Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Hintergrund: Der Morbus Wilson ist eine seltene, angeborene Störung des Kupferstoffwechsels, bei welcher es zu Akkumulationen von Kupfer und infolgedessen zu Schäden in verschiedenen Organen des menschlichen Körpers kommt. Die Therapie besteht vor allem darin, den Kupferspiegel medikamentös zu senken. In einzelnen Fällen wurde der Kupferspiegel während der Therapie so weit gesenkt, dass bei den Patient*innen neurologische Symptome auftraten (sogenannte Kupfermangel-Myeloneuropathien). Kupfer ist ein essenzieller Kofaktor mehrerer Enzyme im menschlichen Körper, so auch der Cytochrom-c-Oxidase, welche einen wichtigen Bestandteil der mitochondrialen Atmungskette und damit der zellulären Energiegewinnung darstellt. Die Bestimmung ihrer Aktivität ist bisher für verschiedene Zellen und Gewebe etabliert worden, ein standardisierter Assay für die Bestimmung in Thrombozyten existiert jedoch nicht. Fragestellung: Für die optimale Bestimmung der Cytochrom-c-Oxidase-Aktivität in Thrombozyten sollen bereits existierende Methoden angepasst werden. Ziel dieser Arbeit ist es, die Aktivität der Cytochrom-c-Oxidase bei Morbus Wilson-Patient*innen unter kupfersenkender Therapie zu untersuchen und auf einen Zusammenhang zum Serum-Kupferspiegel zu prüfen. Die Frage, ob eine zu starke Kupfersenkung durch die Therapie des Morbus Wilson zu einer verringerten Cytochrom-c-Oxidase-Aktivität führt und ob diese Myeloneuropathien hervorruft, soll somit beantwortet werden. Material und Methodik: Es wurden 36 Morbus Wilson-Patient*innen unter kupfersenkender Therapie und 20 gesunde Kontrollproband*innen untersucht. Es erfolgte eine Blutabnahme für die Gewinnung der Thrombozyten sowie für die Bestimmung des Serum-Kupferspiegels. Die Bestimmung der Aktivität der Cytochrom-c-Oxidase erfolgte spektralphotometrisch in Thrombozyten. Des Weiteren wurde die Aktivität des Komplex-II der Atmungskette bestimmt, da dieser nicht kupferabhängig ist und seine Aktivität daher bei Kupfermangel nicht eingeschränkt sein sollte. Zusätzlich ermöglichte die Berechnung des Quotienten der Cytochrom-c-Oxidase-Aktivität und der Komplex-II-Aktivität die Erfassung sehr geringer Aktivitätseinschränkungen der Cytochrom-c-Oxidase. Im Rahmen dieser Dissertation wurde die spektralphotometrische Messung dieser beiden Enzymaktivitäten in Thrombozyten entwickelt und optimiert. Zur Justierung der Enzymaktivitäten bei unbekannter Mitochondrienmenge diente die Aktivität der ausschließlich in Mitochondrien vorkommenden Citratsynthase. Die so bestimmten Enzymaktivitäten wurden mittels SPSS zwischen Wilson-Patient*innen und Kontrollproband*innen verglichen und untereinander sowie mit dem Serum-Kupferspiegel auf Zusammenhänge untersucht. Ferner wurden die Morbus Wilson-Patient*innen klinisch auf Anzeichen für Myeloneuropathien untersucht, um die Untersuchungsergebnisse anschließend auf einen Zusammenhang zu der Cytochrom-c-Oxidase-Aktivität zu prüfen. Ergebnisse: Der auf den Untersuchungen von Kirby et al. beruhende Assay für die spektralphotometrische Bestimmung der Cytochrom-c-Oxidase-Aktivität in isolierten Mitochondrien konnte durch die Zugabe von 0,3 mM Dodecylmaltosid für die Messung in Thrombozyten erfolgreich optimiert werden. Ebenso wurde der Assay für die Komplex-II-Aktivität durch die Zugabe von 1 mg/ml BSA für die Bestimmung in Thrombozyten erweitert (Kirby et al., 2007). Die Aktivität der Cytochrom-c-Oxidase der Wilson-Patient*innen war signifikant niedriger als die der Kontrollgruppe, während die Kontrollgruppe eine signifikant höhere Komplex-II-Aktivität aufwies. Der Quotient von Cytochrom-c-Oxidase-Aktivität und Komplex-II-Aktivität war in der Patient*innengruppe folglich ebenfalls signifikant erniedrigt. In der Analyse aller untersuchten Proben zeigte sich ein signifikanter Zusammenhang zwischen Serum-Kupferspiegel und Cytochrom-c-Oxidase-Aktivität, welcher in der Betrachtung der Subgruppen (Wilson Patient*innen und Kontrollproband*innen) jedoch nicht nachgewiesen werden konnte. Keiner der untersuchten Patient*innen wies klinische Anzeichen für Myeloneuropathien auf. Schlussfolgerung: Der optimierte Assay der Cytochrom-c-Oxidase-Aktivität und Komplex-II-Aktivität in Thrombozyten erlaubt die zuverlässige Bestimmung der Atmungskettenaktivität in einem einfach zugänglichen Gewebe und ist damit für vielfältige Fragestellungen einsetzbar, wenn Einflüsse medizinischer Maßnahmen auf die mitochondriale Funktion untersucht werden sollen. Mit 36 Morbus Wilson-Patient*innen umfasst diese Arbeit eine der bisher größten untersuchten Patient*innengruppen dieses seltenen Krankheitsbildes. Der erniedrigte Quotient der Cytochrom-c-Oxidase-Aktivität und Komplex-II-Aktivität ist als Bestätigung einer Cytochrom-c-Oxidase-Einschränkung bei Morbus Wilson-Patient*innen unter kupfersenkender Therapie zu werten. Die Korrelation zwischen dem Serum-Kupferspiegel und der Cytochrom-c-Oxidase-Aktivität sowie die Aktivitätsreduktion der Cytochrom-c-Oxidase in der Patient*innengruppe ist eine wichtige Erkenntnis für die zukünftige Überwachung und gegebenenfalls Anpassung der Therapie von Morbus Wilson. Der Zusammenhang zwischen der Cytochrom-c-Oxidase-Aktivität und Myeloneuropathien sollte an Patient*innen mit Myeloneuropathien weiter untersucht werden. Es wurde jedoch gezeigt, dass Kupfermangel und niedrige Cytochrom-c-Oxidase-Aktivitäten nicht unbedingt mit Myeloneuropathien einhergehen. Therapie-induzierte Kupfermangel-Myeloneuropathien gilt es weiterhin zu vermeiden. / Background: Wilson’s disease is a rare, congenital disorder of copper metabolism, which leads to accumulations of copper and consequent damage in various organs of the human body. The therapy consists mainly in lowering the copper level by medication. In individual cases, the copper level was lowered during the therapy to such an extent that the patients developed neurological symptoms (so-called copper deficiency myeloneuropathies). Copper is an essential cofactor of several enzymes in the human body, including cytochrome c oxidase, which is an important component of the mitochondrial respiratory chain and thus of cellular energy production. The determination of its activity has been established so far for various cells and tissues, but a standardized assay for its determination in platelets does not exist. Purpose: For the optimal determination of cytochrome c oxidase activity in platelets, existing methods will be adapted. The aim of this work is to investigate the activity of cytochrome c oxidase in Wilson’s disease patients under copper-lowering therapy and to test for a correlation to serum copper levels. The question of whether excessive copper lowering by Wilson’s disease therapy leads to reduced cytochrome c oxidase activity and whether this possibly causes myeloneuropathies will thus be answered. Material and Methods: 36 Wilson’s disease patients under copper-lowering therapy and 20 healthy control subjects were studied. Blood was drawn for platelet collection and determination of serum copper levels. The activity of cytochrome c oxidase was determined spectrophotometrically in platelets. Furthermore, the activity of complex II of the respiratory chain was determined, since this is not copper-dependent and its activity should therefore not be limited in copper deficiency. In addition, calculation of the quotient of cytochrome c oxidase activity and complex II activity allowed detection of very low activity limitations of cytochrome c oxidase. In this dissertation, the spectrophotometric measurement of these two enzyme activities in platelets was developed and optimized. The activity of citrate synthase, which occurs exclusively in mitochondria, was used to adjust the enzyme activities when amount of mitochondria was unknown. The enzyme activities determined in this way were compared between Wilson’s disease patients and control subjects using SPSS and examined for correlations with each other and with serum copper levels. Furthermore, the Wilson’s disease patients were clinically examined for signs of myeloneuropathies, in order to subsequently examine the examination results for a correlation to the cytochrome c oxidase activity. Results: The assay for spectrophotometric determination of cytochrome c oxidase activity in isolated mitochondria, based on studies of Kirby et al, was successfully optimized for measurement in platelets by the addition of 0.3 mM dodecylmaltoside. Similarly, the assay for complex II activity was enhanced by the addition of 1 mg/ml BSA for determination in platelets (Kirby et al., 2007). The activity of cytochrome c oxidase of Wilson patients was significantly lower than that of the control group, with the control group had a significantly higher complex II activity. Consequently, the quotient of cytochrome c oxidase activity and complex II activity was also significantly lower in the Wilson patient group. A significant correlation between serum copper level and cytochrome c oxidase activity was found in the analysis of all samples examined, which, however, could not be proven in the examination of the subgroups (Wilson patients and control subjects). None of the patients examined showed clinical signs of myeloneuropathies. Conclusion: The optimized assay of cytochrome c oxidase activity and complex II activity in platelets allows reliable determination of respiratory chain activity in an easily accessible tissue and is thus applicable to a variety of questions when influences of medical interventions on mitochondrial function are to be investigated. With 36 Wilson's disease patients, this work includes one of the largest groups of patients of this rare disease studied so far. The decreased quotient of cytochrome c oxidase activity and complex II activity is a confirmation of cytochrome c oxidase impairment in Wilson’s disease patients on copper-lowering therapy. The correlation between serum copper level and cytochrome c oxidase activity as well as the reduction of cytochrome c oxidase activity in the patient group is an important finding for future monitoring and, if necessary, adjustment of Wilson’s disease therapy. The relationship between cytochrome c oxidase activity and myeloneuropathies should be further investigated in patients with myeloneuropathies. However, it has been shown that copper deficiency and low cytochrome c oxidase activities are not necessarily associated with myeloneuropathies. Therapy-induced copper deficiency myeloneuropathies should continue to be avoided.

La forme canadienne-française du syndrome de Leigh (LSFC) est une maladie métabolique associée à une déficience en cytochrome oxydase (COX) et caractérisée par des crises d’acidose lactique, menant à une mort prématurée. Les mécanismes qui sous-tendent l’induction des crises restent inconnus et il n’existe aucune thérapie efficace pour les prévenir. Cette étude vise à caractériser l'effet de facteurs métaboliques périphériques potentiellement altérés chez les patients LSFC sur la mort de lignées cellulaires issues de ces patients et de témoins puis, à identifier des agents thérapeutiques pouvant la prévenir. Nous postulons que (i) ces facteurs métaboliques induiront une mort prématurée des cellules de patients et que (ii) les interventions susceptibles de la prévenir pallieront les conséquences de la déficience en COX, soit la diminution des taux d’adénosine triphosphate (ATP) et l’augmentation du stress oxydant, du nicotinamide adénine dinucléotide (NADH) et des lipides toxiques. Un criblage de 8 facteurs sanguins et 10 agents thérapeutiques a été réalisé. Les paramètres mesurés incluent la nécrose, l’apoptose, l’ATP et l’activité de la COX. Les fibroblastes LSFC sont plus susceptibles à la mort par nécrose (39±6%) induite par du palmitate plus lactate, un effet associé à des niveaux d’ATP diminués (53±8%). La mort cellulaire est réduite de moitié par l’ajout combiné d’agents ciblant le NADH, l’ATP et les lipides toxiques, alors que l’ajout d’antioxydants l’augmente. Ainsi, un excès de nutriments pourrait induire la mort prématurée des cellules LSFC et, pour atténuer cette mort, il serait important de combiner plusieurs interventions ciblant différents mécanismes. / Leigh syndrome French-Canadian variant (LSFC) is a metabolic disease associated with cytochrome c oxidase (COX) deficiency and characterized by episodes of lactic acidosis, referred to as “crisis”, leading to death at an early age. The mechanisms underlying a crisis and its cellular consequences remain elusive, and there is no effective therapy. The aim of this study was to characterize the effect of peripheral metabolic factors that are potentially altered in patients with LSFC on their cells death and to identify therapeutic agents able to prevent them using cell-lineage from LSFC patients and controls. The hypothesis are that (i) these metabolic factors can induce premature death in patient cells, and (ii) interventions that could rescue these cells may target potential consequences of COX deficiency, namely low adenosine triphosphate (ATP), high nicotinamide adenine dinucleotide (NADH) and toxic lipids, as well as oxidative stress. A screening of 8 blood factors and 10 therapeutic agents was conducted in fibroblasts. Parameter measured included cell death by necrosis and apoptosis, as well as ATP level and COX activity. LSFC fibroblasts were more susceptible to necrosis (39±6%) induced by high palmitate plus lactate and this was associated with a lower ATP (53±8%). Cell death decreased 2-fold with combined interventions, which presumably act on NADH, ATP, and the accumulation of toxic lipids, but increased with antioxidants. Collectively, our results emphasize the importance of nutrient overload as a factor eliciting premature cell death in LSFC cells and of combining interventions acting through various mechanisms for cell death rescue.

La forme canadienne-française du syndrome de Leigh (LSFC) est une maladie métabolique associée à une déficience en cytochrome oxydase (COX) et caractérisée par des crises d’acidose lactique, menant à une mort prématurée. Les mécanismes qui sous-tendent l’induction des crises restent inconnus et il n’existe aucune thérapie efficace pour les prévenir. Cette étude vise à caractériser l'effet de facteurs métaboliques périphériques potentiellement altérés chez les patients LSFC sur la mort de lignées cellulaires issues de ces patients et de témoins puis, à identifier des agents thérapeutiques pouvant la prévenir. Nous postulons que (i) ces facteurs métaboliques induiront une mort prématurée des cellules de patients et que (ii) les interventions susceptibles de la prévenir pallieront les conséquences de la déficience en COX, soit la diminution des taux d’adénosine triphosphate (ATP) et l’augmentation du stress oxydant, du nicotinamide adénine dinucléotide (NADH) et des lipides toxiques. Un criblage de 8 facteurs sanguins et 10 agents thérapeutiques a été réalisé. Les paramètres mesurés incluent la nécrose, l’apoptose, l’ATP et l’activité de la COX. Les fibroblastes LSFC sont plus susceptibles à la mort par nécrose (39±6%) induite par du palmitate plus lactate, un effet associé à des niveaux d’ATP diminués (53±8%). La mort cellulaire est réduite de moitié par l’ajout combiné d’agents ciblant le NADH, l’ATP et les lipides toxiques, alors que l’ajout d’antioxydants l’augmente. Ainsi, un excès de nutriments pourrait induire la mort prématurée des cellules LSFC et, pour atténuer cette mort, il serait important de combiner plusieurs interventions ciblant différents mécanismes. / Leigh syndrome French-Canadian variant (LSFC) is a metabolic disease associated with cytochrome c oxidase (COX) deficiency and characterized by episodes of lactic acidosis, referred to as “crisis”, leading to death at an early age. The mechanisms underlying a crisis and its cellular consequences remain elusive, and there is no effective therapy. The aim of this study was to characterize the effect of peripheral metabolic factors that are potentially altered in patients with LSFC on their cells death and to identify therapeutic agents able to prevent them using cell-lineage from LSFC patients and controls. The hypothesis are that (i) these metabolic factors can induce premature death in patient cells, and (ii) interventions that could rescue these cells may target potential consequences of COX deficiency, namely low adenosine triphosphate (ATP), high nicotinamide adenine dinucleotide (NADH) and toxic lipids, as well as oxidative stress. A screening of 8 blood factors and 10 therapeutic agents was conducted in fibroblasts. Parameter measured included cell death by necrosis and apoptosis, as well as ATP level and COX activity. LSFC fibroblasts were more susceptible to necrosis (39±6%) induced by high palmitate plus lactate and this was associated with a lower ATP (53±8%). Cell death decreased 2-fold with combined interventions, which presumably act on NADH, ATP, and the accumulation of toxic lipids, but increased with antioxidants. Collectively, our results emphasize the importance of nutrient overload as a factor eliciting premature cell death in LSFC cells and of combining interventions acting through various mechanisms for cell death rescue.

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

Des modifications dans le gène codant pour la sous-unité II du cytochrome c oxydase du génome mâle (Mcox2) ont été recensées chez des espèces de bivalves présentant un mode unique de transmission mitochondriale nommé double transmission uniparentale (DUI). Dans la DUI, les mitochondries paternelles (et leur ADNmt mâle) ainsi que les mitochondries maternelles (et leur ADNmt femelle) sont transmises aux descendants mâles. Scrobicularia plana, une espèce de bivalves présentant ce modèle d'hérédité, possède une insertion importante d'environ 4,8 kb dans son gène Mcox2 qui ne change pas le cadre de lecture et qui est traduite en un polypeptide de 1 892 acides aminés, ce qui en fait la plus grande protéine COX2 connue à ce jour chez les métazoaires. L’objectif de cette étude était de caractériser l'évolution et la fonction potentielle de l'insertion dans Mcox2 chez S. plana par RT-PCR, tests immunologiques et analyses bio-informatiques. L'insertion est présente parmi les individus de différentes populations, contient des variations dans la longueur de sa séquence, est riche en zones de désordre intrinsèque et évolue sous sélection purificatrice. La longue insertion pourrait modifier la structure 3D du complexe IV de la chaîne de transport d'électrons (CTE), affectant sa fonction dans la phosphorylation oxydative (OXPHOS) ce qui pourrait expliquer les faibles taux d'OXPHOS observés dans les mitochondries mâles des bivalves à DUI. L'insertion pourrait également modifier le métabolisme mitochondrial mâle en interagissant avec d'autres complexes de la CTE et avec l'ATP synthase. Comme pour les autres modifications de Mcox2 chez les bivalves à DUI, un rôle potentiel dans la détermination du sexe peut être prédit pour MCOX2 chez S. plana. / Modifications in the cytochrome c oxidase subunit II gene of male-transmitted genome (Mcox2) have been found in some bivalve species that exhibit a unique mode of mitochondrial transmission named doubly uniparental inheritance (DUI). In DUI, paternal mitochondria (and their male mtDNA) as well as maternal mitochondria (and their female mtDNA) are transmitted to male offspring. Scrobicularia plana, a bivalve specie exhibiting this inheritance model possesses an important in-frame insertion of approximately 4,8 kb in its Mcox2 gene that is translated into a polypeptide of 1 892 amino acids making it the largest metazoan COX2 protein known to date. The aim of this study was to characterize the evolution and possible function of the Mcox2 insertion in S. plana through RT-PCRs, immunoassays, and bioinformatic analysis. The insertion is present amongst individuals from different populations, contains some variations in its sequence length, is rich in intrinsically disordered regions and evolves under purifying selection. The long insertion could modify the 3D structure of complex IV in the electron transport chain (ETC), impacting its function in oxidative phosphorylation (OXPHOS) which could explain low OXPHOS rates that were found in male mitochondria of DUI bivalves. The insertion could also alter male mitochondrial metabolism by interacting with other complexes of the ETC and with ATP synthase. As for other modifications of Mcox2 in DUI bivalves, a role in sex determination can also be predicted for MCOX2 in S.plana.

O objetivo deste trabalho foi desenvolver biosensores de membranas baseados na técnica de Ressonância Plasmônica de Superfície (SPR) e aplicá-los no estudo da interação do citocromo c (cit c) com modelos de membranas. SPR é uma técnica ótica, que através de medidas de variações de índice de refração (n) próximas a uma interface mensura com alta sensibilidade a adsorção ou ligação de moléculas. Inicialmente desenvolvemos um sistema de gradiente de fluxo acoplado ao SPR, denominado FIG-SPR, e demonstramos a determinação automatizada da variação de n em função da concentração (dn/dC) de diferentes compostos e biopolímeros. O desenvolvimento dos biosensores de membranas iniciou-se com o estudo dos fatores que afetam a formação de uma membrana de bicamada híbrida (HBM). HBMs são compostas de uma monocamada de alcanotiol adsorvida sobre o ouro, e sobre esta uma camada fosfolipídica. A formação da HBM depende da fusão de vesículas em superfícies hidrofóbicas e que não é bem compreendido no nível molecular. Nossos estudos mostraram que na presença de cálcio e espermina a formação da HBM é favorecida, de tal forma que a monocamada de fosfolipídio alcança valores de espessura próximos àqueles previstos, cerca de 20 Å\'. Além disso, mostramos que em soluções de baixa força iônica a camada lipídica não é homogênea. Demonstramos também que a presença de cálcio na concentração 150 mM diminui o tempo de formação da monocamada lipídica cerca de 14 vezes quando comparado ao tempo indicado na literatura. A homogeneidade da HBM e a carga superficial da mesma foram verificadas com a adsorção e a dissociação de cit c e de albumina bovina (BSA). Utilizando HBMs de composição lipídica variada demonstramos a adsorção e a dissociação de cit c induzida por cálcio em HBMs mistas, incluindo um modelo mimético da membrana mitocondrial interna (IMM) constituído de fosfatidilcolina, fosfatidiletanolamina e cardiolipina (PC/PE/CL) na proporção (4,5:3,5:2,0). Demonstramos que a adsorção de cit c nativo segue um perfil cooperativo e padrões esperados de variação de afinidade e cooperatividade em pHs 6,8, 7,4 e 8,0. Um modelo matemático foi desenvolvido para tratar as curvas de ligação de cit c, que é uma adaptação do modelo de Hill para adsorção de proteínas em superfícies. Os resultados de SPR juntamente com dados obtidos por Microscopia de Força Atômica (AFM) sugerem que a ligação cooperativa de cit c com HBM ocorre devido à reorganização das moléculas de CL e formação de domínios fosfolipídicos. O tratamento dos resultados cinéticos da dissociação de cit c por cálcio indica a existência de duas constantes de velocidade de dissociação (kd), sendo a primeira constante (kd1) relacionada à perda das interações eletrostáticas entre a proteína e a HBM, e a segunda (kd2) à perda das interações hidrofóbicas. Além disso, a dissociação do cit c do modelo estudado requer uma concentração mínima de cálcio de 30 µM para se tornar significativa. O estudo da interação entre moléculas de cit c foto-oxidadas (citc405) e a HBM de PC/PE/CL sugerem que ela ocorre com menor afinidade, nos três pHs estudados, se comparados aos resultados com cit c nativo. Além disso, nossos resultados sugerem que o citc405 não é facilmente dissociado por cálcio devido à perda da cooperatividade na interação. Possíveis implicações em eventos celulares destas descobertas, como a liberação do cit c da IMM e a iniciação da apoptose, são discutidas / The aim of this work was to develop membrane biosensors based on Surface Plasmon Resonance (SPR) and to apply them to study the interactions between cytochrome c (cyt c) and model membranes. SPR is an optical technique that provides high-sensitivity measurements of refractive index (n), allowing the characterization of the adsorption and desorption of molecules near interfaces. Initially we developed a flow gradient system connected to SPR, which was called FIG-SPR, and demonstrated the automated determination of the concentration gradient of refractive index (dn/dC) of different materials and biopolymers. The development of the membrane biosensors was initiated by studying the factors that affect the formation of a hybrid bilayer membrane (HBM). HBMs are composed of two monolayers: an alcanethiol monolayer adsorbed on gold over which is adsorbed a layer of phospholipids. The formation of an HBM depends on the fusion of phospholipid vesicles on hydrophobic surfaces, a process that is not well understood at the molecular level. Our results showed that in the presence of calcium and spermine the complete formation of an HBM is facilitated, i.e., the phospholipid monolayer reaches the expected thickness of about 20Å\'. However, in low ionic strength solutions the lipid layer that is formed is not homogeneous. We have also demonstrated that in the presence of 150 mM of calcium the time necessary for the formation of the lipid monolayer is reduced 14 times when compared to the times suggested in the literature. The homogeneity of the HBM and its superficial charge were verified with the adsorption and desorption of cyt c and bovine serum albumine (BSA). The adsorption and desorption of cyt c in different HBMs were studied including a model of the internal mitochondrial membrane (IMM), which is made of phosphatidylcholine, phosphatidylethanolamine and cardiolipin (PC/PE/CL) in the ratio (4,5: 3,5: 2,0). We demonstrated that the adsorption of native cyt c follows a cooperative profile showing expected changes in affinity and cooperativity in different solution pHs of 6,8, 7,4 and 8,0. A mathematical model, which is an adaptation of the Hill model for adsorption of proteins in surfaces, was developed to treat the binding curves of cyt c. The results of SPR together with those obtained by Atomic Force Microscopy (AFM) suggested that the cooperative binding of cyt c in HBMs occurs due to the reorganization of CL molecules and formation of phospholipid domains. The kinetic results of the dissociation of cyt c induced by calcium indicates the existence of two velocity constants (kd), being the larger (kd1) related to the dissociation of cyt c interacting electrostatically with the HBM, and the smaller (kd2) related to the dissociation of cyt c interacting hydrophobically with the HBM. Moreover, the dissociation of cyt c from the HBM requires a minimum calcium concentration of 30 µM. The study of the interaction between photo-oxidized cyt c molecules (cytc405) and the PC/PE/CL HBM suggests that it occurs with smaller affinity when compared with the results obtained with the native cyt c. Moreover, cytc405 is not easily dissociated by calcium due to the loss of the interaction cooperativity with the HBM. Possible implications of these discoveries in cellular events, such as the release of cyt c from the IMM and the initiation of apoptosis, are discussed

Introdução. A identificação dos flebotomíneos baseia-se principalmente na morfologia do adulto, o que pode ser problemático quando as espécies são morfologicamente muito semelhantes. Psychodopygus é um gênero de flebotomíneos de grande interesse em saúde pública devido ao papel de algumas espécies na veiculação de Leishmania spp. no Brasil. No entanto, este gênero inclui espécies com fêmeas morfologicamente indistinguíveis que pertencem à Série Chagasi, sendo elas: P. chagasi, P. complexus, P. squamiventris maripaensis, P. squamiventris squamiventris e P. wellcomei. Objetivos. Investigar a possibilidade de distinguir essas espécies por meio de análises morfométrica e molecular, além de produzir uma distribuição geográfica atualizada para o grupo analisando a probabilidade de ocorrência das espécies através da análise de modelagem de nicho ecológico. Material e Métodos. Foi realizada a análise discriminante na morfometria geométrica (cabeça e asa) e linear, morfologia (usando microscopia óptica e eletrônica de varredura) e a análise do citocromo c oxidase subunidade 1 (COI), avaliando-se um total de 752 espécimes (460 fêmeas e 292 machos) dos seguintes estados Amapá, Amazonas, Ceará, Mato Grosso, Pará, Rondônia, Roraima e Tocantins. Mapas de distribuição foram produzidos através de dados obtidos do material analisado e de revisão bibliográfica. Resultados. A análise discriminante usando caracteres morfométricos lineares mostrou-se capaz de diferenciar todas as espécies, exceto P. complexus, que apresentou 2,2% de erro de identificação. A morfometria geométrica das asas foi incapaz de separar completamente as espécies através da conformação, mas o tamanho do centróide dos espécimes fêmeas falhou apenas em distinguir P. complexus de P. s. maripaensis. Por outro lado, a morfometria geométrica das cabeças foi capaz de distinguir todas as espécies com grande eficiência ao usar tanto a forma como o tamanho do centróide. A análise morfológica revelou que a coloração torácica, principalmente do pronoto e do pós-noto, pode ser usada para separar as cinco espécies em três grupos: P. chagasi, P. wellcomei / P. complexus e P. s. mariapaensis / P. s. squamiventris. Os resultados da análise de DNA Barcoding, mostraram um agrupamento semelhante ao observado na morfologia; embora os espécimes de P. wellcomei do estado do Ceará mostrem uma grande distância genética da população do estado do Pará, evidenciando que essa espécie possa representar um complexo. Quanto à microscopia eletrônica de varredura, foram avaliadas detalhadamente as estruturas das antenas, tórax e genitália masculina. Salientamos que no anepímero (tórax) foi observada uma escama tipo \"raquete\" modificada apenas em Psychodopygus s. squamiventris. A revisão da distribuição geográfica mostrou que as espécies possuem uma distribuição cis-andina, ocorrendo principalmente no bioma Amazônico. A nítida separação de algumas espécies pelo rio Amazonas, sugere que o surgimento do grupo ocorreu no período que se estende da orogênese dos Andes até a formação deste rio. Conclusões. O estudo possibilitou diferenciar completamente as fêmeas das cinco espécies da Série Chagasi utilizando o conjunto de dados obtidos por morfometria linear e geométrica e análises morfológicas e também apresentar novos caracteres morfológicos e padrões distribucionais que facilitarão a identificação de machos e fêmeas dessas espécies. / Introduction. The identification of sand flies is mainly based on adult morphology, which can be problematic when species are morphologically very similar. Psychodopygus is one of the sand fly genera of great interest in public health, due to the role of some species in the transmission of Leishmania spp. in Brazil. However, this genus includes species with morphologically indistinguishable females that belong to the Chagasi series, which includes: P. chagasi, P. complexus, P. squamiventris maripaensis, P. squamiventris squamiventris and P. wellcomei. Objectives. To investigate the possibility of distinguishing among these species by means of morphometric and molecular analyses in addition to producing an updated geographical distribution for the group, analyzing the probability of the occurrence of the species by the analysis of ecological niche modeling. Material and methods. The analyses of the cytochrome c oxidase subunit 1 (COI), geometrical (head and wing) and of linear morphometry and morphology (using optical microscopy and scanning electron microscopy) were carried out using a total of 752 specimens (460 females and 292 males) from the following states: Amapá, Amazonas, Ceará, Mato Grosso, Pará, Rondônia, Roraima e Tocantins. Distribution maps were produced on the basis of data obtained from the material analyzed and a bibliographical review. Results. The discriminant analysis using linear morphometric characters was able to differentiate among all the species, except for P. complexus, which presented a 2.2% error of identification. The geometric morphometry of the wings was unable to completely separate the species by means of the shape analyses, but the centroid size of the female specimens only failed to distinguish P. complexus from P. s. maripaensis. Otherwise, the geometric morphometry of the heads was sufficient to distinguish all the species with great efficiency, when using both the head-shape and the centroid size. The morphological analysis revealed that the thoracic coloration, mainly of the pronotum and the post-notum, can be used to separate the five species into three groups: P. chagasi, P. wellcomei / P. complexus, P. s. mariapaensis / P. s. squamiventris. The results of the Barcoding DNA analyses showed a cluster similar to that observed in the morphology; however, P. wellcomei specimens from the Ceará population showed a great genetic distance from the population of Pará, evidencing that this species may represent a complex. As for the scanning electron microscopy, the structures of the antennae, thorax and male genitalia were evaluated in detail. In the anepimerum (thorax) a modified \"racket\"-type scale was observed only in Psychodopygus s. squamiventris. The review of the geographical distribution showed that the species have a cis-Andean distribution, occurring mainly in the Amazonian biome. The separation of some species from the others by the Amazon river suggests that the appearance of the Chagasi series occurred in the period from the orogenesis of the Andes to the formation of this river. Conclusions. The results clearly differentiate the females of the five species of the Chagasi series using the data set of linear and geometric morphometry and morphological analyses, providing new morphological and distributional data that will facilitate the identification of the males and females of this group.

Ubiquinol-cytochrome c reductase (complex III) is a central component of the respiratory chain of the inner mitochondrial membrane. It transfers electrons from reduced ubiquinone to ferricytochrome c. Correctly assembled and functional complex III is an essential prerequisite for oxidative energy metabolism. Complex III deficiency has been reported to be associated with several neurodegenerative diseases. Formation and assembly of complex III requires a multitude of specific nuclearly encoded proteins. For example, gene specific translational activators for cytochrome b synthesis as well as three non-subunit proteins, which are important for assembly and/or stability have been detected. The role of Bcs1p in assembly of Rieske FeS protein and Qcr10p into complex III has been clasified recently. The role of the two putative chaperones, Cbp3p and Cbp4p, is not known. In spite of the similar phenotype of cbp3D and cbp4D strains, that suggests the role of both proteins in the same step of complex III assembly, we were able for the first time to demonstrate differences on the molecular level between both deletion mutants. We show by BN-PAGE that cbp3D and cbp4D mutants are disturbed in complex III assembly and accumulate intermediate-sized forms of the complex. Moreover deletion of CBP3 interferes with the formation of complex III/IV supracomplexes. Our studies show that Cbp3p and Cbp4p interact and are present in high molecular weight complexes, some of which might represent intermediates of complex III assembly. Overexpression of Cbp4p cannot substitute for the function of Cbp3p, but high level expression of Cbp3p can partially compensate for the lack of Cbp4p. Because lipids play an important role for complex III assembly and stability, we analysed the mitochondrial lipid composition of cbp3D and cbp4D mutants. Our data show that mitochondria of both mutants exhibit a wild type-like lipid composition, that favors the idea that Cbp3p and Cbp4p are specific assembly factors for complex III rather than components of the mitochondrial lipid metabolism. By complementation studies we have shown that Cbp3 proteins of S. cerevisiae, S. pombe and human are (partially) functional homologues. A yeast model based on chimeric constructs of S. cerevisiae and human proteins was constructed, which allows to test the pathogenicity of human mutations. To define the role/s of Cbp3p and Cbp4p in the assembly pathway of complex III, interactions of selected subunits with both assembly factors were analysed by TAP- or co-immunoprecipitation. Based on the results of Cbp3p and Cbp4p topologies, BN-PAGE analysis of null mutant strains and interaction studies a model for complex III assembly and the roles of Cbp3p and Cbp4p in this process are proposed. I present a hypothesis, according to which Cbp3p and Cbp4p form a ?scaffold? for the assembly of all three putative sub-complexes, may act independently in the first steps of bc1 complex assembly (e. g. the formation of sub-complexes) and interact together to assist the final assembly of sub-complexes into a mature enzyme. / Der Ubiquinol-Cytochrom c Reductase (Komplex III) ist eine zentrale Komponente der Atmungskette der inneren Mitochondrienmembran. Er transferiert Elektronen von reduziertem Ubiquinon auf Ferricytochrom c. Der korrekt assemblierte und funktionale Komplex III ist eine essenzielle Voraussetzung für den oxidativen Energiemetabolismus. Komplex III Defizienz ist assoziiert mit verschiedenen neurodegenerativen Krankheiten...

The thesis concerns polyelectrolyte gels in contact with oppositely charged proteins and surfactant micelles, and includes of four papers (I-IV). In paper I confocal Raman spectroscopy was introduced as a method to trace micelles and investigate the structure of gel-surfactant complexes, in phase separated gel spheres. In paper II, the binding of surfactants to microspheres (~50-100 µm) was investigated by means of a micromanipulator-assisted microscopy method. The two surfactants were found to display qualitative difference respect to degree of swelling, surfactant distribution in the gels, and the difference is discussed in terms of absence/presence of hydrophobic attraction to the polyelectrolyte gel network. Kinetics of volume change in gels were analyzed. Aggregation numbers of micelles in polystyrenesulfonate (PSS) solutions, obtained from fluorescence quenching measurements, are presented. In paper III, phase behaviour, protein assembly and diffusion, was studied in PSS gel microspheres. Interpretation of results was aided by measurements of osmotic swelling of individual gel networks, and by combining the results with studies of protein diffusion in macroscopic (cm-sized) gel spheres. Complexes formed were further analyzed with small angle x-ray spectroscopy. In paper IV phase behaviour of mixed ionic/nonionic surfactant micelles is investigated in cm-sized gel spheres. The coexistence of three phases, the formation of dense shells in the bulk of the gels and other phenomena are described for the first time, and the results are presented along with discussion on the charge-density of spherical micelles and of  network induced hysteresis effects in gels. The composition and microstructure of phases are investigated by confocal Raman spectroscopy and small-angle x-ray scattering respectively. The results are interpreted with aid of highly detailed theoretical model calculations.