Elemetary processes of radiation damage in organic molecules of biological interest

It was observed in the ‘80s that the radiation damage on biological systems strongly depends on processes occurring at the microscopic level, involving the elementary constituents of biological cells. Since then, lot of attention has been paid to study elementary processes of photo- and ion-chemistry of isolated organic molecule of biological interest. This work fits in this framework and aims to study the radiation damage mechanisms induced by different types of radiations on simple halogenated biomolecules used as radiosensitizers in radiotherapy. The research is focused on the photofragmentation of halogenated pyrimidine molecules (5Br-pyrimidine, 2Br-pyrimidine and 2Cl-pyrimidine) in the VUV range and on the 12C4+ ion-impact fragmentation of the 5Br-uracil and its homogeneous and hydrated clusters. Although halogen substituted pyrimidines have similar structure to the pyrimidine molecule, their photodissociation dynamics is quite different. These targets have been chosen with the purpose of investigating the effect of the specific halogen atom and site of halogenation on the fragmentation dynamics. Theoretical and experimental studies have highlighted that the site of halogenation and the type of halogen atom, lead either to the preferential breaking of the pyrimidinic ring or to the release of halogen/hydrogen radicals. The two processes can subsequently trigger different mechanisms of biological damage. To understand the effect of the environment on the fragmentation dynamic of the single molecule, the ion-induced fragmentation of homogenous and hydrated clusters of 5Br-uracil have been studied and compared to similar studies on the isolated molecule. The results show that the “protective effect” of the environment on the single molecule hold in the homogeneous clusters, but not in the hydrated clusters, where several hydrated fragments have been observed. This indicates that the presence of water molecules can inhibit some fragmentation channels and promote the keto-enol tautomerization, which is very important in the mutagenesis of the DNA.

Computational analyses on the structure-function relation in ion channels

Ion channels are protein molecules, embedded in the lipid bilayer of the cell membranes. They act as powerful sensing elements switching chemicalphysical stimuli into ion-fluxes. At a glance, ion channels are water-filled pores, which can open and close in response to different stimuli (gating), and one once open select the permeating ion species (selectivity). They play a crucial role in several physiological functions, like nerve transmission, muscular contraction, and secretion. Besides, ion channels can be used in technological applications for different purpose (sensing of organic molecules, DNA sequencing). As a result, there is remarkable interest in understanding the molecular determinants of the channel functioning. Nowadays, both the functional and the structural characteristics of ion channels can be experimentally solved. The purpose of this thesis was to investigate the structure-function relation in ion channels, by computational techniques. Most of the analyses focused on the mechanisms of ion conduction, and the numerical methodologies to compute the channel conductance. The standard techniques for atomistic simulation of complex molecular systems (Molecular Dynamics) cannot be routinely used to calculate ion fluxes in membrane channels, because of the high computational resources needed. The main step forward of the PhD research activity was the development of a computational algorithm for the calculation of ion fluxes in protein channels. The algorithm - based on the electrodiffusion theory - is computational inexpensive, and was used for an extensive analysis on the molecular determinants of the channel conductance. The first record of ion-fluxes through a single protein channel dates back to 1976, and since then measuring the single channel conductance has become a standard experimental procedure. Chapter 1 introduces ion channels, and the experimental techniques used to measure the channel currents. The abundance of functional data (channel currents) does not match with an equal abundance of structural data. The bacterial potassium channel KcsA was the first selective ion channels to be experimentally solved (1998), and after KcsA the structures of four different potassium channels were revealed. These experimental data inspired a new era in ion channel modeling. Once the atomic structures of channels are known, it is possible to define mathematical models based on physical descriptions of the molecular systems. These physically based models can provide an atomic description of ion channel functioning, and predict the effect of structural changes. Chapter 2 introduces the computation methods used throughout the thesis to model ion channels functioning at the atomic level. In Chapter 3 and Chapter 4 the ion conduction through potassium channels is analyzed, by an approach based on the Poisson-Nernst-Planck electrodiffusion theory. In the electrodiffusion theory ion conduction is modeled by the drift-diffusion equations, thus describing the ion distributions by continuum functions. The numerical solver of the Poisson- Nernst-Planck equations was tested in the KcsA potassium channel (Chapter 3), and then used to analyze how the atomic structure of the intracellular vestibule of potassium channels affects the conductance (Chapter 4). As a major result, a correlation between the channel conductance and the potassium concentration in the intracellular vestibule emerged. The atomic structure of the channel modulates the potassium concentration in the vestibule, thus its conductance. This mechanism explains the phenotype of the BK potassium channels, a sub-family of potassium channels with high single channel conductance. The functional role of the intracellular vestibule is also the subject of Chapter 5, where the affinity of the potassium channels hEag1 (involved in tumour-cell proliferation) and hErg (important in the cardiac cycle) for several pharmaceutical drugs was compared. Both experimental measurements and molecular modeling were used in order to identify differences in the blocking mechanism of the two channels, which could be exploited in the synthesis of selective blockers. The experimental data pointed out the different role of residue mutations in the blockage of hEag1 and hErg, and the molecular modeling provided a possible explanation based on different binding sites in the intracellular vestibule. Modeling ion channels at the molecular levels relates the functioning of a channel to its atomic structure (Chapters 3-5), and can also be useful to predict the structure of ion channels (Chapter 6-7). In Chapter 6 the structure of the KcsA potassium channel depleted from potassium ions is analyzed by molecular dynamics simulations. Recently, a surprisingly high osmotic permeability of the KcsA channel was experimentally measured. All the available crystallographic structure of KcsA refers to a channel occupied by potassium ions. To conduct water molecules potassium ions must be expelled from KcsA. The structure of the potassium-depleted KcsA channel and the mechanism of water permeation are still unknown, and have been investigated by numerical simulations. Molecular dynamics of KcsA identified a possible atomic structure of the potassium-depleted KcsA channel, and a mechanism for water permeation. The depletion from potassium ions is an extreme situation for potassium channels, unlikely in physiological conditions. However, the simulation of such an extreme condition could help to identify the structural conformations, so the functional states, accessible to potassium ion channels. The last chapter of the thesis deals with the atomic structure of the !- Hemolysin channel. !-Hemolysin is the major determinant of the Staphylococcus Aureus toxicity, and is also the prototype channel for a possible usage in technological applications. The atomic structure of !- Hemolysin was revealed by X-Ray crystallography, but several experimental evidences suggest the presence of an alternative atomic structure. This alternative structure was predicted, combining experimental measurements of single channel currents and numerical simulations. This thesis is organized in two parts, in the first part an overview on ion channels and on the numerical methods adopted throughout the thesis is provided, while the second part describes the research projects tackled in the course of the PhD programme. The aim of the research activity was to relate the functional characteristics of ion channels to their atomic structure. In presenting the different research projects, the role of numerical simulations to analyze the structure-function relation in ion channels is highlighted.

Mass transport in polymers

The study of mass transport in polymeric membranes has grown in importance due to its potential application in many processes such as separation of gases and vapors, packaging, controlled drug release. The diffusion of a low molecular weight species in a polymer is often accompanied by other phenomena like swelling, reactions, stresses, that have not been investigated in all their aspects yet. Furthermore, novel materials have been developed that include inorganic fillers, reactive functional groups or ions, that make the scenery even more complicated. The present work focused on the experimental study of systems where the diffusion is accompanied by other processes; suitable models were also developed to describe the particular circumstances in order to understand the underlying concepts and be able to design the performances of the material. The effect of solvent-induced deformation in polymeric films during sorption processes was studied since the dilation, especially in constrained membranes, can cause the development of stresses and therefore early failures of the material. The bending beam technique was used to test the effects of the dilation and the stress induced in the polymer by penetrant diffusion. A model based on the laminate theory was developed that accounts for the swelling and is able to predict the stress that raise in the material. The addition of inorganic fillers affects the transport properties of polymeric films. Mixed matrix membranes based on fluorinated, high free volume matrices show attractive performances for separation purposes but there is a need for deeper investigation of the selectivity properties towards gases and vapors. A new procedure based on the NELF model was tested on the experimental data; it allows to predict solubility of every penetrant on the basis of data for one vapor. The method has proved to be useful also for the determination of the diffusion coefficient and for an estimation of the permeability in the composite materials. Oxygen scavenging systems can overcome lack of barrier properties in common polymers that forbids their application in sensitive applications as food packaging. The final goal of obtaining a membrane almost impermeable to oxygen leads to experimental times out of reach. Hence, a simple model was developed in order to describe the transport of oxygen in a membrane with also reactive groups and analyze the experimental data collected on SBS copolymers that show attractive scavenging capacity. Furthermore, a model for predicting the oxygen barrier behavior of a film formed as a blend of OSP in a common packaging material was built, considering particles capable of reactions with oxygen embedded in a non-reactive matrix. Perfluorosulphonic acid ionomers (PFSI) are capturing attention due to a high thermal and chemical resistance coupled with very peculiar transport properties, that make them appropriate to be used in fuel cells. The possible effect of different formation procedure was studied together with the swelling due to water sorption since both water uptake and dilation can dramatically affect the fuel cells performances. The water diffusion and sorption was studied with a FTIR-ATR spectrometer that can give deeper information on the bonds between water molecules and the sulphonic hydrophilic groups and, therefore, on the microstructure of the hydrated ionomer.

Caratterizzazione di membrane metalliche e ionomeriche per applicazioni energetiche

The work of this thesis has been focused on the characterization of metallic membranes for the hydrogen purification from steam reforming process and also of perfluorosulphonic acid ionomeric (PFSI) membranes suitable as electrolytes in fuel cell applications. The experimental study of metallic membranes was divided in three sections: synthesis of palladium and silver palladium coatings on porous ceramic support via electroless deposition (ELD), solubility and diffusivity analysis of hydrogen in palladium based alloys (temperature range between 200 and 400 °C up to 12 bar of pressure) and permeation experiments of pure hydrogen and mixtures containing, besides hydrogen, also nitrogen and methane at high temperatures (up to 600 °C) and pressures (up to 10 bar). Sequential deposition of palladium and silver on to porous alumina tubes by ELD technique was carried out using two different procedures: a stirred batch and a continuous flux method. Pure palladium as well as Pd-Ag membranes were produced: the Pd-Ag membranes’ composition is calculated to be close to 77% Pd and 23% Ag by weight which was the target value that correspond to the best performance of the palladium-based alloys. One of the membranes produced showed an infinite selectivity through hydrogen and relatively high permeability value and is suitable for the potential use as a hydrogen separator. The hydrogen sorption in silver palladium alloys was carried out in a gravimetric system on films produced by ELD technique. In the temperature range inspected, up to 400°C, there is still a lack in literature. The experimental data were analyzed with rigorous equations allowing to calculate the enthalpy and entropy values of the Sieverts’ constant; the results were in very good agreement with the extrapolation made with literature data obtained a lower temperature (up to 150 °C). The information obtained in this study would be directly usable in the modeling of hydrogen permeation in Pd-based systems. Pure and mixed gas permeation tests were performed on Pd-based hydrogen selective membranes at operative conditions close to steam-reforming ones. Two membranes (one produced in this work and another produced by NGK Insulators Japan) showed a virtually infinite selectivity and good permeability. Mixture data revealed the existence of non negligible resistances to hydrogen transport in the gas phase. Even if the decrease of the driving force due to polarization concentration phenomena occurs, in principle, in all membrane-based separation systems endowed with high perm-selectivity, an extensive experimental analysis lack, at the moment, in the palladium-based membrane process in literature. Moreover a new procedure has been introduced for the proper comparison of the mass transport resistance in the gas phase and in the membrane. Another object of study was the water vapor sorption and permeation in PFSI membranes with short and long side chains was also studied; moreover the permeation of gases (i.e. He, N2 and O2) in dry and humid conditions was considered. The water vapor sorption showed strong interactions between the hydrophilic groups and the water as revealed from the hysteresis in the sorption-desorption isotherms and thermo gravimetric analysis. The data obtained were used in the modeling of water vapor permeation, that was described as diffusion-reaction of water molecules, and in the humid gases permeation experiments. In the dry gas experiments the permeability and diffusivity was found to increase with temperature and with the equivalent weight (EW) of the membrane. A linear correlation was drawn between the dry gas permeability and the opposite of the equivalent weight of PFSI membranes, based on which the permeability of pure PTFE is retrieved in the limit of high EW. In the other hand O2 ,N2 and He permeability values was found to increase significantly, and in a similar fashion, with water activity. A model that considers the PFSI membrane as a composite matrix with a hydrophilic and a hydrophobic phase was considered allowing to estimate the variation of gas permeability with relative humidity on the basis of the permeability in the dry PFSI membrane and in pure liquid water.

Theoretical studies of microfluidics: "walking" drops and "melting" snowflakes.

The present PhD thesis summarizes two examples of research in microfluidics. Both times water was the subject of interest, once in the liquid state (droplets adsorbed on chemically functionalized surfaces), the other time in the solid state (ice snowflakes and their fractal behaviour). The first problem deals with a slipping nano-droplet of water adsorbed on a surface with photo-switchable wettability characteristics. Main focus was on identifying the underlying driving forces and mechanical principles at the molecular level of detail. Molecular Dynamics simulation was employed as investigative tool owing to its record of successfully describing the microscopic behaviour of liquids at interfaces. To reproduce the specialized surface on which a water droplet can effectively “walk”, a new implicit surface potential was developed. Applying this new method the experimentally observed droplet slippage could be reproduced successfully. Next the movement of the droplet was analyzed at various conditions emphasizing on the behaviour of the water molecules in contact with the surface. The main objective was to identify driving forces and molecular mechanisms underlying the slippage process. The second part of this thesis is concerned with theoretical studies of snowflake melting. In the present work snowflakes are represented by filled von Koch-like fractals of mesoscopic beads. A new algorithm has been developed from scratch to simulate the thermal collapse of fractal structures based on Monte Carlo and Random Walk Simulations (MCRWS). The developed method was applied and compared to Molecular Dynamics simulations regarding the melting of ice snowflake crystals and new parameters were derived from this comparison. Bigger snow-fractals were then studied looking at the time evolution at different temperatures again making use of the developed MCRWS method. This was accompanied by an in-depth analysis of fractal properties (border length and gyration radius) in order to shed light on the dynamics of the melting process.

The coupling between electron transfer and Protein/Solvent Dynamics in Photosynthetic Reaction Centers: Spectroscopic Studies in Amorphous Matrices

We investigated at the molecular level protein/solvent interactions and their relevance in protein function through the use of amorphous matrices at room temperature. As a model protein, we used the bacterial photosynthetic reaction center (RC) of Rhodobacter sphaeroides, a pigment protein complex which catalyzes the light-induced charge separation initiating the conversion of solar into chemical energy. The thermal fluctuations of the RC and its dielectric conformational relaxation following photoexcitation have been probed by analyzing the recombination kinetics of the primary charge-separated (P+QA-) state, using time resolved optical and EPR spectroscopies. We have shown that the RC dynamics coupled to this electron transfer process can be progressively inhibited at room temperature by decreasing the water content of RC films or of RC-trehalose glassy matrices. Extensive dehydration of the amorphous matrices inhibits RC relaxation and interconversion among conformational substates to an extent comparable to that attained at cryogenic temperatures in water-glycerol samples. An isopiestic method has been developed to finely tune the hydration level of the system. We have combined FTIR spectral analysis of the combination and association bands of residual water with differential light-minus-dark FTIR and high-field EPR spectroscopy to gain information on thermodynamics of water sorption, and on structure/dynamics of the residual water molecules, of protein residues and of RC cofactors. The following main conclusions were reached: (i) the RC dynamics is slaved to that of the hydration shell; (ii) in dehydrated trehalose glasses inhibition of protein dynamics is most likely mediated by residual water molecules simultaneously bound to protein residues and sugar molecules at the protein-matrix interface; (iii) the local environment of cofactors is not involved in the conformational dynamics which stabilizes the P+QA-; (iv) this conformational relaxation appears to be rather delocalized over several aminoacidic residues as well as water molecules weakly hydrogen-bonded to the RC.

Contribution of Non-Covalent Interactions and Electronic Effects on the Conformational Landscape and Tautomeric Equilibria of Molecules and Molecular Complexes: Structural and Dynamical Data from Rotational Spectroscopy

This thesis concerns the study of complex conformational surfaces and tautomeric equilibria of molecules and molecular complexes by quantum chemical methods and rotational spectroscopy techniques. In particular, the focus of this research is on the effects of substitution and noncovalent interactions in determining the energies and geometries of different conformers, tautomers or molecular complexes. The Free-Jet Absorption Millimeter Wave spectroscopy and the Pulsed-Jet Fourier Transform Microwave spectroscopy have been applied to perform these studies and the obtained results showcase the suitability of these techniques for the study of conformational surfaces and intermolecular interactions. The series of investigations of selected medium-size molecules and complexes have shown how different instrumental setups can be used to obtain a variety of results on molecular properties. The systems studied, include molecules of biological interest such as anethole and molecules of astrophysical interest such as N-methylaminoethanol. Moreover halogenation effects have been investigated on halogen substituted tautomeric systems (5-chlorohydroxypyridine and 6-chlorohydroxypyridine), where it has shown that the position of the inserted halogen atom affects the prototropic equilibrium. As for fluorination effects, interesting results have been achieved investigating some small complexes where a molecule of water is used as a probe to reveal the changes on the electrostatic potential of different fluorinated compounds: 2-fluoropyridine, 3-fluoropyridine and penta-fluoropyridine. While in the case of the molecular complex between water and 2-fluoropyridine and 3-fluoropyridine the geometry of the complex with one water molecule is analogous to that of pyridine with the water molecule linked to the pyridine nitrogen, the case of pentafluoropyridine reveals the effect of perfluorination and the water oxygen points towards the positive center of the pyridine ring. Additional molecular adducts with a molecule of water have been analyzed (benzylamine-water and acrylic acid-water) in order to reveal the stabilizing driving forces that characterize these complexes.

A glassful of graphene: graphene-based materials for drinking water remediation

Modern world suffers from an intense water crisis. Emerging contaminants represent one of the most concerning elements of this issue. Substances, molecules, ions, and microorganisms take part in this vast and variegated class of pollutants, which main characteristic is to be highly resistant to traditional water purification technologies. An intense international research effort is being carried out in order to find new and innovative solutions to this problem, and graphene-based materials are one of the most promising options. Graphene oxide (GO) is a nanostructured material where domains populated by oxygenated groups alternate with interconnected areas of sp2 hybridized carbon atoms, on the surface of a one-atom thick nanosheets. GO can adsorb a great number of molecules and ions on its surface, thanks to the variety of different interactions that it can express, such as hydrogen bonding, p-p stacking, and electrostatic and hydrophobic interaction. These characteristics, added to the high superficial area, make it an optimal material for the development of innovative materials for drinking water remediation. The main concern in the use of GO in this field is to avoid secondary contaminations (i.e. GO itself must not become a pollutant). This issue can be faced through the immobilization of GO onto polymeric substrates, thus developing composite materials. The use of micro/ultrafiltration polymeric hollow fibers as substrates allows the design of adsorptive membranes, meaning devices that can perform filtration and adsorption simultaneously. In this thesis, two strategies for the development of adsorptive membranes were investigated: a core-shell strategy, where hollow fibers are coated with GO, and a coextrusion strategy, where GO is embedded in the polymeric matrix of the fibers. The so-obtained devices were exploited for both fundamental studies (i.e. molecular and ionic behaviour in between GO nanosheets) and real applications (the coextruded material is now at TRL 9).

Microwave and millimeter-wave spectroscopy of sulfur-containing molecules

The rotational spectroscopy of several sulfur bearing molecules and their 1:1 water complex, cysteamine, cysteamine monohydrate, 1-thioglycerol and 1-propanethiol were studied in the micro-wave and (or) millimeter-wave range. Precise laboratory spectra and conformational information were obtained. For cysteamine, the conformational space (at the B3LYP-GD3(BJ)/Def2-TZVP level) and the measurement and analysis of its rotational spectra in the 6 - 18 and 59.6 - 120 GHz are reported. The hyperfine structure of the rotational spectra was observed and analyzed for the first time. Based on the measured spectra, a search of the different conformers of cysteamine was performed toward the G+0.693-0.027 molecular cloud. We computed the upper limit of the ratio of ethanolamine to cysteamine, which is >0.8−5.3. For the cysteamine monohydrate, the conformational space was explored (at the B3LYP-GD3(BJ)/Def2-TZVP level). The rotational spectra of the cysteamine monohydrate complex have been assigned in the frequency range 6 – 18.5 GHz. The global minimum, Conf A1, was the only observed one. The 34S isotopologue of Conf A1 was observed in natural abundance, while 18O isotopologue was detected by introducing the H218O. In this conformer, the water molecule plays both proton donor and acceptor roles, forming a OHw···N interaction, a SH···Ow interaction and a CH···Ow interaction. The conformational space of 1-thioglycerol has been characterized by quantum mechanical calculation and its rotational spectrum has been recorded and analyzed in the frequency range 59.6 - 78.4 GHz. The global minimum of 1-thioglycerol is gTg’Gg’ and were detected together with gTg’Tg and gGgG’g, while the two detected conformers are g’G’gGg’ and tGgGg. The high-resolution rotational spectrum of 1-propanethiol in the frequency range 59.6 – 78.4 GHz was measured. Two conformers, Gg and Tg, were observed and their spectra were analyzed. Considering the overall conformational space calculated at the B3LYP-GD3(BJ)/Def2-TZVP level they are among the lowest energy conformers.

Investigation of organic semiconductor/water interfaces for optobioelectronic devices

The thesis investigates the potential of photoactive organic semiconductors as a new class of materials for developing bioelectronic devices that can convert light into biological signals. The materials can be either small molecules or polymers. When these materials interact with aqueous biological fluids, they give rise to various electrochemical phenomena, including photofaradaic or photocapacitive processes, depending on whether photogenerated charges participate in redox processes or accumulate at an interface. The thesis starts by studying the behavior of the H2Pc/PTCDI molecular p/n thin-film heterojunction in contact with aqueous electrolyte. An equivalent circuit model is developed, explaining the measurements and predicting behavior in wireless mode. A systematic study on p-type polymeric thin-films is presented, comparing rr-P3HT with two low bandgap conjugated polymers: PBDB-T and PTB7. The results demonstrate that PTB7 has superior photocurrent performance due to more effective electron-transfer onto acceptor states in solution. Furthermore, the thesis addresses the issue of photovoltage generation for wireless photoelectrodes. An analytical model based on photoactivated charge-transfer across the organic-semiconductor/water interface is developed, explaining the large photovoltages observed for polymeric p-type semiconductor electrodes in water. Then, flash-precipitated nanoparticles made of the same three photoactive polymers are investigated, assessing the influence of fabrication parameters on the stability, structure, and energetics of the nanoparticles. Photocathodic current generation and consequent positive charge accumulation is also investigated. Additionally, newly developed porous P3HT thin-films are tested, showing that porosity increases both the photocurrent and the semiconductor/water interfacial capacity. Finally, the thesis demonstrates the biocompatibility of the materials in in-vitro experiments and shows safe levels of photoinduced intracellular ROS production with p-type polymeric thin-films and nanoparticles. The findings highlight the potential of photoactive organic semiconductors in the development of optobioelectronic devices, demonstrating their ability to convert light into biological signals and interface with biological fluids.