Sem resumo / Sem abstract

A finalidade desse trabalho foi a análise de aspectos estruturais e biológicos de uma lectina do veneno de B. atrox, denominada galatrox. A purificação da galatrox envolveu dois passos cromatográficos, sendo o primeiro por afinidade em coluna de agarose-lactose e o segundo referente a aplicação do material retido no gel de agarose-lactose (LacR), em coluna de Sephadex G-25. As etapas de purificação foram monitoradas por leitura de absorbância em 280nm e SDS-PAGE. Preparações de galatrox foram submetidas à digestão in situ com tripsina e a massa molecular e o sequenciamento dos peptídeos obtidos foram determinados por espectrometria de massa (MALDI-TOF-MS e ESI-CID-MS/MS). A seqüência N-terminal de aminoácidos da galatrox foi obtida pela reação automatizada de Edman (PROCISE-419®). A atividade lectínica dessa proteína foi caracterizada por meio de testes de aglutinação de eritrócitos humanos, na presença ou ausência de diferentes carboidratos como lactose (4mM) e galactose (20mM), EDTA (5mM) e aquecimento (100ºC/10min). A desgranulação mastocitária foi determinada pela liberação de -Hexosaminidase por células RBL-2H3 sensibilizadas com IgE anti-DNP(dinitrofenil) e estimuladas com galatrox, veneno bruto, fração não retida na coluna de agarose-lactose (Lac-nR), HSA-DNP (controle positivo) ou PBS (controle negativo). O nível de indução de apoptpse e/ou necrose de células RBL-2H3 tratadas com galatrox foi avaliado pela marcação com anexina V-FITC e/ou iodeto de propídeo e analisado por citometria de fluxo (FACScanto®) com o auxílio do software (CBA-DIVA®). A camptotecina foi utilizada como referência de apoptose/necrose. A atividade edematogênica foi testada em camundongos pela injeção intraplantar de galatrox; fração Lac-nR, veneno bruto e PBS. Análise por SDS-PAGE indicou que as preparações de galatrox eram homogêneas e continham bandas de 14.000 e 28.000 de massa molecular relativa em condições redutoras ou não, respectivamente. A seqüência N-terminal foi correspondente aos seguintes aminoácidos: NNXPQDWLPMNGLXYKIFD, e a seqüência de alguns aminoácidos internos corresponderam a KDFSWEWTDR e GHSEVWLGLWDK. A atividade hemaglutinante dessa lectina foi dose-dependente e inibida por EDTA (5mM), aquecimento (100ºC/10min) e lactose (32mM, 16mM, 8mM, 4mM, 2mM, 1mM, 0,5mM). Ao contrário do veneno bruto e da fração Lac-nR, esta lectina não foi edematogênica e nem promoveu apoptose e necrose de células RBL- 2H3. A galatrox induziu uma discreta desgranulação mastocitária em comparação com o veneno bruto e a fração Lac-nR. Com base nos dados obtidos sugere-se que a galatrox é uma lectina homodímérica com 28.000 de massa molecular relativa, ligante de -galactosídeos e que apresenta similaridade estrutural com outras lectinas tipo-C do veneno de Bothrops ssp. Além disso, a galatrox comportou-se como um fraco agente pró-inflamatório e não induziu efeitos apoptótico e necrótico significantes. Finalmente, esse trabalho poderá contribuir para o melhor entendimento do impacto biológico da presença de lectinas no veneno e nos envenenamentos, bem como na geração de novos produtos biotecnológicos. / The aim of this work was the analysis of structural and biological aspects of a B. atrox venom lectin, named galatrox. The galatrox purification involved two chromatographic steps, starting with an affinity column of Lactosyl-Sepharose followed by application of the retained material on a Lactosyl-Sepharose gel (LacR) in Sephadex G-25 column. The purification steps were monitored by absorbance at 280nm and SDSPAGE. Galatrox samples were submitted to digestion in situ with trypsin and the sequencing mass of the obtained peptides were determined by mass spectrometry (MALDI-TOF-MS and ESI-CID-MS/MS). The N-terminal sequence of amino acids from galatrox was obtained by automatized Edman degradation (PROCISE-491®). The lectin activity of this protein was characterized by human erytrocytes agglutination test in presence or absence of different carbohydrates as lactose (4mM) and galactose (20mM), EDTA (5mM) and heat. The mast cells degranulation was determined by -hexosaminidase release in RBL-2H3 cells sensibilized with anti-DNP IgE and challenged with galatrox, crude venom in a non retained fraction in Lactosyl-Sepharose (Lac-nR) , HAS-DNP (positive control) or PBS (negative control). The apoptosis and/or necrosi induced level in treated RBL- 2H3 cells with galatrox was evaluated using AnnexinV and/or Propidium iodide and analysed by flow citometry (FACSCanto®) with the help software (CBA-DIVA®). The camptotecin was used with apoptosi/necrosi reference. The edematogenic was tested in mice by the intraplantar injection of galatrox, Lac-nR fraction, crude venom and PBS. SDS-PAGE analyse indicated that the galatrox preparations were homogenous and contained a single band of 14,000 or 28,000 relative molecular weight showed in reducting or non-reducting conditions, respectively. N-terminal amino acid sequence was determined as following: NNXPQDWLPMNGLXYKIFD, and some internal amino acid sequence was obtained which correspond to KDFSWEWTDR and GHSEVWLGLWDK. The hemagglutination activity of this lectin was dose dependent and inhibited by EDTA (5mM), heating (100ºC/10min) and lactose (32mM, 16mM, 8mM, 4mM, 2mM, 1mM, 0,5mM). In the other hand, the crude venom and Lac-nR fraction, this lectin was not edematogenic nor promoted apoptosis and necrosi of RBL-2H3 cells. The galatrox induced a discret mast cells degranulation compared with crude venom and Lac-nR fraction. Based in obtained datas is suggested that galatrox is a homodimeric lectin with relative molecular mass of 28,000, -galactoside bindings and that shows structural similarity with other Ctype lectins from Bothrops ssp. venom. Even more the galatrox showed to be a weak proinflamatory agent and did not induced significant apoptotic and necrotic effect. Finally this work would contribute to better understanding of the biological impact that the lectin presence in venom and in poisonings as well in the generation of a new biotechnological products.

Objetivos. Verificar a relação entre limiares de detecção dos gostos doce e salgado, concentração ideal e mais aceita de açúcar, sal e gordura, sensibilidade ao 6-n-propiltiouracil (PROP) e sexo, grupo etário e estado nutricional. Verificar a relação entre sensibilidade ao PROP e frequência referida de ingestão alimentar. Verificar a correlação entre aceitação e concentração ideal. Métodos. Estudo transversal com 123 adultos (20 a 58 anos). Variáveis de estudo: limiar de detecção dos gostos doce e salgado, concentração ideal e mais aceita de açúcar, sal e gordura, sensibilidade ao PROP. O estado nutricional foi determinado pelo Índice de Massa Corporal, sendo incluídos indivíduos com valores entre 18,5 e 24,9kg/m2 (peso adequado) e, 25kg/m2(excesso de peso). No teste de limiar de detecção, o participante recebeu 5 soluções de sacarose e 5 de cloreto de sódio (NaCl). A concentração ideal e a mais aceita foram determinadas, utilizando 5 concentrações de açúcar em suco de laranja, 5, de sal e 5, de gordura, em purê de batata industrializado. Para a determinação da concentração ideal, utilizaram-se as médias obtidas a partir de escala de 7 centímetros e, para o teste de aceitação, escala hedônica de 7 pontos. O nível de sensibilidade ao PROP foi determinado a partir da degustação de soluções de PROP e NaCl, com utilização de escala de magnitude rotulada. Para identificar a frequência de ingestão alimentar foi utilizado questionário de frequência alimentar. Análise estatística. As variáveis do 7 estudo foram apresentadas por meio de média, desvio-padrão e amplitude. A análise estatística foi conduzida, utilizando os testes Kruskall-Wallis, Mann-Whitney, análise não paramétrica de dados ordinais com medidas repetidas e correlação de Spearman. O nível de significância adotado foi de 5 por cento. Resultados. Os limiares do gosto doce foram menores para os indivíduos com excesso de peso. As mulheres atribuíram notas médias menores para as concentrações 1 e 3 por cento no teste de concentração ideal de açúcar. Para o ideal de gordura, as notas médias dos indivíduos com peso adequado foram mais altas em relação aos com excesso de peso. Os indivíduos de 31 a 40 anos conferiram notas médias maiores no teste de aceitação para a concentração 1 por cento de açúcar. No teste de concentração ideal de gordura, as médias das mulheres foram mais altas que as dos homens. Houve correlação positiva entre concentração ideal e aceitação. 72 por cento dos indivíduos eram sensíveis ao PROP. Não houve relação entre de sensibilidade ao PROP e sexo, idade, estado nutricional e, frequência de ingestão alimentar, com exceção de biscoito e bolo. Conclusões. Verificou-se relação entre limiar de detecção do gosto doce e estado nutricional, concentração ideal de açúcar e sexo, concentração ideal de gordura e estado nutricional, aceitação de sal e grupo etário. Observou-se relação entre sensibilidade ao PROP e ingestão de biscoito e bolo, e correlação entre aceitação e concentração ideal / Objectives. To investigate the relationship between thresholds detection of sweet and salty tastes, just-about-right and most accepted concentrations of sweet, salty, fat, sensitivity to 6-n-propylthiouracil (PROP) and gender, age and nutritional status. To investigate the relationship between PROP sensitivity and frequency of food intake. To verify the relationship between acceptance and just-about-right concentrations. Methods. Cross-sectional study with 123 adults (20 to 58 years). The variables were: threshold detection of sweet and salty tastes, most accepted and just-about-right concentrations of sugar, salt and fat, sensitivity to PROP. Nutritional status was determined by Body Mass Index (BMI). A BMI between 18.5 and 24.9 kg/m2 correspond to normal weight, and 25kg/m2, to overweight. In the threshold test the participant received 5 solutions of sucrose and 5 of sodium chloride (NaCl). The just-about-right and the most accepted concentrations were determined using 5 concentrations of sugar in orange juice, 5 of salt, 5 of fat in industrialized mashed potato. To determine the just-about-right concentration it was used the scores from the 7 cm scale and for acceptance testing, 7-point scale. The level of sensitivity to PROP was determined from tasting PROP and NaCl solutions, using a labeled magnitude scale. A food frequency questionnaire was used to identify frequency of food intake. Statistical analysis. The study variables were presented by mean, standard deviation and amplitude. Statistical analysis was conducted using Kruskal-Wallis, Mann-Whitney, 9 non-parametric analysis of ordinal data with repeated measures and Spearman correlation. The level of significance was 5 per cent. Results. The sweet taste thresholds were lower for individuals with overweight. Women assigned lower scores than men for the concentrations 1 and 3 per cent of sugar in the just-about-right test. For fat, just-about-right scores of individuals with normal weight were higher in relation to overweight. Individuals aged 31 to 40 years have given higher scores in acceptance test for the concentration 1 per cent of sugar. In just-about-right test of fat, the women\'s scores were higher than those of men. There was perfect correlation between ideal concentration and acceptance. 72 per cent of subjects were sensitive to PROP. There was no relationship between sensitivity to PROP and gender, age, nutritional status, and frequency of food intake, except for biscuits and cake. Conclusions. There was relationship between threshold detection of sweet taste and nutritional status, just-about-right concentration of sugar and gender, just-about-right concentration of fat and nutritional status, acceptance of salt and age. It was also observed relationship between sensitivity to PROP and intake of biscuits and cake and correlation between acceptance and just-about-right test

Consideramos nesse trabalho questões relativas a parte inferior do espectro de energia-momento para o modelo de teoria campos na rede com tempo imaginário, associado ao sistema ferromagnético de spins clássicos de $N$-compontentes definido na rede $d$ dimensional: O Modelo de Spin O$(N)$. Esses sistemas são caracterizados por uma distribuição de probabilidade de spin por sítio. Tratamos apenas da região de altas temperaturas. O espectro de energia e momento deste modelo apresenta curvas de dispersão isoladas, que podem ser interpretadas como quasi-partículas. Em particular, estudaremos os estados de uma e duas quasi-partículas. Para o espectro de uma partícula, obteremos a curva de dispersão e a massa de uma partícula. Esse resultado mostra a existência da chamada 'lacuna espectral'. Ainda trabalhando no espectro de uma partícula, demonstraremos a existência de uma banda de espectro contínuo, associada a estados de duas partículas livres, e determinaremos a largura desta banda. Nossa análise de duas partículas é restrita a uma aproximação em escada da equação Bethe-Salpeter. Usando essa aproximação mostraremos que a existência e a localização de estados ligados depende da verificação da dominação gaussiana para a função de correlação de quatro pontos. É sabido que estados ligados de duas partículas aparecem abaixo da banda de duas partículas se não vale a dominação gaussiana. Mostraremos que estados ligados de duas partículas aparecem acima da banda de duas partículas, caso a dominação gaussiana seja verificada. Além disso, mostramos como o padrão espectral de duas partículas para desses modelos podem ser compreendido através da correspondência entre a equação Bethe-Salpeter e um operador hamiltoniano de Schrödinger de duas partículas na rede com potenciais atrativos ou repulsivos do tipo delta e dependentes dos indices de spin. Uma transformação de staggering é utilizada para relacionar os casos de potenciais atrativos e repulsivos e o espectro dos hamiltonianos e suas autofunções. / In this work, we consider the low-lying energy-momentum spectrum for the imaginary-time lattice quantum field model associated with d-dimensional lattice ferromagnetic classical N-component vector spin systems: The O(N) Spin Model. Each system is characterized by a single site 'a priori' spin probability distribution. We work only at high temperature region (0<&#946;<=1). The energy-momentum spectrum exhibits isolated dispersion curves which are identified as single particles and multi-particle bands. In particular, we study states of one and two-particles. For the single particle spectrum, we obtain the dispersion curve and the particle mass. This result show the existence of the so called 'low spectral gap'. Still working with the single particle spectrum, e show the existence of a continuum spectra band, associated to states of two free partciles, and we obtain the band width. Our two-particle bound state analysis is restricted to a ladder approximation of the Bethe-Salpeter equation, and the existence of bound states depend on whether or not Gaussian domination for the four-point function is verified. It is known that two-particle bound states appear below the two-particle band if Gaussian domination does not hold. Here, we show that two two-particle bound states appear above the two-particle band if Gaussian domination is verified. We also show how the complete two-particle spectral pattern for these models can be understood by making a correspondence between the Bethe-Salpeter equation and a two-particle lattice Schrödinger Hamiltonian operator with attractive or repulsive spin-dependent delta potentials at the origin. A staggering transformation is used to relate the attractive and repulsive potential cases, as well as their associated Hamiltonians spectrum and eigenfunctions.

This thesis describes the development of nickel(I) complexes incorporating ring expanded N-heterocyclic carbene (RE NHC) ligands and examines their electronic characterisation, activation of O2, reactivity and catalytic applications. A series of three coordinate, paramagnetic Ni(I) complexes of the form Ni(RE NHC)(PPh3)Br (1 – 10) were prepared by comproportionation of Ni(COD)2 and Ni(PPh3)2Br2 in the presence of RE NHCs. The RE NHCs employed varied in the degree of ring expansion (6-, 7- and 8-membered), extent of N-substituent steric bulk (Mes, oTol, oAnis) and the donor/acceptor properties of the carbene (diamino vs. diamido). EPR and DFT electronic characterisation of 1 – 10 confirmed that the unpaired electron was located on the nickel ion in a mixed orbital of predominantly 3dz2 character. Yellow solutions of 1 or 6 (RE NHC = 6Mes and 7Mes respectively) immediately became purple in the presence of O2 due to O2 activation and incorporation of the oxygen atoms as bridging ligands resulting from C-H activation/oxygenation of an RE NHC N-substituent. This generated the dimeric Ni(II) complexes Ni(6/7Mes)Br(µ-OH)(µ-O-6/7Mes)’NiBr (6Mes = 13; 7Mes = 14). Mass spectrometry demonstrated that the doubly activated complexes [NiBr(µ-O-6/7Mes)’]2 (15 and 16 respectively) were also formed in the reactions. UV-vis spectroscopy revealed the reactions took place rapidly, even at 190 K. Contrasting reactivity was observed when 2 or 7, bearing the less sterically encumbered N-oTol substituents 6oTol and 7oTol respectively, were exposed to O2, which led to the ligand redistribution products NiII(6/7oTol)(PPh3)Br2 (17 and 18 respectively). The less electron rich diamido analogue containing 6MesDAC (5), underwent dissociation and oxidation of the RE NHC and PPh3 ligands. Attempts to abstract the bromide from 1 generated novel two and three coordinate Ni(I) products. Reaction with additional 6Mes produced the two coordinate cation [Ni(6Mes)2]+ (19), which could be reduced with KC8 to Ni(6Mes)2 (20). SQUID analysis of 19 revealed it to be the first example of a nickel containing mononuclear single molecular magnet (SMM). Addition of [Et3Si]+ to 1 followed by work up in toluene led to the isolation of the Ni(I)-(η2-toluene) complex [Ni(6Mes)(η2-C6H5CH3)]+ (21). Mesitylene generated the analogous [Ni(6Mes)(η2-C6H3(CH3)3)]+ (23), but neither 1,4-xylene nor naphthalene gave isolable products. In all cases, cocrystallisation of [6MesH]+…arene was observed in variable amounts, which compromised reaction studies of the Ni-arene complexes. Removal of bromide from 1 with TlPF6 in THF generated the solvent coordinated cationic species [Ni(6Mes)(PPh3)(THF)]+ (24). Attempts to remove the ligated THF molecule were unsuccessful, however, it could be directly substituted by CO to form [Ni(6Mes)(PPh3)(CO)]+ (26). Similarly to 1, complex 24 activated O2, generating a dimer analogous to the singly activated complex 13 (Ni(6Mes)(PPh3)(µ-OH)(µ-O-6Mes)’NiBr (25)). Reactivity of 1 with NaBH4 produced [Ni(6Mes)(κ2-BH4)]2 (28), a Ni(I) dimer bridged by two BH4 ligands. The catalytic efficiency of neutral 1 in Kumada cross-coupling of aryl halides and PhMgCl or MesMgBr was probed. Of note was the high activity towards both aryl chlorides and aryl fluorides. Comparisons with cationic 24, larger 7- (7) and 8-membered ring (8 and 9) variants and the Ni(II) complexes Ni(6Mes/6oTol/7oTol)(PPh3)Br2 (29, 17 and 18 respectively) revealed that 1 exhibited the highest reactivity of all the precursors.

Les hétérocycles azotés chiraux représentent une classe importante de composés biologiquement actifs. L’hydroamination asymétrique intramoléculaire correspond parfaitement au concept d’économie d’atomes et permet l’accès direct à la formation de nouvelles liaisons carbone-azote. Le développement de catalyseurs pour la formation de pyrrolidines et piperidines reste cependant une tâche importante. Le thème de cette thèse est l’étude de nouveaux complexes chiraux binaphthylamide d’yttrium facilement accessibles et leur application pour promouvoir la réaction d’hydroamination/cyclisation des amines primaires liées à des alcènes stériquement encombrés.Des complexes chiraux binaphthylamidure alkyl ate d’yttrium ont été développés. Ces systèmes catalytiques ont été préparés in situ par une réaction simple stœchiométrique en présence d’un précurseur d’yttrium [Li(THF)4][Y(CH2SiMe3)4] connu et d’une variété de ligands chiraux binaphthyldiamine substitués. Les complexes chiraux hétéroleptiques obtenus se sont révélés actifs et énantiosélectifs pour la réaction d’hydroamination asymétrique intramoléculaire des aminoalcènes 1,2-disubstitués conduisant à la formation d’hétérocycles azotés avec cinq et six châinons. Les catalyseurs d’yttrium préparés à partir du ligand (R)-N-anthrylmethyl-binaphthylamine H2L13 et des ligands (R)-benzyl N-para-substitués H2L4 or H2L6 se sont révélés les plus énantiosélectifs à 70-110°C pour la réaction d’hydroamination/cyclisation des aminoalcènes encombrés. Un excès énantiomérique de 77% a été obtenu comme valeur la plus élevée décrite jusqu’à présent pour l’hydroamination asymétrique intramoléculaire des amines comportant des alcènes 1,2-disubstitués. Les premiers exemples de la réaction d’hydroamination intramoléculaire asymétrique des aminoalcènes 1,1,2-trisubstitués ont été rapportés. Les complexes ate alkyl d’yttrium ont fourni les produits hétérocycliques avec des centres quaternaires énantiomériquement enrichis dans des conditions réactionelles plus sévères que la cyclisation des aminoalcènes 1,2-disubstitués et avec des énantiosélectivités prometteuses atteignant 55%. Les complexes alkyl d’yttrium contenant une molécule de chlorure de lithium [{(R)-C20H12(NSiMe3)2}Y{CH2SiMe3}{LiCl(THF)2}] and [{(R)-C20H12(NC5H9)2}Y{CH2SiMe3}{LiCl(THF)2}] ont été préparés et comparés aux mêmes complexes sans LiCl. Ils se sont révélés être des catalyseurs efficaces pour la réaction d’hydroamination intramoléculaire des aminoalcènes terminaux. / Chiral nitrogen-containing heterocycles represent an important class of biologically active compounds. The asymmetric intramolecular hydroamination perfectly matches the concept of sustainable chemistry and allows the formation of new nitrogen-carbon bonds in an ideal atom efficiency and economy, starting from non activated substrates. The development of catalysts for formation of enantioenriched pyrrolidines and piperidines derivatives remains however a challenging task. The topic of this dissertation is the study of new easily accessible chiral yttrium binaphthylamide complexes and their application for promoting the hydroamination/cyclisation of primary amines tethered to sterically demanding alkenes. Chiral binaphthylamido alkyl ate yttrium complexes have been investigated. These catalytic systems have been prepared by a facile in situ stoichiometric reaction of a well-known yttrium precursor [Li(THF)4][Y(CH2SiMe3)4] with a variety of chiral substituted (R)-binaphthylamine ligands. These chiral heteroleptic complexes are shown to be efficient catalysts for the enantioselective intramolecular hydroamination of 1,2-disubstituted aminoalkenes leading to the formation of five and six-membered N-heterocycles. Yttrium catalysts prepared from (R)-N-anthrylmethyl-binaphthylamine ligand H2L13 and (R)-N-para-substituted benzyl ligands H2L4 or H2L6 proved to be the most enantioselective catalysts at 70-110°C for the hydroamination/cyclisation of challenging aminoalkenes. An enantiomeric excess value of 77 % was indeed disclosed as the highest value reported so far for the asymmetric intramolecular hydroamination of amines tethered to 1,2-disubstituted alkenes. The first examples of asymmetric intramolecular hydroamination of 1,1,2-tri-substituted aminoalkenes have been reported. The alkyl ate yttrium complexes produced the heterocyclic compounds with enantioenriched quaternary centres under harsher reaction conditions than the cyclisation of the corresponding 1,2-disubstituted alkenes, and with promising enantioselectivities of up to 55 %.The neutral alkyl yttrium complexes containing one molecule of lithium chloride [{(R)-C20H12(NSiMe3)2}Y{CH2SiMe3}{LiCl(THF)2}] and [{(R)-C20H12(NC5H9)2}Y{CH2SiMe3}{LiCl(THF)2}] have been prepared and compared with the same complexes lacking LiCl. They have been revealed as efficient catalysts for intramolecular hydroamination of terminal aminoalkenes.

Les cellules épithéliales sont capable de se polariser avec un domaine apical et un basolatéral qui diffèrent nettement en leurs protéines, leurs composition en lipides et donc en fonction. Cette asymétrie reflète la capacité des cellules épithéliales à affecter les protéines nouvellement synthétisées et les lipides à chacune des surfaces de la cellule. Alors que les signaux responsables pour la sélection basolatérale des protéines ont été clairement identifiés, la situation en ce qui concerne la sélection apicale des protéines demeure mal comprise. Nous avons précédemment montré que contrairement aux GPI-APs basolatérales l’oligoisomérisation des protéines ancrées glycosylphosphatidylinositol (GPI-AP) dans l'appareil de Golgi est requise pour le tri apical. Il est intéressent de noter que ce mécanisme est conservé dans deux types de cellules épithéliales, les cellules MDCK et les cellules FRT, qui présentent des différences de tri des GPI-APs. Cependant, le mécanisme précis menant à cet événement n'est pas compris. Nos données précédentes ont démontré que le simple ajout de cholestérol aux cellules MDCK est nécessaire et suffisant pour induire l'oligomérisation et le tri apical d'une GPI-AP basolatérale. Alors que dans l’étude présente sur les cellules FRT, nous avons montré que contrairement aux cellules MDCK, le cholestérol ne joue pas un rôle majeur dans la régulation du tri apicale des GPI-APs. De plus, nous avons également montré que les GPI-APs apicales et basolatérales ne sont pas séparées dans le Golgi en fonction de leur teneur en cholestérol entourant l'environnement membranaire. Par ailleurs, nous avon démontré que la N-glycosylation des protéines de l’ectodomaine est indispensable à l’oligomérisation et au tri apical de GPI-APS. Nos données indiquent qu’il existe au moins deux mécanismes, l’un dépendant du taux de cholestérol et l’autre de la N-glycosylation, qui déterminent l’oligomérisation dans l'appareil de Golgi et le tri des GPI-APs vers la membrane apicale. / Epithelial cells represent the ability to polarize with an apical and basolateral domains which differ markedly in proteins, lipid composition and therefore in function. This asymmetry reflects the ability of epithelial cells to sort newly synthesized proteins and lipid to either cell surface. While the signals responsible for basolateral targeting of the proteins have been clearly understood, the situation regarding the apical sorting of proteins is more obscure. We have previously shown that differently from basolateral GPI-APs oligomerization in the Golgi apparatus is necessary for apical sorting of Glycosylphosphatidylinositol- anchored proteins (GPI-APs). Interestingly this mechanism is conserved in two different kinds of epithelial cells, MDCK and FRT cells, which exhibits a difference in the sorting of GPI-APs. However the precise mechanism leading to this event is not understood. Our previous data demonstrated that simple addition of cholesterol to MDCK cells is necessary and sufficient to induce the oligomerization and apical sorting of a basolateral GPI-AP. Whereas, in this present study in FRT cells we showed that in contrast with MDCK cells cholesterol is not an active player in the regulation of GPI- APs apical sorting. In addition, we also showed that apical and basolateral GPI-APs are not segregated in the Golgi on the bases of the cholesterol content of the surrounding membrane environment. Furthermore, we demonstrated that N- glycosylation of the protein ectodomain is critical for oligomerization and apical sorting of GPI-APs. Our data indicates that at least two mechanisms depending either on cholesterol or on N-glycosylation exist to determine oligomerization in the Golgi and sorting to the apical membrane of GPI-APs.

Un statut pauvre en Acides Gras Poly-Insaturés ω3 (AGPI ω3), favorisé par une alimentation occidentale comportant un faible ratio en ω3/ω6, semble contribuer au déclin cognitif chez les personnes âgées, mais les mécanismes cellulaires impactés sont encore mal connus. Nous avons donc étudié l’influence du statut en ω3 sur l’évolution de la neurotransmission glutamatergique et des fonctions astrocytaires au cours du vieillissement dans l’hippocampe de rats. Ces processus sont impliqués dans la formation de la mémoire et leurs dérégulations participent aux dommages cérébraux conduisant au déclin cognitif. Nous avons comparé 6 groupes de rats agés de 6 et 22 mois nourris avec un régime déficient en ω3, équilibré en ω3/ω6 ou supplémenté en ω3 (huile de poisson) : Jeunes équilibrés (JEq), déficients (JDef) ou supplémentés (JSup) et Agés équilibrés (AEq), déficients (ADef) ou supplémentés (ASup). Nous avons évalué l’efficacité synaptique et la plasticité (enregistrements électrophysiologiques), les fonctions astrocytaires (capture de glutamate et expression de la GFAP), les marqueurs neuronaux (transporteurs et récepteurs du glutamate), les capacités cognitives (Openfield et Labyrinthe de Barnes) et analysé la composition lipidique cérébrale. Les manipulations nutritionnelles d’apport en ω3 modifient efficacement l’incorporation de l’acide docosahexaénoïque (DHA, principale ω3 des membranes cellulaires) dans le cerveau (-50% deficient vs équilibré, +10% supplementé vs équilibré). Le vieillissement induit une diminution de 35% de l’efficacité synaptique en raison d’une baisse de la libération de glutamate pré-synatique, et une diminution de 30% de la capture de glutamate associé à une astrogliose conséquente (+100% GFAP). La déficience en ω3 acentue les effets du vieillissement (rats ADef vs AEq: -35% efficacité synaptique, -15% capture de glutamate, +30% GFAP). Al’inverse, la supplémentation en ω3 améliore l’efficacité synaptique (rats ASup vs AEq +25%) et semble inhiber l’astrogliose chez le rat âgé (ASup vs JEq : pas de modification de la GFAP). Les tests comportementaux montrent que le vieillissement a des effets plus marqués chez les déficients en ω3 et au contraire atténués chez les supplémentés. Nos résultats révèlent des altérations de la synapse glutamatergique de l’hippocampe au cours du vieillissement aggravées par la déficience en ω3 et atténuées par la supplémentation en ω3. Afin d’évaluer l’influence du statut en ω3 sur l’activation astrocytaire, des modèles in vitro d’astrocytes « âgés » et « activés » par des cytokines inflammatoires dont l’augmentation à bas bruit est caractéristique du vieillissement cérébral, ont été développés. / A poor ω3 polyunsaturated fatty acids (ω3 PUFA) status, favored by the low ω3/ω6 ratio in western diets, seems to contribute to cognitive decline in the elderly, but mechanistic evidence is lacking. We therefore explored the impact of ω3 status on the evolution of glutamatergic transmission and astrocytic functions in the hippocampus during ageing in rats. These processes are involved in memory formation and their dysregulation participates to the age-related brain damage leading to cognitive decline. We have compared 6 groups of rats aged 6 to 22 months fed ω3-deficient, ω3/ω6-balanced, or ω3 (fish oil) supplemented diets: Young ω3 Balanced (YB), Deficient (YD) or Supplemented (YS), and Old ω3 Balanced (OB), Deficient (OD) or Supplemented (OS) rats. We have evaluated synaptic efficacy and plasticity (electrophysiological recording), astroglial regulations (glutamate uptake and GFAP expression), neuronal markers (glutamate transporters and receptors), cognitive abilities (Barnes maze and Openfield) and analyzed brain fatty acids composition. Dietary modulation of ω3 intakes efficiently modified the incorporation of docosahexaenoic acid (DHA, the main ω3 in cell membranes) in brain (-50% deficient vs balanced, +10% supplemented vs balanced). Ageing induced a 35% reduction of synaptic efficacy due to decreased pre-synaptic glutamate release, and a 30% decrease in the astroglial glutamate uptake associated to a marked astrogliosis (+100% GFAP). ω3 deficiency further decreased these hallmarks of ageing (OD vs OB rats: -35% synaptic efficacy, -15% glutamate uptake, +30% GFAP). On the opposite, ω3 supplementation increased synaptic efficacy (+25% OS vs OD) and seems to abolish astrogliosis (OS vs YS : no change in GFAP). Behavioural tests showed some increased effects of age in deficient rats and attenuated effects in supplemented ones. Our results characterize some specific age-related alterations of the glutamatergic synapse in the hippocampus that are aggravated by a dietary deficit in ω3 and attenuated by ω3 supplementation. In order to explore ω3 status on astrocytic activation, in vitro models of “old” astrocytes and “activated” by inflammatory cytokines which characterize the low-grade inflammation in brain aging, have been developed.

Les glioblastomes multiformes (GBM) comptent parmi les tumeurs au pronostic le plus sombre. L’extraordinaire capacité invasive des cellules tumorales rend toutes les interventions thérapeutiques actuelles totalement impuissantes. Une sous-population de Cellules Souches de Glioblastome (CSG) hautement invasive est responsable de la récurrence tumorale. Dans le cerveau, les GBM migrent principalement le long des vaisseaux sanguins au sein de l’espace périvasculaire riche en laminine, fibronectine et collagène ainsi qu’en suivant l’alignement des fibres myélinisées du corps calleux. La Matrice Extracellulaire (MEC) de ces régions joue un rôle important dans l’invasion des GBM, mais les mécanismes mis en jeu n’ont pas été complètement dévoilés. De plus, le développement de nouvelles thérapies anti-migratrices ciblant l’interaction des GBM avec la MEC reste encore limité. Dans le but de mimer la composition biochimique et les propriétés mécaniques de la MEC cérébrale et d’étudier leur rôle(s) dans la migration des CSG, nous avons développé un nouveau support de nanofibres (NF) alignées et fonctionnalisées avec de la laminine. Mes travaux de thèse ont montré que les NF génèrent un microenvironnement tridimensionnel (3D) favorisant l’adhésion et la migration des CSG. Cette adhésion est améliorée en comparaison avec les supports planaires (SP) conventionnels (2D) et récapitule mieux les mécanismes d’interaction des CSG avec la MEC au cours de l’invasion dans le modèle murin de tumeurs xénogreffées. Dans ces conditions physiologiques plus convenables générées par les NF, la variation des composantes biochimiques et mécaniques de la MEC affecte la migration des CSG. La présence ou l’absence de laminine régule le mode migratoire et l’orientation de fibres contrôle la direction de migration des CSG. D’un autre coté, l’altération de la glycosylation des protéines de la surface cellulaire module l’interaction des cellules tumorales du cerveau avec la MEC et augmente leur invasion. La deuxième partie de mes travaux de thèse a permis de démontrer que les glycomimétiques phostines « 3.1a » réorganisent le processus de la N-glycosylation des CSG diminuant leur invasivité in vitro et in vivo en inhibant les voies de signalisation de la kinase FAK et du récepteur de TGF-β impliqués dans l’interconnexion cellule-MEC. / Glioblastoma Multiforme (GBM) is a biologically aggressive tumor with an extremely poor prognosis. The highly invasive capacity of a subpopulation of Glioblastoma Initiating Cells (GIC) makes complete surgical resection impossible. GBM dissemination occurs along preexisting brain structures such as the perivascular space rich in laminin, fibronectine and collagen as well as the aligned myelinated fibers of the corpus callosum. The Extracellular Matrix (ECM) of these cerebral regions plays an important role during GBM invasion, but the underlying mechanisms remain largely unknown. Accordingly, the development of new anti-migratory therapies targeting the cell-ECM interactions is lacking. In order to mimic the compositional and physical properties of the cerebral ECM and to investigate their role(s) in GBM invasion, we have set up a new aligned nanofibers (NF)scaffold functionalized with laminin. My work demonstrated that the NFs constitute a tridimensional (3D) microenvironment supporting GIC adhesion and migration. The cell-ECM adhesion is improved on the NF in comparison to the conventional 2D planar surfaces (PS). Furthermore, the mechanisms of GIC interaction with the ECM on the NF are similar to those observed in the human GBM xenograft murine model. In this physiologically more relevant 3D microenvironment reproduced by the NF, the variation of the different biochemical and mechanical components of the ECM affects the migration of GIC. The presence or absence of laminin on the NF regulates the mode of migration and the orientation of the fibers dictates the direction of migration of GIC. On the other hand, the glycosylation that decorates cell surface proteins modulates the interaction of GBM tumor cells with the ECM and its alteration increases their invasion. The second part of my thesis demonstrated that the glycomimetics phostines « 3.1a » remodel the N-glycosylation of GIC and decrease their invasivity in vitro and in vivo via the inhibition of FAK and TGFβ-R signaling pathways known to be implicated in the cell-ECM intercommunication.

This thesis contributes to debates concerning the role of the Muslim Brotherhood and the study of political systems in transition, with a focus on Egypt following the 25 January 2011 revolution. Orthodox studies on political systems within Political Science, including scholarship on large-scale transitions and (liberal) democratisation, often focus on political institution-building, regime elites, civil/political rights, and purely structuralist approaches as explaining political dynamics and change. This study argues that discourse, power relations, and forms of resistance indicate and shape political processes, including change and transition. The originality of this work thus derives from its focus on discourse and power dynamics, analysed within the narrative accounts of informal political actors collected during Egypt's transition. This thesis also argues that the pillars of (liberal) democratisation have made possible the analytical location of the Brotherhood within these debates. For example, political systems have been measured on a (sometimes linear) scale from authoritarian to liberal democracy and this has made possible the framing of Islamic political movements on a scale from 'radical' to 'moderate', or constructed binaries in which movements like the Brothers are categorised. Through this work's analytical and empirical approach, these categories are shown to be simplistic and fragile. Two central themes are developed in relation to discourse, power relations, and the Brotherhood: democratisation as discourse and political violence. Through this study's framework political change and transformation are studied beyond the level of the regime and political elite, as is often the focus within political systems/transitions literature. Conceptions of Egypt's political transition and of the Brothers are grounded in empirical research and the experiences of political actors on the ground.