Food safety and zoonotic enteric pathogens: sources, risk factors and transmission routes of human salmonellosis and campylobacteriosis

Salmonella and Campylobacter are common causes of human gastroenteritis. Their epidemiology is complex and a multi-tiered approach to control is needed, taking into account the different reservoirs, pathways and risk factors. In this thesis, trends in human gastroenteritis and food-borne outbreak notifications in Italy were explored. Moreover, the improved sensitivity of two recently-implemented regional surveillance systems in Lombardy and Piedmont was evidenced, providing a basis for improving notification at the national level. Trends in human Salmonella serovars were explored: serovars Enteritidis and Infantis decreased, Typhimurium remained stable and 4,[5],12:i:-, Derby and Napoli increased, suggesting that sources of infection have changed over time. Attribution analysis identified pigs as the main source of human salmonellosis in Italy, accounting for 43–60% of infections, followed by Gallus gallus (18–34%). Attributions to pigs and Gallus gallus showed increasing and decreasing trends, respectively. Potential bias and sampling issues related to the use of non-local/non-recent multilocus sequence typing (MLST) data in Campylobacter jejuni/coli source attribution using the Asymmetric Island (AI) model were investigated. As MLST data become increasingly dissimilar with increasing geographical/temporal distance, attributions to sources not sampled close to human cases can be underestimated. A combined case-control and source attribution analysis was developed to investigate risk factors for human Campylobacter jejuni/coli infection of chicken, ruminant, environmental, pet and exotic origin in The Netherlands. Most infections (~87%) were attributed to chicken and cattle. Individuals infected from different reservoirs had different associated risk factors: chicken consumption increased the risk for chicken-attributed infections; animal contact, barbecuing, tripe consumption, and never/seldom chicken consumption increased that for ruminant-attributed infections; game consumption and attending swimming pools increased that for environment-attributed infections; and dog ownership increased that for environment- and pet-attributed infections. Person-to-person contacts around holiday periods were risk factors for infections with exotic strains, putatively introduced by returning travellers.

Probiotics for the prevention/treatment of human diseases and ecological study of the intestinal microbiota

The interest in human intestinal microbiota has increased in the last 20 years and significant advances have been achieved with regard to its composition and functions. The gut microbiota contributes to the maintenance of the host health status and, since alterations in the gut microbiota have been involved in the pathogenesis/progression of some diseases, several studies have focused on the manipulation of its composition. Probiotics are a strategy to maintain/restore the correct balance of gut microbial population and to prevent/treat diseases. The aim of this thesis was to explore the possibility of probiotic supplementation for the prevention/treatment of human diseases and the related study of the intestinal microbial environment. After reviewing studies concerning the use of Bifidobacterium breve as probiotic in paediatric diseases, the effectiveness of a probiotic formulation consisting of two strains of B. breve was assessed in paediatric subjects for the prevention or alleviation of gastrointestinal disorders, including coeliac disease and paediatric obesity. As the emerging role of gut microbiota in neurological diseases, the intestinal microbial environment in amyotrophic lateral sclerosis patients compared to healthy controls and the effects of a probiotic administration were examined. Considering the role of viruses in shaping gut microbiota, gut bacteriophages and bacterial community of preterm infants were investigated. The results evidenced differences in gut microbial composition of healthy controls and diseased subjects in coeliac and amyotrophic lateral sclerosis patients. The probiotic approach was effective in restoring the microbial composition in the former, whereas, in the latter, the influence was focused only on some microbial groups. The probiotic intervention was effective in improving the glyco-insulinemic profile in obese children and in preventing gastrointestinal disorders in healthy newborns. The study of the bacterial and phage composition in preterm infants suggested a transkingdom interplay between bacteria and viruses with a reciprocal influence on their composition.

Stream sediments analysis for geochemical mapping of Romagna Apennines (Northern Italy): monitoring and management tool of environmental resources at various scales

Geochemical mapping is a valuable tool for the control of territory that can be used not only in the identification of mineral resources and geological, agricultural and forestry studies but also in the monitoring of natural resources by giving solutions to environmental and economic problems. Stream sediments are widely used in the sampling campaigns carried out by the world's governments and research groups for their characteristics of broad representativeness of rocks and soils, for ease of sampling and for the possibility to conduct very detailed sampling In this context, the environmental role of stream sediments provides a good basis for the implementation of environmental management measures, in fact the composition of river sediments is an important factor in understanding the complex dynamics that develop within catchment basins therefore they represent a critical environmental compartment: they can persistently incorporate pollutants after a process of contamination and release into the biosphere if the environmental conditions change. It is essential to determine whether the concentrations of certain elements, in particular heavy metals, can be the result of natural erosion of rocks containing high concentrations of specific elements or are generated as residues of human activities related to a certain study area. This PhD thesis aims to extract from an extensive database on stream sediments of the Romagna rivers the widest spectrum of informations. The study involved low and high order stream in the mountain and hilly area, but also the sediments of the floodplain area, where intensive agriculture is active. The geochemical signals recorded by the stream sediments will be interpreted in order to reconstruct the natural variability related to bedrock and soil contribution, the effects of the river dynamics, the anomalous sites, and with the calculation of background values be able to evaluate their level of degradation and predict the environmental risk.

The EU’s foreign policy towards the Western Balkans during the Juncker Commission. The cases of Bosnia Herzegovina and Serbia.

In February 2018, four years after the President of the Commission Jean Claude Juncker explicitly deprioritised the enlargement dossier, the European Commission relaunched the enlargement strategy to the region of the Western Balkans. This occurred despite the persistent polarisation around the topic among the EU Member States and the still-present struggles, when not outright regression, of some of the countries in pursuing the demanded reforms. This thesis carries out a multilevel foreign policy analysis of EU foreign policy toward the region of the Western Balkans during the period of the Juncker Commission, through the cases of Bosnia Herzegovina and Serbia. Drawing from Actorness theory (Bretherton and Vogler 2006), combined with perspectives from new institutional leadership (Smeets and Beach 2020), and new intergovernmentalism (Moravcsik 2018; Bickerton et al. 2015), this study seeks to explain the relaunch of enlargement by examining three dimensions: the international context and the role of non-EU actors such as China, Russia, and Turkey; the EU context, through the interaction of the significant EU Member States and Institutions; finally, the local context, through the analysis of the changes in the local perception of the EU and the considered non-EU actors. This study posits two interconnected points: first, that the changes in the international context, specifically the increased presence of non-EU actors such as China, Russia, and Turkey in the region, acted as triggering factors for the relaunch of the strategy. In addition, it argues that this relaunch was successful due to the peculiar combination of Germany’s interests and leadership within the Council, coupled with the Commission’s priorities.

The firm. Techno-organizational changes, industrial relations and performances. An enquiry on Reggio Emilia local industrial system

It is not unknown that the evolution of firm theories has been developed along a path paved by an increasing awareness of the organizational structure importance. From the early “neoclassical” conceptualizations that intended the firm as a rational actor whose aim is to produce that amount of output, given the inputs at its disposal and in accordance to technological or environmental constraints, which maximizes the revenue (see Boulding, 1942 for a past mid century state of the art discussion) to the knowledge based theory of the firm (Nonaka & Takeuchi, 1995; Nonaka & Toyama, 2005), which recognizes in the firm a knnowledge creating entity, with specific organizational capabilities (Teece, 1996; Teece & Pisano, 1998) that allow to sustaine competitive advantages. Tracing back a map of the theory of the firm evolution, taking into account the several perspectives adopted in the history of thought, would take the length of many books. Because of that a more fruitful strategy is circumscribing the focus of the description of the literature evolution to one flow connected to a crucial question about the nature of firm’s behaviour and about the determinants of competitive advantages. In so doing I adopt a perspective that allows me to consider the organizational structure of the firm as an element according to which the different theories can be discriminated. The approach adopted starts by considering the drawbacks of the standard neoclassical theory of the firm. Discussing the most influential theoretical approaches I end up with a close examination of the knowledge based perspective of the firm. Within this perspective the firm is considered as a knowledge creating entity that produce and mange knowledge (Nonaka, Toyama, & Nagata, 2000; Nonaka & Toyama, 2005). In a knowledge intensive organization, knowledge is clearly embedded for the most part in the human capital of the individuals that compose such an organization. In a knowledge based organization, the management, in order to cope with knowledge intensive productions, ought to develop and accumulate capabilities that shape the organizational forms in a way that relies on “cross-functional processes, extensive delayering and empowerment” (Foss 2005, p.12). This mechanism contributes to determine the absorptive capacity of the firm towards specific technologies and, in so doing, it also shape the technological trajectories along which the firm moves. After having recognized the growing importance of the firm’s organizational structure in the theoretical literature concerning the firm theory, the subsequent point of the analysis is that of providing an overview of the changes that have been occurred at micro level to the firm’s organization of production. The economic actors have to deal with challenges posed by processes of internationalisation and globalization, increased and increasing competitive pressure of less developed countries on low value added production activities, changes in technologies and increased environmental turbulence and volatility. As a consequence, it has been widely recognized that the main organizational models of production that fitted well in the 20th century are now partially inadequate and processes aiming to reorganize production activities have been widespread across several economies in recent years. Recently, the emergence of a “new” form of production organization has been proposed both by scholars, practitioners and institutions: the most prominent characteristic of such a model is its recognition of the importance of employees commitment and involvement. As a consequence it is characterized by a strong accent on the human resource management and on those practices that aim to widen the autonomy and responsibility of the workers as well as increasing their commitment to the organization (Osterman, 1994; 2000; Lynch, 2007). This “model” of production organization is by many defined as High Performance Work System (HPWS). Despite the increasing diffusion of workplace practices that may be inscribed within the concept of HPWS in western countries’ companies, it is an hazard, to some extent, to speak about the emergence of a “new organizational paradigm”. The discussion about organizational changes and the diffusion of HPWP the focus cannot abstract from a discussion about the industrial relations systems, with a particular accent on the employment relationships, because of their relevance, in the same way as production organization, in determining two major outcomes of the firm: innovation and economic performances. The argument is treated starting from the issue of the Social Dialogue at macro level, both in an European perspective and Italian perspective. The model of interaction between the social parties has repercussions, at micro level, on the employment relationships, that is to say on the relations between union delegates and management or workers and management. Finding economic and social policies capable of sustaining growth and employment within a knowledge based scenario is likely to constitute the major challenge for the next generation of social pacts, which are the main social dialogue outcomes. As Acocella and Leoni (2007) put forward the social pacts may constitute an instrument to trade wage moderation for high intensity in ICT, organizational and human capital investments. Empirical evidence, especially focused on the micro level, about the positive relation between economic growth and new organizational designs coupled with ICT adoption and non adversarial industrial relations is growing. Partnership among social parties may become an instrument to enhance firm competitiveness. The outcome of the discussion is the integration of organizational changes and industrial relations elements within a unified framework: the HPWS. Such a choice may help in disentangling the potential existence of complementarities between these two aspects of the firm internal structure on economic and innovative performance. With the third chapter starts the more original part of the thesis. The data utilized in order to disentangle the relations between HPWS practices, innovation and economic performance refer to the manufacturing firms of the Reggio Emilia province with more than 50 employees. The data have been collected through face to face interviews both to management (199 respondents) and to union representatives (181 respondents). Coupled with the cross section datasets a further data source is constituted by longitudinal balance sheets (1994-2004). Collecting reliable data that in turn provide reliable results needs always a great effort to which are connected uncertain results. Data at micro level are often subjected to a trade off: the wider is the geographical context to which the population surveyed belong the lesser is the amount of information usually collected (low level of resolution); the narrower is the focus on specific geographical context, the higher is the amount of information usually collected (high level of resolution). For the Italian case the evidence about the diffusion of HPWP and their effects on firm performances is still scanty and usually limited to local level studies (Cristini, et al., 2003). The thesis is also devoted to the deepening of an argument of particular interest: the existence of complementarities between the HPWS practices. It has been widely shown by empirical evidence that when HPWP are adopted in bundles they are more likely to impact on firm’s performances than when adopted in isolation (Ichniowski, Prennushi, Shaw, 1997). Is it true also for the local production system of Reggio Emilia? The empirical analysis has the precise aim of providing evidence on the relations between the HPWS dimensions and the innovative and economic performances of the firm. As far as the first line of analysis is concerned it must to be stressed the fundamental role that innovation plays in the economy (Geroski & Machin, 1993; Stoneman & Kwoon 1994, 1996; OECD, 2005; EC, 2002). On this point the evidence goes from the traditional innovations, usually approximated by R&D investment expenditure or number of patents, to the introduction and adoption of ICT, in the recent years (Brynjolfsson & Hitt, 2000). If innovation is important then it is critical to analyse its determinants. In this work it is hypothesised that organizational changes and firm level industrial relations/employment relations aspects that can be put under the heading of HPWS, influence the propensity to innovate in product, process and quality of the firm. The general argument may goes as follow: changes in production management and work organization reconfigure the absorptive capacity of the firm towards specific technologies and, in so doing, they shape the technological trajectories along which the firm moves; cooperative industrial relations may lead to smother adoption of innovations, because not contrasted by unions. From the first empirical chapter emerges that the different types of innovations seem to respond in different ways to the HPWS variables. The underlying processes of product, process and quality innovations are likely to answer to different firm’s strategies and needs. Nevertheless, it is possible to extract some general results in terms of the most influencing HPWS factors on innovative performance. The main three aspects are training coverage, employees involvement and the diffusion of bonuses. These variables show persistent and significant relations with all the three innovation types. The same do the components having such variables at their inside. In sum the aspects of the HPWS influence the propensity to innovate of the firm. At the same time, emerges a quite neat (although not always strong) evidence of complementarities presence between HPWS practices. In terns of the complementarity issue it can be said that some specific complementarities exist. Training activities, when adopted and managed in bundles, are related to the propensity to innovate. Having a sound skill base may be an element that enhances the firm’s capacity to innovate. It may enhance both the capacity to absorbe exogenous innovation and the capacity to endogenously develop innovations. The presence and diffusion of bonuses and the employees involvement also spur innovative propensity. The former because of their incentive nature and the latter because direct workers participation may increase workers commitment to the organizationa and thus their willingness to support and suggest inovations. The other line of analysis provides results on the relation between HPWS and economic performances of the firm. There have been a bulk of international empirical studies on the relation between organizational changes and economic performance (Black & Lynch 2001; Zwick 2004; Janod & Saint-Martin 2004; Huselid 1995; Huselid & Becker 1996; Cappelli & Neumark 2001), while the works aiming to capture the relations between economic performance and unions or industrial relations aspects are quite scant (Addison & Belfield, 2001; Pencavel, 2003; Machin & Stewart, 1990; Addison, 2005). In the empirical analysis the integration of the two main areas of the HPWS represent a scarcely exploited approach in the panorama of both national and international empirical studies. As remarked by Addison “although most analysis of workers representation and employee involvement/high performance work practices have been conducted in isolation – while sometimes including the other as controls – research is beginning to consider their interactions” (Addison, 2005, p.407). The analysis conducted exploiting temporal lags between dependent and covariates, possibility given by the merger of cross section and panel data, provides evidence in favour of the existence of HPWS practices impact on firm’s economic performance, differently measured. Although it does not seem to emerge robust evidence on the existence of complementarities among HPWS aspects on performances there is evidence of a general positive influence of the single practices. The results are quite sensible to the time lags, inducing to hypothesize that time varying heterogeneity is an important factor in determining the impact of organizational changes on economic performance. The implications of the analysis can be of help both to management and local level policy makers. Although the results are not simply extendible to other local production systems it may be argued that for contexts similar to the Reggio Emilia province, characterized by the presence of small and medium enterprises organized in districts and by a deep rooted unionism, with strong supporting institutions, the results and the implications here obtained can also fit well. However, a hope for future researches on the subject treated in the present work is that of collecting good quality information over wider geographical areas, possibly at national level, and repeated in time. Only in this way it is possible to solve the Gordian knot about the linkages between innovation, performance, high performance work practices and industrial relations.

Burnout and work engagement among teachers: an application of the job demands-resources model

The present dissertation focuses on burnout and work engagement among teachers, with especial focus on the Job-Demands Resources Model: Chapter 1 focuses on teacher burnout. It aims to investigate the role of efficacy beliefs using negatively worded inefficacy items instead of positive ones and to establish whether depersonalization and cynism can be considered two different dimensions of the teacher burnout syndrome. Chapter 2 investigates the factorial validity of the instruments used to measure work engagement (i.e. Utrecht Work Engagement Scale, UWES-17 and UWES-9). Moreover, because the current study is partly longitudinal in nature, also the stability across time of engagement can be investigated. Finally, based on cluster-analyses, two groups that differ in levels of engagement are compared as far as their job- and personal resources (i.e. possibilities for personal development, work-life balance, and self-efficacy), positive organizational attitudes and behaviours (i.e., job satisfaction and organizational citizenship behaviour) and perceived health are concerned. Chapter 3 tests the JD-R model in a longitudinal way, by integrating also the role of personal resources (i.e. self-efficacy). This chapter seeks answers to questions on what are the most important job demands, job and personal resources contributing to discriminate burned-out teachers from non-burned-out teachers, as well as engaged teachers from non-engaged teachers. Chapter 4 uses a diary study to extend knowledge about the dynamic nature of the JD-R model by considering between- and within-person variations with regard to both motivational and health impairment processes.

The Making of a Shiite Bourgeoisie in Lebanon Political Mobilisation, Economic Resources and Formation of a Social Group

The thesis analyses the making of the Shiite middle- and upper/entrepreneurial-class in Lebanon from the 1960s till the present day. The trajectory explores the historical, political and social (internal and external) factors that brought a sub-proletariat to mobilise and become an entrepreneurial bourgeoisie in the span of less than three generations. This work proposes the main theoretical hypothesis to unpack and reveal the trajectory of a very recent social class that through education, diaspora, political and social mobilisation evolved in a few years into a very peculiar bourgeoisie: whereas Christian-Maronite middle class practically produced political formations and benefited from them and from Maronite’s state supremacy (National Pact, 1943) reinforcing the community’s status quo, Shiites built their own bourgeoisie from within, and mobilised their “cadres” (Boltanski) not just to benefit from their renovated presence at the state level, but to oppose to it. The general Social Movement Theory (SMT), as well as a vast amount of the literature on (middle) class formation are therefore largely contradicted, opening up new territories for discussion on how to build a bourgeoisie without the state’s support (Social Mobilisation Theory, Resource Mobilisation Theory) and if, eventually, the middle class always produces democratic movements (the emergence of a social group out of backwardness and isolation into near dominance of a political order). The middle/upper class described here is at once an economic class related to the control of multiple forms of capital, and produced by local, national, and transnational networks related to flows of services, money, and education, and a culturally constructed social location and identity structured by economic as well as other forms of capital in relation to other groups in Lebanon.

Transcriptional regulation of human mu-opioid receptor gene: functional characterization of activating and inhibitory transcription factors

The organization of the nervous and immune systems is characterized by obvious differences and striking parallels. Both systems need to relay information across very short and very long distances. The nervous system communicates over both long and short ranges primarily by means of more or less hardwired intercellular connections, consisting of axons, dendrites, and synapses. Longrange communication in the immune system occurs mainly via the ordered and guided migration of immune cells and systemically acting soluble factors such as antibodies, cytokines, and chemokines. Its short-range communication either is mediated by locally acting soluble factors or transpires during direct cell–cell contact across specialized areas called “immunological synapses” (Kirschensteiner et al., 2003). These parallels in intercellular communication are complemented by a complex array of factors that induce cell growth and differentiation: these factors in the immune system are called cytokines; in the nervous system, they are called neurotrophic factors. Neither the cytokines nor the neurotrophic factors appear to be completely exclusive to either system (Neumann et al., 2002). In particular, mounting evidence indicates that some of the most potent members of the neurotrophin family, for example, nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF), act on or are produced by immune cells (Kerschensteiner et al., 1999) There are, however, other neurotrophic factors, for example the insulin-like growth factor-1 (IGF-1), that can behave similarly (Kermer et al., 2000). These factors may allow the two systems to “cross-talk” and eventually may provide a molecular explanation for the reports that inflammation after central nervous system (CNS) injury has beneficial effects (Moalem et al., 1999). In order to shed some more light on such a cross-talk, therefore, transcription factors modulating mu-opioid receptor (MOPr) expression in neurons and immune cells are here investigated. More precisely, I focused my attention on IGF-I modulation of MOPr in neurons and T-cell receptor induction of MOPr expression in T-lymphocytes. Three different opioid receptors [mu (MOPr), delta (DOPr), and kappa (KOPr)] belonging to the G-protein coupled receptor super-family have been cloned. They are activated by structurallyrelated exogenous opioids or endogenous opioid peptides, and contribute to the regulation of several functions including pain transmission, respiration, cardiac and gastrointestinal functions, and immune response (Zollner and Stein 2007). MOPr is expressed mainly in the central nervous system where it regulates morphine-induced analgesia, tolerance and dependence (Mayer and Hollt 2006). Recently, induction of MOPr expression in different immune cells induced by cytokines has been reported (Kraus et al., 2001; Kraus et al., 2003). The human mu-opioid receptor gene (OPRM1) promoter is of the TATA-less type and has clusters of potential binding sites for different transcription factors (Law et al. 2004). Several studies, primarily focused on the upstream region of the OPRM1 promoter, have investigated transcriptional regulation of MOPr expression. Presently, however, it is still not completely clear how positive and negative transcription regulators cooperatively coordinate cellor tissue-specific transcription of the OPRM1 gene, and how specific growth factors influence its expression. IGF-I and its receptors are widely distributed throughout the nervous system during development, and their involvement in neurogenesis has been extensively investigated (Arsenijevic et al. 1998; van Golen and Feldman 2000). As previously mentioned, such neurotrophic factors can be also produced and/or act on immune cells (Kerschenseteiner et al., 2003). Most of the physiologic effects of IGF-I are mediated by the type I IGF surface receptor which, after ligand binding-induced autophosphorylation, associates with specific adaptor proteins and activates different second messengers (Bondy and Cheng 2004). These include: phosphatidylinositol 3-kinase, mitogen-activated protein kinase (Vincent and Feldman 2002; Di Toro et al. 2005) and members of the Janus kinase (JAK)/STAT3 signalling pathway (Zong et al. 2000; Yadav et al. 2005). REST plays a complex role in neuronal cells by differentially repressing target gene expression (Lunyak et al. 2004; Coulson 2005; Ballas and Mandel 2005). REST expression decreases during neurogenesis, but has been detected in the adult rat brain (Palm et al. 1998) and is up-regulated in response to global ischemia (Calderone et al. 2003) and induction of epilepsy (Spencer et al. 2006). Thus, the REST concentration seems to influence its function and the expression of neuronal genes, and may have different effects in embryonic and differentiated neurons (Su et al. 2004; Sun et al. 2005). In a previous study, REST was elevated during the early stages of neural induction by IGF-I in neuroblastoma cells. REST may contribute to the down-regulation of genes not yet required by the differentiation program, but its expression decreases after five days of treatment to allow for the acquisition of neural phenotypes. Di Toro et al. proposed a model in which the extent of neurite outgrowth in differentiating neuroblastoma cells was affected by the disappearance of REST (Di Toro et al. 2005). The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. Therefore, in the fist part of this thesis, I investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I upregulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 (STAT3) signaling pathway and this transcription factor, binding to the STAT1/3 DNA element located in the promoter, increases OPRM1 transcription. T-cell receptor (TCR) recognizes peptide antigens displayed in the context of the major histocompatibility complex (MHC) and gives rise to a potent as well as branched intracellular signalling that convert naïve T-cells in mature effectors, thus significantly contributing to the genesis of a specific immune response. In the second part of my work I exposed wild type Jurkat CD4+ T-cells to a mixture of CD3 and CD28 antigens in order to fully activate TCR and study whether its signalling influence OPRM1 expression. Results were that TCR engagement determined a significant induction of OPRM1 expression through the activation of transcription factors AP-1, NF-kB and NFAT. Eventually, I investigated MOPr turnover once it has been expressed on T-cells outer membrane. It turned out that DAMGO induced MOPr internalisation and recycling, whereas morphine did not. Overall, from the data collected in this thesis we can conclude that that a reduction in REST is a critical switch enabling IGF-I to up-regulate human MOPr, helping these findings clarify how human MOPr expression is regulated in neuronal cells, and that TCR engagement up-regulates OPRM1 transcription in T-cells. My results that neurotrophic factors a and TCR engagement, as well as it is reported for cytokines, seem to up-regulate OPRM1 in both neurons and immune cells suggest an important role for MOPr as a molecular bridge between neurons and immune cells; therefore, MOPr could play a key role in the cross-talk between immune system and nervous system and in particular in the balance between pro-inflammatory and pro-nociceptive stimuli and analgesic and neuroprotective effects.

Methodological improvement of 3D fluoroscopic analysis for the robust quantification of 3D kinematics of human joints

3D video-fluoroscopy is an accurate but cumbersome technique to estimate natural or prosthetic human joint kinematics. This dissertation proposes innovative methodologies to improve the 3D fluoroscopic analysis reliability and usability. Being based on direct radiographic imaging of the joint, and avoiding soft tissue artefact that limits the accuracy of skin marker based techniques, the fluoroscopic analysis has a potential accuracy of the order of mm/deg or better. It can provide fundamental informations for clinical and methodological applications, but, notwithstanding the number of methodological protocols proposed in the literature, time consuming user interaction is exploited to obtain consistent results. The user-dependency prevented a reliable quantification of the actual accuracy and precision of the methods, and, consequently, slowed down the translation to the clinical practice. The objective of the present work was to speed up this process introducing methodological improvements in the analysis. In the thesis, the fluoroscopic analysis was characterized in depth, in order to evaluate its pros and cons, and to provide reliable solutions to overcome its limitations. To this aim, an analytical approach was followed. The major sources of error were isolated with in-silico preliminary studies as: (a) geometric distortion and calibration errors, (b) 2D images and 3D models resolutions, (c) incorrect contour extraction, (d) bone model symmetries, (e) optimization algorithm limitations, (f) user errors. The effect of each criticality was quantified, and verified with an in-vivo preliminary study on the elbow joint. The dominant source of error was identified in the limited extent of the convergence domain for the local optimization algorithms, which forced the user to manually specify the starting pose for the estimating process. To solve this problem, two different approaches were followed: to increase the optimal pose convergence basin, the local approach used sequential alignments of the 6 degrees of freedom in order of sensitivity, or a geometrical feature-based estimation of the initial conditions for the optimization; the global approach used an unsupervised memetic algorithm to optimally explore the search domain. The performances of the technique were evaluated with a series of in-silico studies and validated in-vitro with a phantom based comparison with a radiostereometric gold-standard. The accuracy of the method is joint-dependent, and for the intact knee joint, the new unsupervised algorithm guaranteed a maximum error lower than 0.5 mm for in-plane translations, 10 mm for out-of-plane translation, and of 3 deg for rotations in a mono-planar setup; and lower than 0.5 mm for translations and 1 deg for rotations in a bi-planar setups. The bi-planar setup is best suited when accurate results are needed, such as for methodological research studies. The mono-planar analysis may be enough for clinical application when the analysis time and cost may be an issue. A further reduction of the user interaction was obtained for prosthetic joints kinematics. A mixed region-growing and level-set segmentation method was proposed and halved the analysis time, delegating the computational burden to the machine. In-silico and in-vivo studies demonstrated that the reliability of the new semiautomatic method was comparable to a user defined manual gold-standard. The improved fluoroscopic analysis was finally applied to a first in-vivo methodological study on the foot kinematics. Preliminary evaluations showed that the presented methodology represents a feasible gold-standard for the validation of skin marker based foot kinematics protocols.

Analysis of human body kinematics using a hybrid markerless video acquisition and processing

A main objective of the human movement analysis is the quantitative description of joint kinematics and kinetics. This information may have great possibility to address clinical problems both in orthopaedics and motor rehabilitation. Previous studies have shown that the assessment of kinematics and kinetics from stereophotogrammetric data necessitates a setup phase, special equipment and expertise to operate. Besides, this procedure may cause feeling of uneasiness on the subjects and may hinder with their walking. The general aim of this thesis is the implementation and evaluation of new 2D markerless techniques, in order to contribute to the development of an alternative technique to the traditional stereophotogrammetric techniques. At first, the focus of the study has been the estimation of the ankle-foot complex kinematics during stance phase of the gait. Two particular cases were considered: subjects barefoot and subjects wearing ankle socks. The use of socks was investigated in view of the development of the hybrid method proposed in this work. Different algorithms were analyzed, evaluated and implemented in order to have a 2D markerless solution to estimate the kinematics for both cases. The validation of the proposed technique was done with a traditional stereophotogrammetric system. The implementation of the technique leads towards an easy to configure (and more comfortable for the subject) alternative to the traditional stereophotogrammetric system. Then, the abovementioned technique has been improved so that the measurement of knee flexion/extension could be done with a 2D markerless technique. The main changes on the implementation were on occlusion handling and background segmentation. With the additional constraints, the proposed technique was applied to the estimation of knee flexion/extension and compared with a traditional stereophotogrammetric system. Results showed that the knee flexion/extension estimation from traditional stereophotogrammetric system and the proposed markerless system were highly comparable, making the latter a potential alternative for clinical use. A contribution has also been given in the estimation of lower limb kinematics of the children with cerebral palsy (CP). For this purpose, a hybrid technique, which uses high-cut underwear and ankle socks as “segmental markers” in combination with a markerless methodology, was proposed. The proposed hybrid technique is different than the abovementioned markerless technique in terms of the algorithm chosen. Results showed that the proposed hybrid technique can become a simple and low-cost alternative to the traditional stereophotogrammetric systems.

Identification and functional characterization of human cytomegalovirus Fc binding proteins

ABSTRACT Human cytomegalovirus (HCMV) employs many different mechanisms to escape and subvert the host immune system surveillance. Among these different mechanisms the role of human IgG Fc receptors (FcγR) in HCMV pathogenesis is still unclear. In mammalians, FcγRs are expressed on the surface of all haematopoietic cells and have a multifaceted role in regulating the activity of antibodies to generate a well-balanced immune response. Viral proteins with Fcγ binding ability are highly diffuse among herpesviruses. They interfere with the host receptors functions in order to counteract immune system recognition. So far, two human HCMV Fcγ binding proteins have been described: UL119 and RL11. This work was aimed to the identification and characterization of HCMV Fcγ binding proteins. The study is divided in two parts: first the characterization of UL119 and RL11; second the identification and characterization of novel HCMV Fcγ binding proteins. Regarding the first part, we demonstrated that both UL119 and RL11 internalize Fcγ fragments from transfected cells surface through a clathrin dependent pathway. In infected cells both proteins were found in the viral assembly complex and on virions surface as envelope associated glycoproteins. Moreover, internalized Fcγ in infected cells do not undergo lysosomal degradation but rather traffic in early endosomes up to the viral assembly complex. Regarding the second part, we were able to identify two novels Fcγ binding protein coded by CMV: RL12 and RL13. The latter was also further characterized as recombinant protein in terms of cellular localization, Fc binding site and IgG internalization ability. Finally binding specificity of both RL12 and RL13 seems to be confined to human IgG1 and IgG2. Taken together, these data show that HCMV codes for up to 4 FcγR and that they could have a double role both on virus and on infected cells.

Data management and data analysis in the large European projects GEHA (GEnetics of Healthy Aging) and NU-AGE (NUtrition and AGEing): a bioinformatic approach

The aging process is characterized by the progressive fitness decline experienced at all the levels of physiological organization, from single molecules up to the whole organism. Studies confirmed inflammaging, a chronic low-level inflammation, as a deeply intertwined partner of the aging process, which may provide the “common soil” upon which age-related diseases develop and flourish. Thus, albeit inflammation per se represents a physiological process, it can rapidly become detrimental if it goes out of control causing an excess of local and systemic inflammatory response, a striking risk factor for the elderly population. Developing interventions to counteract the establishment of this state is thus a top priority. Diet, among other factors, represents a good candidate to regulate inflammation. Building on top of this consideration, the EU project NU-AGE is now trying to assess if a Mediterranean diet, fortified for the elderly population needs, may help in modulating inflammaging. To do so, NU-AGE enrolled a total of 1250 subjects, half of which followed a 1-year long diet, and characterized them by mean of the most advanced –omics and non –omics analyses. The aim of this thesis was the development of a solid data management pipeline able to efficiently cope with the results of these assays, which are now flowing inside a centralized database, ready to be used to test the most disparate scientific hypotheses. At the same time, the work hereby described encompasses the data analysis of the GEHA project, which was focused on identifying the genetic determinants of longevity, with a particular focus on developing and applying a method for detecting epistatic interactions in human mtDNA. Eventually, in an effort to propel the adoption of NGS technologies in everyday pipeline, we developed a NGS variant calling pipeline devoted to solve all the sequencing-related issues of the mtDNA.