The large conductance Ca++-activated K+ channel (BKCa) regulates neuronal excitability through the efflux of K+, in response to membrane depolarization and increases in intracellular Ca++. The activity of the BKCa channel is increased by acute exposure to ethanol (EtOH), which is thought to underlie, in part, the influence of the drug on peptide hormone release from neurohypophysial nerve terminals (Dopico et al., 1996, 1998). Moreover, chronic EtOH exposure attenuates acute drug action on hormone release, and reduces the sensitivity of BKCa channels to acute EtOH exposure (Knott et al., 2002). The factors regulating EtOH action on BKCa channels are not well understood. Several lines of evidence suggest, however, that the lipid composition of the plasma membrane may influence channel sensitivity to the drug. The plasma membrane is highly complex in its organization (Welti and Glaser, 1994; Brown and London, 1998). There is a growing body of literature indicating that the local lipid composition of the membrane can influence the function of ion channels, including BKCa (Chang et al., 1995a, b; Moczydlowski et al., 1985; Park et al., 2003; Turnheim et al., 1999). Interestingly, chronic exposure to EtOH in animal models results in alterations in the composition of synaptic plasma membranes, including changes in the amount and distribution of membrane cholesterol (CHS) (Chin et al., 1978; Chin et al., 1979; Wood et al., 1989). The significance of these alterations is unclear. Here, we set out to determine the ability of membrane lipids to modulate BKCa channel activity and EtOH sensitivity. To address this, we implement the planar lipid bilayer technique, allowing control of both the protein and lipid components of the membrane. Native BKCa channels retain EtOH sensitivity in this reductionist preparation (Chu et al., 1998), and we extend the study here to examine cloned human brain (hslo) BKCachannels. We show here that hslo channels maintain their characteristic large conductance, voltage and Ca++-dependent gating, and sensitivity to 50 mM EtOH in bilayers cast from a 3:1 mixture of 1-pamiltoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-pamiltoyl-2-oleoyl-phosphatidylserine (POPS). The addition of CHS to the bilayer decreases both the basal activity and EtOH sensitivity of the channels, in a concentration-dependent manner. This lends support to the notion that alterations in plasma membrane CHS levels following chronic EtOH exposure may reflect adaptations to the acute actions of the drug on ion channels. Furthermore, the EtOH sensitivity and CHS modulation of these reconstituted hslo channels are greatly reduced in the absence of negatively charged POPS in the bilayer (pure POPE). Based on these findings, we look to gain mechanistic insight into the lipid headgroup and acyl chain properties that may regulate BKCa channel modulation by EtOH and CHS. When POPS is replaced with the uncharged lipid 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), the hslo response to EtOH and CHS is restored, suggesting that the loss of negative surface charge or PS headgroup structure itself cannot explain the lack of channel modulation by these agents in POPE bilayers. Moreover, increases in the proportion of unsaturated acyl chains in the bilayer cannot significantly influence the hslo response to EtOH. The loss of EtOH sensitivity in pure POPE and CHS-containing bilayers may, therefore, reflect the propensity of POPE and CHS to form nonlamellar (nonbilayer) structures. Regarding the basal activity of the channel, we demonstrate that decreases in negative surface charge, increases in the proportion of unsaturated acyl chains, and increases in the complexity of head group interactions can all influence the steady-state activity of reconstituted hslochannels, relative to control POPE/POPS (3:1) bilayers. Overall, these data further suggest the ability of the local lipid environment to regulate the basal function and EtOH sensitivity of an ion channel protein. Parts of this dissertation have appeared in separate publications: Treistman, S.N., O'Connell, R.J., and Crowley, J.J. (2002). Artificial Bilayer Techniques in Ion Channel Study. In Methods in Alcohol-Related Neuroscience Research, D. Lovinger and Y. Liu, eds. (Boca Raton, Florida: CRC Press) Crowley, J.J., Treistman, S.N., and Dopico, A.M. (2003). Cholesterol antagonizes ethanol potentiation of human BKCA channels in binary phospholipid bilayers. Mol. Pharma. 64(2):364-372.

INTRODUÇÃO: As malformações do desenvolvimento cortical (MDC) resultam de distúrbios no dinâmico processo de corticogênese cerebral e são importante causa de epilepsia grave, atraso do desenvolvimento, déficits motores e cognitivos. O papel do metabolismo na epilepsia humana tem sido extensamente debatido, e há inúmeras evidências que apontam para disfunções bioenergéticas como fatores-chave na ictogênese. Distúrbios metabólicos foram identificados nas malformações corticais com outras modalidades de neuroimagem, tais como a espectroscopia de prótons por ressonância magnética. Para o nosso conhecimento, entretanto, o metabolismo de fósforo em pacientes com epilepsia secundária a MDC não foi extensamente investigado até o momento. OBJETIVOS: O objetivo deste estudo foi avaliar o metabolismo de fosfolipídios in vivo em uma série de pacientes com epilepsia e MDC. MÉTODO: Trinta e sete pacientes com MDC e 31 voluntários foram estudados usando espectroscopia de fósforo por ressonância magnética (31P-ERM) tridimensional em aparelho de 3,0 Tesla. Os voxels nas lesões foram comparados ao córtex frontoparietal dos controles (volumes efetivos de 12,5 cm3). O parênquima aparentemente normal foi avaliado em voxels homólogos de pacientes e controles, abrangendo cinco regiões cerebrais: regiões nucleocapsulares direita e esquerda, córtex frontoparietal parassagital, e centros semiovais direito e esquerdo. Foram utilizados métodos de quantificação para ajustar os dados no domínio do tempo para as seguintes ressonâncias: fosfoetanolamina (PE), fosfocolina (PC), glicerofosfoetanolamina (GPE), glicerofosfocolina (GPC), fosfato inorgânico (Pi), fosfocreatina (PCr), e a-, b- e g-adenosina trifosfato (ATP). Também foram calculados o ATP total (ATPt=a-+b-+g-ATP), fosfodiésteres (PDE=GPC+GPE), fosfomonoésteres (PME=PE+PC), e as razões PME/PDE, PCr/ATPt, e PCr/Pi. O magnésio (Mg2+) e os níveis de pH foram calculados com base nos desvios químicos da PCr, Pi, e -ATP. RESULTADOS: Comparativamente aos controles, e assumindo um valor de p < 0,05 estatisticamente significativo, as lesões apresentaram redução dos valores de pH e aumento de Mg2+. Também foram encontrados redução significativa de GPC e PDE, e aumento da relação PME/PDE nas MDC. O parênquima aparentemente normal também demonstrou redução dos valores de pH no córtex frontoparietal e no centro semioval bilateral. As diferenças nos valores de pH, tanto nas lesões como no parênquima aparentemente normal, permaneceram estatisticamente significativas nos subgrupos individuais de MDC (displasia cortical ou hemimegalencefalia; heterotopia; polimicrogiria e/ou esquizencefalia). Não houve correlação entre o tempo da última convulsão e as alterações do pH. CONCLUSÕES: O Mg2+ e o pH são parâmetros muito importantes na regulação bioenergética e estão envolvidos em múltiplas vias da atividade elétrica cerebral. Nossos dados corroboram a ideia de que distúrbios metabólicos ocorrem nas lesões focais de MDC, com propagação para áreas remotas aparentemente normais. As anormalidades de GPC, PDE, e da razão PME/PDE sugerem que há deficiências na renovação das membranas celulares nas lesões dos pacientes com epilepsia e MDC. / INTRODUCTION: Malformations of cortical development (MCD) result from disruptions in the dynamic process of cerebral corticogenesis and are important causes of severe epilepsy, neurodevelopmental delay, motor deficits and cognitive impairment. Metabolism in human epilepsy has been intensely debated, and there are several evidences pointing to brain bioenergetic disturbances as key factors in ictogenesis. Metabolic impairments in cortical malformations have been identified with other neuroimaging tools, such as proton magnetic resonance spectroscopy. To our knowledge, however, phosphorus metabolism in epilepsy caused by MCD has not been thoroughly investigated hitherto. OBJECTIVES: The aim of this study was to evaluate phospholipids metabolism in vivo in a series of patients with epilepsy and MCD. METHODS: Thirty-seven patients with MCD and 31 control subjects were studied using three-dimensional phosphorus magnetic resonance spectroscopy (31P-MRS) at a 3.0 T scanner. The voxels in the lesions were compared to the frontoparietal cortex of the control subjects (the effective volumes were 12.5 cm3). Normal appearing parenchyma was evaluated in homologous voxels of patients and controls encompassing five cerebral regions: right and left nucleocapsular regions, midline frontoparietal cortex and right and left semioval centers. Quantification methods were applied to fit the time-domain data to the following resonances: phosphoethanolamine (PE), phosphocholine (PC), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC), inorganic phosphate (Pi), phosphocreatine (PCr), and a-, b-, and g-adenosine triphosphate (ATP). We also estimated the total ATP (ATPt=a-+b-+g-ATP), phosphodiesters (PDE=GPC+ GPE), phosphomonoesters (PME=PE+PC), and the PME/PDE, PCr/ATPt, and PCr/Pi ratios. The magnesium (Mg2+) levels and pH were calculated based on PCr, Pi, and -ATP chemical shifts. RESULTS: Compared to controls and assuming that a p-value < 0.05 indicates significance, the MCD lesions exhibited lower pH values and higher Mg2+ levels. The lesions also presented significant reduction of GPC and PDE, and an increased PME/PDE ratio. The otherwise normal appearing parenchyma also demonstrated lower pH values in the frontoparietal cortex and bilateral centrum semiovale. The differences in pH values, both in the lesions and in the normal appearing parenchyma, remained statistically significant in individual subgroups of MCD (hemimegalencephaly or cortical dysplasia; heterotopia; polymicrogyria and/or schizencephaly). There was no correlation between the time of the last seizure and the pH abnormalities. CONCLUSIONS: Mg2+ and pH are very important in the regulation of bioenergetics and are involved in many electrical activity pathways in the brain. Our data support the idea that metabolic impairments occur in the lesions of MCD, with propagation to remote normal appearing parenchyma. The GPC, PDE, and PME/PDE abnormalities suggest that there are membrane turnover disturbances in MCD lesions.

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.

La demande en phospholipides ne cessent d'augmenter pour les applications alimentaires, cosmétiques et pharmaceutiques. A l'heure actuelle, les principales sources de phospholipides utilisées proviennent du soja et du jaune d’œuf. Elles sont obtenues par des procédés d'extraction utilisant des solvants organiques. Dans le contexte de la diversification des sources de phospholipides, d'une part, et le développement de techniques d'extraction plus respectueuses de l'environnement, d'autre part, l'ensemble des travaux réalisés a permis : 1) de valoriser les parties non comestibles de la coquille Saint Jacques comme une source de phospholipides, 2) d’identifier les étapes et les paramètres du procédé d'extraction par voie supercritique à l’aide de CO2 (pression, température, pourcentage de co-solvant, nature du co-solvant, type et dimension de réacteurs) sur les rendements d’extraction des phospholipides et la pureté des extraits obtenus, 3) d’étudier le développement d’un procédé supercritique continu couplant l’extraction de phospholipides d’une part et la formation de liposomes, d’autre part. / The demand in phospholipids is increasing because of their use in defferent domains, i.e. pharmaceutics, food industry, and cosmetics. Nowadays, the main sources of phospholipids come from soya and egg yolk classically extracted using organic solvents. The present work was undertaken in order to add value to waste products of fishery and to extract the lipids using a green technology. We developed an alternative green technique for lipid extraction based on the use of GRAS solvents as CO2 and ethanol. The extractions were carried out by flowing supercritical CO2. Varying the operating conditions (pressure, temperature, proportion and nature of the co-solvent, type of reactors) allowed obtaining extracts with different purities and contents in phospholipids. Finally, we explored the possibility of producing liposomes by coupling the phospholipid extraction using supercritical fluids and the vesicle formation in a continuous process.

Dans cette thèse de doctorat, la structure de membranes naturelles et modèles et leurs interactions avec des molécules biologiquement actives ont été étudiées au moyen de la réflectométrie de neutrons. Les lipides naturels ont été extraits de la levure Pichia pastoris, poussée en milieux deutéré et hydrogéné. L’analyse a montré que la quantité relative de phospholipides n’est pas affectée par le changement en composition isotopique du milieu de croissance. Cependant, les cellules de levures deutérées contiennent principalement des acides gras C18 :1 alors que le degré d’insaturation est plus élevé chez les levures hydrogénés. Diminuer la température du milieu de croissance permet d’augmenter le degré d’insaturation des acides gras chez les levures deutérées. Une analyse qualitative des sphingolipides a été réalisée et un protocole pour séparer les fractions phosphocholines et phosphoethanolamine a été établi.<p><p>La structure de bicouches composées des lipides de levures a été étudiée par réflectivité de neutrons. La bicouche composée de lipides deutérés polaires a une épaisseur similaire aux bicouches faites de phosphocholines C18:1 synthétiques. En présence de stérols, la rugosité aux interfaces entre les têtes polaires et les chaînes augmente. La bicouche composée de lipides polaires hydrogénés est plus mince que celle deutérée. Ceci est dû à la composition en acides gras beaucoup plus variée et du plus grand nombre d’insaturations. En présence de stérols, l’épaisseur de la bicouche hydrogénée augmente. <p>L’interaction de ces bicouches avec l’amphotéricine B (AmB) a été étudiée. L’AmB est un antifongique qui interagit fortement avec les membranes contenant de l’ergostérol et moins fortement avec des membranes contenant du cholestérol. Dans tous les cas, les molécules d’AmB forment une couche épaisse et diluée au dessus de la bicouche lipidique. En présence de stérols, les molécules d’AmB pénètrent dans la bicouche et change sa structure selon la composition en acide gras.<p><p>La structure de bicouches lipidiques de plante et leurs interactions avec des intermédiaires de synthèse ont aussi été étudiées par réflectivité de neutrons. Des mélanges ternaires de plantes étaient déposés sur silicium et des mélanges quaternaires sur saphir. L’épaisseur de la bicouche composée de mélange ternaire est de 38 Å, tandis que celle du mélange ternaire est de 28 Å, la différence venant probablement d’un effet de substrat. La présence de diacylglycérol (DAG) a comme conséquence d’augmenter l’aire par lipide, et ainsi de changer la conformation des têtes polaires. L’interaction des bicouches de lipide de plante avec l’acide phosphatidique (PA) dans le but d’observer un flip-flop possible a aussi été étudiée mais le PA a tendance à désorbé les bicouches du substrat et aucun mécanisme de flip flop n’a été détecté.<p><p>Finalement, la localisation d’une petite molécule, le resvératrol, dans des bicouches modèles a été étudiée. Le resvératrol est connu pour être responsable du « paradoxe français » qui est une corrélation inverse entre la consommation d’aliment gras et un faible taux de maladie cardiaque. Quand le resvératrol est adsorbé à partir de la phase liquide, il induit une réorganisation des têtes polaires. Quand il est déposé sur le substrat en présence des lipides, il est présent à l’interface entre les têtes polaires et les chaines.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Abstract Heavy alcohol consumption places a substantial burden on health all over the world. Metabolites of alcohol evoke alterations that lead to tissue damage in many organs. Phosphatidylethanol (PEth) is a unique phospholipid formed in the cellular membranes during the metabolism of ethanol after alcohol consumption. PEth has attracted special attention as it is postulated to be a reliable marker of long term heavy alcohol consumption. The aims of present study were to investigate the immunogenicity of phosphatidylethanol in mice and to analyze the plasma antibodies binding to phosphatidylethanol in humans. In this study a clear immune response was generated in mice immunized with PEth in human low density lipoprotein (LDL) carrier. Mouse monoclonal IgM antibodies binding specifically to phosphoethyl head group of PEth were generated using hybridoma technology. Since PEth was shown to be immunogenic in mice, plasma was analyzed for the presence of antibodies also in humans. PEth-specific antibodies of IgG, IgA and IgM isotypes in plasma were detected in heavy drinkers of alcohol with or without pancreatitis as well as in the controls. The plasma levels of the antibodies binding to PEth were significantly lower in the study subjects with heavy alcohol use and in this present study sample the low IgA levels to PEth were better indicators of heavy alcohol consumption as compared to the some of the traditional markers of heavy alcohol use. The antibody levels to PEth associated significantly to plasma antibodies binding to malondialdehyde-acetaldehyde adducts that are known to be formed during alcohol metabolism but not to antibodies binding to phosphocholine which is generated by lipid oxidation in humans. In conclusion, this study demonstrates that phosphatidylethanol is immunogenic in mice when using carriers such as human LDL in the immunization process. The binding of the monoclonal antibodies specifically to the PEth head group suggests that it would be feasible to develop a diagnostic immunoassay to PEth. The presence of antibodies binding to PEth in plasma indicates that PEth may be a target of humoral immunity in humans. / Tiivistelmä Runsas alkoholinkulutus aiheuttaa maailmanlaajuisesti merkittäviä terveydellisiä haittoja. Alkoholin aineenvaihduntatuotteet muuttavat kudoksien rakenteita ja aiheuttavat kudosvaurioita. Fosfatidyylietanoli on alkoholin aineenvaihdunnan tuloksena solukalvoilla syntyvä fosfolipidi, jota on tutkittu kahdenkymmenen vuoden ajan lupaavana alkoholin suurkulutuksen merkkiaineena. Tutkimuksen tavoitteena oli selvittää fosfatidyylietanolin immunisoinnin aiheuttamaa vasta-aineiden muodostumista koe-eläinmallina käytetyissä hiirissä sekä määrittää ihmisten plasmanäytteistä vasta-aineita, jotka sitoutuvat fosfatidyylietanoliin. Tutkimuksessa havaittiin immuunivasteen muodostuminen hiirissä, jotka immunisoitiin ihmisen LDL hiukkasiin liitetyllä fosfatidyylietanolilla. Hiiren monoklonaalisia fosfatidyylietanoliin sitoutuvia IgM-luokan vasta-aineita tuotettiin tutkimuksessa soluviljelyn avulla. Fosfatidyylietanolin aiheuttama vasta-aineiden muodostuminen hiirillä johdatti mittaamaan fosfatidyylietanoliin sitoutuvia vasta-aineita myös ihmisiltä. Tutkimuksessa havaittiin fosfatidyylietanoliin sitoutuvia IgG-, IgA- ja IgM-luokan vasta-aineita alkoholin suurkuluttajilla, alkoholihaimatulehdusta sairastavilla ja verrokkihenkilöillä. Vasta-aineiden pitoisuudet olivat alkoholia runsaasti käyttävillä koehenkilöillä merkitsevästi pienemmät kuin verrokkiryhmällä. Matalat IgA-vasta-ainepitoisuudet osoittautuivat aineistossa paremmaksi alkoholin suurkulutuksen osoittajiksi kuin eräät tavanomaisesti käytetyt alkoholinkäytön merkkiaineet. Plasman fosfatidyylietanoli-vasta-aineiden ja alkoholin aineenvaihdunnan seurauksena syntyvien malondialdehydi-asetaldehydi-addukteihin sitoutuvien vasta-aineiden määrän välillä havaittiin merkitsevä yhteys, jota ei havaittu rasvojen hapettumisen seurauksena syntyvien fosfokoliini-vasta-aineiden ja fosfatidyylietanoli-vasta-aineiden välillä. Tutkimus osoittaa, että hiirillä voidaan aikaansaada vasta-ainevälitteinen immuunivaste, kun ne rokotetaan ihmisen LDL-hiukkaseen liitetyllä fosfatidyylietanolilla. Fosfatidyylietanoliin spesifisesti sitoutuvien monoklonaalisten vasta-aineiden tuottaminen voi tulevaisuudessa johtaa immunologisen diagnostisen määritysmenetelmän kehittämiseen. Fosfatidyylietanoliin sitoutuvien plasman vasta-aineiden havaitseminen viittaa siihen, että fosfatidyylietanoli on vasta-ainevälitteisen immuunivasteen kohde myös ihmisillä.

Quitosana é um polissacarídeo usado em diversas aplicações biológicas, por exemplo, em liberação controlada de drogas, transfecção, aceleração da cicatrização de feridas e como agente bactericida, entre outras. Em todas essas aplicações, o polímero interage com tecidos e células. Entretanto, embora sua ação seja comprovada, os mecanismos de ação e a interação do polímero com células e biomembranas no nível molecular ainda não são conhecidos. Nesta tese de doutorado, filmes de Langmuir e Langmuir-Blodgett (LB) de lipídios foram usados como modelos de membrana celular para estudar em nanoescala a interação e os efeitos causados pela quitosana. Primeiramente, observou-se que a quitosana, um polieletrólito solúvel em pH ácidos, possui atividade superficial induzida na presença de um filme interfacial de lipídio, demonstrando que o polímero possui interação favorável com membranas. Após adsorver sobre as monocamadas, a quitosana expande as mesmas, o que ocorre apenas até uma determinada concentração de polímero, denominada concentração de saturação. A magnitude dessa expansão é menor para filmes compactos, o que sugere que a quitosana é parcialmente expulsa da interface, localizando-se na subsuperfície. Isso foi comprovado com o uso de filmes LB, que mostraram que filmes mistos com quitosana têm rugosidade cerca de 10 vezes a de filmes puros de ácido dimiristoil fosfatídico (DMPA). Foi possível confirmar que a quitosana penetra na monocamada, formando agregados com até 150 nm de altura. Além disso, a maior orientação das moléculas de fosfolipídios, sugerida por isotermas de potencial de superfície (V-A) para filmes de Langmuir, também foi comprovada para os filmes LB por medidas de espectroscopia de geração de soma de freqüências (SFG). Filmes mistos de DMPA e colesterol também foram estudados, sendo que o colesterol provoca condensação nos filmes de DMPA a baixas pressões, mas expande as monocamadas em altos estágios de compactação. Quando a quitosana interage com os filmes mistos, ela provoca a mesma expansão para todas as monocamadas independentemente da proporção de colesterol na mistura. Embora esse comportamento possa sugerir um papel inerte do colesterol, ele é explicado pela modulação da penetração da quitosana nos filmes pelo colesterol. Isso ocorre porque há um número fixo de pontos de interações eletrostáticas entre os grupos NH3+ da quitosana e PO2- do DMPA, o que foi comprovado por medidas de espectroscopia de reflexão-absorção na região do infravermelho com modulação da polarização (PM-IRRAS). Com esta técnica para filmes de Langmuir, e espectroscopia SFG para filmes LB, pôde ser traçado um panorama dos efeitos da inserção de colesterol na membrana de DMPA, seguido da interação da quitosana com a membrana mista. A adição do colesterol ao filme de fosfolipídio acarreta em diminuição da ordem das cadeias de DMPA, detectado por variações nas bandas de s(CH2) e ass(PO2-) do fosfolipídio no espectro de PM-IRRAS, e pela razão s(CH3)/s(CH2) nos espectros de SFG. Por outro lado, a interação da quitosana com esse filme misto causa recuperação da orientação das caudas polares do fosfolipídio, verificada pela análise das mesmas bandas de PM-IRRAS e pela razão s(CH3)/s(CH2), que diminui de 6,62 para 4,58 com a adição de colesterol, mas volta a 5,97 após a interação com o polímero. De forma geral, a ação da quitosana sobre biomembranas é governada principalmente por interações eletrostáticas com lipídios carregados negativamente, na superfície externa das mesmas. Dentre os principais efeitos causados pelo polímero, destaca-se a diminuição da elasticidade da membrana e o aumento da orientação das moléculas de lipídio, que podem ter importantes implicações biológicas. A observação de uma concentração de saturação dos efeitos, na maioria dos casos, sugere que a dosagem e a estrutura química da quitosana devem ser bem controladas para alcançar o efeito biológico desejado. / Chitosan is a polyssaccharide with many biological applications, as in drug delivery, transfection, wound healing and as bactericidal agent, for instance. In all these applications the polymer interacts with tissues and cells. The efficacy of chitosan has been proven, but the mechanisms of action and the interactions with cells and biomembranes are still unknown. In this thesis, Langmuir and Langmuir-Blodgett (LB) films made of lipids were employed as cell membrane models, in order to investigate the interactions and modulations caused by chitosan at the molecular level. Firstly, the soluble polyelectrolyte chitosan was found to induce surface activity when a lipid monolayer is at the air/water interface, demonstrating that the interaction of chitosan with membranes is favorable. Upon chitosan adsorption, the monolayers were increasingly expanded with increasing chitosan concentration in the subphase up to a saturation concentration. The extension of this expansion was lower for highly packed films, suggesting that chitosan was partially expelled from the interface after the compression, being located at the sub-monolayer region. This was confirmed by the 10-fold increase in film roughness observed for the areas without aggregates in LB films. Also, we could observe aggregates as high as 150 nm on the film surface, thus confirming chitosan penetration in the dimyristoyl phosphatidic acid (DMPA) monolayer. Mixed DMPA-cholesterol Langmuir monolayers were also produced, with cholesterol inducing condensation of the DMPA films at low pressures, and film expansion at high pressures. Regardless of the cholesterol proportion in the film, chitosan always induced the same degree of expansion on the DMPA mixed monolayers as for a neat DMPA monolayer. Although this behaviour may suggest an inert role for cholesterol, it can only be explained if the sterol is assumed to regulate the extension of chitosan penetration into the monolayer. This occurs because there is a fixed number of sites for electrostatic interactions between NH3+ groups from chitosan and PO2- from DMPA, probed by infrared reflection-absorption spectroscopy (PM-IRRAS) measurements. Indeed, with PM-IRRAS measurements for Langmuir monolayers and sum-frequency generation spectroscopy (SFG) measurements for LB films, we could establish an overview of the effects from cholesterol on DMPA films upon interaction with chitosan. The addition of cholesterol to the DMPA monolayer caused a decrease in the chain order, which was detected by changes in the s(CH2) and ass(PO2-) bands from the phospholipid in the PM-IRRAS spectrum, and by the s(CH3)/s(CH2) intensity ratio in SFG measurements. On the other hand, the interaction of chitosan with these mixed monolayers restored chain order, as observed from the analysis of PM-IRRAS bands and the s(CH3)/s(CH2) in SFG. The latter dropped from 6.62 to 4.58 with cholesterol addition, but further increased to 5.97 with the chitosan interaction. Overall, the chitosan action on biomembranes is mainly governed by electrostatic interactions with negatively charged lipids at the external leaflet of the membrane. The main effects from chitosan to the membrane models are the decrease in membrane elasticity and the increase in molecular ordering, which can lead to important biological implications. Moreover, the existence of the so-called concentration of saturation for most systems suggests that the dosage and chemical structure of chitosan must be well controlled to obtain the desired biological effect.

Chronic hypoxia is a state of oxygen limitation that is common in many aquatic and terrestrial environments. Metabolic suppression is an essential strategy that is used by hypoxia-tolerant champions such as goldfish and naked mole-rats to cope with prolonged low oxygen. This thesis examines the physiological processes used by goldfish and naked mole-rats to survive in low oxygen environments. It proposes a novel mechanism - the remodeling of membrane lipids - to reduce ATP use and production. Temperature (homeoviscous adaptation), diet (natural doping in migrant birds) and body mass (membrane pacemaker of metabolism) have an impact on the lipid composition of membranes that, in turn, modulates metabolism. In chapters 2 and 3 of this thesis, I demonstrate that vertebrate champions of hypoxia tolerance undergo extensive changes in membrane lipid composition upon in vivo exposure to low oxygen. These changes and those observed in hibernating mammals can promote the downregulation of Na⁺/K⁺-ATPase (major ATP consumers), mitochondrial respiration capacity [OXPHOS (phosphorylating conditions), proton leak (non-phosphorylating conditions), cytochrome c oxidase], and energy metabolism (β-oxidation and glycolysis) as discussed in chapters 3 and 4. A common membrane signal regulating the joint inhibition of ion pumps and channels could be an exquisite way to preserve the balance between ATP supply and demand in hypometabolic states. In chapter 5, I show that the reduction in ATP turnover is also orchestrated by mechanisms that involve post-translational and post-transcriptional modifications and epigenetic changes. Membrane remodeling, together with these more traditional molecular mechanisms, could work in concert to cause metabolic suppression.

Cílem této práce bylo studium mechanismu oxidace lipidů katalyzované hovězím methemoglobinem a zhodnocení účinků různých experimentálních podmínek a antioxidantů (EDTA, askorbová kyselina, kávová kyselina, a-tokoferol, d-tokoferol, astaxanthin a L-askorbyl-6-palmitát) na methemoglobinem zprostředkovanou oxidaci lipidů v modelovém systému liposomů připravených z fosfolipidů. K monitorování oxidace lipidů při pH 5,5 a teplotě 30 °C bylo použito spotřeby kyslíku. Pro zhodnocení antioxidační aktivity v modelovém systému liposomů se ukázaly být důležitými faktory typ prooxidantu a koncentrace prooxidantu a antioxidantu. Dalšími důležitými faktory jsou struktura molekuly antioxidantu, jeho hydrofilita/lipofilita a umístění v systému. Všechny testované antioxidanty ve všech koncentracích (kromě koncentrace 0.1 % astaxanthinu and 0.1 % askorbyl palmitátu) inhibovaly oxidaci vyvolanou methemoglobinem. Účinnost antioxidantu stoupala s jeho zvyšující se koncentrací. Koncentrace 0.1 % astaxanthinu neměla žádný vliv na oxidaci liposomů. Koncentrace 0.1 % askorbyl palmitátu měla prooxidační efekt, který lze vysvětlit prooxidačním působením radikálu askorbylu, který může urychlit štěpení hydroperoxidů. Volné železo uvolněné z methemoglobinu se podílelo jen velmi málo na oxidaci liposomů, zatímco část prooxidační aktivity methemoglobinu byla přisouzena tvorbě singletového kyslíku (methemoglobin jako fotosenzitizátor). Antioxidační aktivita astaxanthinu, askorbyl palmitátu a tokoferolu byla z části přisouzena schopnosti zhášet singletový kyslík. Ovšem hlavním prooxidačním mechanismem methemoglobinu se ukázal být rozklad lipidových hydroperoxidů, tvorba volných radikálů a hypervalentních forem hemoglobinu. EDTA utlumila oxidaci liposomů díky chelataci přechodných kovů obsažených v liposomech a chelataci volného železa přítomného v methemoglobinovém roztoku. Velmi důležitým antioxidačním mechanismem (který vykazují askorbyl palmitát, askorbová a kávová kyselina) se ukázala být redukce hypervalentních forem hemoglobinu. Askorbová kyselina, kávová kyselina, tokoferoly a astaxanthin inhibovaly methemoglobinem zprostředkovanou oxidaci lipidů odstraňováním volných radikálů. Při použití peroxidu vodíku nebyl pozorován žádný vliv na oxidaci liposomů vyvolanou methemoglobinem. Působení vysoké teploty (tepelná denaturace) mírně utlumilo oxidaci. Významná inhibice oxidace byla pozorována u liposomů obsahujících TPP (triphenylphosphin), což značí, že je methemoglobinem vyvolaná oxidace liposomů závislá na přítomnosti již vzniklých lipidových peroxidů. Výsledky této práce přispívají k hlubšímu pochopení prooxidačních a antioxidačních mechanismů a faktorů, které ovlivňují oxidaci liposomálních roztoků, buněčných membrán a emulzí typu olej ve vodě stabilizovaných fosfolipidy.