Atomic Force Microscopy Studies of the Amyloidogenic Processes of Intrinsically Unstructured Proteins related to Neurodegenerative Diseases

Protein aggregation and formation of insoluble aggregates in central nervous system is the main cause of neurodegenerative disease. Parkinson’s disease is associated with the appearance of spherical masses of aggregated proteins inside nerve cells called Lewy bodies. α-Synuclein is the main component of Lewy bodies. In addition to α-synuclein, there are more than a hundred of other proteins co-localized in Lewy bodies: 14-3-3η protein is one of them. In order to increase our understanding on the aggregation mechanism of α-synuclein and to study the effect of 14-3-3η on it, I addressed the following questions. (i) How α-synuclein monomers pack each other during aggregation? (ii) Which is the role of 14-3-3η on α-synuclein packing during its aggregation? (iii) Which is the role of 14-3-3η on an aggregation of α-synuclein “seeded” by fragments of its fibrils? In order to answer these questions, I used different biophysical techniques (e.g., Atomic force microscope (AFM), Nuclear magnetic resonance (NMR), Surface plasmon resonance (SPR) and Fluorescence spectroscopy (FS)).

Synthesis and characterization of Poly(vinylphosphonic acid) for proton exchange membranes in fuel cells

Vinylphosphonic acid (VPA) was polymerized at 80 ºC by free radical polymerization to give polymers (PVPA) of different molecular weight depending on the initiator concentration. The highest molecular weight, Mw, achieved was 6.2 x 104 g/mol as determined by static light scattering. High resolution nuclear magnetic resonance (NMR) spectroscopy was used to gain microstructure information about the polymer chain. Information based on tetrad probabilities was utilized to deduce an almost atactic configuration. In addition, 13C-NMR gave evidence for the presence of head-head and tail-tail links. Refined analysis of the 1H NMR spectra allowed for the quantitative determination of the fraction of these links (23.5 percent of all links). Experimental evidence suggested that the polymerization proceeded via cyclopolymerization of the vinylphosphonic acid anhydride as an intermediate. Titration curves indicated that high molecular weight poly(vinylphosphonic acid) PVPA behaved as a monoprotic acid. Proton conductors with phosphonic acid moieties as protogenic groups are promising due to their high charge carrier concentration, thermal stability, and oxidation resistivity. Blends and copolymers of PVPA have already been reported, but PVPA has not been characterized sufficiently with respect to its polymer properties. Therefore, we also studied the proton conductivity behaviour of a well-characterized PVPA. PVPA is a conductor; however, the conductivity depends strongly on the water content of the material. The phosphonic acid functionality in the resulting polymer, PVPA, undergoes condensation leading to the formation of phosphonic anhydride groups at elevated temperature. Anhydride formation was found to be temperature dependent by solid state NMR. Anhydride formation affects the proton conductivity to a large extent because not only the number of charge carriers but also the mobility of the charge carriers seems to change.

La diagnostica sonica nell'arboricoltura ornamentale

Il tomografo sonico è uno strumento di recente applicazione nell’analisi morfo-sintomatica delle alberature. Si tratta di uno strumento che sfrutta la propagazione delle onde sonore nel legno per determinarne la densità e le possibili alterazioni interne. Oltre all’applicazione su larga scala in un parco di Imola, per effettuare una valutazione approfondita di tutti gli esemplari, lo strumento è stato applicato per scopi diversi. In prima analisi è stato utilizzato per valutare stadi precoci di alterazione e l’evoluzione delle patologie interne nel tempo. Successivamente si voleva identificare il percorso di sostanze liquide iniettate con mezzi endoterapici nel tronco, attraverso l’applicazione di tomografia sonica sopra e sotto il punto di iniezione. In ultima analisi è stato effettuato un confronto tra tomografia sonica e risonanza magnetica nucleare per identificare patologie invisibili ai normali strumenti utilizzati nell’analisi della stabilità delle piante.

NMR spectroscopy and imaging of hyperpolarized gases

Since the discovery of the nuclear magnetic resonance (NMR) phenomenon, countless NMR techniques have been developed that are today indispensable tools in physics, chemistry, biology, and medicine. As one of the main obstacles in NMR is its notorious lack of sensitivity, different hyperpolarization (HP) methods have been established to increase signals up to several orders of magnitude. In this work, different aspects of magnetic resonance, using HP noble gases, are studied, hereby combining different disciplines of research. The first part examines new fundamental effects in NMR of HP gases, in theory and experiment. The spin echo phenomenon, which provides the basis of numerous modern experiments, is studied in detail in the gas phase. The changes of the echo signal in terms of amplitude, shape, and position, due to the fast translational motion, are described by an extension of the existing theory and computer simulations. With this knowledge as a prerequisite, the detection of intermolecular double-quantum coherences was accomplished for the first time in the gas phase. The second part of this thesis focuses on the development of a practical method to enhance the dissolution process of HP 129Xe, without loss of polarization or shortening of T1. Two different setups for application in NMR spectroscopy and magnetic resonance imaging (MRI) are presented. The continuous operation allows biological and multidimensional spectroscopy in solutions. Also, first in vitro MRI images with dissolved HP 129Xe as contrast agent were obtained at a clinical scanner.

Struktur-Dynamik-Beziehungen in amorphen festen Lösungen

Feste Lösungen homogen dispergierter Wirkstoffmoleküle in amorphen Polymermatrizen sind wichtige Materialien in vielen pharmazeutischen Anwendungen, bei denen eine kontrollierte Abgabe wasserunlöslicher Wirkstoffe in wässrige Systeme eine Rolle spielt. Die intermolekulare Bindungs-stärke zwischen Polymer- und Wirkstoffmolekülgruppen bestimmt die Stabilität der festen Lösung und steuert somit die biologische Aktivität der Wirkstoffmoleküle. In festen Lösungen, die aus acryl-säurehaltigen Copolymeren (Protonendonoren) und basischen Wirkstoffmolekülen (Protonenakzepto-ren) hergestellt werden, sind intermolekulare Wasserstoffbrücken zwischen den Systemkomponenten Triebkraft für die Bildung einer stabilen homogenen Dispersion und für die Entstehung struktureller Merkmale zwischen den Molekülgruppen der Systemkomponenten. Zudem ist die Bindungsstärke der Wasserstoffbrücken im Hinblick auf die kontrollierte Abgabe der Wirkstoffe von Bedeutung. Da dynamische chemische Gleichgewichte bei der Bildung der Wasserstoffbrücken eine wichtige Rolle spielen müssen neben strukturellen Parametern auch dynamische Faktoren beleuchtet werden. Ziel dieser Arbeit ist neben der Ermittlung von intermolekularen Bindungsstärken vor allem die Identifika-tion struktureller Verhältnisse zwischen den Systemkomponenten auf molekularer Ebene. Die Be-stimmung der Abhängigkeit dieser Parameter von der Struktur der verwendeten Polymere und einer Vielzahl weiterer Einflüsse wie z.B. Feuchtigkeit, Lagerdauer oder Wirkstoffkonzentration soll ein kontrolliertes Design fester Lösungen mit definierten anwendungsspezifischen Eigenschaften ermögli-chen. Temperaturabhängige 1H-Festkörper-MAS-NMR (Magic Angle Spinning Nuclear Magnetic Resonance) Experimente an festen Lösungen mit unterschiedlichen Copolymer-Zusammensetzungen weisen die Existenz dynamischer chemischer Gleichgewichte in den komplexen Wasserstoffbrücken-netzwerken nach. Veränderungen in der chemischen Verschiebung und in der Linienform der Reso-nanzlinien acider Protonen erlauben einen tiefen Einblick in die Architektur dieser Netzwerke und legen die Bindungsverhältnisse unter Berücksichtigung der Polymerchemie und der Mobilität der Systemkomponenten dar, wobei die Befunde mithilfe quantenchemischer Rechnungen untermauert werden können. Die Gegenwart acider Protonen ermöglicht einen einfachen 1H-2H-Austausch, wor-aufhin mithilfe rotorsynchronisierter temperaturabhängiger 2H-MAS-NMR Experimente die Wasser-stoffbrückenbindungsstärke bestimmt werden kann. Mit 1H-1H-Korrelationsexperimenten (Doppelquantenspektroskopie) stehen Methoden für die Bestimmung homonuklearer dipolarer 1H-1H-Kopplungen zur Verfügung, die strukturelle Aussagen aufgrund von bevorzugten räumlichen Kontak-ten bestimmter Molekülgruppen ermöglichen. Weiterhin können diese Experimente verwendet werden, um Wasserstoffbrücken zwischen Polymergruppen von Polymer-Wirkstoff-Wasserstoffbrücken zu unterscheiden, wodurch eine quantitative Beschreibung des Bindungsnetzwerks und der Konkurrenz-prozesse zwischen den einzelnen wasserstoffverbrückten Spezies ermöglicht wird. Eine Kristallisation der Wirkstoffmoleküle ist in vielen Anwendungen unerwünscht, da sie die biologische Verfügbarkeit des Wirkstoffs reduzieren. Mit 1H-Festkörper-MAS-NMR Experimenten können kristalline von amorph dispergierten Wirkstoffmolekülen unterschieden werden, wodurch eine Quantifizierung der Destabilisierungsprozesse ermöglicht wird, die durch Exposition der festen Lösungen mit Wasserdampf ausgelöst werden können. Die Zeit- und Konzentrationsabhängigkeit der Wasseraufnahme kann mit NMR-Experimenten verfolgt werden, wobei unterschiedlich mobile Was-serspezies an unterschiedlichen Bindungsorten identifiziert werden können, was zum molekularen Verständnis der Destabilisierungsprozesse beiträgt. Zusätzlich wird die Mobilität der Wirkstoffmole-küle bestimmt, die sich – wie auch die Wirkstoffkonzentration - als wichtige Größe in der Beschrei-bung der Destabilisierung erweist. Aufbauend auf den Beobachtungen wird ein Zusammenhang zwischen der Copolymerzusammensetzung und einer kritischen Wirkstoffkonzentration hergestellt, der für die Anwendungen amorpher fester Lösungen in biologischen Systemen von großer Bedeutung ist.

Anhydrous proton conducting polymer electrolytes based on polymeric ionic liquids

Imidazolium types of ionic liquids were immobilized by tethering it to acrylate backbone. These imidazolium salt containing acrylate monomers were polymerize at 70oC by free radical polymerization to give polymers poly(AcIm-n) with n being the side chain lenght. The chemical structure of the polymer electrolytes obtained by the described synthetic routes was investigated by NMR-spectroscopy. The polymers were doped with various amounts of H3PO4 and LiN(SO2CF3)2, to obtain poly(AcIm-n) x H3PO4 and poly(AcIm-2-Li) x LiN(SO2CF3)2. The TG curves show that the polymer electrolytes are thermally stable up to about 200◦C. DSC results indicates the softening effect of the length of the spacers (n) as well as phosphoric acid. The proton conductivity of the samples increase with x and reaches to 10-2 Scm-1 at 120oC for both poly(AcIm-2)2H3PO4 and poly(AcIm-6)2H3PO4. It was observed that the lithium ion conductivity of the poly(AcIm-2-Li) x LiN(SO2CF3)2 increases with blends (x) up to certain composition and then leveled off independently from blend content. The conductivity reaches to about 10-5 S cm-1 at 30oC and 10-3 at 100oC for poly(AcIm-2-Li) x LiN(SO2CF3)2 where x is 10. The phosphate and phosphoric acid functionality in the resulting polymers, poly(AcIm-n) x H3PO4, undergoes condensation leading to the formation of cross-linked materials at elevated temperature which may improve the mechanical properties to be used as membrane materials in fuel cells. High resolution nuclear magnetic resonance (NMR) spectroscopy was used to obtain information about hydrogen bonding in solids. The low Tg enhances molecular mobility and this leads to better resolved resonances in both the backbone region and side chain region. The mobile and immobile protons can be distinguished by comparing 1H MAS and 1H-DQF NMR spectra. The interaction of the protons which may contribute to the conductivity is observed from the 2D double quantum correlation (DQC) spectra.

Large-scale coupled-cluster calculations

Coupled-cluster theory provides one of the most successful concepts in electronic-structure theory. This work covers the parallelization of coupled-cluster energies, gradients, and second derivatives and its application to selected large-scale chemical problems, beside the more practical aspects such as the publication and support of the quantum-chemistry package ACES II MAB and the design and development of a computational environment optimized for coupled-cluster calculations. The main objective of this thesis was to extend the range of applicability of coupled-cluster models to larger molecular systems and their properties and therefore to bring large-scale coupled-cluster calculations into day-to-day routine of computational chemistry. A straightforward strategy for the parallelization of CCSD and CCSD(T) energies, gradients, and second derivatives has been outlined and implemented for closed-shell and open-shell references. Starting from the highly efficient serial implementation of the ACES II MAB computer code an adaptation for affordable workstation clusters has been obtained by parallelizing the most time-consuming steps of the algorithms. Benchmark calculations for systems with up to 1300 basis functions and the presented applications show that the resulting algorithm for energies, gradients and second derivatives at the CCSD and CCSD(T) level of theory exhibits good scaling with the number of processors and substantially extends the range of applicability. Within the framework of the ’High accuracy Extrapolated Ab initio Thermochemistry’ (HEAT) protocols effects of increased basis-set size and higher excitations in the coupled- cluster expansion were investigated. The HEAT scheme was generalized for molecules containing second-row atoms in the case of vinyl chloride. This allowed the different experimental reported values to be discriminated. In the case of the benzene molecule it was shown that even for molecules of this size chemical accuracy can be achieved. Near-quantitative agreement with experiment (about 2 ppm deviation) for the prediction of fluorine-19 nuclear magnetic shielding constants can be achieved by employing the CCSD(T) model together with large basis sets at accurate equilibrium geometries if vibrational averaging and temperature corrections via second-order vibrational perturbation theory are considered. Applying a very similar level of theory for the calculation of the carbon-13 NMR chemical shifts of benzene resulted in quantitative agreement with experimental gas-phase data. The NMR chemical shift study for the bridgehead 1-adamantyl cation at the CCSD(T) level resolved earlier discrepancies of lower-level theoretical treatment. The equilibrium structure of diacetylene has been determined based on the combination of experimental rotational constants of thirteen isotopic species and zero-point vibrational corrections calculated at various quantum-chemical levels. These empirical equilibrium structures agree to within 0.1 pm irrespective of the theoretical level employed. High-level quantum-chemical calculations on the hyperfine structure parameters of the cyanopolyynes were found to be in excellent agreement with experiment. Finally, the theoretically most accurate determination of the molecular equilibrium structure of ferrocene to date is presented.

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.

Spectroscopic properties from hybrid QM, MM molecular dynamics simulation

The central aim of this thesis work is the application and further development of a hybrid quantum mechanical/molecular mechanics (QM/MM) based approach to compute spectroscopic properties of molecules in complex chemical environments from electronic structure theory. In the framework of this thesis, an existing density functional theory implementation of the QM/MM approach is first used to calculate the nuclear magnetic resonance (NMR) solvent shifts of an adenine molecule in aqueous solution. The findings show that the aqueous solvation with its strongly fluctuating hydrogen bond network leads to specific changes in the NMR resonance lines. Besides the absolute values, also the ordering of the NMR lines changes under the influence of the solvating water molecules. Without the QM/MM scheme, a quantum chemical calculation could have led to an incorrect assignment of these lines. The second part of this thesis describes a methodological improvement of the QM/MM method that is designed for cases in which a covalent chemical bond crosses the QM/MM boundary. The development consists in an automatized protocol to optimize a so-called capping potential that saturates the electronic subsystem in the QM region. The optimization scheme is capable of tuning the parameters in such a way that the deviations of the electronic orbitals between the regular and the truncated (and "capped") molecule are minimized. This in turn results in a considerable improvement of the structural and spectroscopic parameters when computed with the new optimized capping potential within the QM/MM technique. This optimization scheme is applied and benchmarked on the example of truncated carbon-carbon bonds in a set of small test molecules. It turns out that the optimized capping potentials yield an excellent agreement of NMR chemical shifts and protonation energies with respect to the corresponding full molecules. These results are very promising, so that the application to larger biological complexes will significantly improve the reliability of the prediction of the related spectroscopic properties.

Development of methodologies for fish freshness assessment using metabonomics applications.

This study focuses on the use of metabonomics applications in measuring fish freshness in various biological species and in evaluating how they are stored. This metabonomic approach is innovative and is based upon molecular profiling through nuclear magnetic resonance (NMR). On one hand, the aim is to ascertain if a type of fish has maintained, within certain limits, its sensory and nutritional characteristics after being caught; and on the second, the research observes the alterations in the product’s composition. The spectroscopic data obtained through experimental nuclear magnetic resonance, 1H-NMR, of the molecular profiles of the fish extracts are compared with those obtained on the same samples through analytical and conventional methods now in practice. These second methods are used to obtain chemical indices of freshness through biochemical and microbial degradation of the proteic nitrogen compounds and not (trimethylamine, N-(CH3)3, nucleotides, amino acids, etc.). At a later time, a principal components analysis (PCA) and a linear discriminant analysis (PLS-DA) are performed through a metabonomic approach to condense the temporal evolution of freshness into a single parameter. In particular, the first principal component (PC1) under both storage conditions (4 °C and 0 °C) represents the component together with the molecular composition of the samples (through 1H-NMR spectrum) evolving during storage with a very high variance. The results of this study give scientific evidence supporting the objective elements evaluating the freshness of fish products showing those which can be labeled “fresh fish.”

Solid-state NMR investigation of phosphonic acid based proton conducting materials

In this work, new promising proton conducting fuel cell membrane materials were characterized in terms of their structure and dynamic properties using solid-state nuclear magnetic resonance (NMR) spectroscopy and X-ray diffraction. Structurally different, phosphonic acid (PA) containing materials were systematically evaluated for possible high-temperature operation (e.g. at T>100°C). Notably, 1H, 2H and 31P magic angle spinning (MAS) NMR provided insight into local connectivities and dynamics of the hydrogen bonded network, while packing arrangements were identified by means of heteronuclear dipolar recoupling techniques.rnThe first part of this work introduced rather crystalline, low molecular weight ionomers for proton conducting membranes, where six different geometries such as line, triangle, screw, tetrahedron, square and hexagon, were investigated. The hexagon was identified as the most promising geometry with high-temperature bulk proton conductivities in the range of 10-3 Scm-1 at a relative humidity of 50%. However, 2H NMR and TGA-MS data suggest that the bulk proton transport is mainly due to the presence of crystal water. Single crystal X-ray data revealed that in the tetrahedron phosphonic acids form tetrameric clusters isolating the mobile protons while the phosphonic acids in the hexagon form zigzag-type pathways through the sample.rnThe second part of this work demonstrates how acid-base pairing and the choice of appropriate spacers may influence proton conduction. Different ratios of statistical copolymers of poly (vinylphosphonic acid) and poly (4-vinylpyridine) were measured to derive information about the local structure and chemical changes. Though anhydrous proton conductivities of all statistical copolymers are rather poor, the conductivity increases to 10-2 S cm-1 when exposing the sample to relative humidity of 80%. In contrast to PVPA, anhydride formation of phosphonic acids in the copolymer is not reversible even when exposing the sample to a relative humidity of 100%.rnIn addition, the influence of both spacers and degree of backbone crystallinity on bulk proton conductivity was investigated. Unlike in systems such as poly benzimidazole (PBI), spacers were inserted between the protogenic groups along the backbone. It was found that dilution of the protogenic groups decreases the conductivity, but compared to PVPA, similar apparent activation energies for local motions were obtained from both variable temperature 1H NMR and impedance spectroscopy data. These observations suggest the formation of phosphonic acid clusters with high degrees of local proton motion, where only a fraction of motions contribute to the observable bulk proton conductivity. Additionally, it was shown that gradual changes of the spacer length lead to different morphologies.rnIn summary, applying advanced solid-state NMR and X-ray analysis, structural and dynamic phenomena in proton conducting materials were identified on a molecular level. The results were discussed with respect to different proton conduction mechanisms and may contribute to a more rational design or improvement of proton conducting membranes.rn

Sensitivity enhancement in NMR by using parahydrogen induced polarization

Enhancing the sensitivity of nuclear magnetic resonance measurements via hyperpolarization techniques like parahydrogen induced polarization (PHIP) is of high interest for spectroscopic investigations. Parahydrogen induced polarization is a chemical method, which makes use of the correlation between nuclear spins in parahydrogen to create hyperpolarized molecules. The key feature of this technique is the pairwise and simultaneous transfer of the two hydrogen atoms of parahydrogen to a double or triple bond resulting in a population of the Zeeman energy levels different from the Boltzmann equation. The obtained hyperpolarization results in antiphase peaks in the NMR spectrum with high intensities. Due to these strong NMR signals, this method finds arnlot of applications in chemistry e.g. the characterization of short-lived reaction intermediates. Also in medicine it opens up the possibility to boost the sensitivity of medical diagnostics via magnetic labeling of active contrast agents. Thus, further examination and optimization of the PHIP technique is of significant importance in order to achieve the highest possible sensitivity gain.rnrnIn this work, different aspects concerning PHIP were studied with respect to its chemical and spectroscopic background. The first part of this work mainly focused on optimizing the PHIP technique by investigating different catalyst systems and developing new setups for the parahydrogenation. Further examinations facilitated the transfer of the generated polarization from the protons to heteronuclei like 13C. The second part of this thesis examined the possibility to transfer these results to different biologically active compounds to enable their later application in medical diagnostics. Onerngroup of interesting substances is represented by metabolites or neurotransmitters in mammalian cells. Other interesting substances are clinically relevant drugs like a barbituric acid derivative or antidepressant drugs like citalopram which were investigated with regard to their applicability for the PHIP technique and the possibility to achievernpolarization transfer to 13C nuclei. The last investigated substrate is a polymerizable monomer whose polymer was used as a blood plasma expander for trauma victims after the first half of the 20th century. In this case, the utility of the monomer for the PHIP technique as a basis for later investigations of a polymerization reaction using hyperpolarized monomers was examined.rnrnHence, this thesis covers the optimization of the PHIP technology, hereby combining different fields of research like chemical and spectroscopical aspects, and transfers the results to applications of real biologally acitve compounds.

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.

Studies of degradation of organic solar cell materials using electrical scanning probe microscopy

Intense research is being done in the field of organic photovoltaics in order to synthesize low band-gap organic molecules. These molecules are electron donors which feature in combination with acceptor molecules, typically fullerene derivarntives, forming an active blend. This active blend has phase separated bicontinuous morphology on a nanometer scale. The highest recorded power conversionrnefficiencies for such cells have been 10.6%. Organic semiconductors differ from inorganic ones due to the presence of tightly bonded excitons (electron-hole pairs)resulting from their low dielectric constant (εr ≈2-4). An additional driving force is required to separate such Frenkel excitons since their binding energy (0.3-1 eV) is too large to be dissociated by an electric field alone. This additional driving force arises from the energy difference between the lowest unoccupied molecular orbital (LUMO) of the donor and the acceptor materials. Moreover, the efficiency of the cells also depends on the difference between the highest occupied molecular orbital (HOMO) of the donor and LUMO of the acceptor. Therefore, a precise control and estimation of these energy levels are required. Furthermore any external influences that change the energy levels will cause a degradation of the power conversion efficiency of organic solar cell materials. In particular, the role of photo-induced degradation on the morphology and electrical performance is a major contribution to degradation and needs to be understood on a nanometer scale. Scanning Probe Microscopy (SPM) offers the resolution to image the nanometer scale bicontinuous morphology. In addition SPM can be operated to measure the local contact potential difference (CPD) of materials from which energy levels in the materials can be derived. Thus SPM is an unique method for the characterization of surface morphology, potential changes and conductivity changes under operating conditions. In the present work, I describe investigations of organic photovoltaic materials upon photo-oxidation which is one of the major causes of degradation of these solar cell materials. SPM, Nuclear Magnetic Resonance (NMR) and UV-Vis spectroscopy studies allowed me to identify the chemical reactions occurring inside the active layer upon photo-oxidation. From the measured data, it was possible to deduce the energy levels and explain the various shifts which gave a better understanding of the physics of the device. In addition, I was able to quantify the degradation by correlating the local changes in the CPD and conductivity to the device characteristics, i.e., open circuit voltage and short circuit current. Furthermore, time-resolved electrostatic force microscopy (tr-EFM) allowed us to probe dynamic processes like the charging rate of the individual donor and acceptor domains within the active blend. Upon photo-oxidation, it was observed, that the acceptor molecules got oxidized first preventing the donor polymer from degrading. Work functions of electrodes can be tailored by modifying the interface with monomolecular thin layers of molecules which are made by a chemical reaction in liquids. These modifications in the work function are particularly attractive for opto-electronic devices whose performance depends on the band alignment between the electrodes and the active material. In order to measure the shift in work function on a nanometer scale, I used KPFM in situ, which means in liquids, to follow changes in the work function of Au upon hexadecanethiol adsorption from decane. All the above investigations give us a better understanding of the photo-degradation processes of the active material at the nanoscale. Also, a method to compare various new materials used for organic solar cells for stability is proposed which eliminates the requirement to make fully functional devices saving time and additional engineering efforts.

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.