Nesta dissertação estão descritos os resultados obtidos sobre a preparação de nanocascas funcionalizadas (nanopartículas ocas) de ouro/prata de diâmetro de 50 nm e imobilização de diferentes lipases (Burkholderia cepacia (BCL) e pâncreas de porco (PPL)). [Obs.: A imagem do esquema pode ser visto no arquivo PDF] Inicialmente as nanocascas de ouro/prata (NSs AgAu) foram sintetizadas e caracterizadas, através de imagens de MEV e MET. Através destas imagens algumas características das NSs AgAu foram elucidadas, como seu tamanho e sua característica oca. Em seguida, a funcionalização das NSs AgAu com diferentes moléculas mercapto-alcanóicas e uma molécula mercapto-amina foi realizada. Depois de funcionalizadas as NSs-funcionalizadas foram ativadas, com glutaraldeído ou EDC, para que assim elas ficassem aptas à imobilização das lipases, via ligação covalente. Para a BCL foi possível imobilizar 0,155-0,282 mg de proteína/3 mg do suporte. No caso da PPL uma menor quantidade de enzima foi imobilizada (0,035-0,048 mg/3 mg do suporte). A atividade da enzima imobilizada foi testada frente à reação de acetilação enantiosseletiva do (R,S)-1-feniletanol com acetato de vinila (RCE, resolução cinética enzimática). Excelentes resultados de conversão (50%) e seletividade (E > 200) foram conseguidos com a BCL imobilizada em todos os suportes. A PPL livre não catalisava a acetilação deste substrato e quando esta enzima foi imobilizada nas diferentes NSs-funcionalizadas, os resultados apresentados para acetilação enantiosseletiva do (R,S)-1-feniletanol com acetato de vinila foram interessantes. Nesse caso conseguimos alcançar conversões de até 4% do substrato R à sua forma acetilada com excelente enantiosseletividade ( > 99% e.e. do produto). Como uma alternativa de demonstrar a atividade enzimática da BCL imobilizada em reação tradicional de hidrólise, o teste da hidrólise do palmitato de p-Nitrofenila pela BCL livre e imobilizada foi realizado. Este teste revelou que a BCL imobilizada nas NSs-funcionalizadas catalisavam a hidrólise do palmitato de p-Nitrofenila mais rápido que a enzima livre, comprovando os bons resultados obtidos na RCE, mostrando que os sistemas onde tínhamos a BCL imobilizada foram mais eficientes que a enzima livre. As NSs AgAu, NSs-funcionalizadas e até as nanocascas contendo as lipases imobilizadas foram caracterizadas por de imagens de MEV e MET, análises de FT-IR e método de Bradford. Outros estudos com os sistemas contendo a BCL imobilizada foram elucidados. Diferentes funcionalizadores de diferentes tamanhos foram utilizados e a influência de cada um deles sobre a atividade enzimática foi estudado. Por exemplo, quando as NSs AuAg foram funcionalizadas com moléculas mercapto-alcanóicas menores, como por exemplo, o ácido mercapto acético, melhores resultados para a RC do (R,S)-1-feniletanol foram conseguidos. As diferentes formas de ativação da NSs-funcionalizadas utilizando glutaraldeído ou EDC, para consequente imobilização da BCL não resultaram em alterações da atividade enzimática na RC, apresentando valores idênticos para RCE. Experimentos para testar a estabilidade dos sistemas contendo a BCL imobilizada também foram feitos. Descobrimos que é possível armazenar a BCL imobilizada nos diferentes sistemas à - 4 °C por até 28 dias. O estudo da reciclagem destes sistemas revelou que por até 3 ciclos os sistemas conseguiram manter 90% da atividade enzimática. / This dissertation presents the results achieved on the preparation of functionalized gold/silver nanoshells (hollow nanoparticles, 50 nm) and immobilization of different lipases (Burkholderia cepacia (BCL) and porcine pancreatic (PPL)). Initially Gold/Silver nanoshells (NSs Ag Au) were synthesized and characterized through SEM and TEM pictures. By these images some characteristics of NSs AgAu were elucidated, as its size and hollow feature. The functionalization NSs AgAu with different mercapto-alkanoic molecules and mercapto-amine molecule was next step performed. After the functionalized NSs-functionalized were activated with glutaraldehyde or EDC, after that they remained suitable for the immobilization of lipases via covalent bond. BCL was possible immobilized 0.155-0.282 mg protein/3 mg of support. And the PPL a smaller amount of enzyme was immobilized (from 0.035-0.048 mg / 3 mg of support). The activity of the immobilized enzyme was assayed by the reaction enantioselective acetylation of (R,S)-1-phenylethanol with vinyl acetate (KR, kinetic resolution). Excellent conversion results (50%) and selectivity (E > 200) were achieved with the immobilized BCL. The free PLP did not catalyzed acetylation of the substrate and when this enzyme was immobilized on NSs-functionalized the results for enantioselective acetylation of (R,S)-1-phenylethanol with vinyl acetate were interesting. In this case we achieve conversions of 4% of the substrate (R) to acetylated form with excellent enantioselectivity (> 99% e.e. of the product). As alternative to demonstrate the enzymatic activity of BCL immobilized on traditional hydrolysis reaction, the hydrolysis of p-nitrophenyl palmitate by free and immobilized BCL was performed. This test revealed BCL immobilized on NSs-functionalized catalyzed hydrolysis of p-nitrophenyl palmitate faster than free enzyme, confirming the good results obtained in KR, showing that systems which had immobilized BCL were more efficient than the enzyme free. The NSs AgAu, NSs-functionalized and Nanoshells containing the immobilized lipases were characterized by SEM and TEM images , FT-IR analysis , the Bradford method. Other studies with systems containing immobilized BCL were elucidated. Different spacers with different sizes were used for functionalized the nanoshells, and the influence of each of the enzymatic activity was studied. For example, when the NSs were functionalized with smaller molecules mercapto-alkanoic and cysteamine best results for the KR (R,S)-1-(phenyl)ethanol were obtained. The different forms of activation of NSs-functionalized using glutaraldehyde or EDC, for subsequent immobilization of BCL did not result in changes in enzyme activity in the KR . Experiments to test the stability of systems containing immobilized BCL were also made . We found it possible to store the BCL immobilized on different systems at - 4 ° C for 30 days. The study of the recycling of these systems was made and by 3 cycles systems maintain 90% of the enzyme activity.

O estresse tem se mostrado como um agravante de diversas patologias e transtornos psiquiátricos, como o transtorno depressivo maior (TDM). Estudos apontam que o hipocampo, bem como as vias serotonérgicas presentes neste, estão envolvidas com as respostas de estresse bem como nos efeitos deletérios causados por este. O antagonismo do receptor 5-HT 7, presente nestas vias, tem apresentado um efeito antidepressivo e ansiolítico em alguns trabalhos, porém poucos são os estudos que investigam o papel do receptor 5-HT 7 nas estruturas encefálicas em relação às repostas de estresse. Diante desse dado, o presente trabalho utilizou o modelo Labirinto em Cruz Elevado (LCE) 24 horas após a restrição para investigar o papel do receptor 5-HT 7 do hipocampo na consolidação da memória aversiva. A metodologia utilizada consistiu na injeção bilateral de SB-258741, um antagonista do receptor 5-HT7, no hipocampo dorsal de ratos Wistar machos, feita logo após o estresse de restrição de movimento com exposição ao LCE 24 horas depois. Como resultado desta metodologia, foi observado um aumento na porcentagem de entradas dos animais nos braços abertos, mas não na porcentagem de tempo em que o animal permaneceu nestes braços. Este dado mostra que a injeção de um antagonista de 5-HT 7 no hipocampo leva a uma atenuação dos efeitos comportamentais da consolidação de memória aversiva, corroborando com efeito ansiolítico consequente ao antagonismo deste receptor, observado na literatura. Nesse sentido, a investigação sobre o papel desse receptor, nas respostas de estresse, pode ser útil para o desenvolvimento de novos fármacos para os tratamentos dos efeitos prejudiciais do estresse. / Stress has been shown to be an aggravating factor for various diseases and psychiatric disorders, such as major depressive disorder (MDD). Studies suggest that the hippocampus, as well as serotonergic pathways present in this structure, are involved in stress responses as well as in the deleterious effects caused by this factor. The receptor antagonism of 5-HT 7 receptor, present in these pathways, has shown an antidepressant and anxiolytic effect in some studies, but few studies are investigating the role of 5-HT 7 receptor in brain structures related to stress responses. Given this data, this study used the Elevated Plus Maze (EPM) 24 hours after the restrain stress to investigate the role of 5-HT 7 receptor in the hippocampus consolidation of aversive memory. The methodology in this study consisted of bilateral injection of SB-258741, an antagonist of the 5-HT7 receptor, into the dorsal hippocampus of male Wistar rats, made 5 minutes after the restrain stress with exposure to EPM after 24 hours. As a result of this method, an increase in the percentage of animals entries into the open arms, but not at the percentage of time the animal remained in these arms, was observed. This data shows that the injection of a 5-HT 7 antagonist in the hippocampus leads to an attenuation of the aversive memory consolidation behavioral effects, corroborating with the consequent anxiolytic effect to the antagonism of this receptor, presented in the literature. In this sense, research on the role of this receptor in stress responses may be useful for the development of new drugs for the treatment of the harmful effects of stress.

Cell-free protein synthesis (CFPS) is a powerful protein expression platform where protein synthesis machinery is borrowed from living organisms. Target proteins are synthesized in a reaction tube together with cell extract, amino acids, energy source, and DNA. This reaction is versatile, and dynamic optimizations of the reaction conditions can be performed. The "œopen" nature of CFPS makes it a compelling candidate for many technologies and applications. This dissertation reports new and innovative applications of CFPS including 1) enzyme encapsulation in a virus-like particle, 2) detection of endocrine disrupting chemicals in the presence of blood and urine, and 3) expression of a multi-disulfide bond therapeutic protein. Two major limitations of enzymes are their instability and recycling difficulty. To overcome these limitations, we report the first enzyme encapsulation in the CFPS by immobilizing in a virus-like particle using an RNA aptamer. This technique allows simple and fast enzyme production and encapsulation We demonstrate, for the first time, the Rapid Adaptable Portable In vitro Detection biosensor platform (RAPID) for detecting endocrine disrupting chemicals (EDCs) in human blood and urine samples. Current living cell-based assays can take a week to detect EDCs, but RAPID requires only 2 hours. It utilizes the versatile nature of CFPS for biosensor protein complex production and EDC detection. Biotherapeutic protein expression in E. coli suffers from inclusion body formation, insolubility, and mis-folding. Since CFPS is not restricted by a cell wall, dynamic optimization can take place during the protein synthesis process. We report the first expression of full-length tissue plasminogen activator (tPA) using CFPS. These research works demonstrate the powerful and versatile nature of the CFPS.

Abstract Millions of tons of mine tailings are generated worldwide annually. Since many valuable metals such as Ag, Cu, Pb, Zn, Au and Ni are usually incorporated into sulphidic minerals, a large proportion of the tailings generated contain high amounts of sulphates and heavy metals. Some of these tailings are used as paste backfill material at mining sites, but large amounts are still being deposited into the tailings dams under water coverage. Sulphidic minerals are stable underground but after mining of the ore and several processing steps these minerals can be oxidized when they come into contact with water and air. This oxidation generates acid and thus reduces the pH of the surrounding environment. Furthermore, the heavy metals present in the mine tailings can be leached into the environment. This phenomenon, called Acid Mine Drainage (AMD), is one of the most critical environmental issues related to the management of sulphidic-rich tailings. Since AMD generation can still occur hundreds of years after closure of the mine, the mine tailings need stable, sustainable and economically viable management methods in order to prevent AMD production in the long term. The aim of this PhD thesis was to study various solidification/stabilization (S/S) methods for the immobilization of sulphidic mine tailings. The main focus was to develop a suitable chemical environment for achieving effective heavy metal (mainly arsenic) and sulphate immobilization while simultaneously ensuring good mechanical properties. Three treatment methods were tested: alkali activation, stabilization using hydrated lime (Ca(OH)2) and blast furnace slag (GBFS), and calcium sulphoaluminate-belite (CSAB) cement stabilization. The mine tailings used in this study contained large amounts of sulphates and heavy metals such as Cr, Cu, Ni, Mn, Zn, V and As. The leaching of arsenic and sulphates from powdered tailings exceeded the legal limits for regular and inert waste. All treatment methods were found to generate a hardened matrix that was suitable for use as a backfilling or construction material, but the calcium-based binding system was the most suitable for effective immobilization of all the heavy metals (including arsenic) and the sulphates. Precipitation in the form of calcium sulphates/calcium arsenate and the formation of ettringite are the main stabilization methods employed in calcium-based stabilization/solidification (S/S) systems. Some evidence of physical encapsulation occurring simultaneously with chemical stabilization was noted. These results can be exploited further to develop more sustainable mine tailing management systems for use in the future. The tailings could be stored in a dry landfill area instead of in tailing dams, and in this way a long-term decrease in AMD generation could be achieved, together with a high potential for recycling. / Tiivistelmä Monet arvometallit kuten kulta, kupari ja nikkeli ovat sitoutuneena sulfidipitoisiin mineraaleihin. Louhittaessa ja rikastettaessa näitä sulfidimineraaleja syntyy miljoonia tonneja sulfidipitoisia rikastushiekkoja vuosittain. Rikastushiekat voivat sisältää myös runsaasti erilaisia raskasmetalleja. Osa rikastushiekoista hyödynnetään kaivostäytössä, mutta suurin osa rikastushiekoista läjitetään edelleen ympäristöön rikastushiekka-altaisiin veden alle. Kun sulfidipitoinen malmi kaivetaan ja käsitellään, sulfidiset mineraalit hapettuvat ollessaan kosketuksissa veden ja hapen kanssa. Hapettuessaan ne muodostavat rikkihappoa, laskien ympäristön pH:ta jolloin useimmat raskasmetallit liukenevat ympäristöön. Muodostuvia happamia kaivosvesiä voi syntyä vielä pitkään kaivoksen sulkemisen jälkeen ja ovat näin ollen yksi suurimmista kaivosteollisuuteen liittyvistä ympäristöongelmista. Lisäksi suuret rikastushiekka-altaat voivat aiheuttaa vaaraa myös ihmisille, mikäli altaan rakenteet pettävät. Rikastushiekkojen kestäviä ja ympäristöystävällisiä varastointimenetelmiä täytyy kehittää, jotta näitä ongelmia voidaan tulevaisuudessa ehkäistä. Tässä työssä tutkittiin menetelmiä, joilla kultakaivoksella syntyvät sulfidipitoiset vaaralliseksi jätteeksi luokitellut rikastushiekat saataisiin stabiloitua tehokkaasti. Työssä keskityttiin kolmeen erilaiseen menetelmään: alkali-aktivointiin, stabilointiin kalsiumhydroksidin ja masuunikuonan avulla ja stabilointiin CSAB sementin avulla. Valmistettujen materiaalien mekaanisia ja kemiallisia ominaisuuksia arvioitiin. Tavoitteena oli ymmärtää, miten eri menetelmät soveltuvat raskasmetallien (erityisesti arseenin) ja sulfaattien sitoutumiseen ja mikä on eri komponenttien rooli reaktioissa. Alkali-aktivoimalla rikastushiekkaa sopivan sidosaineen kanssa saavutettiin hyvät mekaaniset ominaisuudet ja useimmat haitta-aineet sitoutuivat materiaaliin. Ongelmia aiheuttivat edelleen sulfaatit ja arseeni. Kalsiumpohjaiset menetelmät sitoivat raskasmetallit (myös arseenin) ja sulfaatit tehokkaimmin. Sulfaatit ja arseeni saostuivat muodostaen niukkaliukoisia komponentteja kalsiumin kanssa. Samanaikaisesti rakenteeseen muodostui ettringiittiä, jolla on tutkitusti hyvä kyky sitoa erilaisia raskasmetalleja rakenteeseensa. Raskasmetallit myös kapseloituivat rakenteen sisään. Työn tuloksia voidaan hyödyntää, kehitettäessä rikastushiekkojen turvallista varastointia. Kun materiaalille saavutetaan riittävän hyvä lujuus ja kemiallinen stabiilius, rikastushiekat voitaisiin läjittää tulevaisuudessa kuivalle maalle altaan sijaan. Näin vältyttäisiin rikastushiekka-altaiden rakentamiselta ja voitaisiin vähentää happamien kaivosvesien muodostumista pitkällä ajanjaksolla. Saavutettujen tulosten perusteella rikastushiekkoja voidaan mahdollisesti tulevaisuudessa hyödyntää myös erilaisissa betonin tapaisissa rakennusmateriaaleissa.

Le but de ce travail a été d’élaborer des nanoparticules magnétiques (MNPs) fonctionnalisées avec un groupement maléimide, stables, dispersibles dans l’eau et qui assureront une immobilisation covalente,sélective et efficace de biomolécules. Bien qu’un large choix de MNPs soit disponible dans le commerce, lamodification chimique de surface des MNPs reste une étape indispensable pour l’élaboration de matériauxspécifiques. Un contrôle précis de la fonctionnalisation de surface des MNPs est crucial, car en découlentleurs propriétés physico-chimiques, leur stabilité colloïdale, et la préservation de l’activité biologique de labiomolécule immobilisée. Dans ce travail, nous proposons d’augmenter le nombre de groupes fonctionnels(maléimide) accessibles à la surface de MNPs, en la modifiant par des agents de couplage dendritiques. Deuxtypes de MNPs coeur-écorce de 300 nm (avec un noyau de γ-Fe2O3 et une écorce de polymère ou de silice)ont été utilisés. Afin d’étudier l’effet «dendritique» sur la fonctionnalisation de surface, trois types d’agentsde couplage ont été conçus: des agents de couplage linéaires (contenant un groupe maléimide), des agents decouplage dendritiques à deux branches (contenant deux groupes maléimide) et des agents de couplagedendritiques à quatre branches (contenant quatre groupes maléimide). L’efficacité de ces MNPsfonctionnalisées pour immobiliser des biomolécules ou des modèles de biomolécules a été étudiée. Cetteétude a démontré l’intérêt de la fonctionnalisation de la surface des MNPs coeur-écorce par des structuresdendritiques pour une immobilisation efficace et spécifique de biomolécules. / The purpose of this work is to design stable, water-dispersible, maleimide functionalized magneticnanoparticles (MNPs) that will ensure selective covalent immobilization of biomolecules. While, a largechoice of MNPs is now commercially available, the surface modification of MNPs remains an indispensablestep in the elaboration of such MNPs. A precise control over the surface functionalization of MNPs iscrucial, because it governs their physicochemical properties, their colloidal stability, and their biologicalbehaviour. In this work with the aim to increase the number of functional groups on MNPs’ surfaces, it wasproposed to functionalize MNPs with dendritic coupling agents and to compare their efficiency with thosefunctionalized with a linear analogue. Moreover, it was decided to investigate the “dendritic effect” of thesurface functionalization on two types of core-shell MNPs (300 nm) that consist of a maghemite (γ-Fe2O3)ferrofluid core coated with: (I) polymer shell or (II) silica shell. Therefore, three types of coupling agents(that possess an amino or silane anchoring site) were synthesized: linear coupling agents (containing onemaleimide functional group); two-branched coupling agents (containing two maleimide functional groups)and four-branched dendritic coupling agents (containing four maleimide functional groups). Then, thecapacity of MNPs functionalized with dendritic or linear coupling agents to immobilize biomolecules ormodels of biomolecules was investigated. This study proved the efficiency of the surface functionalizationwith dendritic structures for the immobilization of biomolecules.

<p>The work described in this thesis aims to achieving surface patterning through chemical activation of thiolated silicon oxide surfaces, resulting in a spatially controlled covalent immobilization of biomolecules with high resolution.</p><p>Existing chemical methods to immobilize molecules on surfaces do not reach below the micrometer scale while the ones allowing for spatial control mostly lead to non-covalent adsorption of molecules on surfaces, or require several successive chemical reactions to obtain the final covalent immobilization. Methods with improved chemical processes and novel surface modification techniques had to be developed. </p><p>A basic need for studying interactions of biomolecules on chemically modified surfaces with high resolution is the ability to obtain a simple, inexpensive method resulting in ultraflat densely packed and reproducible organic monolayers. Therefore, a new method for silicon oxide chemical derivatization, fulfilling these requirements, was developed. </p><p>Thiol derivatized silicon oxide surfaces allow for a diversity of activation reactions to occur, resulting in thiol-disulfide exchange. The electrooxidation of surface-bound thiol groups was investigated as a way of generating reactive thiolsulfinates/thiolsulfonates, by application of a positive potential difference to the silicon surfaces. Peptide molecules containing thiol groups were successfully immobilized to the electroactivated surfaces. In addition, this new chemical activation method offers the possibility to release the bound molecules in order to regenerate the surfaces. Subsequently, the thiolated surfaces can be reactivated for further use.</p><p>Since the activated area depends directly on the size of the electrodes used for the oxidation, nanoscale activation of the thiolated surfaces was performed by use of an AFM tip as counter-electrode. Electrooxidized patterns, with a line width ranging from 70 nm to 200 nm, were obtained. A thiol-rich protein, b-galactosidase, was selectively immobilized onto the electroactivated patterns.</p><p>An electrochemical version of microcontact printing was developed in order to activate large surface areas with micrometer scale patterns. Conductive soft polymer stamps were produced using an evaporated aluminum coating. Patterned electroactivation of thiols was achieved, and polystyrene beads were subsequently specifically immobilized onto the patterns.</p><p>As a conclusion, these different projects resulted in a strategy enabling the achievement of nanoscale and microscale positioning and immobilization of biomolecules on silicon surfaces, with potential reversibility and reuse of the surfaces.</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>

Le tiludronate (Tildren, CEVA Santé Animale, Libourne, France) est un bisphosphonate récemment introduit sur le marché dans plusieurs pays européens pour le traitement du syndrome podotrochléaire (syndrome naviculaire) et de lostéoarthrose des articulations intertarsienne distale et tarso-métatarsienne (éparvin) chez le cheval. Il est le premier bisphosphonate disponible en médecine vétérinaire en général, et en médecine équine en particulier. La posologie de tiludronate qui a fait lobjet dun enregistrement dans différents pays dEurope est de 10 administrations quotidiennes consécutives de 0.1 mg/kg sous forme de bolus intraveineux (IV). Or, il ressort de la pratique courante que ladministration de la dose totale de 1 mg/kg en une perfusion lente unique est préférée par les utilisateurs pour des raisons pratiques et financières. Il ressort aussi de différentes études que cette forme dadministration est bien tolérée (Varela et al., 2002) et efficace dans certaines pathologies (Coudry et al., 2007). La première étude de ce travail a consisté en la comparaison pharmacologique de ces deux schémas posologiques. Il en est ressorti quils produisaient une même exposition plasmatique totale et des effets similaires sur le marqueur plasmatique de résorption osseuse carboxy-télopeptide du collagène de type I (CTX-1). Il en a été conclu que les dix administrations quotidiennes consécutives de 0.1 mg/kg pouvaient être remplacées par la perfusion IV lente unique de 1 mg/kg. Les bisphosphonates sont caractérisés par des propriétés pharmacologiques très spécifiques et inhabituelles, de par leur forte affinité pour los et leur stockage à long terme dans ce compartiment profond. De ce fait, les modèles PK-PD classiques qui mettent en relation mathématique les concentrations plasmatiques avec les effets du médicament sont inadaptés pour décrire le comportement pharmacologique des bisphosphonates, et il est suggéré que les concentrations osseuses doivent être davantage prises en compte pour le développement de tels modèles (Cremers et al., 2005). Dans le but final de développer un modèle PK-PD du tiludronate chez le cheval, une méthode de biopsie osseuse permettant la répétition de dosages de tiludronate osseux a été validée dans la seconde étude de cette thèse. Dans celle-ci, huit chevaux ont subi des biopsies osseuses bilatéralement en quatre sites (le tuber coxae, le métacarpien principal, la 13ème côte et los cuboïde) à différentes échéances sétalant de 1 jour à 1 an après 1 (n=4) ou 2 (n=4, à 4 semaines dintervalle) traitement(s) au tiludronate à raison de 1 mg/kg par voie IV. En chaque site, les biopsies osseuses ont été effectuées à la fois à laide dune fraise électrique de 5 mm de diamètre interne (Implanteo, Anthogyr) pour léchantillon test et dun ostéotome ou dune scie oscillante pour léchantillon de référence. Le tuber coxae sest avéré être le meilleur site de biopsie, à la fois accessible, facile à biopsier, et sur lequel les dosages de tiludronate ne présentaient pas de difficultés techniques et étaient fiables. Malheureusement, ces biopsies noffraient pas un matériel qualitativement et quantitativement suffisant que pour permettre des analyses histologiques et histomorphométriques. Dans une troisième étude, a donc été testée sur quatre chevaux la validité pour ce type danalyses dune autre technique de biopsies, utilisant un plus grand trocart que dans létude 2 (15 versus 5 mm de diamètre interne), et de type manuel. Les biopsies ont été réalisées sur cheval debout au niveau du tuber coxae immédiatement avant (côté gauche) et 48 h après (côté droit) ladministration dune perfusion IV lente de 1 mg/kg de tiludronate. Les biopsies ainsi réalisées ont permis deffectuer les analyses escomptées. Cependant, aucun effet précoce du traitement au tiludronate na pu être mis en évidence sur les paramètres histologiques et histomorphométriques étudiés. Aucun modèle expérimental équin relatif aux pathologies pour lesquelles le tiludronate a été enregistré nexiste. Or il est indispensable, pour mieux en cerner les propriétés pharmacologiques et les applications cliniques potentielles, de tester son efficacité en conditions standardisées. Limmobilisation dun membre sous plâtre est connue pour induire de lostéopénie de non usage chez le cheval (Buckingham et Jeffcott, 1991; van Harreveld et al., 2002). Dans la quatrième étude de ce travail, lefficacité clinique du tiludronate versus placebo a été testée dans les conditions standardisées dun modèle équin dostéopénie induite par immobilisation sous plâtre dun membre antérieur pendant huit semaines. Seize chevaux ont ainsi été immobilisés, dont huit ont reçu un placebo, et huit ont été traités au tiludronate à raison de 1 mg/kg en perfusion IV lente deux fois à quatre semaines dintervalle, soit en début et à mi-immobilisation. Après la période dimmobilisation, les chevaux ont été progressivement remobilisés pendant 4 semaines puis ont subi un entraînement standardisé pendant 8 semaines. Le traitement au tiludronate a permis de prévenir la chute significative de densité minérale osseuse (DMO) mesurée par absorptiométrie biphotonique à rayons X (DEXA) observée à long terme au niveau du membre immobilisé des chevaux du groupe placebo. Cet effet préventif ne sest pas marqué sur le membre controlatéral. Le tiludronate a également induit chez les chevaux traités une chute rapide et significative du taux de CTX-1 sérique, contrairement aux chevaux du groupe placebo chez qui ce taux est resté élevé pendant la quasi-totalité de limmobilisation. Aucun effet du tiludronate na été constaté sur lactivité des iso-enzymes osseuses des phosphatases alcalines (bone ALP), marqueur de formation osseuse, ni sur les caractéristiques du cortex superficiel du canon du membre immobilisé évaluées par ultrasonographie quantitative (QUS). De lensemble de ces travaux, il a été conclu que (1) le CTX-1 sanguin est un marqueur de résorption osseuse sensible et adéquat pour le suivi dun traitement au tiludronate chez le cheval ; (2) le tuber coxae est un site de biopsie osseuse adéquat pour effectuer les analyses nécessaires à lélaboration dun modèle PK-PD du tiludronate chez le cheval ; (3) le tiludronate administré deux fois à quatre semaines dintervalle à la dose de 1 mg/kg IV est capable dinhiber la résorption osseuse dans un modèle dimmobilisation chez le cheval, et la DEXA est une méthode suffisamment sensible pour lobjectiver en quelques mois ; (4) de nouvelles études pharmacologiques devraient être menées afin de documenter laccumulation osseuse et les effets du tiludronate sur différents paramètres pharmacodynamiques en cas dadministrations répétées de perfusions de 1 mg/kg chez le cheval. Pour ce faire, des techniques plus sensibles de dosage du tiludronate devraient idéalement être utilisées dans un souci dexactitude des données. Dautre part, de nouvelles études cliniques defficacité sur des chevaux souffrant de pathologies osseuses devraient être menées en incluant des mesures de CTX-1 sanguin, et si possible des mesures de DMO à différents sites dintérêt.

Die Etablierung der Transkription von kompletten Genen auf planaren Oberflächen soll eine Verbindung zwischen der Mikroarraytechnologie und der Transkriptomforschung herstellen. Darüber hinaus kann mit diesem Verfahren ein Brückenschlag zwischen der Synthese der Gene und ihrer kodierenden Proteine auf einer Oberfläche erfolgen. Alle transkribierten RNAs wurden mittels RT-PCR in cDNA umgeschrieben und in einer genspezifischen PCR amplifiziert. Die PCR-Produkte wurden hierfür entweder per Hand oder maschinell auf die Oberfläche transferiert. Über eine Oberflächen-PCR war es möglich, die Gensequenz des Reportergens EGFP direkt auf der Oberfläche zu synthetisieren und anschließend zu transkribieren. Somit war eine Transkription mit weniger als 1 ng an Matrize möglich. Der Vorteil einer Oberflächen-Transkription gegenüber der in Lösung liegt in der mehrfachen Verwendung der immobilisierten Matrize, wie sie in dieser Arbeit dreimal erfolgreich absolviert wurde. Die Oberflächen-Translation des EGFP-Gens konnte ebenfalls zweimal an einer immobilisierten Matrize gezeigt werden, wobei Zweifel über eine echte Festphasen-Translation nicht ausgeräumt werden konnten. Zusammenfassend kann festgestellt werden, dass die Transkription und Translation von immobilisierten Gensequenzen auf planaren Oberflächen möglich ist, wofür die linearen Matrizen direkt auf der Oberfläche synthetisiert werden können. / In vitro mRNA synthesis and in vitro translation are of great interest for biochemical and molecular biological basic research, and also for biotechnology and other applications. Solid phase coupled synthesis is very useful for the development of high throughput procedures to elucidate and manipulate gene products. An artificial gene was constructed combining the T7 promoter and terminator with the EGFP-gene from the plasmid pEGFP. The functionality of the construct was shown by in vitro translation. The gene-construct was immobilised on a planar glass surface. The transcription was performed on the immobilised gene and mRNA was determined by RT-PCR. These results demonstrate that the complete gene is transcribed from the covalently coupled PCR product. Thus, it is possible to transfer a standard transcription technique onto an On-chip reaction. The direct PCR amplification of transcriptionable sequences of EGFP bound on surfaces was successfully used for solid phase transcription. Successful transcriptions were also performed at least to 1 ng of used template. The RNA synthesis was also successful in the second and third reaction on the same slide as observed by signals after RT-PCR. It seems to be possible to transfer the translation of reportergenes in a solid phase coupled synthesis, too. For further integration of cellular procedures on a chip, the cell-free RNA synthesis on immobilised templates is an crucial technical hurdle to conquer. Major advantages of using immobilised templates for transcription are, low risk of contamination occuring in solution, and no necessity of further purification steps for downstream applications of the RNA product.

Nanofabrication techniques were used to manufacture nanometer scale protein templates. The fabrication approach employs electron beam lithography (EBL) patterning on poly(ethylene glycol) (PEG) thiol (CH3O(CH2CH2O)17NHCO(CH2)2SH) self-assembled monolayers (SAM) on Au. The PEG SAM prevented protein surface adhesion and binding sites for protein were created in the SAM by EBL. Subsequent to EBL, the patterns in the PEG SAM were backfilled with 40-nm NeutrAvidin-coated fluorescent spheres (FluoSpheres). The spontaneous and directed immobilization of the spheres from a solution to the patterns resulted in high resolution protein patterns. The FluoSpheres could be arranged in any arbitrary pattern with ultimately only one or a few FluoSpheres at each binding site. Growth dynamics and SAM morphology of PEG on Au were studied by atomic force microscopy (AFM). PEG SAMs on three types of Au with different microstructure were examined: thermally evaporated granular Au and two types of Au films produced by hydrogen flame annealing of granular Au, Au(111) and "terraced" Au (crystal orientation unknown). The different Au surfaces' substructure affected the morphology and mechanical properties of the PEG SAM. On Au(111), AFM imaging revealed monolayer formation through three distinct steps: island nucleation, island growth, and coalescence. The fine-structure of the SAM revealed dendritic island formation - an observation which can be explained by attractive intermolecular interactions and diffusion-limited aggregation. Island growth was not observed on the "terraced" Au. AFM studies of EBL patterned PEG SAMs on Au(111) revealed two different patterning mechanisms. At low doses, the pattern formation occurs by SAM ablation in a self-developing process where the feature depth is directly dose dependent. At higher doses electron beam induced deposition of material, so-called contamination writing, is seen in the ablated areas of the SAM. The balance between these two mechanisms is shown to depend on the geometry of the pattern. In addition to PEG SAMs, fibronectin monolayers on SiO2 surfaces were patterned by EBL. The areas exposed with EBL lose their functionality and do not bind anti-fibronectin. With this approach we constructed fibronectin templates and used them for cell studies demonstrating pattern dependent cell geometries and cell adhesion. / QC 20101008