Дисертації з теми "Native hydrogen"

Thesis (Ph.D.); Submitted to New Mexico State Univ., Las Cruces, NM (US); 1 May 2003.<br>Published through the Information Bridge: DOE Scientific and Technical Information. "JLAB-PHY-03-22" "DOE/ER/40150-2731" David McKee. 05/01/2003. Report is also available in paper and microfiche from NTIS.

Thesis (Ph.D.); Submitted to the Catholic Univ. of America, Washington, DC (US); 1 May 2003.<br>Published through the Information Bridge: DOE Scientific and Technical Information. "JLAB-PHY-03-40" "DOE/ER/40150-2764" Brian Carnahan. 05/01/2003. Report is also available in paper and microfiche from NTIS.

Ce travail de thèse porte sur développement de méthodes en spectrométrie de masse structurale pour l’analyse de protéines recombinantes et de leurs complexes associés. L’objectif central s’est porté sur des développements méthodologiques en échange hydrogène/deutérium couplé à la spectrométrie de masse (HDX-MS). Les techniques biophysiques de caractérisation structurale à haute résolution comme la cristallographie ou la RMN se heurtent régulièrement à des problèmes de productions de cristaux, de taille de complexes analysables ou encore de quantité de matériel nécessaire importante. Le développement de méthodes spécifiques HDX-MS a permis de réaliser une caractérisation structurale de systèmes protéiques variés, et réfractaires aux approches haute résolution. La combinaison de cette approche à différents outils de MS structurale est aussi illustrée, et montre tout son intérêt pour l’obtention d’informations à résolution augmentée<br>This thesis work focuses on development of structural mass spectrometry methods for the analysis of recombinant proteins and their associated complex. The central objective has focused on the development of hydrogen/deuterium exchange coupled to mass spectrometry approaches (HDX-MS). The high resolution biophysical techniques for structural characterization such as crystallography or NMR regularly face problems of crystal productions, size analyzable complex or quantity of material required. The development of specific HDX-MS methods allowed the characterization of various, and refractory protein systems to high resolution approaches. The combination of this approach with complementary structural MS tools is also illustrated, and shows its interest to obtain increased resolution information

La France dans le but de décarboner son mix énergétique et baisser ses émissions de CO2 a décidé d’investir massivement dans la production décarbonée d’hydrogène comme vecteur d’énergie pour des applications de mobilités ou stationnaires[1]. Sur le million de tonnes d’hydrogène produit en France, 96 % est produit par vaporeformage d’hydrocarbures. La stratégie Française vise à développer la filière hydrogène en investissant dans l’installation d’électrolyseurs. De plus les dernières découvertes d’énormes gisements d’hydrogène naturel (46 millions de tonnes d’hydrogène en Lorraine) crée l’enthousiasme et agrandi le champ des perspectives. [2]. Une autre filière de production d’hydrogène décarboné dont on parle le moins est la filière biologique qui présente un grand potentiel de diversifications des voies de productions. L’hydrogène issu de ces filières pose le problème de sa qualité pour une application dans la mobilité ou le stationnaire dans des systèmes de pile à combustible.L’objet de ces travaux de thèse est de définir des stratégies pour l’utilisation du bio hydrogène ou hydrogène naturel par la technologie de pile à combustible à membrane échangeuse de protons (PEMFC) et en passant par les étapes de production de l’hydrogène jusqu’à sa conversion électrochimique.La première partie a consisté à étudier l’impact des impuretés ou diluants (N2, Ar, He, CH4, CO2) contenus dans l’hydrogène issu des filières biologique et natif dans une demi-cellule (cellule de diffusion de gaz, GDE). Ensuite cette étude a été étendue à une mono cellule de pile à combustible à membrane échangeuse de protons. Enfin un réacteur biologique à échelle du laboratoire a permis de produire de l’hydrogène à partir de sources organiques par photo fermentation (PF) qui a ensuite été testé en GDE. Plusieurs techniques de caractérisations électrochimiques et physicochimiques comme la voltammétrie cyclique, la chrono amperommétrie, la mesure de surface électro-active par CO stripping, la microscopie électronique à balayage et à transmission, la chromatographie ionique etc…ont permis d’évaluer les performances de la PEMFC alimenté par du bio hydrogène ainsi que son impact sur les éléments d’une pile à combustible.Les résultats de l’activité des électrodes pour la réaction d’oxydation de l’hydrogène en GDE ont mis évidence des effets de limitations par le transport de matières pour l’ensemble mélange, avec des comportements particuliers observés pour le mélange à l’azote, et les mélanges au méthane et au dioxyde de carbone qui en plus de la dilution ont un effet d’empoisonnement au monoxyde de carbone de l’électrode.Ensuite, les tests en mono cellule alimenté par les mélanges H2/Ar, H2/N2 et H2/CO2 à 30 et 40 % volumique en H2 pour une application stationnaire ont révélé des pertes de performances plus importantes pour le mélange au dioxyde carbone, les mélanges à l’argon et à l’azote ont des performances quasiment équivalentes. Ces pertes de performances sont dues à des pertes de surfaces électro actives.Enfin la production de bio hydrogène par PF a montré que le choix de la biomasse, le prétraitement et la souche bactérienne influençaient la qualité du biogaz produit et les performances électrochimiques obtenues à partir de ce dernier sans étapes de purification.Références[1] « Présentation de la stratégie nationale pour le développement de l’hydrogène décarboné en France ». Consulté le: 11 janvier 2024. [En ligne]. Disponible sur: https://www.economie.gouv.fr/presentation-strategie-nationale-developpement-hydrogene-decarbone-france[2] « Le plus gros gisement d’hydrogène naturel du monde vient d’être découvert en France », SudOuest.fr. Consulté le: 11 janvier 2024. [En ligne]. Disponible sur: https://www.sudouest.fr/economie/energie/le-plus-gros-gisement-d-hydrogene-naturel-du-monde-vient-d-etre-decouvert-en-france-17826239.php<br>With the aim of decarbonizing its energy mix and lowering its CO2 emissions, France has decided to invest massively in the decarbonized production of hydrogen as an energy carrier for mobility and stationary applications [1]. Of the one million ton of hydrogen produced in France, 96% is produced by steam reforming of hydrocarbons. France's strategy is to develop the hydrogen sector by investing in the installation of electrolyzers. What's more, the latest discoveries of huge deposits of natural hydrogen (46 million tons of hydrogen in Lorraine) are creating enthusiasm and expanding the field of prospects. [2]. Another decarbonated hydrogen production sector that is less talked about is the biological sector, which offers great potential for diversifying production routes. Hydrogen from these sources raises the question of its quality for use in mobility or stationary fuel cell systems.The aim of this thesis is to define strategies for the use of bio-hydrogen or natural hydrogen using proton exchange membrane fuel cell (PEMFC) technology, from hydrogen production to electrochemical conversion.The first part consisted in studying the impact of impurities or diluents (N2, Ar, He, CH4, CO2) contained in hydrogen from biological and native processes in a half-cell (gas diffusion electrode, GDE). This study was then extended to a single-cell proton exchange membrane fuel cell. Finally, a laboratory-scale biological reactor was used to produce hydrogen from organic sources by photo fermentation (PF), which was then tested in a GDE. Several electrochemical and physicochemical characterization techniques, such as cyclic voltammetry, chrono amperometry, CO stripping for electroactive surface measurement, scanning and transmission electron microscopy, ion chromatography, etc., were used to assess the performance of the PEMFC fed by bio-hydrogen, and its impact on fuel cell components.The results of the electrode activity for the hydrogen oxidation reaction in GDE revealed mass-transport limitation effects for the mixtures, with a particular behavior observed for the nitrogen mixture, and the methane and carbon dioxide mixtures, which in addition to dilution have a carbon monoxide poisoning effect on the electrode.Next, single-cell tests using H2/Ar, H2/N2 and H2/CO2 mixtures at 30 and 40% H2 by volume for stationary applications revealed greater performance losses for the carbon dioxide mixture, while the argon and nitrogen mixtures performed almost equally well. These performance losses are due to electroactive surface losses.Finally, the production of biohydrogen by PF showed that the choice of biomass, pre-treatment and bacterial strain influenced the quality of the biogas produced and the electrochemical performances obtained from it without purification steps.References[1] « Présentation de la stratégie nationale pour le développement de l’hydrogène décarboné en France ». Consulté le: 11 janvier 2024. [En ligne]. Disponible sur: https://www.economie.gouv.fr/presentation-strategie-nationale-developpement-hydrogene-decarbone-france[2] « Le plus gros gisement d’hydrogène naturel du monde vient d’être découvert en France », SudOuest.fr. Consulté le: 11 janvier 2024. [En ligne]. Disponible sur: https://www.sudouest.fr/economie/energie/le-plus-gros-gisement-d-hydrogene-naturel-du-monde-vient-d-etre-decouvert-en-france-17826239.php

Ce travail de thèse porte sur le développement de méthodes de spectrométrie de masse (MS) structurale pour la caractérisation de systèmes protéiques complexes, souvent réfractaires aux approches biophysiques classiques. Dans ce contexte, les développements entrepris furent notamment focalisés sur la caractérisation de complexes impliqués dans la biogénèse des ribosomes et dans la régulation transcriptionnelle, fonctions cellulaires essentielles pouvant être liées à de nombreuses pathologies humaines dont certains cancers. Ainsi, les approches par MS native, pontage chimique et d’HDX-MS ont permis de renseigner sur la connectivité, les proximités spatiales ou encore la dynamique conformationnelle retrouvées au sein des complexes étudiés. Parmi ces techniques, l’HDX-MS permet une approche comparative basée sur les mesures d’incorporations en deutérium renseignant sur la dynamique conformationnelle d’une protéine sous différents états. Aussi, la combinaison d’approches de MS structurale a permis d’approfondir la caractérisation des systèmes complexes étudiés, démontrant ainsi l’intérêt d’une approche intégrative dans ce contexte<br>This PhD thesis focuses on developing methods in structural mass spectrometry (MS) to characterize complex protein systems, given their size and their heterogeneity, frequently inaccessible by classical biophysic approaches. In this context, methodological developments have particularly focused on the characterization of protein complexes involved in ribosomes biogenesis and transcriptional regulation. These fundamental cellular processes are related to numerous diseases such as cancers and genetic diseases. Thus native MS, crosslink, and hydrogen/deuterium exchange coupled to MS (HDX-MS) allowed gaining insights about the stoechiometry, spatial proximities and conformational dynamics of studied systems. Among these approaches, HDX-MS enables a comparative approach based on deuterium incorporation measurements giving information about the conformational dynamics of labeled proteins in various experimental conditions. Finally, the combination of structural approaches enables to deeply characterize complex protein systems, highlighting the advantages of an integrative approach in this context

Ce travail de thèse porte sur des développements méthodologiques en spectrométrie de masse (MS) structurale, principalement par échange hydrogène/deutérium couplé à la MS (HDX-MS), en MS native couplée ou non à la mobilité ionique (IM-MS) et plus récemment en photométrie de masse, pour la caractérisation de différentes protéines d’intérêt thérapeutique. Ce travail a notamment permis de montrer l’apport d’une combinaison d’approches en MS structurale pour la caractérisation approfondie des protéines membranaires, de l’analyse des protéines entières jusqu’à leur dynamique conformationnelle. La mise en place de nouvelles stratégies analytiques en IM-MS et en HDX-MS a également pu être évaluée pour la caractérisation structurale fine d’anticorps thérapeutiques. Enfin, l’intérêt de l’approche HDX-MS a été illustré pour le screening conformationnel et dynamique de ligands dans le cadre de l’étude d’interactions protéine/ligand impliquant des récepteurs nucléaires<br>This PhD work focuses on methodological developments in structural mass spectrometry (MS), especially by hydrogen/deuterium exchange coupled to MS (HDX-MS), by native MS coupled or not to ion mobility (IM-MS) and more recently mass photometry, for the characterization of various proteins of therapeutic interest. In particular, this work has demonstrated the contribution of a combination of structural MS approaches to the in-depth characterization of membrane proteins, from the analysis of intact proteins to their conformational dynamics. The implementation of new analytical strategies in IM-MS and HDX-MS has also been evaluated for the detailed structural characterization of therapeutic antibodies. Finally, the benefits of HDX-MS approach was illustrated for the conformational and dynamic screening of ligands in the context of the study of protein/ligand interactions involving nuclear receptors

Articular cartilage injuries occur frequently in the knee joint. Several methods have been implemented clinically, to treat osteochondral defects but none have been able to produce a long term, durable solution. Photopolymerizable cartilage tissue engineering approaches appear promising; however, fundamentally, forming a stable interface between the tissue engineered cartilage and native tissue, mainly subchondral bone and native cartilage, remains a major challenge. The overall objective of this research is to find a solution for the current problem of dislodgment of tissue engineered cartilage at the defect site for the treatment of degraded cartilage that has been caused due to knee injuries or because of mild to moderate level of osteoarthritis. For this, an in-vitro model was created to analyze the integration of tissue engineered cartilage with the bone, healthy and diseased cartilage over time. We investigated the utility of hydroxyapatite (HA) nanoparticles to promote controlled bone-growth across the bone-cartilage interface in an in vitro engineered tissue model system using bone marrow derived stem cells. We also investigated the application of HA nanoparticles to promote enhance integration between tissue engineered cartilage and native cartilage both in healthy and diseased states. Samples incorporated with HA demonstrated significantly higher interfacial shear strength (at the junction between engineered cartilage and engineered bone and also with diseased cartilage) compared to the constructs without HA (p < 0.05), after 28 days of culture. These findings indicate that the incorporation of HA nanoparticles permits more stable anchorage of the injectable hydrogel-based engineered cartilage construct via augmented integration between bone and cartilage.

Hydrogels are hydrophilic, three dimensional polymers that imbibe large quantities of water while remaining insoluble in aqueous solutions due to chemical or physical cross-linking. The polymers swell in water or biological fluids, immobilizing the bioactive agent, leading to drug release in a well-defined specific manner. Thus the hydrogels’ elastic properties, swellability and biocompatibility make them excellent formulations for drug delivery. Currently, many drug potencies and therapeutic effects are limited or otherwise reduced because of the partial degradation that occurs before the administered drug reaches the desired site of action. On the other hand, sustained release medications release drugs continually, rather than providing relief of symptoms and protection solely when necessary. In fact, it would be much better if drugs could be administered in a manner that precisely matches physiological needs at desired times and at the desired site (site specific targeting). There is therefore an unmet need to develop controlled drug delivery systems especially for delivery of peptide and protein bound drugs. The purpose of this project is to produce hydrogels for structural drug delivery and time-dependent sustained release of drugs (bioactive agents). We use an innovative polymerisation strategy based on native chemical ligation (NCL) to covalently cross-link polymers to form hydrogels. When mixed in aqueous solution, four armed (polyethylene glycol) amine (PEG-4A) end functionalised with thioester and four branched Nterminal cysteine peptide dendrimers spontaneously conjugated to produce biomimetic hydrogels. These hydrogels showed superior resistance to shear stress compared to an equivalent PEG macromonomer system and were shown to be proteolytically degradable with concomitant release of a model payload molecule. This is the first report of a peptide dendrimers/PEG macromonomer approach to hydrogel production and opens up the prospect of facile hydrogel synthesis together with tailored payload release.

L'interaction hydrogene - sulfure est un processus complexe qui conduit a la formation de lacunes anioniques et de groupements s-h, dont la presence est necessaire pour conferer des proprietes catalytiques. L'objectif de ce travail etait de determiner la nature de l'interaction entre l'hydrogene et ces solides en couplant des caracterisations physico-chimiques a des mesures de proprietes catalytiques. Ce travail a ete realise sur le sulfure de ruthenium depose sur zeolithe ky, afin de completer des etudes anterieures et sur mos#2 non supporte, supporte sur alumine et promu par du ni. La phase ruky est constituee de particules 4 fois plus petites que celles observees pour rus#2 depose sur silice ou alumine. Cette difference entraine une reactivite accrue du solide vis-a-vis de l'hydrogene et la chimisorption de h#2 peut etre heterolytique ou homolytique. L'activite des sulfures de ruthenium est maximale lorsque la surface est fortement depeuplee en soufre. Le nombre de rotation est compris entre 0,5 et 1s#-#1 independamment de la taille des particules. Pour mos#2, un traitement sous h#2 a 1073k ne permet d'eliminer qu'une partie des anions soufre situes sur les bords des feuillets de mos#2. La caracterisation des especes h adsorbees indique que celles-ci ont un caractere protonique. L'absence de liaison mo-h suggere un transfert d'electrons depuis la molecule vers le solide. La chimisorption d'hydrogene s'apparente alors a un processus d'oxydoreduction entre l'adsorbat et l'adsorbant. Le depot de cette phase active sur alumine a montre une nette augmentation de sa reactivite vis-a-vis de h#2 conduisant a des rapports h/mo compris entre 0 et 2. Ces rapports eleves suggerent la migration d'une partie de h#2 sur le support. Enfin, l'ajout d'un promoteur tel que le nickel ne modifie pas les proprietes intrinseques de la phase mo, suggerant ainsi que la synergie observee en hydrotraitement n'est pas liee a l'etape d'adsorption de h#2 ou a son activation.

Pour anticiper la fragilisation par l'hydrogène, il faut connaître l'effet des différentes échelles temporelles et spatiales sur les processus de diffusion et de piégeage dans la microstructure du matériau. Dans ce travail, nous examinons l'impact de différents types de jonctions triples (TJ) et de joints de grains (GB) et de leur connectivité sur la diffusion de l'hydrogène. Tout d'abord, nous proposons quatre algorithmes pour contrôler la distribution des fractions de TJ en fonction d'une fraction donnée de joints de grains aléatoires. Ensuite, à l'aide de la modélisation par éléments finis, nous étudions les effets de la distribution des jonctions triples sur la diffusivité de l'hydrogène. Nous découvrons des relations remarquables entre la connectivité des joints de grains et le coefficient de diffusion effectif en quantifiant les réseaux de joints de grains dans des microstructures idéalisées en 2D. De plus, une plus grande diffusivité est reflétée dans les matériaux nanocristallins. Nos résultats prouvent qu'ils reproduisent les chemins de connectivité des GB en contraignant la distribution. En outre, des microstructures EBSD reconstruites en 2D ont été simulées et comparées aux données expérimentales. Ensuite, des simulations de microstructures idéalisées en 3D ont été réalisées, et les effets de la connectivité des jonctions triple ont été étudiés. Nous montrons qu'il existe de forte corrélation entre la connectivité des jonctions triple et le coefficient de diffusion effectif, en particulier dans les matériaux nanocristallins. Les simulations 3D s'approchent plus précisément des résultats expérimentaux tant que l'impact de la connectivité des TJ est inclus<br>Anticipating hydrogen embrittlement requires knowledge of the effect of different time and spatial scales within diffusion and trapping processes in the material's microstructure. In this work, we examine the impact of various types of triple junctions (TJ) and grain boundaries (GB) and their connectivity on the diffusion of hydrogen. First, we propose four algorithms to control the distribution of TJ fractions depending on a given fraction of Random grain boundaries. Then, using finite element modeling, we investigate the effects of triple junction distribution on hydrogen diffusivity. We discover remarkable relationships between grain boundary connectivity and the effective diffusion coefficient by quantifying grain boundary networks in 2D idealized microstructures. In addition, greater diffusivity is reflected in nanocrystalline materials. Our results prove to reproduce GB connectivity paths by compelling the distribution. Furthermore, 2D reconstructed EBSD microstructures were simulated and compared to experimental data. Following that, 3D simulations of idealized microstructures were performed, and the effects of triple junction connectivity were investigated. We show that there is strong correlation between the connectivity of the triple junction and the effective diffusion coefficient, particularly in nanocrystalline materials. 3D simulations approach the experimental results more accurately as the impact of the connectivity of TJs is included

Made available in DSpace on 2016-06-02T18:55:28Z (GMT). No. of bitstreams: 1 6339.pdf: 1320900 bytes, checksum: ceafdc5598f1fbb6c687c57d58657bc1 (MD5) Previous issue date: 2014-08-07<br>Universidade Federal de Sao Carlos<br>The seasonal semideciduous forests from southeastern Brazil have high plant species richness, but have been strongly affected by human interferences, which let them very degraded: nowadays they are represented by small isolated fragments, immersed in a landscape dominated by agriculture and large urban centers. In this sense, the ecology of restoration arises in order to facilitate, accelerate and direct the processes of secondary succession to manage such areas, considering the knowledge about theoretical concepts that aim to review the effectiveness of natural ecosystem management techniques and practices. In such a perspective, we aimed in this experiment to test the following question: During de production of seedlings of the native forest tree species, Ceiba speciosa (Malvaceae) and Handroanthus heptaphyllus (Bignoniaceae), different dilutions of hydrogel associated to irrigation with reused water, could improve their development? To answer this question, we selected three treatments (1g, 4g and 8g of hydrogel), beyond the witness (no hydrogel). The seedlings of both species were irrigated during one month with water reused and water supply. After this period, we estimated the growth of seedlings: roots, height and diameter. We used ANOVA (p = 0.001), and Tukey s test to verify significative differences between the mean of the treatments. We found significative differences in both height and diameter growth from 4g of hydrogel as to C. speciosa as H. heptaphyllus. In relation to the growth of root system of both tree species, the mean of superficial root area differed significantly from 4g of hydrogel when compared to the witness, confirming our initial assumption that the roots would grow in a lower proportion of area because they are in their hydric comfort zone. With such results added to theoretical knowledge in the ecology of restoration, the association of hydrogels with reused water comes to stand up for the sustainable use of water in the irrigation of native seedlings.<br>Apesar das florestas estacionais semideciduais do interior do estado de São Paulo possuir elevada riqueza de espécies, as interferências humanas deixaram esses ecossistemas degradados e representados por pequenos fragmentos isolados, imersos numa paisagem dominada pela agricultura e centros urbanos. Diante essa problemática, a restauração ecológica surge com o objetivo de facilitar, acelerar e direcionar os processos sucessionais naturais a fim de manejar essas áreas, considerando os novos teóricos da ecologia de comunidades vegetais tropicais, que pretendem revisitar a eficácia de técnicas de manejo em restauração ecológica. Nesta perspectiva, surge como objetivo desse trabalho, testar a pergunta científica: O uso de polímeros hidrogéis em diferentes concentrações na produção de mudas nativas de Ceiba speciosa (Malvaceae) e de Handroanthus heptaphyllus (Bignoniaceae), associados à irrigação com água de reúso tratada, interfere significativamente no crescimento de seu sistema radicular, sua altura e diâmetro, e aumenta a longevidade das mudas? Para responder a essa pergunta, utilizou-se mudas das duas espécies nativas, em três tratamentos (1g, 4g e 8g de hidrogel), além da uma testemunha, contendo apenas o solo. As mudas foram irrigadas por um mês com água de abastecimento e água de reúso tratada; depois de cessadas as irrigações, foram avaliadas os estimadores citados. Foram realizadas análises de variância (p = 0,001), seguidas do teste de Tukey para verificar diferenças significativas entre as médias dos tratamentos. Verificaram-se diferenças significativas no crescimento em altura e diâmetro a partir do uso de 4g de hidrogel, tanto para C. speciosa quanto para H. heptaphyllus. Em relação ao sistema radicular, as médias de área superficial diferiram-se estatisticamente a partir de 4g de hidrogel quando comparadas às testemunhas, tanto para C. speciosa quanto para H. heptaphyllus, confirmando a expectativa inicial de que com hidrogel, as raízes cresceriam em menor proporção em área porque estariam na zona de conforto hídrico. Com esses resultados aliados aos conhecimentos teóricos da ecologia da restauração, a associação de polímeros hidrogéis com água de reúso vem defender a racionalização do uso da água na irrigação de mudas de espécies nativas, reaproveitando águas de reúso que seriam descartadas em águas superficiais.

The global incidence of bone fractures, and subsequently that of non-healing ones, is expected to rise in the coming decades, mostly due to an increased risk of age-related conditions. Currently, the biomaterials field is moving towards the design of scaffolds mimicking the cell microenvironment to guide stem cells differentiation and recapitulate the development of target tissues. Biomimicry is a wide concept and several approaches have been adopted to produce cellinstructive scaffolds. Herein, we have explored the use of citric acid and lysyl oxidase, both of them related to bone nanostructure and mechanical performance, to develop scaffolds resembling the extracellular matrix of developing bone. First, elastin-like recombinamers (ELRs) hydrogels were achieved through a one-step chemical crosslinking reaction with citric acid, a molecule currently considered to be essential for the proper performance of bone tissue. By systematically studying the crosslinking reaction and its contribution to hydrogel properties, we were able to control the architecture and stiffness of citric acid-crosslinked hydrogels while preserving the integrity of adhesion sequences in ELRs. Interestingly, the use of citric acid conferred so-produced hydrogels the ability to nucleate calcium phosphate. Mechanically-tailored citric acid-crosslinked hydrogels were shown to be able to support the growth of human mesenchymal stem cells and to lead to seemingly biocompatible degradation products. Despite in vitro differentiation studies weren¿t conclusive as to their osteogenic potential, both mechanically-tailored and non-tailored citric acid-crosslinked hydrogels were shown to integrate into bone and to be partially degraded upon implantation in critical size defects in mouse calvaria. Despite cell invasion in mechanically-tailored scaffolds was seemingly lower than in non-tailored counterparts, both types of matrices allowed the formation of bone tissue, by intramembranous ossification, to a similar extent by the end of the study. At the time points selected for the in vivo study, both tailored and non-tailored hydrogels were found to be osteoconductive; osteoinduction was not observed in any of the cases. Mechanically-tailored hydrogels not being seemingly superior to control matrices at selected time points could to be due to (i) a high surface polymer density hindering cell invasion and thus delaying osteoinduction, or to (ii) a non-osteoinductive combination of properties (chemical + physical) despite hydrogels possessing theoretically osteoinductive stiffness. These results point out that scaffolds must be seen as a whole given the high complexity of the in vivo cell niche, whose signals act synergistically to define cell behavior. Thus, more complex designs are required if recapitulation of bone development is to be targeted. Additionally, recombinant lysyl oxidase (LOX) from human aorta was successfully produced in Escherichia coli to high purity. Despite achieving LOX with copper cofactor amounts and activity higher than those found in the literature, overall activity was low and the insolubilization of ELRs was not achieved, suggesting that novel expression and purification systems not compromising enzymatic activity are required if LOX is to be used to produce scaffolds.<br>S'espera que la incidència global de fractures òssies, i per extensió la d'aquelles que no són capaces de consolidar per si soles, augmenti en les pròximes dècades, principalment degut a l'increment del risc de patologies associades a l'envelliment. Actualment, el camp dels biomaterials es mou cap al disseny de bastides que mimetitzen el microambient cel·lular per tal de guiar la diferenciació de cèl·lules mare i recapitular el desenvolupament de teixits diana. El biomimetisme és un concepte ampli i diverses aproximacions han sigut dutes a terme per tal de produir bastides capaces de guiar el comportament cel·lular. En aquesta tesi hem explorat l'ús d'àcid cítric i l'enzim lisil oxidasa, ambdós relacionats amb la nanoestructura i propietats mecàniques del teixit ossi, per desenvolupar bastides que mimetitzin la matriu extracel·lular de l'os en desenvolupament. En primer lloc, es va produir hidrogels basats en polímers recombinants de tipus elastina (Elastin-like recombinamers; ELRs) mitjançant una reacció d'entrecreuament en un pas amb àcid cítric, una molècula actualment considerada una peça essencial per l'adequat funcionament mecànic del teixit ossi. Havent fet un estudi sistemàtic de la reacció d'entrecreuament i de la seva contribució a les propietats dels hidrogels, vam ser capaços de controlar l'arquitectura i la rigidesa de les bastides entrecreuades amb àcid cítric, tot preservant la integritat de les seqüències d'adhesió cel·lular contingudes als ELRs. És interessant remarcar que l'ús d'àcid cítric com agent entrecreuant conferí als hidrogels capacitat de nucleació de fosfats de calci. Es va demostrar que els hidrogels entrecreuats amb àcid cítric i amb propietats mecàniques diana permeten el creixement de cèl·lules mare mesenquimals humanes i donen lloc a productes de degradació aparentment biocompatibles. Tot i que els estudis de diferenciació in vitro no van ser concloents pel que fa al potencial osteogènic d'aquestes bastides, tant les matrius amb propietats mecàniques diana com aquelles control van ser capaces d'integrar-se amb l'os natiu i van ser parcialment degradades un cop implantades en defectes de mida crítica en calotes de ratolí. Tot i que la invasió cel·lular en hidrogels amb propietats mecàniques diana va ser inferior en comparació amb l'observada en bastides control, ambdós tipus de matrius van permetre la formació d'os nou, mitjançant ossificació intramembranosa, en quantitats similars al final de l'estudi. Als punts temporals seleccionats, ambdós tipus de bastides van demostrar ser osteoconductives, però no es va observar evidències d'osteoconducció en cap cas. El fet que les bastides amb propietats mecàniques diana no fossin superiors a les matrius control pel que fa a formació òssia, podria ser degut a (i) a una elevada densitat superficial que hauria dificultat la invasió cel·lular i retrassat l'osteoinducció, o (ii) a una combinació de propietats (químiques + físiques) no osteoinductiva tot i que les bastides posseïen una rigidesa teòricament osteoinductiva. Aquests resultats demostren que les bastides han de ser vistes com un tot donada l'elevada complexitat del nínxol de cèl·lules mare in vivo, les senyals del qual actuen de forma sinèrgica per definir el comportament cel·lular. Així, és necessari assolir bastides amb un nivell de complexitat més elevat per tal de recapitular el desenvolupament ossi. Addicionalment, es va produir lisil oxidasa (LOX) d'aorta humana en forma recombinant a elevada puresa a partir de cultius d'Escherichia coli. Tot i que es va aconseguir produir LOX amb un contingut del cofactor coure i una activitat superiors a aquells trobats en la literatura, l'activitat de l'enzim va ser generalment baixa i no es va assolir la insolubilització d'ELRs. Això evidencia la necessitat de desenvolupar nous sistemes d'expressió i purificació de l'enzim per tal que aquest pugui ser aplicat a la producció de bastides.

Made available in DSpace on 2017-07-21T18:53:16Z (GMT). No. of bitstreams: 1 Fernanda Janaina Oliveira Gomes da Costa.pdf: 2530982 bytes, checksum: 65f2e845c0bdca1384a01a29d20c0298 (MD5) Previous issue date: 2010-02-08<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior<br>Starch is an abundant carbohydrate in nature, can be extracted from several botanical sources and has many industrial applications, mainly in the food sector.Due to some limitations of native starch, the production of modified starch has been an alternative to overcome these restrictions and promote increase in use of this polymer. This study used techniques thermoanalitycal TG, DTA and DSC for the evaluation of thermal properties (stability, gelatinization and enthalpy). Also has been applied optical microscopy and X-ray diffraction in samples of native cassava starch, extracted in laboratory and commercial sample wich were modified by action of H2O2 in different concentrations (1, 2 and 3%) with and without addition of the catalyst FeSO4 (0,01%). The results of thermal analysis showed that the onset temperature (To) and peak (Tp), and enthalpy of gelatinization (ΔHgel) vary with the degree of oxidation. The native starch granules showed characteristic morphology and only showed small changes in morphology after treatment with H2O2 in the concentration of 3%. Through X-ray diffraction can be seen the typical crystallinity of starches and were observed few changes in the patterns of crystallinity of the modified starches.<br>O amido é um carboidrato abundante na natureza, pode ser extraído de diversas fontes botânicas e possui muitas aplicações industriais, principalmente no ramo alimentício. Devido a algumas limitações dos amidos nativos, a produção de amidos modificados tem sido uma alternativa para superar tais restrições e promover o aumento da utilidade deste polímero. Neste estudo foram utilizadas técnicas termoanaliticas TG, DTA e DSC para a avaliação das propriedades térmicas (estabilidade, gelatinização e entalpia). Também foram aplicadas microscopia óptica e difratometria de raios X nas amostras de amido de mandioca nativo, tanto extraídos em laboratório quanto comercial, que foram modificados pela ação de H2O2 em diferentes concentrações (1, 2 e 3%) com ou sem a adição do catalisador FeSO4 (0,01%). Os resultados da análise térmica mostraram que as temperaturas onset (To) e de pico (Tp) e a entalpia de gelatinização (ΔHgel) variam de acordo com o grau de oxidação. Os grânulos do amido nativo apresentaram morfologia característica e somente apresentaram pequenas alterações após tratamento com H2O2 na concentração de 3%. Através da difratometria de raios X pode-se perceber a cristalinidade típica de amidos e para os amidos modificados foram observadas poucas alterações nos padrões de cristalinidade.

Il concetto di rete dell'informazione può diventare uno schema logico con cui descrivere l'evoluzione delle politiche sulle energie rinnovabili e sulla sostenibilità? La ricerca è stata svolta analizzando l'architettura delle due reti (internet e reti energetiche) e l'evoluzione del bene prodotto e distribuito nella rete energetica, l'energia, esplicitando l'accessibilità da parte della distribuzione mondiale delle risorse petrolifere tradizionali e delle risorse rinnovabili. La struttura metodologica del progetto di ricerca si basa due tipi di analisi teorica: 1) l'analisi della nascita delle società in rete attraverso le teorie di Manuel Castells (concetto di spazio di flussi) e di Saskia Sassen e l'evoluzione delle città (cap.2 e cap.5) 2) le analisi dei flussi dei materiali e delle energie avendo come riferimento metodologico l'approccio ecologico ideato dai ricercatori dell'istituto per il Clima, l'Ambiente e l'Energia di Wuppertal, Germania (cap.3 e cap.4) La contraddizione tra città innovative e città che sono ai livelli di enormi discariche o di baraccopoli è esposta nel cap.6 attraverso casi studio e progetto dei Programmi Europei. Nell'ultimo capitolo (cap.7) si riassumono le ipotesi di partenza e i risultati della ricerca e si espongono le questioni aperte.<br>Can internet logic scheme be used as a basis to describe public policies evolution on renewable energies production and sharing in urban areas all over the world? The research project analyses the two networks (internet and energetic grids) architectures in actual and future urban areas. This analysis is connected with present and future forecasts energy productions from traditional fuels and from renewable sources. Theoretical analysis is conducted following a double conceptual pathway: - societal networks (Manuel Castells theory) and urban areas evolution (Saskia Sassen and Mike Davis) in order to picture the evolution of cities and towns in modern economies and in developing countries (Chapters 2 and 5); - Material and Energy Flow Analysis (approach by Wuppertal Institute for Climate, Environment and Energy) applied to renewable energy (Chapters 3 and 4) In Chapter 6 case studies are exposed on the deep cleavage between two different worlds: innovative, rich towns on a side and the landfills cities, slums on the other side. In the last part hypothesis and thesis are put together and open questions are explained (Chapter 7).

Il concetto di rete dell'informazione può diventare uno schema logico con cui descrivere l'evoluzione delle politiche sulle energie rinnovabili e sulla sostenibilità? La ricerca è stata svolta analizzando l'architettura delle due reti (internet e reti energetiche) e l'evoluzione del bene prodotto e distribuito nella rete energetica, l'energia, esplicitando l'accessibilità da parte della distribuzione mondiale delle risorse petrolifere tradizionali e delle risorse rinnovabili. La struttura metodologica del progetto di ricerca si basa due tipi di analisi teorica: 1) l'analisi della nascita delle società in rete attraverso le teorie di Manuel Castells (concetto di spazio di flussi) e di Saskia Sassen e l'evoluzione delle città (cap.2 e cap.5) 2) le analisi dei flussi dei materiali e delle energie avendo come riferimento metodologico l'approccio ecologico ideato dai ricercatori dell'istituto per il Clima, l'Ambiente e l'Energia di Wuppertal, Germania (cap.3 e cap.4) La contraddizione tra città innovative e città che sono ai livelli di enormi discariche o di baraccopoli è esposta nel cap.6 attraverso casi studio e progetto dei Programmi Europei. Nell'ultimo capitolo (cap.7) si riassumono le ipotesi di partenza e i risultati della ricerca e si espongono le questioni aperte.<br>Can internet logic scheme be used as a basis to describe public policies evolution on renewable energies production and sharing in urban areas all over the world? The research project analyses the two networks (internet and energetic grids) architectures in actual and future urban areas. This analysis is connected with present and future forecasts energy productions from traditional fuels and from renewable sources. Theoretical analysis is conducted following a double conceptual pathway: - societal networks (Manuel Castells theory) and urban areas evolution (Saskia Sassen and Mike Davis) in order to picture the evolution of cities and towns in modern economies and in developing countries (Chapters 2 and 5); - Material and Energy Flow Analysis (approach by Wuppertal Institute for Climate, Environment and Energy) applied to renewable energy (Chapters 3 and 4) In Chapter 6 case studies are exposed on the deep cleavage between two different worlds: innovative, rich towns on a side and the landfills cities, slums on the other side. In the last part hypothesis and thesis are put together and open questions are explained (Chapter 7).

Contraction of cardiac muscle is a highly regulated event that relies on a delicate balance of ions entering and leaving the cell through ion channels. In particular, voltage gated sodium channels are responsible for the rapid depolarization that leads to a contraction. During an oxidative challenge, sodium channels rapidly activate, but do not fully turn off. This alters the rate of cardiac repolarization and can induce cardiac arrhythmias. It is currently unknown whether the most common sodium channel isoform found in the heart, NaV1.5, generates this oxidant-induced persistent current or if other isoforms are responsible. Therefore, I sought to further explore the biophysical properties NaV1.5, and determine if it can enter this persistent mode. I tested the biophysical properties of native INa in cardiac myocytes and in NaV1.5 transfected HEK293 cells under macro cell-attached voltage-clamp. I used a sodium channel enhancer (Anemonia sulcata toxin II; 10 nM), a sodium channel blocker (tetrodotoxin; 10 nM) and a model of oxidative stress (H2O2; 100 µM, 200 µM, 1000 µM) to compare and contrast the cellular responses between both cell types. I observed that transfected HEK293 cells and cardiac myocytes were unaffected by H2O2 at various concentrations. Given the lack of other isoforms in transfected HEK293 cells, and the low abundance (<5%) of other isoforms in cardiac myocytes, I propose that NaV1.5 function is unaffected by H2O2. Furthermore, ATX II prolonged the inactivation process in both HEK293 cells and cardiac myocytes in a voltage-dependent manner, indicating that NaV1.5 can give rise to persistent sodium current. Finally, by comparing both cell types under control settings, I found that transfected HEK293 cells inactivated at a much slower rate and at more negative potentials compared to the current in cardiac myocytes. My results suggest that NaV1.5 does not underlie oxidant-induced persistent current and that β subunits likely play a significant role in the inactivation process.<br>Thesis (Master, Physiology) -- Queen's University, 2011-08-19 14:46:42.665

Xylanases are xylan-degrading enzymes, belong to glycosyl hydrolases (GH). Xylanases from the two major families 10 (GH10) and 11 (GH11) catalyze the hydrolysis of internal β-, bonds of xylan backbone. Xylan is the second most abundant polysaccharide in nature. Nearly one third of the dry weight of the higher plants is xylan and therefore, xylanases have an important role in biomass conversions. Currently, the most effective application of xylanases is in prebleaching of kraft pulp to minimize the use of environmentally hazardous chemicals in the subsequent treatment stages. In recent years, therefore, attention is focused on to isolate and/or engineer the xylanases for the industrial requirements. The desirable roperties of xylanases in paper industry are stability and activity at high temperatures and alkaline pH. While he factors responsible for the thermal stability of GH10 xylanases have been analyzed, factors governing the alkaline stability of GH10 xylanases remain poorly understood. The present thesis reports the crystal structures of an alkali thermostable GH10 extracellular endo-xylanase (BSX) from an alkalophilic organism, Bacillus sp. NG-27 in free and xylosaccharides-bound form. The enzyme was purified from the native organism and crystallized. The structure was solved by molecular replacement method. The 2.2 Å crystal structure of the native BSX enzyme is the first structure of an alkali thermostable GH10 family xylanase from an alkalophilic organism. It has unveiled unique protein properties that can form the basis for improving the thermal, alkaline stability and activity by site directed mutagenesis. The comparative study, especially in relation to GH10 xylanases, deciphered important structural features which are likely to be responsible for the alkaline stability of the enzyme. The work exemplifies the mechanism of adaptation of enzymes to function under polyextreme conditions through changes in the nature and composition of solvent-exposed residues. As apparent from the comparative study, the enhanced stability of the protein can be attributed to the surface rich in acidic residues and less number of solvent-exposed Asn as seen in BSX. This situation which may be roughly described as “acidic residues outside and Asn inside”, is a notable feature of alkali-stable GH10 xylanases from alkalophilic organisms. In addition, the candidate has carried out the comprehensive database analysis of the occurrence of C-H…O hydrogen bonds in helices and helix termini of globular proteins. The study provides a compelling evidence that the main-chain Cα and the side-chains CH which participate in C-H…O hydrogen bonds collectively augment the cohesive energy and thereby contribute together with the classical N-H…O hydrogen bonds and other interactions to the overall stability of helix and therefore of proteins. Chapter 1 starts with a brief introduction of xylanases, their classifications and overall folds. At present, a little more than a dozen of crystal structures of GH10 xylanases are known and described in the literature. A brief mention about these structures and their optimum pH and temperature is outlined under a separate section. In view of the industrial importance of the study enzyme, the potential industrial and biotechnological applications of xylanases are detailed in this Chapter. A section is dedicated to describe the present study enzyme BSX, an alkali thermostable endo-xylanase from an alkalophilic bacterium, Bacillus sp. NG-27. BSX has a molecular mass of ~41 kDa and is optimally active at 343 K and at a pH of 8.4. The alkaline thermostability of the wild type BSX is likely to be industrially important. At the end, the scope of the present work is detailed. Chapter 2 presents the purification of xylanase (BSX) from Bacillus sp. NG-27, the crystallization of the native and xylosaccharides-bound BSX, the X-ray diffraction data collection on these crystals and processing of the data. Repeated attempts to crystallize the protein expressed in the chloroplast of transgenic tobacco plant were unsuccessful. However, crystallization was achieved with the protein sample purified from the native source by hanging drop vapour diffusion method. Crystals were grown at both acidic (4.6) and basic pH (8.5). The corresponding crystallization conditions are 0.2 M MgCl2, 0.1 M sodium acetate pH 4.6 and 20% PEG 550 MME and 0.1 M aCl, 0.01 M MgCl2, 0.1 M Tris-HCl pH 8.5 and 15% PEG 8000. Crystals grown at acidic pH were not suitable for X-ray diffraction study. Subsequently, crystal obtained at a basic pH of 8.5 was used for X-ray data collection and it diffracted X-rays to better than 2.2 Å at the home source at cryo-temperature (100 K). Native BSX crystals belong to monoclinic space group C2 with unit cell parameters a = 174.5 Å, b = 54.7 Å, c = 131.5 Å and β = 131.2°. Crystals of xylosaccharides-bound enzyme were grown in a slightly modified crystallization condition of native, 0.1 M NaCl, 0.2 M MgCl2, 0.1 M Tris-HCl pH 8.5 and 15% PEG 8000 and the enzyme was incubated with xylan prior to setting up the crystallization. Crystals belong to primitive orthorhombic space group P212121 with unit cell parameters a = 59.2 Å, b = 83.8 Å and c = 174.4 Å. A data set was collected using synchrotron radiation of wave length 1.0 Å from a cryo-cooled crystal at Spring-8 BL26B1 beam line, Japan. The Matthews coefficient VM for native and xylosaccharides- bound crystals was calculated to be 2.8 and 2.7 Å3 Da-1, respectively, suggesting two molecules in each crystal asymmetric unit. No twinning was detected in both the datasets and the overall quality of the data sets was found to be good. Chapter 3 details the application of molecular replacement method to the structure solution of native and xylosaccharides-bound BSX, the course of iterative model building and the refinement carried out, and the quality of the final protein structure models. The native-enzyme structure solution was obtained by the molecular replacement method using as a search model the crystal structure (PDB code 1hiz) of the closest homologous, extracellular xylanase (GSX) from Geobacillus stearothermophilus. No non- crystallographic symmetry (NCS) restraint was applied between the two independent molecules in the crystal asymmetric unit at the final round of refinement. The final positional refinement of native BSX converged to R factors of R = 19.4% and Rfree = 23.5% for data between 20.0 to 2.2 Å. The final native model consists of 5704 protein atoms, two Mg2+ ions and 721 solvent water molecules. The final native model was taken as the search structure for the xylosaccharides-bound BSX and a solution with a correlation coefficient of 70.7% and an R-factor of 32.1% was obtained from the molecular replacement calculation. Unlike the native structure refinement, NCS restraint was imposed at all stages of the refinement. Bound xylosaccharides were clearly visible inthe difference Fourier electron density maps. The last round of refinement gave a model with R and Rfree of 21.8% and 25.7%, respectively. The final xylosaccharides-bound model consists of 5766 protein atoms, four Mg2+ ions, 85 atoms belong to bound xylosaccharides and 523 solvent water molecules. No residues were found in the disallowed region of the Ramachandran (φ, ψ) map for both the structures. Chapter 4 describes the native and xylosaccharides-bound BSX crystal structures and the structural comparison of BSX with other GH10 family xylanase crystal structures for which the optimum temperature and pH are known in the literature. BSX folds as the ubiquitous (β/α)8-barrel, a common structural superfold characteristic of GH10 xylanases. The two active site glutamic acid residues, Glu149 and Glu259, are located on opposite sides of the active site cleft and their side-chains are at a distance of 5.5 Å apart suggesting the enzymatic reaction takes place by the retaining mechanism. From the structural superposition of other xylotriose-bound xylanase structures on to the xylosaccharides-bound BSX, structural plasticity in the xylotriose binding can be inferred, implying that the xylose recognition at the subsite -3 displays plasticity and is less specific as opposed to that at -1 and -2 subsites. The stacking interaction of one of the xylose moieties of the xylobiose with the Trp235 seen in BSX provides, for the first time, a structural evidence for the direct involvement of Trp235 in xylosaccharides binding. The crystal structure revealed a metal binding site, found at the C-terminal end of catalytic domain. The presence of metal binding site was not anticipated from earlier theoretically modeled structure and biochemical studies. Further, we have shown experimentally the requirement of Mg2+ ion for the enzyme activity. We havedescribed a novel WP sequence-structure-interaction motif which is present in the (+) side of the active site region and presumably helps in the efficient binding of the carbohydrate moiety of the xylan in the active site cleft of BSX. The structural comparison of BSX with other GH10 xylanases solved to date and characterized to be active at a pH close to neutral was done for the first time. The comparative study revealed the essential structural features which may responsible for the alkaline stability of GH10 xylanases.Briefly, the alkalophilic GH10 xylanases from alkalophilic organisms have surface abundant in acidic residues, the heat and alkaline susceptible residue Asn depleted on the protein surface and increased number of salt bridges. Our study has unveiled the role of the nature and composition of protein surface amino acids in the adaptation of enzymes to polyextreme conditions. The observations reported in the thesis provide important lessons for engineering alkaline stability in xylanases for industrial applications and in general for the understanding of alkaline stability in related proteins. A comparison of the surface features of the BSX and of halophilic proteins allowed us to predict the activity of BSX at high salt concentrations, which we verified through experiments. This offered us important lessons in polyextremophilicity of proteins, where understanding structural features of a protein stable in one set of extreme conditions provided clues about the activity of the protein in other extreme conditions. Chapter 5 summaries the important findings of the present study from the crystal structural analysis of BSX and its comparison with non-alkalophilic GH10 xylanases. Separate sections are made on conclusions and future prospects for the study on BSX. Chapter 6 describes the comprehensive database analysis of C-H…O hydrogen bond in helices of globular proteins. The C-H…O hydrogen bonds found in helices are predominantly of type 5 → 1 or 4 → 1.Our analysis reveals that the Cγ and Cβ hydrogen atom(s) are frequently involved in such hydrogen bonds. A marked preference is noticed for aliphatic β-branched residue Ile to participate in 5 → 1 C- H…O hydrogen bonds involving methylene Cγ1 atom as donor in α-helices. In addition, C-H…O hydrogen bonds are present along with helix stabilizing salt bridges and to some extent compensate for the side-chain conformational entropy loss. Our analysis highlights that a multitude of local C-H…O hydrogen bondsformed by a variety of amino acid side-chains and Cα hydrogen atoms occur in helices and more so at the helix termini. A majority of the helix favouring residues, Met, Glu, Arg, Lys, Leu and Gln which also have large side-chains with more donatable CH groups, have significant propensity to form side-chain to main-chain C-H…O hydrogen bonds in helix. The large side-chains are marked by their ability to shield from the solvent the polar atoms of the peptide backbone and at the same time participate in weak cohesive C-H…O interactions in the helix. This chapter also details the identification for the first time a novel chain reversal motif stabilized by 1 → 5 Cα-H…O interactions. The importance of these hydrogen bonds with respect to helix stability is discussed in the final section of the chapter. Appendix A details the crystallographic and structural analyses oftwares used for the present thesis work. Appendix B describes, in addition to the crystal structure analysis of BSX, the work carried out by the candidate on a comparative study of a thermostable xylanase from Thermoascus aurantiacus, solved in our laboratory at atomic 1.11 Å (293 K) and ultrahigh 0.89 Å (100 K) resolutions. From the comparison, we have for the first time pointed out the possibility of plasticity of ion pairs in proteins with water molecules mediating some of the alternate arrangements. The αβ-loops are relatively less flexible than the βα-loops. The β-strands are least affected structurally with the increase in temperature. Thus the TIM barrel fold in the study enzyme, though having a single domain, may be dissected into parts based on the relative flexibility and described as having a rigid core constituted by the β-barrel and a less rigid exterior formed by the surrounding α-helices. Appendix C presents the crystallization and the preliminary X-ray characterization work done by the author of the thesis on an alkali thermostable cellulase enzyme from Thermomonospora sp. The protein is an extracellular enzyme with molecular mass of 14.2 kDa and interestingly, has the dual activity for both cellulose and xylan. The primary structure of the enzyme is not known. The enzyme was purified from the source organism and crystallized. A complete diffraction data set was collected and processed to 2.3 Å in an orthorhombic space group P212121. Appendix D contains tables which give details about the analysed 5 → 1 Cα- H…O hydrogen bonds in helices and a novel chain reversal motif with 1 → 5 Cα-H…O hydrogen bonds. Appendix E encloses reprints of publications which have resulted from the work reported in the thesis.

Xylanases are xylan-degrading enzymes, belong to glycosyl hydrolases (GH). Xylanases from the two major families 10 (GH10) and 11 (GH11) catalyze the hydrolysis of internal β-, bonds of xylan backbone. Xylan is the second most abundant polysaccharide in nature. Nearly one third of the dry weight of the higher plants is xylan and therefore, xylanases have an important role in biomass conversions. Currently, the most effective application of xylanases is in prebleaching of kraft pulp to minimize the use of environmentally hazardous chemicals in the subsequent treatment stages. In recent years, therefore, attention is focused on to isolate and/or engineer the xylanases for the industrial requirements. The desirable roperties of xylanases in paper industry are stability and activity at high temperatures and alkaline pH. While he factors responsible for the thermal stability of GH10 xylanases have been analyzed, factors governing the alkaline stability of GH10 xylanases remain poorly understood. The present thesis reports the crystal structures of an alkali thermostable GH10 extracellular endo-xylanase (BSX) from an alkalophilic organism, Bacillus sp. NG-27 in free and xylosaccharides-bound form. The enzyme was purified from the native organism and crystallized. The structure was solved by molecular replacement method. The 2.2 Å crystal structure of the native BSX enzyme is the first structure of an alkali thermostable GH10 family xylanase from an alkalophilic organism. It has unveiled unique protein properties that can form the basis for improving the thermal, alkaline stability and activity by site directed mutagenesis. The comparative study, especially in relation to GH10 xylanases, deciphered important structural features which are likely to be responsible for the alkaline stability of the enzyme. The work exemplifies the mechanism of adaptation of enzymes to function under polyextreme conditions through changes in the nature and composition of solvent-exposed residues. As apparent from the comparative study, the enhanced stability of the protein can be attributed to the surface rich in acidic residues and less number of solvent-exposed Asn as seen in BSX. This situation which may be roughly described as “acidic residues outside and Asn inside”, is a notable feature of alkali-stable GH10 xylanases from alkalophilic organisms. In addition, the candidate has carried out the comprehensive database analysis of the occurrence of C-H…O hydrogen bonds in helices and helix termini of globular proteins. The study provides a compelling evidence that the main-chain Cα and the side-chains CH which participate in C-H…O hydrogen bonds collectively augment the cohesive energy and thereby contribute together with the classical N-H…O hydrogen bonds and other interactions to the overall stability of helix and therefore of proteins. Chapter 1 starts with a brief introduction of xylanases, their classifications and overall folds. At present, a little more than a dozen of crystal structures of GH10 xylanases are known and described in the literature. A brief mention about these structures and their optimum pH and temperature is outlined under a separate section. In view of the industrial importance of the study enzyme, the potential industrial and biotechnological applications of xylanases are detailed in this Chapter. A section is dedicated to describe the present study enzyme BSX, an alkali thermostable endo-xylanase from an alkalophilic bacterium, Bacillus sp. NG-27. BSX has a molecular mass of ~41 kDa and is optimally active at 343 K and at a pH of 8.4. The alkaline thermostability of the wild type BSX is likely to be industrially important. At the end, the scope of the present work is detailed. Chapter 2 presents the purification of xylanase (BSX) from Bacillus sp. NG-27, the crystallization of the native and xylosaccharides-bound BSX, the X-ray diffraction data collection on these crystals and processing of the data. Repeated attempts to crystallize the protein expressed in the chloroplast of transgenic tobacco plant were unsuccessful. However, crystallization was achieved with the protein sample purified from the native source by hanging drop vapour diffusion method. Crystals were grown at both acidic (4.6) and basic pH (8.5). The corresponding crystallization conditions are 0.2 M MgCl2, 0.1 M sodium acetate pH 4.6 and 20% PEG 550 MME and 0.1 M aCl, 0.01 M MgCl2, 0.1 M Tris-HCl pH 8.5 and 15% PEG 8000. Crystals grown at acidic pH were not suitable for X-ray diffraction study. Subsequently, crystal obtained at a basic pH of 8.5 was used for X-ray data collection and it diffracted X-rays to better than 2.2 Å at the home source at cryo-temperature (100 K). Native BSX crystals belong to monoclinic space group C2 with unit cell parameters a = 174.5 Å, b = 54.7 Å, c = 131.5 Å and β = 131.2°. Crystals of xylosaccharides-bound enzyme were grown in a slightly modified crystallization condition of native, 0.1 M NaCl, 0.2 M MgCl2, 0.1 M Tris-HCl pH 8.5 and 15% PEG 8000 and the enzyme was incubated with xylan prior to setting up the crystallization. Crystals belong to primitive orthorhombic space group P212121 with unit cell parameters a = 59.2 Å, b = 83.8 Å and c = 174.4 Å. A data set was collected using synchrotron radiation of wave length 1.0 Å from a cryo-cooled crystal at Spring-8 BL26B1 beam line, Japan. The Matthews coefficient VM for native and xylosaccharides- bound crystals was calculated to be 2.8 and 2.7 Å3 Da-1, respectively, suggesting two molecules in each crystal asymmetric unit. No twinning was detected in both the datasets and the overall quality of the data sets was found to be good. Chapter 3 details the application of molecular replacement method to the structure solution of native and xylosaccharides-bound BSX, the course of iterative model building and the refinement carried out, and the quality of the final protein structure models. The native-enzyme structure solution was obtained by the molecular replacement method using as a search model the crystal structure (PDB code 1hiz) of the closest homologous, extracellular xylanase (GSX) from Geobacillus stearothermophilus. No non- crystallographic symmetry (NCS) restraint was applied between the two independent molecules in the crystal asymmetric unit at the final round of refinement. The final positional refinement of native BSX converged to R factors of R = 19.4% and Rfree = 23.5% for data between 20.0 to 2.2 Å. The final native model consists of 5704 protein atoms, two Mg2+ ions and 721 solvent water molecules. The final native model was taken as the search structure for the xylosaccharides-bound BSX and a solution with a correlation coefficient of 70.7% and an R-factor of 32.1% was obtained from the molecular replacement calculation. Unlike the native structure refinement, NCS restraint was imposed at all stages of the refinement. Bound xylosaccharides were clearly visible inthe difference Fourier electron density maps. The last round of refinement gave a model with R and Rfree of 21.8% and 25.7%, respectively. The final xylosaccharides-bound model consists of 5766 protein atoms, four Mg2+ ions, 85 atoms belong to bound xylosaccharides and 523 solvent water molecules. No residues were found in the disallowed region of the Ramachandran (φ, ψ) map for both the structures. Chapter 4 describes the native and xylosaccharides-bound BSX crystal structures and the structural comparison of BSX with other GH10 family xylanase crystal structures for which the optimum temperature and pH are known in the literature. BSX folds as the ubiquitous (β/α)8-barrel, a common structural superfold characteristic of GH10 xylanases. The two active site glutamic acid residues, Glu149 and Glu259, are located on opposite sides of the active site cleft and their side-chains are at a distance of 5.5 Å apart suggesting the enzymatic reaction takes place by the retaining mechanism. From the structural superposition of other xylotriose-bound xylanase structures on to the xylosaccharides-bound BSX, structural plasticity in the xylotriose binding can be inferred, implying that the xylose recognition at the subsite -3 displays plasticity and is less specific as opposed to that at -1 and -2 subsites. The stacking interaction of one of the xylose moieties of the xylobiose with the Trp235 seen in BSX provides, for the first time, a structural evidence for the direct involvement of Trp235 in xylosaccharides binding. The crystal structure revealed a metal binding site, found at the C-terminal end of catalytic domain. The presence of metal binding site was not anticipated from earlier theoretically modeled structure and biochemical studies. Further, we have shown experimentally the requirement of Mg2+ ion for the enzyme activity. We havedescribed a novel WP sequence-structure-interaction motif which is present in the (+) side of the active site region and presumably helps in the efficient binding of the carbohydrate moiety of the xylan in the active site cleft of BSX. The structural comparison of BSX with other GH10 xylanases solved to date and characterized to be active at a pH close to neutral was done for the first time. The comparative study revealed the essential structural features which may responsible for the alkaline stability of GH10 xylanases.Briefly, the alkalophilic GH10 xylanases from alkalophilic organisms have surface abundant in acidic residues, the heat and alkaline susceptible residue Asn depleted on the protein surface and increased number of salt bridges. Our study has unveiled the role of the nature and composition of protein surface amino acids in the adaptation of enzymes to polyextreme conditions. The observations reported in the thesis provide important lessons for engineering alkaline stability in xylanases for industrial applications and in general for the understanding of alkaline stability in related proteins. A comparison of the surface features of the BSX and of halophilic proteins allowed us to predict the activity of BSX at high salt concentrations, which we verified through experiments. This offered us important lessons in polyextremophilicity of proteins, where understanding structural features of a protein stable in one set of extreme conditions provided clues about the activity of the protein in other extreme conditions. Chapter 5 summaries the important findings of the present study from the crystal structural analysis of BSX and its comparison with non-alkalophilic GH10 xylanases. Separate sections are made on conclusions and future prospects for the study on BSX. Chapter 6 describes the comprehensive database analysis of C-H…O hydrogen bond in helices of globular proteins. The C-H…O hydrogen bonds found in helices are predominantly of type 5 → 1 or 4 → 1.Our analysis reveals that the Cγ and Cβ hydrogen atom(s) are frequently involved in such hydrogen bonds. A marked preference is noticed for aliphatic β-branched residue Ile to participate in 5 → 1 C- H…O hydrogen bonds involving methylene Cγ1 atom as donor in α-helices. In addition, C-H…O hydrogen bonds are present along with helix stabilizing salt bridges and to some extent compensate for the side-chain conformational entropy loss. Our analysis highlights that a multitude of local C-H…O hydrogen bondsformed by a variety of amino acid side-chains and Cα hydrogen atoms occur in helices and more so at the helix termini. A majority of the helix favouring residues, Met, Glu, Arg, Lys, Leu and Gln which also have large side-chains with more donatable CH groups, have significant propensity to form side-chain to main-chain C-H…O hydrogen bonds in helix. The large side-chains are marked by their ability to shield from the solvent the polar atoms of the peptide backbone and at the same time participate in weak cohesive C-H…O interactions in the helix. This chapter also details the identification for the first time a novel chain reversal motif stabilized by 1 → 5 Cα-H…O interactions. The importance of these hydrogen bonds with respect to helix stability is discussed in the final section of the chapter. Appendix A details the crystallographic and structural analyses oftwares used for the present thesis work. Appendix B describes, in addition to the crystal structure analysis of BSX, the work carried out by the candidate on a comparative study of a thermostable xylanase from Thermoascus aurantiacus, solved in our laboratory at atomic 1.11 Å (293 K) and ultrahigh 0.89 Å (100 K) resolutions. From the comparison, we have for the first time pointed out the possibility of plasticity of ion pairs in proteins with water molecules mediating some of the alternate arrangements. The αβ-loops are relatively less flexible than the βα-loops. The β-strands are least affected structurally with the increase in temperature. Thus the TIM barrel fold in the study enzyme, though having a single domain, may be dissected into parts based on the relative flexibility and described as having a rigid core constituted by the β-barrel and a less rigid exterior formed by the surrounding α-helices. Appendix C presents the crystallization and the preliminary X-ray characterization work done by the author of the thesis on an alkali thermostable cellulase enzyme from Thermomonospora sp. The protein is an extracellular enzyme with molecular mass of 14.2 kDa and interestingly, has the dual activity for both cellulose and xylan. The primary structure of the enzyme is not known. The enzyme was purified from the source organism and crystallized. A complete diffraction data set was collected and processed to 2.3 Å in an orthorhombic space group P212121. Appendix D contains tables which give details about the analysed 5 → 1 Cα- H…O hydrogen bonds in helices and a novel chain reversal motif with 1 → 5 Cα-H…O hydrogen bonds. Appendix E encloses reprints of publications which have resulted from the work reported in the thesis.

碩士<br>國立中山大學<br>材料與光電科學學系研究所<br>98<br>This research includes two topics. The first topic is prepared a series of poly(methyl methacrylate) (PMMA)-based copolymers through free radical copolymerizations of methyl methacrylate in the presence of the either 2 - vinyl - 4,6 -diamino - 1, 3, 5triazine(VDAT) or vinylbenzyl -thymine (VBT). Using 1H nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), we investigated the thermal properties and hydrogen-bonding interactions within blends of the two copolymers poly(2-vinyl-4,6-diamino-1,3,5-triazine-co-methyl methacrylate) (PVD AT-co-PMMA) and poly(vinylbenzylthymine-co-methyl methacrylate) (PVBT-co-PMMA). A large positive deviation in the behavior of the glass transition temperature determined by using the Kwei equation and DSC analyses indicated that strong multiple hydrogen-bonding interactions existed between the two copolymers. The FTIR and solid-state NMR spectroscopic analyses provided positive evidence for the presence of three hydrogen bonds between the diamino-1,3,5-triazine groups of PVDAT and thymine groups of PVBT. The second topic is synthesized a series of alkyne end-terminated poly(γ-Benzyl L-glutamate) (alkyne-PBLG), which was prepared by the ring-opening polymerization of the N-carboxyanhydride monomer of γ-Benzyl L-glutamate-carboxyanhydride. Then combination of the alkyne-PBLG with polyhedral oligomeric silsesquioxanes (POSS) with eight function groups are prepared through the click reation and formed a peptide-based block polymer with α-helice and β-sheet conformations. The thermal properties and structure of these polypeptides were characterized by using 1H NMR and FTIR. The FTIR, solid-state NMR and WXRD spectroscopic analyses provided evidence for the change of secondary structure between POSS and terminated peptide group, so that the conformation of β-sheets and α-helices would be influenced by the incorporation of POSS nanoparticle.

In recent years, significant success has been made in the field of regenerative medicine. Tissue engineering scaffolds have been developed to repair and replace different types of tissues. The overall goal of the current work was to develop scaffolds of native extracellular matrix components for soft tissue regeneration, more specifically, neural tissue engineering. To date, much research has been focused on developing a nerve guidance scaffold for its ability to fill and heal the gap between the damaged nerve ends. Such scaffolds are marked by several intrinsic properties including: (1) a biodegradable scaffold or conduit, consisting of native ECM components, with controlled internal microarchitecture; (2) support cells (such as Schwann cells) embedded in a soft support matrix; and (3) sustained release of bioactive factors. In the current dissertation, we have developed such scaffolds of native biomaterials including hyaluronic acid (HA) and collagen. HA is a nonsulphated, unbranched, high-molecular weight glycosaminoglycan which is ubiquitously secreted by cells in vivo and is a major component of extracellular matrix (ECM). High concentrations of HA are found in cartilage tissue, skin, vitreous humor, synovial fluid of joints and umbilical cord. HA is nonimmunogenic, enzymatically degradable, non-cell adhesive which makes HA an attractive material for biomedical research. Here we developed new photopolymerizable HA based materials for soft tissue repair application. First, we developed interpenetrating polymer networks (IPN) of HA and collagen with controlled structural and mechanical properties. The IPN hydrogels were enzymatically degradable, porous, viscoelastic and cytocompatible. These properties were dependent on the presence of crosslinked networks of collagen and GMHA and can be controlled by fine tuning the polymer ratio. We further developed these hydrogel constructs as three dimensional cellular constructs by encapsulating Schwann cells in IPN hydrogels. The hydrogel constructs supported cell viability, spreading, proliferation, and growth factor release from the encapsulated cells. Finally, we fabricated scaffolds of photopolymerizable HA with controlled microarchitecture and developed designer scaffolds for neural repair using layer-by-layer fabrication technique. Lastly, we developed HA hydrogels with unique anisotropic swelling behavior. We developed a dual-crosslinking technique in which a super-swelling chemically crosslinked hydrogel is patterned with low-swelling photocrosslinked regions. When this dual-crosslinked hydrogel is swelled it contorts into a new shape because of differential swelling among photopatterned regions.<br>text

碩士<br>國立中央大學<br>生醫科學與工程學系<br>106<br>Metallogels were established based on coordinative interactions, exhibiting unique abilities, and being applied for various biomedical applications. However, the applications of metallogels for wound healing property has not been explored. In this study, we report a newly nature-derived metallogels developed by coordination of polyphenols and metal ions. Gelation occurs by mixing low-cost polyphenolic tannic acid (TA) and group (IV) metal ions (titanium ions) to form TA-TiIV gel. The TA-TiIV gel exhibits a good ability to incorporate diverse functional metal ions by in situ co-gelations. Herein, we used five metal ions: ferric ions (Fe3+), copper(II) ions (Cu2+), zinc(II) ions (Zn2+), cobalt(II) ions (Co2+) and nickel(II) ions (Ni2+) for antimicrobial tests. The formations and collapse of formed metallogels are sensitive to external pH – and the presence of H2O2, which displays in the healing process of infected wounds. The chemical and coordinative structures of TA-TiIV metallogel were characterized by UV-Vis spectrometer and Fourier-transform infrared (FT-IR) spectroscopy. The release of metal ions was evaluated by inductively coupled plasma mass spectrometry (ICP-MS), indicating the different releasing profiles upon the types of metal ions. For TA-TiIV-CuII metallogel, distinct releasing rates at different pH values of 5.5, 7.4 and 8.4 has been studied. The result indicates the pH-dependent metal ions release manner. Moreover, the bacterial environment has been investigated by UV-Vis spectrometer and pH meter, the result showed the acidic environment during the metabolism process of bacteria. Besides, HRP incorporated with TA-TiIV-CuII HG brought to a significant Cu2+ ions released, where H2O2 is presented. As the results, antibacterial effect of synthesized metallogels againsts Gram-negative Escherichia coli, and Gram-positive Methicillin-Resistant Staphylococcus aureus (USA300), Staphylococcus epidermidis bacteria has been investigated by agar diffusion test. Simultaneously, cytotoxicity of metallogels on NIH 3T3 fibroblast cell line was also explored by MTT assay, and turned out to be excellent biocompatibility. For clinical applications as infected wound dressings, TA-TiIV-CuII HG indicated the antimicrobial effect, led to significant smaller in wound area after 8 days, compared to TA-TiIV HG and Gauze. Consequently, demonstrated HG is promising a potential materials for infected wound applications. Keywords- Metallogels, tannic acid (TA), metal ions, nature-derived

The thesis entitled “Unravelling the Nature of Halogen and Chalcogen Intermolecular Interactions by Charge Density Analysis" consists of five chapters. A basic introductory section describes the topics relevant to the work and the methods and techniques utilized. The main focus of the present work is to characterize the interaction patterns devoid of strong classical hydrogen bonds. The case studies include halogen bonds and hydrogen bonds involving bromine (as a halogen bond donor and hydrogen bond acceptor), intermolecular chalcogen bond formation involving sulphur, type I Br Br contacts, type II F F and F S interactions and S-H S hydrogen bonds. Chapter 1 discusses experimental and theoretical charge density analyses on 2,2-dibromo-2,3-dihydroinden-1-one which has been carried out to quantify the topological features of a short C Br···O halogen bond with nearly linear geometry (2.922Å, C Br···O=172.7) and to assess the strength of the interactions using the topological features of the electron density. The electrostatic potential map indicates the presence of the “- hole” on bromine while the interaction energy is comparable to that of a moderate O-H O hydrogen bond. In addition, the energetic contribution of C-H···Br interaction is demonstrated to be on par with that of the C-Br···O halogen bond in stabilizing the crystal structure. Chapter 2 discusses an organic solid, 4,7-dibromo-5,6-dinitro-2,1,3-benzothiadiazole that has been designed to serve as an illustrative example to quantitatively evaluate the relative merits of halogen and chalcogen bonding in terms of charge density features. The compound displays two polymorphic modifications, one crystall zing in a non-centrosymmetric space group (Z =1) and the other in a centrosymmetric space group with two molecules in the asymmetric unit (=2). Topological analysis based on QTAIM clearly brings out the dominance of chalcogen bond over the halogen bond along with an indication that halogen bonds are more directional compared to chalcogen bonds. The cohesive energies calculated with the absence of both strong and weak hydrogen bonds as well as stacking interaction are indicative of the stabilities associated with the polymorphic forms. Chapter 3 discusses the role of a type I C-Br Br-C contact and what drives the contact i.e. how a dispersive interaction is stabilized by the remaining contacts in the structure. In the process we observe the role the Br2Cl motif which is quite unique in its nature. Also the role of the bromine atoms in stabilizing the stacking interactions has been shown by the electrostatic potentials which are oriented perpendicular to the plane of the benzene ring. Chapter 4 discusses the enigmatic type II C-F F-C and C-FS-C interactions in pentafluorophenyl 2,2- bithiazole. Both the interactions are shown to be realistic “-hole” interactions based on high resolution X-ray charge density analysis. As fluorine is the most electronegative element, its participation in halogen bonding wherein the electrostatic potential around the atom gets redistributed to form regions of electron depletion and accumulation had time and again been speculated but never observed. In this chapter the experimental charge dnsity analysis clearly identifies the “-hole” on fluorine and distinguishes the C-F S-C interaction as a halogen bond rather than the chalcogen bond. Chapter 5 discusses the experimental charge density analysis of the hitherto unexplored S-H S hydrogen bond in crystal structures. The work highlights how relatively small is the number of crystal structures which are constructed by the S-H S hydrogen bond compared to the X-H S hydrogen bond via Cambridge Structural Database (CSD) analysis. The potential S-H S hydrogen bond is studied in three isomeric mercaptobenzoic acids with experimental charge density collected on 2-mercaptobenzoic acid and theoretical estimates made on 3- and 4-mercaptobenzoic acid. The strength and directionality of the S-H S hydrogen bond is demonstrated to be mainly due to the conformation locking potential of intramolecular S O halogen bond.

The thesis entitled “Unravelling the Nature of Halogen and Chalcogen Intermolecular Interactions by Charge Density Analysis" consists of five chapters. A basic introductory section describes the topics relevant to the work and the methods and techniques utilized. The main focus of the present work is to characterize the interaction patterns devoid of strong classical hydrogen bonds. The case studies include halogen bonds and hydrogen bonds involving bromine (as a halogen bond donor and hydrogen bond acceptor), intermolecular chalcogen bond formation involving sulphur, type I Br Br contacts, type II F F and F S interactions and S-H S hydrogen bonds. Chapter 1 discusses experimental and theoretical charge density analyses on 2,2-dibromo-2,3-dihydroinden-1-one which has been carried out to quantify the topological features of a short C Br···O halogen bond with nearly linear geometry (2.922Å, C Br···O=172.7) and to assess the strength of the interactions using the topological features of the electron density. The electrostatic potential map indicates the presence of the “- hole” on bromine while the interaction energy is comparable to that of a moderate O-H O hydrogen bond. In addition, the energetic contribution of C-H···Br interaction is demonstrated to be on par with that of the C-Br···O halogen bond in stabilizing the crystal structure. Chapter 2 discusses an organic solid, 4,7-dibromo-5,6-dinitro-2,1,3-benzothiadiazole that has been designed to serve as an illustrative example to quantitatively evaluate the relative merits of halogen and chalcogen bonding in terms of charge density features. The compound displays two polymorphic modifications, one crystall zing in a non-centrosymmetric space group (Z =1) and the other in a centrosymmetric space group with two molecules in the asymmetric unit (=2). Topological analysis based on QTAIM clearly brings out the dominance of chalcogen bond over the halogen bond along with an indication that halogen bonds are more directional compared to chalcogen bonds. The cohesive energies calculated with the absence of both strong and weak hydrogen bonds as well as stacking interaction are indicative of the stabilities associated with the polymorphic forms. Chapter 3 discusses the role of a type I C-Br Br-C contact and what drives the contact i.e. how a dispersive interaction is stabilized by the remaining contacts in the structure. In the process we observe the role the Br2Cl motif which is quite unique in its nature. Also the role of the bromine atoms in stabilizing the stacking interactions has been shown by the electrostatic potentials which are oriented perpendicular to the plane of the benzene ring. Chapter 4 discusses the enigmatic type II C-F F-C and C-FS-C interactions in pentafluorophenyl 2,2- bithiazole. Both the interactions are shown to be realistic “-hole” interactions based on high resolution X-ray charge density analysis. As fluorine is the most electronegative element, its participation in halogen bonding wherein the electrostatic potential around the atom gets redistributed to form regions of electron depletion and accumulation had time and again been speculated but never observed. In this chapter the experimental charge dnsity analysis clearly identifies the “-hole” on fluorine and distinguishes the C-F S-C interaction as a halogen bond rather than the chalcogen bond. Chapter 5 discusses the experimental charge density analysis of the hitherto unexplored S-H S hydrogen bond in crystal structures. The work highlights how relatively small is the number of crystal structures which are constructed by the S-H S hydrogen bond compared to the X-H S hydrogen bond via Cambridge Structural Database (CSD) analysis. The potential S-H S hydrogen bond is studied in three isomeric mercaptobenzoic acids with experimental charge density collected on 2-mercaptobenzoic acid and theoretical estimates made on 3- and 4-mercaptobenzoic acid. The strength and directionality of the S-H S hydrogen bond is demonstrated to be mainly due to the conformation locking potential of intramolecular S O halogen bond.

In solution, the environment around the solute is determined solely by the solvent molecules, which are present closer to the solute. This interaction between solute and solvent shell is very crucial for equilibrium structure and reactivity of the solute. In the thesis, first we have investigated control of solvent polarity on the excited structure of 2,2,2 trifluroacetophenone and later effect of electronic excitation on the solvent shell organization has been described. It has been reported in literature that the lowest energy triplet configuration of 2,2,2 trifluroacetophenone corresponds to n,π* state. There are some other reports in favor of the probable existence of 2,2,2 trifluroacetophenone in n,π* lowest triplet state. Thus, transient absorption and time resolved resonance Raman spectroscopic methods have been used along with theoretical calculations to investigate the discrepancy in the assignment of the lowest triplet state configuration It has been observed that the lowest triplet state of 2,2,2 trifluroacetophenone is indeed nπ* and there is a solvent polarity induced change in triplet state energy ordering and structure changes. The absorption spectrum of hydrated electron is broad and structureless. So, it was though that the broadening feature could be attributed to homogeneous and inhomogeneous broadening. Transient resonance Raman spectrum of the water bending mode in presence of hydrated electron has been recorded at different excitation wavelengths. Interestingly, it has been observed that, peak position of water bending mode in presence of hydrated electron alters with change of excitation wavelength. A model has been proposed based on the experimental data.

In solution, the environment around the solute is determined solely by the solvent molecules, which are present closer to the solute. This interaction between solute and solvent shell is very crucial for equilibrium structure and reactivity of the solute. In the thesis, first we have investigated control of solvent polarity on the excited structure of 2,2,2 trifluroacetophenone and later effect of electronic excitation on the solvent shell organization has been described. It has been reported in literature that the lowest energy triplet configuration of 2,2,2 trifluroacetophenone corresponds to n,π* state. There are some other reports in favor of the probable existence of 2,2,2 trifluroacetophenone in n,π* lowest triplet state. Thus, transient absorption and time resolved resonance Raman spectroscopic methods have been used along with theoretical calculations to investigate the discrepancy in the assignment of the lowest triplet state configuration It has been observed that the lowest triplet state of 2,2,2 trifluroacetophenone is indeed nπ* and there is a solvent polarity induced change in triplet state energy ordering and structure changes. The absorption spectrum of hydrated electron is broad and structureless. So, it was though that the broadening feature could be attributed to homogeneous and inhomogeneous broadening. Transient resonance Raman spectrum of the water bending mode in presence of hydrated electron has been recorded at different excitation wavelengths. Interestingly, it has been observed that, peak position of water bending mode in presence of hydrated electron alters with change of excitation wavelength. A model has been proposed based on the experimental data.

This thesis titled, “Nature of Local Interactions at cisPro-Aro Peptide Sequences in Proteins: Evidences for van der Waals type Interactions. Design and Synthesis of Novel Covalent Surrogates for the Peptide Hydrogen Bond”, describes two important studies. The first is to gain a thorough understanding of the nature of interactions that govern cisPro stability at Pro-Aro sequences, which described in the first four chapters. The final chapter describes the synthesis of novel 4-carbon covalent surrogates for the peptide H-bonding interaction. Chapter 1: Local Interactions Governing cisPro Stability: Refining the Model Peptides Chapter 1 Section A: Understanding the role of inter-side chain CH•••Aro interaction in cis-trans isomerization at Pro-Aro and Aro-Pro Sequences. This chapter is divided into two sections. In the first section an exhaustive overview of earlier investigations into the nature of local interactions at Xaa-cisPro-Aro and Aro-cisPro-Xaa peptide sequences, by various groups, are discussed. Most studies have found evidence for the close assemblage between side chains of residues flanking cisPro motifs, when at least one of them is an aromatic group. An electronic C-H•••π nature has been proposed for these assemblies and they are proposed to influence the cisPro stability. We highlight those features in these studies that indicate that these interactions are not always electronically tunable, are insensitive to presence of strong chaotropes in the solvent and occur at protein sequences lacking Pro or cisPro; all of which contradict the electronic C-H•••π model for these inter-side chain assemblages and their perceived influence on cisPro stability. Chapter 1 Section B: Investigation of the Nature of H Xaa•••Aro interaction at Xaa-Pro-Pro-Phe-sequences In Section B, we design and synthesize Pro-Aro containing short peptide models to investigate the nature of local C-H•••Aro interactions in them. We synthesize a series of homologous Pro-Pro-Aro containing peptides (modeled based on earlier studies) and investigate the relative populations of its four Xaa-Pro rotamers using extensive 1D and 2D NMR techniques including TOCSY, HSQC and ROESY. We find several drawbacks that make this a relatively deficient model. Firstly, their relative populations of the rotamers (the most important data for current investigation) cannot be determined with high fidelity as they are dependent on the solvent polarity, solute concentration and chemical shift degeneracy of crucial NMR signals for the rotamers. Importantly, the populations of a few rotamers are influenced by strong 13-membered ring backbone H-bonds. Notably, some of the cisPro rotamers do not even contain the inter-side chain assembly, whose nature is under investigation. Design of novel models – unconstrained by H-bonds We design the Acyl-Pro-Pro-Aro-OMe peptides that lack the possibility of forming the 13-membered ring H-bonded structures. Thorough 1D and 2D NMR analyses of these models reveal that strong Type VI β-turn type 10-membered ring H-bonds are formed in the rotamers of these models – hence precluding their applications for current study. Interestingly, the relative rotamer populations are strongly influenced by solvent polarity and are entirely different from those of the corresponding C-terminal amide models. We further discover that the Pro-Pro-Aro motif is not essential to express the inter-side chain interactions – Ala-Pro-Aro are sufficient. Formation of the 10-membered H-bonding interactions, however, are not precluded. Chapter 2: Design and Synthesis of Acyl-Pro-Phe-OMe: Novel models to investigate the role of HαXaa•••Aro interactions on Xaa-cisPro-Aro stability. Chapter 2 Section A: Design, Synthesis and Conformational Analysis of Ibu-Pro-Phe-OMe Chapter 2 is divided into two sections. In Section A, we replace the amino acid at the N-terminal of the putative Pro residue with simple isosteric isobutyryl group, the resulting minimalist dipeptide model shows the exclusive influence of desired inter-side chain interactions in the cisPro rotamer. Solvent polarity and temperature coefficient studies reveal that absence of any intramolecular H-bonding or Oπ* interactions in it. 1D and 2D NMR analyses clearly indicate the close proximity between the side chains of Ibu and Phe exclusively in the cisPro rotamer. The Kc/t value decreases upon mutation of Phe to Ala. All these features favor the Ibu-Pro-Phe-OMe as an ideal minimalistic model for investigating the nature of Ibu•••Ph assemblages in the cisPro rotamer. Chapter 2 Section B: Investigation of CH•••Aro /Alp•••Alp interactions in Ibu-cisPro-Xaa-OMe In Section B, the 1D and 2D NMR analyses of the complete set of the aliphatic and aromatic analogues Ibu-Pro-Xaa-OMe were investigated. DMSO-d6 was found to be the best solvent for mimicking both the folded and the unfolded local environments of these short peptide sequences. The HαIbu•••Aro assemblage is observed in Aro analogues, but cannot be electronically tuned. The aliphatic analogues also surprisingly contain the HαIbu•••Alp interactions! The Kc/t values (cisPro %) increase in the aliphatic analogues too, where the aliphatic side chain is long. Increase in cisPro stability is not due to ring current effects or intramolecular H-bonds or Oπ* interactions. It seems to be due to van der Waals type interactions between the involved side chains, either of which need not be aromatic in nature. Chapter 3: Nature of Inter-Side Chain Interactions at Acyl-cisPro-Aro Sequences: Evidence for van der Waals Interactions Chapter 3 Section A: Investigation of nature of inter-side chain interactions in R-CO-cisPro-Phe-OMe Chapter 3 has two sections. Section A describes the systematic design and synthesis of Acyl-Pro-Phe-OMe homologues where first the steric bulk and hence the surface area of the aliphatic side chain of the acyl group is varied. Interaction of the phenyl ring of Phe seems to occur with the Cα-Cβ σ-bond of the acyl group. Branching at either Cα or Cβ seems to destabilize the cisPro rotamer. Aliphatic•••Aromatic interactions overwhelm the cisPro rotamer population to be greater than that of transPro. In the analogues where the acidity of the acyl Cα-H bond is increased, the Kc/t does not increase correspondingly. The Δδ(trans-cis) ppm shifts of HαAcyl protons are dependent exclusively on its acidity rather than on the Kc/t values. In carbamyl-Pro, which entirely lack the HαAcyl proton, the Kc/t values are significantly high and improve as the aliphatic surface on the alkoxy group increases. Introduction of benzyloxy carbamyl group at Pro renders almost the same Kc/t values as that of ethyloxy carbamate. All these data contradict the C-H•••π interaction model and strongly support a van der Waals type interaction between the Acyl (preceding cisPro) group’s Xα-Yβ σ-bond and the Aro or Alp side chains (succeeding cisPro). Chapter 3 Section B: Evidence for the Van der Waals nature of Inter Side Chain (Acyl•••S.C.Aro/Alp) interactions- Determination of Interactions energies In Section B, a thorough investigation of both aliphatic•••aliphatic and aliphatic•••aromatic interactions on the background of homologous Acyl-Pro-Aro/Alp-OMe peptide models is undertaken. These models uniquely allow the delineation of contribution of the van der Waals interactions and the ring current effects to the cis/trans isomerization in these peptides. We see that the energy of the van der Waals component of these aliphatic•••aliphatic and aliphatic…aromatic interactions increase linearly with increase in Kc/t, in both DMSO-d6 and D2O. On other hand, energy from the ring current effects largely remains invariant. The Acyl•••Aro/Alp interactions are not hydrophobic and are facilitated by conformational effects. Chapter 4: Crystallographic evidence for van der Waals interaction-mediated stabilization of cisPro conformers Chapter 4 Section A: Systematic crystallization and crystal structure analyses of homologous Xaa-cisPro-Alp and Xaa-cisPro-Aro rotamers: Evidence for van der Waals interactions Chapter 4 has two sections, both of which present crystallographic evidence for the van der Waals nature of the Xaa•••Aro interactions at Xaa-cisPro-Aro sequences. Section A describes the unique crystal structures of five of the Acyl-Pro-Alp-OMe analogues that have been synthesized in the current study. All of them remarkably crystallize with two features: 1) the Acyl-Pro peptide bond adopts the cisPro rotamer in all; and 2) the aliphatic side chains of the acyl group and the Alp side chain are involved in van der Waals type interactions. The cisPro rotamers of even the bulkiest motifs, namely Ibu-Pro-Val-OMe, Piv-Pro-Ile-OMe and Piv-Pro-Leu-OMe crystallize, stabilized by van der Waals packing between aliphatic groups of the acyl and the Leu/Ile/Val side chains. Where the side chains are not long enough to make sub-van der Waals contacts with each other, their acyl C′-Cα σ-bond rotations are restricted due to Oσ* interactions involving the charge on the acyl carbonyl O. Where this occurs, the short space between the acyl and Alp side chains are filled in by aliphatic groups from neighbouring molecules at sub van der Waals distances. The Pro, Alp and χ1(Alp) dihedral angles are restricted to narrow range of values, irrespective of the length of Alp side chain, indicating that this backbone conformation is a conformational minimum when i+3i backbone H-bond is removed, with Pro at the i+1st position. This is further substantiated in Piv-Pro-Gly-OMe, which crystallizes in trans-Pro form, but still adopts similar backbone dihedral angles in spite of lacking any Alp side chain for interactions with the acyl group. Three of the Acyl-Pro-Aro-OMe models also crystallize in cisPro rotamer forms – both exhibit van der Waals type contacts between the Acyl group and backbone of Phe, rather than the aromatic ring of Phe. The phenyl ring of Phe may or may not form intramolecular Ph•••Pro inter-side chain contacts – which is not a pre-requisite for cisPro stabilization. No C-H••• interactions are observed anywhere in these peptides – van der Waals type contacts alone predominate in all cases. There are no abnormal distortions in bond angles or lengths even in the most sterically hindered cases, signifying that the conformations of these cisPro rotamers involving aliphatic•••aliphatic type contacts are natural minima. Chapter 4 Section B: Mining the PDB for Statistical Evidence of van der Waals interactions Section B of chapter 4 describes the data mining and statistical analyses of Xaa-cisPro-Phe, Xaa-cisPro-Val and Xaa-cisProLeu sequences in the PDB. The PEARL program was used to mine the PDB data. The overall frequency of 5.3% for appearance of cisPro among all Xaa-Pro peptide bonds, improves when Xaa is Phe or Tyr. However, several anomalies highlight the need for refining the analyses set to only those sequences where the side chains of Xaa and Aro/Alp face each other. In this refined set, clearly, inter side chain Xaa•••Alp/Aro contacts take precedence over even Aro•••Pro interactions at Aro-cisPro sequences (where Xaa is Aro). The Phe and the Leu side chains induce similar conformational effects on the preceding Xaa-Pro backbone. So does Val. Strong aliphatic•••aliphatic inter side chain contacts at van der Waals distances are observed to flank cisPro in several proteins. Substitution at the Cα of Xaa governs the proximity of the approaching side chain of Alp / Aro residue. The Cα-H of Xaa steers away from the Aro side chain at Xaa-Pro-Phe sequences, as the Aro group gets closer to it – implying the absence of ordered C-H••• contacts between them. There is consistent parallel alignment between Cα-Cβ -bond of Xaa and the C -C bond of the approaching side chain of Alp or Aro group – clearly highlighting the presence of van der Waals type interactions between them. All these evidences clearly point towards the van der Waals nature of local interactions at cisPro-Aro/Alp peptide sequences. Chapter 5: A novel 4-carbon covalent surrogate model for peptide H-Bond Chapter 5 describes the design and synthesis of novel 4-carbon covalent surrogates for the peptide H-bond (HBS). These surrogates would allow the unique constraining of two peptide strands in their extended conformations. The covalent HBS contain four orthogonal functional groups for independent extension at all of the four ends – similar to an endogenous inter-strand peptide H-bond. The synthesis of the surrogate is achieved by directly using natural chiral amino acid derivatives, beginning from amino alcohols obtained from reduction of desired amino acids. Suitably N-protected alcohols undergo oxidation to aldehyde followed by Grignard addition of allyl magnesium bromide, TBDMS protection of the homoallylic alcohol and reductive ozonolysis of the olefin to get a primary alcohol which is subject to Fukuyama-Mitsunobu reaction with desire protected peptide. The residue preferences that produce strongest inter-strand H-bonds were explored. The designed 4-carbon covalent HBS was incorporated using this methodology in a Gramicidin-S analogue, its first structural mimic containing only a single turn motif. This HBS model will have wide applications for constraining peptides in a number of secondary structures.

This thesis titled, “Nature of Local Interactions at cisPro-Aro Peptide Sequences in Proteins: Evidences for van der Waals type Interactions. Design and Synthesis of Novel Covalent Surrogates for the Peptide Hydrogen Bond”, describes two important studies. The first is to gain a thorough understanding of the nature of interactions that govern cisPro stability at Pro-Aro sequences, which described in the first four chapters. The final chapter describes the synthesis of novel 4-carbon covalent surrogates for the peptide H-bonding interaction. Chapter 1: Local Interactions Governing cisPro Stability: Refining the Model Peptides Chapter 1 Section A: Understanding the role of inter-side chain CH•••Aro interaction in cis-trans isomerization at Pro-Aro and Aro-Pro Sequences. This chapter is divided into two sections. In the first section an exhaustive overview of earlier investigations into the nature of local interactions at Xaa-cisPro-Aro and Aro-cisPro-Xaa peptide sequences, by various groups, are discussed. Most studies have found evidence for the close assemblage between side chains of residues flanking cisPro motifs, when at least one of them is an aromatic group. An electronic C-H•••π nature has been proposed for these assemblies and they are proposed to influence the cisPro stability. We highlight those features in these studies that indicate that these interactions are not always electronically tunable, are insensitive to presence of strong chaotropes in the solvent and occur at protein sequences lacking Pro or cisPro; all of which contradict the electronic C-H•••π model for these inter-side chain assemblages and their perceived influence on cisPro stability. Chapter 1 Section B: Investigation of the Nature of H Xaa•••Aro interaction at Xaa-Pro-Pro-Phe-sequences In Section B, we design and synthesize Pro-Aro containing short peptide models to investigate the nature of local C-H•••Aro interactions in them. We synthesize a series of homologous Pro-Pro-Aro containing peptides (modeled based on earlier studies) and investigate the relative populations of its four Xaa-Pro rotamers using extensive 1D and 2D NMR techniques including TOCSY, HSQC and ROESY. We find several drawbacks that make this a relatively deficient model. Firstly, their relative populations of the rotamers (the most important data for current investigation) cannot be determined with high fidelity as they are dependent on the solvent polarity, solute concentration and chemical shift degeneracy of crucial NMR signals for the rotamers. Importantly, the populations of a few rotamers are influenced by strong 13-membered ring backbone H-bonds. Notably, some of the cisPro rotamers do not even contain the inter-side chain assembly, whose nature is under investigation. Design of novel models – unconstrained by H-bonds We design the Acyl-Pro-Pro-Aro-OMe peptides that lack the possibility of forming the 13-membered ring H-bonded structures. Thorough 1D and 2D NMR analyses of these models reveal that strong Type VI β-turn type 10-membered ring H-bonds are formed in the rotamers of these models – hence precluding their applications for current study. Interestingly, the relative rotamer populations are strongly influenced by solvent polarity and are entirely different from those of the corresponding C-terminal amide models. We further discover that the Pro-Pro-Aro motif is not essential to express the inter-side chain interactions – Ala-Pro-Aro are sufficient. Formation of the 10-membered H-bonding interactions, however, are not precluded. Chapter 2: Design and Synthesis of Acyl-Pro-Phe-OMe: Novel models to investigate the role of HαXaa•••Aro interactions on Xaa-cisPro-Aro stability. Chapter 2 Section A: Design, Synthesis and Conformational Analysis of Ibu-Pro-Phe-OMe Chapter 2 is divided into two sections. In Section A, we replace the amino acid at the N-terminal of the putative Pro residue with simple isosteric isobutyryl group, the resulting minimalist dipeptide model shows the exclusive influence of desired inter-side chain interactions in the cisPro rotamer. Solvent polarity and temperature coefficient studies reveal that absence of any intramolecular H-bonding or Oπ* interactions in it. 1D and 2D NMR analyses clearly indicate the close proximity between the side chains of Ibu and Phe exclusively in the cisPro rotamer. The Kc/t value decreases upon mutation of Phe to Ala. All these features favor the Ibu-Pro-Phe-OMe as an ideal minimalistic model for investigating the nature of Ibu•••Ph assemblages in the cisPro rotamer. Chapter 2 Section B: Investigation of CH•••Aro /Alp•••Alp interactions in Ibu-cisPro-Xaa-OMe In Section B, the 1D and 2D NMR analyses of the complete set of the aliphatic and aromatic analogues Ibu-Pro-Xaa-OMe were investigated. DMSO-d6 was found to be the best solvent for mimicking both the folded and the unfolded local environments of these short peptide sequences. The HαIbu•••Aro assemblage is observed in Aro analogues, but cannot be electronically tuned. The aliphatic analogues also surprisingly contain the HαIbu•••Alp interactions! The Kc/t values (cisPro %) increase in the aliphatic analogues too, where the aliphatic side chain is long. Increase in cisPro stability is not due to ring current effects or intramolecular H-bonds or Oπ* interactions. It seems to be due to van der Waals type interactions between the involved side chains, either of which need not be aromatic in nature. Chapter 3: Nature of Inter-Side Chain Interactions at Acyl-cisPro-Aro Sequences: Evidence for van der Waals Interactions Chapter 3 Section A: Investigation of nature of inter-side chain interactions in R-CO-cisPro-Phe-OMe Chapter 3 has two sections. Section A describes the systematic design and synthesis of Acyl-Pro-Phe-OMe homologues where first the steric bulk and hence the surface area of the aliphatic side chain of the acyl group is varied. Interaction of the phenyl ring of Phe seems to occur with the Cα-Cβ σ-bond of the acyl group. Branching at either Cα or Cβ seems to destabilize the cisPro rotamer. Aliphatic•••Aromatic interactions overwhelm the cisPro rotamer population to be greater than that of transPro. In the analogues where the acidity of the acyl Cα-H bond is increased, the Kc/t does not increase correspondingly. The Δδ(trans-cis) ppm shifts of HαAcyl protons are dependent exclusively on its acidity rather than on the Kc/t values. In carbamyl-Pro, which entirely lack the HαAcyl proton, the Kc/t values are significantly high and improve as the aliphatic surface on the alkoxy group increases. Introduction of benzyloxy carbamyl group at Pro renders almost the same Kc/t values as that of ethyloxy carbamate. All these data contradict the C-H•••π interaction model and strongly support a van der Waals type interaction between the Acyl (preceding cisPro) group’s Xα-Yβ σ-bond and the Aro or Alp side chains (succeeding cisPro). Chapter 3 Section B: Evidence for the Van der Waals nature of Inter Side Chain (Acyl•••S.C.Aro/Alp) interactions- Determination of Interactions energies In Section B, a thorough investigation of both aliphatic•••aliphatic and aliphatic•••aromatic interactions on the background of homologous Acyl-Pro-Aro/Alp-OMe peptide models is undertaken. These models uniquely allow the delineation of contribution of the van der Waals interactions and the ring current effects to the cis/trans isomerization in these peptides. We see that the energy of the van der Waals component of these aliphatic•••aliphatic and aliphatic…aromatic interactions increase linearly with increase in Kc/t, in both DMSO-d6 and D2O. On other hand, energy from the ring current effects largely remains invariant. The Acyl•••Aro/Alp interactions are not hydrophobic and are facilitated by conformational effects. Chapter 4: Crystallographic evidence for van der Waals interaction-mediated stabilization of cisPro conformers Chapter 4 Section A: Systematic crystallization and crystal structure analyses of homologous Xaa-cisPro-Alp and Xaa-cisPro-Aro rotamers: Evidence for van der Waals interactions Chapter 4 has two sections, both of which present crystallographic evidence for the van der Waals nature of the Xaa•••Aro interactions at Xaa-cisPro-Aro sequences. Section A describes the unique crystal structures of five of the Acyl-Pro-Alp-OMe analogues that have been synthesized in the current study. All of them remarkably crystallize with two features: 1) the Acyl-Pro peptide bond adopts the cisPro rotamer in all; and 2) the aliphatic side chains of the acyl group and the Alp side chain are involved in van der Waals type interactions. The cisPro rotamers of even the bulkiest motifs, namely Ibu-Pro-Val-OMe, Piv-Pro-Ile-OMe and Piv-Pro-Leu-OMe crystallize, stabilized by van der Waals packing between aliphatic groups of the acyl and the Leu/Ile/Val side chains. Where the side chains are not long enough to make sub-van der Waals contacts with each other, their acyl C′-Cα σ-bond rotations are restricted due to Oσ* interactions involving the charge on the acyl carbonyl O. Where this occurs, the short space between the acyl and Alp side chains are filled in by aliphatic groups from neighbouring molecules at sub van der Waals distances. The Pro, Alp and χ1(Alp) dihedral angles are restricted to narrow range of values, irrespective of the length of Alp side chain, indicating that this backbone conformation is a conformational minimum when i+3i backbone H-bond is removed, with Pro at the i+1st position. This is further substantiated in Piv-Pro-Gly-OMe, which crystallizes in trans-Pro form, but still adopts similar backbone dihedral angles in spite of lacking any Alp side chain for interactions with the acyl group. Three of the Acyl-Pro-Aro-OMe models also crystallize in cisPro rotamer forms – both exhibit van der Waals type contacts between the Acyl group and backbone of Phe, rather than the aromatic ring of Phe. The phenyl ring of Phe may or may not form intramolecular Ph•••Pro inter-side chain contacts – which is not a pre-requisite for cisPro stabilization. No C-H••• interactions are observed anywhere in these peptides – van der Waals type contacts alone predominate in all cases. There are no abnormal distortions in bond angles or lengths even in the most sterically hindered cases, signifying that the conformations of these cisPro rotamers involving aliphatic•••aliphatic type contacts are natural minima. Chapter 4 Section B: Mining the PDB for Statistical Evidence of van der Waals interactions Section B of chapter 4 describes the data mining and statistical analyses of Xaa-cisPro-Phe, Xaa-cisPro-Val and Xaa-cisProLeu sequences in the PDB. The PEARL program was used to mine the PDB data. The overall frequency of 5.3% for appearance of cisPro among all Xaa-Pro peptide bonds, improves when Xaa is Phe or Tyr. However, several anomalies highlight the need for refining the analyses set to only those sequences where the side chains of Xaa and Aro/Alp face each other. In this refined set, clearly, inter side chain Xaa•••Alp/Aro contacts take precedence over even Aro•••Pro interactions at Aro-cisPro sequences (where Xaa is Aro). The Phe and the Leu side chains induce similar conformational effects on the preceding Xaa-Pro backbone. So does Val. Strong aliphatic•••aliphatic inter side chain contacts at van der Waals distances are observed to flank cisPro in several proteins. Substitution at the Cα of Xaa governs the proximity of the approaching side chain of Alp / Aro residue. The Cα-H of Xaa steers away from the Aro side chain at Xaa-Pro-Phe sequences, as the Aro group gets closer to it – implying the absence of ordered C-H••• contacts between them. There is consistent parallel alignment between Cα-Cβ -bond of Xaa and the C -C bond of the approaching side chain of Alp or Aro group – clearly highlighting the presence of van der Waals type interactions between them. All these evidences clearly point towards the van der Waals nature of local interactions at cisPro-Aro/Alp peptide sequences. Chapter 5: A novel 4-carbon covalent surrogate model for peptide H-Bond Chapter 5 describes the design and synthesis of novel 4-carbon covalent surrogates for the peptide H-bond (HBS). These surrogates would allow the unique constraining of two peptide strands in their extended conformations. The covalent HBS contain four orthogonal functional groups for independent extension at all of the four ends – similar to an endogenous inter-strand peptide H-bond. The synthesis of the surrogate is achieved by directly using natural chiral amino acid derivatives, beginning from amino alcohols obtained from reduction of desired amino acids. Suitably N-protected alcohols undergo oxidation to aldehyde followed by Grignard addition of allyl magnesium bromide, TBDMS protection of the homoallylic alcohol and reductive ozonolysis of the olefin to get a primary alcohol which is subject to Fukuyama-Mitsunobu reaction with desire protected peptide. The residue preferences that produce strongest inter-strand H-bonds were explored. The designed 4-carbon covalent HBS was incorporated using this methodology in a Gramicidin-S analogue, its first structural mimic containing only a single turn motif. This HBS model will have wide applications for constraining peptides in a number of secondary structures.

This thesis provides biophysical bases of the interactions between two proteins involved in microRNA (miRNA)-mediated silencing: CNOT1 and the silencing domain of GW182. The regulation of gene expression at the post-transcriptional level involves the crucial CCR4-NOT deadenylase complex, which deadenylates mRNA, and can also inhibit translation in an independent fashion. In miRNA-mediated silencing, the CCR4-NOT complex is brought into the vicinity of the target mRNA by the successive actions of the miRNA, the Argonaute protein and finally, the GW182 protein, which interacts directly with CCR4-NOT. In the case of silencing of mRNAs containing AU-rich elements, the same action is performed by the protein called tristetraprolin involved in the regulation of inflammatory processes. The interactions and interplay between all of these high molecular weight proteins are relatively poorly understood. In particular, the interaction sites between GW182 and the CCR4-NOT complex were previously unknown. Molecular biology experiments allowed the identification of CCR4-NOT interaction motifs on GW182. One of them is crucial for deadenylation, while the other is vital in mediating the interaction with CCR4-NOT via CNOT1, the scaffolding subunit of the CCR4-NOT complex. Biophysical experiments based on hydrogen-deuterium exchange mass spectrometry allowed the identification of the corresponding binding site on CNOT1(800-999). Surprisingly, the binding site of the GW182 silencing domain was found to be at the same CNOT1(800-999) surface region as the binding site of tristetraprolin. Biochemical experiments excluded their simultaneous binding to CNOT1. The GW182 and tristetraprolin proteins share a common motif, RLPXφ, that interacts with CNOT1 in a very similar, but not identical, manner. This sequence has been proposed to act as a short linear motif. Thus, the two different gene silencing pathways: miRNA-mediated silencing and ARE-mediated silencing intersect at CNOT1, which serves as a molecular hub. The structural dynamics of the GW182 silencing domain and the CNOT1(800-999) fragments were also studied. The GW182 silencing domain was experimentally proved to be natively unstructured except for an RNA-recognition motif (RRM) domain. The GW182 RRM domain was found to be a loose structure, contrary to the CNOT1(800-999) structure that was found to be very rigid. Experiments performed in this thesis have led to the discovery of the interaction sites between the natively disordered GW182 silencing domain and the helical CNOT1(800-999) protein fragment, contributing to the understanding of the molecular mechanisms of recognition within protein complexes involved in gene silencing in different physiological processes.<br>Niniejsza praca doktorska dotyczy biofizycznych podstaw oddziaływania między białkami zaangażowanymi w wyciszanie ekspresji genów przez mikro-RNA (miRNA), a mianowicie pomiędzy białkiem CNOT1 a domeną wyciszającą białka GW182. W procesie wyciszania ekspresji genów przez miRNA, cząsteczki te wiążą się z białkiem Argonaute i naprowadzają je na cząsteczkę mRNA, która ma ulec wyciszeniu. Z białkiem Argonaute oddziałuje białko GW182, które z kolei wiąże się z kompleksem deadenylaz CCR4-NOT. Kompleks ten deadenyluje mRNA oraz może także blokować jego translację, co łącznie prowadzi do wyciszenia ekspresji danego genu. Z kolei w wyciszaniu mRNA zawierających sekwencje bogate w adeninę i urydynę, rolę miRNA wraz z Argonaute i GW182 pełni białko o nazwie tristetraprolina, które odgrywa kluczową rolę w procesach odpowiedzi na stany zapalne. Oddziaływania pomiędzy składnikami tego skomplikowanego układu białek o wielkich masach cząsteczkowych są jeszcze stosunkowo słabo poznane. W szczególności, nieznane były miejsca odpowiedzialne za tworzenie kompleksu pomiędzy GW182 a CCR4-NOT. Doświadczenia z zakresu biologii molekularnej pozwoliły na identyfikację miejsc wiążących CCR4-NOT w sekwencji domeny wyciszającej białka GW182. Jedno z nich ma kluczowy wpływ na deadenylację, a drugie - kluczowy wpływ na oddziaływanie z kompleksem CCR4-NOT za pośrednictwem jego centralnej podjednostki CNOT1. Badania biofizyczne metodą wymiany wodór-deuter sprzężoną ze spektrometrią mas pozwoliły z kolei na identyfikację miejsca oddziaływania GW182 na białku CNOT1 (we fragmencie 800-999), które, niespodziewanie, okazało się bardzo dobrze pokrywać z miejscem oddziaływania CNOT1(800-999) z tristetraproliną. Eksperymenty biochemiczne wykazały, że białka te konkurują o miejsce oddziaływania na CNOT1(800-999). Białka GW182 i tristetraprolina oddziałują z CNOT1 wykorzystując ten sam motyw sekwencji, RLPXφ, w bardzo podobny, jednak nie identyczny sposób. Sekwencja ta prawdopodobnie działa jako tzw. krótki motyw liniowy (z ang. short linear motif, SLiM). Zatem te dwa szlaki kontroli nad ekspresją genów krzyżują się. W pracy zbadano także dynamikę strukturalną białka CNOT1(800-999) oraz domeny wyciszającej białka GW182. Wykazano eksperymentalnie, że białko GW182 ma nieustrukturyzowany charakter, oprócz domeny wiążącej RNA (RRM), która ma strukturę bardzo dynamiczną. Natomiast białko CNOT1(800-999) charakteryzuje się stabilną, ściśle upakowaną strukturą. Przeprowadzone badania doprowadziły do odkrycia miejsc oddziaływania pomiędzy natywnie nieustrukturyzowaną domeną wyciszającą GW182, a helikalnym fragmentem białka CNOT1(800 999), przyczyniając się do zrozumienia molekularnych mechanizmów rozpoznawania w kompleksach białkowych odpowiedzialnych za regulację ekspresji genów w różnych procesach komórkowych.