the compounds within different varieties before and after hydrolysis. In the third part of the study, we performed experiments dedicated to training and applications of atmospheric pressure gas chromatography mass spectrometry (APGC-MS). The experiment was carried out at the Department of Biological Sciences, University of North Texas, under the supervision of Prof. Vladimir Shulaev. We have established the metabolomics protocol for the analysis of fruit volatiles using APGC-MS with an optimized GC and MS conditions and created novel library of the fruit volatile compounds using APGC-MS system. Six different grape varieties were analyzed as a case study and experimental results showed APGC-MS as a valuable solution for metabolomics analysis. The data processing and statistical evaluation was done using XCMS and progenesis QI© software. Moreover, observations based on injections of pure reference standards showed high abundance of molecular ions with minimal fragmentation at low collision energy that is typically missing in traditional vacuum source GC-MS. Moreover, the use of elevated collision energy data resulted in a spectrum similar to the traditional EI data.

Cell membrane is the biological barrier serving as both territorial defense and the communication hinge for the interior of cell from its surroundings. As building blocks of cellular membranes and also precursor for second messengers, a variety of lipids play essential roles in cellular membrane dynamics as well as important functions such as cell proliferation, apoptosis, signal transduction and membrane trafficking modulation. Lipidomics, representing the systematic and integrative studies of diversified lipids (lipidome) in a biological system, is an emerging yet rapid developing field and hence requires advanced and complementary analytical techniques as well as multiple statistical tools. Our development of reliable analytical methodology (the advanced Mass Spectrometric and high-resolution NMR techniques) and application of multiple statitistical approaches (multivariate data analysis and univariate t-test) enable us to achieve these comprehensive understandings. We have investigated, first of all, the effects induced by hypoxia on cervical cancer derived cells (HeLa cells) to see how and how much the changes in phospholipids profile are able to get light into the targeted biological problem (hypoxia) and provide a preliminary insight into the underlying mechanisms. We found that hypoxia stimulation dramatically reduced the total amount of cellular phosphoinositols (PI) but prominently increased the amount of lyso phosphocholines (lyso-PC) and lyso phosphoethanolamines (lyso-PE). Moreover, our studies suggested the polyunsaturated phospholipids species as stronger biomarkers upon hypoxia treatment. The evaluation of changes in the average unsaturation index (UI) of the membrane lipids acyl chains revealed that UI slightly increased in several lipid classes, thus affecting membrane fluidity and further membrane-dependent functions. The plausible mechanisms by HeLa cells to adapt to hypoxia conditions are briefly reported as well. We have also conducted the comparative lipidomic studies of urothelial cancer cell line RT4 (a model system of a benign tumor) and T24 (a model system of a metastatic tumor) aiming to reveal probable roles and relevant differential changes of membrane lipids with respect to urinary bladder metastasis progress. Significant changes of lipids metabolism were found to correlate with urothelial nonmetastatic and metastatic cell models. The most remarkable finding was that the malignant cell type (T24) showed a strong decrease of ether PC species complemented by a sharp increase of the length and the average unsaturation number of lipids acyl chains. Ceramide-based sphinglipids also showed altered profiles in these two cell types. Such analyses suggest a certain significant re-organization of cellular membrane in malignant cell transformation, involving variations in compositional lipid structures and possible signaling transduction pathways. Observations of such reduction of the 1-alkyl PC species and the chain shortening of lipid species might serve as a tool in urinary bladder cancer intervention.

This PhD project arises from the growing awareness of food quality and safety on the part of citizens and institutions. The increase in the population to feed, the lengthening of the production and distribution chains and the socio-economic risks of a poor diet make it crucial to monitor the quality of food from the producer to the consumer. Traditional methods (sensory panels and laboratory analytical techniques) are too expensive and above all slow to evaluate the quality of fresh foods that deteriorate over the course of a few hours. In this context, it is crucial to develop monitoring devices that are cheap, rapid and non-invasive, in order to be able to evaluate the quality of food products extensively and constantly. Solid-state gas sensors are an ideal candidate, as they are inherently non-invasive and inexpensive. In this context, the project focused on chemoresistive sensors based on semiconductor metal oxides, which are among the simplest and most performing, and have the advantage of being sensitive to almost all gases and VOCs. Initially, nanostructures of different materials (n- and p-type semiconductors) and of different morphologies (nanowires and nanosheets) were studied in order to investigate the performance of individual sensors. In this way, some devices have been optimized with respect to the detection of possible biomarkers of the degradation of specific foods. The sensors have demonstrated a rapid response (from a few seconds to a minute), an intense response and above all a very low detection limit (less than 1ppmv, in some cases a few tens of ppbv), important for agri-food applications. This approach is the simplest since it requires a single sensor that is selective towards a certain molecule (ammonia, ethylene...) which can be considered the only important information parameter in a certain application. In most cases, however, the gaseous emission of a food is composed of a large quantity of volatile compounds, and the low selectivity of resistive sensors makes it difficult to discriminate the molecules most informative regarding the degradation process. For this reason, in the second part of the PhD we used the sensors developed up to then to create electronic noses. Exploiting the dimensions of nanostructures, we have developed a new concept of thermal electronic nose, i.e. with sensors of the same material, but within a thermal gradient. In this way, by exploiting multivariate statistical analysis and machine learning techniques, the devices acquired a greater ability to discriminate and quantify the different gases. The electronic noses have shown that they can perfectly recognize the different gases tested (100%) and estimate their concentration with an error of a few ppmv. Measurements in the laboratory are very useful for testing the performance parameters of sensors and electronic noses, as they make it possible to evaluate the correctness of the classification and the error in estimating the concentration of any gas. On the other hand, measuring the emissions of fresh food is different, as the concentrations are not known, and therefore a different approach is needed. The final stage of the project involved using electronic noses to assess the freshness of certain agri-food products. As the developed sensors were particularly sensitive to ammonia, it was decided to study the degradation of meat and fish, where this gas is an important marker. The electronic noses have been able to accurately recognize the meat from the fish (> 95%), and evaluate the state of degradation by giving a very accurate estimate of the microbial count (>95%), responding in a very short time (tens of seconds). The miniaturized electronic noses developed during this PhD project have therefore successfully demonstrated to be a rapid and non-invasive cross-sectional tool for assessing the freshness of agri-food products.

GC×GC is an efficient tool for the analysis of volatile compound. However, improvements are still required on VOC extraction, GC×GC setup and data processing. Different sample preparation techniques and GC×GC setup were compared based on the literature study and experimental results. Each VOC extraction technology has its own drawbacks and needs new developments. There wasn’t an ideal sample preparation technique to recover all the VOCs from the beverage sample. Furthermore, the VOCs recovered by different techniques were very different. The discussion of the pros and cons of the different techniques in our study can serve as a guide for the further development and improvement of these techniques. Combining the results from different sample preparation techniques is necessary to achieve a higher coverage of global VOC profiling. For the known fermentative aromatic compounds, the best coverage can be reached by using SPME together with SPE for beer, and VALLME for wine and cider. A fine GC×GC method development involves modulator selection, column combination and parameter optimization. Thermal modulator provides high detection sensitivity and allow exceptional trace analysis. Since the analytes coverage is the most important factor of in beverage VOC profiling, thermal modulation is a better choice. In fermented beverages, there are more polar compounds than non-polar compounds. The most suitable column combination is polar-semipolar. Same column diameters shall be used to minimize the column overloading. GC×GC parameters must be optimized. These parameters interact with each other therefore statistical prediction model is required. Response surface model is capable of doing this job while using a small number of experimental tests. The nearest neighbor distance was a suitable measurement for peak dispersion. Column and detector saturations are unavoidable if the metabolic sample is measured at one dilution level, incorrect peak deconvolution and mass spectrum construction may happen. Data processing results can be improved by a two-stage data processing strategy that will incorporate a targeted data processing and cleaning approach upstream of the “standard” untargeted analysis. Our experiments show a significant improvement in annotation and quantification results for targeted compounds causing instrumental saturation. After subtracting the saturate signal of targeted compounds, the MS construction was improved for co-eluted compounds. Incomplete signal subtraction may occur. It leads to the detection of false positive peaks or to interferences with the construction of mass spectra of co-diluted peaks. High-resolution MS libraries and more accurate peak area detection methods should be tested for further improvement.

Mass spectrometry imaging (MSI) is a MS-based technique. It provides a way of ascertaining both spatial distribution and relative abundance of a large variety of analytes from various biological sample surfaces. MSI is able to generate distribution maps of multiple analytes simultaneously without any labeling and does not require a prior knowledge of the target analytes, thus it has become an attractive molecular histology tool. MSI has been widely used in medicine and pharmaceutical fields, while its application in plants is recent although information regarding the spatial organization of metabolic processes in plants is of great value for understanding biological questions such as plant development, plant environment interactions, gene function and regulatory processes. The application of MSI to these studies, however, is not straightforward due to the inherent complexity of the technique. In this thesis, the issues of plant sample preparation, surface properties heterogeneity, fast MSI analysis for spatially resolved population studies and data analysis are addressed. More specifically, two MSI approaches, namely matrix assisted laser desorption ionization (MALDI) imaging and desorption electrospray ionization (DESI) imaging, have been evaluated and compared by mapping the localization of a range of secondary and primary metabolites in apple and grapes, respectively. The work based on MALDI has been focused on the optimization of sample preparation for apple tissues to preserve the true quantitative localization of metabolites and on the development of specific data analysis tool to enhance the chemical identification in untargeted MSI (chapter 3). MALDI imaging allows high-spatial localization analysis of metabolites, but it is not suitable for applications where rapid and high throughput analysis is required when the absolute quantitative information is not necessary as in the case of screening a large number of lines in genomic or plant breeding programs. DESI imaging, in contrast, is suitable for high throughput applications with the potential of obtaining statistically robust results. However, DESI is still in its infancy and there are several fundamental aspects which have to be investigated before using it as a reliable technique in extensive imaging applications. With this in mind, we investigated how DESI imaging can be used to map the distribution of the major organic acids in different grapevine tissue parts, aiming at statistically comparing their distribution differences among various grapevine tissues and gaining insights into their metabolic pathways in grapevine. Our study demonstrated that this class of molecules can be successfully detected in grapevine stem sections, but the surface property differences within the structurally heterogeneous grapevine tissues can strongly affect their semi-quantitative detection in DESI, thereby masking their true distribution. Then we decided to investigate this phenomenon in details, in a series of dedicated imaging studies, and the results have been presented in chapter 4. At the same time, during DESI experiments we have observed the production of the dianions of small dicarboxylates acids. We further studied the mechanism of formation of such species in the ion source proposing the use of doubly charged anions as a possible proxy to visualize the distributions of organic acid salts directly in plant tissues (chapter 5). The structural organization of the PhD thesis is as below: Chapter one and Chapter two describe the general MSI principle, compare the most widely used MSI ion sources, and discuss the current status in MSI data pre-processing and statistical methods. Due to the importance of sample preparation in MSI, sample handling for plant samples is independently reviewed in chapter two, with all the essential steps being fully discussed. The first two chapters describe the comprehensive picture regarding to MSI in plants. Chapter three presents high spatial and high mass resolution MALDI imaging of flavonols and dihydrochalcones in apple. Besides its importance in plant research, our results demonstrate that how data analysis as such Intensity Correlation Analysis could benefit untargeted MSI analysis. Chapter four discusses how sample surface property differences in a structurally/biologically heterogeneous sample affect the quantitative mapping of analytes in the DESI imaging of organic acids in grapevine tissue sections. Chapter five discusses the mechanism of formation of dicarboxylate dianions in DESI and ESI Chapter six summarizes the work in the thesis and discusses the future perspectives.

Resveratrol is a polyphenolic compound produced by various plants and present in dietary sources such as red wine. In recent years, its beneficial effects for human health, including protection from heart diseases and cancer prevention, have attracted increasing interest. Resveratrol acts both as an antioxidant and a prooxidant agent when works in vivo with Cu(II) ions occurring naturally in living organisms. The aim of this work is to study the gas phase reactivity of resveratrol in presence of copper and iron ions, in order to more insights on the role of copper in the proposed biological mechanism. By electrospray ionization (ESI) mass spectrometry we have produced and detected some resveratrol-copper complexes by using a resveratrol/CuSO4 solution in acetonitrile/water, and their most stable structures have been calculated at the B3LYP/6-311G(d) level of theory. The formation of dehydrodimer product was also detected in ESI-MS/MS experiments and its structure assigned with evidences for isomeric compounds from copper and iron reactions with resveratrol. Density Functional Theory (DFT) calculations have been carried out to elucidate reaction mechanisms. Finally, the crucial role of the para-OH group in resveratrol structure has been demonstrated by investigating reactions with copper sulfate of synthetic analogues, bearing different number and position of OH groups.

Scopo del progetto era valutare lo stato di salute e la qualità dei suoli agricoli lombardi e di alcune sue aree caratterizzate da specifiche criticità ambientali, attraverso un monitoraggio multidisciplinare. L’uso di indicatori biologici accoppiato alle classiche tecniche analitiche ha consentito di implementare le informazioni ottenute dal punto di vista chimico-fisico. Contrariamente al passato infatti, la componente vivente del suolo è diventata fondamentale alla comprensione delle sue condizioni, in quanto esso è stato riconosciuto come sistema strettamente connesso agli altri comparti ambientali, influenzato e caratterizzato da tutti gli organismi che lo compongono. Sulla base di ciò sono state indagate la struttura e le caratteristiche delle comunità microbiche, dei lombrichi e dei protozoi ciliati, le attività enzimatiche del suolo, e la genotossicità delle sostanze inquinanti utilizzando il trifoglio come pianta indicatrice. Abbiamo inoltre avuto modo di applicare alcune tecniche che esulano dal classico monitoraggio, ma che forniscono informazioni preziose circa il comportamento di una sostanza o di un elemento. Si tratta dell’applicazione della diluizione isotopica per determinare la presenza del cromo esavalente in suoli potenzialmente contaminati, e di biosaggi con batteri bioluminescenti per valutare la biodisponibilità di arsenico e mercurio in un Sito di Interesse Nazionale. / The aim of the project was to assess health status and quality of agricultural soils of the Lombardia region, and some areas with critical environmental situations, through a multidisciplinary monitoring. The use of biological indicators coupled to classic analytical techniques, has allowed the implementation of the information obtained from the chemical-physical point of view. Contrary to the past, the living component of the soil has become crucial to better understand its condition, as it has been recognized as a system closely related to other environmental media, influenced and characterized by all organisms that compose it. Basing on this it was investigated the structure and characteristics of microbial communities, earthworms and ciliated protozoa, the enzymatic activities of the soil, and the genotoxicity of pollutants using clover plants as indicator. We also had the opportunity to apply some of the techniques that are outside the classic monitoring, but that can provide valuable information about the behavior of a substance or an element. These techniques are: the application of isotope dilution to determine the presence of hexavalent chromium in potentially contaminated soils, and bioassays with bioluminescent bacteria to assess the bioavailability of arsenic and mercury in a Site of National Interest.

Molte attività antropiche danno origine a problemi ambientali per i quali è necessario trovare soluzioni. La presenza di sostanze indesiderabili nell'acqua, infatti, può rappresentare un rischio per la salute e l'igiene sia degli uomini che degli animali. È quindi fondamentale trovare metodi per evitare gli inquinanti di queste matrici, in modo da renderli compatibili con il mantenimento di condizioni ambientali adeguate e salutari. Questa tesi si propone di valutare la qualità delle acque sotterranee di un'area in cui la qualità dell'acqua non è mai stata studiata, in particolare indagando la presenza di pesticidi e nitrati al fine di comprendere l’impatto della viticoltura sull'inquinamento delle acque sotterranee. Questo studio nasce dalla necessità di migliorare la governance delle acque e di implementare le migliori pratiche di gestione e misure di mitigazione per prevenire l'inquinamento delle acque e quello ambientale. I risultati degli studi di monitoraggio hanno mostrato una contaminazione delle acque sotterranee da parte di pesticidi e nitrati e quindi è stata effettuata una valutazione della fonte di contaminazione da pesticidi e nitrati attraverso studi isotopici di N e O di NO3-, e attraverso analisi idrologiche utilizzando il modello CRITERIA 3D. Come una delle principali conclusioni del lavoro si può affermare che la presenza di pesticidi nelle acque sotterranee in un’area ad attività vitivinicola intensa, non può essere correlata solo alle proprietà e al destino chimico-ambientale delle sostanze o alle condizioni pedoclimatiche, ma anche al comportamento dell'utilizzatore finale. In effetti, la fonte di contaminazione valutata, è risultata essere dovuta sia a una contaminazione diffusa che a una contaminazione puntiforme. Per quanto riguarda l'influenza della fertilizzazione a base di azoto sulla presenza di nitrati nelle acque sotterranee, i risultati hanno mostrato un'elevata vulnerabilità dell'acquifero ai cambiamenti esterni. Dalle indagini isotopiche è emerso che la maggior parte dell'NO3- rilevato nelle acque sotterranee, deriva dall'utilizzo di fertilizzanti azotati inorganici, in accordo con le pratiche di uso del suolo e di viticoltura dichiarate dagli agricoltori della zona. Il risultato più importante dello studio, tuttavia, è stato che alla fine del processo, l'approccio multi-actor e la strategia di coinvolgimento adottati, hanno avuto successo nel migliorare gli atteggiamenti verso pratiche più sostenibili. / Many anthropic activities give rise to environmental problems for which it is necessary to find solutions. The presence of undesirable substances in water, indeed, can pose a risk to the health and hygiene of both men and animals. It is, therefore, essential to find methods in order to avoid pollutants from these matrices, so as to make them compatible with the maintenance of healthy environmental conditions. This thesis aims to assessing the groundwater quality of an area in which the quality of water was never studied, particularly investigating the occurrence of pesticides and nitrate in order to understand the grant of viticulture on water pollution. This study derives from the necessity to improve the water governance and to implement the best management practices and mitigation measures to prevent groundwater and environmental pollution. The results of monitoring studies showed a contamination of groundwater by pesticides and nitrate and thus an evaluation of PPPs and nitrates source contamination was carried out through isotopic studies of N and O of NO3- , and through hydrologic analysis by use of the model CRITERIA 3D. As one of the main conclusions of the work, the occurrence of PPPs in groundwater in areas with intensive viticultural activities cannot be related just to chemical environmental fate properties or pedoclimatic conditions, but also to end-user behavior. In fact, the contamination source evaluated, resulted to be due to both diffuse and point-source contamination. For what concerns the influence of nitrogen-based fertilization on nitrate occurrence in groundwater, the results showed a high vulnerability of the aquifer to external changes. Isotopic investigations showed that most of the NO3- detected in the groundwater, derived from the use of inorganic nitrogen fertilizers, in agreement with the land use and viticulture practices declared by the farmers. The most important result of the study, however, was that at the end of the overall process, the multi-actor approach and engagement strategy adopted were successful in improving attitudes to more sustainable practices.