Determination of degree of ionization of poly(allylamine hydrochloride) (PAH) and poly[1-[4-(3-carboxy‑4 hydroxyphenylazo)benzene sulfonamido]-1,2-ethanediyl, sodium salt] (PAZO) in layer-by-layer films using vacuum photoabsorption spectroscopy

Electrostatic and hydrophobic interactions govern most of the properties of supramolecular systems, which is the reason determining the degree of ionization of macromolecules has become crucial for many applications. In this paper, we show that highresolution ultraviolet spectroscopy (VUV) can be used to determine the degree of ionization and its effect on the electronic excitation energies of layer-by-layer (LbL) films of poly(allylamine hydrochloride) (PAH) and poly[1-[4-(3-carboxy-4 hydroxyphenylazo)- benzene sulfonamido]-1,2-ethanediyl, sodium salt] (PAZO). A full assignment of the VUV peaks of these polyelectrolytes in solution and in cast or LbL films could be made, with their pH dependence allowing us to determine the p'K IND. a' using the Henderson-Hasselbach equation. The p'K IND. a' for PAZO increased from ca. 6 in solution to ca. 7.3 in LbL films owing to the charge transfer from PAH. Significantly, even using solutions at a fixed pH for PAH, the amount adsorbed on the LbL films still varied with the pH of the PAZO solutions due to these molecular-level interactions. Therefore, the procedure based on a comparison of VUV spectra from solutions and films obtained under distinct conditions is useful to determine the degree of dissociation of macromolecules, in addition to permitting interrogation of interface effects in multilayer films.

Synthesis and characterization of implants for bone substitutions made of biomedical apatites containing silicon

In the last decades the development of bone substitutes characterized by a superior biomimetism has become of particular interest, owing to the increasing economic and societal impact of the bone diseases. In the present work of research the development of bone substitutes characterized by improved biomimetism, has been faced in a chemical, structural and morphological perspective. From a chemical point of view, it has been developed the synthesis of hydroxyapatite powders, exhibiting multiple ionic substitutions in both cationic and anionic sites, so to simulate the chemical composition of the natural bone. Particular emphasis has been given to the effect of silicon on the chemical-physical and solubility properties of the obtained hydroxyapatites. From a structural point of view, it has been developed the synthesis of ceramic composite materials, based on hydroxyapatite and calcium silicates, employed both as a reinforcing phase, to raise the mechanical strength of the composite compared to hydroxyapatite, and as a bioactive phase, able to increase the bioactivity properties of the whole ceramic. Finally the unique morphological features of the bone were mimicked by taking inspiration by Nature, so that native wood structures were treated in chemical and thermal way to obtain hydroxyapatite porous materials characterized by the same morphology as the native wood. The results obtained in the present work were positive in all the three different areas of investigation, so to cover the three different aspects of biomimetism, chemical, structural and morphological. Anyway, only at the convergence of the three different fields it is possible to find out the best solutions to develop the ideal bone-like scaffold. Thus, the future activity should be devoted to solve the problems at the borderline between the different research lines, which hamper this convergence and in consequence, the achievement of a bone scaffold able to mimic the various aspects exhibited by the bone tissue

Bayesian Analysis of Linear Inverse Problems with Applications in Economics and Finance

In my PhD thesis I propose a Bayesian nonparametric estimation method for structural econometric models where the functional parameter of interest describes the economic agent's behavior. The structural parameter is characterized as the solution of a functional equation, or by using more technical words, as the solution of an inverse problem that can be either ill-posed or well-posed. From a Bayesian point of view, the parameter of interest is a random function and the solution to the inference problem is the posterior distribution of this parameter. A regular version of the posterior distribution in functional spaces is characterized. However, the infinite dimension of the considered spaces causes a problem of non continuity of the solution and then a problem of inconsistency, from a frequentist point of view, of the posterior distribution (i.e. problem of ill-posedness). The contribution of this essay is to propose new methods to deal with this problem of ill-posedness. The first one consists in adopting a Tikhonov regularization scheme in the construction of the posterior distribution so that I end up with a new object that I call regularized posterior distribution and that I guess it is solution of the inverse problem. The second approach consists in specifying a prior distribution on the parameter of interest of the g-prior type. Then, I detect a class of models for which the prior distribution is able to correct for the ill-posedness also in infinite dimensional problems. I study asymptotic properties of these proposed solutions and I prove that, under some regularity condition satisfied by the true value of the parameter of interest, they are consistent in a "frequentist" sense. Once I have set the general theory, I apply my bayesian nonparametric methodology to different estimation problems. First, I apply this estimator to deconvolution and to hazard rate, density and regression estimation. Then, I consider the estimation of an Instrumental Regression that is useful in micro-econometrics when we have to deal with problems of endogeneity. Finally, I develop an application in finance: I get the bayesian estimator for the equilibrium asset pricing functional by using the Euler equation defined in the Lucas'(1978) tree-type models.

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).

NMR, Metabonomics and Molecular Profiles: Applications to the Quality Assessment of Foodstuffs

Nuclear Magnetic Resonance (NMR) is a branch of spectroscopy that is based on the fact that many atomic nuclei may be oriented by a strong magnetic field and will absorb radiofrequency radiation at characteristic frequencies. The parameters that can be measured on the resulting spectral lines (line positions, intensities, line widths, multiplicities and transients in time-dependent experi-ments) can be interpreted in terms of molecular structure, conformation, molecular motion and other rate processes. In this way, high resolution (HR) NMR allows performing qualitative and quantitative analysis of samples in solution, in order to determine the structure of molecules in solution and not only. In the past, high-field NMR spectroscopy has mainly concerned with the elucidation of chemical structure in solution, but today is emerging as a powerful exploratory tool for probing biochemical and physical processes. It represents a versatile tool for the analysis of foods. In literature many NMR studies have been reported on different type of food such as wine, olive oil, coffee, fruit juices, milk, meat, egg, starch granules, flour, etc using different NMR techniques. Traditionally, univariate analytical methods have been used to ex-plore spectroscopic data. This method is useful to measure or to se-lect a single descriptive variable from the whole spectrum and , at the end, only this variable is analyzed. This univariate methods ap-proach, applied to HR-NMR data, lead to different problems due especially to the complexity of an NMR spectrum. In fact, the lat-ter is composed of different signals belonging to different mole-cules, but it is also true that the same molecules can be represented by different signals, generally strongly correlated. The univariate methods, in this case, takes in account only one or a few variables, causing a loss of information. Thus, when dealing with complex samples like foodstuff, univariate analysis of spectra data results not enough powerful. Spectra need to be considered in their wholeness and, for analysing them, it must be taken in consideration the whole data matrix: chemometric methods are designed to treat such multivariate data. Multivariate data analysis is used for a number of distinct, differ-ent purposes and the aims can be divided into three main groups: • data description (explorative data structure modelling of any ge-neric n-dimensional data matrix, PCA for example); • regression and prediction (PLS); • classification and prediction of class belongings for new samples (LDA and PLS-DA and ECVA). The aim of this PhD thesis was to verify the possibility of identify-ing and classifying plants or foodstuffs, in different classes, based on the concerted variation in metabolite levels, detected by NMR spectra and using the multivariate data analysis as a tool to inter-pret NMR information. It is important to underline that the results obtained are useful to point out the metabolic consequences of a specific modification on foodstuffs, avoiding the use of a targeted analysis for the different metabolites. The data analysis is performed by applying chemomet-ric multivariate techniques to the NMR dataset of spectra acquired. The research work presented in this thesis is the result of a three years PhD study. This thesis reports the main results obtained from these two main activities: A1) Evaluation of a data pre-processing system in order to mini-mize unwanted sources of variations, due to different instrumental set up, manual spectra processing and to sample preparations arte-facts; A2) Application of multivariate chemiometric models in data analy-sis.

Liquid crystalline oligofluorenes and their derivatives

Homo-oligofluorenes (OFn), polyfluorenes (PF2/6) and oligofluorenes with one fluorenenone group in the center (OFnK) were synthesized. They were used as model compounds to understand of the structure-property relationships of polyfluorenes and the origin of the green emission in the photoluminescence (after photooxidation of the PFs) and the electroluminescence (EL) spectra. The electronic, electrochemical properties, thermal behavior, supramolecular self-assembly, and photophysical properties of OFn, PF2/6 and OFnK were investigated. Oligofluorenes with 2-ethylhexyl side chain (OF2-OF7) from the dimer up to the heptamer were prepared by a series of stepwise transition metal mediated Suzuki and Yamamoto coupling reactions. Polyfluorene was synthesized by Yamamoto coupling of 2,7-dibromo-9,9-bis(2-ethylhexyl)fluorene. Oligofluorenes with one fluorenone group in the center (OF3K, OF5K, OF7K) were prepared by Suzuki coupling between the monoboronic fluorenyl monomer, dimer, trimer and 2, 7-dibromofluorenone. The electrochemical and electronic properties of homo-oligofluorenes (OFn) were systematically studied by several combined techniques such as cyclic voltammetry, differential pulse voltammetry, UV-vis absorption spectroscopy, steady and time-resolved fluorescence spectroscopy. It was found that the oligofluorenes behave like classical conjugated oligomers, i.e., with the increase of the chain-length, the corresponding oxidation potential, the absorption and emission maximum, ionization potential, electron affinity, band gap and the photoluminescence lifetime displayed a very good linear relation with the reciprocal number of the fluorene units (1/n). The extrapolation of these linear relations to infinite chain length predicted the electrochemical and electronic properties of the corresponding polyfluorenes. The thermal behavior, single-crystal structure and supramolecular packing, alignment properties, and molecular dynamics of the homo-oligofluorenes (OFn) up to the polymer were studied using techniques such as TGA, DSC, WAXS, POM and DS. The OFn from tetramer to heptamer show a smectic liquid crystalline phase with clearly defined isotropization temperature. The oligomers do show a glass transition which exhibits n-1 dependence and allows extrapolation to a hypothetical glass transition of the polymer at around 64 °C. A smectic packing and helix-like conformation for the oligofluorenes from tetramer to heptamer was supported by WAXS experiments, simulation, and single-crystal structure of some oligofluorene derivatives. Oligofluorenes were aligned more easily than the corresponding polymer, and the alignability increased with the molecular length from tetramer to heptamer. The molecular dynamics in a series of oligofluorenes up to the polymer was studied using dielectric spectroscopy. The photophysical properties of OFn and PF2/6 were investigated by the steady-state spectra (UV-vis absorption and fluorescence spectra) and time-resolved fluorescence spectra both in solution and thin film. The time-resolved fluorescence spectra of the oligofluorenes were measured by streak camera and gate detection technique. The lifetime of the oligofluorenes decreased with the extension of the chain-length. No green emission was observed in CW, prompt and delayed fluorescence for oligofluorenes in m-THF and film at RT and 77K. Phosphorescence was observed for oligofluorenes in frozen dilute m-THF solution at 77K and its lifetime increased with length of oligofluorenes. A linear relation was obtained for triplet energy and singlet energy as a function of the reciprocal degree of polymerization, and the singlet-triplet energy gap (S1-T1) was found to decrease with the increase of degree of polymerization. Oligofluorenes with one fluorenone unit at the center were used as model compounds to understand the origin of the low-energy (“green”) emission band in the photoluminescence and electroluminescence spectra of polyfluorenes. Their electrochemical properties were investigated by CV, and the ionization potential (Ip) and electron affinity (Ea) were calculated from the onset of oxidation and reduction of OFnK. The photophysical properties of OFnK were studied in dilute solution and thin film by steady-state spectra and time-resolved fluorescence spectra. A strong green emission accompanied with a weak blue emission were obtained in solution and only green emission was observed on film. The strong green emission of OFnK suggested that rapid energy transfer takes place from higher energy sites (fluorene segments) to lower energy sites (fluorenone unit) prior to the radiative decay of the excited species. The fluorescence spectra of OFnK also showed solvatochromism. Monoexponential decay behaviour was observed by time-resolved fluorescence measurements. In addition, the site-selective excitation and concentration dependence of the fluorescence spectra were investigated. The ratio of green and blue emission band intensities increases with the increase of the concentration. The observed strong concentration dependence of the green emission band in solution suggests that increased interchain interactions among the fluorenone-containing oligofluorene chain enhanced the emission from the fluorenone defects at higher concentration. On the other hand, the mono-exponential decay behaviour and power dependence were not influenced significantly by the concentration. We have ruled out the possibility that the green emission band originates from aggregates or excimer formation. Energy transfer was further investigated using a model system of a polyfluorene doped by OFnK. Förster-type energy transfer took place from PF2/6 to OFnK, and the energy transfer efficiency increased with increasing of the concentration of OFnK. Efficient funneling of excitation energy from the high-energy fluorene segments to the low-energy fluorenone defects results from energy migration by hopping of excitations along a single polymer chain until they are trapped on the fluorenone defects on that chain or transferred onto neighbouring chains by Förster-type interchain energy transfer process. These results imply that the red-shifted emission in polyfluorenes can originate from (usually undesirable) keto groups at the bridging carbon atoms-especially if the samples have been subject to photo- or electro-oxidation or if fluorenone units are present due to an improper purification of the monomers prior to polymerization.

New discotic liquid crystals based on large polycyclic aromatic hydrocarbons as materials for molecular electronics

A series of new columnar discotic liquid crystalline materials based on the superphenalene (C96) core has been synthesized by oxidative cyclodehydrogenation with iron(III) chloride of suitable three-dimensional oligophenylene precursors. These compounds were investigated by means of differential scanning calorimetry (DSC), polarized optical microscopy (POM) and wide angle X-ray scattering (WAXS), and showed highly ordered supramolecular arrays and mesophase behavior over a broad temperature range. Good solubility, through the introduction of long alkyl chains, and the fact that these new superphenalene derivatives were found to be liquid crystalline at room temperature enabled the formation of highly ordered films (using the zone-casting technique), a requirement for application in organic electronic devices. The one-dimensional, intracolumnar charge carrier mobilities of superphenalene derivatives were determined using the pulse-radiolysis time-resolved microwave conductivity technique (PR-TRMC). Electrical properties of different C96-C12 architectures on mica surfaces were examined by using Electrostatic Force Microscopy (EFM) and Kelvin Probe Force Microscopy (KPFM). Hexa-peri-hexabenzocoronene (C42) derivatives substituted at the periphery with six branched alkyl ether chains were also synthesized. It was found that the introduction of ether groups within the side chains enhances the affinity of the discotic molecules towards polar surfaces, resulting in homeotropic self-assembly (as shown by POM and 2D-WAXS) when the compounds are processed from the isotropic state between two surfaces. A new, insoluble, superphenalene building block bearing six reactive sites was prepared, and was further used for the preparation of dendronized superphenalenes with bulky dendritic substituents around the core. UV/Vis and fluorescence experiments suggest reduced π-π stacking of the superphenalene cores as a result of steric hindrance between the peripheral dendritic units. A new family of graphitic molecules with partial ”zig-zag” periphery has been established. The incorporation of ”zig-zag” edges was shown to have a strong influence on the electronic properties of the new molecules (as studied by solution and solid-state UV/Vis, and fluorescence spectroscopy), leading to a significant bathochromic shift with respect to the parent PAHs (C42 and C96). The reactivity of the additional double bonds was examined. The attachment of long alkyl chains to a ”zig-zag” superphenalene core afforded a new, processable, liquid crystalline material.

Advances in understanding of osmotic dehydration and vacuum impregnation of fruits

Osmotic Dehydration and Vacuum Impregnation are interesting operations in the food industry with applications in minimal fruit processing and/or freezing, allowing to develop new products with specific innovative characteristics. Osmotic dehydration is widely used for the partial removal of water from cellular tissue by immersion in hypertonic (osmotic) solution. The driving force for the diffusion of water from the tissue is provided by the differences in water chemical potential between the external solution and the internal liquid phase of the cells. Vacuum Impregnation of porous products immersed in a liquid phase consist of reduction of pressure in a solid-liquid system (vacuum step) followed by the restoration of atmospheric pressure (atmospheric step). During the vacuum step the internal gas in the product pores is expanded and partially flows out while during the atmospheric step, there is a compression of residual gas and the external liquid flows into the pores (Fito, 1994). This process is also a very useful unit operation in food engineering as it allows to introduce specific solutes in the tissue which can play different functions (antioxidants, pH regulators, preservatives, cryoprotectants etc.). The present study attempts to enhance our understanding and knowledge of fruit as living organism, interacting dynamically with the environment, and to explore metabolic, structural, physico-chemical changes during fruit processing. The use of innovative approaches and/or technologies such as SAFES (Systematic Approach to Food Engineering System), LF-NMR (Low Frequency Nuclear Magnetic Resonance), GASMAS (Gas in Scattering Media Absorption Spectroscopy) are very promising to deeply study these phenomena. SAFES methodology was applied in order to study irreversibility of the structural changes of kiwifruit during short time of osmotic treatment. The results showed that the deformed tissue can recover its initial state 300 min after osmotic dehydration at 25 °C. The LF-NMR resulted very useful in water status and compartmentalization study, permitting to separate observation of three different water population presented in vacuole, cytoplasm plus extracellular space and cell wall. GASMAS techniques was able to study the pressure equilibration after Vacuum Impregnation showing that after restoration of atmospheric pressure in the solid-liquid system, there was a reminding internal low pressure in the apple tissue that slowly increases until reaching the atmospheric pressure, in a time scale that depends on the vacuum applied during the vacuum step. The physiological response of apple tissue on Vacuum Impregnation process was studied indicating the possibility of vesicular transport within the cells. Finally, the possibility to extend the freezing tolerance of strawberry fruits impregnated with cryoprotectants was proven.

Spectroscopic signatures from first principles calculations: from surface adsorbates to liquids and polymers

In dieser Arbeit werden drei wasserstoffverbrückte Systeme in der kondensierten Phase mit Hilfe von first-principles-Elektronenstruktur-Rechnungen untersucht, die auf der Dichtefunktionaltheorie (DFT) unter periodischen Randbedingungen basieren. Ihre lokalen Konformationen und Wasserstoffbrückenbindungen werden mittels ab-initio Molekulardynamiksimulationen berechnet und weiterhin durch die Bestimmung ihrer spektroskopischen Parameter charakterisiert. Der Schwerpunkt liegt dabei auf lokalen Strukturen und auf schnellen Fluktuationen der Wasserstoffbrückenbindungen, welche von zentraler Bedeutung für die physikalischen und chemischen Eigenschaften der betrachteten Systeme sind. Die für die lokalen, instantanen Konformationen berechneten Spektren werden verwendet, um die physikalischen Prozesse, die hinter den untersuchten Phänomenen stehen, zu erklären: die Wasseradsorption auf metallischen Oberflächen, die Ionensolvatisierung in wässrigen Lösungen und der Protonentransport in protonleitenden Polymeren, welche Prototypen von Membranen für Brennstoffzellen sind. Die Möglichkeit der Vorhersage spektroskopischer Parameter eröffnet vielfältige Möglichkeiten des Dialogs zwischen Experimenten und numerischen Simulationen. Die in dieser Arbeit vorgestellten Ergebnisse zeigen, dass die Zuverlässigkeit dieser theoretischen Berechnungen inzwischen für viele experimentell relevante Systeme ein quantitatives Niveau erreicht hat.

Synthesis of Modified Amino Acids and Insertion in Peptides and Mimetics. Structural Aspects and Impact on Biological Activity.

I studied the effects exerted by the modifications on structures and biological activities of the compounds so obtained. I prepared peptide analogues containing unusual amino acids such as halogenated, alkylated (S)- or (R)-tryptophans, useful for the synthesis of mimetics of the endogenous opioid peptide endomorphin-1, or 2-oxo-1,3-oxazolidine-4-carboxylic acids, utilized as pseudo-prolines having a clear all-trans configuration of the preceding peptide bond. The latter gave access to a series of constrained peptidomimetics with potential interest in medicinal chemistry and in the field of the foldamers. In particular, I have dedicated much efforts to the preparation of cyclopentapeptides containing D-configured, alfa-, or beta-aminoacids, and also of cyclotetrapeptides including the retro-inverso modification. The conformational analyses confirmed that these cyclic compounds can be utilized as rigid scaffolds mimicking gamma- or beta-turns, allowing to generate new molecular and 3D diversity. Much work has been dedicated to the structural analysis in solution and in the receptor-bound state, fundamental for giving a rationale to the experimentally determined bioactivity, as well as for predicting the activity of virtual compounds (in silico pre-screen). The conformational analyses in solution has been done mostly by NMR (2D gCosy, Roesy, VT, molecular dynamics, etc.). A special section is dedicated to the prediction of plausible poses of the ligands when bound to the receptors by Molecular Docking. This computational method proved to be a powerful tool for the investigation of ligand-receptor interactions, and for the design of selective agonists and antagonists. Another practical use of cyclic peptidomimetics was the synthesis and biological evaluation of cyclic analogues of endomorphin-1 lacking in a protonable amino group. The studies revealed that a inverse type II beta-turn on D-Trp-Phe constituted the bioactive conformation.

Physiological and structural aspects of fruit and vegetable mild processing

Over the past years fruit and vegetable industry has become interested in the application of both osmotic dehydration and vacuum impregnation as mild technologies because of their low temperature and energy requirements. Osmotic dehydration is a partial dewatering process by immersion of cellular tissue in hypertonic solution. The diffusion of water from the vegetable tissue to the solution is usually accompanied by the simultaneous solutes counter-diffusion into the tissue. Vacuum impregnation is a unit operation in which porous products are immersed in a solution and subjected to a two-steps pressure change. The first step (vacuum increase) consists of the reduction of the pressure in a solid-liquid system and the gas in the product pores is expanded, partially flowing out. When the atmospheric pressure is restored (second step), the residual gas in the pores compresses and the external liquid flows into the pores. This unit operation allows introducing specific solutes in the tissue, e.g. antioxidants, pH regulators, preservatives, cryoprotectancts. Fruit and vegetable interact dynamically with the environment and the present study attempts to enhance our understanding on the structural, physico-chemical and metabolic changes of plant tissues upon the application of technological processes (osmotic dehydration and vacuum impregnation), by following a multianalytical approach. Macro (low-frequency nuclear magnetic resonance), micro (light microscopy) and ultrastructural (transmission electron microscopy) measurements combined with textural and differential scanning calorimetry analysis allowed evaluating the effects of individual osmotic dehydration or vacuum impregnation processes on (i) the interaction between air and liquid in real plant tissues, (ii) the plant tissue water state and (iii) the cell compartments. Isothermal calorimetry, respiration and photosynthesis determinations led to investigate the metabolic changes upon the application of osmotic dehydration or vacuum impregnation. The proposed multianalytical approach should enable both better designs of processing technologies and estimations of their effects on tissue.

Specific enzymatic cleavage and payload release from peptide-based hybrid nanocapsules

Within this thesis, new approaches for the concepts of peptide-polymer conjugates and peptide-based hybrid nanomaterials are investigated. In the first part, the synthesis of a triblock polymer-peptide-polymer is carried out following a typical peptide coupling reaction, both in solution and on solid-phase. The peptide sequence is chosen, so that it is cleaved by an enzyme preparation of trypsin. End-functionalized polystyrene is used as a model hydrophobic polymer and coupled to the peptide sequence. The results show successful coupling reactions in both methods, while the solid phase method produced a more defined product. Suspensions, consisting of peptide-polymer conjugates particles, are prepared in water by ultrasonication. In contact with the enzyme, the peptide constituting the conjugated particles is cleaved. This demonstrates the enzymatic cleavage in heterophase of enzymatic sequence bond to hydrophobic polymers, and is of great interest for the encapsulation and delivery of hydrophobic molecules.rnA second approach is the preparation of peptide-based hybrid nanocapsules. This is achieved by interfacial polyaddition in inverse miniemulsion with the peptide sequence functionalized with additional amino acids. A method suitable to the use of a peptide sequence for interfacial polyaddition was developed. It is shown that, the polarity of the dispersed phase influences the structures prepared, from particle-like to polymeric shell with a liquid core.rnThe peptide sequence is equipped with a FRET pair (more exactly, an internally-quenched fluorescent system) which allows the real-time monitoring of the enzymatic cleavage of the recognition site. This system shows the successful cleavage of the peptide-based nanocapsules when trypsin preparation is added to the suspensions. A water-soluble fluorescent polymer is efficiently entrapped and its possible use as marker for the capsules is highlighted. Furthermore, a small water-soluble fluorescent dye (SR-101) is successfully encapsulated and the encapsulation efficiency as a function of the functionality of the peptide and the amount of comonomer equivalent (toluene diisocyanate) is studied. The dye is encapsulated at such a high concentration, that self-quenching occurs. Thus, the release of the encapsulated dye triggered by the enzymatic cleavage of the peptide results in a fluorescence recovery of the dye. The fluorescence recovery of the FRET pair in the peptide and of the encapsulated dye correlate well.rnFinally, nanocapsules based on a hepsin-cleavable peptide sequence are prepared. Hepsin is an enzyme, which is highly upregulated in prostate cancer cells. The cleavage of the nanocapsules is investigated with healthy and “cancerous” (hepsin-expressing) cell cultures. The degradation, followed via fluorescence recovery of the FRET system, is faster for the suspensions introduced in the hepsin expressing cell cultures.rnIn summary, this work tackles the domain of responsive nanomaterials for drug delivery from a new perspective. It presents the adaptation of the miniemulsion process for hybrid peptide-based materials, and their successful use in preparing specific enzyme-responsive nanoparticles, with hydrophilic payload release properties.rn

Silk Fibroin: a biopolymer platform for innovative pharmaceutical formulation and biomedical devices.

The protein silk fibroin (SF) from the silkworm Bombyx mori is a FDA-approved biomaterial used over centuries as sutures wire. Importantly, several evidences highlighted the potential of silk biomaterials obtained by using so-called regenerated silk fibroin (RSF) in biomedicine, tissue engineering and drug delivery. Indeed, by a water-based protocol, it is possible to obtain protein water-solution, by extraction and purification of fibroin from silk fibres. Notably, RSF can be processed in a variety of biomaterials forms used in biomedical and technological fields, displaying remarkable properties such as biocompatibility, controllable biodegradability, optical transparency, mechanical robustness. Moreover, RSF biomaterials can be doped and/or chemical functionalized with drugs, optically active molecules, growth factors and/or chemicals In this view, activities of my PhD research program were focused to standardize the process of extraction and purification of protein to get the best physical and chemical characteristics. The analysis of the chemo-physical properties of the fibroin involved both the RSF water-solution and the protein processed in film. Chemo-physical properties have been studied through: vibrational (FT-IR and Raman-FT) and optical (absorption and emission UV-VIS) spectroscopy, nuclear magnetic resonance (1H and 13C NMR), thermal analysis and thermo-gravimetric scan (DSC and TGA). In the last year of my PhD, activities were focused to study and define innovative methods of functionalization of the silk fibroin solution and films. Indeed, research program was the application of different methods of manufacturing approaches of the films of fibroin without the use of harsh treatments and organic solvents. New approaches to doping and chemical functionalization of the silk fibroin were studied. Two different methods have been identified: 1) biodoping that consists in the doping of fibroin with optically active molecules through the addition of fluorescent molecules in the standard diet used for the breeding of silkworms; 2) chemical functionalization via silylation.

Synthesis and characterization of hydroxyapatite modified with (9r)-9-hydroxystearic acid

(9R)-9-hydroxystearic acid (9R-HSA) has been proven to have antitumoral activity because it is shown to inhibit histone deacetylase 1, an enzyme which activates DNA replication, and the (R)-enantiomer has been shown to be more active than the (S)-enantiomer both in vitro and by molecular docking. Hydroxyapatite is the main mineral component of bone and teeth and has been used for over 20 years in prostheses and their coating because it is biocompatible and bioactive. The goal of incorporating 9R-HSA into hydroxyapatite is to have a material that combines the bioactivity of HA with the antitumoral properties of 9R-HSA. In this work, 9R-HSA and its potassium salt were synthesized and the latter was also incorporated into hydroxyapatite. The content of (R)-9-hydroxystearate ion incorporated into the apatitic structure was shown to be a function of its concentration in solution and can reach values higher than 8.5%. (9R)-9-hydroxystearic acid modified hydroxyapatite was extensively characterized to determine the effect of the incorporation of the organic molecule. This incorporation does not significantly alter the unit cell but reduces the size of both the crystals as well as the coherent domains, mainly along the a-axis of hydroxyapatite. This is believed to be due to the coordination of the negatively charged carboxylate group to the calcium ions which are more exposed on the (100) face of the crystal, therefore limiting the growth mainly in this direction. Further analyses showed that the material becomes hydrophobic and more negatively charged with the addition of 9R-HSA but both of these properties reach a plateau at less than 5% wt of 9R-HSA.

Accretion of Mass and Angular Momentum onto The Discs of Spiral Galaxies

In this Thesis, we study the accretion of mass and angular momentum onto the disc of spiral galaxies from a global and a local perspective and comparing theory predictions with several observational data. First, we propose a method to measure the specific mass and radial growth rates of stellar discs, based on their star formation rate density profiles and we apply it to a sample of nearby spiral galaxies. We find a positive radial growth rate for almost all galaxies in our sample. Our galaxies grow in size, on average, at one third of the rate at which they grow in mass. Our results are in agreement with theoretical expectations if known scaling relations of disc galaxies are not evolving with time. We also propose a novel method to reconstruct accretion profiles and the local angular momentum of the accreting material from the observed structural and chemical properties of spiral galaxies. Applied to the Milky Way and to one external galaxy, our analysis indicates that accretion occurs at relatively large radii and has a local deficit of angular momentum with respect to the disc. Finally, we show how structure and kinematics of hot gaseous coronae, which are believed to be the source of mass and angular momentum of massive spiral galaxies, can be reconstructed from their angular momentum and entropy distributions. We find that isothermal models with cosmologically motivated angular momentum distributions are compatible with several independent observational constraints. We also consider more complex baroclinic equilibria: we describe a new parametrization for these states, a new self-similar family of solution and a method for reconstructing structure and kinematics from the joint angular momentum/entropy distribution.