This thesis provides a thoroughly theoretical background in network theory and shows novel applications to real problems and data. In the first chapter a general introduction to network ensembles is given, and the relations with “standard” equilibrium statistical mechanics are described. Moreover, an entropy measure is considered to analyze statistical properties of the integrated PPI-signalling-mRNA expression networks in different cases. In the second chapter multilayer networks are introduced to evaluate and quantify the correlations between real interdependent networks. Multiplex networks describing citation-collaboration interactions and patterns in colorectal cancer are presented. The last chapter is completely dedicated to control theory and its relation with network theory. We characterise how the structural controllability of a network is affected by the fraction of low in-degree and low out-degree nodes. Finally, we present a novel approach to the controllability of multiplex networks

Plusieurs études théoriques intéressées par l’Afrique subsaharienne, plus particulièrement par la République Démocratique du Congo (RDC), abordent dans une perspective de genre, la problématique de l’accès des femmes à la terre. Elles se concentrent sur les restrictions de l’accès à la terre et exposent les failles de la gouvernance foncière. Elles se focalisent, par exemple, sur la dualité dans la gestion foncière, les discriminations et obstacles auxquels sont sujettes les femmes, la non-application de la loi, les pratiques coutumières, etc. L’originalité de la présente thèse consiste à aborder la problématique de l’accès des femmes à la terre et du contrôle qu’elles peuvent concrètement exercer, par le biais d’une étude empirique à Beni, ville du nord-est de la RDC. D’une part, l’objectif est d’étudier le niveau auquel un outil « hybride », soit le modèle du domaine de la tenure sociale (STDM), a été intégré ou adapté par les acteurs afin d’être plus acceptable pour le système d’administration foncière étatique existant, en RDC, qui est un système rigide. D’autre part, analyser la sensibilité du STDM aux enjeux de genre relatives au foncier. L’étude se fonde sur la revue de la littérature et sur une analyse qualitative des résultats. Ceux-ci ont été classés par thématiques. Les analyses ont révélé que le STDM a fonctionné parallèlement avec le système préexistant. Il a quand même influencé le système d’administration foncière. Pourtant, ce système hybride n’a pas effectivement résolu les conflits fonciers, sécurisé les droits des pauvres et surtout des femmes pauvres. Les documents initiaux du projet ne priorisent pas les questions genre. Ainsi, ce dernier thème suscite une incompréhension ou une indifférence de la part des parties prenantes.

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

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.

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.

L’organisation territoriale de l’Égypte « unifiée » a dans un premier consisté en un découpage des deux grandes entités géographiques qui la constituent, la Haute et la Basse-Égypte, en un nombre variable de régions nommées sp3.t (sépat). Chacune de ces divisions, que nous nommons « province », possède une histoire, une culture et des cultes qui lui sont propres mais qui s’inscrit malgré tout dans celle du pays.L’étude de la XVIe province de Haute-Égypte, ou province de l'Oryx, se fait sur trois niveaux. Tout d’abord la géographie humaine, qui traite de l’administration du territoire comme des ressources ainsi que de leur évolution, mais également des individus vécurent à ces époques et prirent part à l’histoire de la province. Ensuite, la topographie cultuelle qui s’intéresse à la répartition des nécropoles et des lieux de cultes dont l’intérêt concerne autant l’économie que l’influence cultuelle. Enfin la géographie religieuse s’appuie sur les conceptions mythologiques, comme les processions géographiques, qui reprennent des éléments de tradition cultuelle dans le but de développer une véritable somme cultuelle de chaque province alors que, durant le IIe millénaire AEC, le système provincial s’effondre. Dès lors, le découpage des provinces se maintient dans les temples, bien que certains éléments de ces sommes soient de pures inventions destinées à compléter des données inconnues ou inexistantes, mais créant alors de toutes nouvelles traditions.De cette manière, la province de l’Oryx se démarque par une importance relative durant l’Ancien Empire mais surtout durant le Moyen Empire. Au Nouvel Empire, le fractionnement de la province a conduit certaines des localités les plus importantes à nouer des relations avec une métropole voisine, Hermopolis. Ainsi, peu à peu, la province perd en puissance et finit par perdre son identité au profit du nouveau nome hermopolitain. En revanche, durant ce déclin, l’ancienne capitale, Hébénou, voit un regain de popularité à travers l’emblème de la province, l’oryx qui devient une bête séthienne et maléfique. Sa divinité poliade, Horus, acquiert alors un statut de chasseur d’oryx et de restaurateur de l’ordre cosmique, des traits qui deviennent le nouveau visage de cette province autrement oubliée. / The territorial organisation of the « united » Egypt consisted first in a division of the two great geographical entities composing it, Upper and Lower Egypt, in a varying number of regions called sp3.t (sepat). Each one of these divisions we call “province” has its own history, culture and cults which are included in the ones of the country.The study of the 16th Upper Egyptian province, or Oryx province, stands on three levels. First, human geography deals with territory administration and resources and their evolution, but also on the individuals who lived in those times and took action in the history of the province. Then, cultic topography focuses on the distribution of necropolises and sanctuaries, which interest is economy as well as cultic influence. At last, the religious geography is based on mythological constructions, such as geographical processions, which take elements from the cultic traditions in order to develop real cultic summas while, during the 2nd millennium BCE, the provincial system collapses. Therefore, the provincial division remains in the temples, even if some elements of these summas are pure inventions intended to complement unknown or inexistent data, creating so whole new traditions.In this way, the Oryx province stands out with a relative importance during the Old Kingdom, but especially during the Middle Kingdom. In the New Kingdom, the splitting of the province led some of the most important localities to bond with a neighbour metropolis, Hermopolis. Thus, progressively, the province loses power and ends up with losing its own identity in favour of the new Hermopolitan Nome. However, during this demise, the old capital, Hebenu, gets a popular revival through the province emblem, the oryx, which becomes a sethian and maleficent beast. Its poliad divinity, Horus, acquires a new status of oryx hunter and cosmic order restorer, features forming the new face of this otherwise forgotten province.

This thesis examines various methods for estimating the spatial distribution of solar radiation, and in particular its diffuse and direct components in mountainous regions. The study area is the Province of Bolzano (Italy). The motivation behind this work is that radiation components are an essential input for a series of applications, such as modeling various natural processes, assessing the effect of atmospheric pollutants on Earth's climate, and planning technological applications converting solar energy into electric power. The main mechanisms that should be considered when estimating solar radiation are: absorption and scattering by clouds and aerosols, and shading, reflections and sky obstructions by terrain. Ground-based measurements capture all these effects, but are unevenly distributed and poorly available in the Italian Alps. Consequently they are inadequate for assessing spatially distributed incoming radiation through interpolation. Furthermore conventional weather stations generally do not measure radiation components. As an alternative, decomposition methods can be applied for splitting global irradiance into the direct and diffuse components. In this study a logistic function was developed from the data measured at three alpine sites in Italy and Switzerland. The validation of this model gave MAB = 51 Wm^-2, and MBD = -17 Wm^-2 for the hourly averages of diffuse radiation. In addition, artificial intelligence methods, such as artificial neural networks (ANN), can be applied for reproducing the functional relationship between radiation components and meteorological and geometrical factors. Here a multilayer perceptron ANN model was implemented which derives diffuse irradiance from global irradiance and other predictors. Results show good accuracy (MAB in [32,43] Wm^-2, and MBD in [-7,-25] Wm^-2) suggesting that ANN are an interesting tool for decomposing solar radiation into direct and diffuse, and they can reach low error and high generality. On the other hand, radiative transfer models (RTM) can describe accurately the effect of aerosols and clouds. Indeed in this study the RTM libRadtran was exploited for calculating vertical profiles of direct aerosol radiative forcing, atmospheric absorption and heating rate from measurements of black carbon, aerosol number size distribution and chemical composition. This allowed to model the effect of aerosols on radiation and climate. However, despite their flexibility in including as much information as available on the atmosphere, RTM are computationally expensive, thus their operational application requires optimization strategies. Algorithms based on satellite data can overcome these limitations. They exploit RTM-based look up tables for modeling clear-sky radiation, and derive the radiative effect of clouds from remote observations of reflected radiation. However results strongly depend on the spatial resolution of satellite data and on the accuracy of the external input. In this thesis the algorithm HelioMont, developed by MeteoSwiss, was validated at three alpine locations. This algorithm exploits high temporal resolution METEOSAT satellite data (1 km at nadir). Results indicate that the algorithm is able to provide monthly climatologies of both global irradiance and its components over complex terrain with an error of 10 Wm^-2. However the estimation of the diffuse and direct components of irradiance on daily and hourly time scale is associated with an error exceeding 50 Wm^-2, especially under clear-sky conditions. This problem is attributable to the low spatial and temporal resolution of aerosol distribution in the atmosphere used in the clear-sky scheme. To quantify the potential improvement, daily averages of accurate aerosol and water vapor data were exploited at the AERONET stations of Bolzano and Davos. Clear-sky radiation was simulated by the RTM libRadtran, and low values of bias were found between RTM simulations and ground measurements. This confirmed that HelioMont performance would benefit from more accurate local-scale aerosol boundary conditions. In summary, the analysis of different methods demonstrates that algorithms based on geostationary satellite data are a suitable tool for reproducing both the temporal and the spatial variability of surface radiation at regional scale. However better performances are achievable with a more detailed characterization of the local-scale clear-sky atmospheric conditions. In contrast, for plot scale applications, either the logistic function or ANN can be used for retrieving solar radiation components.