Le CD36 est un récepteur de type éboueur de classe B exprimé à la surface de nombreux types cellulaires dont les macrophages, les cellules endothéliales de la microvasculature et les plaquettes. Ce récepteur multiligand est impliqué dans plusieurs processus pathologiques notamment l’athérosclérose, l’angiogénèse et la malaria via la liaison spécifique de ligands comme les lipoprotéines oxydées de basse densité, la thrombospondine-1 et la protéine PfEMP-1, respectivement. Les peptides de la relâche de l’hormone de croissance (GHRP) ont été identifiés comme les premiers ligands synthétiques du CD36. Afin de développer de nouveaux ligands synthétiques du CD36, l’établissement d’une méthode de criblage est essentiel pour découvrir des composés avec une liaison de haute affinité pour ce récepteur. Pour y parvenir, nous avons surexprimé le domaine extracellulaire du CD36 humain dans les cellules d’insectes Sf9. La protéine soluble purifiée par chromatographie d’affinité fut immobilisée à la surface d’une plaque de résonance de plasmons de surface (SPR) pour les études de liaison. La méthodologie développée a permis de caractériser les ligands du CD36 en déterminant leurs constantes de dissociation (KD), et d’établir une relation structure-activité des ligands de la famille des azapeptides, des composés dérivés du GHRP-6. Afin de valider la méthode par spectroscopie SPR, une corrélation a été établie entre les valeurs de KD obtenues en SPR et les valeurs d’CI50 de courbes d’inhibition de la phosphorylation des MAP kinases JNK1/2 induite par un phospholipide oxydé, le POVPC, en présence de concentrations croissantes de ligands du CD36 dans les macrophages RAW 264.7. / CD36 is a class B scavenger receptor expressed at the cell surface of macrophages, endothelial cells and platelets, among others. This multiligand receptor is implicated in various diseases such as atherosclerosis, angiogenesis and malaria through the specific binding of ligands, such as oxidized low-density lipoproteins, thrombospondin-1 and the PfEMP-1 protein, respectively. Growth hormone-releasing peptides (GHRP) were identified as the first CD36 synthetic ligands. In order to identify new CD36 synthetic ligands, the development of a high-throughput method is essential to unveil compounds of high binding affinity. We have expressed a recombinant CD36 ectodomain protein in Sf9 insect cells. The soluble and affinity purified protein was immobilized on a surface plasmon resonance (SPR) sensor for binding studies. Synthetic ligands were analyzed by SPR spectroscopy for determination of their respective dissociation constant (KD). A structure-activity relationship of CD36 ligands was established. To validate the SPR binding signal, a good correlation was observed between KD and the IC50 values obtained from the inhibition curves of the MAPK kinase JNK1/2 phosphorylation induced by an oxidized phospholipid, the POVPC, in the presence of increasing concentrations of CD36 ligands in RAW 264.7 macrophage cells.

L’oxydoréduction de monocouches auto-assemblées (SAMs) de ferrocénylalcanethiolates à la surface d’or (FcRSAu) a été étudiée en temps réel par la spectroscopie de résonance de plasmons de surface couplée avec l’électrochimie (E-SPR). La sensibilité de cette technique permet de déterminer des changements d’épaisseur de couche l’ordre de quelques angström résultant d’un changement de structure de la SAM. Plusieurs études antérieures ont proposé que l’oxydation électrochimique d’une SAM de FcRSAu induit une réorientation moléculaire. L’E-SPR est utilisé pour identifier l’origine de ce changement structurel. D’abord, une calibration du réfractomètre SPR utilisé a été effectuée afin de trouver une équation de conversion du signal SPR obtenu en pixel en angle d’incidence pour que l’on puisse calculer le changement d’épaisseur de monocouche à partir du changement d’angle de résonance avec le modèle de Fresnel. Par la suite, une caractérisation approfondie des SAMs de FcCnSAu (où n = 6, 8, 12, 14) en contact avec du NaClO4 acidifié a été réalisée par électrochimie, éllipsométrie, spectroscopie infrarouge et microscopie à force atomique. Les résultats obtenus montrent que l’augmentation de la longueur des chaînes alkyles donne des SAMs de ferrocènes plus épaisses et moins désordonnées. L’analyse par l’E-SPR de ces SAMs pures montre que le changement d’épaisseur induit par l’électro-oxydation dépend linéairement du nombre de méthylènes sur la chaîne alkyle. En appliquant la déconvolution mathématique aux voltampérogrammes cycliques enregistrés pour les SAM mixtes (FcC12SAu/C11SAu) de différentes compositions, on arrive à la conclusion qu’il y a un redressement des chaînes alkyles dans les domaines des ferrocènes agrégés mais la réorientation des têtes de ferrocène dans les domaines de ferrocènes agrégés ou dispersés ne peut pas être exclue. Enfin, l’effet de l’anion électrolytique sur le changement d’épaisseur de la SAM mesuré par l’E-SPR a été étudié. L’analyse électrochimique montre que la capacité de pairage d’anions avec les ferrocéniums décroit comme suit : PF6- > ClO4- > BF4- > NO3-. Tandis que l’épaisseur de la SAM donnée par le changement d’angle de résonance suit la tendance suivante : NO3- ≥ ClO4- > PF6- ≈ BF4-. Des études plus approfondies seront nécessaire pour clarifier cette tendance observée par E-SPR. / The oxidation and reduction of self-assembled ferrocenylalkanethiolate monolayers formed on gold surfaces (FcRSAu) have been followed in real-time by electrochemical surface plasmon resonance (E-SPR) spectroscopy. Film thickness changes on the order of angstroms resulting from SAM structural changes can be detected by SPR. The electrochemically-induced reorientation of the ferrocenylalkanethiolates has been proposed by several spectroelectrochemistry studies. We have used E-SPR to elucidate the nature of the molecular reorientations. The SPR refractometer was first calibrated to find a pixel to angle equation so that Fresnel modeling could be used to calculate SAM thickness changes from the measured resonance angle changes (Δθmin). SAMs of FcCnSAu (where n = 6, 8, 12, 14) were characterized by electrochemistry, infrared spectroscopy and atomic force microscopy. The results obtained show that an increase in the alkyl chain length gives a thicker and less disordered SAM. Oxidation of the surface-bound ferrocenes to ferroceniums and pairing with ClO4- anions produces a similar change in Δθmin for these SAMs, but the variation in the SAM thickness is linearly dependent on the chain length. Mathematical deconvolution of the cyclic voltammograms of binary SAMs (FcC12SAu/C11SAu) of different compositions was used to separate the contributions of the different ferrocene domains (aggregated vs. isolated ferrocenes). We conclude that the aggregated-ferrocenes undergo a change in the orientation of the alkyl chain orientation. We cannot however exclude that the cyclopentadiene rings also reorient themselves. Anion pairing effect was studied by E-SPR. Our electrochemistry results indicate that the ability of the electrolyte anion to pair with the electrogenereted ferrocenium decreases in the following order: PF6- > ClO4- > BF4- > NO3-. On the other hand, the change in Δθmin does not follow the associated trend observed for the anion pairing capacity but rather NO3- ≥ ClO4- > PF6- ≈ BF4-. We hypothesize that the difference in the uptake water molecules by the oxidized SAM may be the reason for this observation. This hypothesis still needs to be verified.

FGF2 (Fibroblast Growth Factor 2), o fundador da família FGF, tem funções regulatórias na mitogênese, diferenciação, morfogênese e reparo tecidual. Diversas espécies moleculares de FGF2 compartilham uma seqüência C-terminal comum de 155 aminoácidos, pois se originam de diferentes sítios de iniciação de leitura de um único mRNA. O menor, o FGF2-18kDa, é liberado extracelularmente para se ligar a receptores específicos (FGFRs) para disparar as funções parácrinas e autócrinas pelas quais este fator é conhecido. Por outro lado, as espécies maiores (FGF2-21, 22, 22,5 e 34kDa) são intracelulares se ligam a parceiros moleculares desconhecidos para exercer funções intrácrinas ainda indefinidas. O objetivo desta tese foi produzir espécies recombinantes do FGF2-18 e FGF2-22,5, na forma de proteínas de fusão, para analisar funções biológicas e mecanismos de sinalização. Nas células malignas Y1 de camundongo, os recombinantes de FGF2-18kDa (FGF2-18, His-FGF2-18 e His-FGF2-18-ProA) dispararam uma resposta antagônica estimulando as vias de sinalização mitogênica, mas bloqueando o ciclo celular. Nos fibroblastos não tumorigênicos Balb3T3, estes mesmos recombinantes de FGF2-18kDa dispararam apenas a resposta mitogênica clássica. Todos os efeitos biológicos destes recombinantes de FGF2-18kDa foram bloqueados pelo inibidor específico da proteína quinase de tirosina dos FGFRs, PD173074, demonstrando que são respostas intermediadas pelos FGFRs. Portanto, os domínios estruturais adicionados aos recombinantes de FGF2-18kDa não impediram que estas proteínas se ligassem e ativassem os FGFRs. Por outro lado, o recombinante His-FGF2-22,5 dispara apenas as vias de sinalização mitogênica em ambas as células Y1 e 3T3, mas este efeito biológico não é inibido por PD173074. Estes resultados sugerem que a seqüência N-terminal de 55 resíduos, rica em aminoácidos básicos, impede que o FGF2-22,5kDa se ligue e/ou ative os FGFRs. Entretanto, o recombinante His-FGF2-22,5ProA dispara a resposta antagônica característica do FGF2-18kDa. As implicações destes últimos resultados é que o domínio de ProA adicionado ao C-terminal torna o FGF2-22,5kDa um bom ligante dos FGFRs. A interação física entre ligante e receptor das formas recombinantes His-FGF2-18kDa (ou His-FGF2-18ProA) e FGF2-22,5kDa com os putativos FGFRs foi analisada através da técnica de SPR e os resultados mostram KDs aproximados (Kd18=21, 488.10-9 e Kd22,5=20,70393.10-9), enquanto que o número de sítios ligantes em vesículas microssomais das células é significantemente inferior para o FGF2-22,5kDa. Estes resultados são compatíveis com a existência de receptores diferentes para FGF2-18kDa e FGF2-22,5kDa, uma hipótese ainda a ser definitivamente corroborada. Em conclusão, o FGF2-18kDa, mesmo em formas recombinantes como proteína de fusão, dispara todos os efeitos biológicos descritos para FGF2, através dos FGFRs. Diferentemente, o FGF2-22,5kDa, como fator parácrino, só desencadeou a resposta mitogênica clássica de FGF2, provavelmente através de receptores diferentes dos FGFRs. Os resultados e conclusões desta tese têm um potencial indiscutivelmente relevante para a biologia molecular do câncer, com implicações possíveis em terapia oncológica / FGF2 (Fibroblast Growth Factor 2), the founder of the FGF family, has regulatory functions in mitogenesis, differentiation, morphogenesis and tissue repair. Multiple FGF2 molecular species, sharing a C-terminal sequence of 155 amino acids, are translated from different iniciation sites of the same mRNA. The smaller, the FGF2-18kD, is extracellularly released to bind to specific membrane receptors (FGFRs), performing paracrine and autocrine functions. On the other hand, the larger FGF2s (21, 22, 22.5 and 34kDa) are intracellular species that bind to unknown partners to play still undefined intracrine roles. The aim of this thesis was to produce recombinant species of FGF2-18kDa and FGF2-22,5kDa, in the form of fusion proteins, to analyze functions and signaling mechanisms. In mouse Y1 malignant cells, FGF2-18kD recombinants (FGF2-18kDa and His-FGF2-18kDaProA) triggered an antagonistic response activating mitogenic signaling pathways, but blocking the cell cycle. However, in non tumorigenic Balb3T3 fibroblasts, these same FGF2-18kD recombinants only elicited the classical mitogenic response. All biological effects of these FGF2-18kD recombinants were blocked by the specific inhibitor of FGFR-protein-tyrosine-kinases, PD173074, demonstrating that these responses are mediated by FGFRs. Therefore, the new peptide domains added to FGF2-18kD did not prevent these recombinant fusion proteins to bind and activate FGFRs. Conversely, the recombinant His-FGF2-22,5kDa triggered only mitogenic signaling pathways in both Y1 and Balb3T3 cells, a biological effect not inhibited by PD173074. These results suggested that the additional basic-rich N-terminal sequence of 55 amino acid residues, found in FGF2-22,5kDa, prevents this FGF2 species from binding and / or activate FGFRs. However, surprisingly, the recombinant His-FGF2-22kDaProA triggered the antagonistic response characteristic of FGF2-18kDa. These results imply that the ProA-domain added to the C-terminal end rendered the FGF2-22,5kDaProA a good ligand of FGFRs. The physical interaction between recombinants of both His-FGF2-18kD and His-FGF2-22kDa with putative FGFRs, analyzed by SPR, yielded close KD values (KD18=21, 5.10-9 e K D22,5=20,7.10-9), while the number of binding sites in cell microsomal vesicles were significantly lower for the His-FGF2-22,5kDa. These results are consistent with the existence of different receptors for FGF2 and FGF2-18kD-22,5kDa, a hypothesis that has yet to be definitively confirmed. In conclusion, FGF2-18kD, even as recombinant fusion proteins, triggered all biological effects of FGF2, through FGFRs. Conversely, the FGF2-22, 5kDa only triggered the classical mitogenic response, probably via receptors other than FGFRs. The results and conclusions of this thesis are potentially of great interest in cancer molecular biology, with implications in oncologic therapy.

FGF2 (Fibroblast Growth Factor 2), o fundador da família FGF, tem funções regulatórias na mitogênese, diferenciação, morfogênese e reparo tecidual. Diversas espécies moleculares de FGF2 compartilham uma seqüência C-terminal comum de 155 aminoácidos, pois se originam de diferentes sítios de iniciação de leitura de um único mRNA. O menor, o FGF2-18kDa, é liberado extracelularmente para se ligar a receptores específicos (FGFRs) para disparar as funções parácrinas e autócrinas pelas quais este fator é conhecido. Por outro lado, as espécies maiores (FGF2-21, 22, 22,5 e 34kDa) são intracelulares se ligam a parceiros moleculares desconhecidos para exercer funções intrácrinas ainda indefinidas. O objetivo desta tese foi produzir espécies recombinantes do FGF2-18 e FGF2-22,5, na forma de proteínas de fusão, para analisar funções biológicas e mecanismos de sinalização. Nas células malignas Y1 de camundongo, os recombinantes de FGF2-18kDa (FGF2-18, His-FGF2-18 e His-FGF2-18-ProA) dispararam uma resposta antagônica estimulando as vias de sinalização mitogênica, mas bloqueando o ciclo celular. Nos fibroblastos não tumorigênicos Balb3T3, estes mesmos recombinantes de FGF2-18kDa dispararam apenas a resposta mitogênica clássica. Todos os efeitos biológicos destes recombinantes de FGF2-18kDa foram bloqueados pelo inibidor específico da proteína quinase de tirosina dos FGFRs, PD173074, demonstrando que são respostas intermediadas pelos FGFRs. Portanto, os domínios estruturais adicionados aos recombinantes de FGF2-18kDa não impediram que estas proteínas se ligassem e ativassem os FGFRs. Por outro lado, o recombinante His-FGF2-22,5 dispara apenas as vias de sinalização mitogênica em ambas as células Y1 e 3T3, mas este efeito biológico não é inibido por PD173074. Estes resultados sugerem que a seqüência N-terminal de 55 resíduos, rica em aminoácidos básicos, impede que o FGF2-22,5kDa se ligue e/ou ative os FGFRs. Entretanto, o recombinante His-FGF2-22,5ProA dispara a resposta antagônica característica do FGF2-18kDa. As implicações destes últimos resultados é que o domínio de ProA adicionado ao C-terminal torna o FGF2-22,5kDa um bom ligante dos FGFRs. A interação física entre ligante e receptor das formas recombinantes His-FGF2-18kDa (ou His-FGF2-18ProA) e FGF2-22,5kDa com os putativos FGFRs foi analisada através da técnica de SPR e os resultados mostram KDs aproximados (Kd18=21, 488.10-9 e Kd22,5=20,70393.10-9), enquanto que o número de sítios ligantes em vesículas microssomais das células é significantemente inferior para o FGF2-22,5kDa. Estes resultados são compatíveis com a existência de receptores diferentes para FGF2-18kDa e FGF2-22,5kDa, uma hipótese ainda a ser definitivamente corroborada. Em conclusão, o FGF2-18kDa, mesmo em formas recombinantes como proteína de fusão, dispara todos os efeitos biológicos descritos para FGF2, através dos FGFRs. Diferentemente, o FGF2-22,5kDa, como fator parácrino, só desencadeou a resposta mitogênica clássica de FGF2, provavelmente através de receptores diferentes dos FGFRs. Os resultados e conclusões desta tese têm um potencial indiscutivelmente relevante para a biologia molecular do câncer, com implicações possíveis em terapia oncológica / FGF2 (Fibroblast Growth Factor 2), the founder of the FGF family, has regulatory functions in mitogenesis, differentiation, morphogenesis and tissue repair. Multiple FGF2 molecular species, sharing a C-terminal sequence of 155 amino acids, are translated from different iniciation sites of the same mRNA. The smaller, the FGF2-18kD, is extracellularly released to bind to specific membrane receptors (FGFRs), performing paracrine and autocrine functions. On the other hand, the larger FGF2s (21, 22, 22.5 and 34kDa) are intracellular species that bind to unknown partners to play still undefined intracrine roles. The aim of this thesis was to produce recombinant species of FGF2-18kDa and FGF2-22,5kDa, in the form of fusion proteins, to analyze functions and signaling mechanisms. In mouse Y1 malignant cells, FGF2-18kD recombinants (FGF2-18kDa and His-FGF2-18kDaProA) triggered an antagonistic response activating mitogenic signaling pathways, but blocking the cell cycle. However, in non tumorigenic Balb3T3 fibroblasts, these same FGF2-18kD recombinants only elicited the classical mitogenic response. All biological effects of these FGF2-18kD recombinants were blocked by the specific inhibitor of FGFR-protein-tyrosine-kinases, PD173074, demonstrating that these responses are mediated by FGFRs. Therefore, the new peptide domains added to FGF2-18kD did not prevent these recombinant fusion proteins to bind and activate FGFRs. Conversely, the recombinant His-FGF2-22,5kDa triggered only mitogenic signaling pathways in both Y1 and Balb3T3 cells, a biological effect not inhibited by PD173074. These results suggested that the additional basic-rich N-terminal sequence of 55 amino acid residues, found in FGF2-22,5kDa, prevents this FGF2 species from binding and / or activate FGFRs. However, surprisingly, the recombinant His-FGF2-22kDaProA triggered the antagonistic response characteristic of FGF2-18kDa. These results imply that the ProA-domain added to the C-terminal end rendered the FGF2-22,5kDaProA a good ligand of FGFRs. The physical interaction between recombinants of both His-FGF2-18kD and His-FGF2-22kDa with putative FGFRs, analyzed by SPR, yielded close KD values (KD18=21, 5.10-9 e K D22,5=20,7.10-9), while the number of binding sites in cell microsomal vesicles were significantly lower for the His-FGF2-22,5kDa. These results are consistent with the existence of different receptors for FGF2 and FGF2-18kD-22,5kDa, a hypothesis that has yet to be definitively confirmed. In conclusion, FGF2-18kD, even as recombinant fusion proteins, triggered all biological effects of FGF2, through FGFRs. Conversely, the FGF2-22, 5kDa only triggered the classical mitogenic response, probably via receptors other than FGFRs. The results and conclusions of this thesis are potentially of great interest in cancer molecular biology, with implications in oncologic therapy.

<p>In this study the formation of supported membranes onto planar solid supports has been investigated. The stability and activity of reconstituted membrane receptors has been studied. The potential use of such preparations in biosensor applications is discussed.</p><p>The lipid films were made by the Langmuir Blodgett and by the liposome fusion techniques. These supported films were characterised by ellipsometry, atomic force microscopy, surface plasmon resonance (SPR) and resonant mirror techniques. The thickness of the films was in agreement with that of a cell membrane. The kinetics of formation of the lipid films was studied and discussed.</p><p>The proteins, bacteriorhodopsin, cytochrome oxidase, acetylcholinesterase and the nicotinic acetylcholine receptor were reconstituted into the supported membrane. The subsequent analysis showed that the proteins were individually distributed and that the activity was retained, in some cases for several weeks after immobilisation.</p><p>The glycolipids, GM1, GM2, GD1b, asialo-GM1, globotriaosylceramide, lactosylceramide and galactosylceramide, were also reconstituted into the supported membranes. Their specific interaction with the toxin ricin or with its B-chain was examined using SPR. The affinity of intact toxin and of its B-chain differed markedly and was pH dependent. The carbohydrate chain length and charge density of the glycolipids also influenced the affinity.</p>

La détection de séquences génétiques par la technologie des puces à ADN se heurte à des problèmes de fiabilité et de reproductibilité, dus en grande partie à des problèmes de mélange. Dans toute cette thèse, nous montrons à quel point le mélange par advection chaotique améliore les performances de ces puces. Pour cela nous comparons, par une étude principalement numérique, l'efficacité de deux protocoles de mélange basés sur le principe d'injection alternée et périodique de fluide (modèle puits/sources) : l'un utilise des seringues réversibles, l'autre des pompes recyclant le fluide extrait, conduisant globalement à un mélange bien plus efficace et rapide. En outre, nous mettons en évidence le rôle important de la géométrie de la chambre. Dans un second temps, nous introduisons un modèle de capture chimique entre les monobrins d'ADN libres en volume (cibles) et ceux fixés sur la puce (sondes). Nous montrons alors numériquement que la réaction est généralement grandement limitée par la diffusion, mais que l'advection chaotique améliore les choses de manière très significative grâce au mélange. Ceci nous permet d'estimer les constantes de vitesses dans le cas statique (où la diffusion agit seule) et le cas dynamique (avec mélangeur). Enfin, profitant de l'opportunité d'un stage à l'Université de Sherbrooke, j'ai effectué un premier suivi en temps réel par SPR d'une cinétique de type "cibles en solution/sondes sur support" pour tenter de comparer les vitesses d'hybridation statique et dynamique.

<p>Within telecommunication and routing of traffic in IP-networks a protocol named“Open Shortest Path First” (OSPF) is widely used. This means that a server dealswith the routing over a network with given weights by calculating shortest paths touse for routing. If we assume that a desired traffic pattern is given the problem isto find out if it is possible to set the weights so that the desired traffic pattern is apart of a shortest path graph. In this thesis we assume that it is a unique shortestpath. To search for weights that solve the problem leads to a complex LP-model. Analternative is to search in the LP-dual under certain restrictions. These solutions tothe LP-dual are called conflicts and a conflict means that there exists no weights sothat the desired traffic pattern is obtained. The goal of this thesis is to study, classifyand search for conflicts. An algorithm has been developed that finds some kind ofconflicts in polynomial time with respect to the size of the graph.</p> / <p>Inom telekommunikation och ruttning av datatrafik i IP-nätverk så används oftaett protokoll som kallas “Open Shortest Path First” (OSPF). Det innebär att enserver sköter ruttningen över ett nätverk genom att utifrån givna bågkostnaderberäkna billigaste vägar som används för ruttningen. Frågeställningen utgårfrån att vi har ett önskat ruttningsschema och vi vill ta reda på om det gåratt sätta bågkostnader så att det önskade ruttningsschemat ingår i en billigasteväg-graf. I det här examensarbetet splittas inte trafik utan varje billigaste vägär unik mellan två noder. Att söka efter bågkostnader som löser problemet geren krävande LP-modell och ett alternativ är att utgå från LP-dualen undervissa restriktioner. Dessa lösningar till LP-dualen benämns konflikter och enkonflikt motsvarar att det inte finns några bågkostnader så att det önskaderuttningsschemat fås. Målet med examensarbetet är att studera, klassificeraoch söka efter konflikter. En algoritm har tagits fram som hittar vissa typer avsådana konflikter i polynomiell tid, sett till storleken på grafen.</p>

<p>In this thesis, the structure and stability of globular proteins adsorbed onto nanometer-sized hydrophilic silica particles were investigated using differential scanning calorimetry (DSC), hydrogen/deuterium exchange (HDX), and mass spectrometry (MS). The adsorption process itself was characterized with fluorescence and absorption spectroscopy and surface plasmon resonance (SPR). The combination of these methods offered a unique insight into adsorption-induced changes within proteins related to their adsorption characteristics. DSC contributed with thermodynamic information on the overall structural stability within the protein population. HDX in combination with MS contributed information on the structure and stability of adsorbed proteins with focus on changes within the secondary structure elements. In order to increase the structural resolution in this part of the investigation, proteolysis was performed prior to the MS analyzing step. Knowledge on the protein adsorption process was utilized in a practical approach called ligand fishing. In this approach, SPR was used to monitor the chip-based affinity purification of a protein with MS used for protein identification.</p><p>Adsorption isotherms revealed that electrostatic interactions play an important role in the adsorption of proteins to hydrophilic surfaces. DSC investigation revealed that the thermal stability of proteins reduces with increasing electrostatic attraction between the protein and the surface and that this effect diminishes at higher surface coverage. The mass-increase due to exchange between protein hydrogen atoms and deuterium atoms in solution was investigated as a function of time. This gave insight into adsorption-induced changes in the structural stability of proteins. By combining DSC and HDX-MS, it was possible to differentiate between adsorption-induced changes in the secondary and tertiary structure. Additionally, if limited proteolysis was performed, the investigations gave insight into the orientation and protein segment specific changes in the stability of proteins adsorbed to silica surfaces. The adsorption of proteins to silica particles also provided the basis for a new experimental design that allows handling of minute amounts of proteins in a ligand fishing application, as used in the field of functional proteomics.</p>

My doctoral research has focused on the development of surface plasmon resonance (SPR) reflectance imaging technique to detect near-field transport properties such as concentration, temperature, and salinity in micro/nano fluidic phenomena in label-free, real-time, and full-field manner. A label-free visualization technique based on surface plasmon resonance (SPR) reflectance sensing is presented for real-time and full-field mapping of microscale concentration and temperature fields. The key idea is that the SPR reflectance sensitivity varies with the refractive index of the near-wall region of the test mixture fluid. The Fresnel equation, based on Kretschmann’s theory, correlates the SPR reflectance with the refractive index of the test medium, and then, the refractive index correlates with the mixture concentration or temperature. The basic operation principle is summarized and the laboratory-developed SPR imaging/analyzing system is described with the measurement sensitivity, uncertainties and detection limitations of the implemented SPR reflectance imaging. Total five proposed uses of SPR reflectance imaging technique are presented: (1) micromixing concentration field development of ethanol penetrating into water contained in a micro-channel, (2) full-field detection of the near-wall salinity profiles for convective/diffusion of saline droplet into water, (3) full-field and real-time surface plasmon resonance imaging thermometry, (4) correlation of near-field refractive index of nanofluids with surface plasmon resonance reflectance, and (5) unveiling hidden complex cavities formed during nanocrystalline self-assembly.

This thesis describes studies directed towards development of novel antiadhesive agents, with particular emphasis on compounds that prevent attachment of bacteria to a host-cell. Three different proteins involved in the assembly or function of adhesive pili in uropathogenic Escherichia coli have been targeted either by rational structure based design or statistical molecular methods. A library of substituted galabiose (Galα1-4Gal) derivatives was screened for binding to the E. coli adhesin PapG in an assay based on surface plasmon resonance, and for inhibition of Streptococcus suis adhesins PN and PO in a hemagglutination assay. The results were used to generate QSAR models which had good predictive powers and provided further insight in the structural requirements needed for high affinity binding. 2-pyridones and amino acid derivatives were modelled into the binding site of chaperones involved in pilus assembly in E. coli and a heuristic method, VALIDATE, was used for affinity prediction. The affinity of the compounds for the chaperones PapD and FimC were assessed in assays based on surface plasmon resonance and relaxation-edited NMR spectroscopy. Their ability to disrupt chaperone/subunit complexes was investigated in vitro through a FPLC assay and their capacity to inhibit pilus formation in vivo was determined via hemagglutination and confirmed with atomic force microscopy. Statistical molecular design was used to design a diverse peptide library targeting pili subunits, and an ELISA was developed to investigate the ability of the peptides to inhibit chaperone/subunit complexation. The resulting QSAR model provided extensive information regarding binding of the peptides to the subunits. Because the peptides were suggested to bind in an extended β-strand formation, β-strand mimetics consisting of oligomeric enaminones were designed. Finally, new methods to synthesize enaminone building blocks were developed using microwave assisted chemistry. The projects described have generated compounds that besides their value as leads for developing novel antibacterial agents, also constitute new chemical tools to study the mechanisms underlying bacterial virulence.