The tuberous sclerosis-2 (Tsc-2) gene product, tuberin, functions as a renal tumor suppressor. Treatment of Eker (Tsc-2 EK/+) rats and primary renal epithelial cells derived from Tsc-2 EK/+ rats (QTRRE cells) with 2,3,5-tris-(glutathion-S-yl) hydroquinone (TGHQ) results in loss of heterozygosity at the Tsc-2 locus in kidney tumors and QTRRE cells. QTRRE cells are carcinogenic in athymic nude mice. Analysis of kidney tumors formed in Tsc-2 EK/+ + rats following 8-months of TGHQ treatment reveals increases in B-Raf, Raf-1, pERK, cyclin D1, p27Kip1, 4EBP1, p-4EBP1(Thr70), p-4EBP1(Ser65), and p-4EBP1(Thr37/46) protein expression. These data establish the involvement of mTOR and MAPK signaling cascades in tuberin null tumors. Similar increases in 4EBP1 and p4EBP1 are observed in renal tumor QTRRE-xenografts in nude mice. Concomitant with increases in expression of these proteins in TGHQ-induced renal tumors, similar changes are observed in QTRRE cells, which also exhibit high ERK, B-Raf and Raf-1 kinase activity; and increased expression of cyclin D1, p27, p-4EBP1 (Thr70), p-4EBP1 (Ser65), and p-4EBP1 (Thr37/46). Manipulation of the Raf/MEK/ERK kinase cascade in QTRRE cells, with kinase inhibitors and siRNA, indicates that Raf-1/MEK/ERK participates in crosstalk with 4EBP1 to regulate translation of cyclin D1.Cyclin D1 and p27 protein levels are increased in the cytoplasm in our RCC models. In normal HK-2 cells, p27 and cyclin D1 are localized to the nucleus. Due to the instability of the cyclin D1-CDK4 complex, p27 interaction is necessary for cyclin D1-CDK4 complex assembly and stabilization in the nucleus. Manipulation of p27 protein levels in QTRRE cells with phosphodiesterase inhibitors, dibutyryl cAMP, and the proteosome inhibitor MG132, all result in a parallel increase in p27 and cyclin D1. Furthermore, p27 siRNA and sorafenib treatment both cause a decrease in p27 and cyclin D1. Further manipulation of cAMP, Rap1B, and B-Raf proteins, revealed that cAMP/PKA/Rap1B/B-Raf activation and B-Raf//ERK MAPK inhibition both modulate p27 expression and compartmental localization in tuberous sclerosis renal cancer. Phosphodiesterase inhibitors play a role in regulating the expression, degradation, and cytoplasmic localization of p27. Therefore, cytoplasmic p27-cyclin D1 mislocalization and stabilization may have an oncogenic role in the cytosol and play a crucial role in tumor formation.

Les travaux réalisés dans cette thèse concernent les efforts réalisés pour la synthèse de dérivés chiraux à base d'iode hypervalent analogues de l'acide iodoxybenzoïque, appelé IBX. Ces travaux ont permis de réaliser la synthèses de nouveaux dérivés iodés hypervalents qui ont été utilisés lors de réactions de désaromatisation de phénols énantiosélectives. Outre, les nouveaux dérivés hypervalents iodés synthétisés cette thèse a permis de mettre en avant le caractère problématique de la réaction d'oxydation de l'iode. Une alternative à ce problème a été la mise au point de méthode de génération in situ de dérivés iodés hypervalent permettant de réaliser des réactions de désaromatisation de phénols énantiosélectives. / The work presented in this thesis are about the synthesis of new chiral hypervalent iodine compounds analogues of the iodoxybenzoic acid, called IBX. This work shows an access to new chiral hypervalent iodine compounds which were used in enantioselective dearomatisation of phenols. In addition to the new compounds synthesized, this thesis gives a view of the complexity of the oxidation of iodine compounds. An alternative to this problematic synthetic step is given by the development of in situ generation methodology of hypervalent iodine compounds in order to perform an enantioselectiv variant of the dearomatization reaction.

Les études d'interaction protéine-ligand aident à mieux comprendre la structure et la fonction des protéines. Dans la première partie de la thèse, la cyt bd oxydase a été étudiée. La protéine d'E. coli a été immobilisée avec succès sur des électrodes modifiées par des nanoparticules d'or. Ainsi, un biocapteur électrochimique a été créé, permettant de tester certains inhibiteurs potentiels de cyt bd provenant d’E. coli. Le cyt bd issue de G. thermodenitrificans thermophile a également été étudié. En faisant appel aux spectroscopies IR et Raman ainsi que l’électrochimie, il a été démontré que la protéine est distincte du cyt bd d’E. coli. Une influence mutuelle du pH et de la température sur la catalyse a été aussi démontrée. La deuxième partie de la thèse portait sur la protéine mitochondriale mitoNEET. L'influence du pH et de divers ligands (pioglitazone, resvératrol, ions phosphates) a été examinée. / Protein-ligand interaction studies help to better understand the structure and function of proteins. In the first part of the thesis cyt bd oxidase was studied. The protein from E. coli was successfully immobilised at gold nanoparticles modified electrodes. Thus, an electrochemical biosensor was created allowing testing some potential inhibitors of cyt bd from E. coli. The cyt bd from thermophilic G. thermodenitrificans was also studied. By means of IR, Raman spectroscopy and electrochemistry the protein was shown to be distinct from cyt bd from E. coli. A mutual influence of pH and temperature was demonstrated on the electrochemical and catalytical properties. The second part of the thesis focused on mitochondrial mitoNEET protein. The influence of the pH and various ligands was studied.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2018-03-15T12:31:20Z No. of bitstreams: 1 Tese Doutorado Adilio.pdf: 2662669 bytes, checksum: 73c86d6d555c94d57c1f0714b5c2ccbc (MD5) / Approved for entry into archive by Tatiana Lopes (tatiana.lopes@pucrs.br) on 2018-03-23T11:44:45Z (GMT) No. of bitstreams: 1 Tese Doutorado Adilio.pdf: 2662669 bytes, checksum: 73c86d6d555c94d57c1f0714b5c2ccbc (MD5) / Made available in DSpace on 2018-03-23T11:52:37Z (GMT). No. of bitstreams: 1 Tese Doutorado Adilio.pdf: 2662669 bytes, checksum: 73c86d6d555c94d57c1f0714b5c2ccbc (MD5) Previous issue date: 2018-03-02 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Tuberculosis (TB), caused mainly by Mycobacterium tuberculosis, is an infectious disease responsible for a significant number of deaths worldwide. IQG-607 is an analog of isoniazid (INH). According to several experiments carried out by our research group, IQG-607 showed both in vitro and in vivo anti-TB activity. Initial studies showed that the compound INCT-TB551 (a quinoline derivative) also presents in vitro anti-TB activity. The aim of this study was to develop analytical methods by high performance liquid chromatography (HPLC-UV) for quantification of IQG-607 and INCT-TB551 in mice plasma, and therefore to perform pharmacokinetic studies. The analytical method for the determination of IQG-607 in mice plasma showed linearity (r = 0.9992) in 0.5? 50 ?g/mL concentration range. Intra- and inter-day precision was < 15%, and the recovery ranged from 92.07 to 107.68%, showing that the method provides a precise and accurate analysis of the compound. In addition, IQG-607 was stable in plasma for at least 30 days at ?80 ?C, and after plasma processing, for 4 h in the auto-sampler (6 ?C) and maintained on ice (recovery > 85%). The applicability of the method for pharmacokinetic studies was determined after intravenous (i.v.) and oral (fasted and fed conditions) administrations to mice. IQG-607 levels in plasma were quantified at time points for up to 2.5 h. A short half-life (t1/2) (1.14 h), a high clearance (CL) (3.89 L/h/kg), a moderate volume of distribution at steady state (Vdss, 1.22 L/kg), were observed after i.v. (50 mg/kg) administration. Similar results were obtained for oral administration (250 mg/kg) under fasted and fed conditions. The oral bioavailability (F), approximately 4%, was not altered by feeding. Plasma protein binding was 88.87 ? 0.9%. Recently, experiments in mice infected with M. tuberculosis have shown that the compound INCT-TB551 has no in vivo activity, unlike previous in vitro activity studies. An analytical method was developed for the determination of INCT-TB551 in mice plasma to assess compound absorption, if any, after oral administration. The analytical method presented linearity from 0.1-10 ?g/mL (r = 0.9999). After development of the analytical method, the compound was orally administered in mice and the plasma levels were quantified at time points for up to 1 h. The compound INCT-TB551 was detected in the plasma of animals, which indicated that INCT-TB551 was absorbed. Although absorbed when orally administered, the compound is not exhibiting activity against M. tuberculosis in this animal model. This finding may be associated with the formation of inactive metabolites and/or the plasma concentration of INCT-TB551 achieved may be inadequate to exert its therapeutic effect. However, these points require further investigations. The protocols described here may serve as support to initiate pharmacokinetic studies of promising compounds, collaborating to advance in earlier stages of drug development. / A tuberculose (TB) ? uma doen?a infecto-contagiosa, causada principalmente pelo Mycobacterium tuberculosis, respons?vel por um n?mero consider?vel de mortes em todo o mundo. O composto IQG-607 ? um an?logo da isoniazida (INH) que, de acordo com diversos experimentos realizados pelo grupo de pesquisa envolvido no seu estudo, apresenta atividade anti-TB in vitro e in vivo. Estudos iniciais do mesmo grupo demonstraram que o composto INCT-TB551 (um derivado quinol?nico) tamb?m apresenta promissora atividade anti-TB. O objetivo deste estudo foi desenvolver metodologias anal?ticas por cromatografia l?quida de alta efici?ncia (CLAE-UV) para a quantifica??o do composto IQG-607 e do INCT-TB551, em plasma de camundongos, para a realiza??o de estudos de farmacocin?tica. O m?todo anal?tico para a determina??o do composto IQG-607 em plasma de camundongos apresentou linearidade (r = 0.9992) na faixa de 0.5?50 ?g/mL. Os valores de precis?o intra e interensaio (CV < 15%) e de recupera??o (92.07-107.68%) demonstram que o m?todo ? preciso e exato para a an?lise do composto. Al?m disso, o IQG-607 se mostrou est?vel no plasma por at? 30 dias, quando armazenado no freezer -80 ?C, e est?vel ap?s o tratamento da amostra do plasma, por at? 4 horas (h) na bancada (gelo) e no autosampler do equipamento (6 ?C). A aplicabilidade do m?todo anal?tico para o estudo de farmacocin?tica foi determinada ap?s a administra??o i.v. e oral em camundongos (animais em jejum e animais alimentados). As concentra??es plasm?ticas de IQG-607 foram quantificadas por at? 2,5 h ap?s a administra??o nos animais. Quando administrado pela via i.v. foi observado um tempo de meia vida (t1/2) curto (1,14 h), elevado clearance (CL) (3,89 L/h/kg), e um moderado volume de distribui??o (Vdss) (1,22 L/kg). Resultados similares foram obtidos ap?s a administra??o oral (250 mg/kg) em camundongos que estavam em jejum ou alimentados. A biodisponibilidade oral (F) foi de aproximadamente 4 %, valor que n?o foi alterado pela alimenta??o. A taxa de liga??o a prote?nas plasm?ticas do IQG-607 ? de aproximadamente 88 ? 0.9%. Experimentos recentes em camundongos infectados com M. tuberculosis demonstraram que o composto INCT-TB551 n?o apresentou atividade neste modelo in vivo, ao contr?rio dos estudos de atividade in vitro realizados anteriormente. Um m?todo anal?tico para a determina??o do composto INCT-TB551 em plasma de camundongos foi desenvolvido para avaliar se o composto estava sendo absorvido ap?s a administra??o oral. O m?todo anal?tico apresentou linearidade na faixa de 0.1-10 ?g/mL (r = 0.9999). Ap?s o m?todo ter sido padronizado, o composto foi administrado por via oral em camundongos, e as concentra??es plasm?ticas foram quantificadas por at? 1 h ap?s a administra??o. O composto INCT-TB551 foi detectado no plasma dos animais, indicando que estava sendo absorvido. Apesar de absorvido quando administrado por via oral, o composto n?o est? apresentando atividade contra M. tuberculosis neste modelo animal, o que pode estar relacionado, por exemplo, com a forma??o de metab?litos inativos e/ou ainda, o n?vel plasm?tico atingido pode ser inadequado para que o composto consiga exercer o seu efeito terap?utico, e isto precisa ser melhor investigado. Os protocolos apresentados neste trabalho poder?o servir como suporte para estudos de farmacocin?ticoa de compostos que se apresentam promissores, colaborando para o avan?o nas etapas necess?rias para o desenvolvimento de poss?veis f?rmacos.

Le domaine de la bioénergétique concerne l’étude des échanges et des transformations de l’énergie au sein des organismes vivants. Cette thèse propose une étude électrochimique et spectroscopique de protéines issues de la chaine respiratoire, les oxydases terminales, afin de comprendre l’influence de différentes propriétés de ces enzymes (potentiels des cofacteurs, dépendance pH…) sur leur mécanisme réactionnel. La première partie de ce travail décrit le développement d’une méthode d’immobilisation permettant de conserver l’intégrité et l’activité de ces enzymes. Cette technique a d’abord été utilisée pour étudier l’inhibition de la cytochrome aa3 oxydase de P. denitrificans et a permis de mettre en avant l’importance du transfert de protons sur la réaction de réduction de l’oxygène. Une deuxième étude propose de comparer deux isoformes de la cytochrome cbb3 oxydase dont aucune différence n’a été observée à ce jour. La spectroscopie IRTF couplée à l’électrochimie montre l’implication de résidus acides différents au cours de la réaction d’oxydoréduction suggérant des différences mécanistiques. La dernière partie propose une étude comparative d’oxydases terminales de différents types et met en perspective l’influence des potentiels relatifs des hèmes sur la réaction de réduction de l’oxygène. / The field of bioenergetics concerns the study of exchange and transformation of energy in living organisms. This manuscript proposes an electrochemical and spectroscopic study of the fourth complex of the respiratory chain, the terminal oxidases. The aim of this study was to understand the influence of some properties of these enzymes (potential of the cofactors, pH dependency…) on the catalytic mechanism. The first part describes an immobilization procedure which retains the protein activity and structure. This procedure has been applied for the study the inhibition of the proton pathways of cytochrome aa3 oxidase from P. denitrificans and shows the importance of proton transfer on the oxygen reduction. In a second study, two isoforms of cytochrome cbb3 oxidase were compared. No differences were observed between them until now. Our electrochemically induced FTIR spectroscopy study suggests the implication of different acidic residues during the redox reaction implying differences in the mechanism of these enzymes. The last part deals with the comparison of terminal oxidases of different types and shows the influence of the relative order of the midpoint potentials of the hemes on the oxygen reduction.

La réaction de désaromatisation hydroxylante de phénols (HPD) est un outil puissant pour accéder de façon biomimétique, en une seule étape à partir de 2-alkylphénols, aux ortho-quinols, c’est-à-dire les 6-alkyl-6-hydroxycyclohexa-2,4-diénones. Ces synthons peuvent être présentés tels quels par de nombreux produits naturels mais peuvent aussi servir d’intermédiaires hautement fonctionnalisés pour la construction rapide d’architectures structurales complexes. L’acide o-iodoxybenzoïque (IBX), un réactif de type iodane-?5, et sa formulation stabilisée non-explosible (SIBX) se sont révélés particulièrement efficaces pour promouvoir des réactions HPD de manière ortho-sélective. Ces travaux de thèse concernent l’application de cette réaction à l’élaboration d’un intermédiaire ortho-quinolique clé hautement fonctionnalisé pour la synthèse d’un antibiotique de type angucycline, la (+)-aquayamycine, ainsi qu’à la première synthèse totale d’un ortho-quinol naturel non dimérisant, la (+)-wasabidiénone B1. Enfin, le développement d’une version asymétrique de cette réaction a été entrepris. La génération in situ de iodanes à partir de iodoarènes chiraux et de m-CPBA comme co-oxydant permet de préparer soit des ortho-quinols de façon non racémique lorsque des quantités stoechiométriques des deux réactifs sont utilisées, soit des ortho-quinols régio- et diastéréosélectivement époxydés lorsqu’une quantité catalytique de iodoarène chiral est employée. Un suivi de ces réactions par spectrométrie de masse a conduit à la détection d’espèces de type iodanyl-?3 et/ou -?5, et à la proposition d’un mécanisme pour ces réactions de désaromatisation hydroxylante asymétrique. / The hydroxylative phenol dearomatization (HPD) reaction is a powerful tool to access, in one biomimetic step from various 2-alkylphenols, ortho-quinols, i.e., 6-alkyl-6-hydroxycyclohexa-2,4-dienones. These dearomatized moieties can be found as such in few natural products or can be used as highly functionalized intermediates. The ?5-iodane 2-iodoxybenzoic acid (IBX) and its stabilized nonexplosive formulation (SIBX) have proved particularly useful and efficient in mediating HPD reactions in a strictly ortho-selective manner. This PhD work describes the application of our SIBX-mediated HPD reaction to the elaboration of a key ortho-quinolic advanced intermediate for the synthesis of the angucycline-type antibiotic (+)-aquayamycin, and to the first total synthesis of the natural non-dimerizing ortho-quinol (+)-wasabidienone B1. An asymmetric version of this HPD reaction has been also developed. In situ generation of iodanes from chiral iodoarenes and m-CPBA as co-oxidant enables the preparation of either ortho-quinols in a non racemic form when using stoechiometric amounts of both reagents, or regio- and diastereoselectively epoxidized ortho-quinols when a catalytic amount of the chiral iodoarene is used. Monitoring of these reactions by mass spectrometry allowed the detection of ?3- and/or ?5-iodanyl-type species, and the proposition of a mechanism for these asymmetric hydroxylative dearomatization reactions.