Optimization of molecular and crystalline forms of drugs, agrochemicals, pesticides in relation to activity, bioavailability, patentability and to the fabrication of polymorphs, solvates, co-crystals with green chemistry methods

This doctorate was funded by the Regione Emilia Romagna, within a Spinner PhD project coordinated by the University of Parma, and involving the universities of Bologna, Ferrara and Modena. The aim of the project was: - Production of polymorphs, solvates, hydrates and co-crystals of active pharmaceutical ingredients (APIs) and agrochemicals with green chemistry methods; - Optimization of molecular and crystalline forms of APIs and pesticides in relation to activity, bioavailability and patentability. In the last decades, a growing interest in the solid-state properties of drugs in addition to their solution chemistry has blossomed. The achievement of the desired and/or the more stable polymorph during the production process can be a challenge for the industry. The study of crystalline forms could be a valuable step to produce new polymorphs and/or co-crystals with better physical-chemical properties such as solubility, permeability, thermal stability, habit, bulk density, compressibility, friability, hygroscopicity and dissolution rate in order to have potential industrial applications. Selected APIs (active pharmaceutical ingredients) were studied and their relationship between crystal structure and properties investigated, both in the solid state and in solution. Polymorph screening and synthesis of solvates and molecular/ionic co-crystals were performed according to green chemistry principles. Part of this project was developed in collaboration with chemical/pharmaceutical companies such as BASF (Germany) and UCB (Belgium). We focused on on the optimization of conditions and parameters of crystallization processes (additives, concentration, temperature), and on the synthesis and characterization of ionic co-crystals. Moreover, during a four-months research period in the laboratories of Professor Nair Rodriguez-Hormedo (University of Michigan), the stability in aqueous solution at the equilibrium of ionic co-crystals (ICCs) of the API piracetam was investigated, to understand the relationship between their solid-state and solution properties, in view of future design of new crystalline drugs with predefined solid and solution properties.

Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model

The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

Meccanismi evolutivi dei parapoxvirus: caratterizzazione genomica di pseudocowpoxvirus e messa a punto di sistemi per lo studio delle ricombinazioni

The Poxviruses are a family of double stranded DNA (dsDNA) viruses that cause disease in many species, both vertebrate and invertebrate. Their genomes range in size from 135 to 365 kbp and show conservation in both organization and content. In particular, the central genomic regions of the chordopoxvirus subfamily (those capable of infecting vertebrates) contain 88 genes which are present in all the virus species characterised to date and which mostly occur in the same order and orientation. In contrast, however, the terminal regions of the genomes frequently contain genes that are species or genera-specific and that are not essential for the growth of the virus in vitro but instead often encode factors with important roles in vivo including modulation of the host immune response to infection and determination of the host range of the virus. The Parapoxviruses (PPV), of which Orf virus is the prototypic species, represent a genus within the chordopoxvirus subfamily of Poxviridae and are characterised by their ability to infect ruminants and humans. The genus currently contains four recognised species of virus, bovine papular stomatitis virus (BPSV) and pseudocowpox virus (PCPV) both of which infect cattle, orf virus (OV) that infects sheep and goats, and parapoxvirus of red deer in New Zealand (PVNZ). The ORFV genome has been fully sequenced, as has that of BPSV, and is ~138 kb in length encoding ~132 genes. The vast majority of these genes allow the virus to replicate in the cytoplasm of the infected host cell and therefore encode proteins involved in replication, transcription and metabolism of nucleic acids. These genes are well conserved between all known genera of poxviruses. There is however another class of genes, located at either end of the linear dsDNA genome, that encode proteins which are non-essential for replication and generally dictate host range and virulence of the virus. The non-essential genes are often the most variable within and between species of virus and therefore are potentially useful for diagnostic purposes. Given their role in subverting the host-immune response to infection they are also targets for novel therapeutics. The function of only a relatively small number of these proteins has been elucidated and there are several genes whose function still remains obscure principally because there is little similarity between them and proteins of known function in current sequence databases. It is thought that by selectively removing some of the virulence genes, or at least neutralising the proteins in some way, current vaccines could be improved. The evolution of poxviruses has been proposed to be an adaptive process involving frequent events of gene gain and loss, such that the virus co-evolves with its specific host. Gene capture or horizontal gene transfer from the host to the virus is considered an important source of new viral genes including those likely to be involved in host range and those enabling the virus to interfere with the host immune response to infection. Given the low rate of nucleotide substitution, recombination can be seen as an essential evolutionary driving force although it is likely underestimated. Recombination in poxviruses is intimately linked to DNA replication with both viral and cellular proteins participate in this recombination-dependent replication. It has been shown, in other poxvirus genera, that recombination between isolates and perhaps even between species does occur, thereby providing another mechanism for the acquisition of new genes and for the rapid evolution of viruses. Such events may result in viruses that have a selective advantage over others, for example in re-infections (a characteristic of the PPV), or in viruses that are able to jump the species barrier and infect new hosts. Sequence data related to viral strains isolated from goats suggest that possible recombination events may have occurred between OV and PCPV (Ueda et al. 2003). The recombination events are frequent during poxvirus replication and comparative genomic analysis of several poxvirus species has revealed that recombinations occur frequently on the right terminal region. Intraspecific recombination can occur between strains of the same PPV species, but also interspecific recombination can happen depending on enough sequence similarity to enable recombination between distinct PPV species. The most important pre-requisite for a successful recombination is the coinfection of the individual host by different virus strains or species. Consequently, the following factors affecting the distribution of different viruses to shared target cells need to be considered: dose of inoculated virus, time interval between inoculation of the first and the second virus, distance between the marker mutations, genetic homology. At present there are no available data on the replication dynamics of PPV in permissive and non permissive hosts and reguarding co-infetions there are no information on the interference mechanisms occurring during the simultaneous replication of viruses of different species. This work has been carried out to set up permissive substrates allowing the replication of different PPV species, in particular keratinocytes monolayers and organotypic skin cultures. Furthermore a method to isolate and expand ovine skin stem cells was has been set up to indeep further aspects of viral cellular tropism during natural infection. The study produced important data to elucidate the replication dynamics of OV and PCPV virus in vitro as well as the mechanisms of interference that can arise during co-infection with different viral species. Moreover, the analysis carried on the genomic right terminal region of PCPV 1303/05 contributed to a better knowledge of the viral genes involved in host interaction and pathogenesis as well as to locate recombination breakpoints and genetic homologies between PPV species. Taken together these data filled several crucial gaps for the study of interspecific recombinations of PPVs which are thought to be important for a better understanding of the viral evolution and to improve the biosafety of antiviral therapy and PPV-based vectors.

Expression and cellular localization of Copper transporter 2 (Ctr2) in Mus musculus

The Ctr family is an essential part of the copper homeostasis machinery and its members share sequence homology and structural and functional features. Higher eukaryotes express two members of this family Ctr1 and Ctr2. Numerous structural and functional studies are available for Ctr1, the only high affinity Cu(I) transporter thus far identified. Ctr1 holigotrimers mediate cellular copper uptake and this protein was demonstrated to be essential for embryonic development and to play a crucial role in dietary copper acquisition. Instead very little is known about Ctr2, it bears structural homology to the yeast vacuolar copper transporter, which mediates mobilization of vacuolar copper stores. Recent studies using over-expressed epitope-tagged forms of human Ctr2 suggested a function as a low affinity copper transporter that can mediate either copper uptake from the extracellular environment or mobilization of lysosomal copper stores. Using an antibody that recognizes endogenous mouse Ctr2, we studied the expression and localization of endogenous mouse Ctr2 in cell culture and in mouse models to understand its regulation and function in copper homeostasis. By immunoblot we observed a regulation of mCtr2 protein levels in a copper and Ctr1 dependent way. Our observations in cells and transgenic mice suggest that lack of Ctr1 induces a strong downregulation of Ctr2 probably by a post-translational mechanism. By indirect immunofluorescence we observed an exclusive intracellular localization in a perinuclear compartment and no co-localization with lysosomal markers. Immunofluorescence experiments in Ctr1 null cells, supported by sequence analysis, suggest that lysosomes may play a role in mCtr2 biology not as resident compartment, but as a degradation site. In appendix a LC-mass method for analysis of algal biotoxins belonging to the family of PsP (paralytic shellfish poisoning) is described.

New insight on a Group A Streptococcus protective antigen contributing to bacterial cell division

Bioinformatic analysis of Group A Streptococcus (GAS) genomes aiming at the identification of new vaccine antigens, revealed the presence of a gene coding for a putative surface-associated protein, named GAS40, inducing protective antibodies in an animal model of sepsis. The aim of our study was to unravel the involvement of GAS40 in cell division processes and to identify the putative interactor. Firstly, bioinformatic analysis showed that gas40 shares homology with ezrA, a gene coding for a negative regulator of Z-ring formation during cell division process. Both scanning and transmission electron microscopy indicated morphological differences between wild-type and the GAS40 knock-out mutant strain, with the latter showing an impaired capacity to divide resulting in the formation of very long chains. Moreover, when the localization of the antigen on the bacterial surface was analyzed, we found that in bacteria grown at exponential phase GAS40 specifically localized at septum, indicating a possible role in cell division. Furthermore, by ELISA and co-sedimentation assays, we found that GAS40 is able to interact with FtsZ, a protein involved in Z-ring formation during cell division process. These data together with the co-localization of GAS40/FtsZ at bacterial septum demonstrated by by confocal microscopy, strongly support the hypothesis for a key role of GAS40 in bacterial cell division.

Polymorphs solvates and co-crystals of molecular materials

The scope of my research project is to produce and characterize new crystalline forms of organic compounds, focusing the attention on co-crystals and then transferring these notions on APIs to produce co-crystals of potential interest in the pharmaceutical field. In the first part of this work co-crystallization experiments were performed using as building blocks the family of aliphatic dicarboxylic acids HOOC-(CH2)n-COOH, with n= 2-8. This class of compounds has always been an object of study because it is characterized by an interesting phenomenon of alternation of melting points: the acids with an even number of carbon atoms show a melting point higher than those with an odd one. The acids were co-crystallized with four dipyridyl molecules (formed by two pyridine rings with a different number of bridging carbon atoms) through the formation of intermolecular interactions N•••(H)O. The bases used were: 4,4’-bipyridine (BPY), 1,2-bis(4-pyridyl)ethane (BPA), 1,2-(di-4-pyridyl)ethylene (BPE) and 1,2-bis(4-pyridyl)propane (BPP). The co-crystals obtained by solution synthesis were characterized by different solid-state techniques to determine the structure and to see how the melting points in co-crystals change. In the second part of this study we tried to obtain new crystal forms of compounds of pharmaceutical interest. The APIs studied are: O-desmethylvenlafaxine, Lidocaine, Nalidixic Acid and Sulfadiazine. Each API was subjected to Polymorph Screening and Salt/Co-crystal Screening experiments to identify new crystal forms characterized by different properties. In a typical Salt/Co-crystal Screening the sample was made to react with a co-former (solid or liquid) through different methods: crystallization by solution, grinding, kneading and solid-gas reactions. The new crystal forms obtained were characterized by different solid state techniques (X-ray single crystal diffraction, X-ray powder diffraction, Differential Scanning Calorimetry, Thermogravimetric Analysis, Evolved gas analysis, FT-IR – ATR, Solid State N.M.R).

Reverse genetic studies of Benyvirus - Polymyxa betae molecular interaction: Role of the RNA4-encoded protein in virus transmission

Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, is included within viruses transmitted through the soil from plasmodiophorid as Polymyxa betae. BNYVV is the causal agent of Rhizomania, which induces abnormal rootlet proliferation and is widespread in the sugar beet growing areas in Europe, Asia and America; for review see (Peltier et al., 2008). In this latter continent, Beet soil-borne mosaic virus (BSBMV) has been identified (Lee et al., 2001) and belongs to the benyvirus genus together with BNYVV, both vectored by P. betae. BSBMV is widely distributed only in the United States and it has not been reported yet in others countries. It was first identified in Texas as a sugar beet virus morphologically similar but serologically distinct to BNYVV. Subsequent sequence analysis of BSBMV RNAs evidenced similar genomic organization to that of BNYVV but sufficient molecular differences to distinct BSBMV and BNYVV in two different species (Rush et al., 2003). Benyviruses field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs -1 contains a single long ORF encoding polypeptide that shares amino acid homology with known viral RNA-dependent RNA polymerases (RdRp) and helicases. RNAs -2 contains six ORFs: capsid protein (CP), one readthrough protein, triple gene block proteins (TGB) that are required for cell-to-cell virus movement and the sixth 14 kDa ORF is a post-translation gene silencing suppressor. RNAs -3 is involved on disease symptoms and is essential for virus systemic movement. BSBMV RNA-3 can be trans-replicated, trans-encapsidated by the BNYVV helper strain (RNA-1 and -2) (Ratti et al., 2009). BNYVV RNA-4 encoded one 31 kDa protein and is essential for vector interactions and virus transmission by P. betae (Rahim et al., 2007). BNYVV RNA-5 encoded 26 kDa protein that improve virus infections and accumulation in the hosts. We are interest on BSBMV effect on Rhizomania studies using powerful tools as full-length infectious cDNA clones. B-type full-length infectious cDNA clones are available (Quillet et al., 1989) as well as A/P-type RNA-3, -4 and -5 from BNYVV (unpublished). A-type BNYVV full-length clones are also available, but RNA-1 cDNA clone still need to be modified. During the PhD program, we start production of BSBMV full-length cDNA clones and we investigate molecular interactions between plant and Benyviruses exploiting biological, epidemiological and molecular similarities/divergences between BSBMV and BNYVV. During my PhD researchrs we obtained full length infectious cDNA clones of BSBMV RNA-1 and -2 and we demonstrate that they transcripts are replicated and packaged in planta and able to substitute BNYVV RNA-1 or RNA-2 in a chimeric viral progeny (BSBMV RNA-1 + BNYVV RNA-2 or BNYVV RNA-1 + BSBMV RNA-2). During BSBMV full-length cDNA clones production, unexpected 1,730 nts long form of BSBMV RNA-4 has been detected from sugar beet roots grown on BSBMV infected soil. Sequence analysis of the new BSBMV RNA-4 form revealed high identity (~100%) with published version of BSBMV RNA-4 sequence (NC_003508) between nucleotides 1-608 and 1,138-1,730, however the new form shows 528 additionally nucleotides between positions 608-1,138 (FJ424610). Two putative ORFs has been identified, the first one (nucleotides 383 to 1,234), encode a protein with predicted mass of 32 kDa (p32) and the second one (nucleotides 885 to 1,244) express an expected product of 13 kDa (p13). As for BSBMV RNA-3 (Ratti et al., 2009), full-length BSBMV RNA-4 cDNA clone permitted to obtain infectious transcripts that BNYVV viral machinery (Stras12) is able to replicate and to encapsidate in planta. Moreover, we demonstrated that BSBMV RNA-4 can substitute BNYVV RNA-4 for an efficient transmission through the vector P. betae in Beta vulgaris plants, demonstrating a very high correlation between BNYVV and BSBMV. At the same time, using BNYVV helper strain, we studied BSBMV RNA-4’s protein expression in planta. We associated a local necrotic lesions phenotype to the p32 protein expression onto mechanically inoculated C. quinoa. Flag or GFP-tagged sequences of p32 and p13 have been expressed in viral context, using Rep3 replicons, based on BNYVV RNA-3. Western blot analyses of local lesions contents, using FLAG-specific antibody, revealed a high molecular weight protein, which suggest either a strong interaction of BSBMV RNA4’s protein with host protein(s) or post translational modifications. GFP-fusion sequences permitted the subcellular localization of BSBMV RNA4’s proteins. Moreover we demonstrated the absence of self-activation domains on p32 by yeast two hybrid system approaches. We also confirmed that p32 protein is essential for virus transmission by P. betae using BNYVV helper strain and BNYVV RNA-3 and we investigated its role by the use of different deleted forms of p32 protein. Serial mechanical inoculation of wild-type BSBMV on C. quinoa plants were performed every 7 days. Deleted form of BSBMV RNA-4 (1298 bp) appeared after 14 passages and its sequence analysis shows deletion of 433 nucleotides between positions 611 and 1044 of RNA-4 new form. We demonstrated that this deleted form can’t support transmission by P. betae using BNYVV helper strain and BNYVV RNA-3, moreover we confirmed our hypothesis that BSBMV RNA-4 described by Lee et al. (2001) is a deleted form. Interesting after 21 passages we identifed one chimeric form of BSBMV RNA-4 and BSBMV RNA-3 (1146 bp). Two putative ORFs has been identified on its sequence, the first one (nucleotides 383 to 562), encode a protein with predicted mass of 7 kDa (p7), corresponding to the N-terminal of p32 protein encoded by BSBMV RNA-4; the second one (nucleotides 562 to 789) express an expected product of 9 kDa (p9) corresponding to the C-terminal of p29 encoded by BSBMV RNA-3. Results obtained by our research in this topic opened new research lines that our laboratories will develop in a closely future. In particular BSBMV p32 and its mutated forms will be used to identify factors, as host or vector protein(s), involved in the virus transmission through P. betae. The new results could allow selection or production of sugar beet plants able to prevent virus transmission then able to reduce viral inoculum in the soil.

The role of collective action in the provision of agri-environmental public goods: theoretical development through case studies in Italy

The thesis aims at analysing the role of collective action as a viable alternative to the traditional forms of intervention in agriculture in order to encourage the provision of agri-environmental public goods. Which are the main benefits of collective action, in terms of effectiveness and efficiency, compared to traditional market or public intervention policies? What are the drivers that encourage farmers to participate into collective action? To what extent it is possible to incorporate collective aspects into policies aimed at providing agri-environmental public goods? With the objective of addressing these research questions, the thesis is articulated in two levels: a theoretical analysis on the role of collective action in the provision of public goods and a specific investigation of two local initiative,s were an approach collective management of agro-environmental resources was successfully implemented. The first case study concerns a project named “Custodians of the Territory”, developed by the local agency in Tuscany “Comunità Montana Media Valle del Serchio”, which settled for an agreement with local farmers for a collective provision of environmental services related to the hydro-geological management of the district. The second case study is related to the territorial agri-environmental agreement experimented in Valdaso (Marche), where local farmers have adopted integrated pest management practices collectively with the aim of reducing the environmental impact of their farming practices. The analysis of these initiatives, carried out through participatory methods (Rapid Rural Appraisal), allowed developing a theoretical discussion on the role of innovative tools (such as co-production and co-management) in the provision of agri-environmental public goods. The case studies also provided some recommendations on the government intervention and policies needed to promote successful collective action for the provision of agri-environmental public goods.

Local Trigonometric Methods for Time Series Smoothing.

The thesis is concerned with local trigonometric regression methods. The aim was to develop a method for extraction of cyclical components in time series. The main results of the thesis are the following. First, a generalization of the filter proposed by Christiano and Fitzgerald is furnished for the smoothing of ARIMA(p,d,q) process. Second, a local trigonometric filter is built, with its statistical properties. Third, they are discussed the convergence properties of trigonometric estimators, and the problem of choosing the order of the model. A large scale simulation experiment has been designed in order to assess the performance of the proposed models and methods. The results show that local trigonometric regression may be a useful tool for periodic time series analysis.

Potential Analysis for Hypoelliptic Second Order PDEs with Nonnegative Characteristic Form

In this Thesis we consider a class of second order partial differential operators with non-negative characteristic form and with smooth coefficients. Main assumptions on the relevant operators are hypoellipticity and existence of a well-behaved global fundamental solution. We first make a deep analysis of the L-Green function for arbitrary open sets and of its applications to the Representation Theorems of Riesz-type for L-subharmonic and L-superharmonic functions. Then, we prove an Inverse Mean value Theorem characterizing the superlevel sets of the fundamental solution by means of L-harmonic functions. Furthermore, we establish a Lebesgue-type result showing the role of the mean-integal operator in solving the homogeneus Dirichlet problem related to L in the Perron-Wiener sense. Finally, we compare Perron-Wiener and weak variational solutions of the homogeneous Dirichlet problem, under specific hypothesis on the boundary datum.