Apple latent infection caused by Neofabraea alba: host-pathogen interaction and disease management

Apple latent infection caused by Neofabraea alba: host-pathogen interaction and disease management Bull’s eye rot (BER) caused by Neofabraea alba is one of the most frequent and damaging latent infection occurring in stored pome fruits worldwide. Fruit infection occurs in the orchard, but disease symptoms appear only 3 months after harvest, during refrigerated storage. In Italy BER is particularly serious for late harvest apple cultivar as ‘Pink Lady™’. The purposes of this thesis were: i) Evaluate the influence of ‘Pink Lady™’ apple primary metabolites in N. alba quiescence ii) Evaluate the influence of pH in five different apple cultivars on BER susceptibility iii) To find out not chemical method to control N. alba infection iv) Identify some fungal volatile compounds in order to use them as N. alba infections markers. Results regarding the role of primary metabolites showed that chlorogenic, quinic and malic acid inhibit N. alba development. The study based on the evaluation of cultivar susceptibility, showed that Granny Smith was the most resistant apple cultivar among the varieties analyzed. Moreover, Granny Smith showed the lowest pH value from harvest until the end of storage, supporting the thesis that ambient pH could be involved in the interaction between N. alba and apple. In order to find out new technologies able to improve lenticel rot management, the application of a non-destructive device for the determination of chlorophyll content was applied. Results showed that fruit with higher chlorophyll content are less susceptible to BER, and molecular analyses comforted this result. Fruits with higher chlorophyll content showed up-regulation of PGIP and HCT, genes involved in plant defence. Through the application of PTR-MS and SPME GC-MS, 25 volatile organic compounds emitted by N. alba were identified. Among them, 16 molecules were identified as potential biomarkers.

Supramolecular functional assemblies with photo or chemical addressability

Supramolecular self-assembly represents a key technology for the spontaneous construction of nanoarchitectures and for the fabrication of materials with enhanced physical and chemical properties. In addition, a significant asset of supramolecular self-assemblies rests on their reversible formation, thanks to the kinetic lability of their non-covalent interactions. This dynamic nature can be exploited for the development of “self-healing” and “smart” materials towards the tuning of their functional properties upon various external factors. One particular intriguing objective in the field is to reach a high level of control over the shape and size of the supramolecular architectures, in order to produce well-defined functional nanostructures by rational design. In this direction, many investigations have been pursued toward the construction of self-assembled objects from numerous low-molecular weight scaffolds, for instance by exploiting multiple directional hydrogen-bonding interactions. In particular, nucleobases have been used as supramolecular synthons as a result of their efficiency to code for non-covalent interaction motifs. Among nucleobases, guanine represents the most versatile one, because of its different H-bond donor and acceptor sites which display self-complementary patterns of interactions. Interestingly, and depending on the environmental conditions, guanosine derivatives can form various types of structures. Most of the supramolecular architectures reported in this Thesis from guanosine derivatives require the presence of a cation which stabilizes, via dipole-ion interactions, the macrocyclic G-quartet that can, in turn, stack in columnar G-quadruplex arrangements. In addition, in absence of cations, guanosine can polymerize via hydrogen bonding to give a variety of supramolecular networks including linear ribbons. This complex supramolecular behavior confers to the guanine-guanine interactions their upper interest among all the homonucleobases studied. They have been subjected to intense investigations in various areas ranging from structural biology and medicinal chemistry – guanine-rich sequences are abundant in telomeric ends of chromosomes and promoter regions of DNA, and are capable of forming G-quartet based structures– to material science and nanotechnology. This Thesis, organized into five Chapters, describes mainly some recent advances in the form and function provided by self-assembly of guanine based systems. More generally, Chapter 4 will focus on the construction of supramolecular self-assemblies whose self-assembling process and self-assembled architectures can be controlled by light as external stimulus. Chapter 1 will describe some of the many recent studies of G-quartets in the general area of nanoscience. Natural G- quadruplexes can be useful motifs to build new structures and biomaterials such as self-assembled nanomachines, biosensors, therapeutic aptamer and catalysts. In Chapters 2-4 it is pointed out the core concept held in this PhD Thesis, i.e. the supramolecular organization of lipophilic guanosine derivatives with photo or chemical addressability. Chapter 2 will mainly focus on the use of cation-templated guanosine derivatives as a potential scaffold for designing functional materials with tailored physical properties, showing a new way to control the bottom-up realization of well-defined nanoarchitectures. In section 2.6.7, the self-assembly properties of compound 28a may be considered an example of open-shell moieties ordered by a supramolecular guanosine architecture showing a new (magnetic) property. Chapter 3 will report on ribbon-like structures, supramolecular architectures formed by guanosine derivatives that may be of interest for the fabrication of molecular nanowires within the framework of future molecular electronic applications. In section 3.4 we investigate the supramolecular polymerizations of derivatives dG 1 and G 30 by light scattering technique and TEM experiments. The obtained data reveal the presence of several levels of organization due to the hierarchical self-assembly of the guanosine units in ribbons that in turn aggregate in fibrillar or lamellar soft structures. The elucidation of these structures furnishes an explanation to the physical behaviour of guanosine units which display organogelator properties. Chapter 4 will describe photoresponsive self-assembling systems. Numerous research examples have demonstrated that the use of photochromic molecules in supramolecular self-assemblies is the most reasonable method to noninvasively manipulate their degree of aggregation and supramolecular architectures. In section 4.4 we report on the photocontrolled self-assembly of modified guanosine nucleobase E-42: by the introduction of a photoactive moiety at C8 it is possible to operate a photocontrol over the self-assembly of the molecule, where the existence of G-quartets can be alternately switched on and off. In section 4.5 we focus on the use of cyclodextrins as photoresponsive host-guest assemblies: αCD–azobenzene conjugates 47-48 (section 4.5.3) are synthesized in order to obtain a photoresponsive system exhibiting a fine photocontrollable degree of aggregation and self-assembled architecture. Finally, Chapter 5 contains the experimental protocols used for the research described in Chapters 2-4.

Synthesis and Physical-Chemical characterization of Metallic Nanoparticles

The stabilization of nanoparticles against their irreversible particle aggregation and oxidation reactions. is a requirement for further advancement in nanoparticle science and technology. For this reason the research aim on this topic focuses on the synthesis of various metal nanoparticles protected with monolayers containing different reactive head groups and functional tail groups. In this work cuprous bromide nanocrystals haave been synthetized with a diameter of about 20 nanometers according to a new sybthetic method adding dropwise ascorbic acid to a water solution of lithium bromide and cupric chloride under continuous stirring and nitrogen flux. Butane thiolate Cu protected nanoparticles have been synthetized according to three different syntesys methods. Their morphologies appear related to the physicochemical conditions during the synthesis and to the dispersing medium used to prepare the sample. Synthesis method II allows to obtain stable nanoparticles of 1-2 nm in size both isolated and forming clusters. Nanoparticle cluster formation was enhanced as water was used as dispersing medium probably due to the idrophobic nature of the butanethiolate layers coating the nanoparticle surface. Synthesis methods I and III lead to large unstable spherical nanoparticles with size ranging between 20 to 50 nm. These nanoparticles appeared in the TEM micrograph with the same morphology independently on the dispersing medium used in the sample preparation. The stability and dimensions of the copper nanoparticles appear inversely related. Using the same methods above described for the butanethiolate protected copper nanoparticles 4-methylbenzenethiol protected copper nanoparticles have been prepared. Diffractometric and spectroscopic data reveal that decomposition processes didn’t occur in both the 4-methylbenzenethiol copper protected nanoparticles precipitates from formic acid and from water in a period of time six month long. Se anticarcinogenic effects by multiple mechanisms have been extensively investigated and documented and Se is defined a genuine nutritional cancer-protecting element and a significant protective effect of Se against major forms of cancer. Furthermore phloroglucinol was found to possess cytoprotective effects against oxidative stress, thanks to reactive oxygen species (ROS) which are associated with cells and tissue damages and are the contributing factors for inflammation, aging, cancer, arteriosclerosis, hypertension and diabetes. The goal of our work has been to set up a new method to synthesize in mild conditions amorphous Se nanopaticles surface capped with phloroglucinol, which is used during synthesis as reducing agent to obtain stable Se nanoparticles in ethanol, performing the synergies offered by the specific anticarcinogenic properties of Se and the antioxiding ones of phloroalucinol. We have synthesized selenium nanoparticles protected by phenolic molecules chemically bonded to their surface. The phenol molecules coating the nanoparticles surfaces form low ordered arrays as can be seen from the wider shape of the absorptions in the FT-IR spectrum with respect to those appearing in that of crystalline phenol. On the other hand, metallic nanoparticles with unique optical properties, facile surface chemistry and appropriate size scale are generating much enthusiasm in nanomedicine. In fact Au nanoparticles has immense potential for both cancer diagnosis and therapy. Especially Au nanoparticles efficiently convert the strongly adsorbed light into localized heat, which can be exploited for the selective laser photothermal therapy of cancer. According to the about, metal nanoparticles-HA nanocrystals composites should have tremendous potential in novel methods for therapy of cancer. 11 mercaptoundecanoic surface protected Au4Ag1 nanoparticles adsorbed on nanometric apathyte crystals we have successfully prepared like an anticancer nanoparticles deliver system utilizing biomimetic hydroxyapatyte nanocrystals as deliver agents. Furthermore natural chrysotile, formed by densely packed bundles of multiwalled hollow nanotubes, is a mineral very suitable for nanowires preparation when their inner nanometer-sized cavity is filled with a proper material. Bundles of chrysotile nanotubes can then behave as host systems, where their large interchannel separation is actually expected to prevent the interaction between individual guest metallic nanoparticles and act as a confining barrier. Chrysotile nanotubes have been filled with molten metals such as Hg, Pb, Sn, semimetals, Bi, Te, Se, and with semiconductor materials such as InSb, CdSe, GaAs, and InP using both high-pressure techniques and metal-organic chemical vapor deposition. Under hydrothermal conditions chrysotile nanocrystals have been synthesized as a single phase and can be utilized as a very suitable for nanowires preparation filling their inner nanometer-sized cavity with metallic nanoparticles. In this research work we have synthesized and characterized Stoichiometric synthetic chrysotile nanotubes have been partially filled with bi and monometallic highly monodispersed nanoparticles with diameters ranging from 1,7 to 5,5 nm depending on the core composition (Au, Au4Ag1, Au1Ag4, Ag). In the case of 4 methylbenzenethiol protected silver nanoparticles, the filling was carried out by convection and capillarity effect at room temperature and pressure using a suitable organic solvent. We have obtained new interesting nanowires constituted of metallic nanoparticles filled in inorganic nanotubes with a inner cavity of 7 nm and an isolating wall with a thick ranging from 7 to 21 nm.

Ab initio quantum mechanical investigation of structural and chemical-physical properties of selected minerals for minero-petrological, structural ceramic and biomaterial applications

The purpose of this thesis is the atomic-scale simulation of the crystal-chemical and physical (phonon, energetic) properties of some strategically important minerals for structural ceramics, biomedical and petrological applications. These properties affect the thermodynamic stability and rule the mineral-environment interface phenomena, with important economical, (bio)technological, petrological and environmental implications. The minerals of interest belong to the family of phyllosilicates (talc, pyrophyllite and muscovite) and apatite (OHAp), chosen for their importance in industrial and biomedical applications (structural ceramics) and petrophysics. In this thesis work we have applicated quantum mechanics methods, formulas and knowledge to the resolution of mineralogical problems ("Quantum Mineralogy”). The chosen theoretical approach is the Density Functional Theory (DFT), along with periodic boundary conditions to limit the portion of the mineral in analysis to the crystallographic cell and the hybrid functional B3LYP. The crystalline orbitals were simulated by linear combination of Gaussian functions (GTO). The dispersive forces, which are important for the structural determination of phyllosilicates and not properly con-sidered in pure DFT method, have been included by means of a semi-empirical correction. The phonon and the mechanical properties were also calculated. The equation of state, both in athermal conditions and in a wide temperature range, has been obtained by means of variations in the volume of the cell and quasi-harmonic approximation. Some thermo-chemical properties of the minerals (isochoric and isobaric thermal capacity) were calculated, because of their considerable applicative importance. For the first time three-dimensional charts related to these properties at different pressures and temperatures were provided. The hydroxylapatite has been studied from the standpoint of structural and phonon properties for its biotechnological role. In fact, biological apatite represents the inorganic phase of vertebrate hard tissues. Numerous carbonated (hydroxyl)apatite structures were modelled by QM to cover the broadest spectrum of possible biological structural variations to fulfil bioceramics applications.

Semi-synthetic bile acids as novel drug candidate in liver diseases: physico-chemical characterization and HPLC-ES-MS/MS methods for their quali-quantitative analysis in different experimental animal models

The physico-chemical characterization, structure-pharmacokinetic and metabolism studies of new semi synthetic analogues of natural bile acids (BAs) drug candidates have been performed. Recent studies discovered a role of BAs as agonists of FXR and TGR5 receptor, thus opening new therapeutic target for the treatment of liver diseases or metabolic disorders. Up to twenty new semisynthetic analogues have been synthesized and studied in order to find promising novel drugs candidates. In order to define the BAs structure-activity relationship, their main physico-chemical properties (solubility, detergency, lipophilicity and affinity with serum albumin) have been measured with validated analytical methodologies. Their metabolism and biodistribution has been studied in “bile fistula rat”, model where each BA is acutely administered through duodenal and femoral infusion and bile collected at different time interval allowing to define the relationship between structure and intestinal absorption and hepatic uptake ,metabolism and systemic spill-over. One of the studied analogues, 6α-ethyl-3α7α-dihydroxy-5β-cholanic acid, analogue of CDCA (INT 747, Obeticholic Acid (OCA)), recently under approval for the treatment of cholestatic liver diseases, requires additional studies to ensure its safety and lack of toxicity when administered to patients with a strong liver impairment. For this purpose, CCl4 inhalation to rat causing hepatic decompensation (cirrhosis) animal model has been developed and used to define the difference of OCA biodistribution in respect to control animals trying to define whether peripheral tissues might be also exposed as a result of toxic plasma levels of OCA, evaluating also the endogenous BAs biodistribution. An accurate and sensitive HPLC-ES-MS/MS method is developed to identify and quantify all BAs in biological matrices (bile, plasma, urine, liver, kidney, intestinal content and tissue) for which a sample pretreatment have been optimized.