Sviluppo di sistemi per l'analisi della combustione in tempo reale per motori endotermici alternativi

I moderni motori a combustione interna diventano sempre più complessi L'introduzione della normativa antinquinamento EURO VI richiederà una significativa riduzione degli inquinanti allo scarico. La maggiore criticità è rappresentata dalla riduzione degli NOx per i motori Diesel da aggiungersi a quelle già in vigore con le precedenti normative. Tipicamente la messa a punto di una nuova motorizzazione prevede una serie di test specifici al banco prova. Il numero sempre maggiore di parametri di controllo della combustione, sorti come conseguenza della maggior complessità meccanica del motore stesso, causa un aumento esponenziale delle prove da eseguire per caratterizzare l'intero sistema. L'obiettivo di questo progetto di dottorato è quello di realizzare un sistema di analisi della combustione in tempo reale in cui siano implementati diversi algoritmi non ancora presenti nelle centraline moderne. Tutto questo facendo particolare attenzione alla scelta dell'hardware su cui implementare gli algoritmi di analisi. Creando una piattaforma di Rapid Control Prototyping (RCP) che sfrutti la maggior parte dei sensori presenti in vettura di serie; che sia in grado di abbreviare i tempi e i costi della sperimentazione sui motopropulsori, riducendo la necessità di effettuare analisi a posteriori, su dati precedentemente acquisiti, a fronte di una maggior quantità di calcoli effettuati in tempo reale. La soluzione proposta garantisce l'aggiornabilità, la possibilità di mantenere al massimo livello tecnologico la piattaforma di calcolo, allontanandone l'obsolescenza e i costi di sostituzione. Questa proprietà si traduce nella necessità di mantenere la compatibilità tra hardware e software di generazioni differenti, rendendo possibile la sostituzione di quei componenti che limitano le prestazioni senza riprogettare il software.

New materials for electrochemiluminescence

The study of electrochemiluminescence (ECL) involves photophysical and electrochemical aspects. Excited states are populated by an electrical stimulus. The most important applications are in the diagnostic field where a number of different biologically-relevant molecules (e.g. proteins and nucleic acids) can be recognized and quantified with a sensitivity and specificity previously not reachable. As a matter of fact the electrochemistry, differently to the classic techniques as fluorescence and chemiluminescence, allows to control the excited state generation spatially and temporally. The two research visits into A. J. Bard electrochemistry laboratories were priceless. Dr. Bard has been one of ECL pioneers, the first to introduce the technique and the one who discovered in 1972 the surprising emission of Ru(bpy)3 2+. I consider necessary to thank by now my supervisors Massimo and Francesco for their help and for giving me the great opportunity to know this unique science man that made me feel enthusiastic. I will never be grateful enough… Considering that the experimental techniques of ECL did not changed significantly in these last years the most convenient research direction has been the developing of materials with new or improved properties. In Chapter I the basics concepts and mechanisms of ECL are introduced so that the successive experiments can be easily understood. In the final paragraph the scopes of the thesis are briefly described. In Chapter II by starting from ECL experimental apparatus of Dr. Bard’s laboratories the design, assembly and preliminary tests of the new Bologna instrument are carefully described. The instrument assembly required to work hard but resulted in the introduction of the new technique in our labs by allowing the continuation of the ECL studies began in Texas. In Chapter III are described the results of electrochemical and ECL studies performed on new synthesized Ru(II) complexes containing tetrazolate based ligands. ECL emission has been investigated in solution and in solid thin films. The effect of the chemical protonation of the tetrazolate ring on ECL emission has been also investigated evidencing the possibility of a catalytic effect (generation of molecular hydrogen) of one of the complexes in organic media. Finally, after a series of preliminary studies on ECL emission in acqueous buffers, the direct interaction with calf thymus DNA of some complexes has been tested by ECL and photoluminescence (PL) titration. In Chapter IV different Ir(III) complexes have been characterized electrochemically and photophysically (ECL and PL). Some complexes were already well-known in literature for their high quantum efficiency whereas the remaining were new synthesized compounds containing tetrazolate based ligands analogous to those investigated in Chapt. III. During the tests on a halogenated complex was unexpectedly evidenced the possibility to follow the kinetics of an electro-induced chemical reaction by using ECL signal. In the last chapter (V) the possibility to use mono-use silicon chips electrodes as ECL analitycal devices is under investigation. The chapter begins by describing the chip structure and materials then a signal reproducibility study and geometry optimization is carried on by using two different complexes. In the following paragraphs is reported in detail the synthesis of an ECL label based on Ru(bpy)3 2+ and the chip functionalization by using a lipoic acid SAM and the same label. After some preliminary characterizations (mass spectroscopy TOF) has been demonstrated that by mean of a simple and fast ECL measurement it’s possible to confirm the presence of the coupling product SAM-label into the chip with a very high sensitivity. No signal was detected from the same system by using photoluminescence.

Motion control and real-time systems: an approach to trajectory rebuilding in non-deterministic networks

Motion control is a sub-field of automation, in which the position and/or velocity of machines are controlled using some type of device. In motion control the position, velocity, force, pressure, etc., profiles are designed in such a way that the different mechanical parts work as an harmonious whole in which a perfect synchronization must be achieved. The real-time exchange of information in the distributed system that is nowadays an industrial plant plays an important role in order to achieve always better performance, better effectiveness and better safety. The network for connecting field devices such as sensors, actuators, field controllers such as PLCs, regulators, drive controller etc., and man-machine interfaces is commonly called fieldbus. Since the motion transmission is now task of the communication system, and not more of kinematic chains as in the past, the communication protocol must assure that the desired profiles, and their properties, are correctly transmitted to the axes then reproduced or else the synchronization among the different parts is lost with all the resulting consequences. In this thesis, the problem of trajectory reconstruction in the case of an event-triggered communication system is faced. The most important feature that a real-time communication system must have is the preservation of the following temporal and spatial properties: absolute temporal consistency, relative temporal consistency, spatial consistency. Starting from the basic system composed by one master and one slave and passing through systems made up by many slaves and one master or many masters and one slave, the problems in the profile reconstruction and temporal properties preservation, and subsequently the synchronization of different profiles in network adopting an event-triggered communication system, have been shown. These networks are characterized by the fact that a common knowledge of the global time is not available. Therefore they are non-deterministic networks. Each topology is analyzed and the proposed solution based on phase-locked loops adopted for the basic master-slave case has been improved to face with the other configurations.

Nutritional strategies to control mycotoxin damages in swine

Mycotoxins are contaminants of agricultural products both in the field and during storage and can enter the food chain through contaminated cereals and foods (milk, meat, and eggs) obtained from animals fed mycotoxin contaminated feeds. Mycotoxins are genotoxic carcinogens that cause health and economic problems. Ochratoxin A and fumonisin B1 have been classified by the International Agency for Research on Cancer in 1993, as “possibly carcinogenic to humans” (class 2B). To control mycotoxins induced damages, different strategies have been developed to reduce the growth of mycotoxigenic fungi as well as to decontaminate and/or detoxify mycotoxin contaminated foods and animal feeds. Critical points, target for these strategies, are: prevention of mycotoxin contamination, detoxification of mycotoxins already present in food and feed, inhibition of mycotoxin absorption in the gastrointestinal tract, reduce mycotoxin induced damages when absorption occurs. Decontamination processes, as indicate by FAO, needs the following requisites to reduce toxic and economic impact of mycotoxins: it must destroy, inactivate, or remove mycotoxins; it must not produce or leave toxic and/or carcinogenic/mutagenic residues in the final products or in food products obtained from animals fed decontaminated feed; it must be capable of destroying fungal spores and mycelium in order to avoiding mycotoxin formation under favorable conditions; it should not adversely affect desirable physical and sensory properties of the feedstuff; it has to be technically and economically feasible. One important approach to the prevention of mycotoxicosis in livestock is the addition in the diets of the non-nutritionally adsorbents that bind mycotoxins preventing the absorption in the gastrointestinal tract. Activated carbons, hydrated sodium calcium aluminosilicate (HSCAS), zeolites, bentonites, and certain clays, are the most studied adsorbent and they possess a high affinity for mycotoxins. In recent years, there has been increasing interest on the hypothesis that the absorption in consumed food can be inhibited by microorganisms in the gastrointestinal tract. Numerous investigators showed that some dairy strains of LAB and bifidobacteria were able to bind aflatoxins effectively. There is a strong need for prevention of the mycotoxin-induced damages once the toxin is ingested. Nutritional approaches, such as supplementation of nutrients, food components, or additives with protective effects against mycotoxin toxicity are assuming increasing interest. Since mycotoxins have been known to produce damages by increasing oxidative stress, the protective properties of antioxidant substances have been extensively investigated. Purpose of the present study was to investigate in vitro and in vivo, strategies to counteract mycotoxin threat particularly in swine husbandry. The Ussing chambers technique was applied in the present study that for the first time to investigate in vitro the permeability of OTA and FB1 through rat intestinal mucosa. Results showed that OTA and FB1 were not absorbed from rat small intestine mucosa. Since in vivo absorption of both mycotoxins normally occurs, it is evident that in these experimental conditions Ussing diffusion chambers were not able to assess the intestinal permeability of OTA and FB1. A large number of LAB strains isolated from feces and different gastrointestinal tract regions of pigs and poultry were screened for their ability to remove OTA, FB1, and DON from bacterial medium. Results of this in vitro study showed low efficacy of isolated LAB strains to reduce OTA, FB1, and DON from bacterial medium. An in vivo trial in rats was performed to evaluate the effects of in-feed supplementation of a LAB strain, Pediococcus pentosaceus FBB61, to counteract the toxic effects induced by exposure to OTA contaminated diets. The study allows to conclude that feed supplementation with P. pentosaceus FBB61 ameliorates the oxidative status in liver, and lowers OTA induced oxidative damage in liver and kidney if diet was contaminated by OTA. This P. pentosaceus FBB61 feature joined to its bactericidal activity against Gram positive bacteria and its ability to modulate gut microflora balance in pigs, encourage additional in vivo experiments in order to better understand the potential role of P. pentosaceus FBB61 as probiotic for farm animals and humans. In the present study, in vivo trial on weaned piglets fed FB1 allow to conclude that feeding of 7.32 ppm of FB1 for 6 weeks did not impair growth performance. Deoxynivalenol contamination of feeds was evaluated in an in vivo trial on weaned piglets. The comparison between growth parameters of piglets fed DON contaminated diet and contaminated diet supplemented with the commercial product did not reach the significance level but piglet growth performances were numerically improved when the commercial product was added to DON contaminated diet. Further studies are needed to improve knowledge on mycotoxins intestinal absorption, mechanism for their detoxification in feeds and foods, and nutritional strategies to reduce mycotoxins induced damages in animals and humans. The multifactorial approach acting on each of the various steps could be a promising strategy to counteract mycotoxins damages.

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.

New reactants and improved catalysts for maleic anhydride synthesis

The role of the amount of Nb, used as a dopant for VPP, and how its presence may affect the generation of the active and selective δ-VOPO4 at the VPP surface under reaction conditions, was investigated, employing ex-situ and in-situ characterisation techniques. We found that Nb indeed may favour, under specific conditions, the generation of the desired δ-VOPO4 compound; however, its effect of enhancement of catalytic behaviour was not simply proportional to its concentration. In order to better understand how Nb may affect the generation of the active phase, we prepared V/Nb mixed phosphates; the formation of a solid solution was possible only under specific conditions, with a limited reciprocal dissolution of the two elements. We concluded that even though the incorporation of small amounts of Nb5+ in the VOPO4 (and also of V5+ in NbOPO4) cannot be excluded, a phenomenon which might favour the generation of the desired δ-VOPO4 compound, however the main role of Nb5+ was related to a modification of the redox properties of V4+ in the VPP, and specifically of the redox potential associated to the couple V4+/V5+. This led to a catalyst that during reaction was more oxidized than the corresponding undoped VPP, which under specific reaction conditions allowed obtain a better selectivity to MA. Oppositely, an excessive oxidation of VPP (catalysts having high [Nb]) affected negatively the MA selectivity, because of the excessive formation of COx. A preliminary study regarding the oxidehydration of 1-butanol into MA was carried out testing various catalysts: the best catalyst resulted VPP; however the MA selectivity was lower than that obtained from n-butane. With in-situ/operando Raman study of the Nb-doped and undoped catalysts we verified that the redox cycle involves the VPP and the δ-VOPO4 compounds, that the reoxidation step of V4+ in VPP is the rate-determining one.

Control of fruit postharvest diseases by thermotherapy: understanding the mechanisms of action by –omics approaches

In recent years the hot water treatment (HW) represents an effective and safe approach for managing postharvest decay. This study reported the effect of an HW (60°C for 60 s and 45°C for 10 min) on brown rot and blue mould respectively. Peaches was found more thermotolerant compared to apple fruit, otherwise Penicillium expansum was more resistant to heat with respect to Monilinia spp. In semi-commercial and commercial trials, the inhibition of brown rot in naturally infected peaches was higher than 78% after 6 days at 0°C and 3 days at 20°C. Moreover, in laboratory trials a 100% disease incidence reduction was obtained by treating artificially infected peaches at 6-12 h after inoculation revealing a curative effect of HW. The expression levels of some genes were evaluated by qRT-PCR. Specifically, the cell wall genes (β-GAL, PL, PG, PME) showed a general decrease of expression level whereas PAL, CHI, HSP70 and ROS-scavenging genes were induced in treated peaches compared to the control ones. Contrarily, HW applied on artificially infected fruit before the inoculum was found to increase brown rot susceptibility. This aspect might be due to an increase of fruit VOCs emission as revealed by PTR-ToF-MS analysis. In addition a microarray experiment was conducted to analyze molecular mechanisms underneath the apple response to heat. Our results showed a largest amount of induced Heat shock proteins (HSPs), Heat shock cognate proteins (HSCs), Heat shock transcription factors (HSTFs) genes found at 1 and 4 hours from the treatment. Those genes required for the thermotolerance process could be involved in induced resistance response. The hypothesis was confirmed by 30% of blue mold disease reduction in artificially inoculated apple after 1 and 4 hours from the treatment. In order to improve peaches quality and disease management during storage, an innovative tool was also used: Da-meter.

NLGA based Active Control in Aerospace: fault tolerability, disturbance rejection, and parameter estimation

A new control scheme has been presented in this thesis. Based on the NonLinear Geometric Approach, the proposed Active Control System represents a new way to see the reconfigurable controllers for aerospace applications. The presence of the Diagnosis module (providing the estimation of generic signals which, based on the case, can be faults, disturbances or system parameters), mean feature of the depicted Active Control System, is a characteristic shared by three well known control systems: the Active Fault Tolerant Controls, the Indirect Adaptive Controls and the Active Disturbance Rejection Controls. The standard NonLinear Geometric Approach (NLGA) has been accurately investigated and than improved to extend its applicability to more complex models. The standard NLGA procedure has been modified to take account of feasible and estimable sets of unknown signals. Furthermore the application of the Singular Perturbations approximation has led to the solution of Detection and Isolation problems in scenarios too complex to be solved by the standard NLGA. Also the estimation process has been improved, where multiple redundant measuremtent are available, by the introduction of a new algorithm, here called "Least Squares - Sliding Mode". It guarantees optimality, in the sense of the least squares, and finite estimation time, in the sense of the sliding mode. The Active Control System concept has been formalized in two controller: a nonlinear backstepping controller and a nonlinear composite controller. Particularly interesting is the integration, in the controller design, of the estimations coming from the Diagnosis module. Stability proofs are provided for both the control schemes. Finally, different applications in aerospace have been provided to show the applicability and the effectiveness of the proposed NLGA-based Active Control System.