La biochimie médicale est une discipline en constante évolution. L’enjeu du développement de nouvelles techniques est de permettre l’analyse à haut débit, de manière spécifique et à faible coût. Les techniques d’immunoanalyse omniprésentes en laboratoire de biologie médicale (LBM) répondent convenablement à ces critères, mais sont cependant perfectibles en ce qui concerne l’analyse des petites molécules d’intérêt biologique. L’objectif de ce travail est de développer des méthodes de dosage innovantes, pour les petites molécules, en utilisant les aptamères comme nouveaux outils de reconnaissance moléculaire. Il s’agit d’oligonucléotides fonctionnels simple brin, capables de reconnaître de manière spécifique une cible, isolés à partir d’une banque de candidats par une approche combinatoire in vitro nommée SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Ils sont en concurrence avec les anticorps, en particulier dans le domaine du diagnostic. Nous avons dans un premier temps travaillé sur le développement de systèmes de dosage à double reconnaissance pour petite molécule, impliquant la formation d’un complexe boucle-boucle. L’adénosine et la théophylline ont tout d’abord servi de cibles modèles pour le développement en phase hétérogène d’une technique de dosage colorimétrique avec amplification enzymatique du signal. Le développement a ensuite été axé sur un analyte ayant un réel intérêt biologique, l’arginine-vasopressine. Le système a été développé en phase homogène en utilisant la technique d’anisotropie de fluorescence. L’application en milieu biologique a été facilitée par l’utilisation d’oligonucléotides non naturels en série L. Enfin nous avons décrit une méthode particulièrement innovante, sans marquage (« label-free »), permettant l’analyse des cibles de petite taille. Cette méthode basée sur l’utilisation du SYBR Green en solution couplée à la technique d’anisotropie de fluorescence, permet également l’étude des ligands de l’ADN. / Clinical chemistry is a constantly evolving discipline. The challenge of developing new techniques is to enable high throughput analysis specific and at low cost. Immunoassay techniques respond appropriately to these criteria, but are nevertheless perfectible with regards to the detection of small molecules of biological interest. The aim of this work is to develop innovative assay methods for small molecules of biological interest, using aptamers as alternative molecular recognition tools. They are single-stranded functional nucleic acids that are isolated from a very large library of candidates through an in vitro combinatorial approach (SELEX) for their ability to bind a peculiar species. They compete with antibodies, particularly in the area of diagnosis. First, we focused our work on the design of small molecule dual recognition assay systems that involved the formation of a loop-loop complex. Adenosine and theophylline served as model targets for the heterogeneous phase development of a colorimetric assay with enzymatic signal amplification. Subsequent works were performed by using arginine-vasopressin, an analyte with a real biological interest as target. A homogeneous phase fluorescence anisotropy detection system was constructed. Applications in complex matrix were facilitated by the use of non-natural L-oligonucleotides. Finally, a particularly innovative SYBR Green-based fluorescence anisotropy method, was reported allowing the detection of both small targets and DNA ligands.

<p>Deep vein thrombosis (DVT) and Pulmonary embolism (PE) are responsible for over 900,000 cases and 100,000 deaths each year in the US. Direct fibrinolytic agents such as plasmin are being investigated for their treatment. However, plasmin administration is not widely studied as low plasmin concentrations are rapidly inactivated by antiplasmin in vivo, whereas high plasmin doses would deplete endogenous antiplasmin and impose bleeding risks. Thus, a plasmin delivery system that can achieve efficient clot lysis while minimizing inactivation by antiplasmin and has reduced bleeding risks is needed. To address this, we propose using reversible inhibitors of plasmin that can sequester plasmin from antiplasmin and release it on the surface of a fibrin clot to achieve clot lysis. The inhibition must be tuned such that it is strong enough to protect plasmin from antiplasmin and weak enough to release plasmin at the clot for lysis. To achieve this, we utilize principles of multivalency to synthesize three classes of inhibitors with varying potencies and mechanisms of inhibition: (i) Multivalent benzamidines (ii) Multivalent tranexamic acids (TXA), and (iii) Hetero-multivalent inhibitors having both benzamidine and TXA. Benzamidine is a competitive inhibitor of plasmin’s active site. TXA, on the other hand, is an FDA-approved weak active site inhibitor that is primarily used to disrupt plasmin(ogen) from binding to fibrin on the clot by inhibiting plasmin’s kringle domains. Multivalent inhibitors were synthesized using amine-reactive chemistry, purified using RP-HPLC and confirmed with Mass Spectrometry. Inhibition assays were performed to assess inhibition potency by determining Ki values (inhibition constants). Lower Ki values indicate stronger inhibition. With multivalent benzamidine derivatives, it was observed that changing valency and linker length substantially impacted inhibition and resulted in Ki values ranging from 2.1 to 1,395 μM. Inhibitors of higher valencies and shorter linker lengths exhibited stronger inhibition. Multivalent TXAs of valencies 1 to 16 were also tested and they exhibited Ki values varying from 2.5 to 21,370 μM indicating up to 8,548-fold improvement in inhibition due to valency. It was found that monovalent TXA, primarily a kringle inhibitor, can be converted into a stronger active site inhibitor by multivalency. With hetero-bivalent TXA-dPEG36-AMB, simultaneous binding of benzamidine to the active site and TXA to the kringle domains was achieved to attain improved inhibition. These results indicate that multivalency can significantly alter the potency of inhibitors and can modulate plasmin inhibition for drug delivery.</p>

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In dieser Arbeit sollten Metallsiloxidkomplexe als potentielle Modellsysteme für metallbeladene Zeolithe, die in der Oxidationskatalyse eingesetzt werden, detailliert untersucht werden. Für Ti und Cu stand dabei die Entwicklung einer Syntheseroute im Vordergrund. Im Falle von Ti sollte TiCl4 mit Silanol PhSi(OSiPh2OH)3 (PhLH3) umgesetzt werden, während für Cu ein Kupfer(I)aluminosilikat synthetisiert werden sollte. Nach erfolgreicher Darstellung und Charakterisierung sollte deren Verhalten gegenüber Oxidanzien untersucht werden. Der Cu(I)-Komplex schien inert gegenüber O2 und OAT-Reagenzien, während bei der Reaktion der Ti-Komplexe mit H2O2 eine kurzlebige Spezies, vermutlich Ti−OOH, gebildet worden war. Der Komplex [Al3(µ2-OH)3(THF)3PhL2] wurde als Modell für Al−OH−Al Funktionen getestet und es zeigte sich, dass die sauren Al−OH−Al Funktionen in der Lage sind, Alkohole zu protonieren, diese unter Wasserabspaltung in Al−OR−Al Einheiten und anschließend durch beta-H-Eliminierung in verschiedene Olefine zu überführen. Zudem wurde die Azidität mit 15N-Festkörper-NMR-Spektroskopie des Pyridinaddukts untersucht. Der Komplex [L2Fe][Li(THF)2]2 (L = OSiR2OSiR2O, R = Ph) ist das beste Modell für Fe-ZSM-5 und sollte auf dessen O2-Reaktivität untersucht werden. Zum einen sollte der Einfluss des Ligandenrückgrats analysiert werden. Dafür wurden verschiedene Reste (R = Me, iPr, Ph'') verwendet und in einigen Fällen konnte das Phänomen der Allogonisomerie beobachtet werden. Zum anderen sollte der Einfluss der Alkalimetallionen bestimmt werden. Es stellte sich heraus, dass mit zunehmendem Ionenradius eine zunehmende Verzerrung der FeO4-Einheit in Richtung Tetraeder einhergeht. Diese Verzerrung wiederum spiegelte sich in der Zunahme von Delta-EQ und der Reaktionsgeschwindigkeit mit O2 wider. Neben Fe(II)- sollten auch Fe(III)-Komplexe synthetisiert werden. Zu guter Letzt sollte der Einfluss der an den Alkalimetall-Gegenionen koordinierenden Lösungsmitteln untersucht werden. / For the present study metal siloxid complexes considered as model systems for metal loaded zeolites, were investigated. For Ti and Cu, the focus was on developing a synthesis route. In the case of Ti, TiCl4 should therefore be reacted with silanol PhSi(OSiPh2OH)3 (PhLH3), while for Cu a copper(I)aluminosilicate should be synthesized. After the preparation and characterization their behavior towards oxidants was investigated. The Cu(I) complex appeared to be inert towards O2 and OAT reagents. During the reaction of the Ti complexes with H2O2 a reactive species was produced, probably Ti−OOH. The complex [Al3(µ2-OH)3(THF)3PhL2] was tested as a model for Al−OH−Al functions. It was shown that the acidic Al−OH−Al functions can protonate alcohols, thereby initiates the conversion into Al−OR−Al units under release of water and subsequently form different olefins through beta-H elimination. In addition, the acidity was investigated by 15N solid-state NMR spectroscopy of the pyridin adduct. The compound [L2Fe][Li(THF)2]2 (L = OSiR2OSiR2O, R = Ph) is the so far best model for Fe-ZSM-5 and a should be investigated with respect to the O2 reactivity. On the one hand, the influence of the ligand backbone was analyzed. For this purpose different residues (R = Me, iPr, Ph'') were used and in some cases the phenomenon of allogonisomerism was observed. On the other hand, the influence of the alkali metal ions should be investigated. It turned out that increasing ion radius was accompanied by an increasing distortion of the FeO4 unit in the direction of the tetrahedron. This distortion was reflected by an increasing Delta-EQ and reaction rate with O2. In addition to Fe(II)- Fe(III) complexes should be synthesized, too. Finally, the influence of the solvent molecules coordinated to the alkali metal counterions should be investigated.

Boron neutron capture therapy (BNCT) has the potential to specifically destroy tumor cells without damaging the tissues infiltrated by the tumor. BNCT is a binary treatment method based on the combination of two agents that have no effect when applied individually: 10B and thermal neutrons. Exclusively, the combination of both produces an effect, whose extent depends on the amount of 10B in the tumor but also on the organs at risk. It is not yet possible to determine the 10B concentration in a specific tissue using non-invasive methods. At present, it is only possible to measure the 10B concentration in blood and to estimate the boron concentration in tissues based on the assumption that there is a fixed uptake of 10B from the blood into tissues. On this imprecise assumption, BNCT can hardly be developed further. A therapeutic approach, combining the boron carrier for therapeutic purposes with an imaging tool, might allow us to determine the 10B concentration in a specific tissue using a non-invasive method. This review provides an overview of the current clinical protocols and preclinical experiments and results on how innovative drug development for boron delivery systems can also incorporate concurrent imaging. The last section focuses on the importance of proteomics for further optimization of BNCT, a highly precise and personalized therapeutic approach.

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No description available.

Ebselen (Ebs) is considered as a glutathione peroxidase (GPx) mimetic and primarily thought to function by scavenging intracellular reactive oxygen species (ROS). Previous to our work, Deng et al. (2010a) demonstrated complete block of ICl,swell with 15 microM Ebs following endothelin-1 (ET-1) induced activation of the current in cardiomyocytes. This block was presumed to take effect mainly via the quenching of ROS. Nonetheless, our work with DI TNC1 astrocytes strongly emphasizes that Ebs might function by an alternative mechanism based on its kinetic profile in blocking ICl,swell. Our experiments showed that 45 nM Ebs can fully block ICl,swell thus suggesting an apparent IC50 result, we predicted Ebs to possess a high kon with a low koff close to zero. As predicted, Ebs failed to washout in the timescale covered by our patch-clamp experiments. The block was also distal to H2O2, previously considered as the most proximate regulator of ICl,swell. And based on further evidence demonstrating irreversible block of ICl,swell distal to H2O2 with Ebs congeners, complete suppression of native ICl,swell with MTS reagents, and failure of Ebs to block ICl,swell from the cytosol, we concluded that Ebs and its congeners can covalently modify important –SH groups required for current activation while functioning as sulfhydryl reagents. Complete irreversible block of ICl,swell with 110 mM cell impermeant MTSES in native DI TNC1 astrocytes contrasts sharply to SWELL1 (Qiu et al., 2014) or LRRC8A (Voss et al., 2014), the latest molecular entity presumably responsible for ICl,swell, where 3.33 mM MTSES failed to demonstrate block of ICl,swell in the wild-type stably expressing SWELL1 (Qiu et al., 2014). Our data with Ebs, its congeners, and MTS reagents indicate the existence of a common extracellular binding site which involves a selenenylsulfide (Se-S) bond that critically modulates ICl,swell. We, therefore, synthesized a derivative of Ebs called ebselen-para-yne (Ebs-p-yne), which provided an even higher affinity for blocking ICl,swell with a presumed IC50 ~picomolar range. Ebs-p-yne is a promising novel molecule that may serve as a tag in identifying the molecular fingerprint ultimately responsible for ICl,swell. Furthermore, we can take advantage of click chemistry to ultimately pull out the channel or channel component which has remained elusive for greater than two decades.

This thesis reports the viability of E. coli cell extracts prepared using equipment that is both common to biotechnology laboratories and able to process small volume samples and expression of proteins containing unnatural amino acids (UAAs) at higher level using PCR amplified linear DNA templates (LETs) in cell-free protein synthesis (CFPS) system. E. coli-based cell extracts are a vital component of inexpensive and high-yielding CFPS reactions. However, effective preparation of E. coli cell extract is limited to high-pressure homogenizers (French press style or impinge-style) or bead mill homogenizers, which all require a significant capital investment. This work specifically assessed the following capital cost lysis techniques: (1) sonication, (2) bead vortex mixing, (3) freeze-thaw cycling, and (4) lysozyme incubation to prepare E. coli cell extract for CFPS. In this work, simple shake flask fermentation with a commercially available E. coli strain was used. Additionally, the RNA polymerase was over expressed in the E. coli cells prior to lysis which eliminated the need to add independently purified RNA polymerase to the CFPS reaction. As a result, high yielding E. coli-based cell extract was prepared using equipment requiring reduced capital investment and common to biotechnology laboratories. To our knowledge, this is the first successful prokaryote-based CFPS reaction to be carried out with extract prepared by sonication or bead vortex mixing. LETs are an attractive alternative to plasmids for site-specific incorporation of unnatural amino acids in proteins in the CFPS system because of their short preparation time and ease of production. However, major limitations associated with LETs are: (1) their degradation by RecBCD enzyme present in the cell-extract used for CFPS and (2) high CFPS energy costs. In this work, we report the optimization of LET-based CFPS for improved protein yield by inhibiting the RecBCD enzyme with small inhibitor molecules resulting in three fold increment in yield of protein containing UAA. We also assessed alternative energy sources such as glucose, fructose-1,6-bisphospate, creatine phosphate/creatine kinase, and high glutamate salt for cost reduction. This work could be important for high-throughput applications based on linear expression templates. This work demonstrates simple E. coli extract preparation and improved yield with linear expression templates for further advancements of cell-free protein synthesis system.

Mon travail de thèse porte sur l’assemblage supramoléculaire et le transport de charge des multi-composants utilisés dans le domaine de l’électronique à base organique. En particulier, l’étude et l’optimisation des transistors organiques à effet de champ (OFETs), des phototransistors, et des inverseurs organiques. Nous avons démontré que la température de recuit des dispositifs OFETs améliore les performances électriques d’un dérivé de fullerène (ICBA). Ces dispositifs dont les surfaces de SiO2 sont fonctionnalisées par OTS ou HMDS ont montrés des mobilités d’électrons de 0.1cm2V-1s-1, qui est la plus élevée par rapport à la littérature. Aussi, nous avons fabriqué des phototransistors à base de mono- et multifibres de PDIF-CN2 qui ont été optimisés par traitements de surfaces du diélectrique (HMDS ou OTS). Les propriétés optoélectroniques de ces dispositifs ont été comparées à ceux des dispositifs à base des couches minces déposés par spin-coating (éduction centrifuge). Nos dispositifs mono-fibres ont montré des valeurs de mobilité plus élevées (supérieure à 2 cm2V-1s-1) par rapport à ceux des multifibres et couches minces. Une telle efficacité de transport d’électrons est le résultat d’une cristallinité très élevée des fibres, qui permet une collecte efficace des excitons photo-générés qui se traduit par la plus haute sensibilité à la lumière (R) et photosensibilité (P) rapportées pour les phototransistors à base de mono-fibre supérieure à 2 × 103 AW-1, et 5 × 103 AW-1. Enfin, un polymère ambipolaire (DPPT-TT) a été utilisé lors de la fabrication de nouveaux dispositifs multifonctionnels par l’addition des molécules diaryléthènes (DAE_tBu et ou DAE_F), dont les propriétés électriques sont contrôlées par la lumière. Cette approche a permis un contrôle optique de gain en tension des inverseurs organiques, ces dispositifs multi-composants sont caractérisés par des gain en tensions très élevées (jusqu’au 504) comparés à ceux reportés dans la littérature (86). Ces travaux réalisés durant cette thèse offrent de nouvelles perspectives dans le domaine de l’optoélectronique et la conception des mémoires optiques. / This thesis is focused on the investigation of supramolecular assemblies and the charge carriers transport across organic single, bi- and three-component materials, used as the active layer in organic field-effect transistors (OFET), phototransistors (OPT) and complementary inverters. We demonstrated that thermal annealing and duration has high impact in OFET performances based on a fullerene derivative called ICBA. The devices electron mobility enhanced upon HMDS and OTS treated SiO2 surface and reached 0.1 cm2V-1s-1, which is the highest reported value in literature. We have provided evidence for the influence of the order at the supramolecular level in the semiconducting material (PDIF-CN2) on the performance of OPTs. We compared solution processed single crystalline PDIF-CN2 fibers and multifiber assemblies with spin-coated thin films, which revealed that the former exhibited good electron mobility up to 2 cm2s-1V-1. The improved fiber crystallinity allows efficient collection of photogenerated excitons, results in the highest reported responsivity R (>5 × 103 AW-1), and photoswitching ratio P (>2 × 103), which are to date the highest reported in literature for PDI-single crystal OPTs. Finally, we have performed for the first time new multifunctional devices combining an ambipolar polymer (DPPT-TT) with inserted diarylethene molecules in its matrix. The fabricated OFET and organic complementary inverters were optically controlled. The resultant inverters gain values are tuned by ultraviolet and visible light irradiation, reaching 504, which is higher than those reported in literature (86). These findings qualify them as promising potential candidates for the construction of high-performance integrated logic circuits and memory chips.