Multicelled Behaviour in E. coli: Design and experimental characterization of an engineered library of hybrid promoters.

Synthetic biology has recently had a great development, many papers have been published and many applications have been presented, spanning from the production of biopharmacheuticals to the synthesis of bioenergetic substrates or industrial catalysts. But, despite these advances, most of the applications are quite simple and don’t fully exploit the potential of this discipline. This limitation in complexity has many causes, like the incomplete characterization of some components, or the intrinsic variability of the biological systems, but one of the most important reasons is the incapability of the cell to sustain the additional metabolic burden introduced by a complex circuit. The objective of the project, of which this work is part, is trying to solve this problem through the engineering of a multicellular behaviour in prokaryotic cells. This system will introduce a cooperative behaviour that will allow to implement complex functionalities, that can’t be obtained with a single cell. In particular the goal is to implement the Leader Election, this procedure has been firstly devised in the field of distributed computing, to identify the process that allow to identify a single process as organizer and coordinator of a series of tasks assigned to the whole population. The election of the Leader greatly simplifies the computation providing a centralized control. Further- more this system may even be useful to evolutionary studies that aims to explain how complex organisms evolved from unicellular systems. The work presented here describes, in particular, the design and the experimental characterization of a component of the circuit that solves the Leader Election problem. This module, composed of an hybrid promoter and a gene, is activated in the non-leader cells after receiving the signal that a leader is present in the colony. The most important element, in this case, is the hybrid promoter, it has been realized in different versions, applying the heuristic rules stated in [22], and their activity has been experimentally tested. The objective of the experimental characterization was to test the response of the genetic circuit to the introduction, in the cellular environment, of particular molecules, inducers, that can be considered inputs of the system. The desired behaviour is similar to the one of a logic AND gate in which the exit, represented by the luminous signal produced by a fluorescent protein, is one only in presence of both inducers. The robustness and the stability of this behaviour have been tested by changing the concentration of the input signals and building dose response curves. From these data it is possible to conclude that the analysed constructs have an AND-like behaviour over a wide range of inducers’ concentrations, even if it is possible to identify many differences in the expression profiles of the different constructs. This variability accounts for the fact that the input and the output signals are continuous, and so their binary representation isn’t able to capture the complexity of the behaviour. The module of the circuit that has been considered in this analysis has a fundamental role in the realization of the intercellular communication system that is necessary for the cooperative behaviour to take place. For this reason, the second phase of the characterization has been focused on the analysis of the signal transmission. In particular, the interaction between this element and the one that is responsible for emitting the chemical signal has been tested. The desired behaviour is still similar to a logic AND, since, even in this case, the exit signal is determined by the hybrid promoter activity. The experimental results have demonstrated that the systems behave correctly, even if there is still a substantial variability between them. The dose response curves highlighted that stricter constrains on the inducers concentrations need to be imposed in order to obtain a clear separation between the two levels of expression. In the conclusive chapter the DNA sequences of the hybrid promoters are analysed, trying to identify the regulatory elements that are most important for the determination of the gene expression. Given the available data it wasn’t possible to draw definitive conclusions. In the end, few considerations on promoter engineering and complex circuits realization are presented. This section aims to briefly recall some of the problems outlined in the introduction and provide a few possible solutions.

Analysis and development of interface components and procedures for the design and commissioning toolchains in automation systems

The objective of the thesis project, developed within the Line Control & Software Engineering team of G.D company, is to analyze and identify the appropriate tool to automate the HW configuration process using Beckhoff technologies by importing data from an ECAD tool. This would save a great deal of time, since the I/O topology created as part of the electrical planning is presently imported manually in the related SW project of the machine. Moreover, a manual import is more error-prone because of human mistake than an automatic configuration tool. First, an introduction about TwinCAT 3, EtherCAT and Automation Interface is provided; then, it is analyzed the official Beckhoff tool, XCAD Interface, and the requirements on the electrical planning to use it: the interface is realized by means of the AutomationML format. Finally, due to some limitations observed, the design and implementation of a company internal tool is performed. Tests and validation of the tool are performed on a sample production line of the company.

Synthesis, characterization and conformational studies of arylbenzylmaleimides

From the discoveries of Pasteur, stereochemistry has played an increasingly important role in the chemical sciences. In particular conformational study of molecules with axial chirality is object of intense research. Through Dynamic-NMR analysis and simulation of the spectra, the energy rotational barriers value of conformers are obtained. When this barrier is high sufficiently, atropisomeric stable compounds can be reached. They can be separated and used in stereo-synthesis and in packing processes. 3,4-bis-aryl maleimides, in which the aromatic groups are sufficiently bulky, generate atropisomeric stable configurations, that can be isolated at room temperature. The assignment of absolute configurations is performed through ECD analysis and comparison with computational calculations. The biological activities of maleimide derivatives widen the field of atropisomers application also in biological systems.

Theoretical and experimental studies of gold catalysts for glucose oxidation

The glucaric acid (GLA) has been identified as a “top value-added chemical from biomass” that can be employed for many uses; for instance, it could be a precursor of adipic acid, a monomer of Nylon-6,6. GLA can be synthetized by the oxidation of glucose (GLU), passing through the intermediate gluconic acid (GLO). In recent years, a new process has been sought to obtain GLA in an economic and environmental sustainable way, in order to replace the current use of HNO3 as a stoichiometric oxidant, or electrocatalysis and biochemical synthesis, which show several disadvantages. Thereby, this work is focused on the study of catalysts based on gold nanoparticles supported on activated carbon for the oxidation reaction of GLU to GLA using O2 as an oxidant agent and NaOH as base. The sol-immobilization method leads us to obtain small and well dispersed nanoparticles, characterized by UV-Vis, XRD and TEM techniques. Repeating the reaction on different batches of catalyst, both the synthesis and the reaction were confirmed to be reproducible. The effect of the reaction time feeding GLO as reagent was studied: the results show that the conversion of GLO increases as the reaction time increases; however, the yields of GLA and others increase up to 1 hour, and then they remain constant. In order to obtain information on the catalytic mechanism at the atomistic level, a computational study based on density functional theory and atomistic modeling of the gold nano-catalyst were performed. Highly symmetric (icosahedral and cubo-octahedral) and distorted Au55 nanoparticles have been optimized along with Au(111) and Au(100) surfaces. Distorted structures were found to be more stable than symmetrical ones due to relativistic effects. On these various models the adsorptions of various species involved in the catalysis have been studied, including OH- species, GLU and GLO. The study carried out aims to provide a method for approaching to the study of nanoparticellary catalytic systems.

Design and acoustic characterization of a new listening room

Il presente lavoro tratta la progettazione e caratterizzazione di una nuova "listening room" ad acustica controllata partendo dai requisiti dettati dalle norme tecniche ITU-R BS 1116-1 e EBU/UER Tech. doc. 3276. Ad oggi è presente un'ampia letteratura, che tratta approcci per valutazione acustica delle sale di ascolto. Essa inizialmente era volta a trovare proporzioni ideali tra le dimensioni della camera, poi la ricerca si è spostata sull'elaborazione di modelli previsionali. Purtroppo tali metodi spesso non riescono a garantire le prestazioni desiderate, mentre le prove sperimentali dettate dalle norme risultano essere di comprovata validità. L'ambiente oggetto di studio è stato progettato all'interno dello spazio dei laboratori CIRI. La tecnologia costruttiva è frutto di uno studio approfondito, in particolare la scelta di fibre di poliestere termolegate, per il rivestimento delle pareti interne, è stata valutata attraverso misure in camera riverberante secondo UNI-EN-ISO 354. Si è poi seguita una metodologia iterativa che coinvolgesse messa in opera, misurazioni in situ e valutazione tramite confronto con i parametri consigliati dalle normative tecniche sopra citate. In quest'ottica sono state effettuate acquisizioni di risposte all'impulso monoaurali e dei livelli di pressione sonora per verificare la qualità di isolamento e il comportamento alle basse frequenze. La validazione restituisce indicazioni positive per gli utilizzi dell'ambiente ipotizzati e risulta compatibile con le stringenti richieste delle norme tecniche.

Synthesis, characterization and conformational studies of bis-phenothiazine-aryl-boranes

This thesis project presents a work based on the study of a particular class of amino-boranes, called bis-phenothiazine-aryl-boranes. The peculiarity of these compounds is the N-B-N chemical moiety and their complex conformational behaviour, due to the combination of steric hindrance and conjugation of the B-N bond. Our work is focused on two main products with different symmetry: bis-phenothiazine-2-methylnaphthyl-borane (2b) and bis-phenothiazine-anthracenyl-borane (2c). We firstly focused our attention on an effective way of synthesis, by optimizing both reaction conditions and purification. The products and co-products of interest were then characterized with NMR, mass spectroscopy and X-Ray diffraction on single crystals. The products were eventually analysed through conformational studies, by experimental techniques, such as dynamic NMR and EXSY, and by a theorical approach with DFT calculations.

Synthesis, Characterization and Conformational Studies of Modified Carbazole-bis-aryl-boranes

During this project we have synthetized different compounds belonging to the class of amino-boranes for the study of bis-aryl-B=N system. We have decided to keep unchanged the aryl components and change only the amine to observe the effect of that on the B=N bond. The used amines are modified carbazoles with functional groups chosen to amplify or disempower the steric and the conjugation effect. We have found that the evaluation of steric barrier was possible studying the gearing aryls rotation around the C-B bonds, while the conjugation barrier is instead given by the energy needed to break the formal double bond B=N and allow the amine rotation. The work started with a proposed synthesis, improved for every reaction, then the products are characterized by NMR, fluorometric spectroscopy, mass spectrometry and X-Ray diffraction on single crystal. The following study on rotational energy barrier was possible theoretically through DFT calculation and experimentally with techniques like Dynamic NMR and EXSY. The fluorometric analysis was done for the study of the solvatochromic propriety of the products.

Design and Performance Analysis of the Electrical Power System of a Petrochemical Facility

This thesis deals with the sizing and analysis of the electrical power system of a petrochemical plant. The activity was carried out in the framework of an electrical engineering internship. The sizing and electrical calculations, as well as the study of the dynamic behavior of network quantities, are accomplished by using the ETAP (Electrical Transient Analyzer Program) software. After determining the type and size of the loads, the calculation of power flows is carried out for all possible network layout and different power supply configurations. The network is normally operated in a double radial configuration. However, the sizing must be carried out taking into account the most critical configuration, i.e., the temporary one of single radial operation, and also considering the most unfavorable power supply conditions. The calculation of shortcircuit currents is then carried out and the appropriate circuit breakers are selected. Where necessary, capacitor banks are sized in order to keep power factor at the point of common coupling within the preset limits. The grounding system is sized by using the finite element method. For loads with the highest level of criticality, UPS are sized in order to ensure their operation even in the absence of the main power supply. The main faults that can occur in the plant are examined, determining the intervention times of the protections to ensure that, in case of failure on one component, the others can still properly operate. The report concludes with the dynamic and stability analysis of the power system during island operation, in order to ensure that the two gas turbines are able to support the load even during transient conditions.

Design and Characterization of an Active Three-Phase EMI Noise Separator for Three-Phase Power Electronics Systems

One of the major issues for power converters that are connected to the electric grid are the measurement of three phase Conduced Emissions (CE), which are regulated by international and regional standards. CE are composed of two components which are Common Mode (CM) noise and Differential Mode (DM) noise. To achieve compliance with these regulations the Equipment Under Test (EUT) includes filtering and other electromagnetic emission control strategies. The separation of differential mode and common mode noise in Electromagnetic Interference (EMI) analysis is a well-known procedure which is useful especially for the optimization of the EMI filter, to improve the CM or DM attenuation depending on which component of the conducted emissions is predominant, and for the analysis and the understanding of interference phenomena of switched mode power converters. However, separating both components is rarely done during measurements. Therefore, in this thesis an active device for the separation of the CM and DM EMI noise in three phase power electronic systems has been designed and experimentally analysed.

Atmospheric corrosion of quaternary bronzes (Cu-Sn-Zn-Pb): laboratory tests (accelerated ageing in wet & dry conditions)and field studies (the Bottego monument in Parma, Italy).

The interactions between outdoor bronzes and the environment, which lead to bronze corrosion, require a better understanding in order to design effective conservation strategies in the Cultural Heritage field. In the present work, investigations on real patinas of the outdoor monument to Vittorio Bottego (Parma, Italy) and laboratory studies on accelerated corrosion testing of inhibited (by silane-based films, with and without ceria nanoparticles) and non-inhibited quaternary bronzes are reported and discussed. In particular, a wet&dry ageing method was used both for testing the efficiency of the inhibitor and for patinating bronze coupons before applying the inhibitor. A wide range of spectroscopic techniques has been used, for characterizing the core metal (SEM+EDS, XRF, AAS), the corroded surfaces (SEM+EDS, portable XRF, micro-Raman, ATR-IR, Py-GC-MS) and the ageing solutions (AAS). The main conclusions were: 1. The investigations on the Bottego monument confirmed the differentiation of the corrosion products as a function of the exposure geometry, already observed in previous works, further highlighting the need to take into account the different surface features when selecting conservation procedures such as the application of inhibitors (i.e. the relative Sn enrichment in unsheltered areas requires inhibitors which effectively interact not only with Cu but also with Sn). 2. The ageing (pre-patination) cycle on coupons was able to reproduce the relative Sn enrichment that actually happens in real patinated surfaces, making the bronze specimens representative of the real support for bronze inhibitors. 3. The non-toxic silane-based inhibitors display a good protective efficiency towards pre-patinated surfaces, differently from other widely used inhibitors such as benzotriazole (BTA) and its derivatives. 4. The 3-mercapto-propyl-trimethoxy-silane (PropS-SH) additivated with CeO2 nanoparticles generally offered a better corrosion protection than PropS-SH.

Design and optimization of turbo-expanders for organic rankine cycles

In a world focused on the need to produce energy for a growing population, while reducing atmospheric emissions of carbon dioxide, organic Rankine cycles represent a solution to fulfil this goal. This study focuses on the design and optimization of axial-flow turbines for organic Rankine cycles. From the turbine designer point of view, most of this fluids exhibit some peculiar characteristics, such as small enthalpy drop, low speed of sound, large expansion ratio. A computational model for the prediction of axial-flow turbine performance is developed and validated against experimental data. The model allows to calculate turbine performance within a range of accuracy of ±3%. The design procedure is coupled with an optimization process, performed using a genetic algorithm where the turbine total-to-static efficiency represents the objective function. The computational model is integrated in a wider analysis of thermodynamic cycle units, by providing the turbine optimal design. First, the calculation routine is applied in the context of the Draugen offshore platform, where three heat recovery systems are compared. The turbine performance is investigated for three competing bottoming cycles: organic Rankine cycle (operating cyclopentane), steam Rankine cycle and air bottoming cycle. Findings indicate the air turbine as the most efficient solution (total-to-static efficiency = 0.89), while the cyclopentane turbine results as the most flexible and compact technology (2.45 ton/MW and 0.63 m3/MW). Furthermore, the study shows that, for organic and steam Rankine cycles, the optimal design configurations for the expanders do not coincide with those of the thermodynamic cycles. This suggests the possibility to obtain a more accurate analysis by including the computational model in the simulations of the thermodynamic cycles. Afterwards, the performance analysis is carried out by comparing three organic fluids: cyclopentane, MDM and R245fa. Results suggest MDM as the most effective fluid from the turbine performance viewpoint (total-to-total efficiency = 0.89). On the other hand, cyclopentane guarantees a greater net power output of the organic Rankine cycle (P = 5.35 MW), while R245fa represents the most compact solution (1.63 ton/MW and 0.20 m3/MW). Finally, the influence of the composition of an isopentane/isobutane mixture on both the thermodynamic cycle performance and the expander isentropic efficiency is investigated. Findings show how the mixture composition affects the turbine efficiency and so the cycle performance. Moreover, the analysis demonstrates that the use of binary mixtures leads to an enhancement of the thermodynamic cycle performance.

Design and Implementation of a Cloud-based Middleware for Persuasive Android Applications

Negli ultimi decenni, le tecnologie e i prodotti informatici sono diventati pervasivi e sono ora una parte essenziale delle nostre vite. Ogni giorno ci influenzano in maniera più o meno esplicita, cambiando il nostro modo di vivere e i nostri comportamenti più o meno intenzionalmente. Tuttavia, i computer non nacquero inizialmente per persuadere: essi furono costruiti per gestire, calcolare, immagazzinare e recuperare dati. Non appena i computer si sono spostati dai laboratori di ricerca alla vita di tutti i giorni, sono però diventati sempre più persuasivi. Questa area di ricerca è chiamata pesuasive technology o captology, anche definita come lo studio dei sistemi informatici interattivi progettati per cambiare le attitudini e le abitudini delle persone. Nonostante il successo crescente delle tecnologie persuasive, sembra esserci una mancanza di framework sia teorici che pratici, che possano aiutare gli sviluppatori di applicazioni mobili a costruire applicazioni in grado di persuadere effettivamente gli utenti finali. Tuttavia, il lavoro condotto dal Professor Helal e dal Professor Lee al Persuasive Laboratory all’interno dell’University of Florida tenta di colmare questa lacuna. Infatti, hanno proposto un modello di persuasione semplice ma efficace, il quale può essere usato in maniera intuitiva da ingegneri o specialisti informatici. Inoltre, il Professor Helal e il Professor Lee hanno anche sviluppato Cicero, un middleware per dispositivi Android basato sul loro precedente modello, il quale può essere usato in modo molto semplice e veloce dagli sviluppatori per creare applicazioni persuasive. Il mio lavoro al centro di questa tesi progettuale si concentra sull’analisi del middleware appena descritto e, successivamente, sui miglioramenti e ampliamenti portati ad esso. I più importanti sono una nuova architettura di sensing, una nuova struttura basata sul cloud e un nuovo protocollo che permette di creare applicazioni specifiche per smartwatch.