Novel microwave transmitter and receiver circuits have been developed for implementing UWB (Ultra-Wideband) impulse radar imaging sensor operating in frequency band 0.2 to 4 GHz. with tunable operating frequency band. The fundamental system design parameters such as the required transmitting pulse power and the pulse duration were estimated for a presumed specific application, the pavement assessment. The designed transmitter is the tunable monocycle pulse generator with tuning capability for the output pulse duration from 450- to 1200- ps, and has relatively high transmitting pulse power from 200 to 400 mW. Tuning of the pulse duration was implemented by novel PIN diode switch configuration and decoupling circuit, and boosting of transmitting pulse power was made possible by using a high power pulse driving circuit and SRD coupling circuit. The synchronous sampling receiver system was designed by using the integrated sampling mixer and two reference clock oscillators placed in the transmitter and receiver respectively for timing control. A novel integrated CSH (Coupled-Slotline Hybrid)sampling mixer has been developed along with the design of the strobe pulse generator appropriate for the impulse radar system. The integrated sampling mixer has unprecedented conversion loss of 2.5 dB for the pulse signal, bandwidth 5.5 GHz, and dynamic range 50 dB. The introduced UWB LNA (Low Noise Amplifier) design operating up to 4 GHz should be useful for weak signal detection applications. The design of the UWB microstrip quasi-horn antenna was optimized for short pulse transmission with respect to the input return loss and the pulse stretching effect. For signal detection in the signal processing stage, the background subtraction technique and B-scan data format were used. A novel signal monitoring technique was introduced in the signal processing to compensate the frequency modulation effect of the reference clock. The test results for the complete system with respect to some sample multi-layer structures shows good receiving pulse waveform with low distortion, enough pulse penetration depth for 13” pavement sample structure, and minimum 1-in of range resolution.

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.

Although Malaria rates are on the decline due to the efforts of the World Health Organization and other organizations dedicated to the eradication of this disease, a relaxed attitude towards the development of new antimalarial entities would be flawed. Due to the emergence of resistance in the parasite, the almost 50% world-wide reduction in malarial death rates that have been produced over the past 15 years are threatening to be lost New drugs are urgently needed and our approach focuses on the re-evaluation and optimization of the historic antimalarial ICI 56,780. Due to its causal prophylactic activity, along with its ability to prevent transmission and potent blood schizonticidal activities, it was revisited with the hopes of first understanding which functionalities were responsible to the compound's activity. Secondly, we wanted to optimize the substituents in the 3, 6 and 7-positions. Finally and most importantly, we wanted to address the cross-resistance problem of the ICI 56,780 scaffold. Initial, analogues showed the importance of the ester in facilitating the convergence of the RI towards 1. Although those analogues lost activity in W2, TM90-C2B, and Pb, they were our first glimpse at this important trend that was later exploited in our 3-halo-6-butyl-7-(2-phenoxyethoxy)quinolin-4(1H)-one and 3-halo-6-butyl-2-methyl-7-(2-phenoxyethoxy)quinolin-4(1H)-ones which showed RI values of < 5 for our best analogues. Although our lead compound 3-bromo-6-butyl-2-methyl7-(2phenoxyethoxy)quinolin-4(1H) one possessed decreased activities as compared to ICI 56,780 at 2.60 nM for W2, 12.2 nM for TM90-C2B and 2.12 nM for Pb, it had 100% inhibition of parasite development on day 6 PE in our scouting assay and 61% inhibition on day 6in our Thompson model, increased from the < 2% value given by the ICI 56,780. Solubility and unfavorable in vivo stability were still major issues for this scaffold. Therefore, a series of piperazinyl 4(1H)-quinolones with greatly enhanced solubility were designed and tested in detailed structure activity relationships and structure property relationship studies. Initial results showed that 7-piperazinyl-4(1H)-quinolones possessed greatly increased solubilities when compared to ICI 56,780 analogues. Primarily, the linker length and the piperazine core was probed. This showed that compounds with a single carbon spacer were most active. Further testing of the 6-position gave methyl 6-methyl-4-oxo-7-((4-phenylpiperazin-1-yl)methyl)-1,4-dihydroquinoline-3-carboxylate with W2 and TM90-C2B values of 0.435 nM and 147 nM respectively. Substitution on the piperazinyl phenyl gave the most active compounds however the RI of >1500 was unacceptable. Because of this, 3-halo substituents were added to these quinolones with promising results. With RIs of < 3, the compounds were promising, however they were not active in vivo. However, methyl 6-methoxy-4-oxo-7-((4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)methyl)-1,4-dihydroquinoline-3-carboxylate and methyl 6-methyl-4-oxo-7-((4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)methyl)-1,4-dihydroquinoline-3-carboxylate both gave cures in our in vivo Thomson model. These studies highlight the potential in using detailed structural activity and structural property studies to re-evaluate and optimize historic antimalarials. These studies have introduced a new generation of soluble 4(1H)-quinolones with high potency against P. falciparum.

Arf1 est une petite protéine G (pG), essentiellement impliquée dans le trafic vésiculaire. Arf1 oscille entre deux conformations, l'une active liée au GTP et l'autre inactive associée au GDP. Arno est un des facteurs d'échange (GEF) capable d'activer Arf1 en stimulant l'échange GDP/GTP. Suractivée dans les cellules invasives du cancer du sein, Arf1 joue un rôle important dans la migration et la prolifération des cellules cancéreuses.Le but de ma thèse s'inscrit dans l'étude et la modulation de l'interaction pG-GEF, et plus spécifiquement, le couple Arf1-Arno. Mon travail a été planifié autour de deux axes: (1) L'étude fine de l'interaction entre Arf1 et Arno, et sa modulation avec un inhibiteur connu la Bréféldine A (BFA). (2) La mise en place d'une stratégie de conception d'inhibiteurs de l'interaction protéine-protéine du couple Arf1-Arno.Dans un premier temps, nous avons mis en place une méthode basée sur la résonance plasmonique de surface (SPR) permettant la détermination des paramètres cinétiques de l'interaction entre Arf1 et Arno. Nous avons précisé aussi les conséquences des partenaires allostériques (GDP, GTP, et Mg2+) et de la BFA sur les paramètres cinétiques de l'interaction. Ceci a permis une analyse fine de la régulation allostérique et du mode d'action de la BFA. Appliquée à d'autres inhibiteurs, cette méthode permettra d'examiner leur mécanisme d'inhibition.Dans la deuxième partie j'expose, la stratégie que nous avons utilisé pour la conception rationnelle d'inhibiteur de l'interaction entre Arf1 et Arno. Elle est basée sur le criblage virtuel de fragments au niveau des résidus clé « hotspots » de l'interaction, la validation des molécules-touches par des techniques biophysiques, et l'élimination de molécules artefacts. Les structures des complexes fragments-Arno ont été résolues, ce qui confirme la validité de cette stratégie ouvrant la voie vers l'optimisation moléculaire pour obtenir des inhibiteurs plus efficaces. / Arf1 is a small GTPases, essentially involved in the vesicular traffic. Arf1 switch between two conformations, an active form bound to GTP and an inactive form bound to GDP. Arno is one of the exchange factors (GEF) that can activate Arf1, through its catalytic Sec7 domain, promoting the exchange of GDP by GTP. Activated in breast cancer cells, Arf1 plays an important role in the migration and proliferation of cancer cells.The aim of my thesis was the study and the modulation of the interaction between small G proteins and their GEFs, more precisely the Arf1-Arno interaction. My work has been planned around two axes: (1) the study of the interaction between Arf1 and Arno, and its modulation with a known inhibitor Brefeldin A (BFA). (2) The development of a rational strategy for designing inhibitors of protein-protein interaction for the Arf1-Arno complex.In the first part of my PhD work, we set up a Surface Plasmon Resonance (SPR) method allowing to determine the kinetic parameters of the interaction between Arf1 and Arno. We also studied the effects of allosteric partners such as GDP, GTP and Mg2+ as well as the known uncompetitive inhibitor (Brefeldin A). This SPR approach allowed a very informative analysis at qualitative and quantitative levels of the various complexes taking place during the exchange reaction that should help to solve the inhibitory mechanism for the known inhibitors reported in the literature. In the second part of my thesis, we propose a strategy for targeting the interaction between Arf1and Arno. This approach is based on virtual screening of fragments at hotspot regions. Using biophysical techniques such fluorescence techniques, SPR, NMR and X-Ray crystallography, we identified and validated Hits, showing by crystallographic structural data their modes of interaction with the target protein Arno. A fluorescence polarization test was also developed to identify false positive fragments to eliminate promiscuous aggregators. Taken together, our work proposes a method based on SPR allowing the study of known inhibitors of GEFs, understanding at molecular level their mode of action. We also propose a general strategy for finding Hit fragments that designing competitive inhibitor of the interaction small G protein with its GEFs, that can be the scaffold for designing more powerful inhibitors.

As proteínas pequenas ricas em prolina (small proline-rich protein/SPRRs) compreendem uma sub-classe específica de precursores da camada córnea, codificadas por uma família multi-gênica do complexo de diferenciação epidérmica mapeado no cromossomo 1q21. Vários estudos têm sugerido que as SPRs estão relacionadas com proliferação epitelial elevada e processos malignos. O presente trabalho teve como objetivo investigar a participação das SPRs no desenvolvimento de carcinomas epidermóides de cabeça e do pescoço e no fenótipo da célula neoplásica. O perfil de expressão de onze genes SPRRs foi avaliado em cinco linhagens celulares (FADU, HEP-2, UM-SCC-38, SCC-9) e em carcinomas primários da cabeça e pescoço, utilizando PCR em tempo real. Os tumores foram classificados em agressivos (A) e menos agressivos (LA) dependendo da presença ou da ausência de células neoplásicas nos nódulos linfáticos regionais. Os resultados revelaram baixa expressão de genes SPRRs em todas as linhagens celulares, exceto o SPRR4. Por outro lado, foram observados níveis elevados de transcritos de SPRR2G, SPRR4 e SPRR2E e níveis reduzidos de transcritos de SPRR3 tanto nos grupos A e LA de carcinomas. A expressão ectópica de SPRR2E resultou em menor capacidade de invasão celular em ensaio de câmara de Boyden, bem como em mudança do fenótipo epitelial para fibroblastóide, mas não afetou a proliferação celular ou a migração. Após o tratamento das células HEP-2 com a proteína anti-inflamatória anexina A1 (peptídeo AC2-26), ocorreu um aumento substancial de expressão da maioria dos SPRRs, sugerindo uma associação entre SPRs e inflamação. Os genes SPRRs possuem sequências altamente conservadas e, após uma análise filogenética utilizando o método de neighbor-joining, os resultados indicaram um grupo homogêneo, que incluiu SPRR2 e SPRR4, e um grupo mais heterogêneo com SPRR1 e SPRR3. Aparentemente, a diversidade de sequência destes genes é baixa. Isto sugere que o controle da dosagem da proteína pode ser mais importante para o desempenho de sua função que a complexidade estrutural. A compreensão da função das SPRs avançou bastante nos últimos anos, mas muitas questões sobre o seu papel no processo neoplásico ainda permanecem sem resposta. Muitos dados são ainda necessários para identificar sua associação com outras proteínas e com vias de sinalização. Portanto, é importante melhorar nosso conhecimento sobre a regulação e a função de cada RPN, para que as descobertas obtidas na pesquisa básica sejam traduzidas em aplicações clínicas / The small proline rich proteins (SPRs) constitute a specific sub-class of cornified cell envelope precursors, encoded by a multi-gene family which are part of the epidermal differentiation complex on chromosome 1q21. Several studies have suggested that the SPRs are related to increased epithelial proliferation and to malignant processes. The present work aimed to investigate the participation of SPRs in the development of head and neck squamous cell carcinomas and in the phenotype of the neoplastic cell. The expression profile of eleven SPRRs genes was evaluated in five cell lines (FaDu, HEP-2, UM-SCC-38, SCC-9) and in primary carcinomas from head and neck, using real time PCR. The tumors were classified as aggressive (A) and less aggressive (LA) depending on the presence or absence of neoplastic cells in the regional lymph nodes. The results revealed low expression of SPRRs genes in all cell lines, except SPRR4. Otherwise, high levels of SPRR2G, SPRR4 and SPRR2E and low levels of SPRR3 transcripts were observed in both A and LA carcinomas. Ectopic SPRR2E expression resulted in reduced cell invasion evaluated by a Boyden chamber assay together with morphologic changes from epithelial to fibroblast-like phenotype, but did not affected cell proliferation or migration. After treatment of HEP-2 cell line with the anti-inflammatory protein annexin A1 (peptide Ac2-26), there was a substantial increase of expression of most SPRRs, suggesting an association between SPRs and inflammation. The SPRRs genes have highly conserved sequences and, after a phylogenetic analysis using the neighbor-joining method, the results indicated a homogeneous group including SPRR2 and SPRR4, and a more heterogeneous group with SPRR1 and SPRR3. Apparently, the sequence diversity of these genes is low. This suggests that the control of protein dosage may be more important to the performance of their function than the structural complexity. The understanding of the function of SPRs has advanced in the past years but many questions on their role in the tumorigenic process still remain unanswered. Much more data are required to recognize their association to other proteins and signaling pathways. Therefore, it is important to improve our knowledge on the regulation and function of each SPRs in order to translate findings from basic research into clinical applications

La maladie d’Alzheimer est une pathologie neurodégénérative qui amène à une perte progressive de la mémoire et cause des changements comportementaux. Selon plusieurs théories, le développement de cette maladie est associé à l’accumulation du peptide amyloïde beta et de la protéine tau dans des zones précises du cerveau humain. A l’heure actuelle, les approches thérapeutiques testées sont fondées sur l’hypothèse de la cascade amyloïde, mais les résultats n’ont pas été jugés suffisamment efficaces. Pour augmenter les chances de succès des traitements thérapeutiques existants, de meilleures techniques pour un dépistage précoce de l’Alzheimer semblent nécessaires. De ce fait, dans cette thèse, des stratégies innovantes pour l’analyse d’un des biomarqueurs de la maladie d’Alzheimer sont proposées. En particulier le projet porte sur l’analyse de la protéine tau avec des biocapteurs basés sur la Résonance de Plasmons de Surface (SPR). L’augmentation du niveau de ce biomarqueur dans le Liquide Céphalo-Rachidien (LCR) est déjà indicateur d’un processus de neurodégénérescence. De plus, si la mesure de la protéine tau est combinée à celle d’autres biomarqueurs de la pathologie (i.e. : amyloïde beta), les possibilités de dépistage sont fortement augmentées. Les travaux ont portés sur deux aspects : initialement l’interaction antigène-anticorps a été exploitée pour développer un immunocapteur pour la protéine tau. En utilisant cette technologie, nous avons pu caractériser les paramètres analytiques de l’essai direct (avec un seul anticorps) et ceux de l’essai sandwich (avec deux anticorps complémentaires). Dès ces premières approches, nous avons remarqué le besoin d’augmenter la sensibilité de la méthode SPR développée. En effet la limite de détection pour l’essai sandwich était de l’ordre du nM, alors que les niveaux de tau dans le LCR sont de l’ordre du pM. L’utilisation de nanotechnologies, en particulier des nanotubes de carbone, a permis d’atteindre des niveaux proches du pM, avec de bonnes performances en terme de répétabilité de l’essai.Une approche alternative a été conçue dans la deuxième partie du projet. Elle était consacrée à la sélection d’un aptamère pour la protéine tau, afin d’exploiter les avantages de cette classe de récepteurs par rapport aux anticorps. Pour accomplir cet objectif, deux stratégies de sélection ont été mises en place. Premièrement la sélection traditionnelle (SELEX, Systematic Evolution of Ligands by EXponential enrichment) a été appliquée en utilisant l’Electrophorèse Capillaire (EC) comme moyen de séparation. Bien que de nombreuses conditions aient été modifiées, avec le SELEX traditionnel nous n’avons pas observé une évolution significative de l’affinité entre les séquences d’ADN et la protéine tau. Dans la deuxième approche nous avons utilisé la même méthode de séparation pour mener la sélection à travers l’EC-Non-SELEX. En utilisant cette méthode, où les étapes de PCR étaient réduites, une évolution positive a été observée après seulement trois rounds. En effet cinq séquences parmi celles issues du dernier round ont montré une affinité supérieure pour la cible par rapport à la banque. Néanmoins le nombre de séquences analysées à la fois par SPR et par anisotropie de fluorescence reste extrêmement limité par rapport au pool initial. Même si ceci semble être une limite, ce travail est le premier où les aptamères sont appliqués à l’analyse de la protéine tau. Le potentiel de cette classe de récepteurs reste en grande partie inexploré, ce qui laisse entrevoir des améliorations possibles de l’affinité grâce à de meilleurs processus de sélection et au développement de nouveaux outils bioinformatiques.En conclusion la SPR grâce à ses caractéristiques jouera un rôle fondamental dans les prochaines années pour l’analyse des biomarqueurs et pour le screening de nouvelles molécules, qui seront l’objet de futurs essais cliniques pour limiter l’agrégation de la protéine tau. / Alzheimer’s disease (AD) is a widespread pathogenic condition causing memory and behavior impairment mostly in elderlies because of the accumulation of amyloid beta peptide and tau protein in human brain. Current therapeutic approaches, based on the amyloid hypothesis, are unable to arrest the progression of the disease, hence early diagnosis is crucial for an effective intervention. Based on the updated criteria for AD probable diagnosis, and considering the limits associated with the actual analytical techniques, my work in this thesis was dedicated to develop novel strategies for AD diagnosis. The whole project focused on the analysis of tau protein by Surface Plasmon Resonance (SPR) biosensing. Such protein is well known for being relevant as neurodegenerative marker. In particular if the measurement of tau is associated with that of the amyloid beta peptide and that of the phosphorylated tau, the clinical specificity of this protein become significant to detect Alzheimer. Two aspects were studied; first of all an immunosensor was developed taking advantage of the well-established antigen-antibody interaction. After characterization of the analytical parameters of the direct assay (with primary antibody), a sandwich assay (using two monoclonal antibodies mapping on different analyte i.e. protein tau epitopes) was developed, allowing very low sensitivity to be obtained in artificial Cerebrospinal Fluid (aCSF). In particular to enhance the analytical signal Carbon Nano Tubes (CNTs) were used. Secondly, the research was focused on the selection of aptamers for tau. To this aim two SELEX (Systematic Evolution of Ligands by EXponential enrichment) methods were compared, both based on Capillary Electrophoresis (CE) for partitioning step of the process. Whether with CE-SELEX (first method), no significant affinity improvement was measured, using the CE-Non-SELEX (second method) affinity of the DNA library for tau protein was consistently improved. After isolation of a limited population of aptamer candidates, five sequences were chosen to be analyzed for their affinity for the target. Fluorescence Anisotropy (FA) measurements and SPR highlight similar behavior for the selected sequences, despite the detection principles of these techniques are significantly different. In conclusion the work highlight versatility of SPR technology used both for quantitative analysis and for new selected aptamers characterization in terms of affinity for the analyte tau. The above mentioned versatility is of great interest in a field such AD, which is rapidly expanding. Lowering the total tau levels has been recently identified as a new goal for therapy. Therefore many drug candidates are likely going to be tested in the near future. SPR technology is already widely used in pharmaceutical industry to investigate novel molecules, since it gives access to a large panel of information. In this panorama aptamer technology may improve the overall quality of the analytical data, allowing better comparison among drug candidates. With respect of these receptors, the thesis opened the door to new studies for DNA aptamers to recognize tau, with considerable advantages in term of the receptor stability. Moreover the whole potential of DNA aptamers selected in this work still remain to be explored. New selection methodologies, combined with fast progression of bioinformatics tools might give rise to affinity improvement, which will lead to sensitivity improvement for tau detection in the next few years.

Ce travail de thèse porte sur la conception d’un « laboratoire-sur-fibre » original dédié à l’analyse moléculaire à distance, sans marquage et in vivo compatible dans l’avenir avec les examens endoscopiques et dédié à l’assistance aux diagnostiques. Notre approche est basée sur l’utilisation de faisceaux de fibres microstructurés. Lorsqu’ils sont correctement conçus et recouverts d’une couche d’or, ces assemblages de fibres présentent des propriétés plasmoniques intéressantes. Dans un premier temps, le modèle numérique utilisé pour atteindre une meilleure compréhension des phénomènes physiques impliqués dans l’optimisation de la sensibilité des capteurs est expliqué. Les simulations, basées sur l’optique géométrique, ont été utilisées pour optimiser la géométrie des pointes et l’épaisseur de la couche d’or dans le but d’améliorer les performances analytiques et permettre ainsi des détections d’interactions biochimiques. Le processus de fabrication des capteurs est ensuite expliqué depuis leur structuration par gravure chimique effectuée à l’ISM (Bordeaux) jusqu’à leur métallisation réalisée au CEA Grenoble. Une comparaison entre les comportements théoriques et expérimentaux et alors menée pour comprendre l’influence de l’hétérogénéité du dépôt d’or et des surfaces gravées sur la sensibilité optique. Ces propriétés optiques sont ensuite exploitées jusqu’à la preuve de concept d’analyses biochimiques déportées. Cette étape a été réalisée en deux temps : d’abord la sensibilité à l’indice local a été démontrée en détectant l’adsorption d’une couche organique auto-assemblée et ensuite un suivi de l’interaction spécifique entre deux brins d’ADN complémentaires a été effectué. Le manuscrit s’achève par une analyse des aspects plus complexes liés à la nature peu multimodale des fibres présentes dans le faisceau. La théorie des guides d’ondes est alors utilisée pour expliquer l’influence du caractère modal de la propagation de la lumière sur les réponses des fibres optiques. / This Ph.D. thesis focuses on the design of an original “lab-on-fiber” tool for remote, label-free in vivo molecular analysis that could be dedicated in the future to endoscopic diagnosis. Our approach is based on functionalized microstructured optical fiber bundles. When appropriately designed and covered by a gold layer, those fibers exhibit interesting plasmonic properties. First, the numerical model used to reach a better understanding of the physical phenomena involved in the optimization of the sensor’s sensitivity is explained. The simulations based on ray optics were then used to optimize the fiber tip geometry and gold coating thickness to enhance the analytical performances and ultimately allow biochemical detections. The fabrication process of the sensor is then explained going from the chemical etching done by the ISM team (Bordeaux) to the metallization of the tips performed at the CEA Grenoble. A comparison between theoretical and experimental behaviors is then conducted to assess the influence of the heterogeneity of both the gold deposit and the etched surfaces on the optical sensitivity. Afterwards, we take advantage of those optical properties to perform remote biochemical analysis. This was achieved in two steps: we first proved that our sensor was sensitive to local optical index variations by detecting the adsorption of a thin self-assembled organic layer and ultimately a specific interaction between two complementary DNA strands was monitored. The last part of this work tackles the more difficult aspects of the few-modes fibers composing the bundle. Waveguide theory is then used to explain the influence of the modal characteristics of light propagation on the optical fibers responses.

This dissertation consists of two topics. One is a "Multi-pass Fiber Optic Surface Plasmon Resonance Sensor (SPR)" and the other is a "Nano-metallic Surface Plasmon Lens." Since both topics involved surface plasmon, the title of this dissertation is named "Surface plasmon coupled sensor and nanolens." For a multi-pass fiber optic SPR sensor, a fiber optic 4-pass SPR sensor coupled with a field-assist capability for detecting an extremely low concentration of charged particles is first demonstrated. The multipass feature increases the sensitivity by a factor equal to the number of passes. The field-assist feature forces charged particles/molecules to the SPR surface, increasing the sensitivity by an additional factor of about 100. Overall, the sensitivity exceeds the one-pass SPR device by a factor of about 400. A 10 pM concentration of 47 nm diameter polystyrene (PS) latex beads and 1 ?M concentration of salt dissolved in DI water were detected within a few seconds by the combined system. The equivalent index resolution for atomic size corresponding to ionized chlorine in salt is 10-8. This technique offers the potential for sensitive and fast detection of biomolecules in a solution. Secondly, a 44-pass fiber optic surface plasmon resonance (SPR) sensor coupled with a field-assist capability for measurement of refractive index change due to positive and negative ions is shown. The field-assist feature forces ions to the SPR surface, causing the SPR signal response to change which reflects a decrease or increase in refractive index depending on whether positive or negative ions are being attracted to the surface. This technique offers the potential for the sensitive detection of cations and anions in a solution. For a nano-metallic surface plasmon lens, we analyze the transmission of a normally incident plane wave through an Ag/dielectric layered concentric ring structure using finite difference time domain (FDTD) analysis. The dependency of the transmission efficiency on the refractive index in slit is studied. The numerical analysis indicates that the focusing beyond diffraction limit is found even at the extended focal length comparable to the distance of 7" from the exit plane using a circularly polarized coherent plane wave, ?=405 nm. Especially, compared to an Ag-only structure, the Ag/ LiNbO3 structure amplifies the transmission power by a factor of 6. Therefore, this Ag/dielectric layered lens has the potential for significantly higher resolution imaging and optical data storage.

Les protéines eucaryotes Elmo (Engulfment and Cell Motility) forment une famille de régulateurs conservés qui jouent un rôle central dans les processus biologiques reposant sur le remodelage du cytosquelette d'actine comme la phagocytose et la migration cellulaire et régulé par les GTPases de la famille Rho. Les protéines Elmo régulent la fonction des protéines Dock (Downstream of Crk), qui sont des Facteurs d'Echange de Guanine (GEF) atypiques pour les GTPases Rac1 et Cdc42. Le mécanisme de régulation connu à ce jour, repose sur l'interaction entre les 200 résidus C-terminaux d'Elmo et les 180 premiers résidus N-terminaux de Dock. Cependant, le rôle précis des différents domaines et motifs identifiés dans ces régions n'est pas encore défini. En effet, les données fonctionnelles, biochimiques et structurales rapportées à ce jour semblent contradictoires quant à la contribution de l'extrémité C-terminale d'Elmo qui comprend un motif polyproline et le domaine SH3 N-terminal de Dock. Nous avons donc étudié la contribution de l'extrémité C-terminale de Elmo1 à l'interaction entre Elmo1 et le domaine SH3 de Dock1 en utilisant notamment la résonance plasmonique de surface. Nos données démontrent la capacité du domaine SH3 de Dock1 à interagir avec Elmo1, indépendamment de l'extrémité C-terminale contenant le motif polyproline. Toutefois, la présence de cette région conduit à une augmentation significative du temps de demi-vie du complexe Elmo1/Dock1. En parallèle, des expériences de diffusion des rayons X aux petits angles nous ont permis de déterminer des enveloppes tridimensionnelles de la protéine Elmo1. Ces données nous permettent ainsi de proposer un premier modèle à basse résolution dans lequel nous localisons les parties N et C-terminales de Elmo1. De façon surprenante cette étude semble indiquer un changement de conformation de la région N-terminale de Elmo1 ainsi qu'une interaction possible de cette même région avec le domaine SH3 de Dock1. / The eukaryotic Elmo proteins (EnguLfment and cell MOtility) form a conserved regulatory family that plays a central role in a number of processes that depend on actin cytoskeleton remodeling, such as phagocytosis and cell migration. Elmo proteins regulate the function of Dock proteins (Downstream of CrK), a new family of atypical guanine exchange factors (GEF) for Rac1 and Cdc42 GTPases. The regulation of this mechanism is based on the interaction between the 200 C-terminal residues of Elmo and the 180 N-terminal residues of Dock. However, the precise role of the different domains and motifs identified in these regions is still not well defined. Indeed, functional, structural and biochemical data reported to date seem contradictory with respect to the contribution of the C-terminal end of Elmo, which includes a polyproline motif, and the N-terminal SH3 domain of Dock. We have therefore investigated the contribution of the C-terminal region of Elmo1 to the interaction between Elmo1 and the SH3 domain of Dock1 using surface plasmon resonance. Our data demonstrate the ability of the SH3 domain of Dock1 to interact with Elmo1 independently of the C-terminal polyproline containing region. However, the presence of this region induces a significant increase in the half-life of the Elmo1/Dock1 complex. In parallel, small angle X-ray experiments were recorded. These data allowed us to propose the first low-resolution model of Elmo1 in which we can locate N and C-terminal regions. Surprisingly, this study suggests a conformational change of the N-terminal region of Elmo1 and a possible interaction of this same region with the SH3 domain of Dock1

As proteínas pequenas ricas em prolina (small proline-rich protein/SPRRs) compreendem uma sub-classe específica de precursores da camada córnea, codificadas por uma família multi-gênica do complexo de diferenciação epidérmica mapeado no cromossomo 1q21. Vários estudos têm sugerido que as SPRs estão relacionadas com proliferação epitelial elevada e processos malignos. O presente trabalho teve como objetivo investigar a participação das SPRs no desenvolvimento de carcinomas epidermóides de cabeça e do pescoço e no fenótipo da célula neoplásica. O perfil de expressão de onze genes SPRRs foi avaliado em cinco linhagens celulares (FADU, HEP-2, UM-SCC-38, SCC-9) e em carcinomas primários da cabeça e pescoço, utilizando PCR em tempo real. Os tumores foram classificados em agressivos (A) e menos agressivos (LA) dependendo da presença ou da ausência de células neoplásicas nos nódulos linfáticos regionais. Os resultados revelaram baixa expressão de genes SPRRs em todas as linhagens celulares, exceto o SPRR4. Por outro lado, foram observados níveis elevados de transcritos de SPRR2G, SPRR4 e SPRR2E e níveis reduzidos de transcritos de SPRR3 tanto nos grupos A e LA de carcinomas. A expressão ectópica de SPRR2E resultou em menor capacidade de invasão celular em ensaio de câmara de Boyden, bem como em mudança do fenótipo epitelial para fibroblastóide, mas não afetou a proliferação celular ou a migração. Após o tratamento das células HEP-2 com a proteína anti-inflamatória anexina A1 (peptídeo AC2-26), ocorreu um aumento substancial de expressão da maioria dos SPRRs, sugerindo uma associação entre SPRs e inflamação. Os genes SPRRs possuem sequências altamente conservadas e, após uma análise filogenética utilizando o método de neighbor-joining, os resultados indicaram um grupo homogêneo, que incluiu SPRR2 e SPRR4, e um grupo mais heterogêneo com SPRR1 e SPRR3. Aparentemente, a diversidade de sequência destes genes é baixa. Isto sugere que o controle da dosagem da proteína pode ser mais importante para o desempenho de sua função que a complexidade estrutural. A compreensão da função das SPRs avançou bastante nos últimos anos, mas muitas questões sobre o seu papel no processo neoplásico ainda permanecem sem resposta. Muitos dados são ainda necessários para identificar sua associação com outras proteínas e com vias de sinalização. Portanto, é importante melhorar nosso conhecimento sobre a regulação e a função de cada RPN, para que as descobertas obtidas na pesquisa básica sejam traduzidas em aplicações clínicas / The small proline rich proteins (SPRs) constitute a specific sub-class of cornified cell envelope precursors, encoded by a multi-gene family which are part of the epidermal differentiation complex on chromosome 1q21. Several studies have suggested that the SPRs are related to increased epithelial proliferation and to malignant processes. The present work aimed to investigate the participation of SPRs in the development of head and neck squamous cell carcinomas and in the phenotype of the neoplastic cell. The expression profile of eleven SPRRs genes was evaluated in five cell lines (FaDu, HEP-2, UM-SCC-38, SCC-9) and in primary carcinomas from head and neck, using real time PCR. The tumors were classified as aggressive (A) and less aggressive (LA) depending on the presence or absence of neoplastic cells in the regional lymph nodes. The results revealed low expression of SPRRs genes in all cell lines, except SPRR4. Otherwise, high levels of SPRR2G, SPRR4 and SPRR2E and low levels of SPRR3 transcripts were observed in both A and LA carcinomas. Ectopic SPRR2E expression resulted in reduced cell invasion evaluated by a Boyden chamber assay together with morphologic changes from epithelial to fibroblast-like phenotype, but did not affected cell proliferation or migration. After treatment of HEP-2 cell line with the anti-inflammatory protein annexin A1 (peptide Ac2-26), there was a substantial increase of expression of most SPRRs, suggesting an association between SPRs and inflammation. The SPRRs genes have highly conserved sequences and, after a phylogenetic analysis using the neighbor-joining method, the results indicated a homogeneous group including SPRR2 and SPRR4, and a more heterogeneous group with SPRR1 and SPRR3. Apparently, the sequence diversity of these genes is low. This suggests that the control of protein dosage may be more important to the performance of their function than the structural complexity. The understanding of the function of SPRs has advanced in the past years but many questions on their role in the tumorigenic process still remain unanswered. Much more data are required to recognize their association to other proteins and signaling pathways. Therefore, it is important to improve our knowledge on the regulation and function of each SPRs in order to translate findings from basic research into clinical applications