Analytical pyrolysis to study microplastics and other polymers in the environment

Synthetic polymers constitute a wide class of materials which has enhanced the quality of human life, providing comforts and innovations. Anyway, the increasing production and the incorrect waste management, are leading to the occurrence of polymers in the environment, generating concern. To understand the extent of this issue, analytical investigation holds an essential position. Standardised methods have not established yet, and additional studies are required to improve the present knowledge. The main aim of this thesis was to provide comprehensive information about the potential of pyrolysis coupled with gas-chromatography and mass spectrometry (Py-GC-MS) for polymers investigation, from their characterisation to their identification and quantification in complex matrices. Water-soluble (poly(dimethylsiloxanes), PDMS bearing poly(ethylene glycol), PEG, side chains) and water-insoluble polymers (microplastics, MPs, and bioplastics) were studied. The different studies revealed the possibility to identify heterogeneous classes of polymers, fingerprinting the presence of PDMS copolymers and distinguishing chemically different polyurethanes (PURs). The occurrence of secondary reactions in pyrolysis of polymer mixtures was observed as possible drawback. Pyrolysis products indicative of secondary reactions and their reaction mechanisms were identified. Py-GC-MS also revealed its fundamental role for the identification of polymers composing commercial bioplastics items based. The results aided to identify chemicals that have the potential to migrate in sea waters. Investigations of environmental samples demonstrated the capability of Py-GC-MS to provide reliable, reproducible and comparable results about polymers in complex matrices (PEG-PDMS in sewage sludges and PURs and other MPs in road dusts and spider webs). Criticisms were especially found in quantitation, such as the retrieval reference materials, the construction of reliable calibration protocols and the occurrence of bias due to interferences between pyrolysis products. This thesis pursues the greater purpose to develop harmonised and standardised methods for environmental investigations of polymers, that is fundamental to assess the real state of the environment.

Elastic Propagation in random media: applications to the imaging of volcano structures

High-frequency seismograms contain features that reflect the random inhomogeneities of the earth. In this work I use an imaging method to locate the high contrast small- scale heterogeneity respect to the background earth medium. This method was first introduced by Nishigami (1991) and than applied to different volcanic and tectonically active areas (Nishigami, 1997, Nishigami, 2000, Nishigami, 2006). The scattering imaging method is applied to two volcanic areas: Campi Flegrei and Mt. Vesuvius. Volcanic and seismological active areas are often characterized by complex velocity structures, due to the presence of rocks with different elastic properties. I introduce some modifications to the original method in order to make it suitable for small and highly complex media. In particular, for very complex media the single scattering approximation assumed by Nishigami (1991) is not applicable as the mean free path becomes short. The multiple scattering or diffusive approximation become closer to the reality. In this thesis, differently from the ordinary Nishigami’s method (Nishigami, 1991), I use the mean of the recorded coda envelope as reference curve and calculate the variations from this average envelope. In this way I implicitly do not assume any particular scattering regime for the "average" scattered radiation, whereas I consider the variations as due to waves that are singularly scattered from the strongest heterogeneities. The imaging method is applied to a relatively small area (20 x 20 km), this choice being justified by the small length of the analyzed codas of the low magnitude earthquakes. I apply the unmodified Nishigami’s method to the volcanic area of Campi Flegrei and compare the results with the other tomographies done in the same area. The scattering images, obtained with frequency waves around 18 Hz, show the presence of high scatterers in correspondence with the submerged caldera rim in the southern part of the Pozzuoli bay. Strong scattering is also found below the Solfatara crater, characterized by the presence of densely fractured, fluid-filled rocks and by a strong thermal anomaly. The modified Nishigami’s technique is applied to the Mt. Vesuvius area. Results show a low scattering area just below the central cone and a high scattering area around it. The high scattering zone seems to be due to the contrast between the high rigidity body located beneath the crater and the low rigidity materials located around it. The central low scattering area overlaps the hydrothermal reservoirs located below the central cone. An interpretation of the results in terms of geological properties of the medium is also supplied, aiming to find a correspondence of the scattering properties and the geological nature of the material. A complementary result reported in this thesis is that the strong heterogeneity of the volcanic medium create a phenomenon called "coda localization". It has been verified that the shape of the seismograms recorded from the stations located at the top of the volcanic edifice of Mt. Vesuvius is different from the shape of the seismograms recorded at the bottom. This behavior is justified by the consideration that the coda energy is not uniformly distributed within a region surrounding the source for great lapse time.

Liquid crystal polymers: macromolecular design for enhanced performances

During this work, done mainly in the laboratories of the department of Industrial Chemistry and Materials of the University of Bologna but also in the laboratories of the Carnegie Mellon University in collaboration with prof. K. Matyjaszewski and at the university of Zaragoza in collaboration with prof. J. Barberá, was focused mainly on the synthesis and characterization of new functional polymeric materials. In the past years our group gained a deep knowledge about the photomodulation of azobenzene containing polymers. The aim of this thesis is to push forward the performances of these materials by the synthesis of well defined materials, in which, by a precise control over the macromolecular structures, better or even new functionality can be delivered to the synthesized material. For this purpose, besides the rich photochemistry of azoaromatic polymers that brings to the application, the control offered from the recent techniques of controlled radical polymerization, ATRP over all, gives an enormous range of opportunity for the developing of a new generation of functional materials whose properties are determinate not only by the chemical nature of the functional center (e.g. azoaromatic chromophore) but are tuned and even amplified by a synergy with the whole macromolecular structure. Old materials in new structures. In this contest the work of this thesis was focused mainly on the synthesis and characterization of well defined azoaromatic polymers in order to establish, for the first time, precise structure-properties correlation. In fact a series of well defined different azopolymers, chiral and achiral, with different molecular weight and highly monodisperse were synthesized and their properties were studied, in terms of photoexpansion and photomodulation of chirality. We were then able to study the influence of the macromolecular structure in terms of molecular weight and ramification on the studied properties. The huge amount of possibility offered by the tailoring of the macromolecular structure were exploited for the synthesis of new cholesteric photochromic polymers that can be used as a smart label for the certification of the thermal history of any thermosensitive product. Finally the ATRP synthesis allowed us to synthesize a total new class of material, named molecular brushes: a flat surface covered with an ultra thin layer of polymeric chain covalently bond onto the surface from one end. This new class of materials is of extreme interest as they offer the possibility to tune and manage the interaction of the surface with the environment. In this contest we synthesized both azoaromatic surfaces, growing directly the polymer from the surface, and mixed brushes: surfaces covered with incompatible macromolecules. Both type of surfaces acts as “smart” surfaces: the first it is able to move the orientation of a LC cell by simply photomodulation and, thanks to the robustness of the covalent bond, can be used as a command surface overcoming all the limitation due to the dewetting of the active layer. The second type of surface, functionalized by a grafting-to method, can self assemble the topmost layer responding to changed environmental conditions, exposing different functionality according to different environment.

Study of the genes involved in Naphthenic acids (NAs) degradation by Rhodococcus spp.

Naphthenic acids (NAs) are an important group of organic pollutants mainly found in hydrocarbon deposits. Although these compounds are toxic, recalcitrant, and persistent in the environment, we are just learning the diversity of microbial communities involved in NAs- degradation and the mechanisms by which NAs are biodegraded. Studies have shown that naphthenic acids are susceptible to biodegradation, which decreases their concentration and reduces toxicity. Nevertheless, little is still known about their biodegradability. The present PhD Thesis’s work is aimed to study the biodegradation of simple model NAs using bacteria strains belonging to the Rhodococcus genus. In particular, Rh. sp. BCP1 and Rh. opacus R7 were able to utilize NAs such as cyclohexane carboxylic acid and cyclopentane carboxylic acid as the sole carbon and energy sources, even at concentrations up to 1000 mg/L. The presence of either substituents or longer carboxylic acid chains attached to the cyclohexane ring negatively affected the growth by pure bacterial cultures. Moreover, BCP1 and R7 cells incubated in the presence of CHCA or CPCA show a general increase of saturated and methyl-substituted fatty acids in their membrane, while the cis-mono-unsaturated ones decrease, as compared to glucose-grown cells. The observed lipid molecules modification during the growth in the presence of NAs is suggested as a possible mechanism to decrease the fluidity of the cell membrane to counteract NAs toxicity. In order to further evaluate this toxic effect on cell features, the morphological changes of BCP1 and R7 cells were also assessed through Transmission Electron Microscopy (TEM), revealing interesting ultrastructural changes. The induction of putative genes, and the construction of a random transposon mutagenesis library were also carried out to reveal the mechanisms by which these Rhodococcus strains can degrade toxic compounds such as NAs.

Rich media content adaptation in e-learning systems

The wide use of e-technologies represents a great opportunity for underserved segments of the population, especially with the aim of reintegrating excluded individuals back into society through education. This is particularly true for people with different types of disabilities who may have difficulties while attending traditional on-site learning programs that are typically based on printed learning resources. The creation and provision of accessible e-learning contents may therefore become a key factor in enabling people with different access needs to enjoy quality learning experiences and services. Another e-learning challenge is represented by m-learning (which stands for mobile learning), which is emerging as a consequence of mobile terminals diffusion and provides the opportunity to browse didactical materials everywhere, outside places that are traditionally devoted to education. Both such situations share the need to access materials in limited conditions and collide with the growing use of rich media in didactical contents, which are designed to be enjoyed without any restriction. Nowadays, Web-based teaching makes great use of multimedia technologies, ranging from Flash animations to prerecorded video-lectures. Rich media in e-learning can offer significant potential in enhancing the learning environment, through helping to increase access to education, enhance the learning experience and support multiple learning styles. Moreover, they can often be used to improve the structure of Web-based courses. These highly variegated and structured contents may significantly improve the quality and the effectiveness of educational activities for learners. For example, rich media contents allow us to describe complex concepts and process flows. Audio and video elements may be utilized to add a “human touch” to distance-learning courses. Finally, real lectures may be recorded and distributed to integrate or enrich on line materials. A confirmation of the advantages of these approaches can be seen in the exponential growth of video-lecture availability on the net, due to the ease of recording and delivering activities which take place in a traditional classroom. Furthermore, the wide use of assistive technologies for learners with disabilities injects new life into e-learning systems. E-learning allows distance and flexible educational activities, thus helping disabled learners to access resources which would otherwise present significant barriers for them. For instance, students with visual impairments have difficulties in reading traditional visual materials, deaf learners have trouble in following traditional (spoken) lectures, people with motion disabilities have problems in attending on-site programs. As already mentioned, the use of wireless technologies and pervasive computing may really enhance the educational learner experience by offering mobile e-learning services that can be accessed by handheld devices. This new paradigm of educational content distribution maximizes the benefits for learners since it enables users to overcome constraints imposed by the surrounding environment. While certainly helpful for users without disabilities, we believe that the use of newmobile technologies may also become a fundamental tool for impaired learners, since it frees them from sitting in front of a PC. In this way, educational activities can be enjoyed by all the users, without hindrance, thus increasing the social inclusion of non-typical learners. While the provision of fully accessible and portable video-lectures may be extremely useful for students, it is widely recognized that structuring and managing rich media contents for mobile learning services are complex and expensive tasks. Indeed, major difficulties originate from the basic need to provide a textual equivalent for each media resource composing a rich media Learning Object (LO). Moreover, tests need to be carried out to establish whether a given LO is fully accessible to all kinds of learners. Unfortunately, both these tasks are truly time-consuming processes, depending on the type of contents the teacher is writing and on the authoring tool he/she is using. Due to these difficulties, online LOs are often distributed as partially accessible or totally inaccessible content. Bearing this in mind, this thesis aims to discuss the key issues of a system we have developed to deliver accessible, customized or nomadic learning experiences to learners with different access needs and skills. To reduce the risk of excluding users with particular access capabilities, our system exploits Learning Objects (LOs) which are dynamically adapted and transcoded based on the specific needs of non-typical users and on the barriers that they can encounter in the environment. The basic idea is to dynamically adapt contents, by selecting them from a set of media resources packaged in SCORM-compliant LOs and stored in a self-adapting format. The system schedules and orchestrates a set of transcoding processes based on specific learner needs, so as to produce a customized LO that can be fully enjoyed by any (impaired or mobile) student.

Customer Evolution in Sales Channel Migration

This research has been triggered by an emergent trend in customer behavior: customers have rapidly expanded their channel experiences and preferences beyond traditional channels (such as stores) and they expect the company with which they do business to have a presence on all these channels. This evidence has produced an increasing interest in multichannel customer behavior and it has motivated several researchers to study the customers’ channel choices dynamics in multichannel environment. We study how the consumer decision process for channel choice and response to marketing communications evolves for a cohort of new customers. We assume a newly acquired customer’s decisions are described by a “trial” model, but the customer’s choice process evolves to a “post-trial” model as the customer learns his or her preferences and becomes familiar with the firm’s marketing efforts. The trial and post-trial decision processes are each described by different multinomial logit choice models, and the evolution from the trial to post-trial model is determined by a customer-level geometric distribution that captures the time it takes for the customer to make the transition. We utilize data for a major retailer who sells in three channels – retail store, the Internet, and via catalog. The model is estimated using Bayesian methods that allow for cross-customer heterogeneity. This allows us to have distinct parameters estimates for a trial and an after trial stages and to estimate the quickness of this transit at the individual level. The results show for example that the customer decision process indeed does evolve over time. Customers differ in the duration of the trial period and marketing has a different impact on channel choice in the trial and post-trial stages. Furthermore, we show that some people switch channel decision processes while others don’t and we found that several factors have an impact on the probability to switch decision process. Insights from this study can help managers tailor their marketing communication strategy as customers gain channel choice experience. Managers may also have insights on the timing of the direct marketing communications. They can predict the duration of the trial phase at individual level detecting the customers with a quick, long or even absent trial phase. They can even predict if the customer will change or not his decision process over time, and they can influence the switching process using specific marketing tools

La comunicazione chimica in Blattaria: contributo al miglioramento della lotta

CHEMICAL COMMUNICATION IN BLATTARIA: CONTRIBUTION TO THE IMPROVEMENT OF THE CONTROL TECNIQUES The management of cockroach infestations in urban environment has undergone some changes in recent years by moving to the predominant use of baits, thanks to the awareness of the risks connected with the use of spray insecticides. The effectiveness of a bait is determined by the collective performance of its components, including active and inert ingredients, the food attractant and any other attractive odour. This research has focused on the behavioral responses of Italian sinanthropic cockroaches to semiochemicals and food attractants, for the purpose of evaluating a possible practical application and of contributing to the improvement of control techniques. Behavioral and morphological studies have been carried out on Supella longipalpa (F.), a small cockroach that is spreading in Italy. Behavioral assays showed that the fourth and fifth tergites of females are significantly more attractive than other region of the body. Cuticular pores and ducts ending in glandular structures (observed with a S.E.M. = Scanning Electron Microscope) are present in large number on these tergites, suggesting that they could be involved in the production and the release of sexual pheromone. Cockroaches produce an aggregation pheromone that is excreted along with their frass and that consists of volatile and non-volatile compounds, mainly amines and steroidal glycosides. The effectiveness of faecal extracts obtained from excrements of Blattella germanica (L.), Blatta orientalis L., Periplaneta americana (L.) and S. longipalpa was evaluated, both at intraspecific and interspecific level, using a "Y" tube olfactometer. Bioassays showed that faecal extracts obtained with methanol have a good intraspecific attractiveness and, in some cases, they showed also interspecific behavioral responses. A gel was prepared, having physical characteristics that could give a good resistance to dehydration, as a potential basis for a new bait; the gel was then added faecal extracts, obtained with methanol from B. germanica and S. longipalpa frass. Arena-tests showed that the new gel containing faecal extracts is more attractive than some commercial gel formulations used as comparison: it was the only product that could attract 100% of insects placed in the arenas in 4-5 days. In conclusion, the substances involved in chemical communication of Blattaria may be able to effectively increase the attractiveness of products for monitoring and controlling cockroaches.

Ottimizzazione delle reti di distribuzione idrica tramite algoritmi genetici multi-obiettivo

Water distribution networks optimization is a challenging problem due to the dimension and the complexity of these systems. Since the last half of the twentieth century this field has been investigated by many authors. Recently, to overcome discrete nature of variables and non linearity of equations, the research has been focused on the development of heuristic algorithms. This algorithms do not require continuity and linearity of the problem functions because they are linked to an external hydraulic simulator that solve equations of mass continuity and of energy conservation of the network. In this work, a NSGA-II (Non-dominating Sorting Genetic Algorithm) has been used. This is a heuristic multi-objective genetic algorithm based on the analogy of evolution in nature. Starting from an initial random set of solutions, called population, it evolves them towards a front of solutions that minimize, separately and contemporaneously, all the objectives. This can be very useful in practical problems where multiple and discordant goals are common. Usually, one of the main drawback of these algorithms is related to time consuming: being a stochastic research, a lot of solutions must be analized before good ones are found. Results of this thesis about the classical optimal design problem shows that is possible to improve results modifying the mathematical definition of objective functions and the survival criterion, inserting good solutions created by a Cellular Automata and using rules created by classifier algorithm (C4.5). This part has been tested using the version of NSGA-II supplied by Centre for Water Systems (University of Exeter, UK) in MATLAB® environment. Even if orientating the research can constrain the algorithm with the risk of not finding the optimal set of solutions, it can greatly improve the results. Subsequently, thanks to CINECA help, a version of NSGA-II has been implemented in C language and parallelized: results about the global parallelization show the speed up, while results about the island parallelization show that communication among islands can improve the optimization. Finally, some tests about the optimization of pump scheduling have been carried out. In this case, good results are found for a small network, while the solutions of a big problem are affected by the lack of constraints on the number of pump switches. Possible future research is about the insertion of further constraints and the evolution guide. In the end, the optimization of water distribution systems is still far from a definitive solution, but the improvement in this field can be very useful in reducing the solutions cost of practical problems, where the high number of variables makes their management very difficult from human point of view.

Natural patterns and anthropogenic disturbance in north Adriatic marine benthic assemblages: descriptive and methodological studies

Marine soft bottom systems show a high variability across multiple spatial and temporal scales. Both natural and anthropogenic sources of disturbance act together in affecting benthic sedimentary characteristics and species distribution. The description of such spatial variability is required to understand the ecological processes behind them. However, in order to have a better estimate of spatial patterns, methods that take into account the complexity of the sedimentary system are required. This PhD thesis aims to give a significant contribution both in improving the methodological approaches to the study of biological variability in soft bottom habitats and in increasing the knowledge of the effect that different process (both natural and anthropogenic) could have on the benthic communities of a large area in the North Adriatic Sea. Beta diversity is a measure of the variability in species composition, and Whittaker’s index has become the most widely used measure of beta-diversity. However, application of the Whittaker index to soft bottom assemblages of the Adriatic Sea highlighted its sensitivity to rare species (species recorded in a single sample). This over-weighting of rare species induces biased estimates of the heterogeneity, thus it becomes difficult to compare assemblages containing a high proportion of rare species. In benthic communities, the unusual large number of rare species is frequently attributed to a combination of sampling errors and insufficient sampling effort. In order to reduce the influence of rare species on the measure of beta diversity, I have developed an alternative index based on simple probabilistic considerations. It turns out that this probability index is an ordinary Michaelis-Menten transformation of Whittaker's index but behaves more favourably when species heterogeneity increases. The suggested index therefore seems appropriate when comparing patterns of complexity in marine benthic assemblages. Although the new index makes an important contribution to the study of biodiversity in sedimentary environment, it remains to be seen which processes, and at what scales, influence benthic patterns. The ability to predict the effects of ecological phenomena on benthic fauna highly depends on both spatial and temporal scales of variation. Once defined, implicitly or explicitly, these scales influence the questions asked, the methodological approaches and the interpretation of results. Problem often arise when representative samples are not taken and results are over-generalized, as can happen when results from small-scale experiments are used for resource planning and management. Such issues, although globally recognized, are far from been resolved in the North Adriatic Sea. This area is potentially affected by both natural (e.g. river inflow, eutrophication) and anthropogenic (e.g. gas extraction, fish-trawling) sources of disturbance. Although few studies in this area aimed at understanding which of these processes mainly affect macrobenthos, these have been conducted at a small spatial scale, as they were designated to examine local changes in benthic communities or particular species. However, in order to better describe all the putative processes occurring in the entire area, a high sampling effort performed at a large spatial scale is required. The sedimentary environment of the western part of the Adriatic Sea was extensively studied in this thesis. I have described, in detail, spatial patterns both in terms of sedimentary characteristics and macrobenthic organisms and have suggested putative processes (natural or of human origin) that might affect the benthic environment of the entire area. In particular I have examined the effect of off shore gas platforms on benthic diversity and tested their effect over a background of natural spatial variability. The results obtained suggest that natural processes in the North Adriatic such as river outflow and euthrophication show an inter-annual variability that might have important consequences on benthic assemblages, affecting for example their spatial pattern moving away from the coast and along a North to South gradient. Depth-related factors, such as food supply, light, temperature and salinity play an important role in explaining large scale benthic spatial variability (i.e., affecting both the abundance patterns and beta diversity). Nonetheless, more locally, effects probably related to an organic enrichment or pollution from Po river input has been observed. All these processes, together with few human-induced sources of variability (e.g. fishing disturbance), have a higher effect on macrofauna distribution than any effect related to the presence of gas platforms. The main effect of gas platforms is restricted mainly to small spatial scales and related to a change in habitat complexity due to a natural dislodgement or structure cleaning of mussels that colonize their legs. The accumulation of mussels on the sediment reasonably affects benthic infauna composition. All the components of the study presented in this thesis highlight the need to carefully consider methodological aspects related to the study of sedimentary habitats. With particular regards to the North Adriatic Sea, a multi-scale analysis along natural and anthopogenic gradients was useful for detecting the influence of all the processes affecting the sedimentary environment. In the future, applying a similar approach may lead to an unambiguous assessment of the state of the benthic community in the North Adriatic Sea. Such assessment may be useful in understanding if any anthropogenic source of disturbance has a negative effect on the marine environment, and if so, planning sustainable strategies for a proper management of the affected area.

Derivati polimerici organometallici ancorati su matrice inorganica per applicazioni in chimica fine

Organotin compounds have found in the last few decades a wide variety of applications. Indeed, they are used successfully as antifouling paints, PVC stabilizers and ion carriers, as well as homogeneous catalysts. In this context, it has been proved that the Lewis acidity of the metal centre allows these compounds to promote the reaction between alcohol and ester. However their use is now limited by their well-known toxicity, moreover they are hardly removable from the reaction mixture. This problem can be overcome by grafting the organotin derivative onto a polymeric cross-linked support. In this way the obtained heterogeneous catalyst can be easily filtered off from the reaction mixture, thus creating the so-called "clean organotin reagents", avoiding the presence of toxic organotin residues in solution and the tin release in the environment. In the last few years several insoluble polystyrene resins containing triorganotin carboxylate moieties have been synthesized with the aim of improving their catalytic activity: in particular we have investigated and opportunely modified their chemical structure in order to optimize the accessibility to the metal centre and its Lewis acidity. Recently, we replaced the polymeric matrix with an inorganic one, in order to dispose of a relatively cheaper and easily available support. For this purpose an ordered mesoporous silica, characterized by 2D-hexagonal pores, named MCM-41, and an amorphous silica have been selected. In the present work two kinds of MCM-41 silica containing the triorganotin carboxylate moiety have been synthesized starting from a commercial Cab-O-Sil M5 silica. These catalysts have two different spacers between the core and the tin-carboxylate moiety, namely a polyaliphatic chain (compound FT29) or a poliethereal one (compound FT6), with the aim to improve the interaction between catalyst and reacting ester. Three catalysts supported onto an amorphous silica have been also synthesized: the structure is the same as silica FT29, i.e. a compound having a polialiphatic chain, and they have different percentage of organotin derivative grafted on the silica surface (10, 30, 50% respectively for silica MB9, SU27 and SU28). The performances of the above silica as heterogeneous catalysts in transesterification reactions have been tested in a model reaction between ethyl acetate and 1-octanol, a primary alcohol sensitive to the reaction conditions. The alcohol conversion was assessed by gas-chromatography, determining the relative amount of transesterified product and starting alcohol after established time intervals.

Monooxygenases involved in the n-alkanes metabolism by Rhodococcus sp. BCP1: molecular characterization and expression of alkB gene

Bioremediation implies the use of living organisms, primarily microorganisms, to convert environmental contaminants into less toxic forms. The impact of the consequences of hydrocarbon release in the environment maintain a high research interest in the study of microbial metabolisms associated with the biodegradation of aromatic and aliphatic hydrocarbons but also in the analysis of microbial enzymes that can convert petroleum substrates to value-added products. The studies described in this Thesis fall within the research field that directs the efforts into identifying gene/proteins involved in the catabolism of n-alkanes and into studying the regulatory mechanisms leading to their oxidation. In particular the studies were aimed at investigating the molecular aspects of the ability of Rhodococcus sp. BCP1 to grow on aliphatic hydrocarbons as sole carbon and energy sources. We studied the ability of Rhodococcus sp. BCP1 to grow on gaseous (C2-C4), liquid (C5-C16) and solid (C17-C28) n-alkanes that resulted to be biochemically correlated with the activity of one or more monooxygenases. In order to identify the alkane monooxygenase that is involved in the n-alkanes degradation pathway in Rhodococcus sp. BCP1, PCR-based methodology was applied by using degenerate primers targeting AlkB monooxygenase family members. As result, a chromosomal region, including the alkB gene cluster, was cloned from Rhodococcus sp. BCP1 genome. We characterized the products of this alkB gene cluster and the products of the orfs included in the flanking regions by comparative analysis with the homologues in the database. alkB gene expression studies were carried out by RT-PCR and by the construction of a promoter probe vector containing the lacZ gene downstream of the alkB promoter. B-galactosidase assays revealed the alkB promoter activity induced by n-alkanes and by n-alkanes metabolic products. Furthermore, the transcriptional start of alkB gene was determined by primer extension procedure. A proteomic approach was subsequently applied to compare the protein patterns expressed by BCP1 growing on n-butane, n-hexane, n-hexadecane or n-eicosane with the protein pattern expressed by BCP1 growing on succinate. The accumulation of enzymes specifically induced on n-alkanes was determined. These enzymes were identified by tandem mass spectrometry (LC/MS/MS). Finally, a prm gene, homologue to the gene family coding for soluble di-iron monooxygenases (SDIMOs), has been isolated from Rhodococcus sp. BCP1 genome. This gene product could be involved in the degradation of gaseous n-alkanes in this Rhodococcus strain. The versatility in utilizing hydrocarbons and the discovery of new remarkable metabolic activities outline the potential applications of this microorganism in environmental and industrial biotechnologies.

Reaching to a target in the 3-D space

Reaching and grasping an object is an action that can be performed in light, under visual guidance, as well as in darkness, under proprioceptive control only. Area V6A is a visuomotor area involved in the control of reaching movements. V6A, besides neurons activated by the execution of reaching movements, shows passive somatosensory and visual responses. This suggests fro V6A a multimodal capability of integrating sensory and motor-related information, We wanted to know whether this integration occurrs in reaching movements and in the present study we tested whether the visual feedback influenced the reaching activity of V6A neurons. In order to better address this question, we wanted to interpret the neural data in the light of the kinematic of reaching performance. We used an experimental paradigm that could examine V6A responses in two different visual backgrounds, light and dark. In these conditions, the monkey performed an istructed-delay reaching task moving the hand towards different target positions located in the peripersonal space. During the execution of reaching task, the visual feedback is processed in a variety of patterns of modulation, sometimes not expected. In fact, having already demonstrated in V6A reach-related discharges in absence of visual feedback, we expected two types of neural modulation: 1) the addition of light in the environment enhanced reach-related discharges recorded in the dark; 2) the light left the neural response unmodified. Unexpectedly, the results show a complex pattern of modulation that argues against a simple additive interaction between visual and motor-related signals.

Diet of bluefin tuna (Thunnus thynnus) and swordfish (Xhipias gladius) in the Adriatic Sea

The present issue analyses bluefin tuna (Thynnus thunnus) and swordfish (Xiphias glaudis) diet, caught by professional long-line fishing in the middle Adriatic Sea (Pomo pit). These species represent apex predators in pelagic environment that may play key roles in determining food web structure and ecosystem dynamics. The studies about their feedings habits, based upon stomach contents analysis, are important for the comprehension of biological and ecological interaction. Over the years, many studies have been performed on the diet of tuna and swordfish in the Mediterranean Sea. This research is based on a fairly wide number of analyzed stomach contents, in comparison with the previous ones. In this work, the analysis of 340 stomach contents of bluefin tuna caught by long-line in the central Adriatic sea confirms in general the opportunistic behaviour of this species. Finding support the hypothesis that Adriatic tuna and swordfish chase their food over a wide bathymetric zone and probably near the surface at night. No indication of food preference respect to size of predator or sample season are found. It seems that the two species are able to cohabit because their trophic niche are not overlapped, changing during the time and the vertical and horizontal space.

"Crop elicitation": approccio innovativo per la valorizzazione delle proprietà funzionali in leguminose da granella

Crop elicitation: innovative approach for the valorization of grain legume functional properties. In Italy grain legume cultivation has encountered a drastic decrease due to several causes (productive, economic, social). In this regard, studies aimed at the setting up of agronomic techniques able to guarantee high and constant in planta yields of health-promoting compounds may concur at re-launching legume production. In this context, 22 accessions of grain legumes (17 Phaseolus vulgaris, 3 Phaseolus coccineus, 1 Vigna unguiculata and 1 Glycine max genotypes) were screened with the aim of identifying genotypes rich in health beneficial phytochemicals (α-amylase inhibitors, α -glucosidase inhibitors, polyphenols) and with low anti-nutritional compounds (lectins). A wide variability was observed among investigated accessions. Four genotypes (Verdone, Kidney Cina, Roviotto and DG) showed a α -amylase inhibitory activity significantly higher (approximately 30% more) than all other tested accessions. The α -amylase inhibitory activity was not correlated neither with the protein nor with the polyphenol contents. Conversely, the α -glucosidase inhibitory activity was positively correlated with grain color and polyphenol content: dark-colored seeds had a mean inhibitory activity of 83.64 ± 22.07%, whereas light-colored seeds had mean values of 21.11 ± 9.36%. As regards the anti-nutritional compounds, out of all common bean accessions, only DG showed no erythro-agglutination activity (lectins). Preliminary experiments, performed in controlled environment, permitted to highlight that different germination conditions markedly affect the synthesis and accumulation of functional compounds in legume seedlings. Those findings were confirmed with field trials performed in two different locations (Bologna and Pisa), on two bean genotypes (Verdone and Zolfino), during the 2004-2005 cropping season. Results showed that the application of abiotic stresses (no fertilization and /or no irrigation) lead to a significant increase of flavonoids in grains, but a decrease (up to 50%) in legume yields was also observed. Crop elicitation, even if valuable for boosting health-promoting compound synthesis in crops, must necessary cope with economically acceptable crop yields.

Perfluoroalkylated substances in food matrices: development of mass spectrometry based analytical methods and preliminary monitoring

Perfluoroalkylated substances are a group of chemicals that have been largely employed during the last 60 years in several applications, widely spreading and accumulating in the environment due to their extreme resistance to degradation. As a consequence, they have been found also in various types of food as well as in drinking water, proving that they can easily reach humans through the diet. The available information concerning their adverse effects on health has recently increased the interest towards these contaminants and highlighted the importance of investigating all the potential sources of human exposure, among which diet was proved to be the most relevant. This need has been underlined by the European Union through Recommendation 2010/161/EU: in this document, Member States were called to monitor their presence of in food, producing accurate estimations of human exposure. The purpose of the research presented in this thesis, which is the result of a partnership between an Italian and a French laboratory, was to develop reliable tools for the analysis of these pollutants in food, to be used for generating data on potentially contaminated matrices. An efficient method based on liquid chromatography-mass spectrometry for the detection of 16 different perfluorinated compounds in milk has been validated in accordance with current European regulation guidelines (2002/657/EC) and is currently under evaluation for ISO 17025 accreditation. The proposed technique was applied to cow, powder and human breast milk samples from Italy and France to produce a preliminary monitoring on the presence of these contaminants. In accordance with the above mentioned European Recommendation, this project led also to the development of a promising technique for the quantification of some precursors of these substances in fish. This method showed extremely satisfying performances in terms of linearity and limits of detection, and will be useful for future surveys.