La lutte contre le terrorisme et le trafic de narcotiques sont devenus des enjeux sociétaux majeurs. Par exemple, l’identification rapide des colis piégés est aujourd’hui indispensable dans les lieux publics, motivant le développement de systèmes de détection de types nez électroniques. Ce travail de recherche, porte sur l’étude des transducteurs MEMS de type microleviers et SAW (surface acoustic wave), choisis parmi les différentes familles de biocapteurs existants pour leurs nombreux avantages tels que leur grande sensibilité à détecter tous types de molécules. Cette thèse est axée sur l’étude de ces deux types de MEMS en diamant combinés à des biorécepteurs olfactifs spécifiquement impliqués dans la perception et la reconnaissance des odeurs. Les propriétés physiques et chimiques exceptionnelles du diamant déposé sur leur surface ont permis le développement de détecteurs à la fois miniaturisés, robustes et sensibles. La chimie unique du diamant a permis en particulier d’immobiliser sur la surface de ces transducteurs différents types de biorécepteurs de type OBP (Odorant Binding Protein), MUP (Major Urinary Protein) et OR (récepteurs olfactifs) comme couche sensible. L’immobilisation de ces LBP sur les MEMS en diamant a été caractérisée par diverses techniques telles que la spectroscopie électrochimique d’impédance (EIS), la spectroscopie de fluorescence ou encore la spectroscopie XPS. Les performances des capteurs ont été suivies par vibrométrie laser dans le cas des microleviers et à l’aide d’un système d’acquisition commercial dans le cas des SAW, en mesurant les déplacements de fréquence de résonance lors d’exposition aux composés cibles. Une comparaison entre les différents types de LBP provenant de différentes espèces (mammifères, insectes) a été effectuée en termes de sensibilité lors d’exposition à des drogues et des composés explosifs. Les limites de détections (LOD) obtenues dans nos conditions de mesures se situent dans la gamme d’1µg jusqu’à 35 ng par exemple pour l’héroïne, en fonction des espèces cibles testées. La LOD du TNT est d’environ 100 ng. / Over the last decade, the need for resources devoted to counter terrorism as well as narcotic trafficking has grown. Thus the effective fight against those scourges requires the development of advanced physical and chemical detection systems and sensor systems such as electronic noses able to detect drugs and explosives compounds. In this context, ligand binding proteins (LBP) combined to diamond MEMS such as resonant microcantilevers and surface acoustic wave (SAW) sensors are foreseen as highly promising transducers for the design of label free biosensors in particular for the detection of small organic molecules. LBP are small proteins involved in the perception and recognition of odorant molecules. In this study, OBP (Odorant Binding Protein), MUP (Major Urinary Protein) and OR (olfactory receptor) were used to bind selected analytes. The carbon nature of diamond offers wide opportunities for stable grafting of such bioreceptors. The performances of these transducers present a real improvement in terms of speed, miniaturization and sensitivity. Immobilization of LBP on diamond transducers was investigated using fluorescence methods, electrical impedance spectroscopy (EIS) and X-Ray photoelectron spectroscopy (XPS). The sensing performances of the resulting biosensors were assessed by monitoring the frequency shift in real time upon exposure to the target molecules. We compared different kind of LBP, coming from vertebrates, insects, mutant or wild type in terms of sensitivity, selectivity and for their capability to bind odors, explosive and drug compounds. Within our experimental conditions, the detection of a wide variety of drugs and explosives was possible in the concentration range typically from 1 µg to e.g. 35 ng in the case of heroin, depending on the test substances. The limit of detection of TNT compound is about 100 ng.

Substitution of the 1,5-cyclooctadiene (cod) ligand in PtCl2(cod) (1) by the diphosphine ligand 4,5-bis(diphenylphosphino)-4-cyclopenten-1,3-dione (bpcd) yields PtCl2(bpcd) (2). Knoevenagel condensation of 2 with 9-anthracenecarboxaldehyde leads to the functionalization of the bpcd ligand and formation of the corresponding 2-(9-anthracenylidene)-4,5-bis(diphenylphosphino)-4-cyclopenten-1,3-dione (abpcd) substituted compound PtCl2(abpcd) (3), which is also obtained from the direct reaction of 1 with the abpcd ligand in near quantitative yield. The reaction of 3 with disodium maleonitriledithiolate (Na2mnt) affords the chelating dithiolate compound Pt(mnt)(abpcd) (4). The reaction of PtCl2(bpcd) (2) with 4-methyl-1,2-benzene dithiol under basic conditions affords Pt(tdt)(bpcd) (5). Compounds 2-5 have been fully characterized in solution by IR and NMR spectroscopies (1H and 31P), and their molecular structures established by X-ray crystallography. The electrochemical properties of 2‑5 have examined by cyclic voltammetry, and the nature of the HOMO and LUMO levels in systems 2-4 has been established by MO calculations at the extended Hückel level, the results of which are discussed with respect to electrochemical data and related diphosphine derivatives. In addition the new compounds 2-5 have been isolated by column chromatography and characterized by IR, UV-Vis spectroscopy.

The reaction between the formamidine ligand PriN=CHNHPri and the activated cluster Os3(CO)10(MeCN)2 has been studied. A rapid reaction is observed at room temperature, yielding the hydride clusters HOs3(CO)9[μ-OCNPriC(H)NPri] and HOs3(CO)10[μ-NPriC(H)NPri] as the principal products. The spectroscopic data and X-ray diffraction structures of those formamidinate-substituted clusters will be present. The thermal reactivity of the clusters has been investigated, with the face-capped cluster HOs3(CO)9[μ-NPriC(H)NPri] found as the sole observable product. The relationship between these three clusters has been established by kinetic studies, the results of which will be discussed.

The direct conversion of carbon–hydrogen bonds into valuable carbon-carbon and carbon-heteroatom bonds is a significant challenge to synthetic organic chemists. More than ever, chemists are employing Rh(III)-catalysts bearing cyclopentadienyl (Cp) ligands to transform otherwise inert C–H bonds. Furthermore, manipulating the sterics and electronics of the Cp ligand show significant impact on catalytic transformations. Our group has developed a library of CpˣRh(III)-precatalysts in hopes of enhancing known reactivity as well as discovering new C–H bond functionalizations. We have previously reported that N-enoxyphthalimides are a unique one-carbon component for the cyclopropanation of activated alkenes. In an effort to expand the scope to accessible alkenes, we have found a number of symmetrical unactivated alkenes undergo [2+1] annulation to afford intriguing spirocyclic cyclopropanes. Additionally, we have developed a Rh(III)-catalyzed diastereoselective [2+1] annulation onto allylic alcohols to furnish substituted cyclopropyl ketones. Notably, the traceless oxyphthalimide handle serves three functions: directing C–H activation, oxidation of Rh(III), and, collectively with the allylic alcohol, in directing cyclopropanation to control diastereoselectivity. Allylic alcohols are shown to be highly reactive olefin coupling partners leading to a directed diastereoselective cyclopropanation reaction, providing products not accessible by other routes. Next, an artifact of previous cyclopropanation reactions leads to the formation of a Rh-π-allyl complex. Attempts at 1,1-carboamination of alkenes are made using alkenes and nitrenoid precursors toward the 3-component synthesis of allylic amines. Stoichiometric studies help elucidate the mechanism and challenges. Lastly, efforts toward 1,2-carboamination of alkenes initiated by sp³ C–H bond activation are made with two different reactivity manifolds. Isolation of reaction intermediates are discussed as well as providing viable paths toward valuable products.

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Virtually all processes in living organisms are conducted by proteins. Proteins perform their function by binding to other proteins (protein-protein interactions) or small molecules - so called ligands (protein-ligand interactions). Active sites for protein-ligand interactions are pockets in protein structure where ligand can bind. Predicting of ligand binding sites is the first step to study and predict protein functions and structure based drug-design. In this thesis we reviewed current approaches for binding site prediction and proposed our own improvement. We have developed a novel pocket ranking function based on prediction model that predicts ligandability (ability to bind a ligand) of a given point inside of a pocket. Prediction is done considering only a local physicochemical and geometric properties derived from neighbourhood.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Hana Danková Supervisors: Prof. Ago Rinken, PhD. MSc. Tõnis Laasfeld PharmDr. Ivan Vokřál, PhD. Title of diploma thesis: Characetrization of ligand binding to M1 muscarinic receptor using fluorescence anisotropy method Muscarinic acetylcholine receptors (mAChRs), members of the superfamily of G-protein coupled receptors (GPCRs), regulate vital physiological processes and are important targets in drug research. Five different subtypes (M1 - M5) have been identified. M1 mAChR is mainly distributed in the central nervous system and is linked to pathophysiology of neurodegenerative diseases. In recent years, fluorescent methods have been frequently used in studies of ligand binding to receptors. The fluorescence anisotropy (FA) is a homogenous assay to characterize ligand binding to receptors. In this work, we have evaluated the FA method with fluorescent ligand MK342 binding to M1 mAChRs expressed on budded baculovirus (BBV) particles. The fluorescence ligand was binding with the high affinity (4,4 nM) to M1 receptor in constructed BBV preparation. The apparent binding affinities (pKi) of eleven classical and three bitopic muscarinic ligands were screened and compared to previously published...

LIN-12/Notch is a conserved transmembrane receptor that is required during animal development for proper cell-fate decisions and specification. In Caenorhabditis elegans, activation of LIN-12 occurs through binding to ligand expressed by an adjacent cell. This binding event triggers two cleavage steps and results in the release of the LIN-12 intracellular domain [LIN-12(intra)], which translocates to the nucleus to form a ternary complex with two other proteins: LAG-1/Su(H)/Cbf1 and SEL-8/Mastermind/Mastermind-like. This ternary complex will then transcriptionally activate target genes via LAG-1 Binding Sites (LBSs). LAG-1 is the sole DNA-binding component within the complex, and in the absence of LIN-12(intra), can act as a transcriptional repressor. LIN-12 signal transduction can be studied in the C. elegans Vulval Precursor Cells (VPCs), which exhibit precise spatiotemporal patterning regulated by LIN-12 activity. Here, I show that LAG-1 is positively autoregulated by LIN-12 activity in cells where LIN-12 activity is high. Autoregulation is mediated by an enhancer element that contains a cluster of 18 LBSs that are located within a conserved high occupancy target region, which is a span of DNA that is pulled down promiscuously in ChIP-Seq experiments. Mutation of the LBSs abrogates preferential expression mediated by the enhancer in cells with high LIN-12 signal transduction. When the HOT region is deleted from the endogenous lag-1 locus, expression in the VPCs is strongly reduced and no overt Lag phenotype occurs. Instead, cold-sensitive vulval and egg-laying defects, reminiscent of phenotypes seen in lin-12 hypomorphs, are found. Autoregulation of lag-1, therefore, appears to contribute to the robustness of LIN-12 cell fate specification in response to stochastic environmental and genetic perturbations. Under adverse environmental conditions, C. elegans enter a state of diapause in which they form dauer larvae, which are long-lived and stress-resistant. The VPCs of dauer larvae remain developmentally arrested indefinitely until favorable conditions are reintroduced. Experimentally, this arrest can be relieved by depletion of the Forkhead transcription factor DAF-16. I show that expression of the components of the LIN-12/SEL-8/LAG-1 ternary complex are downregulated during the L2d-dauer molt (prior to dauer entry) and that this downregulation is not relieved by DAF-16 depletion. Instead, DAF-16 depletion leads to resumption of LIN-12 signaling and expression of ternary complex only in completely formed dauer larvae. These observations suggest that DAF-16 is required for the maintenance but not the initiation of blocking LIN-12 signaling. The components of the ternary complex are required to effect LIN-12 signaling. This work contributes to better understanding how these components are regulated and how their expression can affect LIN-12 -mediated cell fate decisions.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Hana Danková Supervisors: Prof. Ago Rinken, PhD. MSc. Tõnis Laasfeld PharmDr. Ivan Vokřál, PhD. Title of diploma thesis: Characetrization of ligand binding to M1 muscarinic receptor using fluorescence anisotropy method Muscarinic acetylcholine receptors (mAChRs), members of the superfamily of G-protein coupled receptors (GPCRs), regulate vital physiological processes and are important targets in drug research. Five different subtypes (M1 - M5) have been identified. M1 mAChR is mainly distributed in the central nervous system and is linked to pathophysiology of neurodegenerative diseases. In recent years, fluorescent methods have been frequently used in studies of ligand binding to receptors. The fluorescence anisotropy (FA) is a homogenous assay to characterize ligand binding to receptors. In this work, we have evaluated the FA method with fluorescent ligand MK342 binding to M1 mAChRs expressed on budded baculovirus (BBV) particles. The fluorescence ligand was binding with the high affinity (4,4 nM) to M1 receptor in constructed BBV preparation. The apparent binding affinities (pKi) of eleven classical and three bitopic muscarinic ligands were screened and compared to previously published...