Made available in DSpace on 2017-07-20T12:40:14Z (GMT). No. of bitstreams: 1 Bianca Sandrino.pdf: 3429333 bytes, checksum: e320b1605b6c2c81bf2ecd35d8aa88f5 (MD5) Previous issue date: 2014-09-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / One of the major challenges in drug design is to identify compounds with potential toxicity toward target cells, preferably with molecular-level understanding of their mode of action. In this study, the antitumor property of a ruthenium complex, mer-RuCl3(dppb)(VPy)] (dppb = 1,4-bis (diphenylphosphine) butane and VPy = 4-vinylpyridine),RuVPy) was analyzed. Results showed that this compound led to a mortality rate of 50% of human laryngeal carcinoma HEp-2 cell with 120 ±10 mol L-1, indicating its high toxicity. Toward a better understanding if its mode of action is associated with its interaction with cell membranes, Langmuir monolayers were used as a membrane model. RuVPy had a strong effect on the surface pressure isotherms, especially on the elastic properties of the zwitterionic dipalmitoylphosphatidylcholine (DPPC) and the negatively charged dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidylserine (DPPS) phospholipids. Results of thermodynamic parameters indicated miscibility between the components is not ideal mixed monolayers. Preferably attractive and repulsive interactions between RuVPy and zwitterionic or anionic phospholipids, respectively, are observed with mixed monolayer of DPPS/RuVPy energetically unfavorable. These data were confirmed polarization - modulated infrared reflection-absorption spectroscopy (PM-IRRAS). In addition, interactions between the positive group from RuVPy and the phosphate group from phospholipids were corroborated by density functional theory (DFT) calculations, allowing the determination of the Ru complex orientation at the air-water interface. Proof of interaction was confirmed by electrochemical results of Langmuir-Blodgett films of the phospholipid/RuVPy mixture. The presence of the RuVPy on the conductor substrate, which presents higher electron density, form "defects" in the monolayer of phospholipids increasing accumulation of electrons in the electrode/solution interface making it more permeable material. Although possible contributions from receptors or other cell components cannot be discarded, the results reported here represent evidence for significant effects on the cell membranes which are probably associated with the high toxicity of RuVPy. / Um dos grandes desafios na concepção de medicamentos é a identificação de compostos com potencial toxicidade para as células-alvo e a compreensão do seu modo de ação. Nesta tese, foi analisada a propriedade antitumoral do complexo de rutênio mer-[RuCl3(dppb)(VPy)] (dppb = 1,4-bis (difenilfosfina)butano e VPy = 4-vinilpiridina) (RuVPy), e os resultados mostraram que este composto levou a uma taxa de mortalidade de 50% de células de câncer de laringe (HEp-2) com 120 ± 10 μmol L-1, indicando sua alta toxicidade. Para a compreensão do modo de ação em nível molecular deste complexo, associada à sua interação com membranas celulares, monocamadas de Langmuir foram utilizadas como um modelo simples de membrana. O RuVPy apresentou um forte efeito sobre as isotermas de pressão de superfície, especialmente sobre as propriedades elásticas do zwitteriônico dipalmitoilfosfatidilcolina (DPPC) e dos fosfolipídios carregados negativamente dipalmitoilfosfatidilglicerol (DPPG) e dipalmitoilfosfatidilserina (DPPS). Resultados dos parâmetros termodinâmicos indicaram que há miscibilidade entre os componentes das monocamadas mistas não ideais. Interações preferencialmente atrativas e repulsivas foram constatadas entre o RuVPy e os fosfolipídios zwitteriônico e aniônicos, respectivamente, sendo a monocamada mista de DPPS/RuVPy energeticamente desfavorável. A interação entre o grupo de maior densidade eletrônica do RuVPy, obtido por cálculo de teoria funcional da densidade (DFT), e o grupo fosfato dos fosfolipídios foi confirmada por espectroscopia de infravermelho de reflexão e absorção de modulo polarizado (PM-IRRAS) realizada na interface ar-água. Prova desta interação foi constatada por resultados eletroquímicos dos filmes Langmuir-Blodgett da mistura fosfolipídio/RuVPy. A presença do complexo no substrato condutor, por ter maior densidade eletrônica, forma “defeitos” na monocamada dos fosfolipídios aumentando o acúmulo de elétrons na interface eletrodo/solução tornando o material mais permeável. Desta forma, é evidente que além de eventuais contribuições de outros receptores ou componentes celulares não poderem ser descartadas, os resultados aqui apresentados trazem os efeitos significativos nas membranas celulares que provavelmente estão associados à alta toxicidade do RuVPy.

Neste trabalho estão apresentadas novas estratégias para funcionalizar superfícies de ouro baseadas na sustentação de bicamadas lipídicas em superfícies de sensores de imagem por Ressonância de Plasmons de Superfície (SPRi) e a construção de um biosensor para detecção de citocromo c. SPRi é uma técnica ótica de gravimetria em tempo real. Por meio de medidas de variações de índice de refração (n) próximas a uma interface, a adsorção e desorção de moléculas podem ser mensuradas. Inicialmente testamos várias estratégias para encontrar um suporte adequado que se ligasse na superfície de ouro e que oferecesse sustentação e estabilidade para a bicamada de fosfolipídeo biotinilado. Estudos de FT-IR e MEV mostraram que a quitosana facilita a formação de uma bicamada íntegra de fosfolipídeos, de tal modo, que a mesma alcança valores de espessura próximos àqueles previstos, ~ 34,5 Å. Além disso, mostramos que esse sistema apresenta vantagens perante outros modelos, tais como, (poli-lisina/fosfolipídeos) e (tiol hidrofóbico/fosfolipídeo). Utilizando-se o complexo químico biotina/estreptoavidina conseguimos imobilizar o anticorpo anti cit c na bicamada, mantendo-o afastado da superfície de ouro. A construção do biosensor foi acompanhada com experimentos de SPRi. O limite de detecção de citocromo c atingido foi de 10-11mol/L. Um sensor construído somente com BSA e anticorpo anti cit c apresentou sensibilidade semelhante. Esta sensibilidade é em torno de três ordens de grandeza superior aos experimentos de imunoblotting usualmente utilizados para detecção de cit c. A principal limitação deste biosensor, tal como de outros imunoensaios, está intimamente ligada às vantagens e desvantagens dos anticorpos como ferramentas analíticas. / In this work we developed new strategies to functionalize gold surfaces based on the support of lipid bilayers on the surfaces of surface plasmon resonance imaging sensors (SPRi) and the construction of a biosensor for detection of cytochrome c. SPRi is an optical gravimetric real time technique. Through measurements of changes in refractive index (n) in close proximity to an interface, the adsorption and desorption of molecules can be measured. Initially we tested several strategies for finding a suitable medium that would adsorb on the gold surface and also support and stabilize a biotinylated phospholipid bilayer. Studies of FT-IR and SEM showed that chitosan induces the formation of an intact phospholipid bilayer, so that it reaches thickness values close to those expected, ~ 34.5 Å. Furthermore, we showed that this system has advantages in relation to other models, such as (poli-lisine/phospholipids) and (thiol hydrophobic / phospholipid). Using the chemical complex biotin/streptavidin anti cyt c antibody could be immobilized in the bilayer, keeping it away from the gold surface. The construction of the biosensor was accompanied with SPRi experiments. The limit of detection of cytochrome c was achieved from 10-11mol / L. A sensor built only with BSA and anti cyt c showed similar sensitivity. This sensitivity is about three orders of magnitude higher than the immunoblotting experiments commonly used for detection of cyt c. The main limitation of this biosensor, like in other immunoassays, is linked to the advantages and disadvantages of antibodies as analytical tools.

Muitas aplicações biológicas da quitosana dependem de sua interação com membranas celulares, cujo mecanismo não é conhecido em nível molecular. Nesta tese, empregam-se filmes de Langmuir dos fosfolipídios dipalmitoil fosfatidil colina (DPPC), dipalmitoil fosfatidil glicerol (DPPG) e ácido dimiristoil fosfatídico (DMPA) para mimetizar a membrana, e é avaliada a influência dos grupos hidroxila e amino de quitosana nas propriedades dos filmes. Para tanto, O-acilquitosanas foram produzidas por meio de reação de acilação, gerando os derivados 3,6 - O,O\'- dietanoilquitosana (DEQUI) e 3,6 - O,O\'- dipropanoilquitosana (DPPQUI) solúveis em solução aquosa ácida, e 3,6 - O,O\'- dimiristoilquitosana (DMQUI) e 3,6 - O,O\'- dipalmitoilquitosana (DPQUI), solúveis em clorofórmio. DEQUI e DPPQUI afetam mais fortemente as isotermas de pressão de superfície e elasticidade dos filmes do que quitosana, sendo os efeitos de DPPQUI (mais hidrofóbico) maiores do que para DEQUI. Isso indica que ligações hidrogênio envolvendo as hidroxilas da quitosana não são essenciais na interação. Espectros no infravermelho com modulação de polarização (PM-IRRAS) confirmaram interações hidrofóbicas, com penetração dos derivados entre as moléculas de fosfolipídio. DEQUI causa mais ordenamento das cadeias do fosfolipídio, enquanto o efeito de DPPQUI é oposto. DMQUI e DPQUI formam filmes de Langmuir altamente compactados com agregação de moléculas, inferida das isotermas de pressão e potencial de superfície. Os resultados sobre a influência dos grupos amino foram inconclusivos, pois o comportamento atrativo entre os materiais pode ser devido tanto à existência de grupos com cargas opostas, quanto interações hidrofóbicas. Quitosanas com diferentes massas moleculares (alta - QAMM e baixa - QBMM) foram utilizadas para obter informações sobre a orientação dos grupos químicos da quitosana e fosfolipídios e conformação do polímero em solução. Espectros PM-IRRAS indicam maior efeito de QBMM em monocamadas de DPPG, provocando diminuição na intensidade e deslocamento para maiores números de onda das bandas de CH, inversão na orientação do grupo P=O do DPPG e maior intensidade da banda amida II, sugerindo maior densidade desses grupos na interface. Os espectros de geração de soma de frequência (SFG) mostraram diminuição na ordenação/compactação das caudas de DPPG, aumento do espaçamento entre as moléculas e de defeitos gauche. Conclui-se que derivados O-acilados de quitosana têm maior efeito sobre modelos de membrana, principalmente devido às forças hidrofóbicas, sendo mais adequados em aplicações biológicas que dependam dessa interação. Também favorece a interação com a membrana a atração eletrostática, com efeitos mais relevantes para quitosanas de menores massas moleculares. / Many biological applications of chitosan depend on its interaction with cell membranes, whose mechanism at the molecular level is not known. In this thesis, Langmuir films from the phospholipids dipalmitoyl phosphatidyl choline (DPPC), dipalmitoyl phosphatidyl glycerol (DPPG) and dimyristoyl phosphatidic acid (DMPA) were used to mimic the cell membrane, and effects from the hydroxyl and amine groups in chitosan on the film properties were evaluated. For this, O-acylchitosans were produced by acylation reaction, resulting in the derivatives 3,6 - O,O\' - diacetylchitosan (DECT) and 3,6 - O,O\'- dipropionylchitosan (DPPCT), which are soluble in acidic aqueous solution, and 3,6 - O,O\'- dimyristoylchitosan (DMCT) and 3,6 - O,O\'- dipalmitoylchitosan (DPCT), soluble in chloroform. DECT and DPPCT affect the surface pressure and elasticity of the films more strongly than chitosan, especially DPPCT that is more hydrophobic. This indicates that hydrogen bonds involving the hydroxyl groups from chitosan are not essential for the interaction. Polarization-modulated infrared reflection absorption (PM-IRRAS) spectra confirmed hydrophobic interactions with penetration of derivatives between the phospholipid molecules. DECT induces ordering in the chains, while the opposite occurs for DPPCT. DMCT and DPCT form highly compressed films with aggregation, as shown by surface pressure and surface potential isotherms. The results on the importance of amino groups were inconclusive because the attractive behavior between materials may be due to either the oppositely charged groups or hydrophobic interactions. Chitosans with different molecular weights (high - CHMW and low - CLMW) were used to obtain information about the chitosan and phospholipids chemical groups orientation and polymer conformation in solution. PM-IRRAS spectra indicate greater effect from QBMM on DPPG monolayers, causing a decrease in intensity and shift to higher wavenumbers of the CH bands, inversion in the orientation of the P=O group from DPPG and greater intensity of the amide II band, suggesting greater density of these groups at the interface. The sum-frequency generation (SFG) spectra showed a decrease in ordering/packing of the DPPG chains, increased spacing between molecules and gauche defects. Overall, the O-acyl derivatives of chitosan have greater effect on cell membrane models, owing to hydrophobic forces, being therefore more suitable for biological applications that depend on this interaction. Also important for the interaction is the electrostatic attraction, with more relevant effects observed with low-molecular weight chitosans.

Concentração de apolipoproteína A-1 (1.5±0.3; 1.5±0.2g/l). O tamanho da HDL também foi igual entre os dois grupos (8.8±0.8; 9.0±0.5 nm, respectivamente). A menopausa também não afetou a transferência de lípides da LDE para a HDL (em % total de radioatividade/10mg HDL/h), CE (0.5±0.3; 0.5±0.2, respectivamente), CL (0.9±0.2; 0.9±0.2), TG (0.6±0.2;0.6±0.2) e PL (3.0±0.7; 3.3±1.0). Conclusão: A menopausa não Introdução: A concentração plasmática da HDL é um fator de risco importante e independente para a prevenção da doença aterosclerótica, principalmente na mulher. Seu metabolismo e suas características estruturais e funcionais também têm sido estudados como fatores de risco. Neste estudo, foram comparadas mulheres de mesma faixa etária na pré e na pós-menopausa, para determinar a influência da menopausa sobre o tamanho da HDL e sobre a habilidade desta lipoproteína em receber lipídios de lipoproteínas doadoras, um processo que depende de proteínas de transferência e da composição e estrutura da HDL. Métodos: Vinte e duas mulheres saudáveis, normolipidêmicas na pré e dezoito na pós-menopausa, de idades entre 40-50 anos foram estudadas. Os grupos não diferiam em IMC, glicemia, colesterol total, LDL, triglicérides, apo A1 e apo B. Uma nanoemulsão artificial foi usada como modelo de LDL (LDE) para doar lípides para a HDL. LDE marcada radioativamente com 3 H-triglicérides (TG) e 14 C-colesterol livre (CL) ou 3 H- ésteres colesterol (CE) e 14 C-fosfolipídios (PL) foram incubados com as amostras de plasma por 1 hora. Após a precipitação química do sobrenadante contendo HDL, foi contada a radioatividade. O tamanho da HDL foi medido por espalhamento da luz laser. Resultados: A concentração da HDL nos dois grupos não diferiu, demonstrada pela concentração de HDL colesterol (61±12; 61±14 mg/dl respectivamente) e influenciou o tamanho das partículas HDL e um importante parâmetro funcional que é a habilidade da HDL de receber lipídios. / Objective: HDL levels are important for atherosclerosis prevention especially in the female gender, but functional aspects of the lipoprotein are also important. In this study, post-menopausal were compared to pre-menopausal women in the same age range to determine the influence of menopause upon the HDL size and ability of the lipoprotein to receive lipids from donor lipoproteins, a process that depends on transfer proteins and on HDL composition and structure. Methods: Twenty-two pre and eighteen postmenopausal, healthy and normolipidemic women, aged 40-50 yr. Both groups did not differ in BMI and plasma glucose, total and HDL cholesterol, triglycerides and apo B concentration. An artificial nanoemulsion (LDE) was used as a model of LDL to donate lipids to HDL. LDE labeled with 3 H-triglicerides (TG) and 14 C-free cholesterol (FC) or 3 H-cholesteryl esters (CE) and 14 C-phospholipids (PL) incubated with plasma samples for 1h. After chemical precipitation, the supernatant containing HDL was counted for radioactivity. HDL size was measured by laser-light-scattering. Results: HDL concentration of pre and post menopausal women did not differ as estimated by HDL cholesterol (61±12; 61±14 mg/dl respectively) and apo A1 concentration (1.5±0.3; 1.5±0.2g/l). HDL size also did not differ (8.8±0.8; 9.0±0.5 nm, respectively). Menopause also did not affect the transfer of lipids from LDE to HDL (in % of total radioactivity/10mg HDL/h), namely CE (0.5±0.3; 0.5±0.2, respectively), FC (0.9±0.2; 0.9±0.2), TG (0.6±0.2;0.6±0.2) and PL (3.0±0.7; 3.3±1.0). Conclusion: The menopause does not affect the size and an important functional parameter that is the ability of HDL to receive lipids.

Made available in DSpace on 2015-04-22T22:01:58Z (GMT). No. of bitstreams: 1 Banny Silva Barbosa.pdf: 3257736 bytes, checksum: aa77c1eca753a92a01d30a721f2da598 (MD5) Previous issue date: 2013-01-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Seeing the importance of fish for amazonian people, it potential in the market, it nutritive value, and information shortages relative to the lipid composition of amazonian fish, it was aimed to determinate the lipid composition attendant in dorsal muscle of six amazonia fish species, jaraqui, curimatã, pacu, sardinha, pescada and surubim, through fatty acids analyses constituent of their diferent classes of lipids and attendant steroids in their unsaponifiable lipids. This study involved the development and methodology application for extraction of total lipids, separation of lipid class, extraction of unsaponifiable lipids, derivatization of fatty acids and steroids, analyses of gas chromotagraphy with detector of flames ionization and with mass spectrometry for quantitative and qualitative evaluation of steroids and fatty acids of lipids in class. And also approached the determination of lipids nutritional quality through atherogenicity, thrombogenicity index and quantity of hypercholesterolemic fatty acids. The results indicated the fish in study have interesting lipids, having bigger quantity of total lipids the fishes curimatã and pacu, with bigger participation of neutral lipids, and cholesterol as majority steroid for all fishes. The unsaturated fatty acids, essentials for human health, it was found in bigger quantity in fishes sardinha and pacu in phospholipids class and in pescada (whitefish), curimatã, jaraqui and surubim in neutral lipids class. Besides pacu fish has shown bigger quantity of omega 3 and 6 fatty acids, the fish pescada (whitefish) highlighted for nutritional quality. / Considerando a importância do pescado para o povo amazônico, o seu potencial no mercado, a sua valorização nutritiva, e à escassez de informações referentes à composição lipídica dos peixes amazônicos objetivou-se determinar a composição lipídica presente no músculo dorsal de seis espécies de peixes amazônicos, jaraqui, curimatã, pacu, sardinha, pescada e surubim, através das análises dos ácidos graxos constituintes de suas diferentes classes de lipídeos e dos esteróides presentes em seus lipídeos insaponificáveis. O estudo envolveu o desenvolvimento e a aplicação de metodologias para extração dos lipídeos totais, separação de classes de lipídeos, extração de lipídeos insaponificáveis, derivatização de ácidos graxos e esteróides, análises por cromatografia gasosa com detector de ionização de chamas e com espectrometria de massas para avaliação quanti e qualitativa de esteróides e de ácidos graxos dos lipídeos em classes. E ainda abordou a determinação da qualidade nutricional dos lipídeos através dos índices de aterogenecidade, de trombogenecidade e pela quantidade de ácidos graxos hipocolesterolêmicos. Os resultados indicaram que os peixes em estudo contêm lipídeos interessantes, possuindo maior quantidade de lipídeos totais os peixes curimatã e pacu, com maior participação dos lipídeos neutros, e colesterol como esteróide marjoritário para todos os peixes. Os ácidos graxos insaturados, essenciais para a saúde humana, foi encontrado em maior quantidade nos peixes sardinha e pacu na classe dos fosfolipídeos e nos peixes peixes curimatã, pescada, jaraqui e surubim na classe dos lipídeos neutros. Apesar de o peixe pacu ter mostrado maior quantidade de ácidos graxos de ômega 3 e 6 o peixe pescada se destacou pela qualidade nutricional.

Apoptosis plays a crucial role in multicellular organisms by preserving tissue homeostasis and removing harmful cells. The anti-apoptotic B-cell CLL/lymphoma 2 (Bcl-2) and the pro-apoptotic Bcl-2-associated X protein (Bax) act as major regulators of the mitochondrial apoptotic pathway. Activation of Bax via stress signals causes its translocation to the mitochondrial outer membrane (MOM). There, Bax forms homo-oligomeric pores, leading to the release of apoptogenic factors, caspase activation and ultimately cell death. However, the underlying mechanism for the recruitment and pore forming activity of Bax is still not elucidated. Nevertheless, the mitochondrial membrane system seems to play an active and crucial role, presumably being directly involved in the onset of the mitochondrial apoptosis. Since the formation of reactive oxygen species (ROS) is a common stress signal and one of the hallmarks of the mitochondrial apoptosis, direct damage can occur to these membranes by the generation of oxidized phospholipids (OxPls), whose presence can crucially influence the pro-apoptotic action of Bax there. To better understand the impact of OxPls on membranes as well as their potential role in the mitochondrial apoptotic process, defined OxPl species were incorporated into phospholipid vesicles and studied with various biophysical techniques. Differential scanning calorimetry (DSC) and solid state nuclear magnetic resonance (NMR) spectroscopy were used to gain insight into changes in membrane properties in the presence of OxPls. In addition to circular dichroism (CD) spectroscopy, DSC and solid state NMR were furthermore performed to elucidate the impact of OxPls on Bax-membrane interactions. The occurrence of OxPls gave rise to dramatic changes in membrane organization and dynamics, manifested as lateral phase separation into OxPl-rich and -poor domains and modified hydration at the membrane interface. The presence of OxPls also had a great impact on the interaction between Bax and mitochondria-mimicking vesicles, strongly promoting the association of the protein with the membrane. At the MOM, Bax is believed to be inhibited by Bcl-2. How this inhibition occurs is still a mystery due to the lack of biophysical information on Bcl-2, in particular on the full-length protein variant. Since Bcl-2 is also one of the main culprits in the progression of various forms of cancer, knowledge of the structural and mechanistic properties of the full-length protein is essential for a fundamental understanding of its function at a molecular level. To this end, a method for the production of full-length Bcl-2 was developed. By performing cell-free protein synthesis, preparative amounts of the protein were obtained, which enabled a biophysical characterization of the putative interaction between Bax and Bcl-2 using CD and fluorescence spectroscopy. A protocol for the reconstitution of Bcl-2 into proteoliposomes was also developed, promising for future studies of the full-length protein in its native membrane environment; a prerequisite to fully understand its pro-survival functions as well as providing crucial information for the design of novel anti-cancer drugs.

El objetivo global de esta tesis consiste en mejorar el control de las fermentaciones a bajas temperaturas a partir de los cambios en el metabolismo lipídico. En el primer capítulo, observamos que el almacenamiento en condiciones inadecuadas de una levadura seca activa tenía como consecuencias una menor vitalidad y contenido lipídico. Estos efectos desaparecían tras una recuperación en medio de cultivo óptimo. En el segundo capítulo, la cepa híbrida S. cerevisiae/S. bayanus presentó la mejor cinética fermentativa sin necesidad de aclimatación. Sin embargo, el resto de especies analizadas mostraron una modificación en la composición lipídica y una mejora de su actividad fermentativa a 13 ºC al hacer coincidir la temperatura de fermentación con la de precultivo. En el tercer trabajo, nos centramos en la nutrición lipídica de la levadura consiguiendo una reducción del tiempo de fermentación a bajas temperaturas mediante la adición de ácido palmitoleico en el medio de precultivo. Finalmente, en el último capítulo se analizó el efecto de la supresión de genes del metabolismo de los fosfolípidos sobre la vitalidad a bajas temperaturas y la composición en fosfolípidos. / The global aim of this thesis consists in improving the control of low temperature fermentations by considering the changes in lipid metabolism. In the first chapter, we reported that poor ADWY storage conditions resulted in an impairment of the vitality and a decrease in the lipid content. These effects disappeared after a recovery in optimal medium. In the second chapter, we analysed the strain and specific-response to fermentation temperature, showing that the hybrid S. cerevisiae/S. bayanus presented the highest sugar consumption whatever the preculture temperature used. The rest of the species needed a preadaptation at low temperature involving a change in their lipid composition to improve their fermentation rate at 13 ºC. In the third chapter, we focused on the lipid nutrition of yeast and we saw that palmitoleic acid supplementation reduced significantly the fermentation length at low temperature. In the last chapter various phospholipid mutants were tested to ascertain whether the suppression of some genes could modify the vitality at low temperature and the phospholipid composition.

This study investigated the critical behaviour of ternary mixtures of DOPC and DPPC, with cholesterol. The properties of model membranes such as these are studied in order to provide insight into aspects of complex biological systems. Experiments were performed using the Jeener echo, a static solid-state NMR technique, however no information about the critical phenomena was obtained. Conversely, the sideband linewidths measured from 2H MAS NMR are sensitive to temperature and dependent upon the phase behaviour. By fitting the linewidth data to an equation from Suwelack et al. (J. Chem. Phys., 1980; 73(6):2559-2569), the critical temperature and the critical exponent for the correlation length of the system were calculated. The critical exponent values obtained from these samples ranged between νc = 0.65 and νc = 1.2, which encompasses the critical exponents for both the 2D and 3D Ising models within error. The universality class for these model membranes cannot be unambiguously assigned yet.

No description available.

Les protéines kinases sont responsables de la phosphorylation des protéines, une modification post-traductionnelle qui contrôle ainsi de manière très efficace et presque instantanée une multitude de processus cellulaires. Elles sont impliquées dans la régulation des voies métaboliques dans les organismes permettant leurs adaptations aux changements environnementaux. Chez les procaryotes, l'existence de sérine/thréonine et tyrosine kinases est resté pendant longtemps un sujet de controverse. Cependant, au cours des deux dernières décennies, de nombreuses études ont montré que ces types de phosphorylation (Ser/Thr/Tyr) existent chez les bactéries et sont impliqués dans la régulation d'une grande variété de processus biologiques. En plus de protéines kinases apparentés à des enzymes eucaryotes, les bactéries ont également des protéines kinases spécifiques n’ayant aucune ressemblance structurale avec des protéines eucaryotes ce qui, lorsqu’elles sont essentielles, les qualifient en tant que cible pour de nouveaux agents antimicrobiens. YdiB est une nouvelle protéine kinase bactérienne de Bacillus subtilis. La localisation d’YdiB a été étudiée par immunofluorescence révélant sa présence à proximité de la membrane plasmique. L'interaction d’YdiB avec les phospholipides a été étudiée par des essais de flottation et résonance plasmonique de surface. Ceci révèle la présence d'une interaction stable et forte entre la kinase et deux phospholipides: la phosphatidylsérine et la phosphatidyléthanolamine. En outre, la constante d'affinité (KD) entre YdiB et la phosphatidylsérine a été identifiée et est de l’ordre de 13.3x10-9 M. De plus, ces deux phospholipides, ainsi que leur têtes polaires, sont capables d'induire de manière significative l'autophosphorylation d’YdiB.Par ailleurs, le rôle d’YdiB dans la croissance bactérienne en particulier lors d’un stress oxydant a également été étudié dans la souche sauvage et dans le mutant ∆ydiB. Dans des conditions de stress oxydant, la souche ∆ydiB est incapable de survivre alors qu’une complémentation conditionnelle de ce mutant a permis de restaurer un profil de croissance normale de la bactérie. Cela révèle le rôle important joué par YdiB dans la résistance bactérienne au stress oxydant. En outre, des protéines impliquées dans la résistance au stress oxydant comme la superoxide dismutase F (SodF) et superoxide dismutase A (SodA) ont été testés comme substrats potentiels pour YdiB. Par des tests de trans-phosphorylation, nous avons pu montrer que SodA pourrait être un substrat potentiel pour YdiB expliquant ainsi son implication dans la résistance au stress oxydatif dans Bacillus subtilis. / Protein kinases are responsible of protein phosphorylation, a post-translational modification and a very effective and almost instantaneous control of a multitude of cellular processes. They are involved in the regulation of metabolic pathways in living organisms enabling their adaptation to environmental changes. In prokaryotes, the existence of serine/threonine and tyrosine kinases was, for a long time, a subject of controversy. However, over the past two decades, many studies have shown that these types of phosphorylation (Ser/Thr/Tyr) do exist in bacteria and are involved in the regulation of a wide variety of biological processes. In addition to protein kinases related to eukaryotic enzymes, bacteria also have some specific protein kinases having no structural resemblance to their eukaryotic homologues which, if essential, made them a possible target for new antimicrobial agents. YdiB is a novel bacterial protein kinase from Bacillus subtilis. In situ localisation of YdiB has been studied by immunofluorescence which reveals its presence near the plasma membrane. The interaction of YdiB with the membrane phospholipids was assessed by flotation assay and surface plasmon resonance demonstrating the presence of a stable and strong interaction between the kinase and two phospholipids: phosphatidylserine and phosphatidylethanolamine. In addition, the affinity constant (KD) between YdiB and phosphatidylserine was found to be equal to 13.3 x 10-9 M. Moreover, these two phospholipids or just their charged headgroups were able to significantly induce the autophosphorylation of YdiB.On the other hand, the role of YdiB in the bacterial growth especially under oxidative stress was also studied in the wild-type strain and in ∆ydiB mutant. Under oxidative stress conditions, the deleted strain was unable to survive whereas a conditional complementation of ∆ydiB mutant was able to restore a normal growth profile for the bacterium. This reveals the important role played by YdiB in the bacterial resistance to oxidative stress. In addition, protein involved in the resistance to oxidative stress like Superoxide dismutase F (SodF) and superoxide dismutase A (Sod A) were tested as potential substrates for YdiB. Doing the trans-phosphorylation assay, we were able to prove that Sod B could be a potential substrate for YdiB thus explaining its involvement in the resistance to oxidative stress in Bacillus subtilis.