Human platelets glycoprotein VI (GPVI) is evidenced to be a platelet receptor of major importance in the occurrence of arterial thrombosis. Thus, it can be considered to be of great interest in diagnosis and therapeutic of atheriosclerotic diseases. Antibodies are powerful molecules which can be used in both diagnostic as well as for therapeutic purposes due to their unique characteristics. Monoclonal and recombinant antibodies have antigen restricted specificity, high affinity and can be used in various assays. Moreover, the good knowledge of their structure and molecular engineering facilities now allows the antibody modulation according to desired properties.Our group has already produced several monoclonal antibodies to human GPVI by gene gun immunization against the immunoadhesin hGPVI-Fc, which differ in fine epitopespecificity, affinity and other functional properties (Lecut et al. 2003). One, 3J24, with diagnostic potential while the other, 9O12, has a therapeutic potential because it blocks the binding of GPVI to collagen. Its Fab fragment has been extensively characterized in vitro,ex vivo and in vivo for its antithrombotic properties.Here, we designed and reshaped a single-chain antibody fragment (scFv) based on 3J24variable domains for the quantification of GPVI with diagnostic potential. We were also involved in the design, production and functional evaluation of humanized anti-GPVI recombinant antibody fragments (scFvs and Fabs) with therapeutic properties.

Ce travail a comme objectif d'analyser la voix et le silence dans l'œuvre fragmentaire de deux écrivains, Fernando Pessoa (1888-1935) et Maurice Maeterlinck (1862-1949), héritiers de plusieurs cultures, auteurs dont l'écriture gagne un surplus de sens grâce aux voix qu'elle crée et qui font entendre leurs mots ou bien le silence de leur absence.A travers l'analyse de quelques-unes de leurs oeuvres, nous essayons de comprendre jusqu'à quel point ces deux poètes, rattachés aux mouvements moderniste et symboliste, vécurent l'inquiétude propre à leur siècle, réfléchissant sur les raisons qui les menèrent à transformer la vie en un éternel mouvement vers l'absolu, une quête d'un état d'âme impossible en dehors de leur création poétique ou théâtrale. Nous tenterons ainsi de justifier, dans la mesure du possible, la forme inachevée d'une grande partie de leurs écrits, marqués par la dépersonnalisation et le questionnement concernant l'énigme de l'existence.Fernando Pessoa et ses hétéronymes seront les voix (im)possibles dans le silence de Maeterlinck, deux formes d'une même polyphonie, placée en rapport avec la fragmentation formelle des personnages des oeuvres de chacun des auteurs.

Protein interactions are the basis of synaptic function, and studying these interactions on a molecular level is crucial for understanding basic brain function, as well as mechanisms underlying neurological disorders. In this thesis, kinetic and mechanistic characterization of synaptic protein interactions was performed by using surface plasmon resonance biosensor technology. Fragment library screening against the reverse transcriptase of HIV was included, as it served as an outlook for future drug discovery against ligand-gated ion channels. The protein-protein interaction studies of postsynaptic Ca2+ -binding proteins revealed caldendrin as a novel binding partner of AKAP79. Caldendrin and calmodulin bind and compete at similar binding sites but their interactions display different mechanisms and kinetics. In contrast to calmodulin, caldendrin binds to AKAP79 both in the presence and absence of Ca2+ suggesting distinct in vivo functional properties of caldendrin and calmodulin. Homo-oligomeric β3 GABAA receptors, although not yet identified in vivo, are candidates for a histamine-gated ion channel in the brain. To aid the identification of the receptor, 51 histaminergic ligands were screened and a unique pharmacology was determined. A further requirement for identifying β3 receptors in the brain, is the availability of specific high-affinity ligands. The developed biosensor assay displayed sufficient sensitivity and throughput for screening for such ligands, as well as for being employed for fragment-based drug discovery. AMPA receptors are excitatory ligand-gated ion channels, involved in synaptic plasticity, and modulated by auxiliary proteins. Previous results have indicated that Noelin1, a secreted glycoprotein, interacts with the AMPA receptor. By using biochemical methods, it was shown that Noelin1 interacts directly with the receptor. The kinetics of the interaction were estimated by biosensor analysis, thereby confirming the interaction and suggesting low nanomolar affinity. The results provide a basis for functional characterization of a novel AMPA receptor protein interaction. The results demonstrate how secrets of synaptic protein interactions and function were revealed by using a molecular based approach. Improving the understanding of such interactions is valuable for basic neuroscience. At the same time, the technical advancements that were achieved to study interactions of ligand-gated ion channels by surface plasmon resonance technology, provide an important tool for discovery of novel therapeutics against these important drug targets.

The research work reported in the thesis deals with the development of dynamic covalent systems and their applications in evaluation and screening of protein-ligands and enzyme inhibitors, as well as in synthetic methodologies. The thesis is divided into four parts as described below. In part one, synthetic methodologies to access 3-functionalized phthalides and 3-thioisoindolinones using the concept of cascade reactions are demonstrated. Efficient syntheses of the target products are designed and performed in one-pot process under mild reaction conditions.  In part two, phosphine-catalyzed disulfide metathesis for the generation of dynamic carbohydrate system in aqueous solution is demonstrated. In the presence of biological target (Concanavalin A), the optimal dynamic ligand is successfully identified in situ by the 1H STD-NMR spectroscopy. In part three, lipase-catalyzed resolutions of dynamic reversible systems using reversible cyanohydrin and hemithioacetal reactions in one-pot processes are demonstrated. The dynamic systems are generated under thermodynamic control in organic solution and subsequently resolved by lipase-mediated resolution under kinetic control. The resolution processes resulted in the lipase-selected substrates with high structural and stereochemical specificities. In the last part, dynamic fragment-based strategy is presented using β-galactosidase as a model target enzyme. Based on our previous study, the best dynamic inhibitor of β-galactosidase was identified using 1H STD-NMR technique from dynamic hemithioacetal systems. The structure of the dynamic inhibitor is tailored by fragment linking and optimization processes. The designed inhibitor structures are then synthesized and tested for inhibition activities against β-galactosidase. / QC 20110526

Millions of years of evolution have resulted in an immense number of different proteins, which participate in virtually every process within cells and thus are of utmost importance for allknown forms of life. In addition, there are several examples of natural proteins which have found use in applications outside their natural environment, such as the use of enzymes infood industry and washing powders or the use of antibodies in diagnostic, bioseparation or therapeutic applications. To improve the performance of proteins in such applications, anumber of techniques, all collectively referred to as ‘protein engineering’, are performed in thelaboratory.Traditionally, methods involving ‘rational design’, where a few alterations are introduced atspecific protein locations to hopefully result in expected improvements have been applied.However, the use of more recent techniques involving a simultaneous construction of a large number of candidate variants (protein libraries) by various diversification principles, fromwhich rare clones showing enhanced properties can be isolated have contributed greatly to thefield of protein engineering.In the present thesis, different protein traits of biotechnological importance have beenaddressed for improvements by the use of such methods, in which there is a crucial need tomaintain a clonal link between the genotype and the phenotype to allow an identification of protein library members isolated by virtue of their functional properties. In all protein library investigations included in this thesis this coupling has been obtained by Escherichia coli bacterialcell-membrane compartmental confinement.In a first study, a combination of error prone PCR and gene-shuffling was applied to the Tobacco Etch Virus (TEV)-protease gene in order to produce collections from which genesencoding variants showing an enhanced soluble expression of the enzyme frequently used inbiotechnology to cleave fusion proteins were identified. Using Green Fluorescence Protein(GFP)-based cell fluorescence analysis, a clone with a five-fold increase in the yield of solubly produced protein was successfully isolated. In a second study, a novel and different GFPbased selection system, in addition also involving targeted in vivo protein degradation principles,was employed for investigations of the substrate sequence space of the same protease. In two additional studies, a selection system denoted Protein Fragment Complementation Assay(PCA), based on the affinity driven structural complementation of a genetically split β-lactamase enzyme was used to identify variants having desired target protein binding abilities,including both specificity and affinity. Using Darwinian principles concerning clonal growth advantages, affibody binding proteins showing sub-nanomolar dissociation constants to thehuman cytokine TNF-α were isolated. Taken together, these studies have shown that the bacterial format is very well suited for use in various aspects of protein library selection. / QC 20100729

El objetivo de esta tesis se centra en el estudio de la transferencia de electrones (ET), una de las reacciones más simples y cruciales en bioquímica. Para dichos procesos, obtener información directa de los factores que lo promueves, asi como del camino de transferencia electronica, no es una tarea trivial. Dicha información a un nivel de conocimiento detallado atómico y electrónico, sin embargo, es muy valiosa en términos de una mejor comprensión del ciclo enzimático, que podría conducir, por ejemplo, a un diseño más eficaz de inhibidores. El objetivo principal de esta tesis es el desarrollo de una metodología para el estudio cuantitativo de la ET en los sistemas biológicos. En este sentido, hemos desarrollado un nuevo método para obtener el camino de transferencia electrónico, llamado QM/MM e-­‐ Pathway, que se puede aplicar en sistemas complejos con ET de largo alcance. El método se basa en una búsqueda sucesiva de residuos importantes para la ET, utilizando la modificación de la región quantica en métodos mixtos QM/MM, y siguiendo la evolución de la densidad de espín dentro de la zona de transferencia. Hemos demostrado la utilidad y la aplicabilidad del algoritmo en el complejo P450cam/Pdx, identificando el papel clave de la Arg112 (en P450cam) y del Asp48 (en Pdx), ambos conocidos en la literatura. Además de obtener caminos de ET, hemos cuantificado su importancia en términos del acoplamiento electrónico entre el dador y aceptor para los diferentes caminos. En este sentido, se realizaron dos estudios de la influencia del solvente y de la temperatura en el acoplamiento electrónico para sistemas modelo oligopéptidos. Ambos estudios revelaron que los valores del acoplamiento electrónico fluctúan fuertemente a lo largo de las trayectorias de dinámica molecular obtenidas, y el mecanismo de transferencia de electrones se ve ampliamente afectado por el espacio conformacional del sistema. La combinación del QM/MM e-­‐pathway y de los cálculos de acoplamiento electronico fueron utilizados finalmente para investigar la ET en el complejo CCP/Cytc. Nuestros hallazgos indican el papel fundamental del Trp191 en localizar un estadio intermedio para la transferencia electronica, así como el camino ET principal que incluye Ala194, Ala193, Gly192 y Trp191. Ambos hallazgos fueron confirmados a través de la literatura. Los resultados obtenidos para el muestro de manios de ET, junto con su evaluación a través de cálculos de acoplamiento electrónico, sugieren un enfoque sencillo y prometedor para investigar ET de largo alcance en proteínas. / The focus of this PhD thesis lies on electron transfer (ET) processes, belonging to the simplest but most crucial reactions in biochemistry. Getting direct information of the forces driving the process and the actual electron pathway is not a trivial task. Such atomic and electronic detailed information, however, is very valuable in terms of a better understanding of the enzymatic cycle, which might lead, for example, to more efficient protein inhibitor design. The main objective of this thesis was the development of a methodology for the quantitative study of ET in biological systems. In this regard, we developed a novel approach to map long-­‐range electron transfer pathways, called QM/MM e-­‐Pathway. The method is based on a successive search for important ET residues in terms of modifying the QM region following the evolution of the spin density of the electron (hole) within a given transfer region. We proved the usefulness and applicability of the algorithm on the P450cam/Pdx complex, indicating the key role of Arg112 of P450cam and Asp48 of Pdx for its ET pathway, both being known to be important from the literature. Besides only identifying the ET pathways, we further quantified their importance in terms of electronic coupling of donor and acceptor incorporating the particular pathway residues. Within this regard, we performed two systematic evaluations of the underlying reasons for the influence of solvent and temperature onto electronic coupling in oligopeptide model systems. Both studies revealed that electronic coupling values strongly fluctuate throughout the molecular dynamics trajectories obtained, and the mechanism of electron transfer is affected by the conformational space the system is able to occupy. Combining both ET mapping and electronic coupling calculations, we finally investigated the electron transfer in the CcP/Cytc complex. Our findings indicate the key role of Trp191 being the bridge-­‐localized state of the ET as well as the main pathway consisting of Ala194, Ala193, Gly192 and Trp191 between CcP and Cytc. Both findings were confirmed through the literature. Moreover, our calculations on several snapshots state a nongated ET mechanism in this protein complex. The methodology developed along this thesis, mapping ET pathways together with their evaluation through electronic coupling calculations, suggests a straightforward and promising approach to investigate long-­‐range ET in proteins.

To aid diagnosis, treatment planning, and patient education, clinicians require tools to anal- yse and explore the increasingly large three-dimensional (3-D) datasets generated by modern medical scanners. Direct volume rendering is one such tool finding favour with radiologists and surgeons for its photorealistic representation. More recently, volume illustration — or non-photorealistic rendering (NPR) — has begun to move beyond the mere depiction of data, borrowing concepts from illustrators to visually enhance desired information and suppress un- wanted clutter. Direct volume rendering generates images by accumulating pixel values along rays cast into a 3-D image. Transfer functions allow users to interactively assign material properties such as colour and opacity (a process known as classification). To achieve real-time framerates, the rendering must be accelerated using a technique such as 3-D texture mapping on commod- ity graphics processing units (GPUs). Unfortunately, current methods do not allow users to intuitively enhance regions of interest or suppress occluding structures. Furthermore, addi- tional scalar images describing clinically relevant measures have not been integrated into the direct rendering method. These tasks are essential for the effective exploration, analysis, and presentation of 3-D images. This body of work seeks to address the aforementioned limitations. First, to facilitate the research program, a flexible architecture for prototyping volume illustration methods is pro- posed. This program unifies a number of existing techniques into a single framework based on 3-D texture mapping, while also providing for the rapid experimentation of novel methods. Next, the prototyping environment is employed to improve an existing method—called tagged volume rendering — which restricts transfer functions to given spatial regions using a number of binary segmentations (tags). An efficient method for implementing binary tagged volume rendering is presented, along with various technical considerations for improving the classifi- cation. Finally, the concept of greyscale tags is proposed, leading to a number of novel volume visualisation techniques including position modulated classification and dynamic exploration. The novel methods proposed in this work are generic and can be employed to solve a wide range of problems. However, to demonstrate their usefulness, they are applied to a specific case study. Ischaemic heart disease, caused by narrowed coronary arteries, is a leading healthconcern in many countries including Australia. Computed tomography angiography (CTA) is an imaging modality which has the potential to allow clinicians to visualise diseased coronary arteries in their natural 3-D environment. To apply tagged volume rendering for this case study, an active contour method and minimal path extraction technique are proposed to segment the heart and arteries respectively. The resultant images provide new insight and possibilities for diagnosing and treating ischaemic heart disease.

This thesis examines the nature of the relationship between migrants’ ideology and the dominant political culture in their host country, exploring what happened to German liberal migrant politicians in 19th century Wisconsin and Queensland. It does this by using Louis Hartz’s fragment theory which he developed in The Liberal Tradition in America and The Founding of New Societies. Hartz argues that the crucial factor in the political development of the new settler new societies was the migration of a fragment of European society bound by a common Weltanschauung or world view. In the United States, Hartz identifies the relevant group as the Puritans who fled Britain in the 17th century, and whose Calvinism he links to Lockean liberalism. Hartz and his collaborator Richard Rosecrance, who wrote the Australian section of New Societies, argue Australia was shaped by the lower-middle and working-class migrant fragment, inspired by political reform movement in England, and who arrived in the first half of the 19th century armed with a utilitarian-radical ideology. With no strong opposition these fragments congeal without reference to Europe and stagnate into monolithic political cultures where all the disparate elements merge into a broad - although at times quarrelsome – national consensus. According to Hartz, this consensus is re-enforced by the individualist capitalism of The American Dream or the radical collectivism of The Australian Legend – which become the foundation of the two nations’ respective national character. Hartz acknowledges that the new migrant from Europe is a constant threat to this political-cultural status quo. However, he says by “consciously articulating the fragment ethic”, the new migrant is absorbed, keeping in check the ideological challenges inherent in migration. This thesis argues that, in the case of the German liberals, who left their homeland in the 1840s and 1850s, the process was more complex than the one Hartz describes. In Wisconsin, German liberalism was most aligned to Jeffersonian democracy and the Germans either rejected outright or never fully embraced other strands within the political consensus such as Puritan moralism, Jacksonian democracy and Hamiltonian federalism. In Queensland their German liberalism was most compatible with utilitarianism and the Germans rejected most elements of classical liberalism, the evangelical element within social liberalism and the working-class radicalism of the Labor Party. They accepted Jeffersonian democracy and utilitarianism in their respective new homes because they were closest to their core German liberal principles of secularism, the primacy of the rule of law, romanticism, opposition to the aristocracy, and an aversion to rampant capitalism. Most important, however, were their attitudes towards the Staat and Volk. The Staat was both as a potential enemy and also a vital ally in liberal reform and the Volk were seen as potential colleagues in a liberal state but also as a danger to stability. Over the course of their careers they ideologically realigned, leaving parties and factions whenever challenged and using their German liberal ideals as their political reference point.

This thesis examines the nature of the relationship between migrants’ ideology and the dominant political culture in their host country, exploring what happened to German liberal migrant politicians in 19th century Wisconsin and Queensland. It does this by using Louis Hartz’s fragment theory which he developed in The Liberal Tradition in America and The Founding of New Societies. Hartz argues that the crucial factor in the political development of the new settler new societies was the migration of a fragment of European society bound by a common Weltanschauung or world view. In the United States, Hartz identifies the relevant group as the Puritans who fled Britain in the 17th century, and whose Calvinism he links to Lockean liberalism. Hartz and his collaborator Richard Rosecrance, who wrote the Australian section of New Societies, argue Australia was shaped by the lower-middle and working-class migrant fragment, inspired by political reform movement in England, and who arrived in the first half of the 19th century armed with a utilitarian-radical ideology. With no strong opposition these fragments congeal without reference to Europe and stagnate into monolithic political cultures where all the disparate elements merge into a broad - although at times quarrelsome – national consensus. According to Hartz, this consensus is re-enforced by the individualist capitalism of The American Dream or the radical collectivism of The Australian Legend – which become the foundation of the two nations’ respective national character. Hartz acknowledges that the new migrant from Europe is a constant threat to this political-cultural status quo. However, he says by “consciously articulating the fragment ethic”, the new migrant is absorbed, keeping in check the ideological challenges inherent in migration. This thesis argues that, in the case of the German liberals, who left their homeland in the 1840s and 1850s, the process was more complex than the one Hartz describes. In Wisconsin, German liberalism was most aligned to Jeffersonian democracy and the Germans either rejected outright or never fully embraced other strands within the political consensus such as Puritan moralism, Jacksonian democracy and Hamiltonian federalism. In Queensland their German liberalism was most compatible with utilitarianism and the Germans rejected most elements of classical liberalism, the evangelical element within social liberalism and the working-class radicalism of the Labor Party. They accepted Jeffersonian democracy and utilitarianism in their respective new homes because they were closest to their core German liberal principles of secularism, the primacy of the rule of law, romanticism, opposition to the aristocracy, and an aversion to rampant capitalism. Most important, however, were their attitudes towards the Staat and Volk. The Staat was both as a potential enemy and also a vital ally in liberal reform and the Volk were seen as potential colleagues in a liberal state but also as a danger to stability. Over the course of their careers they ideologically realigned, leaving parties and factions whenever challenged and using their German liberal ideals as their political reference point.

This thesis examines the nature of the relationship between migrants’ ideology and the dominant political culture in their host country, exploring what happened to German liberal migrant politicians in 19th century Wisconsin and Queensland. It does this by using Louis Hartz’s fragment theory which he developed in The Liberal Tradition in America and The Founding of New Societies. Hartz argues that the crucial factor in the political development of the new settler new societies was the migration of a fragment of European society bound by a common Weltanschauung or world view. In the United States, Hartz identifies the relevant group as the Puritans who fled Britain in the 17th century, and whose Calvinism he links to Lockean liberalism. Hartz and his collaborator Richard Rosecrance, who wrote the Australian section of New Societies, argue Australia was shaped by the lower-middle and working-class migrant fragment, inspired by political reform movement in England, and who arrived in the first half of the 19th century armed with a utilitarian-radical ideology. With no strong opposition these fragments congeal without reference to Europe and stagnate into monolithic political cultures where all the disparate elements merge into a broad - although at times quarrelsome – national consensus. According to Hartz, this consensus is re-enforced by the individualist capitalism of The American Dream or the radical collectivism of The Australian Legend – which become the foundation of the two nations’ respective national character. Hartz acknowledges that the new migrant from Europe is a constant threat to this political-cultural status quo. However, he says by “consciously articulating the fragment ethic”, the new migrant is absorbed, keeping in check the ideological challenges inherent in migration. This thesis argues that, in the case of the German liberals, who left their homeland in the 1840s and 1850s, the process was more complex than the one Hartz describes. In Wisconsin, German liberalism was most aligned to Jeffersonian democracy and the Germans either rejected outright or never fully embraced other strands within the political consensus such as Puritan moralism, Jacksonian democracy and Hamiltonian federalism. In Queensland their German liberalism was most compatible with utilitarianism and the Germans rejected most elements of classical liberalism, the evangelical element within social liberalism and the working-class radicalism of the Labor Party. They accepted Jeffersonian democracy and utilitarianism in their respective new homes because they were closest to their core German liberal principles of secularism, the primacy of the rule of law, romanticism, opposition to the aristocracy, and an aversion to rampant capitalism. Most important, however, were their attitudes towards the Staat and Volk. The Staat was both as a potential enemy and also a vital ally in liberal reform and the Volk were seen as potential colleagues in a liberal state but also as a danger to stability. Over the course of their careers they ideologically realigned, leaving parties and factions whenever challenged and using their German liberal ideals as their political reference point.