L'épuisement des ressources d'origine pétrochimique conduit à la recherche de matières premières renouvelables pour l'élaboration des membranes. La gélatine, un biopolymère abondant, sous produit de l'industrie agroalimentaire, représente grâce à ses propriétés filmogènes un candidat de choix pour l'élaboration de membranes “vertes”. L'objectif de cette thèse est l'élaboration de membranes à base de gélatine et l'étude de l'impact de la structure du matériau sur les propriétés mécaniques, thermiques, de résistance à l'eau et de transfert gazeux. Pour cela les mécanismes d'élaboration par TIG-Dry cast process ont d'abord été formalisés par l'établissement du diagramme de phase du système gélatine/eau. Puis des réticulants alternatifs au glutaraldéhyde, toxique, ont été examinés pour augmenter la résistance à l'eau de la gélatine hydrosoluble. L'acide férulique et le téréphthalaldéhyde se sont montrés les plus prometteurs et complémentaires suivant les applications visées. Les films de gélatine se sont révélés barrières aux gaz à cause de la forte cristallinité induite par la renaturation des triples hélices. L'ajout d'un élastomère de la famille des polyéther amines a permis d'augmenter drastiquement les coefficients de perméabilité du CO2 de 1,4 à 250 Barrer. L'influence de la température et de l'humidité relative des flux gazeux sur les perméabilités a également été étudiée. / Petroleum based raw materials shortage leads to investigate renewable raw materials for membrane elaboration. Gelatin, an abundant, industrial by-product is a biosourced polymer with filmogenic properties which makes it an educated choice for “green” membrane production. This thesis work aims at developing gelatin based membrane and studying the influence of the material structure on mechanical and thermal properties, water resistance and gas transport properties. Thus, the elaboration mechanisms by TIG/Dry-cast process were studied in details by establishing the phase diagram of the gelatin/water system. To improve the water resistance of the hydrosoluble gelatin, crosslinking is necessary. Alternative cross-linkers were tested to replace the glutaraldehyde, classified as toxic. Ferulic acid and terephthalaldehyde were promising and showed complementary characteristics. The high crystallinity level of gelatin films, related to their renaturation level, led to rather gas barrier properties. By adding an elastomer, polyetheramine, the permeability to CO2 increased from 1.4 to an outstanding 250 Barrer. The influence of the temperature and relative humidity of the gas flux on permeability was also studied.

Dans une configuration optimale, la couche photo-active d’une cellule solaire organique forme une hétérojonction volumique entre un matériau donneur et un matériau accepteur d’électrons. Cette morphologie optimale se trouve dans un état thermodynamique métastable. En cours de fonctionnement, l’absorption incidente provoque une élévation de la tempéra- ture des dispositifs. L’apport d’énergie thermique au système le fait évoluer vers un état thermodynamique stable correspondant à une micro-séparation de phase entre les deux matériaux nuisible aux performances photovoltaïques du dispositif. La solution à cette problématique envisagée dans cette thèse est de figer la morphologie optimale de la couche photo-active par réticulation chimique. Dans le but de prévenir la diffusion et la cristallisation des dérivés du fullerène, plusieurs approches ont été étudiées : la formation d’une maille de polymères réticulés, l’accroche des dérivés du fullerène aux chaînes du polymères ou la liaison entre plusieurs dérivés du fullerène. Les différentes fonctions réticulantes utilisées dans ces approches sont l’allyle, le cinnamate, l’anthracène et l’azoture. Au cours de ces travaux, des résultats satisfaisants ont été obtenus avec la fonction azoture. Basé sur cette fonction, nous avons développé un additif qui permet d’augmenter notablement la stabilité thermique des cellules solaires réticulées. Cette approche a été validée sur plusieurs couples de matériaux polymères/dérivés du fullerène à l’état de l’art. / Efficient bulk-heterojunction solar cells require an interpenetrating network of electron donating and electron accepting materials. Indeed, the optimum nano-sized phase segregation is a thermodynamically metastable system. Under operating conditions, especially under solar illumination, the temperature of the solar cell increases significantly. Adding thermal energy to the system leads to further micro-phase separation that is harmful for photovoltaic device performances. In order to freeze the optimal morphology, several approaches based on chemical cross-linking have been studied : formation of cross-linked polymer network, binding fullerene derivatives to polymer side chains or cross-linking between fullerene derivatives. Different cross-linkable functions have been used : allyl, cinamate, anthracene and azide. A versatile stabilization strategy of bulk heterojunctions morphology has been found by mean of incorporating a cross-linkable additive in low amounts with different polymer :fullerene blends. This approach is validated on multiple materials at the state-of-the-art.

Chemical biology can be defined as the area of science where chemical tools are used to study biological systems. The simplest way this can be achieved is in the identification of compounds which inhibit or modulate a biological pathway and the consequences studied. However, novel tools are required to enable, for example, the development of assays to allow simpler screening of difficult targets such as membrane proteins and protein-protein interactions. A series of kisspeptin analogues were synthesised for the development of a screening platform compatible with G-protein coupled receptors and tagged one bead one compound (OBOC) combinatorial libraries. Fluorescently labelled kisspeptin showed good affinity for GPCR54 and an on-bead version of the peptide, with the required C-terminal amide presented away from the bead was prepared and used for testing possible screening methods. GPCR54 was expressed in a number of formats and a kisspeptin based OBOC library designed and synthesised. Investigation into the C-terminal RF-amide motif of Kisspeptin was also carried out in order to assess the importance of the carbonyl moiety. The corresponding peptide amine was synthesised and the compound biologically assessed. This led to the development of a novel acid labile benzofuranone (ALBA) linker for anchoring amines to a solid support. For the preparation of fluorescent kisspeptin ligands, a novel general synthetic route which gives direct access to single isomer functionalised rhodamine dyes from phthalides has been developed. This circumvents the arduous task of isomer separation usually associated with the synthesis of functionalised rhodamines. The route has been demonstrated with a range of linkage groups and rhodamine types. This rhodamine material was used as a reporter group in various multifunctional reagents synthesised using a trifunctional orthogonally protected backbone (TOBa), which was prepared on a solid support and enables rapid synthesis of trifunctional reagents. This resin takes advantage of protecting group orthogonality and the high yields of peptide bond formation. A series of trifunctional reagents for screening use were prepared using this resin. A proof of concept study was carried out involving the simultaneous labelling and immobilisation of a protein for applications in probing protein-protein interactions. Development of a trifunctional hydroxamic acid containing cross-linker was carried out which takes advantage of its reaction with boronic acids to enable reversible capture on solid support for enrichment of cross-linked peptides. A new benzophenone based heterobifunctional reagent was prepared for protein cross-linking and mass spectrometry analysis. This was shown to give complimentary reactivity to existing cross-linkers, allowing more structural information to be extracted from protein samples.

3D proteomics combines chemical cross-linking with mass spectrometry to study the structure of protein assemblies and protein-protein interactions both in vitro and in vivo by providing distance constraints that indicate which residues are in close spatial proximity. I addressed the main bottleneck of this technology: the reliable identification of cross-linked peptides. Reporter ion signatures for cross-linked peptides were developed, by fragmenting model compounds containing two lysine residues joined by a cross-linker backbone or a lysine residue modified with a hydrolysed cross-linker. The reporter ion signatures showed 97% specificity at 90% sensitivity and segregated cross-linked from modified and linear peptides. They decreased the false discovery rate of the identification of cross-linked peptides from 5% to 1% in a large dataset. The signatures permit data sorting during and after mass spectrometry acquisition. The advanced 3D proteomics workflow was applied to study the protein-protein interactions in Mycoplasma pneumoniae cells. In lysates of the bacterium we identified 128 protein-protein interactions (of which 24 are novel) and obtained in vivo topological data on 208 proteins, even for cases where high-resolution structures are not yet available. We showed that our data are in excellent agreement with crystal structures of proteins and complexes where available. We defined a network of ribosomal and RNA polymerase proteins that reveals an intricate link between transcription and translation in bacteria. We demonstrated that the method is suitable for identification of homomultimeric protein complexes by exploiting peptide pairs of identical amino acid sequence. The technology has the potential to provide a complete protein interaction network map after the selective enrichment of cross-lined peptides is achieved. The method was next applied to investigate the structure of condensin and cohesin complexes, which play a crucial role in stabilization of chromosome structure during mitosis. The complexes were purified, cross-linked and their linkage map created. The condensin coiled coil cross-linked on the entire length was modeled. The information was used to direct the analysis of in situ cross-linked condensin in intact chromosomes. I found two high confidence linkages between SMC2 and SMC4 coiled coils and identified H2A as a potential condensin receptor on chromosomes.

<p>Predmet doktorske disertacije je izučavanje različitih bioprocesa za modifikovanje biljnih globulina radi unapređenja njihovih funkcionalnih karakteristika. Istraživanja su zasnovana na karakterizaciji i enzimskoj modifikaciji glavnog rezervnog proteina (12S), kukurbitina, iz semena uljane tikve (<em>Cucurbita pepo</em>). Osnova istraživanja je enzimska konverzija globulina i dobijanje proteinskih modifikata delovanjem hidrolaza i transferaza. U okviru istraživanja, enzimski procesi modifikacije globulina izučavani su sa dva aspekta: enzimska hidroliza i enzimsko umrežavanje (cross-linking), primenom komercijalnih enzimskih preparata. Takođe istraživanja obuhvataju i razvoj i kontrolu samih bioprocesa definisanjem i optimizacijom procesnih parametara (temperature, pH, koncentracije enzima i supstrata, vreme reakcije). Ovako definisani procesi eksploatisani su u cilju kreiranja željenih funkcionalnih karakteristika proteina spram njihove potencijalne primene u formulacijama hrane. Odabir i optimizacija procesnih parametara i modelovanje bioprocesa izvedeno je implementiranjem nove kompjuterske i analitičke metodologije</p> / <p>The PhD thesis research is aimed at development of different bioprocesses for modification of plant globulins in order to improve their functional properties. Studies are based on characterization and enzymatic modification of major storage protein (12S), cucurbitin derived from pumpkin oil seed (<em>Cucurbita pepo</em>). The base of research is enzymatic conversion of cucurbitin by hydrolase and transferase. Two different enzymatic processes are used for protein modification: (i) enzymatic hydrolysis and (ii) enzymatic cross-linking. To monitor, control the bioprocesses, and definition of process parameters, such as temperature, pH, enzyme-substrate ratio, reaction time, Response Surface Methodology (RSM) was used. In addition, RSM was employed for production of protein modification with desired functional properties.</p>

No description available.

Primary amine-based plasma polymer films (PPF) attract an increasing interest due to their potential applications as platforms to support cell growth and biomacromolecule immobilization. It has been demonstrated that the biological response of these films is correlated to their primary amine content (%NH2). Control and optimization of the process are thus very important requirements. However, despite the abundant literature related to this attractive technology, plasma polymerization remains very complex so that strong efforts are still needed to understand the relationship between the plasma chemistry and the PPF characteristics. The main part of this work describes the pulsed plasma polymerization of two isomeric precursors, namely allylamine and cyclopropylamine, with the aim to study the influence of the precursor on the plasma and the PPF physico-chemistries. The systems have been compared in similar conditions of mean power injected in the discharge (Pmean). Both experimental and theoretical approaches have been used to shed light on the process. Taking into account the electron energy in the plasma, differences between allylamine and cyclopropylamine plasma chemistries have been rationalized with the help of Density Functional Theory calculations. It is demonstrated that %NH2 can be increased by an appropriate choice of the precursor and the experimental conditions. Indeed, the use of cyclopropylamine can yield to PPF with a larger %NH2 compared to allylamine due to a preferential opening of the ring structure in the plasma. A method is also proposed to discriminate the PPF according to their cross-linking density (÷). While it appears to be independent of the chemical structure of the precursor, ÷ is observed to significantly vary with Pmean. The PPF have further been investigated with the aim to better understand the phenomena taking place when immersed in liquid medium, an essential step for biological applications. The results show that the decrease in the PPF thickness (Äd), conventionally observed upon immersion, directly depends on Pmean; the higher Pmean, the lower Äd. For the first time to our knowledge, the present work allows to unambiguously attribute this phenomenon to an increase of ÷ with Pmean. Furthermore, thanks to the analysis of the resulting solutions, the data give a new insight into the PPF behavior in liquid medium, suggesting that Äd is mostly related to a reorganization of the PPF network and not to material dissolution as usually claimed. Finally, some preliminary experiments indicate that the PPF can be used to support muscle cell culture; the performances of the PPF-coated materials reveal to be at least similar to those of the standard surfaces.

The creation of complex polymer structures has been one of the major research topics over the last couple of decades. This work deals with the synthesis of (block co-)polymers, the creation of complex and stimuli-responsive aggregates by self-assembly, and the cross-linking of these structures. Also the higher-order self-assembly of the aggregates is investigated. The formation of poly-2-oxazoline based micelles in aqueous solution and their simultaneous functionalization and cross-linking using thiol-yne chemistry is e.g. presented. By introducing pH responsive thiols in the core of the micelles the influence of charged groups in the core of micelles on the entire structure can be studied. The charging of these groups leads to a swelling of the core and a decrease in the local concentration of the corona forming block (poly(2-ethyl-2-oxazoline)). This decrease in concentration yields a shift in the cloud point temperature to higher temperatures for this Type I thermoresponsive polymer. When the swelling of the core is prohibited, e.g. by the introduction of sufficient amounts of salt, this behavior disappears. Similar structures can be prepared using complex coacervate core micelles (C3Ms) built through the interaction of weakly acidic and basic polymer blocks. The advantage of these structures is that two different stabilizing blocks can be incorporated, which allows for more diverse and complex structures and behavior of the micelles. Using block copolymers with either a polyanionic or a polycationic block C3Ms could be created with a corona which contains two different soluble nonionic polymers, which either have a mixed corona or a Janus type corona, depending on the polymers that were chosen. Using NHS and EDC the micelles could easily be cross-linked by the formation of amide bonds in the core of the micelles. The higher-order self-assembly behavior of these core cross-linked complex coacervate core micelles (C5Ms) was studied. Due to the cross-linking the micelles are stabilized towards changes in pH and ionic strength, but polymer chains are also no longer able to rearrange. For C5Ms with a mixed corona likely network structures were formed upon the collapse of the thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm), whereas for Janus type C5Ms well defined spherical aggregates of micelles could be obtained, depending on the pH of the solution. Furthermore it could be shown that Janus micelles can adsorb onto inorganic nanoparticles such as colloidal silica (through a selective interaction between PEO and the silica surface) or gold nanoparticles (by the binding of thiol end-groups). Asymmetric aggregates were also formed using the streptavidin-biotin binding motive. This is achieved by using three out of the four binding sites of streptavidin for the binding of one three-arm star polymer, end-functionalized with biotin groups. A homopolymer with one biotin end-group can be used to occupy the last position. This binding of two different polymers makes it possible to create asymmetric complexes. This phase separation is theoretically independent of the kind of polymer since the structure of the protein is the driving force, not the intrinsic phase separation between polymers. Besides Janus structures also specific cross-linking can be achieved by using other mixing ratios. / In den letzten Jahrzehnten war die Herstellung von komplizierten Polymerstrukturen ein wichtiges Forschungsthema für Polymerchemiker. Diese Arbeit behandelt die Synthese von (Blockco-)Polymere, die Herstellung von komplexen und stimulus-responsiven Aggregaten (Mizellen) durch Selbstorganisation, sowie die Vernetzung dieser Strukturen. Auch die Anordnung dieser Mizellen zu Aggregaten mit höherer Ordnung wurde untersucht. Zum Beispiel wird die Bildung von Poly(2-oxazolin) basierter Mizellen in wässriger Lösung und die gleichzeitige Funktionalisierung und Vernetzung dieser Mizellen mittels Thiol-In-Chemie beschrieben. Durch die Einführung von pH-responsiven Gruppen in den Kern der Mizellen konnte der Einfluss von geladenen Gruppen im Kern auf das gesamte Aggregat untersucht werden. Das Einführen von Ladung führt zum Quellen des Mizellkerns und damit zu einer niedrigeren lokalen Konzentration von wasserlöslichem Poly(2-ethyl-2-oxazolin) (PEtOx). Diese niedrigere Konzentration ergibt eine Verschiebung des Trübungspunkt dieses Typ I thermoresponsiven Polymers zu höheren Temperaturen. Wenn die Ausdehnung des Kerns nicht erfolgt, z.B. in Anwesenheit einer hohen Salzkonzentration, findet dieser Effekt nicht statt. Ähnliche Strukturen können mithilfe von Mizellen mit komplexen Koazervatkern (English: Complex Coacervate Core Micelles, C3Ms) durch die Interaktion zwischen Polymeren mit negativ und positiv geladenen Blöcken hergestellt werden. Der Vorteil dieser Strukturen ist, dass zwei verschiedene stabilisierende Polymerblöcke in einem Aggregat vereint werden können, was zur Bildung einer Vielzahl noch komplizierterer Strukturen und zu mehr Responsivität führen kann. Mithilfe von Blockcopolymeren, bestehend aus jeweils einen polyionischen Block und einem neutralen Block (z.B. PEtOx, PEO oder poly(N-isopropylacrylamid) (PNIPAAm)), konnten C3Ms hergestellt werden, in denen zwei neutrale Polymere vereint wurden. Es konnte gezeigt werden, dass diese Polymere sowohl gemischt als auch phasensepariert vorliegen können (letzteres ergibt Janus Mizellen), abhängig welche Polymere gewählt werden. Durch Vernetzung im Kern konnten die Mizellen stabilisiert und fixiert werden (C5Ms). Die Selbstanordnung dieser vernetzten Mizellen zu größeren Aggregaten wurde untersucht. Wenn eine Lösung mit vernetzten Mizellen über den Trübungspunkt von PNIPAAm erhitzt wurde, bildeten sich Netzwerke aus Mizellen mit einer gemischten Korona, während Janus Mizellen sich zu wohldefinierten Aggregaten höherer Ordnung anordneten. Weiterhin konnte gezeigt werden, dass Janus Mizellen sich auf der Oberfläche von anorganischen Nanopartikeln anlagern können; z.B. durch die selektive Wechselwirkung zwischen PEO und Silica oder durch die Adsorption von Thiolgruppen auf Gold-Nanopartikeln. Asymmetrische Aggregate konnten auch mithilfe des Streptavidin-Biotin Komplexes erhalten werden. Durch das Binden der Biotin-Endgruppen eines dreiarmigen Sternpolymeren an eine Streptavidin-Einheit und anschließende Belegung der verbliebenen Bindungsstelle mit der Biotin-gruppe eines Homopolymers, können sehr spezifisch zwei verschiedene Polymere in einem Janus Aggregat vereint werden. Auch die Vernetzung des Streptavidins kann erzielt werden, indem andere Mischverhältnisse gewählt werden.

With growing environmental concerns, efforts are made to replace petroleum based products with renewable alternatives. This is particularly evident in the packaging industry, where replacing synthetic polymers with renewable materials is of considerable interest. Materials for food packaging need to give protection, acting as a barrier against substances that can adversely affect the food quality such as water and oxygen. In this work, barrier dispersion coatings based on starch were used to produce coated papers which act as barrier against water and oxygen. However, since starch is both a hydrophilic and hygroscopic material, this barrier material becomes problematic to use at high relative humidity. In order to reduce this problem and improve the barrier properties enabling starch based barrier materials to be used in food packaging applications, two approaches were studied. Citric acid was utilized as a cross-linker of the starch and it was found to reduce the moisture sorption, the molecular movement and swelling at high relative humidity. It was seen that cross-linking and hydrolysis due to the low pH both affected the barrier properties significantly, but in opposing directions. By controlling these two reactions it was seen that this could lead to reduced gas permeability. It was also seen that cross-linking of starch by citric acid occurs at low temperatures, 70 °C at pH as high as 6.5. Starch nano-composites were produced by incorporating montmorillonite, to the barrier dispersion to improve the barrier properties. It was seen that the suspension viscosity was reduced by poly(ethylene glycol) and citric acid adsorption on the montmorillonite particles. Also, a tendency for improved barrier properties with reduced aggregate volume fraction and reduced swelling was observed. It was also seen that up scaling this formulation to pilot scale was possible and that promising results were achieved. / Baksidestext With growing environmental concerns, efforts are made to replace petroleum based materials with renewable alternatives such as starch. In this work, dispersions based on starch were used to produce coated papers which act as barrier against substances that can adversely affect the food quality such as water and oxygen. However, since starch is both a hydrophilic and hygroscopic material, this barrier material becomes problematic to use at high relative humidity. Citric acid was utilized as cross-linker for starch and it was found to reduce the moisture sorption, diffusion and swelling at high relative humidity. Both cross-linking and hydrolysis due to the low pH affected the barrier properties significantly, but in opposing directions. By controlling these two reactions it was possible to achieve reduced gas permeability. Starch nano-composites were produced by incorporating montmorillonite clay, to the barrier dispersion. It was seen that the suspension viscosity was reduced by poly(ethylene glycol) and citric acid adsorption on the clay. Also, a tendency for improved barrier properties with reduced aggregate volume fraction and reduced swelling was observed. It was also seen that up scaling this formulation to pilot scale was possible and promising results were achieved. / Renewable Functional Barriers

sHSPs maintain partially denaturing substrates in a soluble sHSP-substrate complex. The heterogeneous interaction between sHSPs and substrate within the complex has prevented a detailed study of the mechanism of sHSP substrate protection. Here, purified sHSPs and heat sensitive substrates were used to investigate the mechanism of sHSP chaperone action. Results presented provide new insights into how sHSPs recognize substrates, the architecture of the sHSP-substrate complex and factors contributing to chaperone efficiency.Direct evidence defining the role of the sHSP N-terminal arm and alpha crystallin domain in sHSP-substrate interactions is limited. A photoactivatable probe was site- specifically incorporated into PsHsp18.1, and cross-linking to substrate in sHSP-substrate complexes was quantified. The structurally flexible N-terminal arm of PsHsp18.1 makes strong contacts with both substrates tested, however differences in interaction were seen in the conserved alpha crystallin domain. Regions on the sHSP showing the strongest cross-links to substrates are buried within the dodecamer, supporting the model that the sHSP oligomer undergoes rearrangement or dissociation prior to substrate interactions.The arrangement of sHSPs and substrates whithin the complex is poorly defined. Limited proteolysis and chemical modification was combined with mass spectrometry to probe the sHSP-substrate complex using multiple sHSPs and substrates. This analysis reveals that a similar partially-denatured form of substrate is protected in the complex irrespective of sHSP identity. Further, sHSP in the complex is protected from proteolysis for a longer time compared to free sHSP. These data suggest that sHSPs and substrate are distributed both internally and on the periphery of the sHSP-substrate complex.Exact properties of the sHSP N-terminal arm contributing to protection are poorly defined. A molecular dynamics (MD) study was designed to test the hypothesis that the N-terminal arm could assume multiple conformations that can readily interact with denaturing substrates. Preliminary data suggest that at increased temperatures amino acids in the N-terminal arm form specific clusters which could act as substrate interaction sites. MD simulations, mutagenesis and altering the kinetics of substrate aggregation suggest that the conformational space occupied by the N-terminal arm at increased temperatures, along with flexibility and rate of substrate aggregation contribute to differences in chaperone efficiency.