Development of synthetic molecular circuits for the control of cell systems

Synthetic biology is a young field of applicative research aiming to design and build up artificial biological devices, useful for human applications. How synthetic biology emerged in past years and how the development of the Registry of Standard Biological Parts aimed to introduce one practical starting solution to apply the basics of engineering to molecular biology is presented in chapter 1 in the thesis The same chapter recalls how biological parts can make up a genetic program, the molecular cloning tecnique useful for this purpose, and an overview of the mathematical modeling adopted to describe gene circuit behavior. Although the design of gene circuits has become feasible the increasing complexity of gene networks asks for a rational approach to design gene circuits. A bottom-up approach was proposed, suggesting that the behavior of a complicated system can be predicted from the features of its parts. The option to use modular parts in large-scale networks will be facilitated by a detailed and shared characterization of their functional properties. Such a prediction, requires well-characterized mathematical models of the parts and of how they behave when assembled together. In chapter 2, the feasibility of the bottom-up approach in the design of a synthetic program in Escherichia coli bacterial cells is described. The rational design of gene networks is however far from being established. The synthetic biology approach can used the mathematical formalism to identify biological information not assessable with experimental measurements. In this context, chapter 3 describes the design of a synthetic sensor for identifying molecules of interest inside eukaryotic cells. The Registry of Standard parts collects standard and modular biological parts. To spread the use of BioBricks the iGEM competition was started. The ICM Laboratory, where Francesca Ceroni completed her Ph.D, partecipated with teams of students and Chapter 4 summarizes the projects developed.

Synthetic strategies of new molecular paramagnetic mechanically-interlocked complexes

Supramolecular chemistry is a multidisciplinary field which impinges on other disciplines, focusing on the systems made up of a discrete number of assembled molecular subunits. The forces responsible for the spatial organization are intermolecular reversible interactions. The supramolecular architectures I was interested in are Rotaxanes, mechanically-interlocked architectures consisting of a "dumbbell shaped molecule", threaded through a "macrocycle" where the stoppers at the end of the dumbbell prevent disassociation of components and catenanes, two or more interlocked macrocycles which cannot be separated without breaking the covalent bonds. The aim is to introduce one or more paramagnetic units to use the ESR spectroscopy to investigate complexation properties of these systems cause this technique works in the same time scale of supramolecular assemblies. Chapter 1 underlines the main concepts upon which supramolecular chemistry is based, clarifying the nature of supramolecular interactions and the principles of host-guest chemistry. In chapter 2 it is pointed out the use of ESR spectroscopy to investigate the properties of organic non-covalent assemblies in liquid solution by spin labels and spin probes. The chapter 3 deals with the synthesis of a new class of p-electron-deficient tetracationic cyclophane ring, carrying one or two paramagnetic side-arms based on 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) moiety. In the chapter 4, the Huisgen 1,3-dipolar cycloaddition is exploited to synthesize rotaxanes having paramagnetic cyclodextrins as wheels. In the chapter 5, the catalysis of Huisgen’s cycloaddition by CB[6] is exploited to synthesize paramagnetic CB[6]-based [3]-rotaxanes. In the chapter 6 I reported the first preliminary studies of Actinoid series as a new class of templates in catenanes’ synthesis. Being f-block elements, so having the property of expanding the valence state, they constitute promising candidates as chemical templates offering the possibility to create a complex with coordination number beyond 6.

Modeling and simulation of a synthetic genetic circuit that implements a multicelled behavior in a growing microcolony of E. coli

Synthetic Biology is a relatively new discipline, born at the beginning of the New Millennium, that brings the typical engineering approach (abstraction, modularity and standardization) to biotechnology. These principles aim to tame the extreme complexity of the various components and aid the construction of artificial biological systems with specific functions, usually by means of synthetic genetic circuits implemented in bacteria or simple eukaryotes like yeast. The cell becomes a programmable machine and its low-level programming language is made of strings of DNA. This work was performed in collaboration with researchers of the Department of Electrical Engineering of the University of Washington in Seattle and also with a student of the Corso di Laurea Magistrale in Ingegneria Biomedica at the University of Bologna: Marilisa Cortesi. During the collaboration I contributed to a Synthetic Biology project already started in the Klavins Laboratory. In particular, I modeled and subsequently simulated a synthetic genetic circuit that was ideated for the implementation of a multicelled behavior in a growing bacterial microcolony. In the first chapter the foundations of molecular biology are introduced: structure of the nucleic acids, transcription, translation and methods to regulate gene expression. An introduction to Synthetic Biology completes the section. In the second chapter is described the synthetic genetic circuit that was conceived to make spontaneously emerge, from an isogenic microcolony of bacteria, two different groups of cells, termed leaders and followers. The circuit exploits the intrinsic stochasticity of gene expression and intercellular communication via small molecules to break the symmetry in the phenotype of the microcolony. The four modules of the circuit (coin flipper, sender, receiver and follower) and their interactions are then illustrated. In the third chapter is derived the mathematical representation of the various components of the circuit and the several simplifying assumptions are made explicit. Transcription and translation are modeled as a single step and gene expression is function of the intracellular concentration of the various transcription factors that act on the different promoters of the circuit. A list of the various parameters and a justification for their value closes the chapter. In the fourth chapter are described the main characteristics of the gro simulation environment, developed by the Self Organizing Systems Laboratory of the University of Washington. Then, a sensitivity analysis performed to pinpoint the desirable characteristics of the various genetic components is detailed. The sensitivity analysis makes use of a cost function that is based on the fraction of cells in each one of the different possible states at the end of the simulation and the wanted outcome. Thanks to a particular kind of scatter plot, the parameters are ranked. Starting from an initial condition in which all the parameters assume their nominal value, the ranking suggest which parameter to tune in order to reach the goal. Obtaining a microcolony in which almost all the cells are in the follower state and only a few in the leader state seems to be the most difficult task. A small number of leader cells struggle to produce enough signal to turn the rest of the microcolony in the follower state. It is possible to obtain a microcolony in which the majority of cells are followers by increasing as much as possible the production of signal. Reaching the goal of a microcolony that is split in half between leaders and followers is comparatively easy. The best strategy seems to be increasing slightly the production of the enzyme. To end up with a majority of leaders, instead, it is advisable to increase the basal expression of the coin flipper module. At the end of the chapter, a possible future application of the leader election circuit, the spontaneous formation of spatial patterns in a microcolony, is modeled with the finite state machine formalism. The gro simulations provide insights into the genetic components that are needed to implement the behavior. In particular, since both the examples of pattern formation rely on a local version of Leader Election, a short-range communication system is essential. Moreover, new synthetic components that allow to reliably downregulate the growth rate in specific cells without side effects need to be developed. In the appendix are listed the gro code utilized to simulate the model of the circuit, a script in the Python programming language that was used to split the simulations on a Linux cluster and the Matlab code developed to analyze the data.

Development of new therapeutic approaches in the treatments of Inflammatory Bowel Diseases: functional food and nutraceuticals vs synthetic peptides based vaccines

Inflammatory Bowel Diseases (IBD) are intestinal chronic relapsing diseases which ethiopathogenesis remains uncertain. Several group have attempted to study the role of factors involved such as genetic susceptibility, environmental factors such as smoke, diet, sex, immunological factors as well as the microbioma. None of the treatments available satisfy several criteria at the same time such as safety, long-term remission, histopatological healing, and specificity. We used two different approaches for the development of new therapeutic treatment for Inflammatory Bowel Disease. The first is focused on the understanding of the potential role of functional food and nutraceuticals nutrients in the treatment of IBD. To do so, we investigated the role of Curcuma longa in the treatment of chemical induced colitis in mice model. Since Curcma Longa has been investigated for its antinflammatory role related to the TNFα pathway as well investigators have reported few cases of patients with ulcerative colites treated with this herbs, we harbored the hypothesis of a role of Curcuma Longa in the treatment f IBD as well as we decided to assess its role in intestinal motility. The second part is based on an immunological approach to develop new drugs to induce suppression in Crohn’s disease or to induce mucosa immunity such as in colonrectal tumor. The main idea behind this approach is that we could manipulate relevant cell-cell interactions using synthetic peptides. We demonstrated the role of the unique interaction between molecules expressed on intestinal epithelial cells such as CD1d and CEACAM5 and on CD8+ T cells. In normal condition this interaction has a role for the expansion of the suppressor CD8+ T cells. Here, we characterized this interaction, we defined which are the epitope involved in the binding and we attempted to develop synthetic peptides from the N domain of CEACAM5 in order to manipulate it.

New synthetic bile acid analogue agonists of FXR and TGR5 receptors: Analytical methodologies for the study of their physico-chemical properties, pharmacokinetic activity and metabolism.

This thesis reports an integrated analytical approach for the study of physicochemical and biological properties of new synthetic bile acid (BA) analogues agonists of FXR and TGR5 receptors. Structure-activity data were compared with those previous obtained using the same experimental protocols on synthetic and natural occurring BA. The new synthetic BA analogues are classified in different groups according also to their potency as a FXR and TGR5 agonists: unconjugated and steroid modified BA and side chain modified BA including taurine or glycine conjugates and pseudo-conjugates (sulphonate and sulphate analogues). In order to investigate the relationship between structure and activity the synthetic analogues where admitted to a physicochemical characterization and to a preliminary screening for their pharmacokinetic and metabolism using a bile fistula rat model. Sensitive and accurate analytical methods have been developed for the quali-quantitative analysis of BA in biological fluids and sample used for physicochemical studies. Combined High Performance Liquid Chromatography Electrospray tandem mass spectrometry with efficient chromatographic separation of all studied BA and their metabolites have been optimized and validated. Analytical strategies for the identification of the BA and their minor metabolites have been developed. Taurine and glycine conjugates were identified in MS/MS by monitoring the specific ion transitions in multiple reaction monitoring (MRM) mode while all other metabolites (sulphate, glucuronic acid, dehydroxylated, decarboxylated or oxo) were monitored in a selected-ion reaction (SIR) mode with a negative ESI interface by the following ions. Accurate and precise data where achieved regarding the main physicochemical properties including solubility, detergency, lipophilicity and albumin binding . These studies have shown that minor structural modification greatly affect the pharmacokinetics and metabolism of the new analogues in respect to the natural BA and on turn their site of action, particularly where their receptor are located in the enterohepatic circulation.

Novel Synthetic Procedures in Organocatalysis

The main aim of my PhD project was the design and the synthesis of new pyrrolidine organocatalysts. New effective ferrocenyl pyrrolidine catalysts, active in benchmark organocatalytic reactions, has been developed. The ferrocenyl moiety, in combination with simple ethyl chains, is capable of fixing the enamine conformation addressing the approach trajectory of the nucleophile in the reaction. The results obtained represent an interesting proof-of-concept, showing for the first time the remarkable effectiveness of the ferrocenyl moiety in providing enantioselectivity through conformational selection. This approach could be viably employed in the rational design of ligands for metal or organocatalysts. Other hindered secondary amines has been prepared from alkylation of acyclic chiral nitroderivatives with alcohols in a highly diastereoselective fashion, giving access to functionalized, useful organocatalytic chiral pyrrolidines. A family of new pyrrolidines bearing sterogenic centers and functional groups can be readily accessible by this methodology. The second purpose of the project was to study in deep the reactivity of stabilized carbocations in new metal-free and organocatalytic reactions. By taking advantage of the results from the kinetic studies described by Mayr, a simple and effective procedure for the direct formylation of aryltetrafluoroborate salts, has been development. The coupling of a range of aryl- and heteroaryl- trifluoroborate salts with 1,3-benzodithiolylium tetrafluoroborate, has been attempted in moderate to good yields. Finally, a simple and general methodology for the enamine-mediated enantioselective α-alkylation of α-substituted aldehydes with 1,3-benzodithiolylium tetrafluoroborate has been reported. The introduction of the benzodithiole moiety permit the installation of different functional groups due to its chameleonic behaviour.

In vitro synthesis of the light-harvesting complex into artificial membrane systems

In green plants, the function of collecting solar energy for photosynthesis is fulfilled by a series of light-harvesting complexes (LHC). The light-harvesting chlorophyll a/b protein (LHCP) is synthesized in the cytosol as a precursor (pLHCP), then imported into chloroplasts and assembled into photosynthetic thylakoid membranes. Knowledge about the regulation of the transport processes of LHCP is rather limited. Closely mimicking the in vivo situation, cell-free protein expression system is employed in this dissertation to study the reconstitution of LHCP into artificial membranes. The approach starts merely from the genetic information of the protein, so the difficult and time-consuming procedures of protein expression and purification can be avoided. The LHCP encoding gene from Pisum sativum was cloned into a cell-free compatible vector system and the protein was expressed in wheat germ extracts. Vesicles or pigment-containing vesicles were prepared with either synthetic lipid or purified plant leaf lipid to mimic cell membranes. LHCP was synthesized in wheat germ extract systems with or without supplemented lipids. The addition of either synthetic or purified plant leaf lipid was found to be beneficial to the general productivity of the expression system. The lipid membrane insertion of the LHCP was investigated by radioactive labelling, protease digestion, and centrifugation assays. The LHCP is partially protected against protease digestion; however the protection is independent from the supplemented lipids.

Bioconjugation and synthetic approach towards enantioenriched gem-difluoromethylene compounds through carbenium ions

Bioconjugation of peptides and asymmetric synthesis of gem-difluoromethylene compounds are areas of the modern organic chemistry for which mild and selective methods continue to be developed. This thesis reports new methodologies for these two areas based on the use of stabilized carbenium ions. The reaction that makes the bioconjugation of peptides possible takes place via the direct nucleophilic substitution of alcohols and is driven by the spontaneous formation of stabilized carbenium ions in water. By reacting with the thiol group of cysteine in very mild conditions and with a high selectivity, these carbenium ions allow the site-specific ligation of polypeptides containing cysteine and their covalent derivatization with functionalized probes. The ligation of the indole ring of tryptophan, an emerging target in bioconjugation, is also shown and takes place in the same conditions. The second area investigated is the challenging access to optically active gem-difluoromethylene compounds. We describe a methodology relying on the synthesis of enantioenriched 1,3-benzodithioles intermediates that are shown to be precursors of the corresponding gem-difluoromethylene analogues by oxidative desulfurization-fluorination. This synthesis takes advantage of the highly enantioselective organocatalytic α-alkylation of aldehydes with the benzodithiolylium ion and of the wide possibilities of synthetic transformations offered by the 1,3-benzodithiole group. This approach allows the asymmetric access to complex gem-difluoromethylene compounds through a late-stage fluorination step, thus avoiding the use of fluorinated building blocks.

Fast and accurate numerical solutions in some problems of particle and radiation transport: synthetic acceleration for the method of short characteristics, Doppler-broadened scattering kernel, remote sensing of the cryosphere

The aim of this work is to present various aspects of numerical simulation of particle and radiation transport for industrial and environmental protection applications, to enable the analysis of complex physical processes in a fast, reliable, and efficient way. In the first part we deal with speed-up of numerical simulation of neutron transport for nuclear reactor core analysis. The convergence properties of the source iteration scheme of the Method of Characteristics applied to be heterogeneous structured geometries has been enhanced by means of Boundary Projection Acceleration, enabling the study of 2D and 3D geometries with transport theory without spatial homogenization. The computational performances have been verified with the C5G7 2D and 3D benchmarks, showing a sensible reduction of iterations and CPU time. The second part is devoted to the study of temperature-dependent elastic scattering of neutrons for heavy isotopes near to the thermal zone. A numerical computation of the Doppler convolution of the elastic scattering kernel based on the gas model is presented, for a general energy dependent cross section and scattering law in the center of mass system. The range of integration has been optimized employing a numerical cutoff, allowing a faster numerical evaluation of the convolution integral. Legendre moments of the transfer kernel are subsequently obtained by direct quadrature and a numerical analysis of the convergence is presented. In the third part we focus our attention to remote sensing applications of radiative transfer employed to investigate the Earth's cryosphere. The photon transport equation is applied to simulate reflectivity of glaciers varying the age of the layer of snow or ice, its thickness, the presence or not other underlying layers, the degree of dust included in the snow, creating a framework able to decipher spectral signals collected by orbiting detectors.

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

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

Synthetic strategies of new spin-labelled supramolecular systems

Molecular recognition and self-assembly represent fundamental issues for the construction of supramolecular systems, structures in which the components are held together through non-covalent interactions. The study of host-guest complexes and mechanical interlocked molecules, important examples in this field, is necessary in order to characterize self-assembly processes, achieve more control over the molecular organization and develop sophisticated structures by using properly designed building blocks. The introduction of paramagnetic species, or spin labelling, represents an attractive opportunity that allows their detection and characterization by the Electron Spin Resonance spectroscopy, a valuable technique that provides additional information to those obtained by traditional methods. In this Thesis, recent progresses in the design and the synthesis of new paramagnetic host-guest complexes and rotaxanes characterized by the presence of nitroxide radicals and their investigation by ESR spectroscopy are reported. In Chapter 1 a brief overview of the principal concepts of supramolecular chemistry, the spin labelling approach and the development of ESR methods applied to paramagnetic systems are described. Chapter 2 and 3 are focused on the introduction of radicals in macrocycles as Cucurbiturils and Pillar[n]arenes, due to the interesting binding properties and the potential employment in rotaxanes, in order to investigate their structures and recognition properties. Chapter 4 deals with one of the most studied mechanical interlocked molecules, the bistable [2]rotaxane reported by Stoddart and Heath based on the ciclobis (paraquat-p-phenylene) CBPQT4+, that represents a well known example of molecular switch driven by external stimuli. The spin labelling of analogous architectures allows the monitoring by ESR spectroscopy of the switch mechanism involving the ring compound by tuning the spin exchange interaction. Finally, Chapter 5 contains the experimental procedures used for the synthesis of some of the compounds described in Chapter 2-4.

Synthetic routes toward functional block copolymers and bioconjugates via RAFT polymerization

Synthetic Routes toward Functional Block Copolymers and Bioconjugates via RAFT PolymerizationrnSynthesewege für funktionelle Blockcopolymere und Biohybride über RAFT PolymerisationrnDissertation von Dipl.-Chem. Kerstin T. WissrnIm Rahmen dieser Arbeit wurden effiziente Methoden für die Funktionalisierung beider Polymerkettenenden für Polymer- und Bioanbindung von Polymeren entwickelt, die mittels „Reversible Addition-Fragmentation Chain Transfer“ (RAFT) Polymerisation hergestellt wurden. Zu diesem Zweck wurde ein Dithioester-basiertes Kettentransferagens (CTA) mit einer Aktivestereinheit in der R-Gruppe (Pentafluorphenyl-4-phenylthiocarbonylthio-4-cyanovaleriansäureester, kurz PFP-CTA) synthetisiert und seine Anwendung als universelles Werkzeug für die Funktionalisierung der -Endgruppe demonstriert. Zum Einen wurde gezeigt, wie dieser PFP-CTA als Vorläufer für die Synthese anderer funktioneller CTAs durch einfache Aminolyse des Aktivesters genutzt werden kann und somit den synthetischen Aufwand, der üblicherweise mit der Entwicklung neuer CTAs verbunden ist, reduzieren kann. Zum Anderen konnte der PFP-CTA für die Synthese verschiedener Poly(methacrylate) mit enger Molekulargewichtsverteilung und wohl definierter reaktiver -Endgruppe verwendet werden. Dieses Kettenende konnte dann erfolgreich mit verschiedenen primären Aminen wie Propargylamin, 1-Azido-3-aminopropan und Ethylendiamin oder direkt mit den Amin-Endgruppen verschiedener Peptide umgesetzt werden.rnAus der Reaktion des PFP-CTAs mit Propargylamin wurde ein Alkin-CTA erhalten, der sich als effizientes Werkzeug für die RAFT Polymerisation verschiedener Methacrylate erwiesen hat. Der Einbau der Alkin-Funktion am -Kettenende wurde mittels 1H und 13C NMR Spektroskopie sowie MALDI TOF Massenspektroskopie bestätigt. Als Modelreaktion wurde die Kopplung eines solchen alkin-terminierten Poly(di(ethylenglykol)methylethermethacrylates) (PDEGMEMA) mit azid-terminiertem Poly(tert-butylmethacrylat), das mittels Umsetzung einer Aktivester-Endgruppe erhalten wurde, als kupferkatalysierte Azid-Alkin-Cycloaddition (CuAAC) durchgeführt. Die Aufarbeitung des resultierenden Diblockcopolymers durch Fällen ermöglichte die vollständige Abtrennung des Polymerblocks 1, der im Überschuss eingesetzt wurde. Darüber hinaus blieb nur ein sehr kleiner Anteil (< 2 Gew.-%) nicht umgesetzten Polymerblocks 2, was eine erfolgreiche Polymeranbindung und die Effizienz der Endgruppen-Funktionalisierung ausgehend von der Aktivester--Endgruppe belegt.rnDie direkte Reaktion von stimuli-responsiven Polymeren mit Pentafluorphenyl(PFP)ester-Endgruppen, namentlich PDEGMEMA und Poly(oligo(ethylenglykol)methylethermethacrylat), mit kollagen-ähnlichen Peptiden ergab wohl definierte Polymer-Peptid-Diblockcopolymere und Polymer-Peptid-Polymer-Triblockcopolymer unter nahezu quantitativer Umsetzung der Endgruppen. Alle Produkte konnten vollständig von nicht umgesetztem Überschuss des Homopolymers befreit werden. In Analogie zu natürlichem Kollagen und dem nicht funktionalisierten kollagen-ähnlichen Peptid bilden die PDEGMEMA-basierten, entschützten Hybridcopolymere Trimere mit kollagen-ähnlichen Triple-Helices in kalter wässriger Lösung, was mittels Zirkular-Dichroismus-Spektroskopie (CD) nachgewiesen werden konnte. Temperaturabhängige CD-Spektroskopie, Trübungsmessungen und dynamische Lichtstreuung deuteten darauf hin, dass sie bei höheren Temperaturen doppelt stimuli-responsive Überstrukturen bilden, die mindestens zwei konformative Übergänge beim Aufheizen durchlaufen. Einer dieser Übergänge wird durch den hydrophoben Kollaps des Polymerblocks induziert, der andere durch Entfalten der kollagen-ähnlichen Triple-Helices.rnAls Ausweitung dieser synthetischen Strategie wurde homotelecheles PDEGMEMA mit zwei PFP-Esterendgruppen dargestellt, wozu der PFP-CTA für die Funktionalisierung der -Endgruppe und die radikalische Substitution des Dithioesters durch Behandlung mit einem Überschuss eines funktionellen AIBN-Derivates für die Funktionalisierung der -Endgruppe ausgenutzt wurde. Die Umsetzung der beiden reaktiven Kettenenden mit dem N-Terminus eines Peptidblocks ergab ein Peptid-Polymer-Peptid Triblockcopolymer.rnSchließlich konnten die anorganisch-organischen Hybridmaterialien PMSSQ-Poly(2,2-diethoxyethylacrylat) (PMSSQ-PDEEA) und PMSSQ-Poly(1,3-dioxolan-2-ylmethylacrylat) (PMSSQ-PDMA) für die Herstellung robuster, peptid-reaktiver Oberflächen durch Spin Coaten und thermisch induziertes Vernetzen angewendet werden. Nach saurem Entschützen der Acetalgruppen in diesen Filmen konnten die resultierenden Aldehydgruppen durch einfaches Eintauchen in eine Lösung mit einer Auswahl von Aminen und Hydroxylaminen umgesetzt werden, wodurch die Oberflächenhydrophilie modifiziert werden konnte. Darüber hinaus konnten auf Basis der unterschiedlichen Stabilität der zwei hier verglichenen Acetalgruppen Entschützungsprotokolle für die exklusive Entschützung der Diethylacetale in PMSSQ-PDEEA und deren Umsetzung ohne Entschützung der zyklischen Ethylenacetale in PMSSQ-PDMA entwickelt werden, die die Herstellung multifunktioneller Oberflächenbeschichtungen z.B. für die Proteinimmobilisierung ermöglichen.

Development of synthetic methods of silicon nanocrystals functionalized with photoactive molecules

The thesis is focused on the development of a method for the synthesis of silicon nanocrystals with different sizes, narrow size distribution, good optical properties and stability in air. The resulting silicon nanocrystals have been covalently functionalized with different chromophores with the aim to exploit the new electronic and chemical properties that emerge from the interaction between silicon nanocrystal surface and ligands. The purpose is to use these chromophores as light harvesting antennae, increasing the optical absorption of silicon nanocrystals. Functionalized silicon nanocrystals have been characterized with different analytical techniques leading to a good knowledge of optical properties of semiconductor quantum dots.

Surface-enhanced vibrational techniques for the detection of synthetic organic colorants

The research project object of this thesis is focused on the development of an advanced analytical system based on the combination of an improved thin layer chromatography (TLC) plate coupled with infrared (FTIR) and Raman microscopies for the detection of synthetic dyes. Indeed, the characterization of organic colorants, which are commonly present in mixtures with other components and in a very limited amount, still represents a challenging task in scientific analyses of cultural heritage materials. The approach provides selective spectral fingerprints for each compound, foreseeing the complementary information obtained by micro ATR-RAIRS-FTIR and SERS-Raman analyses, which can be performed on the same separated spot. In particular, silver iodide (AgI) applied on a gold coated slide is proposed as an efficient stationary phase for the discrimination of complex analyte mixtures, such as dyes present in samples of art-historical interest. The gold-AgI-TLC plate shows high performances related both to the chromatographic separation of analytes and to the spectroscopic detection of components. The use of a mid-IR transparent inorganic salt as the stationary phase avoids interferences of the background absorption in FTIR investigations. Moreover, by ATR microscopy measurements performed on the gold-AgI surface, a considerable enhancement in the intensity of spectra is observed. Complementary information can be obtained by Raman analyses, foreseeing a SERS activity of the AgI substrate. The method has been tested for the characterization of a mixture of three synthetic organic colorants widely used in dyeing processes: Brilliant Green (BG1), Rhodamine B (BV10) and Methylene Blue (BB9).