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.

Proteins are one of the most studied biological molecules of the last decades. A great amount of experimental techniques provide to researchers direct or indirect informations on proteins structure and function. In silico simulations can be used as a “computational microscope” giving the possibility to observe protein dynamic properties at atomistic resolution. In this work, various applications of computational methods to biological systems are presented. In particular, all-atom Molecular Dynamics (MD) simulations were employed to investigate the behaviour of proteins at atomstic resolution. The term “Molecular Dynamics” is usually referred to computational methods used for the simulation of classical many-body systems. These techniques are applied to microscopic systems and they represent a powerful approach for the study of physical processes, providing a tool for their interpretation. They have been widely used in the past decades to elucidate a large variety of molecular processes in different fields such as solid state physics, material science, chemistry, biochemistry and biophysics. Here, all-atom MD simulations were employed to observe equilibrium properties of several biologically relevant proteins. This allowed us to direct perform a comparison of molecular mechanisms occurring at the atomistic level as obtained from in silico studies with experimental data, which usually describe processes at larger length and time scales. These MD simulations were also meant as a starting point for the construction of simplified models, as they were processed through coarse-graining procedures to extrapolate crucial systems features, such as informative protein sites, on the basis of information theory approaches. Specifically we studied the dynamics of pembrolizumab, a humanized immunoglobulin of type G4 (IgG4) used as a therapeutic antibody. It is employed for the treatment of lung cancer, melanoma, stomach and head cancer and Hodgkin’s lymphoma. This antibody interacts with the programmed cell death protein 1 (PD-1) receptor, blocking the suppression of the immune response during cancer development. The studied systems are three: the apo state of pembrolizumab, the holo state (i.e. pembrolizumab bound to PD-1) and the glycosylated apo configuration. Each configuration was simulated for 2μs, for a total of 6μs. The analysis of the trajectories was carried out by combining standard structural analysis techniques and information theory-based measures of correlation. From MD trajectories we could extract valuable informations on the connectivity that exists among the structural domains that compose the antibody structure. Moreover, it was possible to infer which regions are involved in the structural rearrangement in the case of the antigen binding. We could observe that the presence of the antigen reduces the conformational variability of the molecule giving a greater stability to it. The second studied system is the P53 protein complex. In this case we focused on the tetramerization domain (TD) region that is composed by 2 identical dimers and has the function of bringing together the four monomers of the p53 complex. Starting from the observation that in case of the mutation of residue R337 several pathologies are developed in humans, we constructed computational models to reproduce the dynamics of the mutants and investigate their behaviour in silico. We performed simulations for a total of 16 μs divided in 8 different cases. In the first part of the study the wild type (WT) protein was compared to the R337C and the R337H mutant in three different protonation states: delta protonated Histidine, epsilon protonated Histidine ad double protonated Histidine. In the second part of the study we highlighted the differences between the WT configuration and three rationally designed mutants: R337D-352D, 337R-D352R, R337D-D352R. In this part of the investigation, the importance of the electrostatic interaction between residues R337 and D352 in the stability of the tetramerization do- main was discussed. Furthermore, we matched the obtained computational results of p53 tetramerization domain with functional experiments in yeasts (performed in collaboration with the CIBIO department) of all the simulated forms. The third simulated protein is the zinc sensing transcriptional repressor (CzrA), an homodimeric protein that binds DNA in Staphylococcus aureus. All-atom MD simulations of two different configurations were performed for a total of 4μs, the first one is the WT apo protein while the second is the WT holo system, where the protein is complexed with two Zn ions. In this case, in addition to standard analysis techniques, we applied the mapping entropy minimization protocol to highlight the most informative protein regions, from the perspective of information theory. Finally, our in silico results were compared to available NMR data of the protein itself.

MACPF/CDCs proteins are a huge family of pore-forming proteins present from the bacteria to the human genera. Cholesterol-dependent cytolysins (CDCs) are a family of toxins that participate in bacterial infection pathway at the membrane level. Great interest in this family is due to their similarity, in structure and in pore-forming mechanism, with some human immune system proteins (MACPF). We focused our attention particularly on two bacterial CDCs, Perfringolysin O and Listeriolysin O, and on the human protein Perforin, which is involved in the apoptotic pathway facilitating Granzyme release. In the literature, two possible configurations of CDCs and Perforin pores are proposed: ring and arc structures that could have different implications on the biological mechanism of action of these pore-forming proteins. By electrophysiological measurements and atomic force microscopy technique on different artificial membrane, we are able to enrich the ring and the arc fraction and demonstrate that both kinds of pore are active, i.e. conduct ions. Thus, my PhD work underlines two physiological structures which are involved in several ways, more than merely by disrupting membrane integrity, in pathogenic attack (bacterial CDCs proteins) as well as in immune response (human Perforin proteins).

Photocatalytic water splitting using solar energy could contribute to the solution of environmental and energy issues related to the hydrogen production. Key research area in this field is the development of photo-catalyst able to provide high energy conversion efficiency. TiO2 has been mostly preferred material as the photo-electrode due to many advantages, mainly related to the cost factor and stability. We have studied on hydrogen production by water splitting in photo-electrochemical cells prepared by using photoanodes made by two different kinds of TiO2: one deposited by RF sputtering and the other one by sol-gel method. Depositions were performed on electrical conducting ITO whose electrical properties plays vital role to reduce the photon energy loss. The photoanodes have been characterised by several techniques to infer on their optical and compositional properties. The observed differences in hydrogen production have been attributed to the peculiarities in absorption properties of the two TiO2 films that in the case of sputter-deposited films are more prone to absorb radiation also because of the produced defects during the deposition process. Metals like Cr and Fe were doped in TiO2 by RF magnetron sputtering and sol-gel methods to increase the efficiency of hydrogen production by water splitting by sensitizing the doped-TiO2 in visible light spectrum. The doping method, dopant concentration, charge transfer from metal dopants to TiO2, and type of dopants used for modification of TiO2 were investigated for their ability to enhance photocatalytic activity. UV-Visible spectra show that the sputter-metaldoped- TiO2 films are much more efficient than the chemically-prepared samples to induce red shift of the absorption edge for absorbing visible light. In addition, we proved that dopant atoms must be located, at low concentration, near the ITO-TiO2 interface to avoid the formation of recombination centers for photo-generated electron-hole pairs. H2 production rate is higher with Fe-doped TiO2 (15.5 μmole/h) than with Cr-doped TiO2 (5.3 μmole/h) because Fe ions trap both electrons and holes thus avoiding recombination. On the other hand, Cr can only trap one type of charge carrier. To increase the light conversion efficiency and reduce the recombination processes of Cr-doped TiO2, a multilayer structure of ITO/Cr-doped-TiO2 (9 at.%) was developed. When the multilayer films were exposed to visible light, we observed that the photocurrent increases as function of the number of bilayers by reaching the maximum with 6-bilayers of ITO/Crdoped-TiO2. The enhanced photocurrent is attributed to: 1) higher absorption of visible light by Cr-doped-TiO2, 2) number of space-charge layers in form of ITO/TiO2 interfaces in multilayer films, and 3) generation of photoelectrons just in/or near to the spacecharge layer by decreasing the Cr-doped-TiO2 layer thickness. The superior photocatalytic efficiency of the 6-bilayers film implies higher hydrogen production rate through water splitting: we obtained indeed 24.4 μmole/h of H2 production rate, a value about two times higher than that of pure TiO2 (12.5 μmole/h). Similar experiment we performed by doing TiO2 with vanadium metal. With 6-ilayers vanadium doped TiO2 film Shows higher hydrogen production rate of about 31.2 μmole/h. This rate is higher than that of CR doped and pure TiO2. A constant H2 generation rate is obtained for long periods of time by all the investigated TiO2 films because of the separate evolution of H2 and O2 gas, thus eliminating the back-reaction effect. Even Ar+ or N+ ion implantation of energy 30 keV was adopted to vary the energy band gap of TiO2 film in order to absorb visible light.The original anatase phase was not changed by implantation. Increase in full visible absorption range was observed for both kinds of ion implanted-TiO2 films which further increases with the ion fluencies, while N+ ion implantation also causes the shift of the absorption edge from UV to visible light range. N+ implanted TiO2 showed narrowing of band gap from 3.2 eV for untreated anatase TiO2 to 2.78 eV for maximum implantation dose. The Ar+ and N+ implantation creates oxygen vacancies related defect energy level in the band gap. In case of N+ implantation, nitrogen also substitutionally replaces the oxygen atoms thus forming an energy level just above the valence band which further interacts with O 2p states resulting in the narrowing of band gap. The black solar absorber material develop over the copper target to absorb concentrated solar radiation and supply heat to the surrounding water. A black copper oxide layer was synthesized over copper substrate by using chemical oxidation treatment. We varied several treatment parameters and optimized the best condition to obtain a black textured layer which has the properties to absorb total solar radiation. The untreated polished copper showed 50 to 60 % reflectance (R) (incidence angle of 15o) and this value decreases to almost zero for whole wavelength range after formation of black copper oxide. The percentage absorption decreases by negligible amount as the angle of incidence increases. The SEM images of the copper oxide layer at high magnification showed a nano-petal like structure which causes the surface texture effect for higher absorption where surface irregularities such as grooves and pores with dimensions similar to the wavelength of the incident radiation simply increase the solar absorptance by multiple reflections. Long time thermal stability and corrosion resistance in hot water was also studied for the copper oxide film. The results revealed that the copper oxide was very stable and showed no changes in optical properties after the test. For the same water heating system a quartz window is used through which the solar radiation is transmitted on the copper target. Thus to acquire high power conversion efficiency it is necessary for quartz window to transmit the entire solar radiation incident on it without much lost due to the reflection on the surface. In general quartz window is able to transmit 90-91 % of the solar radiation while 1-2 % is absorbed and 7-8 % is reflected from the surface. Thus to have nearly complete transmittance it is necessary to cover the surface of quartz window with anti-reflecting (AR) coating: this was the part of my work. We developed single-layer and multi-layer AR coating for single specific wavelength and broad-band wavelength range respectively. Low reflective index material like MgF2 is deposited by e-beam technique to obtain single-layer AR coating. While Al2O3 and ZrO2 layers deposited, by RF-magnetron sputtering, on top of MgF2 forms multi-layer AR coating. The combination of MgF2/ZrO2/Al2O3/MgF2 deposited on both side of quartz showed excellent results with reflectance value of around 0.8% in broad spectral range. The heat exchanger efficiency obtained after using these developed black copper oxide absorber material and AR coating is around 83 % which seems to be significantly higher than the other commercially available water heating system. Concentrating solar power (CSP) systems are utilized to convert sunlight to thermal electric power by using solar absorber. However, the solar absorber are operated at elevated temperature (700-800 oC) and should be spectrally selective to act as perfect absorbers over the solar spectrum (high solar absorptance (α)) and perfect reflectors in the thermal infrared (IR) (low thermal emittance (ε)). Cermet composite solar absorber shows such selective properties at high temperatures. In the present work, we developed Al-AlN based multilayer cermet films by RF magnetron sputtering. We choose combination of Ni/AlxN(1-x)/AlN layers as a solar absorber due to its stability at elevated temperature and high corrosion resistance. In this combination, Ni layer, deposited near to substrate, act as the IR light reflector to provide high thermal emittance. While AlxN(1-x) layer act as an absorber layer for UV-Vis spectrum of solar radiation and transparent AlN layer on top functions as AR coating. To improve absorptance, 3 or 4 layers of AlxN(1-x) film with grading of metal content was synthesized by varying N2 flow during deposition. The optical measurement for these multilayer selective absorber films showed high solar absorptance of 0.92-0.96 and low thermal emittance of around 0.1-0.07. To test the stability of our multilayer coating at high temperature, we annealed these samples at 700 oC with holding time of 2 hrs in air, low vacuum and high vacuum. We observed a slight decrease in solar absorptance value (0.90) for the annealed samples but the results showed that overall performance was not hindered by heat treatment thus proving the thermal stability of our multilayer cermet coating.

In the present thesis two main results are presented. The first is a study of the statistical properties of the finite-time maximum Lyapunov exponent determined out of a time series by using the divergent rate method. To reach this goal, we developed a new, completely automatic algorithm based on the method developed by Gao and Zheng. A main achievement of this part of the work is the interpretation of the uncertainty in the light of the work by Grassberger, Badii e Politi of 1988 on the theoretical distribution of maximum Lyapunov exponents. We showed that the analysis and identification of clusters in diagrams representing uncertainty vs. maximum Lyapunov exponent can provide useful information about the optimal choice of the embedding parameters. In addition, our results allow us to identify systems that can provide suitable benchmarks for the comparison and ranking of different embedding methods. The second main result concerns the development of a new method for the assessment of the optimal embedding parameters. Our method is based on two assumptions: a potential-like quantity is defined on the lattice of points that characterize the embedding; the optimal embedding choice coincides with local extrema (maxima or minima) of this potential. Throughout the work, we used "synthetic" time series generated by numerically integrating the difference and differential equations that describe the following dynamical systems: the Hénon map, the Lorenz attractor, the Rössler attractor and the Mackey-Glass attractor. These four systems are widely used as references in the scientific literature. In the last part of the work, we have started to examine EEG recordings by using the techniques developed in the main part of the work. The EEG recordings are sampled on healthy subjects in resting-state. These investigations are still at a starting phase.