Le travail décrit dans ce manuscrit porte sur la recherche de nouvelles activités enzymatiques nitrilases et plus particulierement α-aminonitrilases. Le but de ce travail était, dans un premier temps, de tester un grand nombre de nitrilases candidates sur un large panel de nitriles pour appréhender leurs activités et établir un catalogue de nouvelles activités nitrilases. Dans un second temps, la recherche de nouvelles activités α-aminonitrilase a été menée dans l’objectif de réaliser la synthèse d’α-aminoacides par dédoublement cinétique dynamique assistée par couplage avec une racemase. 163 protéines ont été sélectionnées par une approche génomique, et testées sous forme de lysat cellulaire à l’aide d’un test enzymatique couplé à la glutamate deshydrogénase sur 28 nitriles répartis en 7 familles (α-aminonitriles exclus). Le grand nombre de résultats positifs obtenus à l’issu du criblage nous a permis d’évaluer les activités nitrilases et d’établir un catalogue de ces activités et d’envisager l’utilisation de certaines de ces nouvelles nitrilases dans la synthèse de synthons chimiques. Après sélection de 38 nitrilases et vérification de leurs activités sur enzymes purifiées, 8 nitrilases ont été testées sur 5 nouveaux nitriles déclinés du 3-phénylpropanenitrile. Une synthèse énantiospécifique de β-ester acide précurseur possible de quinolines fonctionnalisées a été réalisée. La déclinaison des dinitriles a également été abordée, 12 nitrilases sélectionnées ont été testées sur 6 dinitriles. Une nouvelle nitrilase nous a permis de réaliser la désymétrisation de l’iminodiacétonitrile par monohydrolyse. Enfin, de nouvelles activités nitrilases ont été observées sur des cyclooxonitriles pour la formation de plateformes chirales fonctionnalisées. Selon une autre approche génomique, 425 nitrilases candidates ont été selectionnées et testées (en plus les 163 nitrilases du précédent criblage) sous forme de lysat cellulaire grâce à la mise au point d’un suivi de l’activité nitrilase par dérivatisation et analyse par LC-MS sur 6 α-aminonitriles précurseurs d’α-aminoacides naturels. Le criblage a permis de mettre en évidence de nouvelles activités α-aminonitrilases sur le phénylglycinonitrile et le 2-amino pentanenitrile. De nouveaux nitriles déclinés du phénylglycinonitrile, précurseurs d’α-aminoacides d’interêt pharmacologique, ont été téstés sur 4 α-aminonitrilases. / The work described in this manuscript deals with on the search of new nitrilase activities especially α-aminonitrilases. In a first step, the aim was to build a catalog of nitrilase activities. In a second step, the search of new α-aminonitrilase activities was carried out for the synthesis of α-aminoacids by dynamic kinetic resolution with a racemase. Following a genomic approach, 163 proteins were selected and screened as cell lysate. The nitrilase activity was monitored by coupled enzymatic assay with glutamate dehydrogenase on 25 nitriles representative of the structural diversity. The large majority of proteins showed to be promiscuous towards the substrates. The best activities were validated using purified enzymes. Applications to the biocatalyzed synthesis of two building blocks, enantiomerically enriched acid (R)-4-methoxy-4oxo-3-phenylbutanoic acid and (S)-3-oxocyclopentanecarboxylic and the desymmetrization of iminodiacetonitrile by monohydrolyse, were performed. Through selection by a broader genomic approach, 588 candidates nitrilases were then tested on six α-aminonitriles. Analytical monitoring providing access to both conversion and enantiospecificity was developed by LC-MS. Six proteins were found to be active towards two substrates allowing applications to the synthesis of phenylglycine analogs with pharmacological properties.

Submitted by DHARA CARLESSO ZAMPIVA (dhara.zampiva@univates.br) on 2018-06-20T18:04:43Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2018StefaniStoll.pdf: 2349210 bytes, checksum: bf9fc66eaff3e59f76ec3d7a689383f6 (MD5) / Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2018-06-22T20:30:00Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2018StefaniStoll.pdf: 2349210 bytes, checksum: bf9fc66eaff3e59f76ec3d7a689383f6 (MD5) / Made available in DSpace on 2018-06-22T20:30:00Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2018StefaniStoll.pdf: 2349210 bytes, checksum: bf9fc66eaff3e59f76ec3d7a689383f6 (MD5) Previous issue date: 2018-06-20 / CAPES / O câncer se trata de uma das principais causas de mortes no mundo, sendo o câncer de pulmão o primeiro em incidência mundial. No Brasil, o câncer de pulmão é o segundo principal responsável por mortes por câncer em homens (após câncer de próstata) e o quarto em mulheres - sendo o Rio Grande do Sul o segundo estado brasileiro com maior incidência de casos de câncer. Caracterizado pelo crescimento desordenado e desenfreado de células, o desenvolvimento tumoral é definido pela evasão da morte celular e tem sido intimamente correlacionado à inflamação. Tratamentos usuais do câncer, como quimioterapia e radioterapia, apresentam elevados efeitos adversos ao paciente, além do alto custo. Tendo em vista que a maior parte das drogas antineoplásicas produzidas são oriundas de compostos naturais ou de seus derivados, é constante a busca por novos metabólitos de origem natural, em especial os de origem vegetal - que apresentam potencial na busca por novas moléculas para síntese de fármacos. Diversos fitocomponentes, como o terpeno α-terpineol (TPN), apresentam efeito antiproliferativo e anticancerígeno em linhagens de adenocarcinoma de mama, próstata e ovário, e leucêmicas. Neste trabalho foi avaliado o potencial anti-proliferativo do TPN frente à seis linhagens tumorais humanas (A549, MCF-7, HT-29, Caco-2, LNCaP, ACP-03), sendo a A549 (câncer de pulmão) selecionada para experimentos de tratamento repetido por seis dias em associação com o quimioterápico doxorrubicina (DOXO). O mecanismo de morte celular, marcadores moleculares, atividade enzimática (JAK-3, JNK-3, p38-α), migração celular e inibição de TNF-α foram avaliados. Dentre as seis linhagens analisadas, três (A549, MCF-7, HT-29) apresentaram redução significativa da viabilidade celular após tratamento com o TPN por 48 h. A associação do TPN com a DOXO na linhagem A549 potencializou o efeito anti-proliferativo em comparação com a DOXO. Após seis dias de tratamento observou-se comportamento dose- e tempo-dependente da DOXO e do TPN sem apresentar diferença estatística entre eles. O TPN apresentou atividade inibitória da p38-α (IC50=729 μ M) e JAK3 (IC50=6,3 μM). Após 48 h de tratamento, observou-se e necrose nas células tratadas com DOXO (21,5 %) e TPN (7,3 %).Com a associação de ambas as drogas, a taxa de necrose elevou-se para 33,5 %, bem como para os seis dias de tratamento (52,3 %). Observou-se ainda, redução da migração celular após 120 h de tratamento com o TPN em comparação ao controle. Com base no exposto acima, o TPN apresenta potencial como biomolécula para o desenvolvimento de novos fármacos ou mesmo seu uso, como coadjuvante no tratamento de câncer de pulmão humano. / Cancer is one of the leading causes of death worldwide, with lung cancer as the major one. In Brazil, lung cancer is the second leading cause of cancer deaths in men (after the prostate cancer) and fourth in women. Rio Grande do Sul is the second Brazilian state with the highest incidence of cancer. Characterized by disordered cell growth, tumor development is defined by cell death evasion and has been closely correlated to inflammation. Standard cancer treatments, such as chemotherapy and radiotherapy, cause adverse effects on patients. Considering that most of the antineoplastic drugs produced are derived from natural compounds or their derivatives, natural products (especially those of plant origin) present potential in the search for new molecules for drug synthesis. Several phytoconstituents, such as the terpene α-terpineol (TPN), have antiproliferative and anticancer effects in cell lines of breast, prostate and ovary carcinomas, and leukemic cells. In the present study, the anti-proliferative potential of TPN on six human tumor cell lines (A549, MCF-7, HT-29, Caco-2, LNCaP and ACP-03) was evaluated. The A549 cell line (lung adenocarcinoma) was selected for experiments of six days with TPN and doxorubicin (DOXO) co-treatment. Mechanisms of cell death, molecular markers, enzymatic activity (JAK-3, JNK-3, p38-α), cell migration and inhibition of TNF-α were evaluated. Among the six lines, three of them (A549, MCF-7, HT-29) have shown a significant reduction of cell viability after 48 h treatment with TPN. Co-treatment of TPN and DOXO on A549 potentiated the antiproliferative effect compared with DOXO treatment. After six days of treatment, dose and time-dependent effect of DOXO and TPN were observed. TPN showed inhibitory activity on p38-α (IC50 = 729 μM) and JAK3 (IC50 = 6.3 μM). After 48 h of treatment, necrosis was observed in cells treated with DOXO (21,5 %) and TPN (7,3 %). The co-treatment of both drugs increased the necrosis rate up to 33,5 %, as well in the 6th day of treatment (52,3 %). It was also observed a cell migration reduction after 120 h of TPN treatment in comparison with control. Based on that, TPN can be characterized as a potential biomolecule for new drugs development or even its use as adjuvant in the treatment of lung adenocarcinoma.

The effect of chloroethylclonidine on nodrenaline-induced contractions of the rat aorta was studied. Chloroethylclonidine (1.5 × 10-5 M) shifted noradrenaline dose-response curve to the right approximately 5000-fold without depressing the maximum. The response to noradrenaline after chloroethylclonidine was not antagonized by phenoxybenzamine (10-7 M), prazosin (10-7 M), WB 4101 (10-7 M) nor yohimbine (10-5 M) and is therefore not mediated via α1-adrenoceptors. These results would suggest that there is a homogenous population of chloroethylclonidine-sensitive α1-adrenoceptors in the rat aorta and that chloroethylclonidine treatment reveals a non-α-adrenoceptor noradrenaline binding site in this tissue.

Submitted by FERNANDA DA SILVA VON PORSTER (fdsvporster@univates.br) on 2014-10-07T18:57:03Z No. of bitstreams: 3 license_text: 22113 bytes, checksum: 449be06104264f68e4c3e35147dfb393 (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) 2014ElisaPaludo.pdf: 2351032 bytes, checksum: d04a74a436bf66ec47e6bf6c3fb53a49 (MD5) / Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2014-10-08T13:48:20Z (GMT) No. of bitstreams: 3 license_text: 22113 bytes, checksum: 449be06104264f68e4c3e35147dfb393 (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) 2014ElisaPaludo.pdf: 2351032 bytes, checksum: d04a74a436bf66ec47e6bf6c3fb53a49 (MD5) / Made available in DSpace on 2014-10-08T13:48:20Z (GMT). No. of bitstreams: 3 license_text: 22113 bytes, checksum: 449be06104264f68e4c3e35147dfb393 (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) 2014ElisaPaludo.pdf: 2351032 bytes, checksum: d04a74a436bf66ec47e6bf6c3fb53a49 (MD5) / A iontoforese é um método de administração de substâncias através da pele, com propósito terapêutico específico, utiliza-se de potencial ou corrente elétrica para promover a transferência transdermal de um fármaco. A camada córnea corresponde a uma espessura de cerca de 20 μm, sendo reconhecida como a principal barreira à transferência transdermal de fármacos. Em vista disso, cada vez mais técnicas diferentes vêm sendo testadas para a penetração de substâncias através do estrato córneo. Destaca-se ainda o α-tocoferol (vitamina E), um antioxidante natural capaz de reduzir ou bloquear as reações de oxidação induzidas pelos radicais livres nas membranas biológicas em função de exposição solar, por exemplo. O presente estudo tem como objetivo realizar avaliações eletroquímicas do gel + α-tocoferol quando submetido a aplicações de iontoforese, bem como verificar os potenciais de liberação e permeação do α-tocoferol quando associados à iontoforese. Para a realização dos experimentos utilizou-se a célula de difusão tipo Franz, aplicando-se as técnicas de voltametria cíclica e espectrofotometria UV/Vis para avaliação do sistema. A partir dos resultados observa-se um aumento da difusão e degradação do sistema, devido à redução da corrente de pico em 0,78 V das moléculas de α-tocoferol, quando submetido à iontoforese. Nas análises voltamétricas do gel + α-tocoferol, nos diferentes tempos de aplicação de iontoforese o sinal de corrente decai com o tempo, já que parte dele é permeada observa-se uma diminuição da altura do pico e seu consequentemente alargamento. Acredita-se que esteja ocorrendo uma degradação do α-tocoferol pela ação da iontoforese, e que seu produto esteja sendo oxidado no eletrodo de platina num potencial próximo ao do α-tocoferol. Nos ensaios de permeabilidade do α-tocoferol sem e com a aplicação de iontoforese, obteve-se uma diferença significativa de t = 4; p = 0,01 com a aplicação. Nos ensaios de permeação utilizando a muda de pele de cobra Boa constrictor há uma tendência de aumento de fluxo entre os tempos de 2 e 10 min, sendo esta uma diferença significativa, esse fato pode ser devido a utilização da membrana. Desta forma o α-tocoferol possui um comportamento eletroquímico ativo com a aplicação de iontoforese em meio gel de hidroxietilcelulose, avaliando também uma tendência à degradação do ativo com o aumento do tempo de aplicação da iontoforese observado na análise eletroquímica e de difusão. E a aplicação da iontoforese demostrou eficiência no aumento de permeação e liberação do α-tocoferol, comparado a condições sem aplicação.

Abstract Recent decades have experienced a sharp increase in the incidence and prevalence of diabetes mellitus. One antidiabetic therapeutic approach is to reduce gastrointestinal glucose production and absorption through the inhibition of carbohydrate-digesting enzymes such as α- amylase and α-glucosidase and α-amylase. The aim of the current study was to screen six medicinal plant species, with alleged antidiabetic properties for α-glucosidase inhibitory activities. Powdered plant materials were extracted with acetone, and tested for ability to inhibit baker's yeast α-glucosidase and α-amylase activities. The largest mass (440 mg from 10 g) of the extract was obtained from Cassia abbreviata, while both Senna italica and Mormordica balsamina yielded the lowest mass of the extracts. Extracts of stem bark of C. abbreviata inhibited baker's yeast α-glucosidase activity with an IC50 of 0.6 mg/ml. This plant species had activity at low concentrations, with 1.0 mg/ml and above resulting in inhibition of over 70%. The other five plant extracts investigated had IC50 values of between 1.8 and 3.0 mg/ml. Senna italica only managed to inhibit the activity of enzyme-glucosidase at high concentrations with an IC50 value of 1.8 mg/ml, while Tinospora fragosa extracts resulted in about 55% inhibition of the activity of the enzyme at a concentration of 3.5 mg/ml, with an estimated IC50 value of 2.8 mg/ml. The bark extract of C. abbreviata was the most active inhibitor of the enzyme, based on the IC50 values (0.6 mg/ml). The bark extract of C. abbreviata contains non-competitive inhibitor(s) of α-glucosidase, reducing Vmax value of this enzyme from 5 mM·s–1 to 1.67 mM·s–1, while Km remained unchanged at 1.43 mMfor para-nitrophenyl glucopyranoside. Antioxidant activity of the extracts was also investigated. The C. abbreviata extract was more active as an antioxidant than the positive control, trolox. The extracts did not inhibit alphaamylase activity more than about 20% at the highest concentration tested.

PHARMACOLOGIE DU CANAL CFTR : SYNTHESE DE NOUVEAUX COMPOSES ACTIVATEURS DE L'EFFLUX DES IONS CHLORURE.Après la découverte dans notre laboratoire d'une nouvelle réaction entre le méthylglyoxal et les α-aminoazahétérocycles aromatiques, une nouvelle famille de modulateurs de l'activité de la protéine CFTR (« Cystic Fibrosis Transmembrane conductance Regulator ») a été mise en évidence en collaboration avec l'équipe de F. Becq à Poitiers. Les dysfonctionnements de cette protéine transmembranaire résultent de différentes mutations du gène correspondant et sont responsables de plusieurs pathologies dont la mucoviscidose. Les perspectives thérapeutiques de cette maladie grave impliquent l'utilisation et la recherche d'activateurs du canal CFTR.En nous basant sur la structure des composés ayant présenté les meilleurs effets activateurs de la protéine, nous avons conçu et synthétisé une série d'analogues du composé activateur le meilleur mis en évidence précédemment, GPact-11a. Les nouveaux composés préparés sont issus des réactions du méthylglyoxal ou de nouvelles voies de synthèse utilisant des acides aminés. La réactivité de GPact-11a a ensuite été étudiée et exploitée pour préparer des prodrogues potentielles et réaliser la séparation des quatre énantiomères formés lors de la préparation de GPact-11a. Un travail de modélisation des interactions des modulateurs synthétisés avec un modèle de la protéine CFTR construit par homologie de séquences par I. Callebaut, J.-P. Mornon et P. Lehn a également été développé afin de concevoir rationnellement de nouveaux activateurs et de comprendre les effets observés. / PHARMACOLOGY OF CFTR CHANNEL: SYNTHESIS OF NEW ACTIVATORS OF CHLORIDE ION EFFLUX.After the discovery in our laboratory of a new reaction of methylglyoxal with α-aromatic aminoazaheterocycles, a novel family of CFTR modulators (“Cystic Fibrosis Transmembrane conductance Regulator”) has been identified in collaboration with the group of F. Becq in Poitiers. The dysfunctions of this transmembranar protein that result from different genetic mutations lead to several pathologies and among them to the genetic disease Cystic Fibrosis. In the search for more efficient CFTR activators from the structure of the best activator GPact-11a identified previously, a series of GPact-11a analogues was designed and synthesised through reaction of adenine derivatives with methylglyoxal or amino acids. The GPact-11a reactivity was also studied and used for the preparation of potential prodrugs and for the separation of the four enantiomers formed in the reaction leading to GPact-11a. Works were also developed to model the interactions between the synthesised derivatives and a CFTR model built through sequence by I. Callebaut, J.-P. Mornon and P. Lehn in order to design more rationally new activators and understand the biological effects.

The infrared, Raman and ultraviolet spectra of cyclopentanone, cyclopentanone -α,α,α',α'-d₄ and cyclopentanone-d₈ have been studied. The high resolution ultraviolet absorption spectra of the three isotopic species have been analysed in detail. The electronic transition has been identified as a single-singlet transition associated with ṉ→π* orbital electron promotion. The vibrational and rotational structures accompanying this electronic transition have been analysed and the results are in agreement with theoretical predictions. The geometry of the molecule in both ground and excited states has been calculated. In the excited state conformation, the oxygen atom is bent out of the plane of the three adjacent carbon atoms; there is a potential barrier of 705cm⁻¹ with respect to the oxygen out-of-plane bending vibration in the upper state. / Thesis / Doctor of Philosophy (PhD)

La determinación de la estereoquímica en los compuestos orgánicos ha sido un problema que la comunidad científica ha ido dando solución a lo largo de los años. Diferentes métodos han surgido, como la difracción de rayos X o los distintos tipos de Resonancia Magnética (RMN). Sin embargo, se acaba de descubrir que el método utilizado hasta ahora en la determinación de la configuración absoluta de los α-ácidos, que provienen del lúpulo utilizado en la elaboración de las cervezas bitter, parece que no era el correcto. / Determination of the stereochemistry of organic compounds has been a problem that the scientific community has been giving solution to over the years. Different methods have emerged, such as X-ray Diffraction or different types of Nuclear Magnetic Resonance (NMR). However, it just has been discovered that the method used in the determination of the absolute configuration of the α-acids derived from hops used in the manufacture of bitter beers, was not the appropriate.

Antioxidant-rich plant foods can inhibit starch and lipid digestion that are relevant to the management of type-II diabetes. Our objective was to compare the three phenolic substances (total phenolic, total flavonoids, and condensed tannin content) in crude, semi-purified extracts from eight types of foods (purified by XAD-7 column), five fractions (semi-purified extracts fractionated by Sephadex LH-20 column) from black legumes, and to compare their antioxidant capacities. The IC50 values of these crude extracts, semi-purified extracts and fractions against alpha-amylase, alpha-glucosidase and lipase were measured. Results showed that Fraction V from black soybean had the lowest IC50 value (0.25 mg/mL) against alpha-amylase; Fraction V from black bean had the lowest IC50 value (0.25 micro gram/mL) against alpha-glucosidase; Fraction IV of black bean had the lowest IC50 value (76 micro gram/mL) against lipase; myricetin showed the lowest IC50 value against alpha-amylase, alpha-glucosidase and lipase.

Interaction with chlorethylclonidine has been used to subdivide populations of α1-adrenoceptors in some tissues. WB 4101 can distinguish high and low affinity states of the receptor. The present study was carried out to determine if different populations or affinity states of α1-adrenoceptors distinguished by either of these compounds, could explain the variation in α1-adrenoceptor agonist affinity found amongst rabbit arteries. Five arteries were studied whose affinity for noradrenaline vary between 4.8 and 6.4. These were the thoracic aorta, renal, superior mesenteric, ear and ovarian arteries. WB 4101 was found to be equally effective in antagonizing noradrenaline on all arteries. Chlorethylclonidine caused a 20-fold rightward shift of the noradrenaline dose-contraction curve in the thoracic aorta; but had little or no effect on the other vessels. Thus, the combination of different proportionsof subsets of α1-adrenoceptors distinguished by WB 4101 or chlorethylclonidine does not explain the variation in α1-adrenoceptor affinity found in these rabbit arteries.