le antocianine sono una classe di metaboliti secondari nelle piante, appartenenti ai flavonoidi, categoria di molecole antiossidanti utili per la salute di piante, animali e umana. Sono ampiamente studiati nelle Leguminosae poiché implicati in numerose funzioni fisiologiche. In questa tesi, due wild-types e cinque mutanti di Medicago truncatula, coinvolti nel metabolismo dei flavonoidi, sono stati caratterizzati a livello fenotipico, fisiologico e molecolare. La principale differenza tra i wild-type e i mutanti è la riduzione del contenuto di antocianine nelle foglie. In seguito all'esposizione alle radiazioni UV-B, è stata analizzata la loro risposta, evidenziando un diverso comportamento. Inoltre, il metabolismo delle antocianine è stato studiato anche a livello di regolazione genica e un nuovo gene myb è stato isolato e caratterizzato, risultando più espresso nei tessuti che accumulano antocianine. / Anthocyanins are secondary metabolites in plants. They belong to a class of flavonoids synthesized via the phenylpropanoid pathway and they are antioxidant molecules important for plant, animal and human health. Flavonoids are widely studied in legume because they are implicated in several biological and physiological functions. In this thesis seven genotypes (two wild-types and five indipendent mutants) of Medicago truncatula (the model legume plant) affected in flavonoid metabolism have been characterized at phenotypic, physiological and molecular level. The main difference between wild-types and mutants, is the reduction in anthocyanin content in leaves. The anthocyanin accumulation during the leaf life, the flowering time and the fruit formation have been registered and compared. The two wild-types contain 6 μg cyanidin.mg-1 of fresh weight, while in the mutants the anthocyanin amount ranged from 0.12 μg cyanidin mg-1 to 2 μg cyanidin mg-1 of fresh weight. After HPLC-DAD-MS/MS analysis, the main anthocyanin present in the two wild-types is the cyanidin-3-O feruloyl. The Expression profile of genes codifying enzymes and transcriptional factor involved in flavonoid biosynthesis has been investigated. RT-PCR and qPCR results show different possible pathways of reduction of anthocyanins in the five mutants. These mutants, have been exposed to UV-B radiations and their response has been investigated measuring the chlorophyll fluorescence parameters (Fv/Fm, qP and NPQ) in untreated plants, during treatment (after 4hrs and 14hrs of treatment) and in the recovering phase. All genotypes, regardless of the anthocyanins amount, showed a decrease of the photosyntetic efficiency after 14hrs of treatment. This indicates a marginal role of these pigments in the oxidative damages protection. Mutants do not response in the same manner to the UV—B exposure and the anthocyanin amount does not increase equally in all genotypes. The anthocyanin metabolism is studied also at the gene regulation level. A novel Myb gene (MtMYBA) involved in anthocyanin pathway has been isolated and characterized. This gene is more expressed in tissues which accumulate anthocyanins in M. truncatula plants. The functional analysis has been investigated overexpressing this Myb gene under the control of 35S promoter. This construct has been used to transform Arabisopsis thaliana and Medicago truncatula plants. In addition, the MtMYBA promoter has been cloned in a plasmids containing GUS and GFP reporter genes.

La presente attività di ricerca ha avuto come oggetto di studio la formazione di biofilm in condizioni dinamiche: per raggiungere tale obiettivo, è stato costruito un idoneo apparato che permettesse di ottenere una valutazione quantitativa sia delle cellule presenti nella soluzione di crescita sia di quelle adese su tre materiali di prova(acciaio inossidabile, PET e rame). Oltre ad esprimere una valutazione dell’adesione batterica alle superfici, si è proceduto ad indagare sui possibili parametri sperimentali che potessero influenzare la formazione di biofilmdi Listeria su superfici abiotiche. Allo stesso tempo l’efficacia battericida di alcuni biocidi è stata valutata sulle cellule planctoniche così come su biofilm batterici sviluppati su superfici abiotiche. Il ruolo del sistema di quorum sensing basato su luxS nella formazione di biofilm batterici: un ceppo di L. innocua con buone capacità adesive è stato confrontato con il suo ceppo deficiente di luxS per la sua suscettibilità ad agenti biocidi così come per la sua capacità di adesione superficiale L’effetto della precendentemente menzionata inattivazione genetica sulla cinetica di crescita batterica e, quindi, sull’efficienza metabolica complessiva è stata esaminate mediante lo strumento BioscreenC. L’effetto inibitorio di due composti polifenolici è stato valutato sul ceppo deficiente di luxS così come sul ceppo originale. / The current research activity was focused on biofilm formation in dynamic conditions. A suitable device was thus constructed, in order to have a quantitative evaluation of the cells freely suspended in the flowing nutrient solution used as well as the sessile cells which adhered on the three tested surfaces (stainless steel, polyethylene therephthalate and copper). During this project, other than of a quantitative assessment of bacterial adhesion, we investigated also on the parameters which could affect significantly the biofilm formation of Listeria on abiotic surfaces. At the same time antimicrobial efficiency of selected biocides was investigated on planktonic cells as well as on bacterial biofilms grown on abiotic surfaces. The role of luxS-based quorum sensing system in the biofilm development process was evaluated: a luxS-null mutant of an adhesive strain of L. innocua was obtained through Campbell-like genetic inactivation and then evaluated for its sensitivity to biocidal agents and adhesion to surfaces, in comparison with its parental strains. Furthermore the effect of genetic inactivation on bacterial growth kinetic and thus on overall bacterial metabolic efficiency was assessed through the apparatus of BioscreenC. Finally two polyphenolic compounds were investigated for their inhibitory effect on Listeria.

Trichoderma atroviride SC1 (SC1) was isolated from hazelnut wood and it is effective in the biocontrol of soil-borne pathogens. However, its effectiveness decreases as its population declines in the soil over time. To improve its persistency in the soil, lignocellulosic materials (wood pellets) were tested to be used as carriers to sustain the population of SC1 and facilitate its incorporation into the soil. A method was developed to coat wood pellets of fir, beech, and chestnut with a conidial suspension to reach a preset concentration (i.e. 10^4, 10^5, and 10^6 cfu/ g of wood). The growth of SC1 on each type of wood was compared. Chestnut pellets were excluded from further experiments because they had low counts of colony-forming units (cfu) of SC1. Beech pellets were preferred over fir pellets for showing more suitable physicochemical characteristics for soil application. In addition, for the same wood type, increased initial coating concentrations did not impact the final colony counts of SC1 and no significant difference was observed between the counts of 10^4, 10^5, and 10^6 cfu/g of wood at the end of the experiment. The addition of small quantities of nitrogen increased the final cfu on all types of wood pellets. The growth of SC1 on beech pellets was then tested by adding cheap nitrogen sources namely, soy flour, soy protein isolates, and proteins that originated from animal wastes. The best results were obtained with soy protein isolates (1 g/L) and the population of SC1 reached 10^9 cfu/ g of beech wood. Finally, this carrier of coated beech pellets with soy protein isolates was tested in the soil under controlled conditions, in an experimental greenhouse at 25°C and 60% of soil humidity. The pellets were coated to reach a final concentration of 5×10^5 cfu/ g of beech and 10 g of beech coated pellets were mixed with 1 kg of soil in plastic pots to reach the final concentration of 5×10^3 cfu/ g of soil. The carrier increased the bacterial richness and diversity of the soil and decreased the fungal ones. The total Trichoderma population persisted in the first month and then declined after three months with competition from other bacteria such as Massilia spp. and fungi such as Stachybotrys spp. and Mortierella spp.

Nowadays, exciting bioanalytical microsystems are currently receiving increasing attention in biology since they can comply with the considerable demand for reliable, sensitive and low-cost analysis tools. Small reagents volumes, low power consumption, portability, fast analysis, high throughput and systems integration are the key aspects that make these systems more and more appealing within both the academic and industrial communities. In the last years, many microdevices were developed for a wide range of biological applications, particularly dedicated to cellu-lar or molecular analysis. Many efforts were devoted to the realization of Cell-Based Biosensors (CBBs) to monitor the dynamic behaviour of cell cultures for pharmacological screening and basic research. Other researchers focused their interests in the development of so-called Lab-on-a-Chip (LOC) systems for DNA analysis mostly applied to clinical diagnosis. This thesis deals with the investigation of two miniaturized devices – a cell-based biosensor and a DNA amplification system – for the cellular and molecular analysis of wine yeasts, respectively. The first device consists of integrated electrochemical sensors – Ion-Sensitive Field-Effect Transistor (ISFET), impedimetric and temperature sensors – for the real time evaluation of pH and cell settling of yeasts under batch culture conditions. The assessment of yeast performance and robustness has been focused on ethanol tolerance, as it is one of the main stress factors acting in wine, and thus, one of the major causes of stuck fermentations. A good agreement between extracellular acidification and cell growth trends at different ethanol concentration has been demonstrated, significantly reducing the time of the traditional assays. Moreover, resistivity measurements have shown the possibility to follow progressive settling of the cell suspension. Concerning the second system, a Polymerase Chain Reaction (PCR) microdevice has been biologically validated by successfully amplifying yeast genomic DNA fragments. Additionally, the outcome of PCR has been positively assessed with diluted samples and boiled yeast cultures, demonstrating the possibility to skip the time-consuming purification process for potential LOC applications with very little or no pre-PCR sample manipulations. The encouraging results from both microsystems have demonstrated their suitability for wine yeast analysis, aimed at quality improvements of the winemaking process.

Nowadays, both gene mutations detection and function investigation are expected to assume a key role in diseases understanding and in many other biotechnological fields. In fact, gene mutations are often cause of genetic diseases and gene function analysis itself can help to have a broader vision on cells health status. Traditionally, gene mutations detection is carried out at pre-translational/sequence level (transcriptomic approach). On the other hand, the function of innumerable sequenced genes can be investigated by delivering them into cells through transfection methods and observing their expression result at post-translational level (proteomic approach). In this context, Micro-ElectroMechanical Systems (MEMSs) offer the intrinsic advantages of miniaturization: low sample and reagent consumption, reduction of costs, shorter analysis time and higher sensitivity. Their applications range from the whole cell assays to molecular biology investigations. On this subject, the thesis deals with two different tools for gene analysis: a Lab-on-Cell and cantilever-based sensors for in-vitro cell transfection and label-free Single Nucleotide Poly-morphisms (SNPs) detection, respectively. Regarding the first topic, an enhanced platform for single-site electroporation and controlled transfectants delivery has been presented. The device consists of a gold MicroElectrode Array (MEA) with multiple cell compartments, integrated microfluidics based on independent channels and nanostructured titanium dioxide (ns-TiO2) functionalized electrodes. Different activities have been reported, from the study of the microfabrication substrates bioaffinity and device development to the electroporation results. The functional characterization of the system has been carried out by electroporating HeLa cells with a small fluorescent dye and then, in order to validate the approach for gene delivery, with plasmid for the enhanced expression of the Green Fluorescent Protein (pEGFP-N1). The second research activity has been focused on a detection module aimed at the integration in a Lab-on-Chip (LOC) for the early screening of autoimmune diseases. The proposed approach consists of piezoresistive SOI-MEMS cantilever arrays operating in static mode. Their gold surface (aimed at the binding of specific thiolated DNA probes) has been deeply analyzed by means of Atomic Force Microscopy (AFM) and X-ray Photoelectron Spectroscopy (XPS) revealing an evident gold non-uniformity and low content together with oxygen and carbon contaminations. Different technological and cleaning solutions have been chosen in order to optimize the system. However, other improvements will be required. Moreover, the feasibility of the spotting technique has been demonstrated by verifying microcantilever mechanical resistance and good surface coverage without cross-contaminations. Finally, as future perspective, possible biological protocols and procedures have been also proposed and discussed starting from literature.

In agricoltura, i pesticidi sono stati usati molto frequentemente per salvaguardare le colture dagli attacchi di parassiti e dalle malattie. Questi pesticidi, oltre a uccidere gli organismi target, molte volte colpiscono anche gli organismi non-target. Tra gli organismi non-target, possiamo individuare molti microrganismi utili a determinare la fertilità e la qualità del terreno. La presenza di questi xenobiotici nel terreno può influenzare i principali cicli biogeochimici (N, C, S, P) e altre vie metaboliche (es. β-ketoadipate). In questo studio abbiamo analizzato gli effetti di isoproturon, tebuconazole e chlorpyrifos sull’abbondanza, sulla struttura e sulla diversità della comunità microbica. Inoltre, abbiamo anche studiato gli effetti di questi pesticidi sui geni coinvolti nel ciclo dell’azoto. Si è potuto notare che l’abbondanza della comunità batterica è molto influenzata dall’applicazione del fungicida tebuconazole . Per quanto riguarda gli studi sulla funzionalità e diversità della popolazione microbica, l’applicazione di questi pesticidi sembra non indurre una chiara dose-dipendente e un effetto tempo. Diversamente, in relazione all’analisi sulla diversità microbica, possiamo affermare che l’applicazione di questi tre pesticidi ha influenzato il numero di OTU rilevate; tuttavia, l’indice di diversità (H’) ci dice che l’uso di questi pesticidi porta ad un incremento della diversità all’interno dei campioni trattati. In conclusione, è possibile affermare che l’applicazione di questi pesticidi influenza l’abbondanza e la funzionalità della popolazione microbica, ma non induce una diminuzione della diversità all’interno della medesima comunità. / In agriculture, pesticides have been frequently used to protect crops from pest and disease attacks. Many times such pesticides, besides killing the target organisms, hit non-target organisms. Among the non-target organisms, we can find many useful microorganisms that determine fertility and soil quality. The presence of these xenobiotics in soil can influence the main biogeochemical cycles (N, C, S, P) and other metabolic pathways (eg. Β-ketoadipate). In this study, we investigated the effects of isoproturon, tebuconazole and chlorpyrifos on the abundance, the structure and the diversity of the microbial community. We have also studied the effects of these pesticides on the genes involved in the nitrogen cycle. It was observed that the abundance of the bacterial community is significantly affected by the application of the fungicide tebuconazole. As for the studies on the functionality and the diversity of the bacterial population, the application of these pesticides does not seem to induce a clear dose-dependent nor a time effect. On the contrary, with respect to the analysis on microbial diversity, we observed that the application of these three pesticides did influence the number of detected OTU, whereas the diversity index (H') tells us that the use of such pesticides leads to an increase of diversity within the treated samples. Finally, we can conclude that the application of these pesticides affects the abundance and function of the microbial population, but does not lead to lower diversity within the same community.

Recentemente, l’ocratossina A (OTA) è stata segnalata in formaggio grattugiato confezionato: si ritiene sia stata prodotta dallo sviluppo di alcuni funghi durante il periodo di maturazione. Gli studi presentati in questa tesi hanno avuto lo scopo di migliorare la conoscenza dei funghi, Aspergilli e Penicilli, associati al formaggio di tipo grana. Si è dunque provveduto alla loro identificazione tramite approccio multifasico, eseguendo anche prove ecologiche per definire i fabbisogni delle specie presumibilmente dominanti. Obiettivo finale sarà quello di predire il rischio di contaminazione da micotossine alle condizioni ambientali di stagionatura del prodotto. Aspergillus puulaauensis e alcune specie del genere Penicillium (P.crustosum and P. solitum) sono state rilevate con maggior frequenza, mentre non sono state rilevate nei campioni di crosta le specie P. nordicum e P. verrucosum. In ogni caso, le condizioni ambientali dei locali di maturazione (15-22°C; 72-88% UR) sono risultate favorevoli allo sviluppo di queste ultime, dando adito a preoccupazione circa una possibile contaminazione da OTA; tuttavia, il ruolo di altre specie micotossigene, ad esempio P. crustosum, non va sottovalutato. Le azioni di contrasto verso questi funghi micotossigeni dovrebbero, innanzitutto, ridurre l’inoculo presente nell’aria: l’ozono sembra efficace in tal senso; inoltre, l’utilizzo della luce blu potrebbe ridurre notevolmente la crescita fungina sulla superficie dei formaggi e delle scaffalature. Le informazioni fornite in questa tesi saranno utili ai produttori e agli altri operatori della filiera di questo prodotto, aiutandoli ad attuare una miglior gestione del rischio e a garantire un prodotto sicuro e di elevata qualità ai consumatori. / Ochratoxin A (OTA) was detected a few years ago in Italian packed grated cheese, and supposed to be produced by fungal growth during cheese ripening. The works managed and presented in this thesis aimed to improve knowledge about mycotoxigenic Aspergilli and Penicillia associated to Italian grana cheese. A multiphasic approach was applied for their identification and ecological trials were organised to define fungal needs of the expected dominant species. The objective was to predict the risk of mycotoxin contamination during the ripening condition of grana cheese. Aspergillus puulaauensis and some Penicillium spp (P.crustosum and P. solitum) were the prevalent species associated to grana cheese, while neither P. nordicum, nor P. verrucosum were detected in cheese rind. However, the environmental conditions of the ripening rooms (15-22°C; 72-88% RH) are favourable to P. nordicum and P. verrucosum, reinforcing concern about possible OTA contamination, but other mycotoxigenic species such as P. crustosum must not be neglected. Actions against those mycotoxigenic fungi should mainly reduce total inoculum in the environmental air: ozone is reported to be effective in this sense; moreover, blue light may significantly reduce fungal growth on cheese and shelves surfaces. Information provided therein will be useful for producers and stakeholders to perform a better risk management and guarantee a safe, high-quality product to consumers.

The present work has been divided into two parts. At first, research projects attempt to evaluate technologies that increase digestibility of energy and other nutrients in cereal grains and their co-products. Differences in starch digestibility have been attributed to various factors, including the the type of corn endosperm, the presence of proteins and prolamins, the presence of lipids, the amylose-amylopectin ratio, the starch granule structure, the particle size, the conservation and processing methods. Second part of my work was dedicated to investigate the sensors of diet compounds which could be considered as potential markers to monitor nutrition status of organism. Activation of these receptors is believed to influence the hunger-satiety cycle, and their expression levels may be altered by dietary composition. Thus, the aim was to investigate for the first time the effect of arabinoxylans (AX) and β-glucans (BG) on the relative level of expression of carbohydrates, amino and fatty acid nutrient sensors genes in porcine oral and non-oral tissues. / The present work has been divided into two parts. At first, research projects attempt to evaluate technologies that increase digestibility of energy and other nutrients in cereal grains and their co-products. Differences in starch digestibility have been attributed to various factors, including the the type of corn endosperm, the presence of proteins and prolamins, the presence of lipids, the amylose-amylopectin ratio, the starch granule structure, the particle size, the conservation and processing methods. Second part of my work was dedicated to investigate the sensors of diet compounds which could be considered as potential markers to monitor nutrition status of organism. Activation of these receptors is believed to influence the hunger-satiety cycle, and their expression levels may be altered by dietary composition. Thus, the aim was to investigate for the first time the effect of arabinoxylans (AX) and β-glucans (BG) on the relative level of expression of carbohydrates, amino and fatty acid nutrient sensors genes in porcine oral and non-oral tissues.

Qualità e sicurezza degli alimenti, inclusa la loro tracciabilità ed autenticità, è diventato ngli ultimi anni obiettivo primario per la salute e il benessere dei consumatori. Il progetto è diretto allo sviluppo e applicazione di metodiche DNA-based per la tracciabilità di specie vegetali, varietà di frumento duro e microorganismi a difesa della qualita’, salubrita’ ed autenticità della filiera grano e prodotti processati. Le attività progettuali dello studio sono finanziate da industria (Barilla S.P.A.) con il coinvolgimento di enti pubblici di ricerca (Università Cattolica Del Sacro Cuore Di Piacenza, CREA-GB di Fiorenzuola D’arda). Il progetto si articola in tre argomenti principali: WP1.Tracciabilità di specie vegetali nella filiera pasta. Tracciabilità di varietà di frumento duro nella filiera pasta A questo scopo sono state intraprese nel secondo anno di attività due azioni dirette allo sviluppo e validazione di due diverse metodiche di fingerprinting varietale. Partendo dalle direttive UPOV in materia di impiego di marcatori molecolari per la caratterizzazione varietale è stata applicata e validata su di un pool di 26 varietà di interesse per l’industria un’ analisi basata su di una combinazione di marcatori SSR (Simple Sequence Repeats) che ha consentito di identificare in maniera univoca ciascuna delle varietà in esame. Il saggio è stato trasferito ai laboratori dell’industria che lo applica attualmente nella routine per il controllo di partite di granella. A fronte della robustezza dell’analisi SSR si pongono però i lunghi tempi analitici. Per ottimizzare questo aspetto si è completata un’attività di sequenziamento parziale del genoma di 28 varietà presenti in due repliche biologiche (52 campioni) e 12 mix di DNA di due varietà (4 differenti percentuali per ogni coppia di varietà miscelate). I dati ottenuti hanno fornito circa 15.000 marcatori molecolari DArT-seq (Diversity Array Technology ) e SNP (Simple Nucleotide Polimorphisms). Dall’intero set di marcatori è stato quindi individuato, attraverso una procedura bioinformatica, un set ridotto di marcatori ad alta informatività in grado di identificare univocamente le singole varietà e di predire la presenza di altre varietà in miscela e la percentuale di contaminazione. WP2.Tracciabilità Di Microrganismi Fungini Nella Filiera Pasta Questo studio è volto al controllo e identificazione di specie microbiche patogene che possono svilupparsi lungo la filiera grano con conseguente impatto negativo sulla salubrità di granella, di semole e dei prodotti finiti. A questo scopo sono stati prodotti campioni di granella a contaminazione controllata. Si è costituita attraverso l’analisi delle sequenze Barcode una ceppoteca che comprende i maggiori patogeni fungini che possono contaminare la granella durante la crescita della pianta in campo o durante lo stoccaggio della granella. Dopo l’inoculo artificiale dei singoli ceppi in due varietà di frumento duro, sono stati raccolti, a tempi crescenti, campioni di spighe e granella. La metodica è risultata rapida e sensibile nell’identificazione di DNA fungino fin dalle prime fasi dell’infezione, quando i sintomi della malattia risultavano ancora non ancora visibili. Le informazioni di sequenza Barcoding, in prospettiva, potranno essere utilizzate per sviluppare nuovi metodi di identificazione fungina più sensibili e rapidi. WP3. Identificazione molecolare di microrganismi vivi o morti in pesto L’obiettivo di questo lavoro è stato quello di sviluppare metodiche di V-qPCR (Viability-PCR), per permettere l’identificazione, quantificazione e discriminazione cellule microbiche vive o morte in alimentiprocessati, come il pesto. Per lo studio è stato scelto il batterio patogeno B.cereus, microrganismo ubiquitario, patogeno e sporigeno di difficile identificazione soprattutto in matrici complesse. Durante questo lavoro è stato sviluppato un protocollo analitico che prevede l’estrazione del DNA batterico da pesto, matrice interferente e complessa. Parte del lavoro è stata svolta presso l’Istituto di Microbiologia dell’Università Cattolica e l’Istituto IATA CSIC Institut d’Agroquímica i Tecnologia dels Aliments in Valencia. / Food quality and safety, including food traceability and authenticity, have become crucial in the last decades. Today, molecular and genetic progress can support the agri-food industry, due to the improvement of new analytical tools. Among the available applications, DNA-based methods can detect the presence of a particular species or variety along the food supply chain, verify the genetic identity of food and feed ingredients and detecting the presence of contaminating organisms, thus becoming an essential tool to study patterns, causes, and risk factors of diseases and outbreaks. As a consequence, genetic analysis has become increasingly popular even among non-specialists and highly beneficial for consumers, agricultural farmers, governments, and the private sector (Reid, O’Donnell, and Downey 2006). In this framework, the research developed in this thesis arises by active collaboration between the private company Barilla G. & R. Fratelli S.p.A., the public research institute CREA-GB (Consiglio per la Ricerca in agricoltura e l'analisi dell Economia Agraria) and Università Cattolica del Sacro Cuore, to develop a set of DNA-based methods to improve the traceability and authenticity of plant and microbial species and durum wheat varieties applicable from farm to fork. Following these aims, the research developed in this thesis includes: 1. The optimization and validation of qPCR assay for the discrimination of plant species along the pasta production chain through the organization of a ring test involving nine Italian public and private laboratories. The results obtained in this study were published in the Journal of Cereal Science (Chapter 2); 2. The discrimination of durum wheat varieties by selecting SSRs and DarT molecular markers as reliable methods for variety fingerprinting (Chapter 3). The results confirm the sensitivity of the method and the feasibility to 7 protect the food industry from fraud and ensure the consumer a certified pasta quality; 3. The application of the Barcoding technique and the development of qPCR assay for the identification and quantification of field fungi (Fusarium, Alternaria, Michrodochium, Cochliobolus spp.) and saprophytic fungi (Aspergillus, Penicillium, Rhizopus spp) along the wheat chain (Chapter 3). The sensitivity of the method was investigated by inoculating potted durum wheat plants at full anthesis and wheat kernels (pre and postharvest trials). The DNA-based methods demonstrate a key role in pathogen detection and the application in several points of the wheat chain (e.g., for control of both locally and imported grains, for storage lots, to evaluate the environmental risk associated with grain powder for farmers and workers); 4. The optimization of Viability q-PCR (V-qPCR) for the discrimination of dead and alive Bacillus cereus, a spore-forming bacteria (Chapter 4). The results of PMAxx, combined with qPCR, have demonstrated the selective discrimination of B.cereus viable cells, with no false-positive signals determined by dead cells, a peculiar aspect of thermally treated food; 5. The comparison of two DNA extraction kits (FastDNA® SPIN Kit for Soil – MB and NucleoSpin Tissue - Macherey Nagel) by detecting B.cereus spores in basil pesto sauce, selected as a model food matrix. Despite the limit of detection (LOD) achieved (respectively 1.8x102 spores/gr by using Fast DNA TM SPIN and 2.7 x 105 spores/gr by using NucleoSpin®), the principal challenge remains the spores' DNA extraction from the complex matrix. Lastly, the results obtained during the doctoral research project were globally discussed (Chapter 5).

Molecular dating (or molecular clock) is a powerful technique that uses the mutation rate of biomolecules to estimate divergence times among organisms. In the last two decades, the theory behind the molecular clock has been intensively developed, and it is now possible to employ sophisticated evolutionary models on genome-scaled datasets in a Bayesian framework. The molecular clock has been successfully applied to virtually all types of organisms and molecules to estimate timing of speciation, timing of gene duplications, and generation times: this knowledge allows contextualizing past and present events in the light of (paleo)ecological scenarios. Molecular clock studies are routinely used in evolutionary and ecological studies, but their use in applied fields such as agricultural and medical entomology is still scarce in particular because of a paucity of genome data. Genome-scaled clocks have been successfully applied, for example, to various model organisms such as Anopheles and Drosophila, as well as to invasive mosquitoes Aedes aegypti and Aedes albopictus. Many other invasive pests are emerging worldwide aided by global trade, increased connectivity among countries, lack of prevention, and flawed invasive species management. Among them, there is Aedes koreicus and Aedes japonicus, two invasive mosquito species which are monitored for public health concerns because of their harboured human pathogenic viruses. For these, as well as for other insects of societal relevance, such as the parasitoid Trissolcus japonicus, there is a paucity of gene markers and no genome data for large scale molecular clock studies. Invasive pests are typically studied using microevolutionary approaches that tackle events at an intraspecific level: these approaches provide important information for the pest management, for example, by revealing invasion routes and insecticide resistances. Approaches that tackle the deep-time evolution of the pest, such as the molecular clock, are instead less used in pest science. Many important traits associated with invasiveness have evolved by speciation over a long time frame: the molecular clock can reveal the paleo-ecological conditions that favoured these traits helping a better understanding of pest biology. Molecular clock, when coupled with phylogenomics, can further identify genes and patterns that characterize the pest: this knowledge can be used to enhance management practices. Although this is a data-driven thesis, its major aim is to provide new results to demonstrate the utility of the molecular clock in pest science. This has been done by systematically apply the molecular clock to various neglected organisms of medical and agricultural relevance. To this aim, I generated new genome data and/or assembled the largest genome-scaled data to date. I studied the molecular clock in mosquitoes, focusing on the Aedini radiation (Chapter 2) and identified a strong incongruence between the mitochondrial and nuclear phylogeny for what concerns their molecular clock. This result highlighted the importance of employing genome scaled data for these species to exclude stochastic effects due to poor/inaccurate sampling in clock studies. To tackle the absence of data, I further assembled the whole mitogenome of emerging invasive species Aedes koreicus and Aedes japonicus with the aim of producing useful data for molecular typing and of inferring divergence estimates using whole mitogenomes (Chapter 3). Dated phylogenies point toward more recent diversification of Aedini and Culicini compared to estimates from previous works, addressing the issue of taxon sampling sensitivity in dated phylogeny. Although it is possible to perform molecular clock studies on single/few gene markers, the current trend is to couple this methodology with genome-scaled datasets to reduce the stochastic effect of using few genes. For this reason, I sequenced the draft genome of A. koreicus and A. japonicus (Chapter 4). The assemblies were extremely fragmented, highlighting the problem of sequencing large genomes using short reads. The assemblies provided, however enough information for genome skimming allowing extraction of BUSCO genes for downstream analyses, whole mitogenome assemblies (used in Chapter 3), and characterisation of the associated metagenome. These data need to be integrated by long reads; it provides, however a first framework to investigate the genome evolution of these species. I further sequenced and assembled the genome of Trissolcus japonicus, the parasitoid wasp of the invasive pest Halyomorpha halys. To elucidate its divergence, estimate and define an intraspecific typing system to differentiate strains for biocontrol strategies, I reconstructed the mitochondrial genomes of two populations: the mitogenomes were surprisingly identical, suggesting that they belong to the same de facto population. I further provide a detailed clock investigation of Zika, a virus harboured and transmitted by some Aedes species (Chapter 5). Using the largest set of genomes to date, I could set the origin of ZIKV in the middle age and its first diversification in the mid-19th century. From a methodological point of view, the clocking of this virus highlighted the importance of checking for recombination and for cell-passages to obtain correct divergence estimates. I finally show my contributions to molecular clock studies of three other invasive species (Chapter 6): I helped disentangle the divergence times of Bactrocera, a genus of invasive fruit files pest of agriculture; I contributed in performing a phylogenomics study of opsin genes in Diptera; I used chloroplast and nuclear genome data to reconstruct the divergences of the invasive reed Arundo. In the various Chapters of my thesis, I highlighted the limits and the problems of current molecular clock methodologies and identified the best practices for different types of organisms in order to develop a cross-discipline understanding of the molecular clock techniques. The various results presented in this thesis further demonstrate the utility of the molecular clock approach in pest studies.