Application of different instrumental techniques to the selective evaluation of bio-sensible compounds in foodstuff of animal and vegetable origin

This PhD thesis describes the application of some instrumental analytical techniques suitable to the study of fundamental food products for the human diet, such as: extra virgin olive oil and dairy products. These products, widely spread in the market and with high nutritional values, are increasingly recognized healthy properties although their lipid fraction might contain some unfavorable components to the human health. The research activity has been structured in the following investigations: “Comparison of different techniques for trans fatty acids analysis” “Fatty acids analysis of outcrop milk cream samples, with particular emphasis on the content of Conjugated Linoleic Acid (CLA) and trans Fatty Acids (TFA), by using 100m high-polarity capillary column” “Evaluation of the oxidited fatty acids (OFA) content during the Parmigiano-Reggiano cheese seasoning” “Direct analysis of 4-desmethyl sterols and two dihydroxy triterpenes in saponified vegetal oils (olive oil and others) using liquid chromatography-mass spectrometry” “Quantitation of long chain poly-unsatured fatty acids (LC-PUFA) in base infant formulas by Gas Chromatography, and evaluation of the blending phases accuracy during their preparation” “Fatty acids composition of Parmigiano Reggiano cheese samples, with emphasis on trans isomers (TFA)”

Bio-physical interactions and feedbacks in a global climate model

This PhD thesis addresses the topic of large-scale interactions between climate and marine biogeochemistry. To this end, centennial simulations are performed under present and projected future climate conditions with a coupled ocean-atmosphere model containing a complex marine biogeochemistry model. The role of marine biogeochemistry in the climate system is first investigated. Phytoplankton solar radiation absorption in the upper ocean enhances sea surface temperatures and upper ocean stratification. The associated increase in ocean latent heat losses raises atmospheric temperatures and water vapor. Atmospheric circulation is modified at tropical and extratropical latitudes with impacts on precipitation, incoming solar radiation, and ocean circulation which cause upper-ocean heat content to decrease at tropical latitudes and to increase at middle latitudes. Marine biogeochemistry is tightly related to physical climate variability, which may vary in response to internal natural dynamics or to external forcing such as anthropogenic carbon emissions. Wind changes associated with the North Atlantic Oscillation (NAO), the dominant mode of climate variability in the North Atlantic, affect ocean properties by means of momentum, heat, and freshwater fluxes. Changes in upper ocean temperature and mixing impact the spatial structure and seasonality of North Atlantic phytoplankton through light and nutrient limitations. These changes affect the capability of the North Atlantic Ocean of absorbing atmospheric CO2 and of fixing it inside sinking particulate organic matter. Low-frequency NAO phases determine a delayed response of ocean circulation, temperature and salinity, which in turn affects stratification and marine biogeochemistry. In 20th and 21st century simulations natural wind fluctuations in the North Pacific, related to the two dominant modes of atmospheric variability, affect the spatial structure and the magnitude of the phytoplankton spring bloom through changes in upper-ocean temperature and mixing. The impacts of human-induced emissions in the 21st century are generally larger than natural climate fluctuations, with the phytoplankton spring bloom starting one month earlier than in the 20th century and with ~50% lower magnitude. This PhD thesis advances the knowledge of bio-physical interactions within the global climate, highlighting the intrinsic coupling between physical climate and biosphere, and providing a framework on which future studies of Earth System change can be built on.

Structural and functional characterization of Group B Streptococcus class C sortases

In Group B Streptococcus (GBS) three structurally distinct types of pili have been discovered as potential virulence factors and vaccine candidates. The pilus-forming proteins are assembled into high-molecular weight polymers via a transpeptidation mechanism mediated by specific class C sortases. Using a multidisciplinary approach including bioinformatics, structural and biochemical studies and in vivo mutagenesis we performed a broad characterization of GBS sortase C. The high resolution X-ray structure of the enzymes revealed that the active site, located into the β-barrel core of the enzyme, is made of the catalytic triad His157-Cys219-Arg228 and covered by a loop, known as the “lid”. We show that the catalytic triad and the predicted N- and C-terminal trans-membrane regions are required for the enzyme activity. Interestingly, by in vivo complementation mutagenesis studies we found that the deletion of the entire lid loop or mutations in specific lid key residues had no effect on catalytic activity of the enzyme. In addition, kinetic characterizations of recombinant enzymes indicate that the lid mutants can still recognize and cleave the substrate-mimicking peptide at least as well as the wild type protein.

Advanced CMOS Interfaces for Bio-Nanosensors

The improvement of devices provided by Nanotechnology has put forward new classes of sensors, called bio-nanosensors, which are very promising for the detection of biochemical molecules in a large variety of applications. Their use in lab-on-a-chip could gives rise to new opportunities in many fields, from health-care and bio-warfare to environmental and high-throughput screening for pharmaceutical industry. Bio-nanosensors have great advantages in terms of cost, performance, and parallelization. Indeed, they require very low quantities of reagents and improve the overall signal-to-noise-ratio due to increase of binding signal variations vs. area and reduction of stray capacitances. Additionally, they give rise to new challenges, such as the need to design high-performance low-noise integrated electronic interfaces. This thesis is related to the design of high-performance advanced CMOS interfaces for electrochemical bio-nanosensors. The main focus of the thesis is: 1) critical analysis of noise in sensing interfaces, 2) devising new techniques for noise reduction in discrete-time approaches, 3) developing new architectures for low-noise, low-power sensing interfaces. The manuscript reports a multi-project activity focusing on low-noise design and presents two developed integrated circuits (ICs) as examples of advanced CMOS interfaces for bio-nanosensors. The first project concerns low-noise current-sensing interface for DC and transient measurements of electrophysiological signals. The focus of this research activity is on the noise optimization of the electronic interface. A new noise reduction technique has been developed so as to realize an integrated CMOS interfaces with performance comparable with state-of-the-art instrumentations. The second project intends to realize a stand-alone, high-accuracy electrochemical impedance spectroscopy interface. The system is tailored for conductivity-temperature-depth sensors in environmental applications, as well as for bio-nanosensors. It is based on a band-pass delta-sigma technique and combines low-noise performance with low-power requirements.

Development of newly conceived biomimetic nano-structured biomaterials as scaffolds for bone and osteochondral regeneration

The present research thesis was focused on the development of new biomaterials and devices for application in regenerative medicine, particularly in the repair/regeneration of bone and osteochondral regions affected by degenerative diseases such as Osteoarthritis and Osteoporosis or serious traumas. More specifically, the work was focused on the synthesis and physico-chemical-morphological characterization of: i) a new superparamagnetic apatite phase; ii) new biomimetic superparamagnetic bone and osteochondral scaffolds; iii) new bioactive bone cements for regenerative vertebroplasty. The new bio-devices were designed to exhibit high biomimicry with hard human tissues and with functionality promoting faster tissue repair and improved texturing. In particular, recent trends in tissue regeneration indicate magnetism as a new tool to stimulate cells towards tissue formation and organization; in this perspective a new superparamagnetic apatite was synthesized by doping apatite lattice with di-and trivalent iron ions during synthesis. This finding was the pin to synthesize newly conceived superparamagnetic bone and osteochondral scaffolds by reproducing in laboratory the biological processes yielding the formation of new bone, i.e. the self-assembly/organization of collagen fibrils and heterogeneous nucleation of nanosized, ionically substituted apatite mimicking the mineral part of bone. The new scaffolds can be magnetically switched on/off and function as workstations guiding fast tissue regeneration by minimally invasive and more efficient approaches. Moreover, in the view of specific treatments for patients affected by osteoporosis or traumas involving vertebrae weakening or fracture, the present work was also dedicated to the development of new self-setting injectable pastes based on strontium-substituted calcium phosphates, able to harden in vivo and transform into strontium-substituted hydroxyapatite. The addition of strontium may provide an anti-osteoporotic effect, aiding to restore the physiologic bone turnover. The ceramic-based paste was also added with bio-polymers, able to be progressively resorbed thus creating additional porosity in the cement body that favour cell colonization and osseointegration.

Hybrid portable systems for bio-nanosensors

The promising development in the routine nanofabrication and the increasing knowledge of the working principles of new classes of highly sensitive, label-free and possibly cost-effective bio-nanosensors for the detection of molecules in liquid environment, has rapidly increased the possibility to develop portable sensor devices that could have a great impact on many application fields, such as health-care, environment and food production, thanks to the intrinsic ability of these biosensors to detect, monitor and study events at the nanoscale. Moreover, there is a growing demand for low-cost, compact readout structures able to perform accurate preliminary tests on biosensors and/or to perform routine tests with respect to experimental conditions avoiding skilled personnel and bulky laboratory instruments. This thesis focuses on analysing, designing and testing novel implementation of bio-nanosensors in layered hybrid systems where microfluidic devices and microelectronic systems are fused in compact printed circuit board (PCB) technology. In particular the manuscript presents hybrid systems in two validating cases using nanopore and nanowire technology, demonstrating new features not covered by state of the art technologies and based on the use of two custom integrated circuits (ICs). As far as the nanopores interface system is concerned, an automatic setup has been developed for the concurrent formation of bilayer lipid membranes combined with a custom parallel readout electronic system creating a complete portable platform for nanopores or ion channels studies. On the other hand, referring to the nanowire readout hybrid interface, two systems enabling to perform parallel, real-time, complex impedance measurements based on lock-in technique, as well as impedance spectroscopy measurements have been developed. This feature enable to experimentally investigate the possibility to enrich informations on the bio-nanosensors concurrently acquiring impedance magnitude and phase thus investigating capacitive contributions of bioanalytical interactions on biosensor surface.

La migrazione del chagas: Costruzione bio-medica e socio-politica delle malattie tropicali dimenticate

Questo studio propone un'esplorazione dei nessi tra processi migratori ed esperienze di salute e malattia a partire da un'indagine sulle migrazioni provenienti dall'America latina in Emilia-Romagna. Contemporaneamente indaga i termini del dibattito sulla diffusione della Malattia di Chagas, “infezione tropicale dimenticata” endemica in America centro-meridionale che, grazie all'incremento dei flussi migratori transnazionali, viene oggi riconfigurata come 'emergente' in alcuni contesti di immigrazione. Attraverso i paradigmi teorico-metodologici disciplinari dell'antropologia medica, della salute globale e degli studi sulle migrazioni, si è inteso indagare la natura della relazione tra “dimenticanza” ed “emergenza” nelle politiche che caratterizzano il contesto migratorio europeo e italiano nello specifico. Si sono analizzate questioni vincolate alla legittimità degli attori coinvolti nella ridefinizione del fenomeno in ambito pubblico; alle visioni che informano le strategie sanitarie di presa in carico dell'infezione; alle possibili ricadute di tali visioni nelle pratiche di cura. Parte della ricerca si è realizzata all'interno del reparto ospedaliero ove è stato implementato il primo servizio di diagnosi e trattamento per l'infezione in Emilia-Romagna. È stata pertanto realizzata una etnografia fuori/dentro al servizio, coinvolgendo i principali soggetti del campo di indagine -immigrati latinoamericani e operatori sanitari-, con lo scopo di cogliere visioni, logiche e pratiche a partire da un'analisi della legislazione che regola l'accesso al servizio sanitario pubblico in Italia. Attraverso la raccolta di narrazioni biografiche, lo studio ha contribuito a far luce su peculiari percorsi migratori e di vita nel contesto locale; ha permesso di riflettere sulla validità di categorie come quella di “latinoamericano” utilizzata dalla comunità scientifica in stretta correlazione con il Chagas; ha riconfigurato il senso di un approccio attento alle connotazioni culturali all'interno di un più ampio ripensamento delle forme di inclusione e di partecipazione finalizzate a dare asilo ai bisogni sanitari maggiormente percepiti e alle esperienze soggettive di malattia.

Physical land degradation and loss of soil fertility: soil structural stability and bio-physical indicators

This study investigates the changes in soil fertility due to the different aggregate breakdown mechanisms and it analyses their relationships in different soil-plant systems, using physical aggregates behavior and organic matter (OM) changes as indicators. Three case studies were investigated: i) an organic agricultural soil, where a combined method, aimed to couple aggregate stability to nutrients loss, were tested; ii) a soil biosequence, where OM chemical characterisation and fractionation of aggregates on the basis of their physical behaviour were coupled and iii) a soils sequence in different phytoclimatic conditions, where isotopic C signature of separated aggregates was analysed. In agricultural soils the proposed combined method allows to identify that the severity of aggregate breakdown affected the quantity of nutrients lost more than nutrients availability, and that P, K and Mg were the most susceptible elements to water abrasion, while C and N were mainly susceptible to wetting. In the studied Chestnut-Douglas fir biosequence, OM chemical properties affected the relative importance of OM direct and indirect mechanisms (i.e., organic and organic-metallic cements, respectively) involved in aggregate stability and nutrient losses: under Douglas fir, high presence of carboxylate groups enhanced OM-metal interactions and stabilised aggregates; whereas under Chestnut, OM directly acted and fresh, more C-rich OM was preserved. OM direct mechanism seemed to be more efficient in C preservation in aggregates. The 13C natural abundance approach showed that, according to phytoclimatic conditions, stable macroaggregates can form both around partially decomposed OM and by organic-mineral interactions. In topsoils, aggregate resistance enhanced 13C-rich OM preservation, but in subsoils C preservation was due to other mechanisms, likely OM-mineral interactions. The proposed combined approach seems to be useful in the understanding of C and nutrients fate relates to water stresses, and in future research it could provide new insights into the complexity of soil biophysical processes.

Electrochemical surface modification of Single walled carbon nanotubes and graphene-based electrodes for (bio)sensing applications

Sensors are devices that have shown widespread use, from the detection of gas molecules to the tracking of chemical signals in biological cells. Single walled carbon nanotube (SWCNT) and graphene based electrodes have demonstrated to be an excellent material for the development of electrochemical biosensors as they display remarkable electronic properties and the ability to act as individual nanoelectrodes, display an excellent low-dimensional charge carrier transport, and promote surface electrocatalysis. The present work aims at the preparation and investigation of electrochemically modified SWCNT and graphene-based electrodes for applications in the field of biosensors. We initially studied SWCNT films and focused on their topography and surface composition, electrical and optical properties. Parallel to SWCNTs, graphene films were investigated. Higher resistance values were obtained in comparison with nanotubes films. The electrochemical surface modification of both electrodes was investigated following two routes (i) the electrografting of aryl diazonium salts, and (ii) the electrophylic addition of 1, 3-benzodithiolylium tetrafluoroborate (BDYT). Both the qualitative and quantitative characteristics of the modified electrode surfaces were studied such as the degree of functionalization and their surface composition. The combination of Raman, X-ray photoelectron spectroscopy, atomic force microscopy, electrochemistry and other techniques, has demonstrated that selected precursors could be covalently anchored to the nanotubes and graphene-based electrode surfaces through novel carbon-carbon formation.

Fully bio-based epoxy resins

Epoxy resins are mainly produced by reacting bisphenol A with epichlorohydrin. Growing concerns about the negative health effects of bisphenol A are urging researchers to find alternatives. In this work diphenolic acid is suggested, as it derives from levulinic acid, obtained from renewable resources. Nevertheless, it is also synthesized from phenol, from fossil resources, which, in the current paper has been substituted by plant-based phenols. Two interesting derivatives were identified: diphenolic acid from catechol and from resorcinol. Epichlorohydrin on the other hand, is highly carcinogenic and volatile, leading to a tremendous risk of exposure. Thus, two approaches have been investigated and compared with epichlorohydrin. The resulting resins have been characterized to find an appropriate application, as epoxy are commonly used for a wide range of products, ranging from composite materials for boats to films for food cans. Self-curing capacity was observed for the resin deriving from diphenolic acid from catechol. The glycidyl ether of the diphenolic acid from resorcinol, a fully renewable compound, was cured in isothermal and non-isothermal tests tracked by DSC. Two aliphatic amines were used, namely 1,4-butanediamine and 1,6-hexamethylendiamine, in order to determine the effect of chain length on the curing of an epoxy-amine system and determine the kinetic parameters. The latter are crucial to plan any industrial application. Both diamines demonstrated superior properties compared to traditional bisphenol A-amine systems.

Photophysical investigation of light-harvesting systems for solar-to-fuel conversion

In recent years, an increasing attention has been given to the optimization of the performances of new supramolecular systems, as antennas for light collection. In such background, the aim of this thesis was the study of multichromophoric architectures capable of performing such basic action. A synthetic antenna should consist of a structure with large UV-Vis absorption cross-section, panchromatic absorption, fixed orientation of the components and suitable energy gradients between them, in order to funnel absorbed energy towards a specific site, through fast energy-transfer processes. Among the systems investigated in this thesis, three suitable classes of compounds can be identified: 1) transition metal-based multichromophoric arrays, as models for antenna construction, 2) free-base trans-A2B-phenylcorroles, as self-assembling systems to make effective mimics of the photosynthetic system, and 3) a natural harvester, the Photosystem I, immobilized on the photoanode of a solar-to-fuel conversion device. The discussion starts with the description of the photophysical properties of dinuclear quinonoid organometallic systems, able to fulfil some of the above mentioned absorption requirements, displaying in some cases panchromatic absorption. The investigation is extended to the efficient energy transfer processes occurring in supramolecular architectures, suitably organized around rigid organic scaffolds, such as spiro-bifluorene and triptycene. Furthermore, the photophysical characterization of three trans-A2B-phenylcorroles with different substituents on the meso-phenyl ring is introduced, revealing the tendency of such macrocycles to self-organize into dimers, by mimicking natural self-aggregates antenna systems. In the end, the photophysical analysis moved towards the natural super-complex PSI-LHCI, immobilized on the hematite surface of the photoanode of a bio-hybrid dye-sensitized solar cell. The importance of the entire work is related to the need for a deep understanding of the energy transfer mechanisms occurring in supramolecules, to gain insights and improve the strategies for governing the directionality of the energy flow in the construction of well-performing antenna systems.