Content-based image retrieval for visual big data analysis

The Content-Based Image Retrieval (CBIR) task is a computer vision problem. The growth of the digital images on the Internet allows to encourage the proposal of solution for this task more than before. The access to this huge quantity of data has allowed the creation of big datasets, that brought with them lots of new challenges. Briefly, the objective of the task is simply to retrieve and rank the similar images to the query one, called retrieval accuracy, that need to be as high as possible. Moreover, there are secondary targets as retrieval time and memory occupancy that need to be as low as possible. The problem is trivial for humans that simply execute this task through experience and semantic perception, but it is not so easy for a computer. This is known as semantic gap, which refers to the gap between low-level image pixels and high-level semantic concepts. Furthermore, the images may contain noisy patches (e.g. trees, person, cars, ...), be taken with different lightning conditions, viewpoints and resolution. In order to solve this problem it is crucial to develop algorithms and techniques with the objective of reducing the weight of the unnecessary patches of the images and that work well with a vast quantity of data. There are several applications of CBIR systems: libraries and museum applications, fashion application for the search of certain clothes, advanced electronic tourist guides. In this thesis a complete pipeline for the resolution of the CBIR problem is presented and then all the steps of the process are evaluated with a particular focus on CNN transfer learning, embeddings, large-scale retrieval and methods based on graphs as diffusion mechanism. All the methods presented are tested on several public image datasets in order to compare the final retrieval results

Sviluppo e testing biologico di nanostrutture per rigenerazione tissutale

L’ingegneria tissutale si sta concentrando sempre più verso lo sviluppo di nuove strategie per la rigenerazione ossea, target che rappresenta un focus di interesse centrale per migliorare la gestione di casi clinici complessi nell’ambito della chirurgia orale e maxillo-facciale. Uno degli approcci più utilizzati in questo ambito è lo sviluppo di micro- e nanotopografie superficiali che consentano di aumentare le proprietà osteoinduttive dei biomateriali e nello specifico dei materiali implantari. È noto da letteratura che negli ultimi anni è aumentata notevolmente la consapevolezza dell’importanza non solo della microtopografia, ma anche della nanotopografia superficiale dei biomateriali. Infatti, la disposizione nanotopografica dei segnali extracellulari è di fondamentale importanza per controllare adesione, proliferazione e differenziamento cellulare, eventi che portano alla rigenerazione del tessuto. Nonostante non siano ancora note le distribuzioni spaziali ideali per favorire la risposta cellulare, a causa dell’intricata composizione della matrice extracellulare, è noto che la loro disposizione debba essere nel range dimensionale dei suoi componenti. È stato inoltre sottolineato come le cellule rispondano specificamente a differenti nanopatterns superficiali attivando specifici pathways di trasduzione del segnale topografia-dipendenti (ad esempio i cambiamenti conformazionali del citoscheletro, la motilità cellulare, ecc.). In questo lavoro sono state utilizzate nanostrutture create ad hoc per ottimizzare l’adesione, la proliferazione ed il differenziamento delle cellule ossee, con l’intenzione finale di aumentare l’osteointegrazione degli impianti dentali. In particolare, sono stati sviluppati e caratterizzati, sia dal punto di vista fisico-chimico, sia dal punto di vista biologico, differenti tipologie di nanofili, in presenza ed assenza di una funzionalizzazione con 3-mercaptopropil-trimetossisilano. Questa scelta è basata sul fatto che la molecola che favorisce il differenziamento osteoblastico. Gli studi di citocompatibilità effettuati su nanofili di ossicarburo di silicio non mostrano tossicità cellulare, sia per quanto riguarda i nanofili di controllo, sia per i funzionalizzati. È stato notato un aumento della proliferazione in seguito alla funzionalizzazione con 3-mercaptopropil-trimetossisilano, probabilmente dovuta al differente pattern di adsorbimento proteico, oltre che una migliore adesione ed una maggiore espressione dei geni tipicamente legati al differenziamento osteoblastico precoce. Infine, i nanofili di ossido di titanio hanno mostrato buone potenzialità sia per lo stimolo del differenziamento osteoblastico, sia per fungere da substrato di ancoraggio per gli osteoblasti, a conferma del fatto che su superfici nanostrutturate la risposta cellulare viene aumentata, in favore delle funzioni rigenerative dell’osso. In conclusione, possiamo affermare che entrambi, nanofilii di ossicarburo di silicio e nanofili di ossido di titanio, sono substrati promettenti per il miglioramento dell’osteointegrazione degli impianti dentali.Nowadays, tissue engineering has been focusing on the development of promising strategies for bone regeneration, which is the fundamental process to consider in order to improve the success of complex clinical cases in the field of oral and maxillo-facial surgery. One of the most common approaches has been the development of micro- and nanotopographies leading to an increase of osteoconductive properties of biomaterials and, in particular, of implantable materials. It is well known that the importance of biomaterials surfaces micro- and nanotopography has been increased in the last decades. Indeed, nanotopographic disposition of extracellular signals has revealed to be central in the control of cellular adhesion, proliferation and differentiation, all events having a positive impact on tissue regeneration. Although the lack of information about the ideal spatial distribution promoting cellular response, it has been demonstrated that this disposition should be in the dimensional range of its components. Furthermore, it has been underlined the cellular ability to specifically respond to different nanopatterns in order to activate particular topography-dependent signal transduction pathways (for instance conformational changes of the cytoskeleton, cellular motility). The aim of this project was to improve dental implants osteointegration with the use of nanostructures created ad hoc to optimize bone cells adhesion, proliferation and differentiation. Particularly, different categories of nanowires have been developed and characterized on the basis of their physico-chemical and biological properties in presence and in absence of functionalization with 3-mercaptopropyltrimethoxysilane. This kind of functional group has been chosen as the molecule can advance osteoblastic differentiation. Results of cytocompatibility studies performed on silicon oxycarbide nanowires demonstrated no toxic effects for control nanowires as well as functionalized. It has been shown an increase of cells proliferation after functionalization, probably due to factors as different protein absorption pattern, improved adhesion and expression of genes having a fundamental role in the early stages of osteoblastic differentiation. In addition, titanium dioxide nanowires revealed to have great potential both for osteoblastic differentiation and focal adhesion formation, confirming the idea that nanostructured surfaces can promote cellular response supporting bone regeneration. In conclusion, silicon oxycarbide nanowires and titanium dioxide nanowires have been confirmed to be promising substrates to improve dental implants osteointegration

Application of lca (life cycle assessment) for the valorization of food waste collected from the distribution and retail phases in Emilia Romagna region

La presente tesi ha avuto come oggetto di studio la valutazione della sostenibilità ambientale di un nuovo sistema per la valorizzazione dello scarto alimentare generato durante la fase di distribuzione e vendita al dettaglio all’interno della regione Emilia-Romagna. Per la realizzazione di questo documento è stato adottato il metodo denominato Life Cycle Assessment (LCA), metodo sempre più utilizzato per quantificare gli impatti ambientali di attività, prodotti e sistemi produttivi attraverso l’identificazione e la quantificazione dell’energia e dei materiali impiegati e delle emissioni rilasciate nell’ambiente durante l’intero ciclo di vita. Dopo una introduzione generale relativa al metodo LCA, dalle sue origini, alle normative che lo regolamentano e alle fasi in cui è strutturato (Capitolo 1), è stata eseguita poi una panoramica sul problema del Food Waste a livello globale, sulla sua evoluzione la sua quantificazione lungo tutta la filiera agroalimentare (Capitolo 2). Nel terzo capitolo è stato descritto un nuovo progetto - denominato “SORT” - che ha come fine ultimo la valorizzazione dello scarto alimentare generato durante le fasi di distribuzione e vendita al dettaglio tramite lo spacchettamento e la valorizzazione delle diverse matrici alimentari. Nel capitolo 4 è stata valutata la sostenibilità ambientale dell’utilizzo di scarti alimentari a base farina (come pane, biscotti e pasta) per la produzione di mangimi animali. Nel complesso, questo studio dimostra chiaramente che il metodo di produzione di mangimi per animali che utilizzano in parte lo scarto alimentare (in questo caso prodotti da forno e pasta) può essere un'opzione più rispettosa nei confronti dell’ambiente rispetto alla produzione tradizionale di mangimi dove vengono utilizzati ad esempio cereali coltivati appositamente. Il capitolo 5 è incentrato su uno studio di impatto ambientale condotto seguendo la metodologia LCA e relativo alla produzione di Pet Food tramite l’utilizzo di scarti alimentari a base carne. I prodotti a base di carne, infatti, hanno un impatto ambientale significativo (dovuto, in particolare, alle fasi di allevamento e macellazione) e lo spreco di questi prodotti alimentari è un problema enorme dal punto di vista ambientale. Il problema è aggravato dal fatto che la durata di conservazione dei prodotti a base di carne è spesso limitata, poiché la maggior parte di questi prodotti sono freschi e non trasformati. Sulla base di queste considerazioni, questo studio ha valutato i benefici ambientali derivanti dall'uso della frazione di carne proveniente dai rifiuti alimentari confezionati, in sostituzione della carne allevata appositamente che viene impiegata attualmente per la produzione di alcuni alimenti per animali domestici. La principale conclusione di questi risultati è che le fasi aggiuntive di raccolta, conservazione, smistamento e disimballaggio dei rifiuti alimentari hanno un impatto limitato sull'ambiente rispetto all'impatto causato dall'allevamento e dalla macellazione del tessuto muscolare, che è particolarmente rilevante nel caso di pet food a base di carne bovina. Nel caso del prodotto paté, l'attuale uso di sottoprodotti provenienti dai macelli ha un effetto piuttosto limitato sull'impatto ambientale. Il capitolo 6 valuta l’impatto ambientale di un nuovo processo industriale per il recupero e valorizzazione dei materiali di imballaggio derivati da rifiuti alimentari confezionati. Ogni anno nella Regione Emilia-Romagna 14.300 tonnellate di alimenti confezionati raccolti dai negozi al dettaglio e durante la fase di distribuzione vengono sprecati e inviati alle discariche. Tra queste quantità, circa il 13,8% (ovvero 1.973 tonnellate / anno) è costituito da materiale da imballaggio che, al momento, non è né smistato né riciclato. Un modo per risolvere questo problema è introdurre un complesso sistema di raccolta e smistamento in grado di spacchettare il food waste e quindi separare la matrice organica dal proprio packaging. Sulla base di queste premesse, lo studio effettua una valutazione ambientale di un sistema in grado di raccogliere il food waste dalle strutture di distribuzione e punti vendita e inviarlo a una struttura di smistamento, dove i materiali di imballaggio verranno suddivisi e inviati alla migliore opzione di fine vita. Nel complesso, questo studio dimostra che il processo proposto, che comprende la selezione e una valorizzazione del fine dei materiali di imballaggio come ad esempio il riciclaggio e la termovalorizzazione, può offrire vantaggi ambientali rispetto allo scenario attuale in cui i materiali di imballaggio di scarti alimentari vengono smaltiti in discarica. Nel capitolo 7 si propone una valutazione ambientale di diversi scenari di logistica inversa per la raccolta di rifiuti alimentari confezionati dalla catena di vendita al dettaglio della regione Emilia-Romagna (Italia), spedendoli a una serie di centri di distribuzione per lo stoccaggio e ad una struttura di smistamento dove viene spacchettato, con l'obiettivo per deviarlo dallo smaltimento in discariche verso canali alternativi. L’ottimizzazione dei trasporti costituisce un elemento significativo per il miglioramento della sostenibilità ambientale del settore agroalimentare. Attualmente è in fase di sviluppo uno studio finale sull’analisi ambientale globale del progetto SORT che va a riassumere e completare gli studi descritti in questo documento. Grazie ad uno studio globale che comprende la valorizzazione di tutte le frazioni divise e del packaging, si avrà una visione completa a livello ambientale delle performance di questo ambizioso progetto pilota che potrà essere replicato anche in altre regioni italiane con alcuni accorgimenti. In conclusione, la sostenibilità ambientale della filiera agroalimentare costituisce una tematica centrale dei giorni nostri, rispetto alla quale il dibattito rimane aperto e l’attenzione da parte di organizzazioni, istituzioni e mondo della ricerca dovrebbe essere sempre crescente. Il lavoro da fare è tanto ma la strada, che pure rimane in salita, non è invalicabile e per percorrerla è necessario il contributo di tutti gli attori del settore che hanno la responsabilità di rendere reale quel concetto di sviluppo sostenibile la cui attuazione rappresenta una necessità inderogabile

Power scaling ed effetti termici in componenti fotonici e a microonde

Many industrial and research realities have to face the power scaling impact on a wide range of devices and applications. During this Ph.D. project, numerical simulations, analysis and experimental activities have been performed in order to investigate the thermal effects due to the high power demand in electromagnetic devices. In addition, study and development of proper cooling systems, aimed at limiting the detrimental heat load effects on different components, have been carried out. The present work is divided into two main sections: the first one deals with the development activities conducted in collaboration with an Italian company, leader in the production of household appliances; the second one concerns the research activity carried out in the academic context. Within the company, a new microwave generation system for domestic ovens is studied and developed. It involves the use of an inverter, instead of a transformer, for driving the magnetron, the actual microwave generator. The standard transformer works at a single power level, which is regulated by switching pulses. In contrast, the inverter technology can linearly control the microwave output power. This allows more precise cooking and a better defrosting quality. A drawback of the inverter system is its sensitiveness to high temperatures, due to the high power demand during operation and to the oven environment. The temperature distribution on the inverter components is analysed and a proper cooling system, that allows their correct operation in all conditions, is developed. In parallel, the research group of applied electromagnetics of the University of Parma is studying the effects of high power demand in fiber lasers. High power fiber laser operation is illustrated and the onset of the transverse mode instability (TMI) detrimental phenomenon is described. Different strategies to increase the TMI threshold, based on fiber geometry variations, are listed. A new class of fiber lasers, based on photonic crystal fiber (PCF), is introduced to overcome these limitations. Three different types of fibers, Symmetry-free PCFs, Fully-aperiodic PCFs and Multi-core PCFs, characterized by specific geometries and guiding properties, have been analysed. For this purpose, a modal solver based on the finite element method is used, together with a custom-developed software. The fibers are thermally simulated, both individually and as part of an optical amplifier, and their maximum thermal load is defined, considering different cooling conditions

Innovative sources of neural crest - derived ectomesenchymal stem cells for hepatic bioengineering

Liver disorders affect more than 100 million people worldwide and the therapeutic approaches for severe forms such as fulminant toxic hepatitis, cirrhosis, cancer and autoimmune liver disease remain palliative, awaiting for an organ transplant from a donor. An innovative approach involves intrahepatic transplantation of adult, multipotent stem cells of extrahepatic origin, whose up to now the mostly widely used have been mesenchymal stromal cells (MSC) from the bone marrow (bm-MSC). Specifically, MSC have been preliminarily in vitro or post-transplant in vivo induced to hepatocyte differentiation, and then associated with a biocompatible heterologous three-dimensional (3D) support like a decellularized or biostamped collagen matrix. Indeed, the 3D geometry of the matrix is believed to regulate the 3D self-assembly and differentiation of hepatic progenitors, as predicted by Gerald Edelman's Topobiological Theory. Since MSC from bone marrow and other sources exhibit a number of limitations in hepatic differentiation, the aim of my study has been the identify and evaluate innovative sources of adult stem cells suitable for in vitro and ex situ (i.e. once used for a 3D matrix recellularization) hepatic differentiation. My interest has been focussed on the still poorly investigated differentiation potential of adult ectomesenchymal cells of neural crest origin (i.e. adult neuroectodermal stem cells), like those expected to be found in the adult thyroid gland and the cartilage of the nasal septum. In particular, recent evidence collected by the group of research leaded by Prof. Roberto Toni at the University of Parma, in Italy has shown that adult stem cells can in vitro be isolated and expanded using long-term (2-4 months) primary cultures from the male rat thyroid. Preliminary data obtained in collaboration with Prof. Stephen Pennington of the UCD – Ireland on their proteomic signature have shown a very high expression of mesenchymal and, to a lesser extent endodermal protein markers. However, a minor fraction of these markers also includes proteins from a neuroectodermal lineage, in agreement with very recent evidence that the neural crest contribute to the development of the thyroid gland giving rise to its stromal matrix and, possibly to other still unknown progenitor cells. Since these cultures of adult rat stem cells can be in vitro differentiated to functional thyrocytes (that are classical endodermal cells like the hepatocytes), I reasoned that they could represent an innovative model source for hepatic differentiation. To reach this goal, in the first year of my PhD program I preliminary widened the previously acquired data on the proteomic signature of our rat thyroid stem cells / progenitors (TSC/P) using an LC-MS/MS SRM technique based on in silico sequences of not previously investigated proteins of endodermal, mesodermal, and neuroectodermal lineage as well as markers of epithelial-mesenchymal transition. A semiquantitative evaluation based on score values depicted an increase in the number of markers of neuroectodermal lineage, and an evidence of some proteins of epithelial-mesenchymal transition. These results support the original assumption of our group that a large fraction of our rat TSC/P are ectomesenchymal cells of neural crest origin, although a minor effect on the induction of a mesenchymal phenotype was triggered by the monolayer culture per se, as suggested by the presence of few markers of epithelial-mesenchymal transition. In addition, using light microscopic histochemistry, immunocytochemistry, and scanning electron microscopy I evaluated the hepatic differentiation of our rat TSC/P tested with two protocols of in vitro differentiation, both originally developed in our lab. at UNIPR. Of these, only one (n.1) was able to induce a satisfactory hepatocyte differentiation. A key role in this result was likely played by the use of Hepatocyte Growth Factor (HGF), whose Met receptor is expressed in neural crest and neural crest- derived cells like the ectomesenchyme, and also in differentiated hepatocytes and thyrocytes suggesting a great affinity of our TSC/P for an hepatic lineage. As a comparison, in the second year of my PhD program I studied a similar differentiation procedure using the currently believed, cellular gold standard for hepatic differentiation i.e. adult male rat bm-MSCs. Based on the same technologies as during the first year of work but in contrast to TSC/P, none of the two differentiation protocols used provided in vitro satisfactory development of bm-MSCs to hepatocytes. Since HGF may result in downregulation of MSC differentiation to hepatocytes when administered for long periods of times (as in our models), and use of Epidermal Growth Factor should be delivered at periods earlier than those chosen in our protocols, it is concluded that the weak differentiation results obtained depend on differentiation protocols for MSC not efficient enough when their multipotentiality is lowered by repeated culture passages from P3 ahead. In the third year of my PhD program, I substantiated the results on the proteomic signature of TSC/P and bm-MSC following their hepatic differentiation using qualitative LC-LIT-Orbitrap XL. Remarkably, hepatic differentiation of TSC/P with the protocol that had shown the best immunocytochemical performances during the first year of work (n.1) confirmed upregulation of the hepatic marker albumin, and revealed presence of S100-A6, a protein associated with activated hepatic stellate cells. Since the latter are a normal component of the liver in vivo, and are of mesodermal origin, the present in vitro evidence supports the original assumption that the large majority of our TSC/P are cell from an ectomesenchymal, neural-crest derived lineage easily driven to hepatocytes once triggered with adequate differentiation factors. In contrast, MSC differentiated with both previous protocols exhibited a proteomic profile with numerous common markers, confirming their immaturity and resistance to hepatic differentiation in the experimental setting applied. Finally, in search for a new source of neural crest-derived, ectomesenchymal cells potentially suitable for hepatic differentiation in humans (as those found in the rat and previously described), I engaged myself in the detailed lineage and molecular characterization of a very peculiar type of adult stem cells, obtained from the cartilage of the human nasal septum. This work has been developed between 2017 and 2018 under the tenure of my PhD Mobility Program, and in collaboration with the group of Prof. Ivan Martin at the Laboratory of Tissue Engineering / Department of Surgery and Biomedicine of the University Hospital in Basel, Switzerland. Prof. Martin and his group originally studied the gene expression of these cells, and used them as a new mean for repair of articular cartilage defects. They are adult nasal chondrocytes, taken following in vivo surgical sampling, and then in vitro de-differentiated. Recent collaborative studies between the groups of Prof. Toni and Prof. Martin have shown that a number of neuroectodermal markers of these cells can be found in the nasal septum of the human embryo, and can be recognized in the adult nasal chondrocytes by flow cytometry. During this last year of my PhD and using immunocytochemistry, I have evaluated the distribution of the majority of those embryonic markers also in primary cultures of adult nasal chondrocytes. In addition, using statistical analysis I have re-analyzed and re-calculated the quantitative results previously obtained by flow cytometry in nasal chondrocytes on the distribution of markers of mesodermal and neuroectodermal origin. Finally, using qualitative LC-LIT-Orbitrap XL I have obtained a preliminary profile of these adult stem cells showing a molecular setting based on downregulation of glycolytic enzymes and some DNA-associated histones as opposed to upregulation of a number of proteins involved in morphogenetic and developmental processes. Collectively, these data suggest that nasal chondrocytes are prone to a high plasticity of their phenotype, as expected for multipotent stem cells. Since flow cytometry showed that more than 80% of them express cytoplasmic and membrane markers consistent with ectomesenchymal neural crest-derived cells, the current proteomic profile (although still incomplete) supports the original hypothesis that they might successfully be induced to differentiate to cell lineages similar to those observed with ectomesenchymal, neural crest-derived rat thyroid stem cells. As such, I expect that these adult human stem cells may represent a new source for differentiation to human hepatocytes, and become a very innovative tool for regenerative medicine and tissue engineering in disorders of the human liver

Simulazione microscopica del traffico ed esecuzione di manvore attraverso tecniche di multi-agent deep reinforcement learning

In questa tesi vengono fornite le basi, gli algoritmi ed alcuni esempi pratici per la realizzazione di un simulatore sintetico, i cui guidatori, seppur virtuali, sono in grado di seguire comportamenti complessi e hanno capacità di negoziazione, avvicinando quindi i comportamenti dei guidatori umani; inoltre, può essere assegnato a ciascun agente un differente comportamento, così che possa essere simulata anche la diversità nello stile di guida insita in ciascun individuo. Questo simulatore, che ha portato all'ideazione di un nuovo algoritmo multiagente di Deep Reinforcement Learning, è stato principalmente pensato per lo sviluppo di algoritmi di machine-learning il cui obiettivo è quello di essere efficienti nel mondo reale nonostante siano stati addestrati in simulazione. Nella seconda parte della tesi, infatti, questo simulatore viene utilizzato per lo sviluppo di un modulo di alto livello per l'esecuzione della manovra relativa all'inserimento in rotatoria da parte di un veicolo autonomo usando tecniche di Deep Reinforcement Learning, che sia in grado di interagire con altri utenti della strada e guidare il veicolo anche attraverso situazioni di traffico denso dove algoritmi classici porterebbero ad attese indefinite.In this thesis, the basis, algorithms and practical examples are given for the development of a synthetic simulator, whose drivers, even if virtual, are endowed with human-like behaviors and able to navigate complex situations; moreover, to each agent can be assigned a different behavior, so that also the natural diversity among human driving styles can be simulated. This simulator, which brought to the design of a novel multi-agent Deep Reinforcement Learning algorithm, has been thought for being deployed for the development of machine-learning algorithms, whose goal is to be efficient in real-world despite being trained in simulation. Indeed, in the second part of the thesis, this simulator has been used for the development of a high-level module able to guide self-driving vehicles in roundabout insertions; this module, based on Deep Reinforcement Learning techniques, is able to negotiate with humans and accomplishing the maneuver even in dense traffic conditions where classical approaches would lead to undefinite waits

Novel HDPE-based bio-composites for bones replacement application

In the present thesis, Alumina toughened Zirconia (ATZ) and Chitosan have been studied as filler of high density polyethylene (HDPE) with the purpose to achieve novel bio-composites intended for bone replacement applications. The composites have been prepared by melt extrusion technique. Several composites were prepared by melt extrusion technique using different amount of ATZ (1, 2, 3, 5, 7 and 12wt%), obtaining a good dispersion in the polymer matrix, especially at low concentrations. Big aggregates were never formed during the melt processing, suggesting that the procedure was suitable to obtain a good dispersion of the filler in the composites. ATZ presence up to 3wt% leads to an increase of the mechanical properties of the matrix (young modulus and tensile strength), whereas for higher amount a decrease of tensile strength was recorded. ATZ was also effective in reducing the COF and enhancing the wear resistance of the polymer matrix. Among the many interesting properties that chitosan can give to a biomaterial, its proven antibacterial activity can play a fundamental role to avoid possible complication due to infections onset in the surgical site. Chitosan in form of both microparticles and nanoparticles (chitosan M and N) was considered. Two set of composites (i.e. a set for each type of chitosan) were then prepared by melt extrusion technique using different amount of filler. Both chitosan M and N lead to an improving of the composites mechanical properties. In particular, the storage modulus at 40°C for the HDPE/chitosan 98/2 strongly increases with respect to the neat HDPE. In the last part of this thesis work, radiation-induced grafting of HDPE-ATZ composites with chitosan was studied. The induced surface modification was aimed at promoting the interaction between chitosan and composites, in order to match the reinforcing effect of ATZ with the antimicrobial activity of chitosan. The grafting was initiated by irradiation with a low energy electron beam system. Three different type of samples were selected for grafting process (1/99, 2/98 and 7/93 ATZ/HDPE), based on their promising mechanical properties, under the most efficient reaction time assessed on the HDPE (1 hours and 3 hours). Contact angle and ATR measurements have shown evidence of successful grafting process only on the 1/99 ATZ/HDPE composite. In the end, among the materials prepared in this work, the most interesting composites in terms of mechanical properties were subjected to fibroblastic cell adhesion and viability, in order to evaluate the influence of ATZ, chitosan and radiation processing on the biological behavior of the HDPE. Although further research is needed to assess the interaction with more specific cell lines such as osteoblasts and chondrocytes (in order to simulate bones and cartilages), results indicate that both ATZ and chitosan are promising fillers for composites intended for biomedical applications. Indeed, materials with a few amount of filler (2%), were able to improve both cell adhesion and cell viability of the neat HDPE. However, a content of chitosan higher than 2% resulted in a viability lower than the pure polymer. This phenomenon could be related to the ascertained antimicrobial activity of chitosan, that, probably, can results in a detrimental effect on the cells only when the percentage overcomes a threshold value

Studi biofisici su fotorecettori da batteri associati alle piante

The project here presented aims at understanding the photophysical and photochemical properties of: a. biliverding-binding phytochromes, i.e. photoreceptors for red light, from the plant-associated bacterial species Pseudomonas syringae pv tomato (Pst), Pseudomonas aeruginosa (Pa), Xanthomonas campestris pv. campestris (Xcc) and the fungus Aspergillus nidulans: b. photoreceptors for red and blue light from Methylobacterium radiotolerans (Mr), belonging to the plant microbiota. The former are important and well-studied pathogens of agronomic plants, while Mr is a methylotrophic phytosymbiont of great industrial and agronomical interest. This work starts with biliverdin-bindig photoreceptors, 1 as recent works have shown that the photoreceptors from Pst and Xcc play a role in controlling infectivity, virulence and invasiveness towards the model plant Arabidopsis thaliana and citrus plants. In the case of Mr, instead, the functional role of these photoactive proteins still awaits investigation, despite the finding that a genomic survey indicates that these organisms may possess quite a large number of potential and diverse photoreceptors for visible light. The proteins were studied at the molecular level with an array of biophysical techniques: primarily steady-state and time-resolved optical spectroscopy, then time-resolved photoacoustics. Beside the molecular and spectroscopic characterisation of these photoreceptors and their interest as photosensors in prokaryots, the blue-light photoreceptor Mr4511 showed a particular feature: in contrast to the majority of LOV domains, this protein lacks the, in other LOV photoreceptors highly conserved, tryptophan residue, which was previously identified as the major quencher for the FMN triplet-state in LOV-based singlet oxygen (SO) photosensitisers. This experimental work demonstrates that “for Mr4511 it is sufficient to only mutate the reactive cysteine responsible for the photocycle (Cys71) in the native protein to generate an efficient SO photosensitiser: both C71S and C71G variants exhibit SO quantum yields of formation ΦΔ around 0.2 in air-saturated solutions. Under oxygen saturated conditions, ΦΔ reaches ~ 0.5 in deuterated buffer. Also, this protein showed to be exceedingly robust against denaturation with urea and it is more photostable than free FMN.” 2 As a whole, future continuation on this work could reveal the novel potential of photoreceptors from bacteria that are part of plant microbiota, i.e. for environmental, agronomical and biotechnological applications

Reliable molecular markers to assess the potential genotoxicity of metal based ENM in plants

Risk assessment of potential deleterious effects of engineered nanomaterials (ENMs) on human health and environment requires implementation of reliable and versatile tests. Genotoxicity deriving from acute and chronic exposure is one of the main issues related to ENMs. In the first and second year of my PhD, analyses on CdS QDs were conducted using the model plant A. thaliana, accession Landsberg erecta (Ler-0). In particular, Random Amplified polymorphic DNA (34 primers, to investigate potential genotoxic effects on genomic DNA with the use of End Point PCR) and Real Time Quantitative PCR (6 Cp and 5 Mt gene primers, to investigate genotoxic effects on chloroplast and mitochondrion) analyses were performed on plants exposed to a range of CdS QDs concentrations (0, 40, 80, 150, 250 mg/L), and compared with effects of CdSO4 (50, 100 µM) after 0, 10 and 20 days of exposure. While RAPDs analysis permit to highlights that CdS QDs are not responsible of visible genotoxic effects on genomic DNA, RT-qPCR permitted to measure how genes change their relative quantities (RQ) in the different test conditions. Genes YCF1 and PSAC (both Cp genes) decrease significantly in RQ in plants exposed to higher concentration of CdS QDs, while Cp genes PSBA and PSBD increase significantly in RQ at higher time of exposure to CdS QDs. Mt genes COB and COX were find to be statistically more abundant in plants stressed with higher CdSO4 concentrations. In the third year of my PhD the same set of RAPDs, Cp and Mt genes were studied on plants of A. thaliana stressed with new types of ENMs and their relative salts: CeO2 NPs and CeCl3; Fe2O3 NPs, Fe3O4 NPs and FeCl3; ZnS QDs and ZnSO4. Tested concentration of each ENMs and salts was determined after repeated toxicity test to identify minimum inhibition concentration (MIC). ½ MICs was used to stress plants for 20 d of exposure. For all ENMs ½ MIC corresponds to 500 mg/L, for CeCl3 and ZnSO4 salts ½ MIC = 175 mg/L, and for FeCl3 ½ MIC = 75 mg/L. All genotoxic analyses were accompanied with physiological analyses to determine chlorophyll a, b and carotenoids concentration, respiration activity and lipid peroxidation. Even in this cases RAPDs analyses permit to highlight how ENMs exposition don’t cause visible genotoxic effects on genomic DNAs. Contrarily, RT-qPCR permits to highlight how CeO2 ENMs don’t influence in significative way the RQ of both Cp and Mt genes (although a general increase in RQ was observed). In the case of plants exposed to Fe based ENMs and Fe salt, all Cp and Mt genes appears statistically more abundant although differences in RQs were observed between ENMs; these results is explicable considering differences in Fe ion charges released by the ENMs. Finally, ZnO2 QDs seem to cause a general significant increase in RQ for both Cp and Mt genes, and results similar for what observed in plant exposed to Zn salt. Principal Component Analyses (PCA) were performed to highlights possible trends between physiological parameters and genes RQs. PCA performed between data from photosynthetic pigment concentrations and Cp genes RQs permit to see how an increase in pigments concentrations was related with an increase in RQs. This result, in conformity with literature, is explicable with the fact that ENMs can damage chlorophylls and carotenoids forcing plants to increase the number of chloroplast, and consequently the number of Cp genomes with respectively genes. PCA performed between respiration rates and Mt genes RQs highlights how an increase of respiration activity is related to an increase in RQs; similarly, at the previous situation, ENMs can damage mitochondria functionality forcing plants to increase the number of mitochondria and their relative genomes. Finally, PCA performed between lipid peroxidation data and Mt RQs highlights how an increase of oxidative stress was associated with a decrease in Mt genes RQs; according to the literature, ENMs toxicity seem to be related principally with ROS production that can damage lipids, proteins and genetic materials. In all PCAs it’s clearly possible to see how more unstable ENMs (ZnO2 QDs, Fe2O3 NPs and Fe3O4 NPs) cluster together and with their relative salts, indicating how their behavior it’s similar one each other and the release of respective metal ions it’s similar to salts. Contrarily, CeO2 NP cluster principally with control, indicating its relative stability and limited toxicity

Mixing processes and buoyancy transport in stratified turbulent flows

This thesis attempts to increase the overall understanding of turbulent mixing by exploring the controls on the vertical buoyancy transport in a stratified turbulent Taylor-Couette flow. The inner cylinder of the tank, of radius R1, rotates, while the outer cylinder, of radius R2, is fixed, and the gap \Delta R=R2-R1 is filled with fluid up to a depth H, so that the aspect ratio H/ \Delta R=2.7-2.8. To simulate the global overturning circulation that perpetually carries and redistributes heat, salt and carbon between ocean basins at different rates, we conduct a series of experiments where we vary the rotation of the inner cylinder, \Omega. In addition, we model rainfall and ice melting on the ocean surface by supplying a fresh water flux at the top of the tank, while the dense currents released by ice formation at the Antartica are modeled by a saline water flux supplied at the base of the tank. At the same time, we vent the same flux as the supply by two sinks located at the same depth of the respective sources. In our stylized experiments, the diapycnal mixing through isopycnal surfaces can be associated to the salt flux extracted at the top, whereas the upwelling flux can be measured by the fluid extracted at the bottom. In our experiments, we also vary the salinity of the bottom source, in order to simulate different interglacial periods. Firstly, we found that in the unsaturated regime, the vertical buoyancy flux is rate-limited by the salinity of the bottom source and it depends linearly on the buoyancy frequency, N, while in the saturated regime, it is rate-limited by turbulence, it is independent on N and proportional to \Omega^3, matching the equivalent flux through a two-layer or multi-layer stratification, or through the interfaces that spontaneously form in a linear stratified fluid, for sufficiently high initial stratification. In this thesis, we will also discuss the influence of the initial condition and of the position of the sources and sinks on the steady-state stratification, as well as the effects of a coupling between turbulence and advection on the diffusivity D. Secondly, we look in more detail at the mixing mechanisms responsible for mixing and buoyancy transport across a density interface. The density interfaces are very common in the natural environment. A typical example is the thermocline, which separates the upper mixed region of the ocean from the stratified ocean interior. In our experiments, in order to prevent homogenization, we stabilize the density interface by adding a source of fresh water and a source of dense water at the top and bottom of the tank respectively. Analogously to the prior set-up, we withdraw the same volume flux as the supply by two sinks, and wait for the steady state before measuring the density and velocity field and recording videos of the visible mixing phenomena. We find that the dominant mixing mechanism is localized at the interface and it is diffusive, as the time averaged correlation of density and vertical velocity fluctuations at the interface is a good approximation of the total vertical salt transport. We will also present the velocity and turbulence field, as well as the macro (integral) and micro (Taylor) length scales in the vertical, radial and azimuthal direction. The mixing mechanism in a two-layer fluid also involves a single wakelike perturbation that originates at the interface, close to the inner cylinder, and spreads out radially and azimuthally, with a peak period Tp \propto 1/\Omega, and a coeffcient of proportionality equal to 12\pi. From the shadowgraph images we infer that the mixing phenomenon, able to break the sharp gradients and to promote classical diffusion, is intermittent and lasts for approximately 40% of the peak period, causing the interface to fade away and letting parcels of intermediate density be vertically transported. Finally, we develop a model for the salt diffusivity based on diffusion inside the trailing edges of the wake