In un contesto generale i cui contorni risultano ancora non ben deï¬ niti e i cui principali problemi non hanno ancora trovato soluzioni certe o ricette “standard†, il lavoro descritto in questa tesi vuole presentarsi come il tentativo di testare ed esplorare approcci differenti e diversiï¬ cati — sia in relazione allo scopo dellâ€TManalisi, sia in relazione alle fondamenta teoriche cui fanno riferimento — per affrontare il fenomeno e il problema radon indoor: lâ€TMintenzione à ̈ stata quella di ricercare punti di vista alternativi e complementari dai quali poter osservare un problema comune secondo prospettive differenti.

Protein byosynthesis is performed by ribosomes, that translate the genetic information contained in a strand of mRNA and assemble the peptide chain. During translation, several ribosomes associate to a single strand of mRNA, forming supramolecular complexes known as polyribosomes (polysomes).This project is aimed at developing and studying a miniaturized purification system able to isolate and extract polysome-associated mRNA, namely mRNA under active translation. The resulting microdevice will constitute a faster, simpler and low-cost alternative to the time-consuming traditional laboratory procedures for polysome purification and mRNA extraction (sucrose gradient centrifugation and phenol/ethanol RNA extraction). Polysome purification on microdevice will be based on the immobilization of polysomes to the device surfaces, opportunely treated to enhance polysome adhesion. Surface funtionalization will be achieved by formation of Self-Assembled Monolayers (SAM) of organic molecules. In particular, since both ribosomes and nucleic acids expose an high quantity of electrical charged moieties towards the environment [Anger et al., 2013], organic molecules containing charged functional groups will be used as SAM constituents. In this thesis a characterization of gold and silicon oxide plane samples functionalized with different alkanethiols and alkylsilanes SAMs will be presented as well as a quantitative and qualitative evaluation of polysome adhesion performed mainly by Atomic Force Microscopy (AFM). A proof of principle of the purification and extraction of RNA from polysomes using a silicon/Pyrex microdevice will be also reported. [Anger et al., 2013] Anger A.M., Armache J.P., Berninghausen O., Habeck M., Subklewe M., Wilson D.N. and Beckmann R. (2013) Structures of the human and drosophila 80S ribosome. Nature, 497(7447):80-85

The continuing difficulty of integrating Muslims, especially immigrants, into the European nations, has led many ruling political leaders to question the merits of multiculturalism and simultaneously to promote integration through the strengthening and inculcation in the Muslim of a more general set of political values. This thesis aims to examine how these national debates are interconnected and how they attempt to frame a common discourse on integration of Muslims. In order to analyse this debate, I have selected three national debates in France, Germany and the UK and applied a Critical Discourse Analysis (CDA) in order to compare similarities and the degree of convergence within the European public sphere. The analysis has sought to identify convergence by looking into the shared strategies of discourse through which civic integration is articulated, depending on the cultural and political particularities of each national context. The most significant finding to emerge from this study is that Muslims are asked to accept integration with their host countries, through a normative construction of collective identity that cuts across the different national discourses of Europe. These discursive constructions are based on similar definitions of the national values that belong to the European identity and, perhaps more crucially, those that do not belong. Accordingly, the normative and cultural assumptions that underlie various national discourses on civic integration are, in fact, based on European universalism, redefined at the national level. At the same time, this debate on integration systematically excludes the important economic and social problems of Muslim newcomers. Specifically, the discourse on civic integration avoids reference to any welfare program that attempts to produce economic security and social solidarity; Muslims become exclusively responsible for their own integration and civic integration is reduced to only their ability to internalise dominant values.

ClopHensor, a new fluorescent ratiometric GFP-based biosensor, is a powerful tool for non-invasive pH and chloride quantification in cells. ClopHensor is a chimeric construct, with the pH- and chloride-sensing E2GFP linked to the reference red protein DsRed-monomer, whose fluorescence is used as reference signal. E2GFP dissociation constant of about 50 mM (at pH=7.3) makes it ideal for quantifying physiological chloride concentration. However, chloride affinity of E2GFP strongly depends on pH value in solution: precise chloride measurement requires also a pH measurement. By ratio-imaging technique, three different excitation wavelengths are necessary for a pH and chloride concentration estimation. With the goal to reduce the number of excitation wavelengths required for ratio-imaging technique, in this thesis I present a detailed study of H148G-V224L-E2GFP, selected among several E2GFP-variants for its improved photophysic and spectroscopic characteristics. H148G-V224L-E2GFP exhibits a chloride affinity and a pH sensitivity similar to ClopHensor. Its emission spectra interestingly display two distinct emission peaks at 480 nm and 520 nm after excitation at 415 nm. Importantly, fluorescence emission spectra collected at various pH values also display a clear isosbestic point at 495 nm. This property allows the innovative possibility of pH and chloride concentration determination using only two excitation wavelengths. Moreover, while being chloride independent, the 520-to-495 (nm) ratio displays a pKa value of about 7.3, centered in the physiological pH range. These characteristics make it ideal for quantifying intracellular pH changes and chloride fluxes in physiological conditions. Applications in living cells of this new biosensor demonstrated its usefulness for ratio-imaging analysis. H148G-V224L-E2GFP+DsRed was successfully expressed in neuron-like cells, as proof-of-concept that ratio-imaging analysis can be performed also in neuron-like cells. These results are very promising for H148G-V224L-E2GFP+DsRed future expression in brain neurons, where chloride plays a crucial role in neuronal activity. Purified H148G-V224L-E2GFP was successfully uploaded in polymeric vaterite nanospheres to characterize their endocytosis pathways in cells.

Proton therapy is a well-established technology in radiotherapy, whose benefits stem from both physical and biological properties. Ions deposit the maximum dose, i.e. the ratio between the energy absorbed by the tissue and its mass (Gy = J/k g), in a localized region close to the end of their range (called the Bragg Peak BP). The combination of the favorable depth-dose profile with advanced delivery techniques translates into a high dose conformality in the tumor, as well as into a superior sparing of normal tissue compared to conventional radiotherapy with photons. Today, there are 107 proton therapy and 14 carbon ion centers operating worldwide, and many new ones are under construction. In Italy, the Trento proton therapy center and the proton and carbon ion center - Centro Nazionale per l’Adronterapia Oncologica - CANO in Pavia are already operational, while a third one is under construction at the Istituto Europeo Oncologico (IEO) in Milan and will be in operation in 2023. Although clinical results have been encouraging, numerous treatment uncertainties remain major obstacles to the full exploitation of proton therapy. One of the crucial challenges is monitoring the dose delivered during the treatment, both in terms of absolute value and spatial distribution inside the body. Ideally, the actual beam range in the patient should be equal to the value prescribed by the Treatment Planning System (TPS). However, there are sizeable uncertainties at the time of irradiation due to anatomical modifications, patient alignment, beam delivery, and dose calculation. Treatment plans are optimized to be conformal in terms of target coverage, healthy tissue spearing, and robust towards uncertainties. For this reason, the irradiation target is defined as a geometrical volume (Planning Target Volume PTV) corresponding to the physical tumor volume, to which safety margins of a few millimeters are added isotropically. Range errors determine the selection of the safety margins applied to the tumor volume, whose values depend on clinical protocols as well as on the treated area. For example, the Massachusetts General Hospital (MGH) prescribes safety margins equal to 3.5% of the nominal range +1 mm, while the University of Florida proton therapy center considers 2.5% of the nominal range + 1.5 mm. Decreasing the range uncertainties would reduce the safety margins, and hence the dose delivered to the normal tissue surrounding the tumor. In addition, a reduction of the proton range uncertainty could lead to the use of novel beam arrangements making greater use of the distal beam edge. Therefore it would be possible to maintain target coverage while reducing OAR and healthy tissue doses when the range uncertainty is low. Monitoring the proton range in vivo is a key tool to achieve this goal, and thus to improve the overall treatment effectiveness. Several techniques have been proposed to address the fundamental issue of in vivo proton verification, most of which exploit secondary particles produced by the interaction of protons and target nuclei, and are detectable outside the patient. Using these techniques, pre-clinical and clinical tests have obtained promising results in terms of absolute proton estimation. However, none of the investigated techniques are currently employed in the daily clinical workflow. A method already tested on patients is based on PET (Positron Emission Tomography) photon detection. The amount and emission distribution of PET photons depend on the target activity induced by the beam, as well by the delivered dose. Although this method has been clinically tested on patients, it has several limitations. The yield of annihilation photons produced during treatment depends on several factors, including the activity produced by the beam, which is fairly limited (up to two orders of magnitude lower than the diagnostic PET), the metabolic biological washout, and the background due to prompt radiation originated from other reaction channels. These issues have been partly resolved by the use of in-beam PET scanners, which measure annihilation photons during the treatment. One of the most advanced versions is the INSIDE (INnovative Solution for In-beam Dosimetry in hadronthErapy) PET scanner installed at CNAO (Centro Nazionale di Adronterapia Oncologica) in Pavia, Italy. Currently, it is part of a clinical trial and has acquired in-beam PET data during the treatment of various patients. Although encouraging results were obtained, still some limitations in its clinical applicability remain. In-beam PET is designed to work with low-duty-cycle accelerators, and so far it has only been installed in a fixed beam line. The other promising approach for in-vivo range monitoring is based on the prompt gammas (PGs) detection from nuclear de-excitation due to beam interactions in the tissue. The adjective prompt reflects the fact that they are emitted just a few pico-nano seconds after the impact of the proton on the target nucleus. The PGs escaping the patient have energy up to approximately 8 MeV, and their production is spatially correlated to the proton range. The feasibility of using an in vivo prompt gamma-based range verification for proton therapy has been demonstrated by numerous experimental and Monte Carlo studies, as well as by its recent application to the clinical practice for inter-fractional range variations. The current accuracy achieved on patients for retrieving the range of a single pencil beam is 2-3 mm. A major limitation identified by all studies that prevent the full exploitation of any prompt-gamma based approach for single spot range verification is the low statistics of the events produced. This issue is caused by: i) the short duration of a single spot delivery, ii) the immense gamma-ray production rate during delivery, iii) the finite rate capability of detectors, iv) the electronic throughput limits and v) the signal-to-background ratio. A particular PG range verification technique is prompt gamma spectroscopy (PGS). It relies on the analysis of the prompt gamma energy spectrum, which is characterized by specific energy lines corresponding to the reaction channels of the irradiated protons with the elements of the human body. The most common reactions are those with Oxygen and Carbon atoms, which become excited and eventually emit prompt gamma rays up to 8 MeV. Different studies on simplified geometries demonstrated that, by using the PGS technique, it is possible to estimate not only proton range variations, but also differences in the elemental composition of tissues. In this study, we present a novel approach for in vivo range verification via prompt gamma spectroscopy, based on creating signature gammas emitted only when protons traverse the tumor, and whose yield is directly related to the beam range. We propose to achieve this goal by loading the tumor with a drug-delivered stable element, that emits characteristic de-excitation PG following nuclear interactions with the primary protons. The use of tumor marker elements is not new in clinics: an example is a diagnostic PET which employs β+ emitter isotopes linked to a drug carrier, that is uptaken by the tumor allowing its diagnosis via PET scans (e.g. 18-FDG). In our approach, the radioisotope is substituted by a stable element, which decays via PG emission only when the proton interacts with it. By detecting signature gamma lines emitted by the tumor marker element, it is possible to assess if the beam has interacted or not with the tumor and increase the accuracy of the proton range estimation. Selection and characterization of candidate tumor markers The first part of this work focused on the identification of potential candidate elements following three criteria: i) emission of signature gamma energy lines following the proton irradiation, different from the characteristic emission of 12-Carbon and 16-Oxygen; ii) it should not be toxic for the patient iii) selection of an element whose carrier maximizes the tumor selectivity. While (i) is a purely physical constraint, and was deeply investigated in this work, points ii) and iii) depend also on several biological parameters, such as the achievable element concentration in the tumor, molecular carrier, tumor physiology, etc. To fulfill these criteria, we looked at elements that are already employed in medicine, either for diagnostic or therapeutic purposes and for which a drug carrier already exists. This allowed the applicability of our methodology in the clinic. Combining these criteria with simulations from the code TALYS, we identified three candidate tumor markers: 31-Phosphorous, 63-Copper and 89-Yttrium.We employed TALYS to characterize the elements in terms of the energy spectrum and gamma production cross section, and compared the results to Carbon and Oxygen, which are the two most abundant elements in the body. TALYS indicates that the three candidate elements produce signature gamma lines between 1 and 2 MeV, while Carbon and Oxygen signatures are between 3 and 8 MeV. Furthermore, the gamma yield per incident proton generated by the labeling elements is on average one order of magnitude higher than Carbon and Oxygen. To verify TALYS theoretical calculations, we designed an experimental campaign of prompt gamma spectroscopy measurements to characterize the emission of these elements when irradiated with a therapeutic proton beam. We irradiated two types of targets: solids made of 99.99% of candidate elements, and water-based solutions containing the label elements. While solids were used to characterize the PG energy spectrum emitted by the elements without background, the liquid targets were used to study the methodology in a setup closer to the clinical scenario, i.e by investigating the gamma emission of a compound material with a well-defined concentration of the marker element. Furthermore, using water-based solutions we were able to characterize the PG spectrum emitted by different element concentrations (from 2 M to 0.1 M), and evaluate the minimum value that provides a detectable signature. We characterized the elements by irradiating the different targets by using monoenergetic proton beam at 25 MeV and 70 MeV. Due to the thickness of the target, the beam looses all its energy inside the target, thus, these energies can be representative of a proton beam stopping in the first 5 mm of the tumor and after 4 cm depth, respectively. The 70 MeV proton beam was available at the experimental room of the Trento proton therapy center (Italy), while the Cyrcé cyclotron (Institut Pluridisciplinaire Hubert CURIEN-IPHC) in Strasbourg (France) accelerates protons up to 25 MeV. In the experiments performed in Trento and Strasbourg, we employed a LaBr3:Ce gamma-ray detector, which is suitable for our measurements as it is characterized by a fast detection response and high energy resolution. The data confirmed that all candidates emit signature PGs different from water (here used as a proxy for normal tissue), and that the gamma yield is directly proportional to the element concentration in the solution. We detected four specific gamma lines for 31P (1.14, 1.26, 1.78 and 2.23 MeV) and 63Cu (0.96, 1.17, 1.24, 1.326 MeV), while only one for 89Y (1.06 MeV). We compared all experiments with TOPAS MC. It is one of the leading toolkits for simulating particle interaction in the matter for medical physics applications. The comparison between simulations and experiments suggested that TOPAS is able to predict the energy of all characteristic gammas detected in the experimental spectrum, while the yield is either underestimated or overestimated, depending on the gamma-ray energy and element. Previous works had already shown TOPAS limited accuracy in reproducing nuclear de-excitation gammas, even for the most common materials like 16-Oxygen and 12-Carbon, and suggested that this discrepancy stems from the nuclear reaction models implemented in the physics list. Our findings support the hypothesis that the nuclear reaction cross section models available in TOPAS MC predict results with limited accuracy also for 31P, 63Cu and 89Y. Prompt-gamma yield and proton range correlation The finding of the first part of this work indicated that loading the tumor with 31P, 63Cu and 89Y generates a signature PG energy spectrum when irradiated with protons at therapeutic energies. In the second part of the project, we experimentally showed how the PG yield correlates with the proton range. We designed a multilayer phantom to mimic the irradiation of a deep-seated tumor. The phantom was composed of 15.5 cm of solid water (proxy of normal tissue), followed by 5 cm of liquid target filled with water-based solutions containing the marker element (tumor region) and an additional 2 cm of solid water for protons stopping downstream of the tumor.We irradiated the phantom with protons of energy ranging from 154 MeV (16.3 cm range in water) to 184 MeV (22.5 cm range) in order to build an experimental curve of the PG yield of different gamma-ray lines versus the proton range. We also acquired a blind spectrum at an unknown proton energy and used the curve to predict the range. By using the de-excitation peaks of 6.12 MeV from 16O, 4.44 MeV from 12C and 1.26 MeV from 31P, we successfully predicted the proton range of the blind data within 2 mm from the nominal value. The same test was repeated using a 63-Copper target, but due to the signature gamma lower yield, we overestimated the proton range prediction of 5 mm. As already observed for the liquid targets, large discrepancies were found between the experimental data and the simulation. This confirmed that TOPAS MC is not an accurate tool for predicting the PG yield. Toward the clinical application In the last part of the thesis, we discuss the applicability of the presented approach to patients. All experimental measurements were performed in conditions not clinically realistic because they investigated the basic principles of the methodology and provided a proof-of-principle. Using the measurements acquired at 70 MeV with liquid targets, we evaluated the expected PGs produced during a proton therapy treatment if the tumor were irradiated with 109 protons, the elements were loaded with a concentration of 0.4 mM (possible value when a glucose-based carrier is used) and a detection system with a larger solid angle acceptance (5sr) than the one used in our experiments (0.13 sr). We also started a preliminary in-silico investigation of our methodology applied to a real patient geometry. All experimental and simulated results so far presented were obtained by irradiating only homogeneous phantoms without taking into account patient heterogeneity and complex elemental compositions of the different tissues. To reproduce the patient geometry, we used a Computed Tomography (CT) image (3D map of the patient’s anatomy and tissue densities). The tumor region was localized on the prostate organ and its elemental composition and was artificially modified to achieve a homogeneous 31-Phosphorus, 63-Copper and 89-Yttrium concentration at a 5% percentage mass fraction for speeding up the computational time. TOPAS MC was used to simulate the irradiated of the tumor region with a 174 MeV proton beam and we simulated different beam position shifts from the nominal plan of 0.2, 0.4, 0.7, 1.0, 1.2, 1.4, 1.7 and 2 cm. Following the approach of the Massachusetts General Hospital group for prompt gamma spectroscopy range verification, we estimated the voxel-based gamma-ray yield from the elemental composition of the patient (CT scan) and from the gamma-ray production cross sections. TOPAS MC was used only for the calculation of the proton kinetic energy in each voxel of the patient. This analysis highlighted that gammas generated by the label elements are strongly correlated to the elemental composition of tissues traversed by the beam. When the beam partially misses the tumor region, the number of signature PGs emitted by the marker element decreases. Several aspects of the methodology still require further investigation and optimization from a physical, engineering and biological point of view. in vitro and in vivo toxicity studies must be conducted to determine the best carrier molecule that maximizes the tumor’s element concentration. Furthermore, to increase the accuracy of proton range estimation a novel gamma spectroscopy detection system must be designed to be fully integrated with the gantry treatment room. In conclusion, in this work, we demonstrated that loading the tumor with a label element before proton treatment generates signature gammas that can be used to verify the beam range in vivo. We selected three candidate elements already used in the clinic as promising tumor markers. We successfully employed these elements to simulate a proton range verification methodology on a homogeneous phantom. We showed how the current nuclear reaction models for prompt gamma spectroscopy applications are not accurate in predicting the PG yield from all the elements investigated. Further work is necessary to investigate the effect of a non-homogeneous element uptake due to tumor physiology on the proton range accuracy, as well as the diffusion of the label element on the normal tissue surrounding the tumor.

Ce travail se situe dans le domaine de la sociologie des religions : il s’agit d’une monographie issue d’une enquête de terrain conduite avec des méthodes qualitatives. L’enjeu est de saisir la croyance religieuse à partir de l’étude d’un phénomène spécifique : le lien social qui s’est créé entre un ancien sanctuaire et une petite ville. La séculière dévotion à l’égard des saints patrons Victor et Couronne qui s’est développée dans un territoire de l’Italie du Nord donne lieu à une sociabilité qui franchit la limite des motivations et des fins purement religieuses. Le recueil d’une vaste documentation historique d’archives complète les sources et les données que l’on a collectées selon une approche interdisciplinaire qui mobilise la sociologie, l’anthropologie et l’histoire.

MicroRNAs (miRNAs) are short non-coding RNAs, whose primary function consists in mRNA silencing. Mature miRNAs are found in the cytoplasm as single-stranded molecules but there is growing evidence that miRNAs can be excreted by cells, mainly encapsulated inside exosomes, in almost all body fluids. It has also been shown that the level of expression of some of these circulating miRNAs (e.g. miR-21) varies significantly under pathological conditions such as in the presence of cancer. Circulating miRNAs are therefore emerging as promising non-invasive diagnostic and prognostic tumour biomarkers. Nevertheless, current methods for the purification of circulating miRNAs are challenging, mainly due to low body fluid concentration, variability, and quantification limits. This thesis aimed at developing and studying an innovative miniaturised strategy for the purification and detection of cancer-related circulating miRNAs. The employment of microdevices could provide a faster, simpler and low-cost alternative to the current laboratory procedures for the analysis of extracellular miRNAs. The solid-state miRNA purification method shown here is based on the introduction of chemical and morphological modifications on the surface of an adequate substrate (silicon, PDMS). In particular, surface functionalisation with organic molecules carrying charged functional groups was employed to establish specific interactions with the electrical charged moieties of miRNAs. Modulation of the charge density and morphology will be allowed by the additional introduction of neutral organosilanes characterised by different chain length. In this thesis, a detailed chemical and morphological characterisation of the modified planar surfaces is presented and correlated with the capacity to selectively purify miRNAs from a complex biological sample. The most efficient condition was implemented on a PDMS microdevice and further coupled with a sensitive detection technique (RT-qPCR). The performances of our purification system will be eventually tested with both synthetic miRNAs and biological samples.

Proteins are biological macromolecules that consist of long chains of small building blocks, called amino acids. These long sequences of amino acids are unique for each protein, define a specific three-dimensional structure that allows the protein to carry out a specific function in a living organism. In fact, they can catalyse metabolic reactions, respond to stimuli, provide structure and transportation routes within the cell [1]. In a cell proteins are ubiquitous. They can be soluble in water and have usually a globular shape; they can be arranged in fibers, give structural integrity to their host, and provide the infrastucture upon which small molecules are transported where needed; they can be embedded, partially or totally, in the membrane, a wall of a lipidic bilayer, of the cell and mediate the exchange of matter with the environment. In particular, membrane proteins are categorised into three groups: permanently attached to the membrane, integral membrane proteins have several structural elements that span the width of the membrane; peripheral membrane proteins are temporarily attached to the lipid bilayer by hydrophobic and electrostatic interactions, usually following a post-translational modification of a soluble protein; water-soluble proteins, like toxins, that upon aggregation, attack the membrane and cause the disrupture of the cell. In the last decades, the availability of structural information on proteins and their three-dimensional conformation enabled the rapid development of a computational tool, molecular dynamics (MD), that allows to explore biological processes and systems at a sub-nanometer scale. The idea behind MD is to integrate Newton’s equations of motion to describe the evolution of a protein within its biological environment. The refinement of the empirical potentials, called force fields, that defines the interactions of the system of interest and the increase in the computational resources of modern computers have enhanced scientists to investigate and characterise dynamics and functions of protein with high predictive power. This methodology is nowadays widely established as an in silico technique and can be considered a real computational microscope [2, 3]. Despite its successes, the complexity and the timescale involved in realisation of a biological process required the development of new techniques that accelerate the dynamics of the system under scrutiny and the sampling of conformations of the macromolecule [4]. Enhanced sampling methods are, therefore, essential for the study of conformational transitions, key events that trigger the function of a protein. In this thesis I will focus mainly on three membrane proteins I studied in my research that span different functions and interactions with the lipid bilayer. The presence of the membrane slows down the dynamics of an em- bedded protein with respect to the water-soluble counterpart [5]. In addition, it requires a specific treatment of the system and the biological conditions necessary to mimic the experiments as close as possible. Therefore, the first chapter will be devoted to introduce molecular dynamics as a computational technique to shed light on proteins dynamics and the undelying mechanisms of the functions they perform. I will discuss the algorithms that allow a predictive use of molecular dynamics in the presence of the membrane, and a better approximation of the experimental conditions in which biological data are gathered [6]. In addition, I will briefly describe the enhanced sampling methods used to investigate large conformational changes, and the analysis techniques used to extract meaningful information from the simulations. The rest of the thesis will describe the systems that I studied in my research work. In the second chapter I will digress on the prestin protein. Prestin is a motor protein and it is present in arrays in the cochlear outer cells in the mammalian hearing mechanism. Due to its coordinated contraction and elongation in response to external stimuli, this protein changes the shape of the cell allowing the transduction of the signal. This mechanism is mediated by a ligand, but there is no evidence of the transport of the ligand across the membrane. The non-mammalian ortholog of this protein is highly similar in the amino acid sequence, but it does not perform the same function. In fact it is a transporter that allows the exchange of chloride ions, and oxalate molecules, from the intracellular to extracellular environment, and viceversa. To investigate this difference, first I performed the simulation of two proteins, the expression of prestin in the rat and in the zebrafish species, in two conformations, inward open and outward open, for 700 ns each starting from homology models, due to the absence of experimental crystal structures. I assessed the relaxation of the four structures toward a stationary state, and the equilibrated systems were simulated under the action of an external electric field to mimic the cellular environment. with this second step I was able to determine the different paths of chloride ions in the two homologs in the binding to a conserved residue, S398 in rat and S401 in the zebrafish. Finally, each expression of the protein underwent biased simulations to explore possible pathways in the change from the inward to the outward conformation. The data are not definitive to draw a conclusion, although the elevator mechanism seems to favour the elevator-like transport, a mechanism proper of other proteins in the same family of the prestin. In the third chapter I will discuss the insertion of the recoverin protein, a peripheral membrane protein, in a membrane patch. Recoverin is a calcium sensor protein expressed in the vertebrate retina. The binding of two calcium ions triggers the extrusion of a myristoyl group, a post-translational modification of the N-terminus of the protein that adds a hydrophobic chain. This extrusion gives the protein an anchor to bind the lipidic bilayer, and this insertion leads to the formation of a complex with rhodopsin kinase. In collaboration with a master student, I simulated the recoverin in two conditions, both isolated and in the complex with a peptide from the rhodopsin kinase, to investigate its unbiased anchoring. We found that the insertion of the myristoyl is highly enhanced by the electrostatic interaction of the lipidic charged group and arginines of the surface of the protein. The same pattern were found in both setups, and the abovementioned interactions were no longer required to keep the protein in contact with the membran after the myristoyl penetrated the lipidic patch. In addition we analysed the communication networks of the systems and how it was affected by the presence the peptide. This could shed a light on how the recoverin-rhodopsin kinase complex assemblies itself. The last chapter will be devoted to the conformational changes of aquaporin type 4 upon aggregation. This membrane protein is a water channel, assembled in tetramers. In the human species it is present in two isoforms, M1and M23, named after the starting residue of the N-terminus. Studies shows that in the isoform M23, AQP4 aggregates and is more likely to form large orthogonal array of particles (OAPs) that are target for the antibody AQP4-IgG. This leads to an inflammatory disease, neuromyelitis optica [7]. Although the AQP4 has already been studied as a pharmaceutical target, there is no in silico study of the protein in the isonform M23. In order to mimic the OAPs, I created an assembly of four tetramers and simulated it for 800 ns. I analysed the influence of the N-terminus after the aggregation, and no evidence of a significant difference in the global behaviour of the protein were found. New insights are instead evident in the arrangement of the transmembrane segments of the protein. Further developments are being studied to have a better understanding of the aggregation mechanism.

Questa tesi cerca di fornire un quadro di analisi delle azioni e interazione che caratterizzano la produzione di musica indipendente a Milano. Il punto di partenza del progetto è stato uno studio della letteratura collegata alle ‘scene musicali’ (Peterson & Bennett, 2004), termine utilizzato per comprendere una vasta gamma di studi che condividono una visione comune di scena musicale come legata a affinità di genere o a un contesto territoriale. Facendo riferimento a prospettive diverse, la tesi critica la nozione di scena musicale (Webb, 2007) perché non permette di analizzare la complessità e la mobilità delle pratiche musicali (Urry, 2005), e non considera le interazioni esterne con forze politiche ed economiche (Bourdieu, 1993). Per questo motivo, la tesi fornisce un modello per l'analisi di circuiti di produzione di musica indipendente. Il modello comprende tre livelli di analisi, uno relativo alla necessità di analizzare la scena musicale per le sue caratteristiche produttive e organizzative, come circuiti di produzione culturale; il secondo considera la scena come interconnessa a un contesto mediale, economico, normativo, politico e culturale; e il terzo livello cerca di analizzare la natura complessa del territorio in cui pratiche si svolgono, che non può essere limitata ai confini delle città. / This thesis attempts to provide a new analytical framework for the study of actions and interactions of small-scale independent live music production in Milan. The starting point of the project has been the literature connected to the music scene perspective (e.g. Peterson & Bennett, 2004), which comprises a wide range of studies sharing a common understanding of scenes as either genre-based or locally specific. Referring to different frameworks, the thesis criticizes the music scene perspective (e.g Webb, 2007) because it doesn’t allow to analyze the complexity and mobility of music-making practices (Urry, 2005), and the need of considering the external interactions with political and economic forces (Bourdieu, 1993). For this reason, the thesis provides a multi-layered model for the analysis of circuits of independent music production. The model comprises three levels of analysis, one concerning the need to analyze the music scene for its productive and organizational characteristics as circuits of cultural production, the second one considers the scene as interconnected to the media, economic, regulatory, political and cultural environment and the third level that seeks to analyze the complex nature of the territory in which such practices take place which cannot be restricted to physical space of the city.

La tesi ha come oggetto la street art italiana. Questa, sebbene comunemente associata a realtà contro o subculturali, specie nell’ultimo decennio ha visto diversi suoi esponenti percorrere una parabola che da un underground riservato a una nicchia di appassionati, li ha portati a confrontarsi tanto con il mondo dell'arte quanto con il mercato. Una parabola che, in senso diacronico, questa tesi ha cercato di ripercorrere, servendosi degli strumenti propri delle teorie della produzione culturale, analizzando quindi la molteplicità di trasformazioni sociali e culturali che l'hanno accompagnata e resa possibile. Allo stesso tempo, attraverso una metodologia “grounded”, si è tentato di dare conto, su un piano sincronico, dello scenario attuale della street art nazionale, attraversato da tensioni che, di fatto, hanno portato il ricercatore a misurarsi con il processo di crescente “individualizzazione” e “de-standardizzazione” del mercato del lavoro. La tesi si suddivide quindi in quattro macro-sezioni: la prima finalizzata alla descrizione del processo di ricerca; la seconda tesa a ricostruire l’“emersione” dei fenomeni subculturali da cui gli street artists provengono; la terza, volta alla descrizione del panorama contemporaneo della street art italiana, attraverso l'individuazione di “profili” nelle cui caratteristiche si è cercato di far luce su quello che, a tutti gli effetti, può considerarsi un modello di “socialità reticolare”; la quarta, in cui si sono messi a confronto l'attuale fisionomia agerarchica della street art, caratterizzata da una differente distribuzione di opportunità a seconda del diverso capitale sociale posseduto dai suoi protagonisti, e alcune ipotetiche “soluzioni” a tali squilibri. / Street art has always been associated with underground phenomena like sub or counter-cultures. But, especially during the last ten years, many street artists got more and more involved in the institutional field of cultural production, also working for marketing, advertising and graphic design agencies. Starting from a perspective close to the theories of cultural production, the dissertation focused on the social, cultural and economic transformations that have contributed to such an “explosion” of the street art movement. At the same time, using a “grounded” approach, it studied the contemporary world of the Italian street art, as a field crossed by my many tensions, generated by a different distribution of what Pierre Bourdieu defined “social capital”.