Résumé : Les maladies cardiovasculaires représentent, par une large mesure, la première cause de morbidité et de mortalité chez les diabétiques. L’activation de RAGE par les produits de glycation avancée (AGE) générés en conditions hyperglycémiques est associée à une multitude de complications diabétiques vasculaires, notamment par une signalisation favorisant l’inflammation chronique ainsi que la mort des cellules formant les tissus et les organes exposés aux AGE. La surexpression de RAGE dans les cellules musculaires lisses des plaques athérosclérotiques vulnérables suggère que le récepteur pourrait contribuer à la survenue des accidents vasculaires. Nous avons donc émis l’hypothèse que l’activation de RAGE dans les cellules musculaires lisses était impliquée dans leur apoptose. Pour le vérifier, nous avons, dans un premier temps, mis au point une nouvelle méthode de détection sans marqueur basée sur le principe de la résonance des plasmons de surface (SPR) pour mesurer l’apoptose d’une monocouche cellulaire en temps réel et caractériser avec précision les paramètres cinétiques des phases d’initiation et d’exécution. Cet essai a permis de montrer que l’activation de RAGE induit l’apoptose dans plus de 75,6% des cellules musculaires lisses stimulées avec le CML-HSA pendant 20 heures. De surcroît, nous avons remarqué que l’activation de RAGE générait un fort stress du réticulum endoplasmique, indiqué par la formation d’un grand nombre de granules de stress ainsi que par l’augmentation de l’expression du marqueur de stress réticulaire HuR et de la caspase-9, deux importants régulateurs de l’apoptose induite par le stress réticulaire endoplasmique. Afin de vérifier le potentiel d’un antagoniste à bloquer l’activation du récepteur, nous avons ensuite synthétisé le peptide iRAGE dont la séquence est dérivée d’un site de liaison du CML-HSA ayant la particularité de posséder de nombreuses charges négatives à pH physiologique. Le prétraitement avec iRAGE s’est montré efficace pour prévenir l’activation de NF-κB, l’induction de l’apoptose et l’augmentation du stress réticulaire endoplasmique. Nous suggérons un modèle de fonctionnement par lequel iRAGE inhibe la signalisation de RAGE en empêchant la liaison des ligands multimériques et en stabilisant les récepteurs sous forme de monomères. À terme, la synthèse d’un antagoniste de RAGE utilisable en clinique pourrait constituer une avancée majeure dans la prévention des complications vasculaires et l’amélioration de la qualité de vie chez les diabétiques. / Abstract : Cardiovascular diseases represent, to a large extent, the first cause of morbidity and mortality among people with diabetes. RAGE activation by advanced glycation end products (AGE) generated in hyperglycemic conditions is associated to a multitude of vascular diabetic complications, in particular by a signaling promoting chronic inflammation as well as death of cells forming tissues and organs exposed to AGE. Overexpression of RAGE in smooth muscle cells of vulnerable atheromatous plaques suggests the receptor could contribute to heart attacks and strokes. Therefore, we hypothesize that RAGE activation in smooth muscle cells is involved in apoptosis. To verify this hypothesis, we first designed a new label-free assay based of surface plasmon resonance (SPR) to measure apoptosis of a cell monolayer in real-time and to characterize precisely the kinetic parameters of the initiation and execution phases. This assay showed that RAGE activation induces apoptosis of more than 75.6% of smooth muscle cells stimulated with CML-HSA for 20 hours. Moreover, we noticed that RAGE activation generated strong endoplasmic reticular stress, indicated by the formation of a great number of stress granules as well as the increased expression of stress marker HuR and caspase-9, two important regulators of reticular stress-induced apoptosis. In order, to assess the potential of an antagonist to block RAGE activation, we then synthesized the iRAGE peptide whose sequence is derived from a binding site of CML-HSA that has the particularity of owning numerous negative charges at physiological pH. Pretreatment with iRAGE was successful to prevent activation of NF-κB, induction of apoptosis and generation of endoplasmic reticular stress. We suggest a model by which iRAGE inhibits RAGE signaling by hindering the binding of multimeric ligands and by stabilizing the receptors in a monomer state. Ultimately, the synthesis of a RAGE antagonist usable in clinic could constitute a major progress in the prevention of vascular complications and in the quality of life of people with diabetes.

This thesis presents the first example of reversible photoregulation of the binding of a protease, α-chymotrypsin, to a surface. A modular approach is used involving the azobenzene photoswitch group, a surface linker and an enzyme binding group. This approach is designed to be easily extended to the photoregulation of binding of other proteases to surfaces by use of enzyme binding groups selective to these proteases. Chapter one gives a brief outline of some of the important areas involved in to this work, including molecular switches, proteases and surface modification. Chapter two describes the synthesis of azobenzene-containing boronate esters designed as photoswitch inhibitors of α-chymotrypsin. Boronate esters were prepared containing the aminophenylboronate group or the peptidomimetic borophenylalanine group for enzyme binding and a range of substituents designed for enzyme affinity and/or surface attachment. Syntheses primarily involved peptide coupling reactions and azobenzene formation by condensation of nitrosobenzenes and anilines. Coupling reactions were successfully carried out using EDCI or isobutyl chlorofomate in several cases where other reagents gave unacceptable decomposition. Chapter three describes the syntheses and HPLC stability studies of derivatives of a noncovalent α-chymotrypsin inhibitor. Several dipeptide-based compounds containing either an amide group for surface attachment or an azobenzene group for photoswitching were prepared, primarily using peptide coupling reactions. Each compound was incubated with α-chymotrypsin to assess its stability, and all were found by HPLC monitoring to be stable to α-chymotrypsin catalysed hydrolysis. Chapter four describes syntheses of azobenzene-containing trifluoromethylketones and α-ketoesters designed as photoswitch inhibitors of α-chymotrypsin. Trifluoromethylketones/α-ketoesters containing amine groups for surface attachment were prepared, primarily using peptide coupling reactions, but could not be isolated due to the incompatibility of the electrophilic ketone and primary amine groups. Trifluoromethylketones/α-ketoesters containing terminal alkynes for surface attachment were prepared either by the attachment of an alkyne substituent group to a symmetrical azobenzene core or by Pd-catalysed reaction of a protected alkyne with an azobenzene having a halide substitutent. Chapter five describes syntheses of sulfur-containing surface linkers for use in surface attachment of the photoswitch inhibitors described in chapters 2-4. A range of compounds containing disulfide or protected thiol groups for surface attachment and azide or carboxylic acid groups for inhibitor attachment were prepared. Syntheses primarily involved coupling of functionalised alcohols/amides to carboxylic acid-containing disulfides/thioacetates. Selected linkers were attached to azobenzenes by amide coupling or azide-alkyne cycloaddition for surface attachment, photoswitching and/or enzyme assay. Azide-alkyne cycloaddition yields were initially poor, but were improved by use of stoichiometric amounts of copper catalyst. Chapter six describes UV/vis photoisomerisation studies and enzyme assays carried out to assess enzyme photoswitching of the compounds described in chapters 2-5. The trifluoromethylketones and α-ketoesters described in chapter 4 gave the best results, with moderate inhibition of α-chymotrypsin (µM affinity constants) and up to 5.3 fold changes in inhibition on UV/vis irradiation. Many of the boronate esters described in chapter 2 were found to inhibit α-chymotrypsin, but were somewhat unstable to irradiation. The dipeptide-based compounds described in chapter 3 were inactive against α-chymotrypsin. Good photoisomerisation was obtained for an azobenzene containing a symmetrical disulfide surface linker and poor photoisomerisation was obtained for an azobenzene containing a lipoic acid surface linker. Chapter seven describes surface attachment of selected photoswitch inhibitors and studies of photoregulated enzyme binding to the resultant functional surfaces. Self assembled monolayers (SAMs) of disulfides were formed on gold surfaces and characterised by electrochemistry and contact angle measurements. Binding of α-chymotrypsin to SAMs containing a photoswitch inhibitor was detected by quartz crystal microbalance (QCM), but was found to be largely irreversible. An alkyne-containing photoswitch inhibitor was attached to a surface plasmon resonance (SPR) chip in a two step procedure involving generation of an azide modified surface followed by azide-alkyne cycloaddition. Binding of α-chymotrypsin to the resultant modified surface was detected by SPR and successfully regulated by UV/vis irradiation. Chapter eight provides conclusions for the work described in this thesis and suggests future directions. Chapter nine gives experimental details for the work described in this thesis.

Discrete changes in the amplitude, frequency, and cellular localisation of calcium ion (Ca2+) transients encode information about numerous stimuli and function to mediate stimulus-specific responses. Cytoplasmic Ca2+ (Ca2+cyt) undergoes circadian oscillations in concentration that appear to be under the control of the same endogenous oscillator that regulates expression of genes in the photoperiodic-flowering pathway. It is currently not known whether these circadian [Ca2+cyt] oscillations are biochemical artefacts or are decoded and function to transduce clock dependent responses. Calmodulin (CaM) is a primary node in Ca2+ signalling in plants and as such is a promising target for investigating the role of Ca2+ in clock-controlled processes. In this study, Arabidopsis thaliana were treated with experimentally validated concentrations of pharmacological CaM inhibitors. Under inductive photoperiods (16 h light : 8 h dark), CaM inhibition was found to increase developmental flowering time, whilst under non-inductive photoperiods no such changes were evident. Inhibition of CaM led to changes in expression of the key clock gene TIMING OF CAB EXPRESSION 1 and flowering time genes, CONSTANS and FLOWERING LOCUS T and removed repression of flowering in darkness. These observations are consistent with CaM modulating the activity of the putative clock component GIGANTEA and the proteasomal targeting protein SUPPRESSOR OF PHYA-105. Due to the unwanted side effects often generated by chemical CaM inhibitors, a peptide inhibitor of CaM comprising a green fluorescent protein / calspermin fusion and labelled smGN was developed. Surface plasmon resonance analysis and affinity chromatography showed smGN to have extremely high selectivity for, and affinity to, CaM and to function as a powerful inhibitor of CaM in vitro. Further work on the methodology used to deploy smGN as a recombinant alternative to chemical CaM inhibitors in planta is also described.

BACKGROUND Inducible costimulator (ICOS) and its ligand (ICOSL) are a pair of costimulatory molecules that co-localize in germinal centers (GC). This interaction is critical for the maturation of GC B cells to affinity-matured memory B cells and long-lived plasma cells. Both ICOS and ICOSL are implicated in systemic lupus erythematosus (SLE). It is known that ICOSL sheds from the cell membrane and that the soluble form of ICOSL (sICOSL) is elevated in SLE; though the function of sICOSL is poorly understood. While it is known that binding of ICOSL on antigen-presenting cells (APC) to ICOS on T cells leads to cell signaling resulting in T cell activation and differentiation, there is also some preliminary evidence that reverse signaling may also occur through ICOSL in APCs. The binding interaction between ICOS and sICOSL has not been fully characterized and is important to understand if either molecule is to be targeted therapeutically. The hypothesis evaluated in this study was that the ICOS: ICOSL interaction is a potent and critical mediator of proinflammatory signaling and immune activation that functions both via activated T cell-mediated forward signaling and APC-mediated reverse signaling mechanisms and that ectodomain shedding of ICOSL is a protective mechanism that leads to down-regulation of the proinflammatorysignaling cascade initiated by this interaction. The aim of this thesis is to characterize the binding interaction between ICOS and ICOSL and to provide a review of the literature and discuss future work that would enhance the biological understanding of this interaction and its role in lupus and other autoimmune diseases. METHODS The binding interaction between ICOS and ICOSL was characterized using both soluble proteins and cells with expressed recombinant proteins. Purified soluble ICOSL (sICOSL) was characterized using size-exclusion chromatography multiangle light scattering (SEC-MALS). Surface plasmon resonance (SPR) was used to measure the binding affinity between sICOSL and human ICOS fused to the fragment crystallizable (Fc) portion of an immunoglobulin molecule (hICOS.Fc). The binding interaction was further characterized to account for avidity between hICOS.Fc and sICOSL and between hICOS.Fc and ICOSL expressed recombinant on the cell surface using a solution-based binding method. RESULTS Expressed recombinant and purified sICOSL dimerized over time and with increasing temperatures. The sICOSL: hICOS.Fc interaction did not follow a typical 1:1 binding interaction. In-solution binding experiments resulted in a tighter equilibrium dissociation binding constant (KD) than the surface-based results obtained by SPR. The KD for hICOS.Fc binding to human ICOSL(hICOSL) expressed on cells agreed well with the KD for hICOS.Fc to the soluble protein, indicating that the in-solution binding measurement may measure binding avidity rather than affinity and that this may be the more physiologically relevant interaction. CONCLUSIONS I show in the experimental part of this study that the interaction between ICOS and ICOSL is quite potent and that much of the binding strength is due to avidity, or the combined strength of multiple parts of the molecules interacting with one another, rather than the affinity alone. As this interaction is implicated in SLE pathogenesis, it would be useful to develop a clearer understanding of the most relevant physiological form of these molecules (soluble or transmembrane) and of the biological signaling events that are initiated via this interaction in order to determine whether targeting ICOS or ICOSL may be therapeutically viable approaches.

Surface plasmon resonance (SPR) biosensors have attracted great attention in scientific research in the past three decades. Extensive studies on the immobilisation of biorecognition elements have been conducted in pursuit of higher sensitivity, but trialled formats have focussed on a thin layer modification next to the plasmon film, which usually requires in situ derivatization. This thesis investigates an 'off-chip' immobilisation strategy for SPR biosensing using silica particles and considers the implications of a particle-modified evanescent field on the signal amplitude and kinetics, for an exemplar affinity binding between immobilised IgG and its anti-IgG complement. Submicron silica particles were synthesized as carriers for the bio-recognition elements. They were then immobilised to form a sub-monolayer on the gold film of an SPR biosensor using two methods: thiolsilane coupling and physical adsorption aided by mechanical pressure. The bio-sensitivity towards an antigen/antibody interaction was lower than an SPR biosensor with an alkanethiolate SAM due to the difference in ligand capacity and position in the evanescent field. The binding kinetics of antigen/antibody pair was found to follow the Langmuir model closely in a continuous flow configuration but was heavily limited by the mass transport from the bulk to the sensor surface in a stop-flow configuration. A packed channel configuration was designed with larger gel particles as ligand carriers, packed on top of a gold film to create a column-modified SPR biosensor. This sensor has comparable bio-sensitivity to the previous sub-monolayer particle-modified systems, but the binding and dissociation of the analyte was heavily dependent on mass transport and binding equilibria across the column. A bi-directional diffusion mechanism was proposed based on a two-compartment mass transport model and the expanded model fitted well with the experimental data. The column-modified sensor was also studied by SPR imaging and analyte band formation was observed and analysed. Using the lateral resolution, a multiplexing particle column configuration was explored, and its potential in distinguishing a multicomponent analyte.

<p>A proof-of-concept for a novel microarray used to study protein-ligand interaction in real-time using label-free detection is presented. Many of todays commercially available instruments lack the ability to immobilize membrane proteins. At the same time, the pharmaceutical industry develops drugs directed towards membrane-bound receptors. The need to study drug-target kinetics and to be able to screen for new medical substances is high. To study the biomolecular interactions in real-time, imaging surface plasmon resonance (iSPR) is used. A patterned sensor surface with hydrophobic barriers assisting in the piezodispensing of NeutrAvidin with complex-bound biotin-ssDNA is created. Histidine-tagged proteins are immobilized at the vesicle surface using divalent nitrilotriacetic acid. The concept of the vesicle immobilization, the protein-binding to vesicles and the protein-ligand interaction is initially studied using a Biacore instrument. The dissociation of the ligand IFNα2 from its receptor ifnar-2 (wt) are in accordance with the literature. In the imaging SPR experiments, it is found that the dissociation of IFNα2 from the ifnar-2 (wt) receptor is slower than expected, probably due to rebinding of the ligand. It is also found that imidazole is needed to avoid vesicle-vesicle interaction. The immobilization of proteins had to be done on-line i.e. when the vesicles were bound to the surface. Depending on the mixture of receptors at the vesicle surface the affinity for the ligand was changed. The results achieved were reproducible.</p>

No description available.

A proof-of-concept for a novel microarray used to study protein-ligand interaction in real-time using label-free detection is presented. Many of todays commercially available instruments lack the ability to immobilize membrane proteins. At the same time, the pharmaceutical industry develops drugs directed towards membrane-bound receptors. The need to study drug-target kinetics and to be able to screen for new medical substances is high. To study the biomolecular interactions in real-time, imaging surface plasmon resonance (iSPR) is used. A patterned sensor surface with hydrophobic barriers assisting in the piezodispensing of NeutrAvidin with complex-bound biotin-ssDNA is created. Histidine-tagged proteins are immobilized at the vesicle surface using divalent nitrilotriacetic acid. The concept of the vesicle immobilization, the protein-binding to vesicles and the protein-ligand interaction is initially studied using a Biacore instrument. The dissociation of the ligand IFNα2 from its receptor ifnar-2 (wt) are in accordance with the literature. In the imaging SPR experiments, it is found that the dissociation of IFNα2 from the ifnar-2 (wt) receptor is slower than expected, probably due to rebinding of the ligand. It is also found that imidazole is needed to avoid vesicle-vesicle interaction. The immobilization of proteins had to be done on-line i.e. when the vesicles were bound to the surface. Depending on the mixture of receptors at the vesicle surface the affinity for the ligand was changed. The results achieved were reproducible.

<p>When developing applications for self-assembling nanostructures, a challenge is to organize the self assembling components within integrated nano-microsystems. One approach is to impart nanostructure recognition properties to patterned surfaces, such that nanostructure placement could be thermodynamically driven. This research focuses upon self assembling nanostructures composed of DNA and their reversible specific assembly upon functionalized planar surfaces. Assembly strategies that have been developed for solution phase assembly are herein demonstrated as potentially appropriate for heterogeneous nanosystem integration.</p><p>The assembly of DNA nanostructures relies upon unique base pair interactions between single strands. While DNA hybridization that involves many base pairs results in structures that are strongly bound, an assembly strategy that underlies much DNA nanostructure engineering is formation of nanostructures at temperatures at which the interactions are weak. Here, DNA specific nanostructure immobilization is driven by weak forces. Association is characterized using surface sensitive surface plasmon resonance and quartz crystal microbalance methods. The results suggest that future strategies for nanostructure - system integration that require precise nanostructure placement may be accomplished using specific molecular recognition under thermodynamic control.</p><p>Several methods of solution phase nanostructure characterization are explored. The diffusive properties of DNA nanostructures are examined using dynamic light scattering. Effective hydrodynamic radii are found to be large relative to the nanostructure geometric size. The temperature dependence of light scattering from nanostructures is investigated using both resonance light scattering and nonresonant laser light scattering. Additionally, DNA nanostructure building block and superstructure geometry are interrogated in solution using small angle x-ray scattering. Results derived from comparison of small angle data with simulations of scattering from coarse-grained models are compared with structural information derived from imaging immobilized nanostructures with atomic force microscopy. </p><p>Finally, plasmon coupling in systems comprised of metal particles of unlike composition is described. Through simulation, three phenomena that contribute to interparticle coupling are explored. Off resonant metal particles positioned in between pairs of particles near resonance are found to promote optical coupling in a manner similar to that provided by bulk dielectric media.</p> / Dissertation

The microstructure and residual stress of Ni thin film coating on Si influence the properties significantly, which play an important role in advanced applications of the electric and magnetic properties. The properties of Ni thin film deposited on Si at various temperatures and for different thickness have been studied in this work. Samples were characterized by nanoindentation, Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), electrical measurement, grazing angle X-ray diffraction (XRD) and photo reflection spectroscopy of white light. The nanoindentation measurements reveal similar loading curves and young¡¦s modulus for Ni thin films on Si at different deposition temperatures. However, the higher the deposition temperature, the lower is the hardness of the Ni thin film on Si. A maximum stress occurs at deposition temperature of 88-122 ¢XC. From FTIR spectra an unusual IR oscillating absorption of the Ni/Si film was observed from the samples which was deposited at 230 ¢XC for 15 min (23 nm) and for 30 min (52 nm) compared to other deposition duration and deposition temperatures at room temperature, 88 ¢XC, and 122 ¢XC. Furthermore, annealing experiments of the samples were performed after deposited at room temperature, and then annealed at respective temperatures of 88, 122, 230 ¢XC for the durations of 15 min and 30 min for comparison. However, the unmoral IR oscillation doesn¡¦t occur else where. The phase change of Ni/Si was analyzed by grazing angle XRD. A single phase of NiSi (103) structure was observed only in the samples deposited at 230 ¢XC. Further study of the oscillation in the FTIR spectra shows its origin should be related to surface plasmon resonance (SPR) mode. The SPR absorption peaks at 471 nm and 616 nm are analyzed by a photo reflection experiment. The SPR absorption is due to the nano structure of nickel silicide on Ni/Si surface formed during deposition at 230 ¢XC.