Dissertations / Theses on the topic 'Bio-Safety'

This PhD thesis deals with the selection and characterization of Lactic Acid Bacteria strains to be applied in different stages of supply chain of bakery and cereal production, in order to set up bio-control strategies, against the cereal-related fungal contaminants. We studied and highlighted some interesting functional and nutritional features of Lactobacillus plantarum CE84, which can be exploited as new starter for food fermentations, in bakery and/or cereal sector, delaying or inhibiting fungal contaminants. Moreover, we demonstrated that could be possible to prolong the mould-free shelf life of the bread, using selected hetero-fermentative Lactobacillus strains in sourdough preparation with wheat flour and sucrose. Further studies will allow to verify the possible application of these strategies for improving food quality and safety.

Microbial contamination and survival during storage of bread are a cause of both health concerns and economic losses. Traditional fermentation systems were studied as sources of lactic acid bacteria (LAB) with antagonistic potential against foodborne pathogens and spoilage organisms, with the aim to improve the safety and shelf life of bakery products. The antagonistic activity of four types of buttermilk (BM) products fermented with Lactococcus lactis subsp. lactis was evaluated against a number of pathogenic bacteria to select the best fermented-BM for application as bio-preservatives in bread crumpets, showing up to 9 µg/ml of nisin equivalent antimicrobial activity. These food ingredients could be suitable to be used in crumpet formulations, BM fermented with Lc. lactis subsp. lactis and nisin influenced the quality and shelf life of crumpets; the pH value and firmness of products with fermented BM was lower and the acidity and springiness was higher than for unfermented BM treatment and control withouth additive. The nisin and fermented BM treatment had beneficial effects on the pore size and colour in comparison with the control, and improved microbial shelf life by 2 days. Commercial and traditional sourdough and bread samples (n=18) were collected to assess the diversity of LAB strains and potential properties when applied to dough and bread. DGGE followed by sequencing showed that Lactobacillus was the predominant genus in the studied sourdoughs. Lb. plantarum and Lb. brevis strains accounted for 69% of the 32 isolates, out of which 10 were amylolytic and 12 had proteolytic activity. Most were also good acid producers after 24 h at 30°C. Some LAB strains presented a strong in vitro inhibitory activity against five indicator strains, showing potential as starter cultures to ferment sourdough. In subsequent experiments, the properties of 24 sourdoughs were evaluated, and one of them, fermented with Lb. plantarum (SIN3) yielded low pH value, high lactic acid production, and suitable microbial growth, and was selected for further bread making performance trials. The bread with fast fermentation and high sourdough concentration (FFHSD) had a lower pH, higher acidity and increased the quality attributes with significantly better shelf life comparing to the other treatments during the storage period. Sensory evaluation demonstrated that fast-fermented breads were more acceptable than the slow-fermented counterparts. Bread prepared with high level (18%) of sourdough fast-fermented with the selected culture (SIN3) had a good eating quality and shelf life. The approach of this study is likely to yield feasible improvements of the current methods of preparation of baking goods.

La valutazione combinata ed integrata della fisiologia e del comportamento permette una completa e coerente valutazione pre-clinica delle funzioni del sistema nervoso centrale. E’ stato sviluppato un sistema integrato di elettroencefalografia per via telemetrica e di video (Video-tEEG), che permette la registrazione simultanea e continua per lunghi periodi delle tracce EEG e delle immagini video. La presente ricerca e’ stata focalizzata sul set-up strumentale e sulla messa a punto della tecnica chirurgia per la registrazione combinata dei segnali EEG corticale, EEG ippocampale e elettromiogramma (EMG) mediante telemetria nel ratto. Il recupero post-operatorio degli animali e’ stato monitorato per periodi di 24 ore circa in occasione del Giorno 1, 6 e 15 dopo la chirurgia, mediante la registrazione delle trace EEG e dell’attivita’ motoria generale utilizzando i video. I risultati ottenuti suggeriscono che la tecnica chirurgica applicata per l’impianto del trasmettitoro telemetrico consente un recupero graduale degli animali entro 15 giorni dall’intervento chirurgico. Durante tutto il periodo di recupero post-chirurgico, i paramateri comportamentali e locomotori non hanno messo in evidenza cambiamenti del ritmo circadiano-giornaliero di buoi-luce e ritornano ai loro valori basali entro un periodo di 15 giorni. Utilizzando una connessine meccanica tra l’elletrodo penetrante in area profonda del cervello e l’elettrodo telemetrico, il sistema e’ in grado di acquisire segnali di EEG ippocampale di buona qualita’ a partire da 15 giorni dopo l’impianto. La ricerca ha dimostrato il possibile uso di questa tecnica di impianto chirurgico e l’applicazione del sistema integrato di Video-tEEG in diverse aree di ricerca pre-clinica compresa la Safety Pharmacology, con vantaggi per un uso etico degli animali nella ricerca.<br>The combined evaluation of physiology and behaviour allows a complete and comprehensive pre-clinical assessment of central nervous system (CNS) functions. An integrated video-telemetric electroencephalography (Video-tEEG) system, which allows the simultaneous and continuous recording of EEG and video images for long periods, was developed. This research work focuses on the equipment set-up and surgical methodology for combined recording of cortical, hippocampal EEG and electromyogram (EMG) waveforms by telemetry in freely moving rats. The post-operative recovery of animals was monitored by recording of EEGs and the general activity by videos for approximately 24 hours on Day 1, 6 and 15 after surgery. The results suggest that the applied surgical technique for the implantation of telemetric transmitter allows a gradual recovery of animals within 15 days. During all the recovery period the behavioural and locomotor parameters showed that there were no changes of the light-dark circadian cycle and they return to background values within a 15-day period. Using a mechanical connection between the deep and the telemetric electrodes, the recording system is able to acquire hippocampal EEG of good quality starting from 15 days after surgical implantation. The present research work demonstrated the possible use of this surgical technique and the application of the integrated Video-tEEG system in different pre-clinical research areas and also in Safety Pharmacology, with advantages for an ethical use of animals.

Background Nanoscience and nanotechnology are highly promising and rapidly emerging areas for research and industrial innovation. Due to the remarkable physicochemical properties of manufactured nanomaterials, several promising applications were recently developed in the areas of agriculture and food production. Recently, a new method to synthesize superparamagnetic nanoparticles has been developed. These nanoparticles consist of stoichiometric maghemite, γ-Fe2O3, with unique spectroscopic properties and well-defined crystalline structure, and have been called “surface-active maghemite nanoparticles” (SAMNs). They form stable colloidal suspensions in water without any organic or inorganic coating to prevent their aggregation. At the same time, they are able to specifically and reversibly bind organic molecules, leading to composite colloidal materials, which can be exploited for biotechnological applications. In recent years nanotechnology was combined with various sensing techniques to develop the so-called "nano-sensors". Several promising applications were recently developed in the areas of agriculture and food production, with the capacity to impact both food industry and consumers. These sensors can be an effective alternative to the traditional methods for the detection of toxins and pathogens in food. Electrochemical detection is a popular method involving nanomaterial-based sensors with applications in the food industry. SAMNs show remarkable electrocatalytic properties and were used for the development of electrodes and biosensors. In this thesis, a set of different hybrids that include SAMNs are presented and the electrochemical features of the hybrids are reported. Results Tannic acid (TA), Quaternized Carbon Dots (Q-CD) and hexavalent chromium (CrVI) were successfully immobilized on SAMN surface. SAMN@TA was characterized using electrical impedance spectroscopy, voltammetry and chronoamperometry. The nanostructured ferric tannate interface showed improved conductivity and selective electrocatalytic activity toward the oxidation of polyphenols. A carbon-paste electrode modified with SAMN@TA was used for the determination of polyphenols in blueberry extracts by square-wave voltammetry. Q-CD @SAMN revealed specific electrocatalytic behavior toward the oxidation of phenols and the system was applied to develop a sensor for the coulometric determination of polyphenols from plant extracts. which displayed peculiar electrocatalytic properties attributable to the influence of the strong electrostatic interactions exerted by Q-CDs on the SAMN surface. The sensor is composed of a simple carbon paste electrode in a small volume electrochemical flow cell (1 μL), and is used for the complete direct electro-oxidation of polyphenols from plant extracts. Finally, SAMNs were successfully applied to remove CrVI from water. The hybrid SAMN@CrVI was used to immobilize bovine serum amine oxidase (BSAO) and this complex was electrochemical showed good performances toward H2O2 detection. SAMN@CrVI-BSAO was applied for the development of a polyamine biosensor, which was successfully exploited for the discrimination of tumorous and healthy tissues obtained from liver extracts. Conclusions Sensing strategies based on SAMNs offer unique advantages over other techniques. They are produced by a low-cost procedure, they are physically and chemically stable, biocompatible and environmentally safe. In this thesis, SAMNs were utilized to prepare three different hybrids and were successfully applied for the construction of three different electrochemical sensors, showing good performances, and successfully applied to real samples.<br>La nanoscienza e la nanotecnologia sono aree altamente promettenti e rapidamente emergenti per la ricerca e l'innovazione industriale. A causa delle notevoli proprietà fisico-chimiche dei nanomateriali prodotti, sono state recentemente sviluppate diverse applicazioni promettenti nelle aree dell'agricoltura e della produzione alimentare. Recentemente, è stato sviluppato un nuovo metodo per sintetizzare nanoparticelle superparamagnetiche. Queste nanoparticelle consistono di maghemite stechiometrica, γ-Fe2O3, con proprietà spettroscopiche uniche e struttura cristallina ben definita, e sono state chiamate surface active maghemite nanoparticles (SAMN). Formano sospensioni colloidali stabili in acqua senza alcun rivestimento organico o inorganico per impedire la loro aggregazione. Allo stesso tempo, sono in grado di legare in modo specifico e reversibile molecole organiche, portando a materiali colloidali compositi, che possono essere sfruttati per applicazioni biotecnologiche. Negli ultimi anni la nanotecnologia è stata combinata con varie tecniche di rilevamento per sviluppare i cosiddetti "nano-sensori". Diverse applicazioni promettenti sono state recentemente sviluppate nei settori dell'agricoltura e della produzione alimentare, con la capacità di influenzare sia l'industria alimentare che i consumatori. Questi sensori possono essere un'alternativa efficace ai metodi tradizionali per la rilevazione di tossine e patogeni negli alimenti. Il rilevamento elettrochimico è un metodo popolare che coinvolge sensori basati su nanomateriali con applicazioni nell'industria alimentare. Le SAMN mostrano notevoli proprietà elettrocatalitiche e sono stati utilizzati per lo sviluppo di elettrodi e biosensori. In questa tesi vengono presentati un insieme di diversi ibridi che includono SAMN e vengono riportate le caratteristiche elettrochimiche degli ibridi. Risultati L'acido tannico (TA), quantum dots di carbonio (Q-CD) e il cromo esavalente (CrVI) sono stati immobilizzati con successo sulla superficie del SAMN. SAMN@TA è stato caratterizzato mediante spettroscopia di impedenza elettrica, voltammetria e cronoamperometria. L'interfaccia di tannato ferrico nanostrutturata mostrava conduttività migliorata e attività elettrocatalitica selettiva verso l'ossidazione dei polifenoli. Un elettrodo di pasta di carbone modificato con SAMN@TA è stato utilizzato per la determinazione dei polifenoli negli estratti di mirtillo mediante voltammetria. Q-CD@SAMN ha rivelato un comportamento elettrocatalitico specifico verso l'ossidazione dei fenoli e il sistema è stato applicato per sviluppare un sensore per la determinazione coulometrica dei polifenoli dagli estratti vegetali. che mostrava proprietà elettrocatalitiche peculiari attribuibili all'influenza delle forti interazioni elettrostatiche esercitate dai Q-CD sulla superficie del SAMN. Il sensore è composto da un semplice elettrodo di pasta di carbone in una cella a flusso elettrochimico di piccolo volume (1 μL), ed è utilizzato per l'elettro-ossidazione diretta completa di polifenoli da estratti vegetali. Infine, i SAMN sono stati applicati con successo per rimuovere CrVI dall'acqua. L'ibrido SAMN@CrVI è stato utilizzato per immobilizzare l'ammina ossidasi del siero bovino (BSAO) e questo complesso era elettrochimico ha mostrato buone prestazioni per il rilevamento di H2O2. SAMN@CrVI-BSAO è stato applicato per lo sviluppo di un biosensore in poliammide, che è stato sfruttato con successo per la discriminazione dei tessuti tumorali e sani ottenuti da estratti di fegato. Conclusioni Le strategie di sensing basate sui SAMN offrono vantaggi unici rispetto ad altre tecniche. Sono prodotti con una procedura a basso costo, sono fisicamente e chimicamente stabili, biocompatibili e sicuri per l'ambiente. In questa tesi, i SAMN sono stati utilizzati per preparare tre diversi ibridi e sono stati applicati con successo per la costruzione di tre diversi sensori elettrochimici, mostrando buone prestazioni e applicati con successo a campioni reali.

This is a case study dissertation to research the socio-political conflict surrounding Gilles Eric Séralini’s et al (2012) research on the toxicity of Monsanto’s NK603 line of corn and the herbicide Roundup. The study analyzes this conflict as a system of interconnected and often conflicting interests, assumptions and ideologies about how knowledge is created and transformed from the research stage to the policy implementation stage. The goal of this study is to: 1.) analyze critical surface level and underlying factors that contribute to the conflict; 2.) analyze systemic processes between national and international researchers, private interests and government policymakers in developing and implementing research protocols, policies and regulations pertaining (but not limited) to Monsanto’s NK603 corn and Roundup; 3.) identify potential patterns of knowledge transformation from the research stage to policy implementation. The theoretical approach used in this study considers social construction, critical theory and Kuhn’s theory of scientific revolution. In utilizing case study methodology, this study incorporates internal analysis of Séralini’s case with a basic comparative analysis of DDT and lead policy processes and knowledge transformation, using mainly secondary data sources supplemented with primary interview material from two select researchers using purposive sampling. By conducting this research, it is hoped that this study reveals a better understanding of the complex interconnected systems that help create and transform food safety policies and the science that supports and/or transforms them.

La sicurezza e la qualità degli alimenti sono tutt’ora un problema critico per i paesi in via di sviluppo. Le diete a basso contenuto di acido folico, per esempio, possono causare gravi problemi di salute, soprattutto nei bambini. Gravi disturbi legati al tubo neurale (DTN) nei neonati possono derivare infatti da madri che hanno insufficiente apporto di acido folico (400-600 g / giorno) durante il periodo di gravidanza. Inoltre, se non adeguatamente protetti o trattati, I prodotti alimentari possono essere vettori di funghi e batteri patogeni rappresentando una fonte potenziale di malattie per l’uomo e una perdita economica per le industrie agro-alimentari. Nella seguente tesi si è quindi quindi studiato il ruolo di batteri lattici selezionati (LAB) in grado di aumentare il valore nutrizionale del latte attraverso la produzione di acido folico durante il processo di fermentazione. Inoltre, ci si è concentrati sul loro uso come "bio-conservanti" contro funghi e batteri, attraverso la sintesi di composti antimicrobici (batteriocine) in grado di inibire la crescita di funghi filamentosi e/o batteri patogeni.<br>The safety and quality of food are still a critical issue in developing countries. Diets with a low content of folic acid, for example, may cause serious health problems, especially in children. Severe disorders related to neural tube (NTD) in infants may arise from mothers having inadequate intakes of folic acid (400-600 g/dia) during the mother pregnancy period. Moreover foods, when not properly protected or treated, can be vectors of pathogenic fungi and bacteria thereby representing a potential source of human diseases and an economical loss for the food industry. In the following thesis we have therefore investigated the role of selected lactic acid bacteria (LAB) in increasing the nutritional value of milk through the production of folic acid during the fermentation process. In addition, we focused on their use as “bio-preservatives” against fungal and bacterial spoilage, through the synthesis of antimicrobial compounds (bacteriocins) able to inhibit the growth of filamentous fungi and /or pathogenic bacteria.

La sicurezza e la qualità degli alimenti sono tutt’ora un problema critico per i paesi in via di sviluppo. Le diete a basso contenuto di acido folico, per esempio, possono causare gravi problemi di salute, soprattutto nei bambini. Gravi disturbi legati al tubo neurale (DTN) nei neonati possono derivare infatti da madri che hanno insufficiente apporto di acido folico (400-600 g / giorno) durante il periodo di gravidanza. Inoltre, se non adeguatamente protetti o trattati, I prodotti alimentari possono essere vettori di funghi e batteri patogeni rappresentando una fonte potenziale di malattie per l’uomo e una perdita economica per le industrie agro-alimentari. Nella seguente tesi si è quindi quindi studiato il ruolo di batteri lattici selezionati (LAB) in grado di aumentare il valore nutrizionale del latte attraverso la produzione di acido folico durante il processo di fermentazione. Inoltre, ci si è concentrati sul loro uso come "bio-conservanti" contro funghi e batteri, attraverso la sintesi di composti antimicrobici (batteriocine) in grado di inibire la crescita di funghi filamentosi e/o batteri patogeni.<br>The safety and quality of food are still a critical issue in developing countries. Diets with a low content of folic acid, for example, may cause serious health problems, especially in children. Severe disorders related to neural tube (NTD) in infants may arise from mothers having inadequate intakes of folic acid (400-600 g/dia) during the mother pregnancy period. Moreover foods, when not properly protected or treated, can be vectors of pathogenic fungi and bacteria thereby representing a potential source of human diseases and an economical loss for the food industry. In the following thesis we have therefore investigated the role of selected lactic acid bacteria (LAB) in increasing the nutritional value of milk through the production of folic acid during the fermentation process. In addition, we focused on their use as “bio-preservatives” against fungal and bacterial spoilage, through the synthesis of antimicrobial compounds (bacteriocins) able to inhibit the growth of filamentous fungi and /or pathogenic bacteria.

INTRODUCTION Dronedarone is a multichannel blocker and it was approved in 2009 by the European Commission in the treatment of arrhythmias (atrial fibrillation, paroxysmal or persistent). Initially, it was assessed as a less efficacious but possibly safer anti-arrhythmic drug than amiodarone, but soon unfavourable safety issues were raised (doubling of cases of death in patients with severe heart failure and left ventricular systolic dysfunction, life-threatening cases of liver failure requiring liver transplantation, increased rate of heart failure, stroke and death from cardiovascular causes in patients with permanent atrial fibrillation and at risk for major cardiovascular events, reports of possible lung toxicity). In 2012, during the periodic assessment of reported adverse drug reactions in the Italian database, a possible association between dronedarone exposure and acute renal failure (ARF) occurrence was identified. To better investigate the potential association between renal damage and dronedarone, a retrospective cohort study using regional health service claim databases from Lombardy and Emilia Romagna was performed. METHODS From the outpatients drugs prescription database we extracted all patients receiving at least one prescription of amiodarone or dronedarone between 1st September 2010 and 31th December 2012. Each member of the cohort was followed from the first prescription until the earliest of the following events: first hospitalization for ARF (outcome of interest), discontinuation or switching from the election therapy, death of any cause, emigration and end of the follow-up (31th December 2012). We undertook Cox regression models to estimate the hazard ratios (HRs), with 95% confidence intervals (CIs) for the association between dronedarone and the risk of ARF, taking amiodarone group as baseline comparison. We estimated HRs adjusted for high-dimensional propensity score, taking the drug and diagnosis codes from large health care claims databases. RESULTS During the study period, 59,881 patients without previous episodes of renal failure, started a new treatment with dronedarone (1,751) or amiodarone (58,130), and generated 30 and 1831 events of ARF, respectively. The mean age of patients treated with dronedarone was significantly lower than that of amiodarone ones (70.1 vs. 74.8). About 50% of patients in dronedarone group were males compared with 56% of the amiodarone group. Patients on dronedarone group had typically less comorbidities than those starting with amiodarone. The risk of hospitalization for renal failure for dronedarone compared to amiodarone was 0.79 (IC 95% 0.47-1.33). CONCLUSIONS Our results revealed a lower risk of hospitalization for renal failure for dronedarone compared to amiodarone. Since the increasing number of reports collected from pharmacovigilance databases worldwide, it is advisable for clinicians and patients to be aware of the risk of kidney damage during dronedarone therapy. The safety information on renal toxicity provided on the labels of amiodarone-containing products marketed in different countries is not standardized, therefore harmonization would be helpful for the healthcare practitioner and patients.<br>UTILIZZO DI DRONEDARONE, AMIODARONE E RISCHIO DI INSUFFICIENZA RENALE ACUTA: UNO STUDIO DI COORTE SULLA POPOLAZIONE DI LOMBARDIA ED EMILIA ROMAGNA INTRODUZIONE Il dronedarone è un bloccante multicanale approvato dalla Commissione europea nel 2009 per il trattamento di alcune forme di aritmia (fibrillazione atriale, parossistica o persistente). A sostegno della sua autorizzazione al commercio, si dichiarava che il farmaco fosse un po’ meno efficace ma più sicuro dell’amiodarone, ma ben presto il farmaco è stato associato a gravi problemi di sicurezza (raddoppio dei decessi in pazienti con grave insufficienza cardiaca e disfunzione ventricolare sistolica sinistra, casi di insufficienza epatica con necessità di trapianto, di insufficienza cardiaca, ictus, decessi per cause cardiovascolari in pazienti con fibrillazione atriale permanente e a rischio di eventi cardiovascolari maggiori, casi di tossicità polmonare). Nel 2012, alcuni dati derivanti dalla segnalazione spontanea in Italia avevano fatto emergere una associazione causale tra esposizione a dronedarone e insufficienza renale acuta (IRA). Al fine di approfondire tale segnale, è stato eseguito uno studio di coorte utilizzando i database delle prescrizioni farmaceutiche e delle schede di dimissioni ospedaliere delle regioni Lombardia ed Emilia Romagna. METODI Sono stati selezionati i pazienti con una nuova prescrizione di amiodarone o di dronedarone nel periodo compreso tra il 1 settembre 2010 e il 31 dicembre 2012. Ogni paziente è stato seguito dalla prima prescrizione fino alla comparsa di uno dei seguenti eventi: primo episodio di ricovero per IRA (evento di interesse), morte per altra causa, emigrazione, interruzione o switching della terapia, fine del follow-up (31 dicembre 2012). Il rischio di IRA nei trattati con dronedarone rispetto a quelli con amiodarone è stato valutato tramite l’hazard ratio (HR) con intervallo di confidenza (IC) al 95%, stimato da un modello di regressione di Cox. Le stime sono state corrette utilizzando l’high dimensional propensity score, costruito utilizzando i database delle prescrizioni e dei ricoveri. RISULTATI Nel periodo di osservazione, sono stati reclutati 59.881 soggetti senza precedenti episodi di insufficienza renale, di cui 1.751 nuovi trattati con dronedarone e 58.130 con amiodarone, con 30 e 1831 eventi, rispettivamente. I trattati con dronedarone sono risultati più giovani (età media 70,1 vs. 74,8), di sesso femminile (50,1 % vs. 44%) e con meno comorbilità. Il rischio di ricovero per IRA nei trattati con dronedarone rispetto al gruppo trattato con amiodarone è stato pari a 0,79 (IC 95% 0,47-1,33). CONCLUSIONI I nostri risultati hanno rivelato un minor rischio di ospedalizzazione per insufficienza renale da dronedarone rispetto all’amiodarone. Tuttavia, dato il crescente numero di segnalazioni di danno renale da dronedarone raccolte nelle banche dati di farmacovigilanza a livello mondiale, è consigliabile che i medici e i pazienti siano consapevoli di questa associazione. Le informazioni di sicurezza sulla tossicità renale nelle schede tecniche dei medicinali contenenti amiodarone commercializzati in diversi paesi non sono standardizzate, e sarebbe quindi opportuno procedere a una armonizzazione nell’interesse di medici e pazienti.

Gli alimenti fermentati sono stati consumati per più di 10 000 anni. La fermentazione degli alimenti è probabilmente una delle più antiche tecnologie alimentari implementate dall'uomo, sebbene le conoscenze scientifiche alla base del suo ruolo e dei meccanismi di azione siano state studiate solo negli ultimi 150 anni. Attualmente, si stima che i prodotti alimentari fermentati rappresentino un terzo della nostra assunzione di cibo. La microbiologia alimentare ha fornito molte risposte dietro i ruoli, le modalità di azione, la nutrizione e gli effetti sulla salute degli alimenti fermentati. Tuttavia, uno dei principali temi di preoccupazione rimane la dimostrazione di sicurezza delle colture alimentari microbiche. Più di recente, alla fine del 2017, la Cina ha bloccato l'importazione di formaggi dall'Europa, a causa della presenza di colture alimentari microbiche non presenti nell'elenco cinese positivo 2010. Anche l'Europa (Biohazard Panel – Qualified Presumption of Safety) e gli Stati Uniti (GRAS – Generalmente riconosciuto come sicuro) dispongono rispettivamente di una procedura per la valutazione del rischio microbico delle specie microbiche aggiunte volontariamente alla catena alimentare. L'International Dairy Federation (IDF), in collaborazione con l'associazione europea Food and Feed Cultures, ha lavorato negli ultimi 20 anni su un inventario delle specie microbiche con proprietà tecnologiche negli alimenti fermentati. Durante il ciclo di 3 anni, abbiamo proseguito il lavoro dell'IDF, aggiornando due volte l'inventario, nel 2018 con l'introduzione di specie alimentari microbiche da prodotti alimentari indigeni asiatici, e nel 2021 considerando le varie matrici alimentari e l'utilizzo alimentare riconducibili a un singole specie microbiche. Pertanto, abbiamo concentrato il lavoro sul ruolo delle colture alimentari e sulla bioconservazione degli alimenti, come inizialmente suggerito dalla logica iniziale della dimostrazione delle colture alimentari dell'IDF. A dimostrazione del concetto dell'approccio della dimostrazione di sicurezza di una coltura alimentare, il lavoro analitico è stato svolto su una raccolta di alimenti e isolati clinici della specie microbica Weissella confusa, che possono essere sia isolati in prodotti da forno (Europa), piante a base di prodotti derivati ​​(Asia) e con studi preliminari per il suo utilizzo nei prodotti lattiero-caseari. Il presente studio proporrà una dimostrazione di sicurezza delle specie microbiche per il loro uso attraverso l'inoculazione in una matrice alimentare per l'uso nella catena alimentare. Ha lo scopo di evitare scambi commerciali tra paesi in cui non è possibile stabilire una storia di utilizzo sicuro per un prodotto alimentare fermentato autoctono sul mercato internazionale, oltre a evitare le insidie ​​dell'incrocio di fermentazione, modificando al contempo la matrice alimentare in cui viene effettuata la fermentazione alimentare , e possibilmente omettendo le attività dannose dei metaboliti.<br>Fermented foods have been consumed for more than 10 000 years. Food fermentation is probably one of the oldest food technologies implemented by man, although the scientific knowledge behind its role and mechanisms of actions have only been studied in the past 150 years. Presently, fermented food products are estimated to represent a third of our food intake. Food Microbiology has provided many answers behind the roles, modes of action, nutrition and health effects of fermented foods. One of the major topics of concern nevertheless remains the safety demonstration of the microbial food cultures. Most recently, in late 2017, China blocked the importation of cheeses from Europe, due to the presence of microbial food cultures not present in the Chinese 2010 positive list. Europe (Biohazard Panel – Qualified Presumption of Safety) and United States (GRAS – Generally Recognized as Safe) also have a procedure in place respectively for the microbial risk assessment of microbial species voluntarily added to the food chain. The International Dairy Federation (IDF), in collaboration with the European Food and Feed Cultures association, has been working for the past 20 years on an inventory of microbial species with technological properties in fermented foods. During the 3 years cycle, we have continued the work of the IDF, updating twice the inventory, in 2018 with the introduction of microbial food species from indigenous Asian food products, and in 2021 while considering the various food matrices and food usage attributable to a single microbial species. As such, we have focused the work on the role of food cultures and food bio-preservation, as initially suggested by the initial rationale of demonstration of food cultures of the IDF. As a proof of concept of the approach of safety demonstration of a food culture, the analytical work has been done of a collection of food and clinical isolates of the microbial species Weissella confusa, which can be both isolated in bakery products (Europe), plant-based products (Asia) and with preliminary studies for its used in dairy food products. The present study will propose a safety demonstration of microbial species for its use through inoculation in a food matrix for use in the food chain. It is aimed to avoid barrier trades between countries where a history of safe use cannot be established for an indigenous fermented food products on international market, as well as avoiding the pitfalls of cross over fermentation, while changing the food matrix where the food fermentation is done, and possibly omitting deleterious metabolites activities.

Modern buildings increasingly include the usage of innovative materials aimed at improving sustainability and reducing the carbon footprint of the built environment. Phase Change Materials (PCMs) are one such group of novel materials which reduce building energy consumption. These materials are typically flammable and contained within wall linings yet there has been no detailed assessment of their fire performance. Current standard fire test methods provide means to compare similar materials but do not deliver knowledge on how they would behave in the event of a real fire. Thus, the aim of this thesis is to develop a novel testing framework to assess the behaviour of these materials in realistic fire scenarios. For PCMs, a flammability study is conducted in the bench-scale cone calorimeter to evaluate the fire risk associated with these materials. Then, micro-scale Thermogravimetric Analysis (TGA) is used to identify the fundamental chemical reactions to be able to confidently interpret the flammability results. Finally, intermediate-scale standard fire tests are conducted to evaluate the applicability of the bench-scale results to realistic fire scenarios. These take the form of modified Lateral Ignition and Flame spread Test (LIFT) and Single Burning Item (SBI) tests to understand flame spread and compartment fires respectively. Finally, a simplified method to combine this knowledge for use in building design is proposed. This method allows the balancing of potential energy benefits with quantified fire performance to achieve the specified goals of the designer. Hemp-lime insulation is a material which has also becoming increasingly popular in the drive towards sustainability. The porous nature of the material means that smouldering combustions are the dominant reaction mode but there is currently no standardised test method for this type of behaviour. Thus, hemp-lime materials also represent an unquantified risk. The work in this thesis defines a simple, accessible and economically viable bench-scale method for quantifying the fire risk associated with rigid porous materials. This is applicable for both downward opposed flow and upward forward flow smoulder propagation conditions. The behaviour is then interpreted using micro-scale thermogravimetric analysis to understand the underlying pyrolysis and oxidation reactions. Designers can utilise this framework to quantify the smouldering risk associated with hemp-lime materials to enable their usage in the built environment. The holistic fire risk assessment performed in this thesis has quantified the behaviour of PCMs and hemp-lime insulation applicable to realistic fire scenarios. The simplified design method empowers designers to be able to realise innovative buildings through fundamental understanding of the fire behaviour of these materials. The outcomes of this thesis allow designers to mitigate the fire risk associated with these materials and achieve optimised engineering solutions. Furthermore, the novel fire testing frameworks provide the economically viable means to assess the fire performance of future PCMs and hemp-lime products which ensures lasting relevance of this research in the future.

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.<br>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).

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.<br>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).

The effects of two SAR inducers (benzothiadiazole and chitosan) were investigated on volatile isoprenoid (mono- and sesquiterpene) and phytosterol (β-sitosterol, stigmasterol and campesterol) content in a grapevine cultivar (Groppello, autochthonous of Lombardy) and in its respective experimental wines. The efficacy of plant activators has been also assayed on the control of the potentially toxigenic grape mycoflora, by measuring the mycotoxin levels in wines.

La malattia di Pompe è una malattia neuromuscolare grave causata da mutazioni nell’enzima lisosomiale -glucosidasi acida (GAA), le quali determinano l'accumulo patologico di glicogeno in tutti i tessuti. La terapia di sostituzione enzimatica (ERT) è disponibile per la suddetta malattia, ma essa ha solo una limitata efficacia, mentre mostra un’elevata immunogenicità, e non riesce a correggere gruppi di tessuti muscolari e nervosi più refrattari alla cross-csorrezione. Usando l'analisi bioinformatica e ingegnerizzando l’enzima, abbiamo sviluppato transgeni GAA secrenibili per una maggiore cross-correzione della malattia di Pompe tramite virus adeno associati (AAV) attraverso l’espressione epato-specifica della GAA. I topi Pompe sono stati trattati con vettori AAV ottimizzati per l'espressione dei transgeni secrenibili della GAA e fatto un follow-up di 10 mesi. Il trasferimento genico ha evidenziato una correzione dose e tempo dipendente di entrambi i difetti biochimici e funzionali nei muscoli, sistema nervoso centrale e del midollo spinale, con normalizzazione di ipertrofia cardiaca, muscolare e funzione respiratoria, e la sopravvivenza indistinguibile dai topi wild-type. In questi esperimenti, transgeni GAA secernibili hanno mostrato un’efficacia terapeutica superiore e decisamente una bassa immunogenicità rispetto alla loro controparte GAA non secreta. La dimostrazione di un grande potenziale terapeutico e’ evidenziata dal fatto che l’AAV iniettato in primati era in grado di eprimere e secernere discreti livelli di GAA in circolo, ed utilizzando nuovi serotipi di AAV potevamo esprimere la GAA anche in epatociti primari. Considerando che l'immunogenicità della -glucosidasi acida ricombinante (rhGAA) nella terapia enzimatica sostitutiva (ERT) è un problema cosi come l'efficacia nella gestione della malattia di Pompe nei pazienti adulti (LOPD), effetti a lungo termine del ERT sulle risposte umorali e cellulari alla GAA sono ancora poco compresi. Per capire meglio l'impatto dell’immunogenicità del rhGAA sull'efficacia della ERT, dati clinici e campioni di sangue di pazienti LOPD sottoposti a ERT per più di 4 anni o non trattati sono stati raccolti e analizzati. Nei pazienti trattati LOPD, anticorpi anti-GAA avevano un picco entro i primi 1000 giorni di ERT, mentre l'esposizione a lungo termine per rhGAA comportava una clearance degli anticorpi con una produzione residua di IgG non neutralizzanti. Analisi di risposte delle cellule T al rhGAA mostravano una rilevabile reattività delle cellule T solo dopo una ristimolazione in vitro. Incremento di diverse citochine e chemochine era rilevabile sia in soggetti LOPD trattati e non trattati, mentre la secrezione di IL-2 era rilevabile solo nei soggetti che avevano ricevuto ERT. Questi risultati indicano che l’ERT a lungo termine in pazienti LOPD comporta una diminuzione dei titoli anticorpali ed una produzione residua di IgG non inibitorie. Una risposta immunitaria alla GAA a lungo termine in soggetti sottoposti a ERT non sembra influenzare l'efficacia della terapia e sono coerenti con un effetto immunomodulante eventualmente mediata da cellule T regolatorie.<br>Pompe disease is a severe neuromuscular disorder caused by mutations in the lysosomal enzyme acid -glucosidase (GAA), which result in the pathological accumulation of glycogen in all tissues. Enzyme replacement therapy (ERT) is available for Pompe disease, however it has only limited efficacy, high immunogenicity, and fails to correct nervous tissue and muscle groups more refractory to cross-correction. Using bioinformatics analysis and protein engineering, we developed secretable GAA transgenes for enhanced cross-correction of Pompe disease via adeno-associated virus (AAV) vector liver gene transfer. Pompe mice were treated with AAV vectors optimized for hepatic expression of secretable GAA transgenes and followed for up to 10 months post-gene transfer. Gene transfer resulted in dose- and time- dependent whole-body correction of both the biochemical and functional defects in muscle, central nervous system and spinal cord, with normalization of cardiac hypertrophy, muscle and respiratory function, and survival undistinguishable from wild-type littermates. In these experiments, secretable GAA transgenes showed superior therapeutic efficacy and markedly low immunogenicity compared with their native GAA counterpart. Scale up to non-human primates, and modeling of GAA expression in primary hepatocytes using novel AAV vector serotypes, demonstrate the therapeutic potential of AAV vector-mediated liver expression of secretable GAA transgenes, and support the feasibility of the approach in Pompe patients. Considering that immunogenicity of recombinant human acid-alpha glucosidase (rhGAA) in enzyme replacement therapy (ERT) is a safety and efficacy concern in the management of late-onset Pompe disease (LOPD), long-term effects of ERT on humoral and cellular responses to GAA are still poorly understood. To better understand the impact of immunogenicity of rhGAA on the efficacy of ERT, clinical data and blood samples from LOPD patients undergoing ERT for more than 4 years or untreated were collected and analyzed. In treated LOPD patients, anti-GAA antibodies peaked within the first 1000 days of ERT, while long-term exposure to rhGAA resulted in clearance of antibodies with residual production of non-neutralizing IgG. Analysis of T cell responses to rhGAA showed detectable T cell reactivity only after in vitro restimulation. Upregulation of several cytokines and chemokines was detectable both treated and untreated LOPD subjects, while IL2 secretion was detectable only in subjects who received ERT. These results indicate that long-term ERT in LOPD patients results in a decrease in antibody titers and residual production of non-inhibitory IgGs. Immune responses to GAA following long-term ERT do not seem to affect efficacy of ERT and are consistent with an immunomodulatory effect possibly mediated by regulatory T cells.

Several risk factors have been associated with Multiple Sclerosis onset (MSo). However, the role of previous drug exposure in MSo is far to be completely elucidated. In this intricate scenario, another important issue is represented by the safety profile of MS therapies. This thesis aimed to explore the role of drugs as possible risk factor in MSo by performing an evidence mapping of literature and a disproportionality analysis by using the FDA-Adverse Event Reporting System (FAERS) database. In the last part of the thesis, signal of Drug Induced Liver Injury (DILI) events during MS therapies was assessed by using in the same database. The analysis of the literature showed that drugs acting on immune system, hormone balance and infections were suspected as possible cause of MSo in case reports and case series, whereas contrasting results emerged from the few longitudinal studies. These data were in part in line with FAERS analysis, which resulted in disproportionality for drugs acting on immune system, insulin, bisphosphonates and contraceptives. Analysis of DILI events in MS treated patients suggests that a careful clinical monitoring should be applied to these patients. In addition, longitudinal studies should be planned in order to evaluate safety profile of MS drugs in monotherapy as well as in combination with other drugs. In conclusion, the submission of exhaustive reports of adverse drug reactions would permit stratified analyses and a better characterization of rare and unpredictable adverse events. Moreover, case-control studies performed on electronic medical records could confirm or refuse safety signals emerged from this thesis.

Background and purpose: Parkinson’s disease (PD) is a chronic neurodegenerative disorder characterized by the motor symptoms of bradykinesia, tremor, rigidity and postural instability. However, non-motor symptoms such as depression, apathy and rapid eye movement sleep behavior disorder (RBD) are also frequent and play a significant role. Several studies have indicated that altered serotonergic neurotransmission may contribute to the motor and non-motor features commonly associated with PD such as levodopa-induced motor complications, sleep disorders, apathy and depression.The 5-Hydroxytryptophan (5-HTP) is the intermediate metabolite of L-tryptophan in the production of serotonin. Studies in other populations indicate that 5-HTP might be useful in the treatment of depression, chronic headache and sleep disturbances. Furthermore, a recent study demonstrated the antidyskinetic effect of 5-HTP in the rat model. To date, there has been inconsistent research on the use of 5-HTP in PD. The purpose of this study was to compare the effects of 5-HTP to placebo on RBD, depression, apathy and levodopa-induced motor complications in patients with PD. Materials and Methods: A single-center, randomized, double-blind placebo-controlled cross-over trial was employed; 36 subjects were subsequently enrolled into the study, 32 subjects completed the 16-week protocol. Patients received placebo and 50 mg of 5-HTP daily in a cross-over design over a period of 4 weeks. There were 4-weeks washouts between treatments. For the assessment of efficacy on the RBD, video-polysomnography (v-PSG) was performed at baseline, and home-based polysomnography was performed at weeks 4 and 12. For the assessment of efficacy on the overall functional status, depression, apathy and motor complications, the UPDRS (parts I, II, III), Beck Depression Inventory II (BDI-II), 21-item version of Hamilton Depression Rating Scale (HDRS21), Apathy Scale (AS), Unified Dyskinesia Rating Scale (UDyRS), UPDRS IV, Wearing-Off Questionnaire (WOQ-19) and the self-reported 24-hour home dyskinesia diaries were respectively obtained at baseline and weeks 4, 8, 12 and 16 (T-end). Primary efficacy outcomes were the effect of 5-HTP on the the percentage of REM Sleep without Atonia (RSWA) and other polysomnographic sleep measures compared to placebo as well as the comparison of 5-HTP to placebo in mean change from baseline to weeks 4, 8, 12 and 16 in total score on the AS, BDI-II, HDRS21, UDysRS, UPDRS IV and WOQ-19. Results: Repeated measures analysis revealed a significant improvement of levodopa-induced dyskinesias (LIDs) and depressive symptoms during the 50 mg 5-HTP treatment compared to placebo as assessed by the UDyRS, UPDRS-IV and HDRS21. Treatment with 5-HTP significantly reduced the percentage of time in stage N3 compared to placebo. Furthermore, the 5-HTP produced a marginal, non-significant reduction in arousal index, wake after sleep onset (WASO) as well as an increase in total percentage of REM sleep. There were no other significant effects of 5-HTP at dose 50 mg compared to placebo on nighttime sleep parameters evaluated in our study. Conclusions: This study provides preliminary evidence of clinical benefit with 5-HTP for treating LIDs and depressive symptoms in PD. Larger studies with a longer treatment duration need to corroborate these early findings.

Enterococcus faecium è un componente fondamentale del microbiota di diversi alimenti fermentati quali formaggi e salumi e viene spesso isolato in alto numero in alimenti pronti al consumo. É inoltre largamente utilizzato come probiotico sia per l’uomo che per gli animali. Allo stesso tempo, però, questa specie batterica rappresenta una delle cause principali di infezioni nosocomiali quali endocarditi ed infezioni al tratto urinario. Studi recenti hanno dimostato che la specie E. faecium è costituita da due sub-popolazioni principali: la prima è denominate hospital associated (HA) clade “A” ed include la maggior parte dei ceppi responsabili di infezioni umane; la seconda è chiamata community associated (CA) clade “B”, e contiene principalmente ceppi commensali dell’uomo. Analisi più approfondite hanno rivelato un ulteriore suddivisione all’interno del clade A, nel sub-clade A1 (che raggruppa la maggioranza dei ceppi clinici) e nel sub-clade A2, associato agli animali e più sporadicamente ad infezioni umane. Nel 2012, EFSA ha redatto una linea guida per la valutazione della sicurezza di E. faecium usato come probiotico per gli animali, concludendo che i cepi appartenenti all’hospital-associated clade non devono essere utilizzati in nutrizione animale. Comunque, la distinzione tra le due sub-popolazioni è stata fatta utilizzando dati ottenuti prevalentemente da isolati umani e animali e solo un numero limitato di ceppi isolati dagli alimenti è stato considerato. Obiettivo di questa tesi di dottorato è stato quello di valutare la sicurezza di E. faecium negli alimenti fermentati, considerando ceppi isolati da formaggi artigianali e prodotti carnei e utilizzando sia tecniche di genomica che analisi fisiologiche. Nessuno dei ceppi alimentari studiati è risultato parte del clade A1, ma un ceppo isolato da un salame stagionato pronto al consumo ha rivelato diversi tratti tipici dei ceppi A1, tra cui particolari IS, transposase e geni di resistenza agli antibiotici. Questi risultati, così come altri dati, sottolineano la necessità di approfondire le conoscenze circa il ruolo dei ceppi di E. faecium isolati da alimenti come fattore di rischio per la salute umana.<br>Enterococcus faecium is commonly found in high numbers in ready to eat foods, being a member of the bacterial communities of a variety of fermented foods, including cheese and sausages, and is widely used as human and animal probiotic. However, this bacterial species is a leading cause of nosocomial infection, mainly endocarditis and urinary tract infections. Recent studies have demonstrated that E. faecium species consists of two very distinct clades: the hospital associated (HA) clade “A”, which includes most of the strains responsible for human infections, and the community associated (CA) clade “B”, that contains primarily human commensal isolates. Deeper analysis revealed a further split within clade A into sub-clade A1 (which groups the vast majority of clinical isolates), and sub-clade A2, associated with animals and sporadic human infections. In 2012, the European Food Safety Authority has issued a guideline for the safety assessment of E. faecium used as animal probiotics, concluding the strains belonging to the hospital-associated clade should not be used in animal nutrition. However, the differentiation of the two clades has been performed using data mainly deriving from human and animal isolates, and only a limited number of strains from the food chain were considered. Aim of this doctoral thesis was to assess the safety of E. faecium in fermented food, considering strains isolated from artisanal cheese and meat products, and using both whole genome-based techniques and physiological studies. None of the food isolates studied in this work belong to the epidemic clade A1, however a strain isolated from a ready to eat salami revealed several A1-specific traits, such as specific IS, transposases and antibiotic resistance genes. These results, as well as other data, underline the emergency of deeper understanding the role of E. faecium isolated from fermented foods as risk factor for human health.

Enterococcus faecium è un componente fondamentale del microbiota di diversi alimenti fermentati quali formaggi e salumi e viene spesso isolato in alto numero in alimenti pronti al consumo. É inoltre largamente utilizzato come probiotico sia per l’uomo che per gli animali. Allo stesso tempo, però, questa specie batterica rappresenta una delle cause principali di infezioni nosocomiali quali endocarditi ed infezioni al tratto urinario. Studi recenti hanno dimostato che la specie E. faecium è costituita da due sub-popolazioni principali: la prima è denominate hospital associated (HA) clade “A” ed include la maggior parte dei ceppi responsabili di infezioni umane; la seconda è chiamata community associated (CA) clade “B”, e contiene principalmente ceppi commensali dell’uomo. Analisi più approfondite hanno rivelato un ulteriore suddivisione all’interno del clade A, nel sub-clade A1 (che raggruppa la maggioranza dei ceppi clinici) e nel sub-clade A2, associato agli animali e più sporadicamente ad infezioni umane. Nel 2012, EFSA ha redatto una linea guida per la valutazione della sicurezza di E. faecium usato come probiotico per gli animali, concludendo che i cepi appartenenti all’hospital-associated clade non devono essere utilizzati in nutrizione animale. Comunque, la distinzione tra le due sub-popolazioni è stata fatta utilizzando dati ottenuti prevalentemente da isolati umani e animali e solo un numero limitato di ceppi isolati dagli alimenti è stato considerato. Obiettivo di questa tesi di dottorato è stato quello di valutare la sicurezza di E. faecium negli alimenti fermentati, considerando ceppi isolati da formaggi artigianali e prodotti carnei e utilizzando sia tecniche di genomica che analisi fisiologiche. Nessuno dei ceppi alimentari studiati è risultato parte del clade A1, ma un ceppo isolato da un salame stagionato pronto al consumo ha rivelato diversi tratti tipici dei ceppi A1, tra cui particolari IS, transposase e geni di resistenza agli antibiotici. Questi risultati, così come altri dati, sottolineano la necessità di approfondire le conoscenze circa il ruolo dei ceppi di E. faecium isolati da alimenti come fattore di rischio per la salute umana.<br>Enterococcus faecium is commonly found in high numbers in ready to eat foods, being a member of the bacterial communities of a variety of fermented foods, including cheese and sausages, and is widely used as human and animal probiotic. However, this bacterial species is a leading cause of nosocomial infection, mainly endocarditis and urinary tract infections. Recent studies have demonstrated that E. faecium species consists of two very distinct clades: the hospital associated (HA) clade “A”, which includes most of the strains responsible for human infections, and the community associated (CA) clade “B”, that contains primarily human commensal isolates. Deeper analysis revealed a further split within clade A into sub-clade A1 (which groups the vast majority of clinical isolates), and sub-clade A2, associated with animals and sporadic human infections. In 2012, the European Food Safety Authority has issued a guideline for the safety assessment of E. faecium used as animal probiotics, concluding the strains belonging to the hospital-associated clade should not be used in animal nutrition. However, the differentiation of the two clades has been performed using data mainly deriving from human and animal isolates, and only a limited number of strains from the food chain were considered. Aim of this doctoral thesis was to assess the safety of E. faecium in fermented food, considering strains isolated from artisanal cheese and meat products, and using both whole genome-based techniques and physiological studies. None of the food isolates studied in this work belong to the epidemic clade A1, however a strain isolated from a ready to eat salami revealed several A1-specific traits, such as specific IS, transposases and antibiotic resistance genes. These results, as well as other data, underline the emergency of deeper understanding the role of E. faecium isolated from fermented foods as risk factor for human health.

I farmaci di neo-immissione in commercio vengono approvati sulla base delle prove di efficacia e sicurezza dimostrate nel corso dei trial clinici pre-registrativi. Gli studi clinici randomizzati e controllati di fase 3 sono infatti considerati il mezzo più rigoroso per studiare il profilo di efficacia/sicurezza dei farmaci. Vari aspetti legati alla sicurezza di un medicinale possono però non essere individuati durante la conduzione di tali studi clinici e vengono resi noto solo quando il prodotto viene utilizzato nella pratica clinica quotidiana. Le attività di ricerca del farmacologo clinico nel prevedere la sicurezza e l'efficacia dei farmaci richiedono un approccio multidimensionale. Un tale approccio multidimensionale dovrebbe includere tutte le discipline correlata alla farmacologia clinica, come la farmacoepidemiologia, la farmacovigilanza e la farmacia clinica, che possono fornire gli strumenti per una valutazione rigorosa dei benefici e dei possibili effetti collaterali dei farmaci. Le evidenze scaturite dai dati del mondo reale (RWD) forniscono il potenziale per affrontare gli aspetti legati all'efficacia e sicurezza dei trattamenti. Grazie al numero crescente di fonti di RWD, il farmacologo clinico potrebbe sfruttare appieno ed efficacemente la possibilità di utilizzare queste fonti, in accordo allo specifico quesito clinico. A tal fine, l’elaborato di tesi è incentrato sulla proposta e validazione di un modello di approccio multidisciplinare per la farmacologia clinica, in modo tale da verificare se tali strategie possono produrre prove affidabili per rispondere a determinate quesiti clinici sfruttando e combinando prove da diverse fonti di dati, vale a dire i sistemi di segnalazione spontanea, la letteratura e la conduzione di studi osservazionali. Sono stati condotti diversi studi per affrontare determinati quesiti clinici. Il lavoro descritto in questa tesi si basa su cinque studi originali. Negli studi I-II, è stata studiata l’associazione tra l'uso di farmaci psicotropi (antipsicotici/antidepressivi) e l'insorgenza di iponatriemia. La fisiopatologia dell'iponatriemia indotta da farmaci psicotropi rimane poco chiara. Per acquisire conoscenze su questa patologia rara e grave, abbiamo eseguito uno studio che combina i dati di monitoraggio post-marketing con le proprietà farmacodinamiche dei farmaci in oggetto. Questi studi evidenziano i potenziali ruoli dei bersagli recettoriali dei vari agenti psicotropi nella comparsa di iponatriemia. Nel complesso, questi studi contribuiscono a una migliore comprensione ti tale associazione e all’identificazione di potenziali bersagli di interesse che devono essere ulteriormente esplorati. Nello studio III, è stata analizzata la potenziale associazione tra l'uso di inibitori della dipeptidil peptidasi-4 (DPP-4) e la comparsa di pemfigoide bolloso. A partire dal 2012, è stato segnalato un aumento del rischio di pemfigoide bolloso tra gli utilizzatori di farmaci inibitori della DPP-4 nell’ambito di studi di farmacovigilanza e caso-controllo. Ad oggi, il meccanismo con cui gli inibitori della DPP-4 inducono la BP non è stato caratterizzato. Questo è il primo studio volto a valutare il ruolo potenziale delle proprietà farmacologiche delle diverse gliptine nella comparsa di pemfigoide bolloso a seguito dell'esposizione a tali farmaci, utilizzando i dati provenienti dal sistema di segnalazione spontanea internazionale. Nell’analizzare diversi bersagli molecolari, abbiamo riscontrato una rilevanza clinica della selettività delle gliptine per la isoforma DDP-4 nello sviluppo del pemfigoide bolloso come risultato dell'esposizione a questi farmaci. Negli studi IV-V, abbiamo studiato l'associazione tra i cambiamenti antropometrici nei pazienti con malattia infiammatoria intestinale (IBD) e la terapia con farmaci inibitori del fattore di necrosi tumorale (TNF)-α utilizzando tre diverse metodologie. Nello studio IV, abbiamo eseguito una revisione sistematica e una meta-analisi della letteratura sull’utilizzo dei farmaci anti-TNF-α negli adulti e nei pazienti pediatrici con IBD che riportavano cambiamenti nei parametri antropometrici. Successivamente, è stata eseguita un’analisi longitudinale di case series per valutare i cambiamenti antropometrici e glicometabolici nei pazienti pediatrici con IBD dopo il trattamento con anti-TNF-α nella pratica clinica quotidiana. Infine, per mappare un profilo di sicurezza più ampio della terapia anti-TNF-α in un contesto reale, in parallelo, abbiamo effettuato un'analisi del database di farmacovigilanza per quantificare l'associazione tra la terapia anti-TNF-α e il tasso di cambiamenti antropometrici. In conclusione, la ricerca presentata in questo elaborato di tesi ha prodotto diversi nuovi spunti di discussione e ha fornito nuove risposte ai numerosi quesiti clinici irrisolti sulla sicurezza dei farmaci, sviluppando contestualmente un approccio multidisciplinare che include metodi tecnici e statistici per esaminare gli ADE e/o gli effetti benefici dei farmaci che potrebbero verificarsi tra nuovi e vecchi utilizzatori. Questo lavoro fornisce al farmacologo clinico una strategia di intervento pluridisciplinare per la valutazione dei rischi e benefici dei farmaci da adattare potenzialmente ad altri ambiti di ricerca prioritari, così come ad altre popolazioni e fonti dati in grado di contribuire alla caratterizzazione dei profili di sicurezza dei farmaci.<br>New therapeutic products are typically approved based on their demonstrated efficacy and safety in a series of clinical trials. Randomized, controlled, phase 3 studies are considered to be the most rigorous means for studying the efficacy/safety profile of drugs. Various aspects of a medicinal product’s safety may not be known from clinical trials and until the product is used in routine clinical settings. Research efforts by clinical pharmacologist towards predicting safety, efficacy and effectiveness of drugs require a multidimensional approach. Such a multidimensional approach should encompass related discipline of clinical pharmacology, such as pharmacoepidemiology, pharmacovigilance and clinical pharmacy that can provide the tools for rigorous assessment of the good and harm that specific medications provide. Real-world data (RWD) and evidence provide the potential to address the effectiveness and safety of drugs. With the emerging RWD sources, clinical pharmacologist should able to effectively and comprehensively exploit RWD sources depending on the research problem. Towards this goal, this dissertation has proposed and investigated a research framework for clinical pharmacology, if this would produce reliable evidence to answer given research question by exploiting and combining evidence from different data sources, namely pharmacovigilance and observational studies. To answer the initial research questions, several studies were conducted. The work described in this thesis is based on five original studies. In study I-II, an association between psychotropic drugs (anti-psychotics/antidepressants) use and hyponatremia was studied. The pathophysiology of hyponatremia induced by psychotropic drug remains unclear. To gain knowledge into this rare and severe pathology, we performed a study combining a real-world pharmacovigilance safety data and the pharmacodynamics properties of given drug class. These studies highlight the potential roles of receptor targets in psychotropic induced by hyponatremia. Overall, these studies contribute to a better understanding of hyponatremia induced psychotropic and to identify the potential target of interest that needs to be further explored. In study III, the potential association between the use of dipeptidyl peptidase-4 (DPP-4) inhibitors and bullous pemphigoid was analysed. Since 2012, an increased risk of bullous pemphigoid among DPP-4 inhibitor users has been reported in case series, pharmacovigilance reports, and case-control studies. To date, the mechanism by which DPP-4 inhibitors induce BP is not well understood. This is the first study aimed at evaluating the potential role of pharmacological properties of different gliptins in the occurrence of BP as a result of exposure to DPP-4 inhibitors using the global SRS. Several molecular targets were analysed and we found a clinical relevance of gliptins selectivity for DDP-4 in the development of BP as a result of exposure to these drugs. In study IV-V, we studied the association between anthropometric changes in inflammatory bowel disease (IBD) patients following anti-tumour necrosis factor (TNF)- α therapy using three different methodologies. In study IV, we performed a systematic review and meta-analysis of the studies on anti-TNF-α in adults and paediatric IBD patients reporting changes in anthropometric parameters. Subsequently, A longitudinal case series analysis was performed to assess anthropometric and glucometabolic changes in paediatric IBD patients following anti-TNF treatment. Finally, to map a broader safety profile of anti-TNF-α therapy in a real-world setting, we carried out a comprehensive analysis of pharmacovigilance database in parallel to quantify the association between TNF-α therapy and the rate of body-related changes. In conclusion, the research presented in this dissertation has produced several novel insights and provided new answers towards the challenging drug safety questions. This dissertation developed research framework including technical and statistical methods for examining ADEs and/or beneficial effect of drugs that might occur among new or established users of pharmaceuticals. This work provides a template to clinical pharmacologist in establishing a continuous learning system for other priority conditions and drug classes, other populations, and other databases which could potentially unveil drug safety profiles and novel adverse events (AEs).

2011/2012<br>SUMMARY Biotoxins produced by harmful algae during their proliferation can be accumulated by filter feeding organisms, such as bivalve shellfish, within their flesh. Furthermore, these toxins gradually are transferred to the higher trophic levels in the food chain, posing a threat to human health, after consumption of contaminated seafood. Filter-feeding invertebrates are organisms in which the toxin accumulation is a well-known phenomenon, especially during harmful algal blooms. Mussels, cockles, oysters, and scallops feed on toxic dinoflagellates, transferring them from the gills to digestive organs where the toxins accumulate. Different algal toxins can simultaneously contaminate edible shellfish, representing a world-wide sanitary and economic problem. Among them, Palytoxin (PLTX) is a highly toxic polyhydroxylated compound associated to human seafood intoxications in tropical and subtropical areas, but recently it has been detected also in microalgae and shellfish from temperate areas, as Mediterranean Sea. In the last years, also Yessotoxin (YTX) was frequently detected in mussels from Mediterranean Sea and a possible co-exposure to both PLTX and YTX can occur through contaminated seafood consumption. Therefore, the research was initially focused on the detection and quantification of PLTX and YTX in mussels collected in the Gulf of Trieste in order to verify the simultaneous shellfish contamination by these toxins and, subsequently, to study their toxic effects after simultaneous oral exposure. Analyses by liquid chromatography coupled mass spectrometry (LC-MS) did not reveal the presence of PLTX in mussels but identified the presence of YTX together with the diarrheic toxins okadaic acid (OA) and its acyl esters. Consequently, the final goal of this part of the research was the determination of these toxins in mussels from the Gulf of Trieste by LC-MS as well as that of OA and its esters by a functional assay, the protein phosphatase 2A (PP2A) inhibition assay (comparing the results obtained from two different protocols for the PP2A assay). These assays were used to analyse both cooked and uncooked mussel samples, to verify the influence of the heating procedure on the toxin content of mussels. Globally, no significant difference in toxins concentration between uncooked and cooked mussels was observed. However, comparing the data of single samples, a slight increase (not statistically significant) of toxin concentrations was detected in the cooked mussels with respect to the uncooked ones. The mussel analysis by LC-MS/MS detected also the presence of significant amounts of yessotoxin. Contrary to OA group toxins, yessotoxin was slightly less concentrated in the cooked mussels, probably because of its higher polarity that allows a dissolution in the water lost during the cooking procedure. In the other hand and as it was mentioned before, the palytoxin produced by Ostreopsis sps. microalgae have become a problem in more occasions for the attendance of the coastal environment, both for recreation and for business, representing a loss, as well as to public health, even for the tourism and the aquaculture industry. World market globalization, climatic changes and increasing overseas traffic are considered the main responsible for the appearance of these toxins. The expand of these toxins to temperate regions may be due in part to ballast water of ships and also to general changes in climate conditions, enough to induce bloom formation. Despite the extent of the contaminated area, few methods are currently available for palytoxins detection and quantitation in seafood. Moreover, among palytoxins, only palytoxin is commercially available, though expensive, and no certified standard material is currently sold. For monitoring purposes, a combination of screening methods followed by a chemical confirmatory analysis, such as LC-MS, is commonly used to detect palytoxins. Other methods for PLTX analysis include mouse bioassay, cytotoxicity assays, haemolysis assays, receptor binding assays, and immunoassays. Even if there were no food poisoning from palytoxin in the Mediterranean countries, the toxin was detected in shellfish, which gave positivity to the official test for the lipophilic toxins of algal origin (okadaic acid and derivatives, azaspiracids, yessotoxins and pectenotoxins). Some different experiments were applied in the evaluation of palytoxin toxicity in this study (Haemolytic assay, sandwich ELISA, LC-MS/MS and cytotoxicity studies were carried out). Haemolytic assay, carried out incubating mouse erythrocytes with palytoxin for 4 h (standard assay) or for 1 h in diluted PBS (abbreviated assay) is able to detect palytoxin at picomolar concentrations. Nevertheless, with the aim to detect the toxin in mussels, a significant matrix effect impairing the toxin quantification was observed already at the mussel extract concentration of 0.048 mg edible tissues equivalents/mL, which was more evident by the abbreviated assay. However, most of the experiments lack specificity or have other limitations. Thus, an indirect sandwich ELISA has been set up. The ELISA assay (indirect sandwich) was developed using the monoclonal antibody 73D3, and a rabbit polyclonal antibody produced at the University of Trieste. The assay detects the PLTX in a range of concentrations ranging from 1.25 to 40 ng/ml and is able to quantify with very similar sensitivity also biotinilated PLTX as well as 42-OH-PLTX, this latter isolated and characterized from the chemical point of view during the latter years from the group of prof. E. Fattorusso (University of Naples Federico II), in a sample of palytoxin kindly provided by Dr. M. Poly (Maryland, USA). The incapacity to detect okadaic acid (OA), domoic acid (DA), brevetoxin-3 (PbTx-3), saxitoxin (STX) and yessotoxin (YTX)(toxins that may be present along with PLTX in fish contaminated) indicates the specificity of the assay. The structure of Palytoxins is very complex. In addition to this structural complexity, there is still a lack of knowledge about the different congeners involved in this contamination and therefore there is still a very limited availability of standards and reference materials. These issues made difficult the advances in the development and optimization of analytical methods, particularly in the case of LC-MS/MS. Despite of this, a significant progress has been made over the last few years in the development of analytical techniques, particularly on LC-MS/MS approaches. In this part of the study, a LC-MS/MS method was optimized for the analysis of PLTXs in order to be able to detect, quantify and confirm the presence of this toxins in natural samples. During the PhD period, there was the possibility to get some natural contaminated samples to be evaluated by ELISA developed assay and then compare the results with the analysis by the developed LC-MS/MS method. The study was carried out with samples from 3 distinct sites characterized by having different coastal morphologies and continental hydrodynamic conditions: i) Madeira Islands’ archipelago in the NE Atlantic Ocean, Selvagens island in particular (Long, Lat) during the upwelling of August 2008; ii) Cascais, on the west coast of Portugal mainland, located at the northern side of Lisbon bay during the upwelling occurred during the favourable northerly wind periods (from April to September 2011); and iii) Algarve, Lagos, on the South Portuguese coast, also in 2011.<br>RIASSUNTO Biotossine prodotte da alghe nocive durante la loro proliferazione possono essere accumulati da organismi di alimentazione per filtrazione, come molluschi bivalvi, nell'ambito della loro carne. Inoltre, queste tossine vengono trasferite gradualmente ai livelli trofici superiori della catena alimentare, che rappresenta una minaccia per la salute umana, dopo il consumo di frutti di mare contaminati. Invertebrati con filtro-alimentazione sono organismi in cui l'accumulo di tossine, è un fenomeno ben noto, soprattutto durante fioriture algali nocive. Cozze, vongole, ostriche e capesante si nutrono di dinoflagellati tossici, trasferendoli dalle branchie agli organi digestivi, dove le tossine si accumulano. Diverse tossine algali possono contemporaneamente contaminare molluschi commestibili, che rappresenta un problema mondiale sanitario ed economico. Tra questi, Palitossina (PLTX) è un composto altamente tossico poliossidrilato associato ad intossicazioni ittici dell'uomo nelle zone tropicali e subtropicali, ma recentemente è stato rilevato anche in microalghe e molluschi dalle zone temperate, come il Mare Mediterraneo. Negli ultimi anni, anche yessotossina (YTX) è stato spesso rilevato nei mitili dal Mare Mediterraneo e una possibile co-esposizione sia PLTX e YTX può avvenire attraverso il consumo di frutti di mare contaminati. Pertanto, la ricerca è stata inizialmente concentrata sul rilevamento e la quantificazione di PLTX e YTX nei mitili raccolti nel Golfo di Trieste, al fine di verificare la contaminazione simultanea nei frutti di mare da queste tossine e, in seguito, per studiare i loro effetti tossici dopo esposizione orale simultanea. Analisi mediante cromatografia liquida accoppiata alla spettrometria di massa (LC-MS) non ha rivelato la presenza di PLTX in mitili ma è stata identificata la presenza di YTX insieme alle tossine diarroiche acido okadaico (AO) e suoi esteri. Di conseguenza, l'obiettivo finale di questa parte della ricerca è stata la determinazione di queste tossine nei mitili del Golfo di Trieste mediante LC-MS così come quella di AO e suoi esteri da un saggio funzionale, il Saggio d’inibizione Proteina Fosfatasi 2A (PP2A)(confrontando i risultati ottenuti da due diversi protocolli per il saggio PP2A). Questi test sono stati utilizzati per l'analisi di campioni di mitili sia crude e cotte, per verificare l'influenza della procedura di riscaldamento sul contenuto di tossina nei mitili. A livello globale, nessuna differenza significativa nella concentrazione di tossine tra cozze crude e cotte è stata osservata. Tuttavia, confrontando i dati dei singoli campioni, un lieve aumento (non statisticamente significativo) delle concentrazioni di tossine è stato rilevato nelle cozze cotte rispetto a quelli crude. L'analisi dei mitili per LC-MS/MS rilevò anche la presenza di quantità significativa di yessotossina. Contrariamente alle tossine gruppo OA, yessotossina era leggermente meno concentrata nelle cozze cotte, probabilmente a causa della sua polarità superiore che permette una dissoluzione in acqua persa durante la cottura. Altrimenti e come è stato detto prima, la palitossina prodotta da Ostreopsis sps. microalghe sono diventate un problema in più occasioni per la partecipazione dell'ambiente costiero, sia per la ricreazione e per le imprese, con una perdita, così come per la salute pubblica, anche per il turismo e l'industria dell'acquacoltura. Globalizzazione del mercato mondiale, i cambiamenti climatici e l'aumento del traffico all'estero sono considerati il principale responsabile della comparsa di queste tossine. L'espansione di queste tossine per le regioni temperate può essere dovuto in parte alla acque di zavorra delle navi e anche a cambiamenti delle condizioni climatiche generali, tanto da indurre la formazione di fioritura. Nonostante e dovuto alla estensione dell'area contaminata, alcuni metodi sono disponibili per il rilevamento e la quantificazione di palitossina in frutti di mare. Inoltre, tra palitossine, solo palitossina è disponibile in commercio, anche se costoso, e nessun materiale standard certificato è attualmente venduto. A scopo di monitoraggio, una combinazione di metodi di screening seguita da una analisi chimica di conferma, ad esempio LC-MS, è comunemente utilizzato per rilevare palitossine. Altri metodi di analisi includono PLTX biotest sui topi, saggi di citotossicità, saggi emolici, saggi di legame al recettore e saggi immunologici. Anche se non ci sono stati identificate intossicazione alimentare da palitossina nei paesi del Mediterraneo, la tossina è stata rilevata nei molluschi, che ha dato positività al test ufficiale per le tossine lipofile di origine algale (acido okadaico e derivati, azaspiracidi, yessotossine e pectenotossine). Alcuni esperimenti diversi sono stati applicati nella valutazione della tossicità della palitossina in questo studio (saggio emolitico, ELISA, LC-MS/MS e studi di citotossicità sono state effettuate). Saggio emolitico, effettuato incubando eritrociti di topo con palitossina per 4 h (saggio standard) o per 1 h in PBS diluito (saggio abbreviato) è in grado di rilevare la palitossina a concentrazioni picomolari. Tuttavia, con lo scopo di rilevare la tossina nelle cozze, un significativo effetto matrice ledere la quantificazione di tossina è stata osservata già alla concentrazione di estratto di cozze 0,048 mg equivalenti tessuti commestibili/mL, che è stato più evidente con il saggio abbreviato. Tuttavia, per la maggior parte degli esperimenti mancano specificità o hanno altre limitazioni. Così, un indiretto sandwich ELISA è stato istituito. Il saggio ELISA (sandwich indiretto) è stato sviluppato utilizzando gli 73D3 anticorpi monoclonali, e un anticorpo policlonale di coniglio prodotto nella Università di Trieste. Il saggio rileva la PLTX in un intervallo di concentrazioni variabili 1,25-40 ng / ml ed è in grado di quantificare con sensibilità molto simile anche PLTX biotinilata così come 42-OH-PLTX, quest'ultimo isolata e caratterizzata dal punto di vista chimico durante gli ultimi anni dal gruppo del prof. E. Fattorusso (Università degli Studi di Napoli Federico II), in un campione di palitossina gentilmente fornito dal Dr. M. Poli (Maryland, USA). L'incapacità di individuare acido okadaico (AO), acido domoico (AD), brevetossina-3 (PbTx-3), saxitossina (STX) e yessotossina (YTX) (tossine che possono essere presenti insieme a PLTX nel pesce contaminato) indica la specificità del dosaggio. La struttura della palitossina è molto complessa. In aggiunta a questa complessità strutturale, vi è ancora una mancanza di conoscenza sui diversi congeneri coinvolti in questa contaminazione e quindi c'è ancora molto limitata disponibilità di standard e materiali di riferimento. Questi problemi reso difficili gli progressi nello sviluppo e ottimizzazione di metodi analitici, in particolare nel caso di LC-MS/MS. Nonostante, un progresso significativo è stato compiuto negli ultimi anni allo sviluppo di tecniche analitiche, in particolare su approcci LC-MS/MS. In questa parte dello studio, un metodo LC-MS/MS stato ottimizzato per l'analisi di PLTXs per essere in grado di rilevare, quantificare e confermare la presenza di queste tossine in campioni naturali. Durante il periodo di dottorato di ricerca, c’è stata la possibilità di ottenere alcuni campioni naturali contaminati da valutare tramite il saggio ELISA sviluppato e poi confrontare i risultati con l'analisi con il metodo sviluppato di LC-MS/MS. Lo studio è stato effettuato con campioni da 3 posti diversi caratterizzati d’avere diverse morfologie e condizioni idrodinamiche costiere continentali: i) arcipelago Isole Madeira nel nord-orientale dell'Oceano, Selvagens isola in particolare durante il mese di agosto 2008; ii) Cascais, sulla costa occidentale del Portogallo continentale, che si trova sul lato settentrionale della baia di Lisbona durante i periodi favorevoli di vento dal nord (da aprile a settembre 2011), e iii) Algarve, Lagos, sulla costa sud-portoghesa, anche nel 2011.<br>XXV Ciclo<br>1983

This thesis deals with use of qualitative and quantitative probabilistic models for the animal-derived food safety management. Four unrelated models are presented: three quantitative and one qualitative. Two of the quantitative models concern the risk posed by pathogens in raw milk, in the first study, a probabilistic approach for the inclusion of the variability and the uncertainty in the consumers’ habits and the bacterial pathogenic potential is proposed while the second study, demonstrate how the overlook of the relationship between the storage time and temperature has led to overestimated results in raw milk-related models published so far and an equation to address the issue is provided. In the third study, quantitative modelling techniques are used to simulate the dynamics underlying the spread of Campylobacter in broiler flocks and quantify the potential effects that different on-farm mitigation strategies or management measures have on the microbial load in the intestine of infected birds at the end of the rearing period. In the qualitative study, a general approach for the estimation of the likelihoods of introduction of live parasites in aquaculture implants and the commercialization of infested product is outlined by using the example of Anisakids in farmed Atlantic salmon.

Dans ce travail de doctorat, une étude théorique et expérimentale systématique a été menée sur l'isolation du mélange de gaz C4F7N-CO2-O2 respectueux de l'environnement et ses caractéristiques de décomposition et de biosécurité sous défauts électriques et thermiques. Sur la base de la méthode de dynamique moléculaire ReaxFF, le processus de décomposition thermique du mélange gazeux sous différentes teneurs en O2 et températures est simulé. En combinant les résultats simulés avec des essais de décomposition thermique, le processus cinétique de décomposition thermique du mélange gazeux et le mécanisme d'évolution de ses sous-produits dans différentes conditions sont révélés. En même temps, le mécanisme d'influence de la teneur en O2 sur la tension de claquage et les valeurs caractéristiques statistiques du mélange C4F7N-CO2-O2 pour des décharges partielles sont analysés et le mécanisme d'influence de différents facteurs sur la génération et l'inhibition des gaz et des sous-produits solides au cours du processus de décomposition par décharge du mélange gazeux est clarifié. En conclusion, sur la base des résultats de simulation et expérimentaux, nous proposons la quantité optimale d'additif O2 et les composants caractéristiques de diagnostic du mélange gazeux C4F7N-CO2-O2 pour les défauts des équipements isolés au gaz moyenne tension ; nous testons la biosécurité du C4F7N et de ses produits de décomposition après l'exposition à l'arc, puis évaluons la faisabilité, la sécurité de l'application du mélange de gaz C4F7N-CO2-O2 dans l'équipement en combinant avec les caractéristiques isolantes et de décomposition électrique et thermique du mélange de gaz C4F7N-CO2-O2 et, les résultats de la biosécurité<br>In this doctoral work, a systematic theoretical and experimental study has been carried out on the insulation of environmentally friendly C4F7N-CO2-O2 gas mixture and on its decomposition characteristics and biosafety under electrical and thermal faults. Based on the ReaxFF molecular dynamics method, the thermal decomposition process of the gas mixture under different O2 contents and temperatures is simulated. The kinetic process of the thermal decomposition of the gas mixture and the evolution mechanism of its by-products under different conditions are revealed by combining with thermal decomposition tests. Meanwhile, the influence mechanism of O2 content on the breakdown voltage and partial discharge statistical characteristic values of the C4F7N-CO2-O2 mixture is analyzed, and the influence mechanism of different factors on the generation and inhibition of gas and solid by-products during the discharge decomposition process of the gas mixture is clarified. In conclusion, based on the simulation and experimental results, we propose the optimal O2 additive amount and fault diagnosis characteristic components of C4F7N-CO2-O2 gas mixture for medium-voltage gas-insulated equipmentwe test the biosafety of C4F7N and its arc decomposition products, and then evaluate the feasibility and safety of applying C4F7N-CO2-O2 gas mixture in equipment by combining with the insulating and electrical and thermal decomposition characteristics of C4F7N-CO2-O2 gas mixture and the results of the biosafety

L'essor récent de l'intelligence artificielle (IA) a trouvé un large éventail d'applications qui l'intègrent essentiellement dans presque tous les domaines de notre vie. Avec une telle intégration, il est raisonnable que des préoccupations surgissent. Celles-ci doivent être éliminées avant l'utilisation de l'IA sur le terrain, en particulier dans les applications critiques en termes de mission et de sécurité, comme les véhicules autonomes. Les réseaux neuronaux à impulsions (Spiking Neural Networks, SNNs), bien que d'inspiration biologique, n'héritent que partiellement des remarquables capacités de résistance aux pannes de leurs homologues biologiques, car ils sont vulnérables aux défauts électroniques et aux pannes survenant au niveau du matériel. Par conséquent, une exploration méthodologique des caractéristiques de fiabilité des accélérateurs matériels d'IA et des plateformes neuromorphiques est de la plus haute importance. Cette thèse aborde les sujets du test et de la tolérance aux fautes dans les SNNs et leurs implémentations neuromorphiques sur le matériel<br>The recent rise of Artificial Intelligence (AI) has found a wide range of applications essentially integrating it gaining more and more ground in almost every field of our lives. With this steep integration of AI, it is reasonable for concerns to arise, which need to be eliminated before the employment of AI in the field, especially in mission- and safety-critical applications like autonomous vehicles. Spiking Neural Networks (SNNs), although biologically inspired, inherit only partially the remarkable fault resilience capabilities of their biological counterparts, being vulnerable to electronic defects and faults occurring at hardware level. Hence, a methodological exploration of the dependability characteristics of AI hardware accelerators and neuromorphic platforms is of utmost importance. This thesis tackles the subjects of testing and fault tolerance in SNNs and their neuromorphic implementations on hardware

Per inquinamento causato dalle materie plastiche si intende l’accumulo in ambiente di prodotti plastici in grado di indurre problemi sia all’ambiente che alle specie selvatiche. Il Bisfenolo A (BPA) è uno dei distruttori endocrini maggiormente prodotti a livello mondiale durante la lavorazione della plastica. Numerosi studi hanno mostrato la capacità di questo inquinante di creare effetti dannosi sia sull’uomo che sulle specie selvatiche, per cui nel tempo sono stati compiuti diversi tentativi volti a trovare delle valide alternative. Tra i possibili sostituti, in questo studio di dottorato, l’attenzione è stata focalizzata sul Dhietylene dibenzoato (DGB) ed il Diisononilftalato (DiNP). Lo scopo principale di questo progetto è stato quindi quello di studiare l’impatto di questi plastificanti, focalizzando l’attenzione sui loro effetti nella riproduzione e nel metabolismo lipidico. Gli effetti del nonilfenolo, dell’ottilfenolo (t-OP) e del BPA sul metabolismo lipidico, sono stati inoltre analizzati in giovanili di orata. I risultati ottenuti hanno dimostrato come, sia il BPA, che il DiNP, siano in grado di interferire con il processo di oogenesi e che, sia il BPA che il DGB, sono in grado di interferire con il metabolismo lipidico nello zebrafish. La somministrazione a giovanili di orata di mangimi contaminati con diversi inquinanti tra cui il BPA, ha mostrato la loro capacità di indurre disordini metabolici, mostrando come oltre all’esposizione ambientale, anche il consumo di cibi contaminati possa essere considerata una fonte di esposizione importante ai contaminanti. Concludendo, il presente progetto di dottorato, mostra la capacità di alcuni distruttori endocrini, utilizzati nella lavorazione della plastica e nei detergenti, di interferire con la riproduzione ed il metabolismo lipidico nei teleostei, agendo prevalentemente in maniera dose dipendente, secondo curve non-monotoniche e, nel caso del BPA, interferendo con i meccanismi epigenetici.<br>Plastic pollution involves the accumulation of plastic products in the environment that adversely affects wildlife and wildlife habitat. Bisphenol A (BPA) is one of the highest volume endocrine disruptor produced worldwide during the manufacturing of plastic. Due to his well-documented detrimental effects, several substitute for BPA, but also for other plasticizer of common use, have been suggested, such as Diethylene glycol dibenzoate (DGB) and Diisononyl Phthalate (DiNP). The aim of this project is therefore to investigate the impact of several plasticizers, which are supposed to act as endocrine disruptor compounds (EDCs), focusing the attention on their effects on metabolic and reproductive system. Due to the necessity of testing a different range of concentrations in various plastic pollutants, an experimental model easy to reproduce, treat and analyse was needed; thus the choice to introduce zebrafish as experimental model for these studies. Concomitantly, the effects of nonylpnenol (NP), 4-tert-octylphenol (t-OP) and BPA on lipid metabolism were studied in sea bream juveniles. The results we obtained, using a multidisciplinary approach ranging from molecular to spectroscopic techniques, showed the ability of BPA and DiNP to interfere with female’s reproduction in a dose dependent manner. Concerning BPA its epigenetic effects were also demonstrated and eventually both BPA and DGB were found able to interfere with lipid metabolism in a dose dependent manner. Furthermore t-OP, NP and BPA were demonstrated to give rise to hepatic metabolic disorders in sea bream juveniles. In conclusion, the present PhD project, demonstrates that endocrine disruptors compounds, used in the manufacturing of plastics, are able to interfere with reproductive and metabolic system of teleost fish. Furthermore, we demonstrates the capacity of BPA to affect gene expression through the deregulation of epigenetic patterns.

Le travail de recherche présenté dans ce manuscrit fait partie intégrante d’un projet de recherche collaborative financé par l’Union-Européenne (Il s’agit d’un projet H2020 de type « Marie-Curie Action »), dénommé HUGS (pour « HUmins as Green and Sustainable precursors for eco-friendly building-blocks and materials »). Ce projet de recherche implique 5 partenaires (INERIS/UTC, France, Avantium, Pays-Bas, Université de Sophia Antipolis/CNRS, France, l’université de Cordoue, Espagne et le LIKAT de l’université de Rostock en Allemagne). La recherche menée dans ce projet est essentiellement structurée via la mise en place de 5 programmes sous-jacents de doctorat (intitulé « Doctorat industriel européen » dans l’appel d’offre H2020 (H2020-MSCA-ITN-2015) auquel a répondu le consortium de recherche), mis en place lors du lancement du projet « HUGS » en 2016. L’objectif premier du projet HUGS concerne l’étude de divers chemins de valorisation à haute valeur ajoutée des humines. Ces résidus de biomasse, à l’instar des lignines se présentent comme des sources de carbone renouvelable à faible coût, en émergence dans nombre de bioraffineries modernes. Les humines sont des résidus complexes résultant du procédé de déshydratation par catalyse acide des polysaccharides (sucres en C5 et C6) contenus dans la biomasse lignocellulosique, ayant des cycles furaniques dans sa structure polymère. Le travail présenté ici est centré essentiellement sur les questionnements de sécurité soulevés par la phase de développement du projet. De manière plus ciblée, des actions prioritaires ont été définies, à savoir l’obtention d’un premier profilage des risques à caractère physicochimique des humines, ainsi qu’une première évaluation des risques des composés furaniques, lesquels constituent une famille de composés potentiellement très grande et représentent une voie encourageante vers le développement de nouveaux synthons au service d’une économie biosourcée. Les humines étant des résidus fatals, leur réutilisation sure et durable constitue aussi une étape stratégique dans le contexte de l’économie circulaire. De manière opérationnelle, le travail a compris les principaux axes de recherche suivants : • Revue bibliographique continue tout au long du travail de thèse concernant les humines, les composés furaniques et les matériaux associés (polymères) en termes de données relatives à la sécurité et ayant conduit aux principales informations suivantes: o Rareté /absence d’études sur les dangers physiques des humines et nombres de composés furaniques, car ces produits sont souvent au premier stade de leur développement o Malgré une la disponibilité très limitée de données pertinentes sur la sécurité, le constat est fait que les aspects de toxicité (par ingestion) sont le plus souvent le point focal des études, au détriment de l’examen des dangers physiques.o Seuls quelques composés furaniques (ethers, esters) ont spécifiquement fait l’objet de l’étude de certaines caractérisations en lien avec la sécurité (par exemple en termes de stabilité thermique), dans le cas d’application comme composants biosourcés de carburants innovants o De nombreuses variables influent sur les caractéristiques des humines et notamment leur méthode de production : ce qui signifie que les résultats obtenus sur les humines dans le cadre de ce projet (une seule source d’approvisionnement) mériteraient des travaux de consolidation dans le futur • Développements analytiques intégrant un premier examen de la distribution des points d’éclair en fonction des chaleurs de combustion des composés furaniques et une analyse des chaleurs de combustion de ces mêmes composés furaniques<br>The present research work was integrated as part of the EU-funded project named HUGS (HUmins as Green and Sustainable precursors for eco-friendly building blocks and materials), involving 5 main partners (Institut national de l'environnement industriel et des risques - France, Avantium - the Netherlands, Institut de Chimie de Nice - France, Universidad De Cordoba- Spain and Leibniz - Institut Fur Katalyse Ev An Der Universitat Rostock- Germany). The project is essentially supported through five European Industrial Doctorate fellowships put in place when the HUGS-MSCA-ITN-2015 program was launched in 2016. The primary objective of the HUGS project was to explore several valorization pathways of so-called “humins” in order to add value and create better business cases. Humins (and similarly lignins) are the side products that may become low-cost feedstock resulting from a number of future biorefineries and sugar conversion processes. Humins are complex residues resulting from the Acid-Catalyzed Dehydration and condensation of sugars, having furan-rings in their polymeric structures. The work presented in this specific part of the HUGS project is essentially focusing on safety-related topics of all components and subsequent applications related to sugar dehydration technology. Priority actions were devoted to a first insight on the characterization of physicochemical safety profiles of the side-product humins and main (parent) furanic products. Some members of this large family of compounds (e.g. RMF and FDCA) have high volume potential which results in opening new doors towards the development of furanbased building blocks and a bio-based economy. Humins are residues or side products which can be burnt for energy. However, its safe and sustainable use in high-value applications could also become a key milestone in the so-called circular economy. In practice, the work has been developed in two main locations: primarily at the INERIS lab, located in Verneuil-en-Halatte and at Avantium, located in Amsterdam. Nearly all experimental research after the production of the components at Avantium was performed at INERIS. This involved the evaluation of physicochemical hazards of both humins (crude industrial humins and humin foams obtained by thermal curing) and a series of furanic compounds. Avantium is involved in the commercialization of humins, furanics and furanic polymers/materials as novel chemicals and materials. The work has encompassed: An extensive bibliographical review of humins, furanics, and their related products (polymers, composites) resulted in the following main conclusions o A lack of physicochemical safety-oriented studies for many furanic compounds and for humins was observed as these products are still in the early stage of development and only a few may be commercialized in the next 5 years.o Despite the limited availability of safety-related data, more studies on toxicity aspects have been conducted for a selected number of furanics than physicochemical safety-related aspects. o A few furanic family members that have been evaluated as biofuel components were found to have given better emphasis on addressing some physicochemical safety attributes. o Every modification of the process for acid-catalyzed sugar dehydration (such as solvent, temperature, residence time and sugar concentration) will result in different humins, which would certainly demand further characterization and safety profiling of the resulting humins. • Analytical development integrating the first examination of flash point distribution versus the Net Heating Values, and analysis of total heats of combustion of furanic compounds. • Design and development of experimental plan addressing the safety-related key parameters such as thermal stability, self-heating risks, fire-risk-assessment and flammability limits depending on the need for specific tests and availability of the test samples

The Food Safety and Quality Control System in Czech RepublicThe thesis describes the way how is the food safety control and quality protection provided in Czech republic. There is the law definition of the all forms of food during the food production chain from the primary production up to sale to the consumer in the work. The thesis focuses on the requirements laid down in the Czech and European food law on the food producers and sellers. The thesis also deals with the food contamination and quality aspects - nutrition value and quality branding. There is also chapter about the quality management system certification and chapter about the current cases related with the safe food area in the Czech republic in 2012 (Methanol, Polish technical salt and horse lasagne).

碩士<br>國立臺灣大學<br>生物環境系統工程學系暨研究所<br>90<br>The research presents a tunnel type pipe structure designed for bio-production. Under the principles of safety and economy, the data bases of the design were estimated. The research also illustrates the comparison of 54 modeling structure including 3 different height and span ratios, 3 structure scales, 3 frame spacing and 2 truss pitch angles. The computer program STAAD-III, with the demanal of 12th degree wind force and 5th degree earthquake force resistance, calculates and estimates way of constructing while checking the fundamental allowable force. The results find that the minimum material needed is 2.44kg/m3 with height and span ratio equals 1：3,frame spacing is 1m ,trust allocation pitch is 20 degree .This saves 33﹪of the material than module with height and span ratio is 1：2 or 47﹪ than it is 1：4.The average cost of the material to create a 1m3 inner space is NT＄114.67/ m3 ,while is 32﹪ less than NT＄168.67/ m3 with height and span ratio is 1：2 or 54﹪less than NT＄253.90/ m3 with height and span ratio reaches 1：4. When the height and span ratio is 1：3, the inner area extends to its maximum and the cost is most economical as well. It is recommended that the buildings stays 10 meter long with inner space between 195 to 550 m3 and inner area between 90 to 150m2, height and span ratio keeps 1：3, main frame spacing is 1 meter and trust allocation inter remains 20 degree. This combination is the most efficient and safe design founded in this study.

博士<br>國立臺灣大學<br>土木工程學研究所<br>100<br>Most of the architectural designers, the field workers and the inspectors lack of the concepts and the techniques of building biosafety facilities and isolation wards. They rely on the client’s experiences and the requirement instruction, which cannot coordinate both the medical and the construction disciplines in an efficient manner. Without the efficient cooperation, it is impossible to build the biosafety level compliance isolation wards in time to accommodate the infected patients to stop the spreading of the diseases. This study firstly coordinates the various demanding systems within the construction interfaces and management issues, and links the product quality and the management quality, so that the RBS of building the biosafety level isolation wards can be established. The study secondly coordinates the management mechanism in order to ensure the implementing efficiency. The management mechanism is to identify the work items of the core techniques with the IIW process and the IMM. As a result, every functional system and issue of interfaces is clarified by various building stages and the IMD is therefore possibly to be established. This study combines the results of above mentioned approaches, and eventually derives the “6Q+3V” validation management methodology. The validation management integrates the concepts of biosafety, affection prevention techniques, construction techniques, and medical facility installation techniques, and provides the appropriate procedures and means, such as CoSD and CVT, for the building and the operation. The 6Q, including DQ, CQ, EQ, IQ, OQ and PQ, are a series of validations of various building and operation stages. The 3V, including BV, PV and MV, are implemented in the whole life cycle. These validations and qualifications guarantee that the demands will be met, and the expected functions will be stable and continuous. The validation management program constructed in this study may be applied in every kind of buildings to improve the total quality, and bring the present quality management system to the level of Total Quality Management (TQM).

博士<br>國立臺北科技大學<br>能源與光電材料專班<br>106<br>Two-Dimensional (2D) Layered Materials play a key role in nanotechnology and emerging as new generation materials for electrochemical sensors. Particularly, nanocomposites of graphene-based 2D layered materials are considered as an ideal matrix to construct reliable electrochemical (bio) sensors, due to their unique physico-chemical properties. This thesis is focusing on the fabrication of such 2D layered materials based modified electrodes for analytes that are linked to food safety and clinical diagnostics; chloramphenicol, methyl parathion, hydrogen peroxide, nitrite, dopamine and oxidative stress biomarker. The major materials used in this work are graphene, reduced graphene oxide (RGO), molybdenum disulfide (MoS2), multiwalled carbon nanotube (MWCNT), and reduced graphene nanoribbon (rGONR). A sensitive chloramphenicol sensor was developed using MoS2 nanoflowers coated functionalized MWCNTs modified electrode and it was found to be practical applicable in tracing chloramphenicol in milk, honey and powdered milk. MoS2 and graphene nanocomposite had been prepared via simple hydrothermal route for sensitive determination of organophosphate pesticide, methyl parathion in spiked samples of homogenized apple, kiwi, tomato and cabbage; the method is applicable in food safety. A robust nanobiocomposite based on reduced graphene nanoribbons (rGONRs)/chitosan was developed for the fabrication of a sensitive, selective, reproducible and durable biosensor for H2O2 and nitrite in contact lens cleaning solution and meat samples. An interconnected network of graphene oxide encapsulated Co3O4 polyhedrons modified electrode was developed for the sensitive determination of H2O2 in contact lens and disinfectant cleaning solutions. A hybrid material that consists of MoS2, graphene, and MWCNTs was hydrothermally synthesized for dopamine quantification in biological (human serum, rat brain) and pharmaceutical samples (Dopamine hydrochloride injection). A sensitive 3-nitrotyrosine (oxidative stress biomarker) sensor was developed using copper ferrite nanodots (CuFe2O4) entrapped porous RGO, the method was validated in human urine and serum samples.

In the last ten years developmental disorders and its psychopharmacology have profoundly changed the approach in child and adolescent psychiatry. Research in neuroscience have focused the attention to improve diagnosis of psychiatric disorders in children and its neurobiological pathway underlying that contribute to the continuation of diseases to adulthood. The early stages of life strongly affect the development of the central nervous system and can produce persistent effects on functioning, so the purpose is to stop the negative process before the “damage” may modify neurogenic process irreparably. To date there is no curative pharmacological intervention for psychiatric disorders in pediatrics. Most therapeutic interventions aim to reduce the most impairing symptoms of the targeted psychiatric disorders. Two main clusters of symptoms can be identified in childhood: externalizing symptoms with impulsivity, hyperactivity, and disruptive behavior and internalizing symptoms with mood and affective problems, anxiety, etc. Externalizing disorders include symptoms of ADHD, disruptive behavior, and impulsivity, as well as symptoms of tic disorder. Depressive disorders, anxiety disorders, and obsessive-compulsive disorders (OCD) are summarized as internalizing disorders (Kolch & Plener 2016). In general, pharmacotherapy of psychiatric disorders in children should be embedded in psychosocial treatment, which is recommended by many guidelines (Banaschewski et al., 2006; NICE 2005). In child psychiatry, pharmacotherapy represent a challenge: (i) treatment of a developing body and brain needs special caution; (ii) data concerning effectiveness, safety, tolerability, and long-term outcome are still scarce for many psychopharmacotherapeutic interventions within this age-group; (iii) parents often remain insecure about their treatment decision; and (iv) identifying adverse events can be challenging for caregivers and minors. To add to this, most of psychodrugs are used off-label. However, off-label use does not mean that there is no data on efficacy, effectiveness, or safety of medication in children (Kolch & Plener 2016). Since their introduction into clinical practice, antipsychotic medications have been used in the treatment of children and adolescents with a variety of psychiatric conditions especially for externalizing symptoms (Findling et al., 2005). In recent years, the pediatric use of antipsychotics has substantially increased, due to an increment in prescription of second generation antipsychotics (SGA). These drugs are often preferred to traditional antipsychotic drugs (AP) in reason of a better tolerability and safety profile (Briles et al., 2012; Germanò et al., 2014). The use of atypical AP in pediatric age has increased significantly in the last few years. Between 1993–1998 and 2005–2009, in the United States, visits with a prescription of antipsychotic medications per 100 persons increased from 0.24 to 1.83 for children and from 0.78 to 3.76 for adolescents (Olfson et al., 2012). The proportion of total visits including a prescription of antipsychotics increased during this period from 0.16% to 1.07% for youths (Olfson et al., 2012). Off-label use of AP which have not obtained clear pediatric indications is very common in clinical practice (Bazzano et al., 2009). However, only four drugs, such as aripiprazole, olanzapine, quetiapine, and risperidone have received FDA-approved pediatric indications, in particular for schizophrenia (age 13–17 years) and for bipolar mania (age 10–17 years; olanzapine, 13–17 years). In addition, risperidone is also indicated for irritability and aggression associated with autistic disorder (age 6–17 years), and controlled trial data exist for disruptive behavior disorders and tic disorders (FDA, Psychopharmacologic Drugs Advisory Committee, 2009). Moreover, the duration of treatment with these agents has been increasing (Kalverdijk et al., 2008; Rani et al., 2008). This rapid increase and the recognition that many antipsychotics induce metabolic adverse effects, thus increasing the risk for obesity, diabetes type II, and associated cardiovascular morbidity (Guo et al., 2006; Bobes et al., 2007), have raised concerns about the proper utilization of these agents and stirred controversy among both experts and the general public (Elias, 2006, Harris, 2008). After having been hailed as a safer alternative to first-generation antipsychotics because of their lower tendency to induce neurological effects, the SGA are now recognized to have a high propensity for causing other, equally problematic, adverse effects, thus triggering a reconsideration of their benefit/risk ratio, especially in children (Tyrer and Kendall, 2008; Correll, 2008a,b; Sikich et al., 2008). The clinicians’ perception that second-generation antipsychotics are safer than first-generation antipsychotics (FGA) and their expectation of better effectiveness may be one of the principal causes of the significantly increased use in the pediatric population in recent years (Vitiello et al. 2009; Zuddas et al. 2011). However, there are few safety studies of SGA in pediatric populations (Fraguas et al., 2011; Caccia et al., 2011; Arango 2014 et al., 2014; Correll et al., 2014; Alda et al., 2016). In the past decade, various AP such as sertindole, thioridazine, and droperidol were removed from the market because of cardiac side effects (Buckley and Sanders 2000; Glassman and Bigger 2001; Fraguas et al. 2008). Other AP drugs such as ziprasidone were examined in further specific safety studies (Ziprasidone Observational Study of Cardiac Outcomes [ZODIAC Study]) (Strom et al. 2011), triggering an extensive debate regarding the cardiac safety of specific AP drugs (Fraguas et al. 2008). Various studies found an association between AP use and increase in corrected QT interval (QTc) (Buckley and Sanders 2000; Glassman and Bigger 2001). Many AP drugs can, in various ways, prolong the QTc interval of the electrocardiogram and, in the presence of tachycardia, can provoke an increased risk of potentially lethal arrhythmias known as ‘‘Torsade de pointes’’ (Glassman and Bigger 2001; Zuddas et al. 2011). In children and adolescents, cardiovascular side effects associated with the use of SGA and FGA include orthostatic hypotension, tachycardia, QTc prolongation, and arrhythmias (Cheng-Shannon et al. 2004; Jones et al. 2013). In a study with a pediatric population, AP group, gender, age, smoking status, substance abuse, and diagnosis were unrelated to QTc change after introduction of the AP (de Castro et al. 2008). In a recent meta-analysis of 55 studies with >5000 youth patients, the risk of pathological QTc prolongation seemed low during treatment with the nine AP studied in otherwise healthy young people. Nevertheless, because individual risk factors interact with medication related QTc effects, both medication and patient factors need to be considered when choosing AP treatment ( Jensen et al. 2015). Although the cardiovascular side effects during treatment with AP drugs may be less common in children and adolescents than in adults (Masi and Liboni 2011, Arango et al. 2016), the benefit of the electrocardiogram (ECG) screening is not yet clear in the pediatric population. The purpose of the study is to evaluate cardiac symptoms of SGA in a large sample of treatment-naıve (no previous AP) or quasi-naıve (AP exposure for <30 days) pediatric patients for a period of 24 months.

The aging population of the world has consequences for public health, for example in the increasing prevalence of dementia. Since there are no pharmacological options to treat dementia or the psychiatric symptoms associated with it effectively, many prescribers recourse to antipsychotic drugs. These drugs are used to treat symptoms such as aggression, hallucinations and agitation in older persons with dementia. However, antipsychotics are often used off-label and inappropriately. It is therefore of great importance to understand and describe the use of these drugs in persons with dementia. The aim of this thesis precisely to quantify the use of antipsychotics in persons with dementia using healthcare databases and to explore related issues in depth, through literature reviews and exploratory analyses.

Yes<br>Much of the development of model-based design and dependability analysis in the design of dependable systems, including software intensive systems, can be attributed to the application of advances in formal logic and its application to fault forecasting and verification of systems. In parallel, work on bio-inspired technologies has shown potential for the evolutionary design of engineering systems via automated exploration of potentially large design spaces. We have not yet seen the emergence of a design paradigm that effectively combines these two techniques, schematically founded on the two pillars of formal logic and biology, from the early stages of, and throughout, the design lifecycle. Such a design paradigm would apply these techniques synergistically and systematically to enable optimal refinement of new designs which can be driven effectively by dependability requirements. The paper sketches such a model-centric paradigm for the design of dependable systems, presented in the scope of the HiP-HOPS tool and technique, that brings these technologies together to realise their combined potential benefits. The paper begins by identifying current challenges in model-based safety assessment and then overviews the use of meta-heuristics at various stages of the design lifecycle covering topics that span from allocation of dependability requirements, through dependability analysis, to multi-objective optimisation of system architectures and maintenance schedules.

<div>Decades of scaling in semiconductor technology has resulted in a drastic reduction in the cost and size of unit computing. This has enabled computing capabilities in small form factor wearable and implantable devices. These devices communicate with each other to form a network around the body, commonly known as the Wireless Body Area Network (WBAN). Radio wave transmission over air is the commonly used method of communication among these devices. However, the human body can be used as the communication medium by utilizing its electrical conductivity property. This has given rise to Human Body Communication (HBC), which provides higher energy efficiency and enhanced security compared to over the air radio wave communication enabling applications like remote health monitoring, secure authentication. In this thesis we characterize the human body channel characteristics at low frequencies, utilize the insight obtained from the channel characterization to build high energy-efficiency, interference-robust circuits and demonstrate the security and selectivity aspect of HBC through a Common Off the Shelf (COTS) component-based system. First, we characterize the response of the human body channel in the 10KHz1MHz frequency range with wearable transmitter/ receiver to study the feasibility of using it as a broadband communication channel. Voltage mode measurements with capacitive termination show almost at-band response in this frequency range, establishing the body as a broadband channel. The body channel response is also measured across different interaction scenario between two wearable devices and a wearable and a computer. A bio-physical model of the HBC channel is developed to explain the measurement results and the wide discrepancies found in previous studies.We analyze the safety aspect of different type of HBC by carrying out theoretical circuit and FEM based simulations. A study is carried out among multiple subjects to assess the effect of HBC on the vital parameters of a subject. A statistical analysis of the results shows no signicant change in the vital parameters before and during HBC transmission, validating the theoretical simulations showing >!000x safety margin compared to the established ICNIRP guidelines. Next, an HBC transceiver is built utilizing the wire-like, broadband human body channel to enable high energy efficiency. The transceiver also provides robustness to ambient interference picked up by the human body through integration followed by periodic sampling. The transceiver achieves 6.3pJ/bit energy effciency while operating at a maximum data rate of 30Mbps, while providing -30dB interference tolerant operation. Finally, a COTS based HBC prototype is developed, which utilizes low frequency operation to enable selective and physically secure communication strictly during touch for Human Computer Interaction (HCI) between two wearable devices for the rst time. A thorough study of the effect of different parameters such as environment, posture, subject variation, on the channel loss has also been characterized to build a robust HBC system working across different use cases. Applications such as secure authentication (e.g. opening a door, pairing a smart device) and information exchange (e.g. payment, image, medical data, personal profile transfer) through touch is demonstrated to show the impact of HBC in enabling new human-machine interaction modalities.</div>