Computational methods for the analysis of protein structure and function

The vast majority of known proteins have not yet been experimentally characterized and little is known about their function. The design and implementation of computational tools can provide insight into the function of proteins based on their sequence, their structure, their evolutionary history and their association with other proteins. Knowledge of the three-dimensional (3D) structure of a protein can lead to a deep understanding of its mode of action and interaction, but currently the structures of <1% of sequences have been experimentally solved. For this reason, it became urgent to develop new methods that are able to computationally extract relevant information from protein sequence and structure. The starting point of my work has been the study of the properties of contacts between protein residues, since they constrain protein folding and characterize different protein structures. Prediction of residue contacts in proteins is an interesting problem whose solution may be useful in protein folding recognition and de novo design. The prediction of these contacts requires the study of the protein inter-residue distances related to the specific type of amino acid pair that are encoded in the so-called contact map. An interesting new way of analyzing those structures came out when network studies were introduced, with pivotal papers demonstrating that protein contact networks also exhibit small-world behavior. In order to highlight constraints for the prediction of protein contact maps and for applications in the field of protein structure prediction and/or reconstruction from experimentally determined contact maps, I studied to which extent the characteristic path length and clustering coefficient of the protein contacts network are values that reveal characteristic features of protein contact maps. Provided that residue contacts are known for a protein sequence, the major features of its 3D structure could be deduced by combining this knowledge with correctly predicted motifs of secondary structure. In the second part of my work I focused on a particular protein structural motif, the coiled-coil, known to mediate a variety of fundamental biological interactions. Coiled-coils are found in a variety of structural forms and in a wide range of proteins including, for example, small units such as leucine zippers that drive the dimerization of many transcription factors or more complex structures such as the family of viral proteins responsible for virus-host membrane fusion. The coiled-coil structural motif is estimated to account for 5-10% of the protein sequences in the various genomes. Given their biological importance, in my work I introduced a Hidden Markov Model (HMM) that exploits the evolutionary information derived from multiple sequence alignments, to predict coiled-coil regions and to discriminate coiled-coil sequences. The results indicate that the new HMM outperforms all the existing programs and can be adopted for the coiled-coil prediction and for large-scale genome annotation. Genome annotation is a key issue in modern computational biology, being the starting point towards the understanding of the complex processes involved in biological networks. The rapid growth in the number of protein sequences and structures available poses new fundamental problems that still deserve an interpretation. Nevertheless, these data are at the basis of the design of new strategies for tackling problems such as the prediction of protein structure and function. Experimental determination of the functions of all these proteins would be a hugely time-consuming and costly task and, in most instances, has not been carried out. As an example, currently, approximately only 20% of annotated proteins in the Homo sapiens genome have been experimentally characterized. A commonly adopted procedure for annotating protein sequences relies on the "inheritance through homology" based on the notion that similar sequences share similar functions and structures. This procedure consists in the assignment of sequences to a specific group of functionally related sequences which had been grouped through clustering techniques. The clustering procedure is based on suitable similarity rules, since predicting protein structure and function from sequence largely depends on the value of sequence identity. However, additional levels of complexity are due to multi-domain proteins, to proteins that share common domains but that do not necessarily share the same function, to the finding that different combinations of shared domains can lead to different biological roles. In the last part of this study I developed and validate a system that contributes to sequence annotation by taking advantage of a validated transfer through inheritance procedure of the molecular functions and of the structural templates. After a cross-genome comparison with the BLAST program, clusters were built on the basis of two stringent constraints on sequence identity and coverage of the alignment. The adopted measure explicity answers to the problem of multi-domain proteins annotation and allows a fine grain division of the whole set of proteomes used, that ensures cluster homogeneity in terms of sequence length. A high level of coverage of structure templates on the length of protein sequences within clusters ensures that multi-domain proteins when present can be templates for sequences of similar length. This annotation procedure includes the possibility of reliably transferring statistically validated functions and structures to sequences considering information available in the present data bases of molecular functions and structures.

Structure determination of proteins and peptides in solution: simulation, chirality and NMR studies

The study of protein fold is a central problem in life science, leading in the last years to several attempts for improving our knowledge of the protein structures. In this thesis this challenging problem is tackled by means of molecular dynamics, chirality and NMR studies. In the last decades, many algorithms were designed for the protein secondary structure assignment, which reveals the local protein shape adopted by segments of amino acids. In this regard, the use of local chirality for the protein secondary structure assignment was demonstreted, trying to correlate as well the propensity of a given amino acid for a particular secondary structure. The protein fold can be studied also by Nuclear Magnetic Resonance (NMR) investigations, finding the average structure adopted from a protein. In this context, the effect of Residual Dipolar Couplings (RDCs) in the structure refinement was shown, revealing a strong improvement of structure resolution. A wide extent of this thesis is devoted to the study of avian prion protein. Prion protein is the main responsible of a vast class of neurodegenerative diseases, known as Bovine Spongiform Encephalopathy (BSE), present in mammals, but not in avian species and it is caused from the conversion of cellular prion protein to the pathogenic misfolded isoform, accumulating in the brain in form of amiloyd plaques. In particular, the N-terminal region, namely the initial part of the protein, is quite different between mammal and avian species but both of them contain multimeric sequences called Repeats, octameric in mammals and hexameric in avians. However, such repeat regions show differences in the contained amino acids, in particular only avian hexarepeats contain tyrosine residues. The chirality analysis of avian prion protein configurations obtained from molecular dynamics reveals a high stiffness of the avian protein, which tends to preserve its regular secondary structure. This is due to the presence of prolines, histidines and especially tyrosines, which form a hydrogen bond network in the hexarepeat region, only possible in the avian protein, and thus probably hampering the aggregation.

De Novo Design of secondary structures: Synthesis and conformational studies

Chemists have long sought to extrapolate the power of biological catalysis and recognition to synthetic systems. These efforts have focused largely on low molecular weight catalysts and receptors; however, biological systems themselves rely almost exclusively on polymers, proteins and RNA, to perform complex chemical functions. Proteins and RNA are unique in their ability to adopt compact, well-ordered conformations, and specific folding provides precise spatial orientation of the functional groups that comprise the “active site”. These features suggest that identification of new polymer backbones with discrete and predictable folding propensities (“foldamers”) will provide a basis for design of molecular machines with unique capabilities. The foldamer approach complements current efforts to design unnatural properties into polypeptides and polynucleotides. The aim of this thesis is the synthesis and conformational studies of new classes of foldamers, using a peptidomimetic approach. Moreover their attitude to be utilized as ionophores, catalysts, and nanobiomaterials were analyzed in solution and in the solid state. This thesis is divided in thematically chapters that are reported below. It begins with a very general introduction (page 4) which is useful, but not strictly necessary, to the expert reader. It is worth mentioning that paragraph I.3 (page 22) is the starting point of this work and paragraph I.5 (page 32) isrequired to better understand the results of chapters 4 and 5. In chapter 1 (page 39) is reported the synthesis and conformational analysis of a novel class of foldamers containing (S)-β3-homophenylglycine [(S)-β3-hPhg] and D- 4-carboxy-oxazolidin-2-one (D-Oxd) residues in alternate order is reported. The experimental conformational analysis performed in solution by IR, 1HNMR, and CD spectroscopy unambiguously proved that these oligomers fold into ordered structures with increasing sequence length. Theoretical calculations employing ab initio MO theory suggest a helix with 11-membered hydrogenbonded rings as the preferred secondary structure type. The novel structures enrich the field of peptidic foldamers and might be useful in the mimicry of native peptides. In chapter 2 cyclo-(L-Ala-D-Oxd)3 and cyclo-(L-Ala-DOxd) 4 were prepared in the liquid phase with good overall yields and were utilized for bivalent ions chelation (Ca2+, Mg2+, Cu2+, Zn2+ and Hg2+); their chelation skill was analyzed with ESI-MS, CD and 1HNMR techniques and the best results were obtained with cyclo-(L-Ala-D-Oxd)3 and Mg2+ or Ca2+. Chapter 3 describes an application of oligopeptides as catalysts for aldol reactions. Paragraph 3.1 concerns the use of prolinamides as catalysts of the cross aldol addition of hydroxyacetone to aromatic aldeydes, whereas paragraphs 3.2 and 3.3 are about the catalyzed aldol addition of acetone to isatins. By means of DFT and AIM calculations, the steric and stereoelectronic effects that control the enantioselectivity in the cross-aldol addition of acetone to isatin catalysed by L-proline have been studied, also in the presence of small quantities of water. In chapter 4 is reported the synthesis and the analysis of a new fiber-like material, obtained from the selfaggregation of the dipeptide Boc-L-Phe-D-Oxd-OBn, which spontaneously forms uniform fibers consisting of parallel infinite linear chains arising from singleintermolecular N-H···O=C hydrogen bonds. This is the absolute borderline case of a parallel β-sheet structure. Longer oligomers of the same series with general formula Boc-(L-Phe-D-Oxd)n-OBn (where n = 2-5), are described in chapter 5. Their properties in solution and in the solid state were analyzed, in correlation with their attitude to form intramolecular hydrogen bond. In chapter 6 is reported the synthesis of imidazolidin-2- one-4-carboxylate and (tetrahydro)-pyrimidin-2-one-5- carboxylate, via an efficient modification of the Hofmann rearrangement. The reaction affords the desired compounds from protected asparagine or glutamine in good to high yield, using PhI(OAc)2 as source of iodine(III).

Studio di biomateriali usati come scaffold per Tissue Engineering e loro caratterizzazione con tecniche spettroscopiche vibrazionali e di analisi termica

This research investigated someone of the main problems connected to the application of Tissue Engineering in the prosthetic field, in particular about the characterization of the scaffolding materials and biomimetic strategies adopted in order to promote the implant integration. The spectroscopic and thermal analysis techniques were usefully applied to characterize the chemico-physical properties of the materials such as – crystallinity; – relative composition in case of composite materials; – Structure and conformation of polymeric and peptidic chains; – mechanism and degradation rate; – Intramolecular and intermolecular interactions (hydrogen bonds, aliphatic interactions). This kind of information are of great importance in the comprehension of the interactions that scaffold undergoes when it is in contact with biological tissues; this information are fundamental to predict biodegradation mechanisms and to understand how chemico-physical properties change during the degradation process. In order to fully characterize biomaterials, this findings must be integrated by information relative to mechanical aspects and in vitro and in vivo behavior thanks to collaborations with biomedical engineers and biologists. This study was focussed on three different systems that correspond to three different strategies adopted in Tissue Engineering: biomimetic replica of fibrous 3-D structure of extracellular matrix (PCL-PLLA), incorporation of an apatitic phase similar to bone inorganic phase to promote biomineralization (PCL-HA), surface modification with synthetic oligopeptides that elicit the interaction with osteoblasts. The characterization of the PCL-PLLA composite underlined that the degradation started along PLLA fibres, which are more hydrophylic, and they serve as a guide for tissue regeneration. Moreover it was found that some cellular lines are more active in the colonization of the scaffold. In the PCL-HA composite, the weight ratio between the polymeric and the inorganic phase plays an essential role both in the degradation process and in the biomineralization of the material. The study of self-assembling peptides allowed to clarify the influence of primary structure on intermolecular and intermolecular interactions, that lead to the formation of the secondary structure and it was possible to find a new class of oligopeptides useful to functionalize materials surface. Among the analytical techniques used in this study, Raman vibrational spectroscopy played a major role, being non-destructive and non-invasive, two properties that make it suitable to degradation studies and to morphological characterization. Also micro-IR spectroscopy was useful in the comprehension of peptide structure on oxidized titanium: up to date this study was one of the first to employ this relatively new technique in the biomedical field.

De Novo Design of Foldamers for Preparation of Nanostructured Materials

This thesis deals with the synthesis and the conformation analysis of hybrid foldamers containing the 4-carboxyoxazolidin-2-one unit or related molecules, in which an imido-type function is obtained by coupling the nitrogen of the heterocycle with the carboxylic acid moiety of the next unit. The imide group is characterized by a nitrogen atom connected to an endocyclic and an exocyclic carbonyl, which tend always to adopt the trans conformation. As a consequence of this locally constrained disposition effect, these imide-type oligomers are forced to fold in ordered conformations. The synthetic approach is highly tuneable with endless variations, so, simply by changing the design and the synthesis, a wide variety of foldamers with the required properties may be prepared “on demand”. Thus a wide variety of unusual secondary structures and interesting supramolecular materials may be obtained with hybrid foldamers. The behaviour in the solid state of some of these compounds has been analyzed in detail, thus showing the formation of different kinds of supramolecular materials that may be used for several applications. A winning example is the production of a bolaamphiphilic gelators that may also be doped with small amounts of dansyl containing compounds, needed to show the cellular uptake into IGROV-1 cells, by confocal laser scanning microscopy. These gels are readily internalized by cells and are biologically inactive, making them very good candidates in the promising field of drug delivery. In the last part of the thesis, a particular attention was directed to the search of new scaffolds that behave as constrained amino acid mimetics, showing that tetramic acids derivatives could be good candidates for the synthesis and applications of molecules having an ordered secondary structure.

Protein-based nanomaterials as protein heterogeneous nucleants and structural studies on enzymes from two photosynthetic organisms

Proteins, the most essential biological macromolecules, are involved in nearly every aspect of life. The elucidation of their three-dimensional structures through X-ray analysis has significantly contributed to our understanding of fundamental mechanisms in life processes. However, the obstacle of obtaining high-resolution protein crystals remains significant. Thus, searching for materials that can effectively induce nucleation of crystals is a promising and active field. This thesis work characterizes and prepares albumin nanoparticles as heterogeneous nucleants for protein crystallization. These stable Bovine Serum Albumin nanoparticles were synthesized via the desolvation method, purified efficiently, and characterized in terms of dimension, morphology, and secondary structure. The ability of BSA-NPs to induce macromolecule nucleation was tested on three model proteins, exhibiting significant results, with larger NPs inducing more nucleation. The second part of this work focuses on the structural study, mainly through X-ray crystallography, of five chloroplast and cytosolic enzymes involved in the fundamental cellular processes of two photosynthetic organisms, Chlamydomonas reinhardtii and Arabidopsis thaliana. The structures of three enzymes involved in the Calvin-Benson-Bassham Cycle, phosphoribulokinase, troseposphatisomerase, and ribulosiophosphate epimerase from Chlamydomonas reinhardtii, were solved to investigate their catalytic and regulatory mechanisms. Additionally, the structure of nitrosylated-CrTPI made it possible to identify Cys14 as a target for nitrosylation, and the crystallographic structure of CrRPE was solved for the first time, providing insights into its catalytic and regulatory properties. Finally, the structure of S-nitrosoglutathione reductase, AtGSNOR, was compared with that of AtADH1, revealing differences in their catalytic sites. Overall, seven crystallographic structures, including partially oxidized CrPRK, CrPRK/ATP, CrPRK/ADP/Ru5P, CrTPI-nitrosylated, apo-CrRPE, apo-AtGSNOR, and AtADH1-NADH, were solved and are yet to be deposited in the PDB.

Enzimi, acidi organici ed altri metaboliti coinvolti nella patogenesi di Penicillium spp

Blue mould caused by Penicillium expansum Link is one of the most destructive rot of pome fruit in all growing areas (Snowdon, 1990; Jones and Aldwinckle, 1991; Tonini,1996) In the past, Penicillium rot has been controlled by fungicide postharvest treatment mainly by thiabendazole (TBZ) and benomyl (Hardenburg and Spalding, 1972), but their intense use produced the appearance of resistant strains with a great reduction of their activity The aims of the present study were to characterize the isolates of Pencillium sp causing blue mold on pear in Italy by physiological and biochemical parameters. In particular differencing also the behavior of isolates to relationship with sensitivity or resistance to TBZ treatments. We have examined the early stage of infection in relation to enzyme activity, local modulation of pH, production of organic acids, and to secondary metabolism of pathogen. The results described here confirm that the majority of P. expansum isolates from pears packing houses are resistant to TBZ, Among the TBZ-resistant isolates scored in this work, different isolates (RR) showed higher percentage of conidial germination on TBZ-amended medium compared to non amended medium. This may indicate a stimulatory effect of TBZ on conidial germination. Therefore TBZ treatments are not only ineffective for controlling P. expansum, but they may also increase the severity of blue mould on fruits. In the absence of fungicide, isolates showed a significant difference for infection severity, R and RR isolates are characterized by higher pathogenic fitness on fruits, producing larger lesions than S isolates. These data are supported by the study with laboratory-induced resistant isolates, which shows the lack of correlation between TBZ resistance and osmotic sensitivity, and highlights the association between TBZ resistance and infection severity (Baraldi et al 2003). Enzymatic screening gave a positive reaction to esterase, urease, pectinase activity, in addition, the pathogen is able to synthesize a complex enzyme act to degrade the main components of the cell wall especially pectin and cellulose. Isolated sensitive and resistant are characterized by a good activity of pectinase, especially from poligactoronase, which, as already reported by several studies (D'hallewin et al, 2004; Prusky et al, 2004), are the basis of degradative process of cell wall. Also, although the measure was minor also highlighted some activities of cellulase, but even note in the production of this kind of cellulase and hemicellulase P. Expansum were not targeted, studies have found no other source of information in this regard. Twenty isolates of Penicillium expansum, were tested in vitro ad in vivo for acid production ability and pH drop. We have found that modulation of pH and the organic acids extrusion were influence to various parameter:  Initial pH: in general, the greatest reduction of pH was observed in isolates grown at pH 7, except for four isolates that maintained the pH of the medium close to 7, the others significantly decreased the pH, ranging from 5.5 to 4.1.. In extreme acid condition (pH 3,0) growth and modulation of pH is most lower respect optimal condition (pH 5,0). Also isolates R and RR have showed a greater adaptation to environmental condition more than isolates S.  Time: although the acidification continues for some days, PH modulation is strongest in early hours (48-72 hours)of inoculation process. Time also affects the quality of organic acids, for example in vitro results showed an initial abundant production of succinc acid, followed to important production of galacturoinc acid.  Substrates: there are many differences for the type of acids produced in vitro and in vivo. Results showed in vivo an abundant production of galacturonic, malic, and citric acids and some unknown organic acids in smaller concentrations. Secondary metabolite analysis revealed intra-specific differences, and patulin was found in all isolates, but most significant reduction was observed between in vitro and in vivo samples. There was no correlation between the concentration of patulin, and the percentage of infected fruits, but sample with a lower infection severity of rotten area than the others, showed a significantly lower mycotoxin concentration than samples with a higher lesion diameter of rotten area. Beyond of patulin was detected the presence of another secondary metabolite, penitrem A.

Eterogeneità delle proprietà fisico – chimiche della proteina prionica patologica e variabilità fenotipica ceppo specifica nella malattia di Creutzfeldt – Jakob

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are neurodegenerative disorders that affect humans and mammals. Creutzfeldt-Jakob disease (CJD), the most common TSE in humans, can be sporadic (sCJD), genetic (gCJD), or acquired by infection. All TSEs are characterised by the accumulation of PrPSc, a misfolded form of the cellular protein PrPC. PrPSc is insoluble in detergents, partially resistant to proteolysis and shows a highly enriched β-sheet secondary structure. Six clinico-pathological phenotypes of sCJD have been characterized which correlate at the molecular level with two types (1 or 2) of PrPSc with distinctive physicochemical properties and the genotype at the polymorphic (methionine or valine) codon 129 of the prion protein gene. According to the protein-only hypothesis, which postulates that prions are composed exclusively of PrPSc, the strains of prions that are largely responsible for the wide spectrum of TSE phenotypes are enciphered in PrPSc conformation. In support to this view, studies mainly conducted in experimental scrapie, have shown that several prion strains can be identified based on distinguishing PrPSc biochemical properties. To further contribute to the understanding of the molecular basis of strains and to develop more sensitive strain typing assays in humans we have analyzed PrPSc biochemical properties in two experimental setting. In the first we compared the size of the core after protease digestion and the glycoform pattern of PrPSc before and after transmission of human prions to non human primates or bank voles, whereas in the second we analyzed the conformational stability of PrPSc associated with sCJD, vCJD or fCJD using guanidine hydrochloride (GdnHCl) as denaturant. Combining the results of the two studies, we were able to distinguish five human strains for at least one biochemical property. The present data extend our knowledge about the extent of strain variation and its relationship with PrPSc properties in human TSEs.

Hsp90 characterization and inhibition: a multi-methodological study

Many physiological and pathological processes are mediated by the activity of proteins assembled in homo and/or hetero-oligomers. The correct recognition and association of these proteins into a functional complex is a key step determining the fate of the whole pathway. This has led to an increasing interest in selecting molecules able to modulate/inhibit these protein-protein interactions. In particular, our research was focused on Heat Shock Protein 90 (Hsp90), responsible for the activation and maturation and disposition of many client proteins [1], [2] [3]. Circular Dichroism (CD) spectroscopy, Surface Plasmon Resonance (SPR) and Affinity Capillary Electrophoresis (ACE) were used to characterize the Hsp90 target and, furthermore, its inhibition process via C-terminal domain driven by the small molecule Coumermycin A1. Circular Dichroism was used as powerful technique to characterize Hsp90 and its co-chaperone Hop in solution for secondary structure content, stability to different pHs, temperatures and solvents. Furthermore, CD was used to characterize ATP but, unfortunately, we were not able to monitor an interaction between ATP and Hsp90. The utility of SPR technology, on the other hand, arises from the possibility of immobilizing the protein on a chip through its N-terminal domain to later study the interaction with small molecules able to disrupt the Hsp90 dimerization on the C-terminal domain. The protein was attached on SPR chip using the “amine coupling” chemistry so that the C-terminal domain was free to interact with Coumermycin A1. The goal of the experiment was achieved by testing a range of concentrations of the small molecule Coumermycin A1. Despite to the large difference in the molecular weight of the protein (90KDa) and the drug (1110.08 Da), we were able to calculate the affinity constant of the interaction that was found to be 11.2 µm. In order to confirm the binding constant calculated for the Hsp90 on the chip, we decided to use Capillary Electrophoresis to test the Coumermycin binding to Hsp90. First, this technique was conveniently used to characterize the Hsp90 sample in terms of composition and purity. The experimental conditions were settled on two different systems, the bared fused silica and the PVA-coated capillary. We were able to characterize the Hsp90 sample in both systems. Furthermore, we employed an application of capillary electrophoresis, the Affinity Capillary Electrophoresis (ACE), to measure and confirm the binding constant calculated for Coumermycin on Optical Biosensor. We found a KD = 19.45 µM. This result compares favorably with the KD previously obtained on biosensor. This is a promising result for the use of our novel approach to screen new potential inhibitors of Hsp90 C-terminal domain.

Learning with Kernels on Graphs: DAG-based kernels, data streams and RNA function prediction.

In many application domains data can be naturally represented as graphs. When the application of analytical solutions for a given problem is unfeasible, machine learning techniques could be a viable way to solve the problem. Classical machine learning techniques are defined for data represented in a vectorial form. Recently some of them have been extended to deal directly with structured data. Among those techniques, kernel methods have shown promising results both from the computational complexity and the predictive performance point of view. Kernel methods allow to avoid an explicit mapping in a vectorial form relying on kernel functions, which informally are functions calculating a similarity measure between two entities. However, the definition of good kernels for graphs is a challenging problem because of the difficulty to find a good tradeoff between computational complexity and expressiveness. Another problem we face is learning on data streams, where a potentially unbounded sequence of data is generated by some sources. There are three main contributions in this thesis. The first contribution is the definition of a new family of kernels for graphs based on Directed Acyclic Graphs (DAGs). We analyzed two kernels from this family, achieving state-of-the-art results from both the computational and the classification point of view on real-world datasets. The second contribution consists in making the application of learning algorithms for streams of graphs feasible. Moreover,we defined a principled way for the memory management. The third contribution is the application of machine learning techniques for structured data to non-coding RNA function prediction. In this setting, the secondary structure is thought to carry relevant information. However, existing methods considering the secondary structure have prohibitively high computational complexity. We propose to apply kernel methods on this domain, obtaining state-of-the-art results.

Rural governance and livelihoods systems diversification in the Western Balkans: comparative case studies of Bosnia and Herzegovina, Montenegro and Serbia

Agriculture is still important for socio-economic development in rural areas of Bosnia, Montenegro and Serbia (BMS). However, for sustainable rural development rural economies should be diversified so attention should be paid also to off-farm and non-farm income-generating activities. Agricultural and rural development (ARD) processes and farm activity diversification initiatives should be well governed. The ultimate objective of this work is to explore linkages between ARD governance and rural livelihoods diversification in BMS. The thesis is based on an extended secondary data analysis and surveys. Questionnaires for ARD governance and coordination were sent via email to public, civil society and international organizations. Concerning rural livelihood diversification, the field questionnaire surveys were carried out in three rural regions of BMS. Results show that local rural livelihoods are increasingly diversified but a significant share of households are still engaged in agriculture. Diversification strategies have a chance to succeed taking into consideration the three rural regions’ assets. However, rural households have to tackle many problems for developing new income-generating activities such as the lack of financial resources. Weak business skills are also a limiting factor. Fully exploiting rural economy diversification potential in BMS requires many interventions including improving rural governance, enhancing service delivery in rural areas, upgrading rural people’s human capital, strengthening rural social capital and improving physical capital, access of the rural population to finance as well as creating a favourable and enabling legal and legislative environment fostering diversification. Governance and coordination of ARD policy design, implementation and evaluation is still challenging in the three Balkan countries and this has repercussions also on the pace of rural livelihoods diversification. Therefore, there is a strong and urgent need for mobilization of all rural stakeholders and actors through appropriate governance arrangements in order to foster rural livelihoods diversification and quality of life improvement.

Spatially defined peptidomimetics for pain relief and for targeted cancer therapy and diagnostics

Cancer is a disease that has plagued scientists for decades, and how to treat cancer and its complications are inevitable topics in current scientific research. Cancer pain is a major factor that reduces the quality of life of patients. Therefore, the development of analgesic agents with minimal adverse side effects, especially with low addiction, has attracted more and more attention. Among them, opioid analgesics are widely used to alleviate cancer pain and improve the quality of life of patients with advanced cancer, such as in the palliative therapy. Although peptide drugs are efficient, selective and safe, they have several unignorable disadvantages such as poor biological stability, rapid excretion, difficulty in penetrate blood brain barrier. In order to solve these problems, peptidomimetics were developed by introducing unnatural/modified amino acids, decorated peptide backbone, conformational restrictions and secondary structure mimics in peptide sequence. Compared with peptides, peptidomimetics have improved biological stability, increased bioavailability, high affinity and selectivity for receptor binding, and decreased adverse side effects. As the second part of this thesis, I explored the opportunity to design peptide-functionalized responsive biomaterials for the detection of cancer cell and the selective delivery of cytotoxic drugs. The conjugation of peptides with biomaterials enhanced the stability of the loaded drugs, improved targeted delivery, decreased side effects, and increased bioavailability. The precise and controllable drug delivery platform has profound application prospects in cancer treatment. Grafting specific peptides sequence on the surface of biomaterials can satisfy different drug delivery demands according to the characteristics of both peptides and biomaterials. For example, the introduction of tumor-targeting peptides can guide biomaterials into tumor lesions, and blood-brain barrier (BBB) shuttle peptides can lead biomaterials to penetrate the BBB, etc.

Characterization of the chestnut germoplasm of Emilia-Romagna region and ancient fruit tree varieties valorization

Molecular characterization represents a valid support for the recovery of germoplasm, also motivated by the interest for the valorization of local productions in order to make their traceability possible. Molecular characterization is also fundamental for the individuation of misnomers in collection fields in which the different varieties are preserved. In particular, microsatellites have been used in this research to investigate the genetic diversity, inside a population and at an individual level, and the correct varietal correspondence. The research is mainly based on the study of European chestnut (Castanea sativa Mill.) cultivars to evaluate the genetic diversity and relationships in Emilia-Romagna region (Italy). A STRUCTURE analysis was carried out at European level with the allelic frequencies of the samples collected in Emilia-Romagna. Variation found at group and subgroup level may reflect a combination of historical migration/selection processes and adaptive factors to different environments between Italian and Spanish regions. In addition, a case study for the valorization of an old local variety and its re-introduction in the cultivation areas was proposed. This research was carried out by a morphological and molecular characterization of the local apple variety 'Rosa Romana'. The conservation of this variety entails the discrimination of different accessions with very similar phenotype that are present in the original cultivation area. The identification of historical trees and most adequate reference plants are fundamental steps for the correct propagation of this old variety and for the development of nursery activities. This will also promote and re-evaluate the exploitation and protection of such ancient Italian apple cultivars. This model could be in future also carried out for chestnut varieties. In conclusion, analysis with molecular markers is of fundamental importance for the protection and the maintenance of local and ancient varieties which allow to increase the allelic variability available for breeding programs.

Genes involved in germline regulative pathways in metazoa: an integrative approach to investigate a key feature of animal biology

In Metazoa, the germline represents the cell lineage devoted to transmission of genetic heredity across generations. Its functions intuitively evoke the crucial roles that it plays in the development of a new organism and in the evolution of the species. Germline establishment is tightly tied to animal multicellularity itself, in which the complex differentiation of cell lineages is favoured by the confinement of totipotency in specific cell populations. In the present thesis, I addressed the subject of germline characterization in animals through different approaches, in an attempt to cover different sides and scales. First, I investigated the extent and nature of shared differentially transcribed molecular factors in 10 different species germline-related lineages. I observed that newly evolved genes are less likely to be involved in germline-related mechanisms and that the mostly shared transcriptional signal across the species considered was the upregulation of genes associated to proper DNA replication, instead of the expected transcriptional and post-transcriptional regulation, that apparently have a higher level of lineage-specificity. I then focused on the evolutionary history of Tudor domain containing proteins, a gene family that underwent germline-associated expansions in animals. Using data from 24 holozoan phyla, I could confirm the previously proposed evolution of the Tudor domain secondary structure. Also, I associated lineage-specific family reductions and expansions to peculiar genomic dynamics and to the evolution of germline-associated piRNA pathway of retrotransposon silencing. Lastly, I characterized and investigated the expression of the Tudor protein TDRD7 in the clam Ruditapes philippinarum. Through immunolocalization, I could compare its expression profiles in gametogenic specimens to the previously characterized germline marker vasa. Combining results with literature, I proposed that, in this species, TDRD7 is involved in the assembly of germ granules, i.e. cytoplasmic structures associated to germline differentiation in virtually all animals, but whose assemblers can be taxon specific.

Velocity structure and seismic anisotropy in the crust and upper mantle from Receiver Function analysis: three case studies in Italy

The research for this PhD project consisted in the application of the RFs analysis technique to different data-sets of teleseismic events recorded at temporary and permanent stations located in three distinct study regions: Colli Albani area, Northern Apennines and Southern Apennines. We found some velocity models to interpret the structures in these regions, which possess very different geologic and tectonics characteristics and therefore offer interesting case study to face. In the Colli Albani some of the features evidenced in the RFs are shared by all the analyzed stations: the Moho is almost flat and is located at about 23 km depth, and the presence of a relatively shallow limestone layer is a stable feature; contrariwise there are features which vary from station to station, indicating local complexities. Three seismic stations, close to the central part of the former volcanic edifice, display relevant anisotropic signatures­­­ with symmetry axes consistent with the emplacement of the magmatic chamber. Two further anisotropic layers are present at greater depth, in the lower crust and the upper mantle, respectively, with symmetry axes directions related to the evolution of the volcano complex. In Northern Apennines we defined the isotropic structure of the area, finding the depth of the Tyrrhenian (almost 25 km and flat) and Adriatic (40 km and dipping underneath the Apennines crests) Mohos. We determined a zone in which the two Mohos overlap, and identified an anisotropic body in between, involved in the subduction and going down with the Adiratic Moho. We interpreted the downgoing anisotropic layer as generated by post-subduction delamination of the top-slab layer, probably made of metamorphosed crustal rocks caught in the subduction channel and buoyantly rising toward the surface. In the Southern Apennines, we found the Moho depth for 16 seismic stations, and highlighted the presence of an anisotropic layer underneath each station, at about 15-20 km below the whole study area. The moho displays a dome-like geometry, as it is shallow (29 km) in the central part of the study area, whereas it deepens peripherally (down to 45 km); the symmetry axes of anisotropic layer, interpreted as a layer separating the upper and the lower crust, show a moho-related pattern, indicated by the foliation of the layer which is parallel to the Moho trend. Moreover, due to the exceptional seismic event occurred on April 6th next to L’Aquila town, we determined the Vs model for two station located next to the epicenter. An extremely high velocity body is found underneath AQU station at 4-10 km depth, reaching Vs of about 4 km/s, while this body is lacking underneath FAGN station. We compared the presence of this body with other recent works and found an anti-correlation between the high Vs body, the max slip patches and earthquakes distribution. The nature of this body is speculative since such high velocities are consistent with deep crust or upper mantle, but can be interpreted as a as high strength barrier of which the high Vs is a typical connotation.