Micro and Macro essays in Applied Fiscal Policy

This thesis analysis micro and macro aspect of applied fiscal policy issues. The first chapter investigates the extent to which local budget spending composition reacts to fiscal rules variations. I consider the budget of Italian municipalities and exploit specific changes in the Domestic Stability Pact’s rules, to perform a difference-in-discontinuities analysis. The results show that imposing a cap on the total amount of consumption and investment is not as binding as two caps, one for consumption and a different one for investment. More specifically, consumption is triggered by changes in wages and services spending, while investment relies on infrastructure movements. In addition, there is evidence that when an increase in investment is achieved, there is also a higher budget deficit level. The second chapter intends to analyze the extent to which fiscal policy shocks are able to affect macrovariables during business cycle fluctuations, differentiating among three intervention channels: public taxation, consumption and investment. The econometric methodology implemented is a Panel Vector Autoregressive model with a structural characterization. The results show that fiscal shocks have different multipliers in relation to expansion or contraction periods: output does not react during good times while there are significant effects in bad ones. The third chapter evaluates the effects of fiscal policy announcements by the Italian government on the long-term sovereign bond spread of Italy relative to Germany. After collecting data on relevant fiscal policy announcements, we perform an econometric comparative analysis between the three cabinets that followed one another during the period 2009-2013. The results suggest that only fiscal policy announcements made by members of Monti’s cabinet have been effective in influencing significantly the Italian spread in the expected direction, revealing a remarkable credibility gap between Berlusconi’s and Letta’s governments with respect to Monti’s administration.

New high nuclearity carbonyl clusters

The thesis reports the synthesis, and the chemical, structural and spectroscopic characterization of a series of new Rhodium and Au-Fe carbonyl clusters. Most new high-nuclearity rhodium carbonyl clusters have been obtained by redox condensation of preformed rhodium clusters reacting with a species in a different oxidation state generated in situ by mild oxidation. In particular the starting Rh carbonyl clusters is represented by the readily available [Rh7(CO)16]3- 9 compound. The oxidized species is generated in situ by reaction of the above with a stoichiometric defect of a mild oxidizing agents such as [M(H2O)x]n+ aquo complexes possessing different pKa’s and Mn+/M potentials. The experimental results are roughly in keeping with the conclusion that aquo complexes featuring E°(Mn+/M) < ca. -0.20 V do not lead to the formation of hetero-metallic Rh clusters, probably because of the inadequacy of their redox potentials relative to that of the [Rh7(CO)16]3-/2- redox couple. Only homometallic cluster s such as have been fairly selectively obtained. As a fallout of the above investigations, also a convenient and reproducible synthesis of the ill-characterized species [HnRh22(CO)35]8-n has been discovered. The ready availability of the above compound triggered both its complete spectroscopic and chemical characterization. because it is the only example of Rhodium carbonyl clusters with two interstitial metal atoms. The presence of several hydride atoms, firstly suggested by chemical evidences, has been implemented by ESI-MS and 1H-NMR, as well as new structural characterization of its tetra- and penta-anion. All these species display redox behaviour and behave as molecular capacitors. Their chemical reactivity with CO gives rise to a new series of Rh22 clusters containing a different number of carbonyl groups, which have been likewise fully characterized. Formation of hetero-metallic Rh clusters was only observed when using SnCl2H2O as oxidizing agent because. Quite all the Rh-Sn carbonyl clusters obtained have icosahedral geometry. The only previously reported example of an icosahedral Rh cluster with an interstitial atom is the [Rh12Sb(CO)27]3- trianion. They have very similar metal framework, as well as the same number of CO ligands and, consequently, cluster valence electrons (CVEs). .A first interesting aspect of the chemistry of the Rh-Sn system is that it also provides icosahedral clusters making exception to the cluster-borane analogy by showing electron counts from 166 to 171. As a result, the most electron-short species, namely [Rh12Sn(CO)25]4- displays redox propensity, even if disfavoured by the relatively high free negative charge of the starting anion and, moreover, behaves as a chloride scavenger. The presence of these bulky interstitial atoms results in the metal framework adopting structures different from a close-packed metal lattice and, above all, imparts a notable stability to the resulting cluster. An organometallic approach to a new kind of molecular ligand-stabilized gold nanoparticles, in which Fe(CO)x (x = 3,4) moieties protect and stabilize the gold kernel has also been undertaken. As a result, the new clusters [Au21{Fe(CO)4}10]5-, [Au22{Fe(CO)4}12]6-, Au28{Fe(CO)3}4{Fe(CO)4}10]8- and [Au34{Fe(CO)3}6{Fe(CO)4}8]6- have been isolated and characterized. As suggested by concepts of isolobal analogies, the Fe(CO)4 molecular fragment may display the same ligand capability of thiolates and go beyond. Indeed, the above clusters bring structural resemblance to the structurally characterized gold thiolates by showing Fe-Au-Fe, rather than S-Au-S, staple motives. Staple motives, the oxidation state of surface gold atoms and the energy of Au atomic orbitals are likely to concur in delaying the insulator-to-metal transition as the nuclearity of gold thiolates increases, relative to the more compact transition-metal carbonyl clusters. Finally, a few previously reported Au-Fe carbonyl clusters have been used as precursors in the preparation of supported gold catalysts. The catalysts obtained are active for toluene oxidation and the catalytic activity depends on the Fe/Au cluster loading over TiO2.

Low-dimensional carbon allotropes: an electron microscopy investigation

The research reported in this manuscript concerns the structural characterization of graphene membranes and single-walled carbon nanotubes (SWCNTs). The experimental investigation was performed using a wide range of transmission electron microscopy (TEM) techniques, from conventional imaging and diffraction, to advanced interferometric methods, like electron holography and Geometric Phase Analysis (GPA), using a low-voltage optical set-up, to reduce radiation damage in the samples. Electron holography was used to successfully measure the mean electrostatic potential of an isolated SWCNT and that of a mono-atomically thin graphene crystal. The high accuracy achieved in the phase determination, made it possible to measure, for the first time, the valence-charge redistribution induced by the lattice curvature in an individual SWCNT. A novel methodology for the 3D reconstruction of the waviness of a 2D crystal membrane has been developed. Unlike other available TEM reconstruction techniques, like tomography, this new one requires processing of just a single HREM micrograph. The modulations of the inter-planar distances in the HREM image are measured using Geometric Phase Analysis, and used to recover the waviness of the crystal. The method was applied to the case of a folded FGC, and a height variation of 0.8 nm of the surface was successfully determined with nanometric lateral resolution. The adhesion of SWCNTs to the surface of graphene was studied, mixing shortened SWCNTs of different chiralities and FGC membranes. The spontaneous atomic match of the two lattices was directly imaged using HREM, and we found that graphene membranes act as tangential nano-sieves, preferentially grafting achiral tubes to their surface.

Progress in x-ray spectroscopies for the study of advanced materials

This thesis work is focused on the use of selected core-level x-ray spectroscopies to study semiconductor materials of great technological interest and on the development of a new implementation of appearance potential spectroscopy. Core-level spectroscopies can be exploited to study these materials with a local approach since they are sensitive to the electronic structure localized on a chemical species present in the sample examined. This approach, in fact, provides important micro-structural information that is difficult to obtain with techniques sensitive to the average properties of materials. In this thesis work we present a novel approach to the study of semiconductors with core-level spectroscopies based on an original analysis procedure that leads to an insightful understanding of the correlation between the local micro-structure and the spectral features observed. In particular, we studied the micro-structure of Hydrogen induced defects in nitride semiconductors, since the analysed materials show substantial variations of optical and electronic properties as a consequence of H incorporation. Finally, we present a novel implementation of soft x-ray appearance potential spectroscopy, a core-level spectroscopy that uses electrons as a source of excitation and has the great advantage of being an in-house technique. The original set-up illustrated was designed to reach a high signal-to-noise ratio for the acquisition of good quality spectra that can then be analyzed in the framework of the real space full multiple scattering theory. This technique has never been coupled with this analysis approach and therefore our work unite a novel implementation with an original data analysis method, enlarging the field of application of this technique.

Reactivity of activated electrophiles and nucleophiles: labile intermediates and properties of the reaction products

The main topic of my Ph.D. thesis is the study of nucleophilic and electrophilic aromatic substitution reaction, in particular from a mechanistic point of view. The research was mainly focused on the reactivity of superactivated aromatic systems. In spite of their high reactivity (hence the high reaction’s rate), we were able to identify and in some case to isolate -complexes until now only hypothesized. For example, interesting results comes from the study of the protonation of the supernucleophiles tris(dialkylamino)benzenes. However, the best result obtained in this field was the isolation and structural characterization of the first stables zwitterionic Wheland-Meisenheimer complexes by using 2,4-dipyrrolidine-1,3-thiazole as supernucleophile and 4,6-dinitrobenzofuroxan or 4,6-dinitrotetrazolepyridine as superelectrophile. These reactions were also studied by means of computational chemistry, which allowed us to better investigate on the energetic and properties of the reactions and reactants studied. We also discovered, in some case fortuitously, some relevant properties and application of the compounds we synthesized, such as fluorescence in solid state and nanoparticles, or textile dyeing. We decided to investigate all these findings also by collaborating with other research groups. During a period in the “Laboratoire de Structure et Réactivité des Systèmes Moléculaires Complexes-SRSMC, Université de Lorraine et CNRS, France, I carried out computational studies on new iron complexes for the use as dyes in Dye Sensitized Solar Cells (DSSC). Furthermore, thanks to this new expertise, I was involved in a collaboration for the study of the ligands’ interaction in biological systems. A collaboration with University of Urbino allowed us to investigate on the reactivity of 1,2-diaza-1,3-dienes toward nucleophiles such as amino and phosphine derivatives, which led to the synthesis of new products some of which are 6 or 7 member heterocycles containing both phosphorus and nitrogen atoms.

E-cadherin and Choline Kinase: two challenging drug discovery targets

The data presented in this thesis was generated using molecular biology, protein chemistry and X-ray crystallography techniques. However, while the methodologies employed are essentially the same, the research work presented here refers to two different proteins, which are part of different research projects in the laboratory. For this reason, the content of this thesis is divided in two independent parts, each provided with an introduction and a general overview of the research topic and state-ofthe- art, a materials and methods section discussing the techniques used and the protocols followed, and a section where the results are presented and discussed in detail. The first half of the thesis deals with the structural characterization of the complex between human E-cadherin and three different small molecule potential inhibitors identified via a fragment-based drug discovery (FBDD) screening campaign that was conducted using a library of commercially available small fluorinated chemical fragments. For this screening phase, we used 19F-NMR as readout. The NMR experiments were done by our collaborator Dr. Marina Veronesi at the D3 PharmaChemistry division of the Italian Institute of Technology (IIT) in Genova (Italy). Functional cell adhesion assays to validate the inhibitory effects of the fragments thus identified were carried out in collaboration with Prof. Frédéric André at the University of Marseille (France). The second half of the thesis describes the structural characterization of Plasmodium falciparum Choline Kinase (PfChoK), an important pharmaceutical target in the fight against malaria, as well as the biochemical characterization of a library of potential inhibitors of PfChoK. These inhibitors were synthetized in the group of Prof. Luisa Carlota López-Cara at the Department of Pharmaceutical and Organic Chemistry of the University of Granada (Spain) in the framework of an ongoing collaboration between the two groups.

Light & Electron beam - the perfect combination for the observation and application of photo active materials

The aim of the present PhD thesis is to investigate the properties of innovative nanomaterials for energy conversion. The materials have been deeply studied by means of a wide spectrum of different techniques based on both light and electron sources, in order to get an insight into the correlation between the properties of each material and the activity towards different energy conversion applications. The activity has been carried out in the framework of a collaboration between the “G.Ciamician” Chemistry Department of the University of Bologna and the CNR-IMM Bologna. Four main topics have been explored: in the first part, luminescent silicon nanocrystals (SiNCs) have been discussed, suggesting a new approach to improve their optical properties as active material in complementary optoelectronic devices and photovoltaic cells. The luminescence of SiNCs have been exploited to increase the efficiency of conventional photovoltaic cells by means of an innovative architecture. Specifically, SiNCs were shown to be very promising light emitters in luminescent solar concentrators (LSC). The second part of the work has been focused on the study of high phosphorescent molecular chromophores, suggesting a new approach in their use as optical sensors successfully applied to the field of polymeric materials. This is due to the enhanced emission of light that appears in rigid, constrained or crystalline state, that is commonly called: "Aggregation-Induced Emission (AIE)". Such phenomenon is characteristic for molecular structures such as persulfurated benzene chromophores, hereafter named asterisks. The last two parts were focused on conventional and in-situ Transmission Electron Microscopy (TEM) morphological and structural characterization of photoactive and catalytic materials for energetic applications and in particular water splitting.

Structural and functional characterization of Group B Streptococcus class C sortases

In Group B Streptococcus (GBS) three structurally distinct types of pili have been discovered as potential virulence factors and vaccine candidates. The pilus-forming proteins are assembled into high-molecular weight polymers via a transpeptidation mechanism mediated by specific class C sortases. Using a multidisciplinary approach including bioinformatics, structural and biochemical studies and in vivo mutagenesis we performed a broad characterization of GBS sortase C. The high resolution X-ray structure of the enzymes revealed that the active site, located into the β-barrel core of the enzyme, is made of the catalytic triad His157-Cys219-Arg228 and covered by a loop, known as the “lid”. We show that the catalytic triad and the predicted N- and C-terminal trans-membrane regions are required for the enzyme activity. Interestingly, by in vivo complementation mutagenesis studies we found that the deletion of the entire lid loop or mutations in specific lid key residues had no effect on catalytic activity of the enzyme. In addition, kinetic characterizations of recombinant enzymes indicate that the lid mutants can still recognize and cleave the substrate-mimicking peptide at least as well as the wild type protein.

Modeling, design and experimental characterization of Micro-Electro-Mechanical-Systems for gas chromatographic applications

Design parameters, process flows, electro-thermal-fluidic simulations and experimental characterizations of Micro-Electro-Mechanical-Systems (MEMS) suited for gas-chromatographic (GC) applications are presented and thoroughly described in this thesis, whose topic belongs to the research activities the Institute for Microelectronics and Microsystems (IMM)-Bologna is involved since several years, i.e. the development of micro-systems for chemical analysis, based on silicon micro-machining techniques and able to perform analysis of complex gaseous mixtures, especially in the field of environmental monitoring. In this regard, attention has been focused on the development of micro-fabricated devices to be employed in a portable mini-GC system for the analysis of aromatic Volatile Organic Compounds (VOC) like Benzene, Toluene, Ethyl-benzene and Xylene (BTEX), i.e. chemical compounds which can significantly affect environment and human health because of their demonstrated carcinogenicity (benzene) or toxicity (toluene, xylene) even at parts per billion (ppb) concentrations. The most significant results achieved through the laboratory functional characterization of the mini-GC system have been reported, together with in-field analysis results carried out in a station of the Bologna air monitoring network and compared with those provided by a commercial GC system. The development of more advanced prototypes of micro-fabricated devices specifically suited for FAST-GC have been also presented (silicon capillary columns, Ultra-Low-Power (ULP) Metal OXide (MOX) sensor, Thermal Conductivity Detector (TCD)), together with the technological processes for their fabrication. The experimentally demonstrated very high sensitivity of ULP-MOX sensors to VOCs, coupled with the extremely low power consumption, makes the developed ULP-MOX sensor the most performing metal oxide sensor reported up to now in literature, while preliminary test results proved that the developed silicon capillary columns are capable of performances comparable to those of the best fused silica capillary columns. Finally, the development and the validation of a coupled electro-thermal Finite Element Model suited for both steady-state and transient analysis of the micro-devices has been described, and subsequently implemented with a fluidic part to investigate devices behaviour in presence of a gas flowing with certain volumetric flow rates.

Structural and kinetic characterization of DNA polymerases I and III from Escherichia coli

DNA elongation is performed by Pol III α subunit in E. coli, stimulated by the association with ε and θ subunits. These three subunits define the DNA Pol III catalytic core. There is controversy about the DNA Pol III assembly for the simultaneous control of lagging and leading strands replication, since some Authors propose a dimeric model with two cores, whereas others have assembled in vitro a trimeric DNA Pol III with a third catalytic core, which increases the efficiency of DNA replication. Moreover, the function of the PHP domain, located at the N-terminus of α subunit, is still unknown. Previous studies hypothesized a possible pyrophosphatase activity, not confirmed yet. The present Thesis highlights by the first time the production in vivo of a trimeric E. coli DNA Pol III by co-expressing α, τ, ε and θ subunits. This trimeric complex has been enzymatically characterized and a molecular model has been proposed, with 2 α subunits sustaining the lagging-strand replication whereas the third core replicates the leading strand. In addition, the pyrophosphatase activity of the PHP domain has been confirmed. This activity involves, at least, the H12 and the D19 residues, whereas the D201 regulates phosphate release. On the other hand, an artificial polymerase (HoLaMa), designed by deleting the exonuclease domain of Klenow Fragment, has been expressed, purified and characterized for a better understanding of bacterial polymerases mechanism. The absence of exonuclease domain impaired enzyme processivity, since this domain is involved in DNA binding. Finally, Klenow enzyme, HoLaMa, α subunit and DNA Pol III αεθ have been characterized at the single-molecule level by FRET analysis, combining ALEX and TIRF microscopy. Fluorescently-labeled DNA molecules were immobilized, and changes in FRET efficiency enabled us to study polymerase binding and DNA polymerization.

Structural and functional characterization of Group B Streptococcus pilus 2b

Group B Streptococcus (GBS) is a Gram-positive human pathogen representing one of the most common causes of life-threatening bacterial infections such as sepsis and meningitis in neonates. Covalently polymerized pilus-like structures have been discovered in GBS as important virulence factors as well as vaccine candidates. Pili are protein polymers forming long and thin filamentous structures protruding from bacterial cells, mediating adhesion and colonization to host cells. Gram-positive bacteria, including GBS, build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates that are the backbone protein forming the pilus shaft and two ancillary proteins. Also the cell-wall anchoring of the pilus polymers made of covalently linked pilin subunits is mediated by a sortase enzyme. GBS expresses three structurally distinct pilus types (type 1, 2a and 2b). Although the mechanisms of assembly and cell wall anchoring of GBS types 1 and 2a pili have been investigated, those of pilus 2b are not understood until now. Pilus 2b is frequently found in ST-17 strains that are mostly associated with meningitis and high mortality rate especially in infants. In this work the assembly mechanism of GBS pilus type 2b has been elucidated by dissecting through genetic, biochemical and structural studies the role of the two pilus-associated sortases. The most significant findings show that pilus 2b assembly appears “non-canonical”, differing significantly from current pilus assembly models in Gram-positive pathogens. Only sortase-C1 is involved in pilin polymerization, while the sortase-C2 does not act as a pilin polymerase, but it is involved in cell-wall pilus anchoring. Our findings provide new insights into pili biogenesis in Gram-positive bacteria. Moreover, the role of this pilus type during host infection has been investigated. By using a mouse model of meningitis we demonstrated that type 2b pilus contributes to pathogenesis of meningitis in vivo.

Computational resources for structural and metagenomic characterization of functions in bacteria and bacterial communities. Antimicrobial resistance, pathways for xenobiotic degradation and relationship between gut microbiome and ageing.

Prokaryotic organisms are one of the most successful forms of life, they are present in all known ecosystems. The deluge diversity of bacteria reflects their ability to colonise every environment. Also, human beings host trillions of microorganisms in their body districts, including skin, mucosae, and gut. This symbiosis is active for all other terrestrial and marine animals, as well as plants. With the term holobiont we refer, with a single word, to the systems including both the host and its symbiotic microbial species. The coevolution of bacteria within their ecological niches reflects the adaptation of both host and guest species, and it is shaped by complex interactions that are pivotal for determining the host state. Nowadays, thanks to the current sequencing technologies, Next Generation Sequencing, we have unprecedented tools for investigating the bacterial life by studying the prokaryotic genome sequences. NGS revolution has been sustained by the advancements in computational performance, in terms of speed, storage capacity, algorithm development and hardware costs decreasing following the Moore’s Law. Bioinformaticians and computational biologists design and implement ad hoc tools able to analyse high-throughput data and extract valuable biological information. Metagenomics requires the integration of life and computational sciences and it is uncovering the deluge diversity of the bacterial world. The present thesis work focuses mainly on the analysis of prokaryotic genomes under different aspects. Being supervised by two groups at the University of Bologna, the Biocomputing group and the group of Microbial Ecology of Health, I investigated three different topics: i) antimicrobial resistance, particularly with respect to missense point mutations involved in the resistant phenotype, ii) bacterial mechanisms involved in xenobiotic degradation via the computational analysis of metagenomic samples, and iii) the variation of the human gut microbiota through ageing, in elderly and longevous individuals.

Development, mechanical characterization and qualification in liquid lead of stainless steels for structural applications in the future fission and fusion nuclear reactors

The research activity carried out in the Brasimone Research Center of ENEA concerns the development and mechanical characterization of steels conceived as structural materials for future fission reactors (Heavy Liquid Metal IV Generation reactors: MYRRHA and ALFRED) and for the future fusion reactor DEMO. Within this framework, two parallel lines of research have been carried out: (i) characterization in liquid lead of steels and weldings for the components of the IV Generation fission reactors (GIV) by means of creep and SSRT (Slow Strain Rate Tensile) tests; (ii) development and screening on mechanical properties of RAFM (Reduced Activation Ferritic Martensitic) steels to be employed as structural materials of the future DEMO fusion reactor. The doctoral work represents therefore a comprehensive report of the research carried out on nuclear materials both from the point of view of the qualification of existing (commercial) materials for their application in the typical environmental conditions of 4th generation fission reactors operating with lead as coolant, and from the point of view of the metallurgical study (with annexed microstructural and mechanical characterization of the selected compositions / Thermo Mechanical Treatment (TMT) options) of new compositional variants to be proposed for the “Breeding Blanket” of the future DEMO Fusion Reactor.

Nickel dependent carcinogenesis and infections: structural and biophysical characterization of NDRG1 and SgSrnR

In prokaryotic organisms, lower eukaryotes and plants, some important biological reactions are catalyzed by nickel-dependent enzymes, making this metal ion essential microelement for their life. On the other hand, excessive concentration of nickel into the cell, or prolonged exposure to nickel compounds, has toxic effects in living organisms. In addition, nickel has been classified by IARC as Group I human carcinogen, because of the correlation between its inhalation and increased incidence of nasal and lung cancers. The aim of this work was to investigate the nickel impact on human health, considering both its direct role on human cells and its indirect effect as essential element for human important bacteria. In humans, nickel induces N-myc downstream regulated gene 1 (NDRG1) expression, recently proposed as new target in cancer therapy. CD, light scattering and ITC were applied on the recombinant full-length protein and its C-terminal intrinsically disordered domain, for studying the NDRG1 structural and functional properties. In particular, the fold and dynamics of the C-terminal region were examined by NMR spectroscopy and site-directed spin labeling coupled to EPR, showing the features of an intrinsically disordered region. In nickel-dependent bacteria, nickel metabolism is strictly regulated, through the activity of different transcription factors. In Streptomyces griseus the expression of two superoxide dismutases (SODs) is antagonistically regulated by nickel thanks to the transcriptional complex SgSrnR/SgSrnQ. The SgSrnR protein was heterologously expressed and its activity as possible nickel sensor studied. DNaseI footprinting and β-galactosidase gene reporter assays revealed that SgSrnR functions as transcriptional activator, prompting the hypothesis of a new model to describe the activity of this complex. In addition, ITC, NMR and X-ray crystallography demonstrated that SgSrnR presents the fold typical of ArsR/SmtB transcription factors and low metal binding affinity, non compatible with a role as a nickel-sensor, function probably played by its partner SgSrnQ.

Structural and biophysical characterization of RAD52 as novel pharmacological target for synthetic lethality

RAD52 is a protein involved in various DNA reparation mechanisms. In the last few years, RAD52 has been proposed as a novel pharmacological target for cancer synthetic lethality strategies. Hence, this work has the purpose to investigate RAD52 protein, with biophysical and structural tools to shed light on proteins features and mechanistic details that are, up to now poorly described, and to design novel strategies for its inhibition. My PhD work had two goals: the structural and functional characterization of RAD52 and the identification of novel RAD52 inhibitors. For the first part, RAD52 was characterized both for its DNA interaction and oligomerization state together with its propensity to form high molecular weight superstructures. Moreover, using EM and Cryo-EM techniques, additional RAD52 structural hallmarks were obtained, valuable both for understanding protein mechanism of action and for drug discovery purpose. The second part of my PhD project focused on the design and characterization of novel RAD52 inhibitors to be potentially used in combination therapies with PARPi to achieve cancer cells synthetic lethality, avoiding resistance occurrence and side effects. With this aim we selected and characterized promising RAD52 inhibitors through three different approaches: 19F NMR fragment-based screening; virtual screening campaign; aptamers computational design. Selected hits (fragments, molecules and aptamers) were investigated for their binding to RAD52 and for their mechanism of inhibition. Collected data highlighted the identification of hits worthy to be developed into more potent and selective RAD52 inhibitors. Finally, a side project carried out during my PhD is reported. GSK-3β protein, an already validated pharmacological target was investigated using biophysical and structural biology tools. Here, an innovative and adaptable drug discovery screening pipeline able to directly identify selective compounds with binding affinities not higher than a reference binder was developed.