Food safety in wine: optimization of analytical controls and evaluation of production technologies

This PhD thesis has been proposed to validate and then apply innovative analytical methodologies for the determination of compounds with harmful impact on human health, such as biogenic amines and ochratoxin A in wines. Therefore, the influence of production technology (pH, amino acids precursor and use of different malolactic starters) on biogenic amines content in wines was evaluated. An HPLC method for simultaneous determination of amino acids and amines with precolumnderivatization with 9-Fluorenyl-methoxycarbonyl chloride (FMOC-Cl) and UV detection was developed. Initially, the influence of pH, time of derivatization, gradient profile were studied. In order to improve the separation of amino acids and amines and reduce the time of analysis, it was decided to study the influence of different flows and the use of different columns in the chromatographic method. Firstly, a C18 Luna column was used and later two monolithic columns Chromolith in series. It appeared to be suitable for an easy, precise and accurate determination of a relatively large number of amino acids and amines in wines. This method was then applied on different wines produced in the Emilia Romagna region. The investigation permitted to discriminate between red and white wines. Amino acids content is related to the winemaking process. Biogenic amines content in these wines does not represent a possible toxicological problem for human health. The results of the study of influence of technologies and wine composition demonstrated that pH of wines and amino acids content are the most important factors. Particularly wines with pH > 3,5 show higher concentration of biogenic amines than wines with lower pH. The enrichment of wines by nutrients also influences the content of some biogenic amines that are higher in wines added with amino acids precursors. In this study, amino acids and biogenic amines are not statistically affected by strain of lactic acid bacteria inoculated as a starter for malolactic fermentation. An evaluation of different clean-up (SPE-MycoSep; IACs and LLE) and determination methods (HPLC and ELISA) of ochratoxin A was carried out. The results obtained proved that the SPE clean-up are reliable at the same level while the LLE procedures shows lowest recovery. The ELISA method gave a lower determination and a low reproducibility than HPLC method.

Indagine sulla nozione di fatto urbano ne L'architettura della città di Aldo Rossi

Questa tesi di dottorato di ricerca ha come oggetto la nozione di fatto urbano elaborata e presentata da Aldo Rossi nel libro L’architettura della città edito nel 1966. Ne L’architettura della città sono molteplici le definizioni e le forme con cui è enunciata la nozione di fatto urbano. Nel corso della tesi si è indagato come la costruzione nel tempo di questo concetto è stata preceduta da diversi studi giovanili intrapresi dal 1953, poi riorganizzati e sintetizzati a partire dal 1963 in un quaderno manoscritto dal titolo “Manuale di urbanistica”, in diversi appunti e in due quaderni manoscritti. Il lavoro di ricerca ha ricostruito la formulazione della nozione di fatto urbano attraverso gli scritti di Rossi. In questa direzione la rilevazione della partecipazione di Rossi a dibattiti, seminari, riviste, corsi universitari o ricerche accademiche è apparsa di fondamentale importanza, per comprendere la complessità di un lavoro non riconducibile a dei concetti disciplinari, ma alla formazione di una teoria trasmissibile. Il tentativo di comprendere e spiegare la nozione di fatto urbano ha condotto ad esaminare l’accezione con cui Rossi compone L’architettura della città, che egli stesso assimila ad un trattato. L’analisi ha identificato come la composizione del libro non è direttamente riferibile ad un uso classico della stesura editoriale del trattato, la quale ha tra i riferimenti più noti nel passato la promozione di una pratica corretta come nel caso vitruviano o un’impalcatura instauratrice di una nuova categoria come nel caso dell’Alberti. La mancanza di un sistema globale e prescrittivo a differenza dei due libri fondativi e il rimando non immediato alla stesura di un trattato classico è evidente ne L’architettura della città. Tuttavia la possibilità di condurre la ricerca su una serie di documenti inediti ha permesso di rilevare come negli scritti a partire dal 1953, sia maturata una trattazione delle questioni centrali alla nozione di fatto urbano ricca di intuizioni, che aspirano ad un’autonomia, sintetizzate, seppure in modo non sistematico, nella stesura del celebre libro. Si è così cercato di mettere in luce la precisazione nel tempo della nozione di fatto urbano e della sua elaborazione nei molteplici scritti antecedenti la pubblicazione de L’architettura della città, precisando come Rossi, pur costruendo su basi teoriche la nozione di fatto urbano, ne indichi una visione progressiva, ossia un uso operativo sulla città. La ricerca si è proposta come obiettivo di comprendere le radici culturali della nozione di fatto urbano sia tramite un’esplorazione degli interessi di Rossi nel suo percorso formativo sia rispetto alla definizione della struttura materiale del fatto urbano che Rossi individua nelle permanenze e che alimenta nella sua definizione con differenti apporti derivanti da altre discipline. Compito di questa ricerca è stato rileggere criticamente il percorso formativo compiuto da Rossi, a partire dal 1953, sottolinearne gli ambiti innovativi e precisarne i limiti descrittivi che non vedranno mai la determinazione di una nozione esatta, ma piuttosto la strutturazione di una sintesi complessa e ricca di riferimenti ad altri studi. In sintesi la tesi si compone di tre parti: 1. la prima parte, dal titolo “La teoria dei fatti urbani ne L’architettura della città”, analizza il concetto di fatto urbano inserendolo all’interno del più generale contesto teorico contenuto nel libro L’architettura della città. Questo avviene tramite la scomposizione del libro, la concatenazione delle sue argomentazioni e la molteplicità delle fonti esplicitamente citate da Rossi. In questo ambito si precisa la struttura del libro attraverso la rilettura dei riferimenti serviti a Rossi per comporre il suo progetto teorico. Inoltre si ripercorre la sua vita attraverso le varie edizioni, le ristampe, le introduzioni e le illustrazioni. Infine si analizza il ruolo del concetto di fatto urbano nel libro rilevando come sia posto in un rapporto paritetico con il titolo del libro, conseguendone un’accezione di «fatto da osservare» assimilabile all’uso proposto dalla geografia urbana francese dei primi del Novecento. 2. la seconda parte, dal titolo “La formazione della nozione di fatto urbano 1953-66”, è dedicata alla presentazione dell’elaborazione teorica negli scritti di Rossi prima de L’architettura della città, ossia dal 1953 al 1966. Questa parte cerca di descrivere le radici culturali di Rossi, le sue collaborazioni e i suoi interessi ripercorrendo la progressiva definizione della concezione di città nel tempo. Si è analizzato il percorso maturato da Rossi e i documenti scritti fin dagli anni in cui era studente alla Facoltà di Architettura Politecnico di Milano. Emerge un quadro complesso in cui i primi saggi, gli articoli e gli appunti testimoniano una ricerca intellettuale tesa alla costruzione di un sapere sullo sfondo del realismo degli anni Cinquanta. Rossi matura infatti un impegno culturale che lo porta dopo la laurea ad affrontare discorsi più generali sulla città. In particolare la sua importante collaborazione con la rivista Casabella-continuità, con il suo direttore Ernesto Nathan Rogers e tutto il gruppo redazionale segnano il periodo successivo in cui compare l’interesse per la letteratura urbanistica, l’arte, la sociologia, la geografia, l’economia e la filosofia. Seguono poi dal 1963 gli anni di lavoro insieme al gruppo diretto da Carlo Aymonino all’Istituto Universitario di Architettura di Venezia, e in particolare le ricerche sulla tipologia edilizia e la morfologia urbana, che portano Rossi a compiere una sintesi analitica per la fondazione di una teoria della città. Dall’indagine si rileva infatti come gli scritti antecedenti L’architettura della città sviluppano lo studio dei fatti urbani fino ad andare a costituire il nucleo teorico di diversi capitoli del libro. Si racconta così la genesi del libro, la cui scrittura si è svolta nell’arco di due anni, e le aspirazioni che hanno portato quello che era stato concepito come un “manuale d’urbanistica” a divenire quello che Rossi definirà “l’abbozzo di un trattato” per la formulazione di una scienza urbana. 3. la terza parte, dal titolo “La struttura materiale dei fatti urbani: la teoria della permanenza”, indaga monograficamente lo studio della città come un fatto materiale, un manufatto, la cui costruzione è avvenuta nel tempo e del tempo mantiene le tracce. Sul tema della teoria della permanenza è stato importante impostare un confronto con il dibattito vivo negli anni della ricostruzione dopo la guerra intorno ai temi delle preesistenze ambientali nella ricostruzione negli ambienti storici. Sono emersi fin da subito importanti la relazione con Ernesto Nathan Rogers, le discussioni sulle pagine di Casabella-Continuità, la partecipazione ad alcuni dibatti e ricerche. Si è inoltre Rilevato l’uso di diversi termini mutuati dalle tesi filosofiche di alcune personalità come Antonio Banfi e Enzo Paci, poi elaborati dal nucleo redazionale di Casabella-Continuità, di cui faceva parte anche Rossi. Sono così emersi alcuni spostamenti di senso e la formulazione di un vocabolario di termini all’interno della complessa vicenda della cultura architettonica degli anni Cinquanta e Sessanta. 1. Si è poi affrontato questo tema analizzando le forme con cui Rossi presenta la definizione della teoria della permanenza e i contributi desunti da alcuni autori per la costruzione scientifica di una teoria dell’architettura, il cui fine è quello di essere trasmissibile e di offrire strumenti di indagine concreti. Questa ricerca ha permesso di ipotizzare come il lavoro dei geografi francesi della prima metà del XX secolo, e in particolare il contributo più rilevante di Marcel Poëte e di Pierre Lavedan, costituiscono le fonti principali e il campo d’indagine maggiormente esplorato da Rossi per definire la teoria della permanenza e i monumenti. Le permanenze non sono dunque presentate ne L’architettura della città come il “tutto”, ma emergono da un metodo che sceglie di isolare i fatti urbani permanenti, consentendo così di compiere un’ipotesi su “ciò che resta” dopo le trasformazioni continue che operano nella città. Le fonti su cui ho lavorato sono state quelle annunciate da Rossi ne L’architettura della città, e più precisamente i testi nelle edizioni da lui consultate. Anche questo lavoro ha permesso un confronto dei testi che ha fatto emergere ne L’architettura della città l’uso di termini mutuati da linguaggi appartenenti ad altre discipline e quale sia l’uso di concetti estrapolati nella loro interezza. Presupposti metodologici Della formulazione della nozione di fatto urbano si sono indagate l’originalità dell’espressione, le connessioni presunte o contenute negli studi di Rossi sulla città attraverso la raccolta di fonti dirette e indirette che sono andate a formare un notevole corpus di scritti. Le fonti dirette più rilevanti sono state trovare nelle collezioni speciali del Getty Research Institute di Los Angeles in cui sono conservati gli Aldo Rossi Papers, questo archivio comprende materiali inediti dal 1954 al 1988. La natura dei materiali si presenta sotto forma di manoscritti, dattiloscritti, quaderni, documenti ciclostilati, appunti sparsi e una notevole quantità di corrispondenza. Negli Aldo Rossi Papers si trovano anche 32 dei 47 Quaderni Azzurri, le bozze de L’architettura della città e dell’ Autobiografia Scientifica. Per quanto riguarda in particolare L’architettura della città negli Aldo Rossi Papers sono conservati: un quaderno con il titolo “Manuale d’urbanistica, giugno 1963”, chiara prima bozza del libro, degli “Appunti per libro urbanistica estate/inverno 1963”, un quaderno con la copertina rossa datato 20 settembre 1964-8 agosto 1965 e un quaderno con la copertina blu datato 30 agosto 1965-15 dicembre 1965. La possibilità di accedere a questo archivio ha permesso di incrementare la bibliografia relativa agli studi giovanili consentendo di rileggere il percorso culturale in cui Rossi si è formato. E’ così apparsa fondamentale la rivalutazione di alcune questioni relative al realismo socialista che hanno portato a formare un più preciso quadro dei primi scritti di Rossi sullo sfondo di un complesso scenario intellettuale. A questi testi si è affiancata la raccolta delle ricerche universitarie, degli articoli pubblicati su riviste specializzate e degli interventi a dibattiti e seminari. A proposito de L’architettura della città si è raccolta un’ampia letteratura critica riferita sia al testo in specifico che ad una sua collocazione nella storia dell’architettura, mettendo in discussione alcune osservazioni che pongono L’architettura della città come un libro risolutivo e definitivo. Per quanto riguarda il capitolo sulla teoria della permanenza l’analisi è stata svolta a partire dai testi che Rossi stesso indicava ne L’architettura della città rivelando i diversi apporti della letteratura urbanistica francese, e permettendo alla ricerca di precisare le relazioni con alcuni scritti centrali e al contempo colti da Rossi come opportunità per intraprendere l’elaborazione dell’idea di tipo. Per quest’ultima parte si può precisare come Rossi formuli la sua idea di tipo in un contesto culturale dove l’interesse per questo tema era fondamentale. Dunque le fonti che hanno assunto maggior rilievo in quest’ultima fase emergono da un ricco panorama in cui Rossi compie diverse ricerche sia con il gruppo redazionale di Casabella-continuità, sia all’interno della scuola veneziana negli anni Sessanta, ma anche negli studi per l’ILSES e per l’Istituto Nazionale d’Urbanistica. RESEARCH ON THE NOTION OF URBAN ARTIFACT IN THE ARCHITECTURE OF THE CITY BY ALDO ROSSI. Doctoral candidate: Letizia Biondi Tutor: Valter Balducci The present doctoral dissertation deals with the notion of urban artifact that was formulated and presented by Aldo Rossi in his book The Architecture of the City, published in 1966. In The Architecture of the City, the notion of urban artifact is enunciated through a wide range of definitions and forms. In this thesis, a research was done on how the construction of this concept over time was preceded by various studies started in 1953 during the author’s youth, then re-organized and synthesized since 1963 in a manuscript titled “Manual of urban planning” and in two more manuscripts later on. The work of research re-constructed the formulation of the notion of urban artifact through Rossi’s writings. In this sense, the examination of Rossi’s participation in debates, seminars, reviews, university courses or academic researches was of fundamental importance to understand the complexity of a work which is not to be attributed to disciplinary concepts, but to the formulation of a communicable theory. The effort to understand and to explain the notion of urban artifact led to an examination of the meaning used by Rossi to compose The Architecture of the City, which he defines as similar to a treatise. Through this analysis, it emerged that the composition of the book is not directly ascribable to the classical use of editorial writing of a treatise, whose most famous references in the past are the promotion of a correct practice as in the case of Vitruvio’s treatise, or the use of a structure that introduces a new category as in the Alberti case. Contrary to the two founding books, the lack of a global and prescriptive system and the not immediate reference to the writing of a classical treatise are evident in The Architecture of the City. However, the possibility of researching on some unpublished documents allowed to discover that in the writings starting from 1953 the analysis of the questions that are at the core of the notion of urban artifact is rich of intuitions, that aim to autonomy and that would be synthesized, even though not in a systematic way, in his famous book. The attempt was that of highlighting the specification over time of the notion of urban artifact and its elaboration in the various writings preceding the publication of The Architecture of the City. It was also specified that, despite building on theoretical grounds, Rossi indicates a progressive version of the notion of urban artifact, that is a performing use in the city. The present research aims to understand the cultural roots of the notion of urban artifact in two main directions: analyzing, firstly, Rossi’s interests along his formation path and, secondly, the definition of material structure of an urban artifact identified by Rossi in the permanences and enriched by various contributions from other disciplines. The purpose of the present research is to revise the formation path made by Rossi in a critical way, starting by 1953, underlining its innovative aspects and identifying its describing limits, which will never lead to the formulation of an exact notion, but rather to the elaboration of a complex synthesis, enriched by references to other studies. In brief, the thesis is composed of three parts: 1. The first part, titled “The Theory of urban artifacts in The Architecture of the City”, analyzes the concept of urban artifact in the more general theoretical context of the book The Architecture of the City. Such analysis is done by “disassembling” the book, and by linking together the argumentations and the multiplicity of the sources which are explicitly quoted by Rossi. In this context, the book’s structure is defined more precisely through the revision of the references used by Rossi to compose his theoretical project. Moreover, the author’s life is traced back through the various editions, re-printings, introductions and illustrations. Finally, it is specified which role the concept of urban artifact has in the book, pointing out that it is placed in an equal relation with the book’s title; by so doing, the concept of urban artifact gets the new meaning of “fact to be observed”, similar to the use that was suggested by the French urban geography at the beginning of the 20th century. 2. The second part, titled “The formation of the notion of urban artifact 1953-66”, introduces the theoretical elaboration in Rossi’s writings before The Architecture of the City, that is from 1953 to 1966. This part tries to describe Rossi’s cultural roots, his collaborations and his interests, tracing back the progressive definition of his conception of city over time. The analysis focuses on the path followed by Rossi and on the documents that he wrote since the years as a student at the Department of Architecture at the Politecnico in Milan. This leads to a complex scenario of first essays, articles and notes that bear witness to the intellectual research aiming to the construction of a knowledge on the background of the Realism of the 1950s. Rossi develops, in fact, a cultural engagement that leads him after his studies to deal with more general issues about the city. In particular, his important collaboration with the architecture magazine “Casabella-continuità”, with the director Ernesto Nathan Rogers and with the whole redaction staff mark the following period when he starts getting interested in city planning literature, art, sociology, geography, economics and philosophy. Since 1963, Rossi has worked with the group directed by Carlo Aymonino at the “Istituto Universitario di Architettura” (University Institute of Architecture) in Venice, especially researching on building typologies and urban morphology. During these years, Rossi elaborates an analytical synthesis for the formulation of a theory about the city. From the present research, it is evident that the writings preceding The Architecture of the City develop the studies on urban artifacts, which will become theoretical core of different chapters of the book. In conclusion, the genesis of the book is described; written in two years, what was conceived to be an “urban planning manual” became a “treatise draft” for the formulation of an urban science, as Rossi defines it. 3. The third part is titled “The material structure of urban artifacts: the theory of permanence”. This research is made on the study of the city as a material fact, a manufacture, whose construction was made over time, bearing the traces of time. As far as the topic of permanence is concerned, it was also important to draw a comparison with the debate about the issues of environmental pre-existence of re-construction in historical areas, which was very lively during the years of the Reconstruction. Right from the beginning, of fundamental importance were the relationship with Ernesto Nathan Rogers, the discussions on the pages of Casabella-Continuità and the participation to some debates and researches. It is to note that various terms were taken by the philosophical thesis by some personalities such as Antonio Banfi and Enzo Paci, and then re-elaborated by the redaction staff at Casabella-Continuità, which Rossi took part in as well. Through this analysis, it emerged that there were some shifts in meaning and the formulation of a vocabulary of terms within the complex area of the architectonic culture in the 1950s and 1960s. Then, I examined the shapes in which Rossi introduces the definition of the theory of permanence and the references by some authors for the scientific construction of an architecture theory whose aim is being communicable and offering concrete research tools. Such analysis allowed making a hypothesis about the significance for Rossi of the French geographers of the first half of the 20th century: in particular, the work by Marcel Poëte and by Pierre Lavedan is the main source and the research area which Rossi mostly explored to define the theory of permanence and monuments. Therefore, in The Architecture of the City, permanencies are not presented as the “whole”, but they emerge from a method which isolates permanent urban artifacts, in this way allowing making a hypothesis on “what remains” after the continuous transformations made in the city. The sources examined were quoted by Rossi in The Architecture of the City; in particular I analyzed them in the same edition which Rossi referred to. Through such an analysis, it was possible to make a comparison of the texts with one another, which let emerge the use of terms taken by languages belonging to other disciplines in The Architecture of the City and which the use of wholly extrapolated concepts is. Methodological premises As far as the formulation of the notion of urban artifact is concerned, the analysis focuses on the originality of the expression, the connections that are assumed or contained in Rossi’s writings about the city, by collecting direct and indirect sources which formed a significant corpus of writings. The most relevant direct sources were found in the special collections of the Getty Research Institute in Los Angeles, where the “Aldo Rossi Papers” are conserved. This archive contains unpublished material from 1954 to 1988, such as manuscripts, typescripts, notebooks, cyclostyled documents, scraps and notes, and several letters. In the Aldo Rossi Papers there are also 32 out of the 47 Light Blue Notebooks (Quaderni Azzurri), the rough drafts of The Architecture of the City and of the “A Scientific Autobiography”. As regards The Architecture of the City in particular, the Aldo Rossi Papers preserve: a notebook by the title of “Urban planning manual, June, 1963”, which is an explicit first draft of the book; “Notes for urban planning book summer/winter 1963”; a notebook with a red cover dated September 20th, 1964 – August 8th, 1965; and a notebook with a blue cover dated August 30th, 1965 – December 15th, 1965. The possibility of accessing this archive allowed to increase the bibliography related to the youth studies, enabling a revision of the cultural path followed by Rossi’s education. To that end, it was fundamental to re-evaluate some issues linked to the socialist realism which led to a more precise picture of the first writings by Rossi against the background of the intellectual scenario where he formed. In addition to these texts, the collection of university researches, the articles published on specialized reviews and the speeches at debates and seminars were also examined. About The Architecture of the City, a wide-ranging critical literature was collected, related both to the text specifics and to its collocation in the story of architecture, questioning some observations which define The Architecture of the City as a conclusive and definite book. As far as the chapter on the permanence theory is concerned, the analysis started by the texts that Rossi indicated in The Architecture of the City, revealing the different contributions from the French literature on urban planning. This allowed to the present research a more specific definition of the connections to some central writings which, at the same time, were seen by Rossi as an opportunity to start up the elaboration of the idea of type. For this last part, it can be specified that Rossi formulates his idea of type in a cultural context where the interest in this topic was fundamental. Therefore, the sources which played a central role in this final phase emerge from an extensive panorama in which Rossi researched not only with the redaction staff at Casablanca-continuità and within the School of Venice in the 1960s, but also in his studies for the ILSES (Institute of the Region Lombardia for Economics and Social Studies) and for the National Institute of Urban Planning.

Innovative analytical methods for Central Nervous System Drug analysis in biological fluids

During recent years a consistent number of central nervous system (CNS) drugs have been approved and introduced on the market for the treatment of many psychiatric and neurological disorders, including psychosis, depression, Parkinson disease and epilepsy. Despite the great advancements obtained in the treatment of CNS diseases/disorders, partial response to therapy or treatment failure are frequent, at least in part due to poor compliance, but also genetic variability in the metabolism of psychotropic agents or polypharmacy, which may lead to sub-therapeutic or toxic plasma levels of the drugs, and finally inefficacy of the treatment or adverse/toxic effects. With the aim of improving the treatment, reducing toxic/side effects and patient hospitalisation, Therapeutic Drug Monitoring (TDM) is certainly useful, allowing for a personalisation of the therapy. Reliable analytical methods are required to determine the plasma levels of psychotropic drugs, which are often present at low concentrations (tens or hundreds of nanograms per millilitre). The present PhD Thesis has focused on the development of analytical methods for the determination of CNS drugs in biological fluids, including antidepressants (sertraline and duloxetine), antipsychotics (aripiprazole), antiepileptics (vigabatrin and topiramate) and antiparkinsons (pramipexole). Innovative methods based on liquid chromatography or capillary electrophoresis coupled to diode-array or laser-induced fluorescence detectors have been developed, together with the suitable sample pre-treatment for interference removal and fluorescent labelling in case of LIF detection. All methods have been validated according to official guidelines and applied to the analysis of real samples obtained from patients, resulting suitable for the TDM of psychotropic drugs.

Numerical and analytical vibro-acoustic modelling of porous materials: the Cell Method for poroelasticity and the case of inclusions

Porous materials are widely used in many fields of industrial applications, to achieve the requirements of noise reduction, that nowadays derive from strict regulations. The modeling of porous materials is still a problematic issue. Numerical simulations are often problematic in case of real complex geometries, especially in terms of computational times and convergence. At the same time, analytical models, even if partly limited by restrictive simplificative hypotheses, represent a powerful instrument to capture quickly the physics of the problem and general trends. In this context, a recently developed numerical method, called the Cell Method, is described, is presented in the case of the Biot's theory and applied for representative cases. The peculiarity of the Cell Method is that it allows for a direct algebraic and geometrical discretization of the field equations, without any reduction to a weak integral form. Then, the second part of the thesis presents the case of interaction between two poroelastic materials under the context of double porosity. The idea of using periodically repeated inclusions of a second porous material into a layer composed by an original material is described. In particular, the problem is addressed considering the efficiency of the analytical method. A analytical procedure for the simulation of heterogeneous layers based is described and validated considering both conditions of absorption and transmission; a comparison with the available numerical methods is performed. ---------------- I materiali porosi sono ampiamente utilizzati per diverse applicazioni industriali, al fine di raggiungere gli obiettivi di riduzione del rumore, che sono resi impegnativi da norme al giorno d'oggi sempre più stringenti. La modellazione dei materiali porori per applicazioni vibro-acustiche rapprensenta un aspetto di una certa complessità. Le simulazioni numeriche sono spesso problematiche quando siano coinvolte geometrie di pezzi reali, in particolare riguardo i tempi computazionali e la convergenza. Allo stesso tempo, i modelli analitici, anche se parzialmente limitati a causa di ipotesi semplificative che ne restringono l'ambito di utilizzo, rappresentano uno strumento molto utile per comprendere rapidamente la fisica del problema e individuare tendenze generali. In questo contesto, un metodo numerico recentemente sviluppato, il Metodo delle Celle, viene descritto, implementato nel caso della teoria di Biot per la poroelasticità e applicato a casi rappresentativi. La peculiarità del Metodo delle Celle consiste nella discretizzazione diretta algebrica e geometrica delle equazioni di campo, senza alcuna riduzione a forme integrali deboli. Successivamente, nella seconda parte della tesi viene presentato il caso delle interazioni tra due materiali poroelastici a contatto, nel contesto dei materiali a doppia porosità. Viene descritta l'idea di utilizzare inclusioni periodicamente ripetute di un secondo materiale poroso all'interno di un layer a sua volta poroso. In particolare, il problema è studiando il metodo analitico e la sua efficienza. Una procedura analitica per il calcolo di strati eterogenei di materiale viene descritta e validata considerando sia condizioni di assorbimento, sia di trasmissione; viene effettuata una comparazione con i metodi numerici a disposizione.

Development of original analytical methods for the therapeutic drug monitoring of CNS druges: Antipsychotics, Antidepressants and Anxiolytics-hypnotics

Great strides have been made in the last few years in the pharmacological treatment of neuropsychiatric disorders, with the introduction into the therapy of several new and more efficient agents, which have improved the quality of life of many patients. Despite these advances, a large percentage of patients is still considered “non-responder” to the therapy, not drawing any benefits from it. Moreover, these patients have a peculiar therapeutic profile, due to the very frequent application of polypharmacy, attempting to obtain satisfactory remission of the multiple aspects of psychiatric syndromes. Therapy is heavily individualised and switching from one therapeutic agent to another is quite frequent. One of the main problems of this situation is the possibility of unwanted or unexpected pharmacological interactions, which can occur both during polypharmacy and during switching. Simultaneous administration of psychiatric drugs can easily lead to interactions if one of the administered compounds influences the metabolism of the others. Impaired CYP450 function due to inhibition of the enzyme is frequent. Other metabolic pathways, such as glucuronidation, can also be influenced. The Therapeutic Drug Monitoring (TDM) of psychotropic drugs is an important tool for treatment personalisation and optimisation. It deals with the determination of parent drugs and metabolites plasma levels, in order to monitor them over time and to compare these findings with clinical data. This allows establishing chemical-clinical correlations (such as those between administered dose and therapeutic and side effects), which are essential to obtain the maximum therapeutic efficacy, while minimising side and toxic effects. It is evident the importance of developing sensitive and selective analytical methods for the determination of the administered drugs and their main metabolites, in order to obtain reliable data that can correctly support clinical decisions. During the three years of Ph.D. program, some analytical methods based on HPLC have been developed, validated and successfully applied to the TDM of psychiatric patients undergoing treatment with drugs belonging to following classes: antipsychotics, antidepressants and anxiolytic-hypnotics. The biological matrices which have been processed were: blood, plasma, serum, saliva, urine, hair and rat brain. Among antipsychotics, both atypical and classical agents have been considered, such as haloperidol, chlorpromazine, clotiapine, loxapine, risperidone (and 9-hydroxyrisperidone), clozapine (as well as N-desmethylclozapine and clozapine N-oxide) and quetiapine. While the need for an accurate TDM of schizophrenic patients is being increasingly recognized by psychiatrists, only in the last few years the same attention is being paid to the TDM of depressed patients. This is leading to the acknowledgment that depression pharmacotherapy can greatly benefit from the accurate application of TDM. For this reason, the research activity has also been focused on first and second-generation antidepressant agents, like triciclic antidepressants, trazodone and m-chlorophenylpiperazine (m-cpp), paroxetine and its three main metabolites, venlafaxine and its active metabolite, and the most recent antidepressant introduced into the market, duloxetine. Among anxiolytics-hypnotics, benzodiazepines are very often involved in the pharmacotherapy of depression for the relief of anxious components; for this reason, it is useful to monitor these drugs, especially in cases of polypharmacy. The results obtained during these three years of Ph.D. program are reliable and the developed HPLC methods are suitable for the qualitative and quantitative determination of CNS drugs in biological fluids for TDM purposes.

Analytical challenges in the fields of Nanobioscience and Nanobiotecnology: integrated methods for separation and characterization

Nano(bio)science and nano(bio)technology play a growing and tremendous interest both on academic and industrial aspects. They are undergoing rapid developments on many fronts such as genomics, proteomics, system biology, and medical applications. However, the lack of characterization tools for nano(bio)systems is currently considered as a major limiting factor to the final establishment of nano(bio)technologies. Flow Field-Flow Fractionation (FlFFF) is a separation technique that is definitely emerging in the bioanalytical field, and the number of applications on nano(bio)analytes such as high molar-mass proteins and protein complexes, sub-cellular units, viruses, and functionalized nanoparticles is constantly increasing. This can be ascribed to the intrinsic advantages of FlFFF for the separation of nano(bio)analytes. FlFFF is ideally suited to separate particles over a broad size range (1 nm-1 μm) according to their hydrodynamic radius (rh). The fractionation is carried out in an empty channel by a flow stream of a mobile phase of any composition. For these reasons, fractionation is developed without surface interaction of the analyte with packing or gel media, and there is no stationary phase able to induce mechanical or shear stress on nanosized analytes, which are for these reasons kept in their native state. Characterization of nano(bio)analytes is made possible after fractionation by interfacing the FlFFF system with detection techniques for morphological, optical or mass characterization. For instance, FlFFF coupling with multi-angle light scattering (MALS) detection allows for absolute molecular weight and size determination, and mass spectrometry has made FlFFF enter the field of proteomics. Potentialities of FlFFF couplings with multi-detection systems are discussed in the first section of this dissertation. The second and the third sections are dedicated to new methods that have been developed for the analysis and characterization of different samples of interest in the fields of diagnostics, pharmaceutics, and nanomedicine. The second section focuses on biological samples such as protein complexes and protein aggregates. In particular it focuses on FlFFF methods developed to give new insights into: a) chemical composition and morphological features of blood serum lipoprotein classes, b) time-dependent aggregation pattern of the amyloid protein Aβ1-42, and c) aggregation state of antibody therapeutics in their formulation buffers. The third section is dedicated to the analysis and characterization of structured nanoparticles designed for nanomedicine applications. The discussed results indicate that FlFFF with on-line MALS and fluorescence detection (FD) may become the unparallel methodology for the analysis and characterization of new, structured, fluorescent nanomaterials.

Development of muliplexed analytical methods based on bioluminescent whole-cell biosensors

The subject of this Ph.D. research thesis is the development and application of multiplexed analytical methods based on bioluminescent whole-cell biosensors. One of the main goals of analytical chemistry is multianalyte testing in which two or more analytes are measured simultaneously in a single assay. The advantages of multianalyte testing are work simplification, high throughput, and reduction in the overall cost per test. The availability of multiplexed portable analytical systems is of particular interest for on-field analysis of clinical, environmental or food samples as well as for the drug discovery process. To allow highly sensitive and selective analysis, these devices should combine biospecific molecular recognition with ultrasensitive detection systems. To address the current need for rapid, highly sensitive and inexpensive devices for obtaining more data from each sample,genetically engineered whole-cell biosensors as biospecific recognition element were combined with ultrasensitive bioluminescence detection techniques. Genetically engineered cell-based sensing systems were obtained by introducing into bacterial, yeast or mammalian cells a vector expressing a reporter protein whose expression is controlled by regulatory proteins and promoter sequences. The regulatory protein is able to recognize the presence of the analyte (e.g., compounds with hormone-like activity, heavy metals…) and to consequently activate the expression of the reporter protein that can be readily measured and directly related to the analyte bioavailable concentration in the sample. Bioluminescence represents the ideal detection principle for miniaturized analytical devices and multiplexed assays thanks to high detectability in small sample volumes allowing an accurate signal localization and quantification. In the first chapter of this dissertation is discussed the obtainment of improved bioluminescent proteins emitting at different wavelenghts, in term of increased thermostability, enhanced emission decay kinetic and spectral resolution. The second chapter is mainly focused on the use of these proteins in the development of whole-cell based assay with improved analytical performance. In particular since the main drawback of whole-cell biosensors is the high variability of their analyte specific response mainly caused by variations in cell viability due to aspecific effects of the sample’s matrix, an additional bioluminescent reporter has been introduced to correct the analytical response thus increasing the robustness of the bioassays. The feasibility of using a combination of two or more bioluminescent proteins for obtaining biosensors with internal signal correction or for the simultaneous detection of multiple analytes has been demonstrated by developing a dual reporter yeast based biosensor for androgenic activity measurement and a triple reporter mammalian cell-based biosensor for the simultaneous monitoring of two CYP450 enzymes activation, involved in cholesterol degradation, with the use of two spectrally resolved intracellular luciferases and a secreted luciferase as a control for cells viability. In the third chapter is presented the development of a portable multianalyte detection system. In order to develop a portable system that can be used also outside the laboratory environment even by non skilled personnel, cells have been immobilized into a new biocompatible and transparent polymeric matrix within a modified clear bottom black 384 -well microtiter plate to obtain a bioluminescent cell array. The cell array was placed in contact with a portable charge-coupled device (CCD) light sensor able to localize and quantify the luminescent signal produced by different bioluminescent whole-cell biosensors. This multiplexed biosensing platform containing whole-cell biosensors was successfully used to measure the overall toxicity of a given sample as well as to obtain dose response curves for heavy metals and to detect hormonal activity in clinical samples (PCT/IB2010/050625: “Portable device based on immobilized cells for the detection of analytes.” Michelini E, Roda A, Dolci LS, Mezzanotte L, Cevenini L , 2010). At the end of the dissertation some future development steps are also discussed in order to develop a point of care (POCT) device that combine portability, minimum sample pre-treatment and highly sensitive multiplexed assays in a short assay time. In this POCT perspective, field-flow fractionation (FFF) techniques, in particular gravitational variant (GrFFF) that exploit the earth gravitational field to structure the separation, have been investigated for cells fractionation, characterization and isolation. Thanks to the simplicity of its equipment, amenable to miniaturization, the GrFFF techniques appears to be particularly suited for its implementation in POCT devices and may be used as pre-analytical integrated module to be applied directly to drive target analytes of raw samples to the modules where biospecifc recognition reactions based on ultrasensitive bioluminescence detection occurs, providing an increase in overall analytical output.

Bioactive and toxic compounds in different foodstuffs: analytical techniques and sensory analysis

Foods that provide medical and health benefits or have a role in disease risk prevention are termed functional foods. The functionality of functional foods is derived from bioactive compounds that are extranutritional constituents present in small quantities in food. Bioactive components include a range of chemical compounds with varying structures such as carotenoids, flavonoids, plant sterols, omega-3 fatty acids (n-3), allyl and diallyl sulfides, indoles (benzopyrroles), and phenolic acids. The increasing consumer interest in natural bioactive compounds has brought about a rise in demand for these kinds of compounds and, in parallel, an increasing number of scientific studies have this type of substance as main topic. The principal aim of this PhD research project was the study of different bioactive and toxic compounds in several natural matrices. To achieve this goal, chromatographic, spectroscopic and sensorial analysis were performed. This manuscript reports the main results obtained in the six activities briefly summarized as follows: • SECTION I: the influence of conventional packaging on lipid oxidation of pasta was evaluated in egg spaghetti. • SECTION II: the effect of the storage at different temperatures of virgin olive oil was monitored by peroxide value, fatty acid activity, OSI test and sensory analysis. • SECTION III: the glucosinolate and phenolic content of 37 rocket salad accessions were evaluated, comparing Eruca sativa and Diplotaxis tenuifolia species. Sensory analysis and the influence of the phenolic and glucosinolate composition on sensory attributes of rocket salads has been also studied. • SECTION IV: ten buckwheat honeys were characterised on the basis of their pollen, physicochemical, phenolic and volatile composition. • SECTION V: the polyphenolic fraction, anthocyanins and other polar compounds, the antioxidant capacity and the anty-hyperlipemic action of the aqueous extract of Hibiscus sabdariffa were achieved. • SECTION VI: the optimization of a normal phase high pressure liquid chromatography–fluorescence detection method for the quantitation of flavanols and procyanidins in cocoa powder and chocolate samples was performed.

Functional components and lipid quality assessment in vegetable foods: analytical and technological approaches

Lipids are important components that contribute very significantly to nutritional and technological quality of foods because they are the least stable macro-components in foods, due to high susceptibility to oxidation. When rancidity take place, it makes food unhealthy and unacceptable for consumers. Thus, the presence of antioxidants, naturally present of added to foods, is required to enhance shelf life of foods. Moreover, antioxidant like phenolic compounds play an important role in human health enhancing the functionality of foods. The aim of this PhD project was the study of lipid quality and lipid oxidation in different vegetable foods focusing on analytical and technological aspects in order to figure out the effects of lipid composition and bioactive compounds (phenolic compounds, omega-3 fatty acids and dietary fiber) addition on their shelf life. In addition, bioavailability and antioxidant effects of phenolic compounds in human and animals, respectively, were evaluated after consumption of vegetable foods. The first section of the work was focused on the evaluation of lipid quality impact on technological behaviour of vegetable foods. Because of that, cocoa butter with different melting point were evaluated by chromatographic techniques (GC, TLC) and the sample with the higher melting point showed the presence of fatty acids, triglycerides, 2-monoglycerides and FT-IR profile different from genuine cocoa butter, meaning an adding of foreign fat (lauric-fat) not allowed by the law. Looking at lipid quality of other vegetable foods, an accelerated shelf life test (OXITEST®), was used to evaluate of lipid stability to oxidation in tarallini snacks made up using different lipid matrices (sunflower oil, extravirgin olive oil and a blend of extravirgin olive oil and lard). The results showed a good ability of OXITEST® to discriminate between lipid unsaturation and different cooking times, without any samples fat extraction. In the second section, the role of bioactive compounds on cereal based food shelf life was studied in different bakeries by GC, spectrophotometric methods and capillary electrophoresis. It was examined the relationships between phenolic compounds, added with flour, and lipid oxidation of tarallini and frollini. Both products showed an increase in lipid oxidation during storage and antioxidant effects on lipid oxidation were not as expected. Furthermore, the influence of enrichment in polyunsaturated fatty acids on lipid oxidation of pasta was evaluated. The results proved that LC n-3 PUFA were not significantly implicated in the onset of oxidation in spaghetti stored under daylight and accelerated oxidation in a laboratory heater. The importance of phenolic compounds as antioxidant in humans and rats was also studied, by HPLC/MS in the latter section. For this purpose, apigenin and apigenin glycosides excretion was investigated in six women’s urine in a 24 hours study. After a single dose of steamed artichokes, both aglicone and glucuronide metabolites were recovered in 24 h urine. Moreover, the effect of whole grain durum wheat bread and whole grain Kamut® khorasan bread in rats were evaluated. Both cereals were good sources of antioxidants but Kamut® bread fed animals had a better response to stress than wheat durum fed, especially when a sourdough bread was supplied.

An advanced analytical study on linseed oil paint binder

Drying oils, and in particular linseed oil, were the most common binding media employed in painting between XVI and XIX centuries. Artists usually operated some pre-treatments on the oils to obtain binders with modified properties, such as different handling qualities or colour. Oil processing has a key role on the subsequent ageing of and degradation of linseed oil paints. In this thesis a multi-analytical approach was adopted to investigate the drying, polymerization and oxidative degradation of the linseed oil paints. In particular, thermogravimetry analysis (TGA), yielding information on the macromolecular scale, were compared with gas-chromatography mass-spectrometry (GC-MS) and direct exposure mass spectrometry (DEMS) providing information on the molecular scale. The study was performed on linseed oils and paint reconstructions prepared according to an accurate historical description of the painting techniques of the 19th century. TGA revealed that during ageing the molecular weight of the oils changes and that higher molecular weight fractions formed. TGA proved to be an excellent tool to compare the oils and paint reconstructions. This technique is able to highlight the different physical behaviour of oils that were processed using different methods and of paint layers on the basis of the different processed oil and /or the pigment used. GC/MS and DE-MS were used to characterise the soluble and non-polymeric fraction of the oils and paint reconstructions. GC/MS allowed us to calculate the ratios of palmitic to stearic acid (P/S), and azelaic to palmitic acid (A/P) and to evaluate effects produced by oil pre-treatments and the presence of different pigments. This helps to understand the role of the pre-treatments and of the pigments on the oxidative degradation undergone by siccative oils during ageing. DE-MS enabled the various molecular weight fractions of the samples to be simultaneously studied, and thus helped to highlight the presence of oxidation and hydrolysis reactions, and the formation of carboxylates that occur during ageing and with the changing of the oil pre-treatments and the pigments. The combination of thermal analysis with molecular techniques such as GC-MS, DEMS and FTIR enabled a model to be developed, for unravelling some crucial issues: 1) how oil pre-treatments produce binders with different physical-chemical qualities, and how this can influence the ageing of an oil paint film; 2) which is the role of the interaction between oil and pigments in the ageing and degradation process.

The bursting behavior of gas slugs: laboratory and analytical insights into Strombolian volcanic eruptions.

The aim of this thesis is to study how explosive behavior and geophysical signals in a volcanic conduit are related to the development of overpressure in slug-driven eruptions. A first suite of laboratory experiments of gas slugs ascending in analogue conduits was performed. Slugs ascended into a range of analogue liquids and conduit diameters to allow proper scaling to the natural volcanoes. The geometrical variation of the slug in response to the explored variables was parameterised. Volume of gas slug and rheology of the liquid phase revealed the key parameters in controlling slug overpressure at bursting. Founded on these results, a theoretical model to calculate burst overpressure for slug-driven eruptions was developed. The dimensionless approach adopted allowed to apply the model to predict bursting pressure of slugs at Stromboli. Comparison of predicted values with measured data from Stromboli volcano showed that the model can explain the entire spectrum of observed eruptive styles at Stromboli – from low-energy puffing, through normal Strombolian eruptions, up to paroxysmal explosions – as manifestations of a single underlying physical process. Finally, another suite of laboratory experiments was performed to observe oscillatory pressure and forces variations generated during the expansion and bursting of gas slugs ascending in a conduit. Two end-member boundary conditions were imposed at the base of the pipe, simulating slug ascent in closed base (zero magma flux) and open base (constant flux) conduit. At the top of the pipe, a range of boundary conditions that are relevant at a volcanic vent were imposed, going from open to plugged vent. The results obtained illustrate that a change in boundary conditions in the conduit concur to affect the dynamic of slug expansion and burst: an upward flux at the base of the conduit attenuates the magnitude of the pressure transients, while a rheological stiffening in the top-most region of conduit changes dramatically the magnitude of the observed pressure transients, favoring a sudden, and more energetic pressure release into the overlying atmosphere. Finally, a discussion on the implication of changing boundary on the oscillatory processes generated at the volcanic scale is also given.

InSAR deformation measurements of the earthquake cycle in transcurrent tectonic domains, analytical and analog modeling

I applied the SBAS-DInSAR method to the Mattinata Fault (MF) (Southern Italy) and to the Doruneh Fault System (DFS) (Central Iran). In the first case, I observed limited internal deformation and determined the right lateral kinematic pattern with a compressional pattern in the northern sector of the fault. Using the Okada model I inverted the observed velocities defining a right lateral strike slip solution for the MF. Even if it fits the data within the uncertainties, the modeled slip rate of 13-15 mm yr-1 seems too high with respect to the geological record. Concerning the Western termination of DFS, SAR data confirms the main left lateral transcurrent kinematics of this fault segment, but reveal a compressional component. My analytical model fits successfully the observed data and quantifies the slip in ~4 mm yr-1 and ~2.5 mm yr-1 of pure horizontal and vertical displacement respectively. The horizontal velocity is compatible with geological record. I applied classic SAR interferometry to the October–December 2008 Balochistan (Central Pakistan) seismic swarm; I discerned the different contributions of the three Mw > 5.7 earthquakes determining fault positions, lengths, widths, depths and slip distributions, constraining the other source parameters using different Global CMT solutions. A well constrained solution has been obtained for the 09/12/2008 aftershock, whereas I tested two possible fault solutions for the 28-29/10/08 mainshocks. It is not possible to favor one of the solutions without independent constraints derived from geological data. Finally I approached the study of the earthquake-cycle in transcurrent tectonic domains using analog modeling, with alimentary gelatins like crust analog material. I successfully joined the study of finite deformation with the earthquake cycle study and sudden dislocation. A lot of seismic cycles were reproduced in which a characteristic earthquake is recognizable in terms of displacement, coseismic velocity and recurrence time.

Development of instrumental and sensory analytical methods of food obtained by traditional and emerging technologies

The consumer demand for natural, minimally processed, fresh like and functional food has lead to an increasing interest in emerging technologies. The aim of this PhD project was to study three innovative food processing technologies currently used in the food sector. Ultrasound-assisted freezing, vacuum impregnation and pulsed electric field have been investigated through laboratory scale systems and semi-industrial pilot plants. Furthermore, analytical and sensory techniques have been developed to evaluate the quality of food and vegetable matrix obtained by traditional and emerging processes. Ultrasound was found to be a valuable technique to improve the freezing process of potatoes, anticipating the beginning of the nucleation process, mainly when applied during the supercooling phase. A study of the effects of pulsed electric fields on phenol and enzymatic profile of melon juice has been realized and the statistical treatment of data was carried out through a response surface method. Next, flavour enrichment of apple sticks has been realized applying different techniques, as atmospheric, vacuum, ultrasound technologies and their combinations. The second section of the thesis deals with the development of analytical methods for the discrimination and quantification of phenol compounds in vegetable matrix, as chestnut bark extracts and olive mill waste water. The management of waste disposal in mill sector has been approached with the aim of reducing the amount of waste, and at the same time recovering valuable by-products, to be used in different industrial sectors. Finally, the sensory analysis of boiled potatoes has been carried out through the development of a quantitative descriptive procedure for the study of Italian and Mexican potato varieties. An update on flavour development in fresh and cooked potatoes has been realized and a sensory glossary, including general and specific definitions related to organic products, used in the European project Ecropolis, has been drafted.

Perfluoroalkylated substances in food matrices: development of mass spectrometry based analytical methods and preliminary monitoring

Perfluoroalkylated substances are a group of chemicals that have been largely employed during the last 60 years in several applications, widely spreading and accumulating in the environment due to their extreme resistance to degradation. As a consequence, they have been found also in various types of food as well as in drinking water, proving that they can easily reach humans through the diet. The available information concerning their adverse effects on health has recently increased the interest towards these contaminants and highlighted the importance of investigating all the potential sources of human exposure, among which diet was proved to be the most relevant. This need has been underlined by the European Union through Recommendation 2010/161/EU: in this document, Member States were called to monitor their presence of in food, producing accurate estimations of human exposure. The purpose of the research presented in this thesis, which is the result of a partnership between an Italian and a French laboratory, was to develop reliable tools for the analysis of these pollutants in food, to be used for generating data on potentially contaminated matrices. An efficient method based on liquid chromatography-mass spectrometry for the detection of 16 different perfluorinated compounds in milk has been validated in accordance with current European regulation guidelines (2002/657/EC) and is currently under evaluation for ISO 17025 accreditation. The proposed technique was applied to cow, powder and human breast milk samples from Italy and France to produce a preliminary monitoring on the presence of these contaminants. In accordance with the above mentioned European Recommendation, this project led also to the development of a promising technique for the quantification of some precursors of these substances in fish. This method showed extremely satisfying performances in terms of linearity and limits of detection, and will be useful for future surveys.