Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The definitive diagnosis of strongyloidiasis is normally done by detection of larvae on faecal samples; however, the number of parasites is limited in most cases and the elimination of larvae is irregular. Thus, developing reliable serological methods for the diagnosis of strongyloidiasis becomes imperative. The aim of this study was to establish a coproantigen ,antigen, antibody and immune complex detection by enzyme-linked immunosorbent assay in serum and bronchoalveolar lavage fluid (BALF) samples of non immunosuppressed or immunosuppressed rats experimentally infected with Strongyloides venezuelensis. For kinetics of coproantigen detection (0 and 5, 8, 13 and 21 days post-infection (d.p.i)), we used an anti-L3 polyclonal antibody produced in rabbits. For antigen and immune complex detection in serum and BALF samples (0 and 2, 5, 8, 13 and 21d.p.i), the microtitre plates were coated with IgG anti-S. venezuelensis and with alkaline parasite extract for antibody detection. The statistical analysis were analyzed using Two Way ANOVA, followed by the Bonferroni test. The criterion for statistical significance was set at p<0,05. The number of eggs/g of faeces recovered at 8 d.p.i was significantly higher for non immunosuppressed and immunosuppressed animals (p<0.01). The coproantigen detection was significantly higher at 13° d.p.i in non immunosuppressed (p<0.05) and in immunosuppressed it was anticipated to the 5th d.p.i. It was observed that antigen detection in serum samples was not a good approache for evaluating the infection however in BALF samples it showed superior results. In immunosuppressed animals, IgG specific for S. venezuelensis was preferentially detected during the 5° and 13° d.p.i and in immunosuppressed animals, during the entire experimental kinetics. In BALF samples, antibodies detection was observed from the 8° to the 21° d.p.i in non immunosuppressed animals and in immunosuppressed animals it was anticipatedto the 2° d.p.i, with higher reactivity at 5° d.p.i (p<0.05). The immune complex detection in serum samples of the non immunosuppressed animals was observed from the 5° to the 13° d.p.i and in immunosuppressed animals, during the entire kinetics. In BALF samples, immune complex detection was higher in non immunosuppressed animals. In conclusion, coproantigen and immune complex detection in serum and BALF samples are alternatives for early strongyloidiasis diagnosis, mainly in immunocompromised cases. / O diagnóstico definitivo da estrongiloidíase normalmente é realizado mediante a detecção de larvas nas fezes; porém a quantidade de parasitos é limitada e a eliminação de larvas é reduzida e irregular. Sendo assim, o desenvolvimento de testes sorológicos confiáveis para o diagnóstico da estrongiloidíase torna-se uma alternativa necessária. O objetivo deste estudo foi demonstrar a cinética de detecção de coproantígenos e de antígenos, anticorpos e imunocomplexos circulantes em amostras de soro e de lavado bronco alveolar (LBA) de ratos imunossuprimidos e experimentalmente infectados por Strongyloides venezuelensis. Para a cinética (0 e 5, 8, 13 e 21 dias pós-infecção (d.p.i)) de coproantígenos utilizou-se anticorpo policlonal anti-L3 produzido em coelhos. Para a detecção de antígenos e de imunocomplexos em amostras de soro e de LBA (0 e 2, 5, 8, 13 e 21 d.p.i), placas de microtitulação foram sensibilizadas com IgG anti-S. venezuelensis e com extrato alcalino de larvas para a detecção de anticorpos. A análise estatística foi realizada por Two Way ANOVA, seguida pela teste de Bonferroni, considerando p<0,05 significativo. A cinética de eliminação de ovos/g de fezes mostrou que o pico ocorre no 8° d.p.i sendo significativamente maior nos animais imunossuprimidos (p<0,01). O pico de detecção de coproantígenos nos animais não imunossuprimidos foi no 13° d.p.i (p<0,05), sendo que nos animais imunossuprimidos a detecção foi antecipada para o 5° d.p.i. A detecção de antígeno em amostras de soro não foi uma boa ferramenta diagnóstica para avaliar a infecção enquanto que em amostras de LBA mostrou ser ferramenta auxiliar. A detecção de IgG específica para S. venezuelensis em amostras de soro de animais não imunossuprimidos foi preferencialmente durante o 5° e o 8° d.p.i. e em animais imunossuprimidos, durante toda a cinética experimental. Nas amostras de LBA, a detecção de anticorpos ocorreu do 8° ao 21° d.p.i em animais não imunossuprimidos e em animais imunossuprimidos, foi antecipada para o 2° d.p.i, como pico de reatividade no 5° d.p.i (p<0,05). A detecção de imunocomplexos em amostras de soro de animais não imunossuprimidos foi possível do 5° aos 13° d.p.i e em animais imunossuprimidos, durante toda a cinética. Em amostras de LBA, a detecção de imunocomplexo foi maior em animais não imunossuprimidos. Concluiu-se que a detecção de coproantígeno e de imunocomplexos circulantes em amostra de soro e em amostras de LBA são uma alternativa para o diagnóstico precoce da estrongiloidíase principalmente nos casos de imunossupressão. / Doutor em Imunologia e Parasitologia Aplicadas

Este trabalho trata da otimização de painéis alveolares e vigotas protendidas utilizando Algoritmos Genéticos (AGs). A proposta de tal algoritmo foi inspirada no princípio da seleção natural de indivíduos, onde o mais ‘apto’ tende a permanecer na população e se reproduzir, passando seu código genético para a próxima geração. Em alguns casos, esse método pode alcançar melhores soluções se comparados aos métodos tradicionais de otimização. O principal objetivo do trabalho é investigar o uso de AG como uma técnica para a minimização da função custo da aplicação de painéis alveolares e vigotas protendidas. Na análise estão incluídas as verificações dos elementos nas etapas transitórias referentes à produção, transporte e montagem. A função custo é avaliada considerando valores da realidade brasileira. O trabalho de pesquisa compara os resultados obtidos utilizando AGs com aqueles obtidos utilizando o método de otimização convencional conhecido como método do Lagrangiano Aumentado. Os resultados obtidos por ambos os métodos evidenciam a eficácia dos AGs com relação ao método convencional. Foram propostas e analisadas três famílias do AG simples, buscando identificar, dentre seus elementos, quais variantes mais adequados na busca da solução dos problemas. / This work aims to optimize the production cost of hollow core panels and prestressed joists using Genetic Algorithms (GAs). The proposal of such an algorithm was inspired by the principle of natural selection of individuals, where the most ‘capable’ tends to remain in the population and reproduce, passing its genetic code onto the next generation. In some cases, this method can achieve good solutions when compared with conventional methods of optimization. The main goal of the work is to investigate AG as a technique for the minimization of the function cost of hollow core panel and prestressed joist applications. The analysis takes account of the verifications of the precast elements in the transitory stages as production,transportation and erection. The function cost is evaluated within the Brazilian context. The research compares the results using GAs with those using a conventional method, the Augmented Lagrangian. The results provide evidence the effectiveness of the GAs with relation to a conventional method. The research considers three families of the simple GA, searching to identify, among them, the adjusted variant in the search of the solution of the problems.

Essa pesquisa apresenta um estudo da composição de concreto reforçado com fibras de polipropileno e objetiva avaliar seu comportamento estrutural em um tipo de painel alveolar de parede fina. A adição de alto teor de fibras de polipropileno ao concreto modifica algumas de suas propriedades mecânicas, conferindo ao compósito maior resistência à tração, resistência à flexão, resistência ao impacto e tenacidade. Inicialmente foram realizados ensaios de caracterização do compósito, sendo eles: consistência, resistência à compressão axial, módulo de elasticidade, resistência à tração por compressão diametral, resistência à tração na flexão e tenacidade. Estudou-se o comportamento de todas essas propriedades mecânicas para os teores de fibras de polipropileno de 0, 1 e 2% em volume de concreto. Com intuito de estudar o comportamento mecânico dos painéis alveolares de parede fina pela adição de fibras, foram realizados ensaios de carga concentrada e flexão. Este estudo foi realizado para os teores de fibra de 0 e 2%. Foram produzidos dois tipos de corpos de prova, ambos com seção transversal de 300 mm x 140 mm, e comprimentos de 600 mm e 1200 mm, para cada teor de fibra e respectivamente para os ensaios de carga concentrada e flexão. Para os ensaios de caracterização, não houve indícios de resultados significantes da adição dos teores de fibras 1 e 2% para a resistência à compressão e módulo de elasticidade. Já os ensaios de consistência, resistência à tração e tenacidade, apresentaram resultados expressivos, principalmente para o teor de fibra de 2%. Para os ensaios nos corpos de prova no painel quanto à carga concentrada, observou-se aproximadamente dez vezes maior capacidade de carga, cinco vezes maior capacidade de deslocamento e sete vezes maior resistência à punção. Os ensaios à flexão no painel apresentaram tensões resistentes pouco menores para os corpos de prova do painel contendo 2% de fibras. Conclui-se então que as propriedades do material cimentício com elevado teor de fibras são apropriadas para aplicação em painéis alveolares de parede fina. No entanto, é necessário melhorar a trabalhabilidade da mistura, pois devido às pequenas espessuras das paredes do painel, o adensamento do material não foi realizado de forma satisfatória. / This research presents a study on the composition of polypropylene fiber reinforced concrete and intents to evaluate its structural behavior in a type of thin-walled hollow core panel. The addition of high content of polypropylene fibers to the concrete modifies some of its mechanical properties, giving the composite greater split tensile strength, flexural strength, impact strength and toughness. Firstly, characterization tests of the composite were performed: consistency, compressive strength, modulus of elasticity, split tensile strength, flexural tensile strength and toughness. It was studied the behavior of all these mechanical properties for the contents of polypropylene fibers of 0,1 and 2% by volume of concrete. In order to study the mechanical behavior of the thin-walled hollow core panels by the addition of fibers, were performed concentrated load and flexural tests. This study was performed for fiber contents of 0 and 2%. Two types of specimens were produced, both with a cross section of 300 mm x 140 mm, and lengths of 600 mm and 1200 mm, for each fiber content and to be tested respectively for the concentrated load and flexural tests. For the characterization tests, there was no evidence of significant results arising on the addition of the fiber contents 1 and 2% for the compressive strength and modulus of elasticity. The tests of consistency, tensile strength and toughness presented significant results, especially for the fiber content of 2%. The flexural tests on the panel showed slightly lower tensile strengths for specimens containing 2% fibers. It is then concluded that the properties of the high fiber cementitious material are suitable for application in thin-walled alveolar panels. However, it is necessary to improve the workability of the composite, because due to the small thicknesses of the panel walls, a satisfactorily consolidation of the material has not been achieved.

Este trabalho analisa, por meio de método experimental, a viabilidade de apropriar a produção de painel alveolar de concreto armado para aplicação em habitações de interesse social. Compara os processos industriais praticados para a produção deste elemento com alternativas inovadoras proporcionadas pela incorporação de fôrma interna, ou núcleo alveolar, que permite apropriar a tecnologia para aplicação em diferentes escalas conforme as características de cada empreendimento. Avalia a execução, a conectividade e o desempenho mecânico em relação à resistência à compressão do painel alveolar. Contribui com contexto de construção sustentável e o aproveitamento de resíduos industriais e conclui que o núcleo alveolar incorporado configura inovação tecnicamente viável no processo de produção do painel alveolar que pode ser utilizado em sistemas de fechamento e superestruturas (paredes portantes e lajes) em habitações. / This work analyzes the viability of appropriate the hollow core slab production for social housing construction, by means of an experimental method procedure. It compares the industrial process with innovated alternatives proportionate by internal hollow nuclear mould incorporate which provides technology appropriation for several production scales according to construction portage. Evaluate the execution, connectivity (with hydraulic and electric systems) and compressive strength of hollow core slabs. The researchs contributions is relate with the context of sustainable construction and the use of industrial waste as construction raw materials and it concludes that internal hollow nuclear mould incorporate is technically viable in hollow core slabs production´s process and this precast element could be applied in wall´s and slabs for housing construction.

Este trabalho apresenta dois tipos de ensaios não destrutivos para a correlação do módulo dinâmico com a resistência à compressão para elementos de concreto. Os métodos de ensaio são a ultrassonografia e o método de excitação por impulso utilizando o equipamento Sonelastic®. Neste trabalho estão descritos seus funcionamentos, aplicações e limitações. A ultrassonografia, através da propagação de ondas sonoras, fornece, indiretamente, o módulo de elasticidade dinâmico. Com o método de excitação por impulso obtêm-se as frequências naturais e os modos de vibração do elemento estudado, o que permite determinar seu módulo dinâmico. Primeiramente, estes ensaios foram utilizados em corpos-de-prova cilíndricos e lajes alveolares produzidas em laboratório para a obtenção das curvas de correlação e, em seguida, estes mesmos ensaios foram realizados na fábrica de concreto pré-moldado. Para elementos de geometria complexa, como é o caso das lajes alveolares, apresenta-se uma metodologia para a obtenção de uma equação analítica para o cálculo do módulo dinâmico no ensaio de excitação por impulso. Estes métodos tiveram o objetivo final de avaliar a resistência à compressão do concreto na pista de protensão da fábrica, e então determinar o melhor momento para a desforma e corte do cabo de protensão. Com ambos os métodos, obteve-se ótimas correlações do módulo dinâmico com a resistência à compressão dos elementos em laboratório. Na fábrica de concreto pré-moldado não foi possível obter uma curva de correlação representativa de toda a laje na pista de protensão, porém foi possível registrar um bom indicativo de que é possível obter boas correlações para futuras pesquisas no assunto / This work presents two types of non-destructive testing for the correlation of the dynamic elastic modulus with the compressive strength. The test methods are the ultrasonography and the impulse excitation using the Sonelastic® equipment. In this work the equipaments operations, applications and limitations are also described. The ultrasound test indirectly supplies the dynamic elastic modulus through the propagation of sound waves. The natural frequencies and the vibration modes of the studied elements are obtained through impulse excitation method allowing to determine its dynamic modulus. To start, these tests were used in cylindrical specimens and hollow core slabs produced in the laboratory to obtain the correlation curves, and then these same methods were performed in the pre-cast concrete plant. As for complex geometric elements, as in the case of hollow core slabs, a methodology is applied in order to obtain an analytic equation to calculate the dynamic modulus in the impulse excitation test. These methods had the final goal the evaluation of the strength of prestressed concrete lying on track of the plant, so as to determine the best moment to demold and cut the prestressed cable. Excellent correlations of the dynamic modulus with compressive strength of the elements made in laboratory were obtained using both methods. It was not possible to obtain in the precast concrete plant a representative correlation curve of the whole slab on the track, nevertheless, it was possible to record a good indication that it is possible to obtain good correlations for future research.

Paradoxalement, la sécurité routière est assurée entre autre par la production de particules d’usure ! Ainsi, près de 20 000 tonnes de garnitures de frein sont usées par an en France, dont 9 000 tonnes sous forme de particules d’usure aéroportées. Ces particules posent des problèmes de santé car leur composition chimique et leur morphologie font qu’elles interagissent avec la paroi alvéolaire entrainant des pathologies. Au cours de ces pathologies la phase la plus étudiée est la phase inflammatoire qui s’installe une fois que la particule a passé la première barrière de protection qui est le film de surfactant pulmonaire. En revanche, très peu d’études portent sur l’interaction directe des particules aéroportées avec le film de surfactant pulmonaire à cause de difficultés liées aux résolutions des moyens d’investigations cliniques. Alors-que ces études sont d’un intérêt fondamental puisque, de par ses propriétés physicochimiques de surfactant, ce film contrôle la mécanique respiratoire donc la capacité pulmonaire. Dans ce contexte, cette thèse analyse les mécanismes d’action de particules d’usure aéroportées modèles sur les propriétés physicochimiques et mécaniques des parois alvéolaires et plus particulièrement du film de surfactant pulmonaire. Pour cela, un modèle ex vivo de paroi alvéolaire reproduisant la composition, la microstructure du surfactant ainsi que les sollicitations mécaniques pendant les cycles respiratoires, a été mis au point. L’utilisation de ce modèle et les mesures associées ont permis d’élaborer une démarche d’identification des paramètres significatifs des particules qui déterminent leurs interactions avec le film de surfactant pulmonaire. Cela a permis de montrer que l’électronégativité des particules aéroportées est l’un des paramètres significatifs qui induit des changements couplés à différentes échelles, qui vont de la conformation moléculaire (nano), à la microstructure (micro) et aux propriétés mécaniques (macro) de la paroi alvéolaire, conduisant à la diminution de la capacité respiratoire. Ce modèle et les premiers résultats permettront à court terme, d’identifier les autres paramètres significatifs qui caractérisent les actions de particules d’usure aéroportées sur les propriétés mécaniques et physicochimiques des parois alvéolaires. Ceci permettra de connaitre leurs effets sur la capacité pulmonaire. Par conséquent, à plus long terme, cette connaissance permettra de modifier les matériaux en contact et leurs conditions de frottement pour générer des particules satisfaisant les exigences tribologiques et biologiques, donc tribo-bio-compatibles. / Paradoxically, road safety is assured among others by the production of wear particles! Thus, almost 20 000 tons of brake linings are worn each year in France. 9000 tons are airborne wear particles. Due to their size, chemical composition and morphology these particles will interact with the alveolar wall causing pathologies. In these pathologies the most studied is the inflammatory phase that appear after the particle has passed the first protective barrier which is the pulmonary surfactant film. However, very few studies have examined the direct interaction of airborne wear particles with pulmonary surfactant film. These studies are of fundamental interest because, by its physicochemical properties, the pulmonary surfactant film control the respiratory mechanics, hence the pulmonary capacity. In this context, this thesis analyzes the interaction mechanism of model airborne wear particles on the physicochemical and mechanical properties of the alveolar wall and more particularly of pulmonary surfactant film. For this, an ex vivo model of alveolar wall reproducing the composition of the surfactant, its microstructure and the mechanical stresses during the breathing cycles has been developed. This model and the associated measures allowed to develop a method for identifying significant parameters of the particles that determine their interaction with the pulmonary surfactant film. The results showed that the electronegativity of airborne particles is one of the significant parameters which induces changes at different scales ranging from molecular conformation (nano), microstructure (micro) and mechanical properties (macro) of the alveolar wall, leading to the diminution of the pulmonary capacity. This model and the first results will allow, at short term, to identify other significant parameters which characterize the actions of airborne wear particles on mechanical and physicochemical properties of alveolar walls, allowing to know their effects on lung capacity. Therefore, at longer term, this knowledge will permit to change the materials in contact and their friction conditions to generate wear particles satisfying tribological and biological requirements, so tribo-bio-compatibles.

Bei einem akuten Lungenversagen senkt maschinelle Beatmung mit niedrigen Atemzugvolumina das Risiko für einen Ventilationsassoziierten Lungenschaden (VALI). Allerdings sind weitere Beatmungseinstellungen (Positive End-Exspiratory Pressure [PEEP], Recruitment Maneuver [RM]) zur Reduktion eines VALI Gegenstand aktueller klinischer und grundlagentechnischer Forschung. Studiendaten produzieren diesbezüglich ein heterogenes Meinungsbild. Ein restriktives Management von PEEP und Lungenrekrutierung (ARDSnet-PEEP-Tabelle) findet gleichermaßen Befürworter wie Studien, welche die Minimierung von tidaler Rekrutierung (TR) durch RM und/oder PEEP-Titrierung als lungenprotektives Management propagieren. In diesem experimentellen 24-Stunden-Langzeitversuch mit akutem Lungenversagen (ARDS) durch ein klinisch relevantes, experimentelles Salzsäure-Aspirationsmodell am Schwein wurden drei verschiedene, randomisierte Beatmungsstrategien durch individuelle PEEP-Einstellung anhand der ARDSnet-PEEP-Tabelle (ARDSnet-Gruppe), des Open- Lung-Concept (OLC-Gruppe) und des EIT-generierten SDRVD (EIT-Gruppe) untersucht. Hinsichtlich der Frage nach lungenprotektiver, individueller Beatmung und der Vermeidung eines VALI wurden Parameter der quantitativen Computertomographie (CT), der non-invasiven bettseitigen Elektroimpedanztomographie (EIT) und aus histologischen Untersuchungen (Diffuse Alveolar Damage [DAD] Score) herangezogen und verglichen.:I. Inhaltsverzeichnis II. Abkürzungsverzeichnis 1. Einführung in die Thematik 1.1 Akutes Lungenversagen – Acute Respiratory Distress Syndrome (ARDS) 1.1.1 Definition, Epidemiologie, Ätiologie und Pathogenese des ARDS 1.1.2 Lungenprotektive maschinelle Beatmung und Ventilationsassoziierter Lungenschaden (VALI) bei Patienten mit ARDS 1.2 In der Studie verglichene Beatmungsstrategien 1.2.1 ARDS Network Protokoll (ARDSnet-Gruppe) 1.2.2 Open Lung Concept (OLC-Gruppe) 1.2.3 Elektroimpedanztomographie (EIT-Gruppe) 1.3 ARDS – Bildmorphologie (CT und EIT) und Histologie (DAD-Score) 1.3.1 Elektroimpedanztomographie (EIT) – Standard deviation of regional ventilation delay (SDRVD) 1.3.2 Computertomographie (CT) – Lungenkollaps und Tidale Rekrutierung 1.3.3 Histologie – Diffuse Alveolar Damage Score (DAD-Score) 1.4 Statistische Methoden 1.5 Wissenschaftliche Zielsetzung 2. Publikationsmanuskript 3. Zusammenfassung 4. Literaturverzeichnis 5. Anlagen (Supplemental Material) 6. Darstellung des eigenen Beitrags 7. Selbstständigkeitserklärung 8. Teilnahmebescheinigung: Vorlesung zur „Guten wissenschaftlichen Praxis“ an der Medizinischen Fakultät der Universität Leipzig 9. Wissenschaftlicher Lebenslauf 10. Publikationen 11. Danksagung

Die Inzidenz des Atemnotsyndroms ist bei männlichen Neugeborenen etwa 1,7-mal so hoch wie bei weiblichen. Zur Erforschung der Ursachen dieser Tatsache wurden in der vorliegenden Arbeit geschlechtsabhängige Unterschiede im transepithelialen Natriumtransport an fetalen distalen Lungenepithelzellen von Ratten untersucht. Die zugrunde liegende Versuchsanordnung stellt ein Modell der Typ II Pneumozyten des späten Frühgeborenen dar. In Ussing Kammer Messungen wurde ein höherer Natriumtransport in weiblichen Zellen im Vergleich zu männlichen Zellen nachgewiesen. Des Weiteren zeigten Genexpressionsanalysen eine höhere Expression der am Natriumtransport beteiligten Kanäle und Transporter in weiblichen Zellen. Um mögliche Ursachen der festgestellten Geschlechtsunterschiede zu eruieren, wurde die Genexpression von Hormonrezeptoren untersucht. Die Ergebnisse lassen vermuten, dass die Rezeptoren weiblicher Geschlechtshormone dabei eine wichtige Rolle spielen. Abschließend betrachtet diese Arbeit die absolute Zahl fetaler distaler Lungenepithelzellen in Rattenfeten beider Geschlechter. Hierbei fanden sich ebenfalls Geschlechtsdifferenzen. Zusammenfassend kann die vorliegende Arbeit zu einem besseren Verständnis der Pathogenese und der Inzidenz des Atemnotsyndroms des Frühgeborenen beitragen.

Gegenstand der vorliegenden Arbeit sind die purinergen Rezeptoren P2X4R und P2X7R. Die P2XR werden durch ATP aktiviert und stellen unselektive Kationenkanäle dar, die auch für Ca2+ durchlässig sind. Beiden P2XR-Subtypen werden in den Alveolarepithel Typ I (AT I)-Zellen der Lunge exprimiert und aufgrund ihrer Kanalaktivitäten in Zusammenhang mit der alveolären Flüssigkeitshomöostase gebracht. Bei bisherigen Untersuchungen wurde jedoch die mögliche Assoziation und Modulation der P2XR durch Mikrodomänen der Zellmembran außer Acht gelassen. Ein Modell von Garcia-Marcos zeigt, dass P2X7R in Zellen der Glandula submandibularis zum Teil mit Mikrodomänen assoziiert ist. Die funktionellen Eigenschaften von P2X7R sind dabei von der Lokalisation in der Zellmembran abhängig (Garcia-Marcos et al., 2006). Die Caveolen sind eine spezielle Form von Mikrodomänen, die in der Zellmembran der AT I-Zellen auftreten. Das Hauptstrukturprotein der Caveolen im Lungenepithel ist Caveolin-1 (Cav-1). Über die Verteilung von P2X4R und P2X7R in den AT I-Zellen war bislang sehr wenig bekannt. Unsere Arbeitsgruppe identifizierte bei einer Sequenzanalyse potentielle Cav-1-Bindemotive in der Aminosäureabfolge beider P2XR (Couet et al., 1997). Die Assoziation mit den Caveolen würde die P2XR in die räumliche Nähe verschiedener Signalmoleküle bringen und die Beteiligung an downstream Events ermöglichen. Für die folgenden Analysen wurde die Alveolarepithelzelllinie E10 genutzt, da die E10-Zellen AT I-typische Eigenschaften besitzen und P2X4R, P2X7R sowie die Caveoline Cav-1 und Cav-2 aufweisen. Die Untersuchungen konzentrierten sich auf die Assoziation von P2X4R und P2X7R mit Mikrodomänen der Zellmembran sowie die wechselseitige Beziehung der P2XR. Besonders wurde dabei auf die Assoziation der P2XR mit Cav-1 eingegangen. Zusätzlich wurde in vitro die Interaktion der C-terminalen Bereiche der beiden P2XR mit Membranlipiden untersucht. Einige Membranlipide sind eng mit weiteren Signalmolekülen verknüpft. Aus diesem Grund wurde die Auswirkungen der Reduzierung von P2X4R und P2X7R auf den Proteingehalt der Ca2+-aktivierbaren downstream-Effektoren PKCβI und CaM analysiert. Die Auswertungen der Ergebnisse ergaben Folgendes: P2X4R und P2X7R sind Subtyp-spezifisch in den Mikrodomänen der Zellmembran von E10-Zellen verteilt. Mit Hilfe von biochemischen und immunfluoreszenz-mikroskopischen Methoden konnte die Assoziation von P2X4R und P2X7R mit Mikrodomänen nachgewiesen werden. P2X7R ist zum Teil mit Cav-1 assoziiert, wobei Förster Resonanz Energie Transfer (FRET)-Analysen ergaben, dass beide Proteine partiell einen Abstand von kleiner als 10 nm zueinander aufweisen. Durch die Subtyp-spezifische Verteilung könnte die Funktionalität der P2XR-Subtypen spezifisch durch die Bestandteile der Mikrodomänen moduliert und reguliert werden (Martens et al., 2001). P2X4R und P2X7R sind in hochmolekularen Proteinkomplexen assoziiert. Die Ausbildung von hochmolekularen Proteinkomplexen wird in Zusammenhang mit der Assoziation von Proteinen mit Mikrodomänen diskutiert (Zurzolo et al., 2003). Die Untersuchung der molekularen Organisation von P2X4R und P2X7R in E10-Zellen mittels blue native- und high resolution clear native-PAGE zeigte, dass beide P2XR mit hochmolekularen Proteinkomplexen assoziiert sind. P2X7R konnte in drei Komplexen nachgewiesen werden. Im ersten Komplex von ~760 kDa liegt P2X7R mit Cav-1 assoziiert vor, während der dominant auftretende, zweite P2X7R-Subkomplex von ~580 kDa vermutlich nicht mit dem co-migrierten Cav-1/Cav-2-Komplex in Verbindung steht. Der dritte P2X7R-assoziierte Komplex war zusammen mit P2X4R bei ~430 kDa nachweisbar und Immunpräzipitationen bestätigten, dass P2X4R und P2X7R in einem Komplex miteinander assoziiert sind (Weinhold et al., 2010). P2X4R und P2X7R stehen in Wechselbeziehung zueinander. Diese Ergebnisse der siRNA-induzierte Herabregulation von P2X4R und P2X7R lassen vermuten, dass die beiden Rezeptoren direkt oder indirekt miteinander verbunden sind. So führte die Reduzierung von P2X4R zur Erhöhung des P2X7R-Proteingehaltes. Dabei nimmt P2X7R in der Zellmembran zu und verändert seine Verteilung nicht. Umgekehrt nimmt der Proteingehalt von P2X4R in den E10-Zellen zu, wenn P2X7R herabreguliert wird. Die Zunahme von P2X4R in der Zellmembran konnte zwar durch die Biotinylierung der Oberflächenproteine nachgewiesen werden, aber die Verteilung von P2X4R verschob sich zugunsten des intrazellulären P2X4R-Anteils. Vermutlich führt die Reduzierung von P2X7R zu Störungen im exo-/endozytotischen System. Die wechselseitige Zunahme der P2XR in den Mikrodomänen weist zudem auf einen kompensatorischen Mechanismus hin. Negativ geladene Phospholipide interagieren direkt mit den C-terminalen Abschnitten der P2XR. Mit den in vitro Bindetests konnte gezeigt werden, dass die C-terminalen Enden von P2X4R und P2X7R direkt mit den negativ geladenen Phosphoinositiden PI(4)P, PI(4,5)P2, PI(3,4,5)P3 sowie mit Phosphatidsäure, Phosphatidylserin, Phosphatidylglycerol, Cardiolipin und 3 Sulfogalactosylceramid interagieren können. Die Regulation der P2XR durch diese Phospholipide, vor allem PI(4,5)P2, und die Beteiligung der P2XR an Lipid-vermittelten Signalwegen in Epithelzellen, stellen einen möglichen Link zu weiteren downstream-Signalen dar. Die Reduzierung von P2X7R beeinflusst den Proteingehalt der downstream-Effektoren PKCβI und CaM. Sowohl im Lungengewebe von P2rx7(-/-) Mäusen als auch nach der Reduzierung von P2X7R in den E10-Zellen zeigte sich, dass der Proteingehalt der Signalmoleküle PKCβI und CaM vermindert war. Reduzierung von P2X4R hatte dagegen kaum Einfluss auf PKCβI und führte zur Erhöhung des CaM-Proteingehaltes, vermutlich hervorgerufen durch die Zunahme von P2X7R. Beide downstream-Effektoren sind in Mikrodomänen (Caveolen) der Zellmembran lokalisiert und können sowohl durch Lipid-vermittelte Signale als auch durch einen Kanal-vermittelten Ca2+-Einstrom aktiviert und reguliert werden. Die Ergebnisse der vorliegenden Arbeit zeigten, dass P2X4R und P2X7R in AT I-Zellen der Lunge nicht nur Kanaleigenschaften besitzen, sondern durch die Assoziation mit unterschiedlichen Mikrodomänen an verschiedene Signalwege gekoppelt sind. Trotzdem ist bisher wenig über die Funktionen der P2XR in AT I-Zellen hinsichtlich der Beteiligung an apoptotischen Prozessen, der Proliferation, der Differenzierung oder Migration und Wundheilung bekannt (Barth and Kasper, 2009). Aufgrund der komplexen Funktion, vor allem durch die Assoziation mit Cav-1 und der Wechselbeziehung mit dem P2X4R, wird der P2X7R für zukünftige Forschungen im alveolären Lungenepithel von Bedeutung sein. Barth K, Kasper M (2009) Membrane compartments and purinergic signalling: occurrence and function of P2X receptors in lung. FEBS J 276:341-353. Couet J, Li S, Okamoto T, Ikezu T, Lisanti MP (1997) Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins. J Biol Chem 272:6525-6533. Garcia-Marcos M, Perez-Andres E, Tandel S, Fontanils U, Kumps A, Kabre E, Gomez-Munoz A, Marino A, Dehaye JP, Pochet S (2006) Coupling of two pools of P2X7 receptors to distinct intracellular signaling pathways in rat submandibular gland. J Lipid Res 47:705-714. Martens JR, Sakamoto N, Sullivan SA, Grobaski TD, Tamkun MM (2001) Isoform-specific localization of voltage-gated K+ channels to distinct lipid raft populations. Targeting of Kv1.5 to caveolae. J Biol Chem 276:8409-8414. Weinhold K, Krause-Buchholz U, Rödel G, Kasper M, Barth K (2010) Interaction and interrelation of P2X7 and P2X4 receptor complexes in mouse lung epithelial cells. Cell Mol Life Sci 67:2631-2642. Zurzolo C, van Meer G, Mayor S (2003) The order of rafts. Conference on microdomains, lipid rafts and caveolae. EMBO Rep 4:1117-1121.

Introduction : Le but de cette étude rétrospective est de comparer les changements squelettiques et dento-alvéolaires causés par trois appareils myofonctionnels, soit le Twin Block (TB), le Xbow (Xb) et le Carrière® Motion 3D™ Appliance (CMA), durant la première phase de traitement pour corriger une classe II. Méthodes : Les radiographies céphalométriques latérales prétraitement (T0) et post-traitement myofonctionnel (T1) de 43 adolescents traités par un TB (15), un Xb (7) ou un CMA (21) ont été récupérées et tracées; 12 mesures squelettiques et 10 mesures dentaires ont été analysées. Les différences (T1-T0) ont ensuite été comparées entre les appareils en utilisant l’analyse ANOVA, le test-t indépendant ou le test U de Mann-Whitney. Résultats : Le traitement était, en moyenne, d’une durée de 10,1 ± 3,2 mois avec le TB et de 7,4 ± 2,7 mois avec le CMA. Le groupe TB a connu de plus grands changements au niveau de la longueur et de la position sagittale de la mandibule en augmentant Cd-Pg (3,4 mm), Pg-Go (1,3 mm), N-B (//FH) (1,3 mm) et S-N-B (1,2°) plus que le CMA. Le TB a amélioré le Wits de 2,2 mm plus que le CMA. Le TB a également démontré une plus grande rétroclinaison des incisives supérieures en diminuant U1–FH (4,2°) plus que le CMA. Le CMA a restreint le mouvement antéro-postérieur de la première molaire supérieure tandis que le TB n’a pas eu d’effet significatif sur cette variable. Le seul changement cliniquement significatif résultant du CMA était en lien avec la proclinaison des incisives inférieures, L1–MP (4,1°). Les résultats associés au groupe Xb ont été exclus en raison de la petite taille de l’échantillon. Conclusion : La correction de la classe II est obtenue à l’aide d’une combinaison d’effets squelettiques et dento-alvéolaires avec le TB, mais principalement grâce à des effets dento-alvéolaires avec le CMA. Une taille d’échantillon plus grande est nécessaire afin de mieux comparer les effets du Xb. / Introduction: The objective of this retrospective study is to compare the skeletal and dento-alveolar changes caused by three functional appliances, the Twin Block (TB), the Xbow (Xb), and the Carrière® Motion 3D™ Appliance (CMA), during the first phase of treatment for class II correction. Methods: The pre-treatment (T0) and post-functional treatment (T1) lateral cephalometric radiographs of 43 adolescent patients treated with a TB (15), an Xb (7) or a CMA (21) were recovered and traced, and 12 skeletal and 10 dental measurements were analyzed. The differences (T1-T0) between the appliances will be compared using the ANOVA analysis, independent samples t-tests, or Mann-Whitney U-tests. Results: On average, the treatments lasted 10.1 ± 3.2 months with the TB and 7.4 ± 2.7 months with the CMA. The TB group experienced greater mandibular length and sagittal position changes by increasing Cd-Pg (3.4 mm), Pg-Go (1.3 mm), N-B (//FH) (1.3 mm), and S-N-B (1.2°) more than the CMA. The TB improved the Wits appraisal 2.2 mm more than the CMA. The TB also had a superior retroclining effect on the maxillary incisor inclination and position by reducing U1–FH (4.2°) more than the CMA. The CMA restrained the antero-posterior movement of the maxillary first molar while the TB had no significant effect on this variable. The only clinically significant change that the CMA displayed was in relation to mandibular incisor proclination, L1–MP (4.1°). The results related to the Xb group were excluded due to its very small sample size. Conclusion: Class II correction is achieved by a combination of skeletal and dento-alveolar changes with the TB, but primarily through dento-alveolar effects with the CMA. A bigger sample size is required in order to properly compare the effects of the Xb.