Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model

The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

Non-Markovian stochastic processes and their applications: from anomalous diffusion to time series analysis

This work provides a forward step in the study and comprehension of the relationships between stochastic processes and a certain class of integral-partial differential equation, which can be used in order to model anomalous diffusion and transport in statistical physics. In the first part, we brought the reader through the fundamental notions of probability and stochastic processes, stochastic integration and stochastic differential equations as well. In particular, within the study of H-sssi processes, we focused on fractional Brownian motion (fBm) and its discrete-time increment process, the fractional Gaussian noise (fGn), which provide examples of non-Markovian Gaussian processes. The fGn, together with stationary FARIMA processes, is widely used in the modeling and estimation of long-memory, or long-range dependence (LRD). Time series manifesting long-range dependence, are often observed in nature especially in physics, meteorology, climatology, but also in hydrology, geophysics, economy and many others. We deepely studied LRD, giving many real data examples, providing statistical analysis and introducing parametric methods of estimation. Then, we introduced the theory of fractional integrals and derivatives, which indeed turns out to be very appropriate for studying and modeling systems with long-memory properties. After having introduced the basics concepts, we provided many examples and applications. For instance, we investigated the relaxation equation with distributed order time-fractional derivatives, which describes models characterized by a strong memory component and can be used to model relaxation in complex systems, which deviates from the classical exponential Debye pattern. Then, we focused in the study of generalizations of the standard diffusion equation, by passing through the preliminary study of the fractional forward drift equation. Such generalizations have been obtained by using fractional integrals and derivatives of distributed orders. In order to find a connection between the anomalous diffusion described by these equations and the long-range dependence, we introduced and studied the generalized grey Brownian motion (ggBm), which is actually a parametric class of H-sssi processes, which have indeed marginal probability density function evolving in time according to a partial integro-differential equation of fractional type. The ggBm is of course Non-Markovian. All around the work, we have remarked many times that, starting from a master equation of a probability density function f(x,t), it is always possible to define an equivalence class of stochastic processes with the same marginal density function f(x,t). All these processes provide suitable stochastic models for the starting equation. Studying the ggBm, we just focused on a subclass made up of processes with stationary increments. The ggBm has been defined canonically in the so called grey noise space. However, we have been able to provide a characterization notwithstanding the underline probability space. We also pointed out that that the generalized grey Brownian motion is a direct generalization of a Gaussian process and in particular it generalizes Brownain motion and fractional Brownain motion as well. Finally, we introduced and analyzed a more general class of diffusion type equations related to certain non-Markovian stochastic processes. We started from the forward drift equation, which have been made non-local in time by the introduction of a suitable chosen memory kernel K(t). The resulting non-Markovian equation has been interpreted in a natural way as the evolution equation of the marginal density function of a random time process l(t). We then consider the subordinated process Y(t)=X(l(t)) where X(t) is a Markovian diffusion. The corresponding time-evolution of the marginal density function of Y(t) is governed by a non-Markovian Fokker-Planck equation which involves the same memory kernel K(t). We developed several applications and derived the exact solutions. Moreover, we considered different stochastic models for the given equations, providing path simulations.

Numerical simulations of magma chamber dynamics at Campi Flegrei, and associated seismicity, deformation and gravity changes

Understanding the complex relationships between quantities measured by volcanic monitoring network and shallow magma processes is a crucial headway for the comprehension of volcanic processes and a more realistic evaluation of the associated hazard. This question is very relevant at Campi Flegrei, a volcanic quiescent caldera immediately north-west of Napoli (Italy). The system activity shows a high fumarole release and periodic ground slow movement (bradyseism) with high seismicity. This activity, with the high people density and the presence of military and industrial buildings, makes Campi Flegrei one of the areas with higher volcanic hazard in the world. In such a context my thesis has been focused on magma dynamics due to the refilling of shallow magma chambers, and on the geophysical signals detectable by seismic, deformative and gravimetric monitoring networks that are associated with this phenomenologies. Indeed, the refilling of magma chambers is a process frequently occurring just before a volcanic eruption; therefore, the faculty of identifying this dynamics by means of recorded signal analysis is important to evaluate the short term volcanic hazard. The space-time evolution of dynamics due to injection of new magma in the magma chamber has been studied performing numerical simulations with, and implementing additional features in, the code GALES (Longo et al., 2006), recently developed and still on the upgrade at the Istituto Nazionale di Geofisica e Vulcanologia in Pisa (Italy). GALES is a finite element code based on a physico-mathematical two dimensional, transient model able to treat fluids as multiphase homogeneous mixtures, compressible to incompressible. The fundamental equations of mass, momentum and energy balance are discretised both in time and space using the Galerkin Least-Squares and discontinuity-capturing stabilisation technique. The physical properties of the mixture are computed as a function of local conditions of magma composition, pressure and temperature.The model features enable to study a broad range of phenomenologies characterizing pre and sin-eruptive magma dynamics in a wide domain from the volcanic crater to deep magma feeding zones. The study of displacement field associated with the simulated fluid dynamics has been carried out with a numerical code developed by the Geophysical group at the University College Dublin (O’Brien and Bean, 2004b), with whom we started a very profitable collaboration. In this code, the seismic wave propagation in heterogeneous media with free surface (e.g. the Earth’s surface) is simulated using a discrete elastic lattice where particle interactions are controlled by the Hooke’s law. This method allows to consider medium heterogeneities and complex topography. The initial and boundary conditions for the simulations have been defined within a coordinate project (INGV-DPC 2004-06 V3_2 “Research on active volcanoes, precursors, scenarios, hazard and risk - Campi Flegrei”), to which this thesis contributes, and many researchers experienced on Campi Flegrei in volcanological, seismic, petrological, geochemical fields, etc. collaborate. Numerical simulations of magma and rock dynamis have been coupled as described in the thesis. The first part of the thesis consists of a parametric study aimed at understanding the eect of the presence in magma of carbon dioxide in magma in the convection dynamics. Indeed, the presence of this volatile was relevant in many Campi Flegrei eruptions, including some eruptions commonly considered as reference for a future activity of this volcano. A set of simulations considering an elliptical magma chamber, compositionally uniform, refilled from below by a magma with volatile content equal or dierent from that of the resident magma has been performed. To do this, a multicomponent non-ideal magma saturation model (Papale et al., 2006) that considers the simultaneous presence of CO2 and H2O, has been implemented in GALES. Results show that the presence of CO2 in the incoming magma increases its buoyancy force promoting convection ad mixing. The simulated dynamics produce pressure transients with frequency and amplitude in the sensitivity range of modern geophysical monitoring networks such as the one installed at Campi Flegrei . In the second part, simulations more related with the Campi Flegrei volcanic system have been performed. The simulated system has been defined on the basis of conditions consistent with the bulk of knowledge of Campi Flegrei and in particular of the Agnano-Monte Spina eruption (4100 B.P.), commonly considered as reference for a future high intensity eruption in this area. The magmatic system has been modelled as a long dyke refilling a small shallow magma chamber; magmas with trachytic and phonolitic composition and variable volatile content of H2O and CO2 have been considered. The simulations have been carried out changing the condition of magma injection, the system configuration (magma chamber geometry, dyke size) and the resident and refilling magma composition and volatile content, in order to study the influence of these factors on the simulated dynamics. Simulation results allow to follow each step of the gas-rich magma ascent in the denser magma, highlighting the details of magma convection and mixing. In particular, the presence of more CO2 in the deep magma results in more ecient and faster dynamics. Through this simulations the variation of the gravimetric field has been determined. Afterward, the space-time distribution of stress resulting from numerical simulations have been used as boundary conditions for the simulations of the displacement field imposed by the magmatic dynamics on rocks. The properties of the simulated domain (rock density, P and S wave velocities) have been based on data from literature on active and passive tomographic experiments, obtained through a collaboration with A. Zollo at the Dept. of Physics of the Federici II Univeristy in Napoli. The elasto-dynamics simulations allow to determine the variations of the space-time distribution of deformation and the seismic signal associated with the studied magmatic dynamics. In particular, results show that these dynamics induce deformations similar to those measured at Campi Flegrei and seismic signals with energies concentrated on the typical frequency bands observed in volcanic areas. The present work shows that an approach based on the solution of equations describing the physics of processes within a magmatic fluid and the surrounding rock system is able to recognise and describe the relationships between geophysical signals detectable on the surface and deep magma dynamics. Therefore, the results suggest that the combined study of geophysical data and informations from numerical simulations can allow in a near future a more ecient evaluation of the short term volcanic hazard.

Aldo Rossi, autobiografia scientifica: scrittura come progetto. Indagine critica tra scrittura e progetto di architettura

«In altri termini mi sfuggiva e ancora oggi mi sfugge gran parte del significato dell’evoluzione del tempo; come se il tempo fosse una materia che osservo dall’esterno. Questa mancanza di evoluzione è fonte di alcune mie sventure ma anche mi appartiene con gioia.» Aldo Rossi, Autobiografia scientifica. The temporal dimension underpinning the draft of Autobiografia scientifica by Aldo Rossi may be referred to what Lucien Lévy-Bruhl, the well-known French anthropologist, defines as “primitive mentality” and “prelogical” conscience : the book of life has lost its page numbers, even punctuation. For Lévy-Bruhl, but certainly for Rossi, life or its summing up becomes a continuous account of ellipses, gaps, repetitions that may be read from left to right or viceversa, from head to foot or viceversa without distinction. Rossi’s autobiographical writing seems to accept and support the confusion with which memories have been collected, recording them after the order memory gives them in the mental distillation or simply according to the chronological order in which they have happened. For Rossi, the confusion reflects the melting of memory elements into a composite image which is the result of a fusion. He is aware that the same sap pervades all memories he is going to put in order: each of them has got a common denominator. Differences have diminished, almost faded; the quick glance is prevalent over the distinction of each episode. Rossi’s writing is beyond the categories dependent on time: past and present, before and now. For Rossi, the only repetition – the repetition the text will make possible for an indefinite number of times – gives peculiarity to the event. As Gilles Deleuze knows, “things” may only last as “singleness”: more frequent the repetition is, more singular is the memory phenomenon that recurs, because only what is singular magnifies itself and happens endlessly forever. Rossi understands that “to raise the first time to nth forever”, repetition becomes glorification . It may be an autobiography that, celebrating the originality, enhances the memory event in the repetition; in fact it greatly differs from the biographical reproduction, in which each repetition is but a weaker echo, a duller copy, provided with a smaller an smaller power in comparison with the original. Paradoxically, for Deleuze the repetition asserts the originality and singularity of what is repeated. Rossi seems to share the thought expressed by Kierkegaard in the essay Repetition: «The hope is a graceful maiden slipping through your fingers; the memory of an elderly woman, indeed pretty, but never satisfactory if necessary; the repetition is a loved friend you are never tired of, as it is only the new to make you bored. The old never bores you and its presence makes you happy [...] life is but a repetition [...] here is the beauty of life» . Rossi knows well that repetition hints at the lasting stability of cosmic time. Kierkegaard goes on: «The world exists, and it exists as a repetition» . Rossi devotes himself, on purpose and in all conscience, to collect, to inventory and «to review life», his own life, according to a recovery not from the past but of the past: a search work, the «recherche du temps perdu», as Proust entitled his masterpiece on memory. If you want the past time to be not wasted, you must give it presence. «Memoria e specifico come caratteristiche per riconoscere se stesso e ciò che è estraneo mi sembravano le più chiare condizioni e spiegazioni della realtà. Non esiste uno specifico senza memoria, e una memoria che non provenga da un momento specifico; e solo questa unione permette la conoscenza della propria individualità e del contrario (self e non-self)» . Rossi wants to understand himself, his own character; it is really his own character that requires to be understood, to increase its own introspective ability and intelligence. «Può sembrare strano che Planck e Dante associno la loro ricerca scientifica e autobiografica con la morte; una morte che è in qualche modo continuazione di energia. In realtà, in ogni artista o tecnico, il principio della continuazione dell’energia si mescola con la ricerca della felicità e della morte» . The eschatological incipit of Rossi’s autobiography refers to Freud’s thought in the exact circularity of Dante’s framework and in as much exact circularity of the statement of the principle of the conservation of energy: in fact it was Freud to connect repetition to death. For Freud, the desire of repetition is an instinct rooted in biology. The primary aim of such an instinct would be to restore a previous condition, so that the repeated history represents a part of the past (even if concealed) and, relieving the removal, reduces anguish and tension. So, Freud ask himself, what is the most remote state to which the instinct, through the repetition, wants to go back? It is a pre-vital condition, inorganic of the pure entropy, a not-to-be condition in which doesn’t exist any tension; in other words, Death. Rossi, with the theme of death, introduces the theme of circularity which further on refers to the sense of continuity in transformation or, in the opposite way, the transformation in continuity. «[...] la descrizione e il rilievo delle forme antiche permettevano una continuità altrimenti irripetibile, permettevano anche una trasformazione, una volta che la vita fosse fermata in forme precise» . Rossi’s attitude seems to hint at the reflection on time and – in a broad sense – at the thought on life and things expressed by T.S. Eliot in Four Quartets: «Time present and time past / Are both perhaps present in time future, / And time future is contained in time past. / I all time is eternally present / All time is unredeemable. / What might have been is an abstraction / Remaining perpetual possibility / Only in a word of speculation. / What might have been and what has been / Point to one end, which is always present. [...]» . Aldo Rossi’s autobiographical story coincides with the description of “things” and the description of himself through the things in the exact parallel with craft or art. He seems to get all things made by man to coincide with the personal or artistic story, with the consequent immediate necessity of formulating a new interpretation: the flow of things has never met a total stop; all that exists nowadays is but a repetition or a variant of something existing some time ago and so on, without any interruption until the early dawnings of human life. Nevertheless, Rossi must operate specific subdivisions inside the continuous connection in time – of his time – even if limited by a present beginning and end of his own existence. This artist, as an “historian” of himself and his own life – as an auto-biographer – enjoys the privilege to be able to decide if and how to operate the cutting in a certain point rather than in another one, without being compelled to justify his choice. In this sense, his story is a matter very ductile and flexible: a good story-teller can choose any moment to start a certain sequence of events. Yet, Rossi is aware that, beyond the mere narration, there is the problem to identify in history - his own personal story – those flakings where a clean cut enables the separation of events of different nature. In order to do it, he has to make not only an inventory of his own “things”, but also to appeal to authority of the Divina Commedia started by Dante when he was 30. «A trent’anni si deve compiere o iniziare qualcosa di definitivo e fare i conti con la propria formazione» . For Rossi, the poet performs his authority not only in the text, but also in his will of setting out on a mystical journey and handing it down through an exact descriptive will. Rossi turns not only to the authority of poetry, but also evokes the authority of science with Max Plank and his Scientific Autobiography, published, in Italian translation, by Einaudi, 1956. Concerning Planck, Rossi resumes an element seemingly secondary in hit account where the German physicist «[...] risale alle scoperte della fisica moderna ritrovando l’impressione che gli fece l’enunciazione del principio di conservazione dell’energia; [...]» . It is again the act of describing that links Rossi to Planck, it is the description of a circularity, the one of conservation of energy, which endorses Rossi’s autobiographical speech looking for both happiness and death. Rossi seems to agree perfectly to the thought of Planck at the opening of his own autobiography: «The decision to devote myself to science was a direct consequence of a discovery which was never ceased to arouse my enthusiasm since my early youth: the laws of human thought coincide with the ones governing the sequences of the impressions we receive from the world surrounding us, so that the mere logic can enable us to penetrate into the latter one’s mechanism. It is essential that the outer world is something independent of man, something absolute. The search of the laws dealing with this absolute seems to me the highest scientific aim in life» . For Rossi the survey of his own life represents a way to change the events into experiences, to concentrate the emotion and group them in meaningful plots: «It seems, as one becomes older. / That the past has another pattern, and ceases to be a mere sequence [...]» Eliot wrote in Four Quartet, which are a meditation on time, old age and memory . And he goes on: «We had the experience but missed the meaning, / And approach to the meaning restores the experience / In a different form, beyond any meaning [...]» . Rossi restores in his autobiography – but not only in it – the most ancient sense of memory, aware that for at least 15 centuries the Latin word memoria was used to show the activity of bringing back images to mind: the psychology of memory, which starts with Aristotele (De Anima), used to consider such a faculty totally essential to mind. Keith Basso writes: «The thought materializes in the form of “images”» . Rossi knows well – as Aristotele said – that if you do not have a collection of mental images to remember – imagination – there is no thought at all. According to this psychological tradition, what today we conventionally call “memory” is but a way of imagining created by time. Rossi, entering consciously this stream of thought, passing through the Renaissance ars memoriae to reach us gives a great importance to the word and assumes it as a real place, much more than a recollection, even more than a production and an emotional elaboration of images.

In situ real-time investigation of Organic Ultra-Thin-Film transistors: growth, electrical properties and biosensing applications

Organic electronics has grown enormously during the last decades driven by the encouraging results and the potentiality of these materials for allowing innovative applications, such as flexible-large-area displays, low-cost printable circuits, plastic solar cells and lab-on-a-chip devices. Moreover, their possible field of applications reaches from medicine, biotechnology, process control and environmental monitoring to defense and security requirements. However, a large number of questions regarding the mechanism of device operation remain unanswered. Along the most significant is the charge carrier transport in organic semiconductors, which is not yet well understood. Other example is the correlation between the morphology and the electrical response. Even if it is recognized that growth mode plays a crucial role into the performance of devices, it has not been exhaustively investigated. The main goal of this thesis was the finding of a correlation between growth modes, electrical properties and morphology in organic thin-film transistors (OTFTs). In order to study the thickness dependence of electrical performance in organic ultra-thin-film transistors, we have designed and developed a home-built experimental setup for performing real-time electrical monitoring and post-growth in situ electrical characterization techniques. We have grown pentacene TFTs under high vacuum conditions, varying systematically the deposition rate at a fixed room temperature. The drain source current IDS and the gate source current IGS were monitored in real-time; while a complete post-growth in situ electrical characterization was carried out. At the end, an ex situ morphological investigation was performed by using the atomic force microscope (AFM). In this work, we present the correlation for pentacene TFTs between growth conditions, Debye length and morphology (through the correlation length parameter). We have demonstrated that there is a layered charge carriers distribution, which is strongly dependent of the growth mode (i.e. rate deposition for a fixed temperature), leading to a variation of the conduction channel from 2 to 7 monolayers (MLs). We conciliate earlier reported results that were apparently contradictory. Our results made evident the necessity of reconsidering the concept of Debye length in a layered low-dimensional device. Additionally, we introduce by the first time a breakthrough technique. This technique makes evident the percolation of the first MLs on pentacene TFTs by monitoring the IGS in real-time, correlating morphological phenomena with the device electrical response. The present thesis is organized in the following five chapters. Chapter 1 makes an introduction to the organic electronics, illustrating the operation principle of TFTs. Chapter 2 presents the organic growth from theoretical and experimental points of view. The second part of this chapter presents the electrical characterization of OTFTs and the typical performance of pentacene devices is shown. In addition, we introduce a correcting technique for the reconstruction of measurements hampered by leakage current. In chapter 3, we describe in details the design and operation of our innovative home-built experimental setup for performing real-time and in situ electrical measurements. Some preliminary results and the breakthrough technique for correlating morphological and electrical changes are presented. Chapter 4 meets the most important results obtained in real-time and in situ conditions, which correlate growth conditions, electrical properties and morphology of pentacene TFTs. In chapter 5 we describe applicative experiments where the electrical performance of pentacene TFTs has been investigated in ambient conditions, in contact to water or aqueous solutions and, finally, in the detection of DNA concentration as label-free sensor, within the biosensing framework.

Dynamic identification of structures: experimental assessment of modal parameteres through methods in frequency domain, in time-frequency domain and model updating

Among the experimental methods commonly used to define the behaviour of a full scale system, dynamic tests are the most complete and efficient procedures. A dynamic test is an experimental process, which would define a set of characteristic parameters of the dynamic behaviour of the system, such as natural frequencies of the structure, mode shapes and the corresponding modal damping values associated. An assessment of these modal characteristics can be used both to verify the theoretical assumptions of the project, to monitor the performance of the structural system during its operational use. The thesis is structured in the following chapters: The first introductive chapter recalls some basic notions of dynamics of structure, focusing the discussion on the problem of systems with multiply degrees of freedom (MDOF), which can represent a generic real system under study, when it is excited with harmonic force or in free vibration. The second chapter is entirely centred on to the problem of dynamic identification process of a structure, if it is subjected to an experimental test in forced vibrations. It first describes the construction of FRF through classical FFT of the recorded signal. A different method, also in the frequency domain, is subsequently introduced; it allows accurately to compute the FRF using the geometric characteristics of the ellipse that represents the direct input-output comparison. The two methods are compared and then the attention is focused on some advantages of the proposed methodology. The third chapter focuses on the study of real structures when they are subjected to experimental test, where the force is not known, like in an ambient or impact test. In this analysis we decided to use the CWT, which allows a simultaneous investigation in the time and frequency domain of a generic signal x(t). The CWT is first introduced to process free oscillations, with excellent results both in terms of frequencies, dampings and vibration modes. The application in the case of ambient vibrations defines accurate modal parameters of the system, although on the damping some important observations should be made. The fourth chapter is still on the problem of post processing data acquired after a vibration test, but this time through the application of discrete wavelet transform (DWT). In the first part the results obtained by the DWT are compared with those obtained by the application of CWT. Particular attention is given to the use of DWT as a tool for filtering the recorded signal, in fact in case of ambient vibrations the signals are often affected by the presence of a significant level of noise. The fifth chapter focuses on another important aspect of the identification process: the model updating. In this chapter, starting from the modal parameters obtained from some environmental vibration tests, performed by the University of Porto in 2008 and the University of Sheffild on the Humber Bridge in England, a FE model of the bridge is defined, in order to define what type of model is able to capture more accurately the real dynamic behaviour of the bridge. The sixth chapter outlines the necessary conclusions of the presented research. They concern the application of a method in the frequency domain in order to evaluate the modal parameters of a structure and its advantages, the advantages in applying a procedure based on the use of wavelet transforms in the process of identification in tests with unknown input and finally the problem of 3D modeling of systems with many degrees of freedom and with different types of uncertainty.

The Simon effect and its time courses: an investigation

The objective of the current thesis is to investigate the temporal dynamics (i.e., time courses) of the Simon effect, both from a theoretical and experimental point of view, for a better understanding of whether a) one or more process are responsible for the Simon effect and b) how this/these mechanism/s differently influence performance. In the first theoretical (i.e., “Theoretical Overview”) part, I examined in detail the process and justification for analyzing the temporal dynamics of the Simon effect and the assumptions that underlie interpretation of the results which have been obtained in the existing literature so far. In the second part (“Experimental Investigations”), though, I experimentally investigated several issues which the existing literature left unsolved, in order to get further evidence in favor or in contrast with the mainstream models which are currently used to account for the different Simon effect time courses. Some points about the experiments are worth mentioning: First, all the experiments were conducted in the laboratory, facing participants with stimuli presented on a PC screen and then recording their responses. Both stimuli presentation and response collection was controlled by the E-Prime software. The dependent variables of interest were always behavioral measures of performance, such as velocity and accuracy. Second, the most part of my experiments had been conducted at the Communication Sciences Department (University of Bologna), under Prof. Nicoletti’s supervision. The remaining part, though, had been conducted at the Psychological Sciences Department of Purdue University (West Lafayette, Indiana, USA), where I collaborated for one year as a visiting student with Prof. Proctor and his team. Third, my experimental pool was entirely composed by healthy and young students, since the cognitive functioning of elderly people was not the target of my research.

The spatial representation of time

Numerosi studi mostrano che gli intervalli temporali sono rappresentati attraverso un codice spaziale che si estende da sinistra verso destra, dove gli intervalli brevi sono rappresentati a sinistra rispetto a quelli lunghi. Inoltre tale disposizione spaziale del tempo può essere influenzata dalla manipolazione dell’attenzione-spaziale. La presente tesi si inserisce nel dibattito attuale sulla relazione tra rappresentazione spaziale del tempo e attenzione-spaziale attraverso l’uso di una tecnica che modula l’attenzione-spaziale, ovvero, l’Adattamento Prismatico (AP). La prima parte è dedicata ai meccanismi sottostanti tale relazione. Abbiamo mostrato che spostando l’attenzione-spaziale con AP, verso un lato dello spazio, si ottiene una distorsione della rappresentazione di intervalli temporali, in accordo con il lato dello spostamento attenzionale. Questo avviene sia con stimoli visivi, sia con stimoli uditivi, nonostante la modalità uditiva non sia direttamente coinvolta nella procedura visuo-motoria di AP. Questo risultato ci ha suggerito che il codice spaziale utilizzato per rappresentare il tempo, è un meccanismo centrale che viene influenzato ad alti livelli della cognizione spaziale. La tesi prosegue con l’indagine delle aree corticali che mediano l’interazione spazio-tempo, attraverso metodi neuropsicologici, neurofisiologici e di neuroimmagine. In particolare abbiamo evidenziato che, le aree localizzate nell’emisfero destro, sono cruciali per l’elaborazione del tempo, mentre le aree localizzate nell’emisfero sinistro sono cruciali ai fini della procedura di AP e affinché AP abbia effetto sugli intervalli temporali. Infine, la tesi, è dedicata allo studio dei disturbi della rappresentazione spaziale del tempo. I risultati ci indicano che un deficit di attenzione-spaziale, dopo danno emisferico destro, provoca un deficit di rappresentazione spaziale del tempo, che si riflette negativamente sulla vita quotidiana dei pazienti. Particolarmente interessanti sono i risultati ottenuti mediante AP. Un trattamento con AP, efficace nel ridurre il deficit di attenzione-spaziale, riduce anche il deficit di rappresentazione spaziale del tempo, migliorando la qualità di vita dei pazienti.

Ground penetrating radar early-time technique for soil electromagnetic parameters estimation

In recent years, thanks to the technological advances, electromagnetic methods for non-invasive shallow subsurface characterization have been increasingly used in many areas of environmental and geoscience applications. Among all the geophysical electromagnetic methods, the Ground Penetrating Radar (GPR) has received unprecedented attention over the last few decades due to its capability to obtain, spatially and temporally, high-resolution electromagnetic parameter information thanks to its versatility, its handling, its non-invasive nature, its high resolving power, and its fast implementation. The main focus of this thesis is to perform a dielectric site characterization in an efficient and accurate way studying in-depth a physical phenomenon behind a recent developed GPR approach, the so-called early-time technique, which infers the electrical properties of the soil in the proximity of the antennas. In particular, the early-time approach is based on the amplitude analysis of the early-time portion of the GPR waveform using a fixed-offset ground-coupled antenna configuration where the separation between the transmitting and receiving antenna is on the order of the dominant pulse-wavelength. Amplitude information can be extracted from the early-time signal through complex trace analysis, computing the instantaneous-amplitude attributes over a selected time-duration of the early-time signal. Basically, if the acquired GPR signals are considered to represent the real part of a complex trace, and the imaginary part is the quadrature component obtained by applying a Hilbert transform to the GPR trace, the amplitude envelope is the absolute value of the resulting complex trace (also known as the instantaneous-amplitude). Analysing laboratory information, numerical simulations and natural field conditions, and summarising the overall results embodied in this thesis, it is possible to suggest the early-time GPR technique as an effective method to estimate physical properties of the soil in a fast and non-invasive way.

Early Warning For Large Earthquakes: Observations, Models and Real-Time Data Analysis

This thesis is a collection of works focused on the topic of Earthquake Early Warning, with a special attention to large magnitude events. The topic is addressed from different points of view and the structure of the thesis reflects the variety of the aspects which have been analyzed. The first part is dedicated to the giant, 2011 Tohoku-Oki earthquake. The main features of the rupture process are first discussed. The earthquake is then used as a case study to test the feasibility Early Warning methodologies for very large events. Limitations of the standard approaches for large events arise in this chapter. The difficulties are related to the real-time magnitude estimate from the first few seconds of recorded signal. An evolutionary strategy for the real-time magnitude estimate is proposed and applied to the single Tohoku-Oki earthquake. In the second part of the thesis a larger number of earthquakes is analyzed, including small, moderate and large events. Starting from the measurement of two Early Warning parameters, the behavior of small and large earthquakes in the initial portion of recorded signals is investigated. The aim is to understand whether small and large earthquakes can be distinguished from the initial stage of their rupture process. A physical model and a plausible interpretation to justify the observations are proposed. The third part of the thesis is focused on practical, real-time approaches for the rapid identification of the potentially damaged zone during a seismic event. Two different approaches for the rapid prediction of the damage area are proposed and tested. The first one is a threshold-based method which uses traditional seismic data. Then an innovative approach using continuous, GPS data is explored. Both strategies improve the prediction of large scale effects of strong earthquakes.

A new method for the estimation of the reverberation time from measured room impulse responses with application to Italian opera houses

The Schroeder's backward integration method is the most used method to extract the decay curve of an acoustic impulse response and to calculate the reverberation time from this curve. In the literature the limits and the possible improvements of this method are widely discussed. In this work a new method is proposed for the evaluation of the energy decay curve. The new method has been implemented in a Matlab toolbox. Its performance has been tested versus the most accredited literature method. The values of EDT and reverberation time extracted from the energy decay curves calculated with both methods have been compared in terms of the values themselves and in terms of their statistical representativeness. The main case study consists of nine Italian historical theatres in which acoustical measurements were performed. The comparison of the two extraction methods has also been applied to a critical case, i.e. the structural impulse responses of some building elements. The comparison underlines that both methods return a comparable value of the T30. Decreasing the range of evaluation, they reveal increasing differences; in particular, the main differences are in the first part of the decay, where the EDT is evaluated. This is a consequence of the fact that the new method returns a “locally" defined energy decay curve, whereas the Schroeder's method accumulates energy from the tail to the beginning of the impulse response. Another characteristic of the new method for the energy decay extraction curve is its independence on the background noise estimation. Finally, a statistical analysis is performed on the T30 and EDT values calculated from the impulse responses measurements in the Italian historical theatres. The aim of this evaluation is to know whether a subset of measurements could be considered representative for a complete characterization of these opera houses.