Objetivos: comparar o telescópio com lente de contato (TLC) com o telescópio convencional (TC) em pacientes com degeneração macular relacionada à idade (DMRI) e baixa visão, com respeito a: acuidade visual (AV), campo de visão (CV), satisfação do paciente com a visão oferecida pelo telescópio (SV), dificuldade de uso do telescópio (DU) e satisfação do paciente com o aspecto estético do telescópio (SE). Materiais e Métodos: Em ensaio clínico randomizado mascarado, foram incluídos 12 pacientes com DMRI e baixa visão, formando 2 grupos de 6 pacientes cada: grupo 1 (uso de TLC) e grupo 2 (uso de TC). Os telescópios usados no estudo foram padronizados para que tivessem a mesma magnificação (2,8x). A AV e o CV foram aferidos antes e durante o uso do telescópio, enquanto a SV, a DU e a SE foram obtidas após a intervenção. Resultados: Não houve diferenças entre os grupos na linha de base. Os dois telescópios demonstraram melhora da AV em relação à linha de base (P=0,002 com o TLC e P<0,001 com o TC) e não houve diferença entre os grupos a este respeito. O CV foi reduzido em 15° com o TLC (P<0.001) e em 54.3° com o TC (P<0.001), em comparação com a linha de base, e tais diferenças também foram significativas entre os grupos durante a intervenção (P<0.001). Os escores de SV foram semelhantes entre os grupos testados. Já os escores de SE, foram maiores com o TLC (P<0.001), assim como os de DU (P=0.003), em relação ao TC. Conclusões: Os dois telescópios promoveram melhora semelhante da AV em pacientes com DMRI e baixa visão, mas o TLC acarretou menor perda de CV em relação ao TC. A SE foi maior com o TLC, mas a DU também foi maior com o TLC, em relação ao TC. / Purpose: To compare the conventional telescope (CT) with the contact lens telescope (CLT) in patients with age-related macular degeneration (AMD) and low-vision, with respect to visual acuity (VA), visual field (VF), patient satisfaction with the vision provided by the telescope (VS), telescope use difficulties (UD) and patient satisfaction with the cosmetic appearance of the telescope (CS). Methods: In a masked randomized clinical trial, 12 patients with AMD and low-vision were enrolled in 2 groups with 6 patients each: group 1 (CLT use) and group 2. (CT use) The telescopes used in this study were standardized to have the same magnification power. (2.8x) Visual field and VA were obtained before and during the telescope use, while VS, UD and CS were obtained after the telescope use. Results: There were no significant differences between groups at baseline. Both groups achieved VA improvement with telescopes compared to baseline (P=0.002 in CLT group and P<0.001 in CT group) and there were no significant differences between groups in this regard. Visual field was reduced by 15° in CLT group (P<0.001) and by 54.3° in CT group (P<0.001) compared to baseline, and VF differences between groups were also significant during telescope use. (P<0.001) Scores observed in both groups were similar in regard to VS. Telescope use difficulties were significant higher in CLT group (P=0.003) as well as CS scores (P<0.001) compared to CT group. Conclusions: Both telescopes provide similar improvement in VA in AMD patients with low-vision, but CLT caused less VF reduction than CT use. Patient satisfaction with the cosmetic appearance of the telescope was higher in CLT group, but UD was also higher in this group compared to CT group.

Orientador: Vicente Lopes Júnior / Banca: Nobuo Oki / Banca: José Manoel Balthazar / Resumo: Este trabalho tem como propósito projetar controladores para aplicação em tempo real em uma estrutura flexível, objetivando a redução de vibração estrutural. Os controladores são projetados segundo o enfoque de otimização convexa, com formulações envolvendo desigualdades matriciais lineares (LMIs). Duas diferentes sínteses de realimentação são consideradas. A primeira é o projeto de controladores por realimentação de estados, estimados por um observador. A segunda metodologia é baseada no controle H∞ via realimentação do sinal de saída. O modelo matemático da estrutura, usado no projeto dos controladores, foi obtido utilizando o método de Lagrange. A estrutura considerada representa um modelo de um edifício flexível controlado por uma massa móvel (Active Mass Damper -AMD) localizada no topo. A estrutura é submetida a dois tipos de excitações, sísmica e senoidal. Uma mesa de vibração (Shake Table) foi usada para aplicar as excitações. Para rodar o experimento de controle foi usado uma placa de aquisição (MultiQ - PCI) e o software de controle Wincon. Os controladores foram desenvolvidos usando o Simulink e executado em tempo real usando o Wincon. Testes experimentais foram realizados para comprovação e avaliação das metodologias propostas. / Abstract: The proposal of this work is to design real time controllers for application in flexible structure, aiming the structural vibration reduction. The controllers are designed by convex optimization involving linear matrix inequalities (LMIs) approaches. Two different methodologies to feedback the system are explained. The first one is the design controller by state feedback based on observer. The second one is based on H∞ output feedback control. The mathematical model of the structure, used in the controller design, was obtained by Lagrange's method. The structure can represent a flexible building, and it is controlled by a driving mass located at the top. The structure is submitted to seismic and sinusoidal excitations. A vibration table (Shake Table) was used to apply the excitations. The experimental tests were realized using an acquisition board (MultiQ - PCI) and the Wincon control software. The controllers were developed using Simulink, and it run in real time using the Wincon software. Experimental tests were accomplished to validate and evaluate the proposal methodologies. / Mestre

O presente trabalho mostra os resultados obtidos no estudo da utilização do rejeito bruto da extração de carvão mineral como agregado miúdo na produção de blocos de concreto para pavimentação de vias públicas. Neste trabalho os blocos de concreto foram produzidos na fábrica da prefeitura de Criciúma, que está situada nas dependências do Presídio Municipal Santa Augusta. Esta fábrica proporciona uma opção de trabalho aos apenados que adquiriram o direito de cumprirem suas penas em regime semiaberto, cumprindo assim, um importante papel social na ressocialização destas pessoas, além de produzir o material de revestimento, que é aplicado nas vias públicas do município, a um custo bastante atraente. Durante a produção das amostras dos blocos de concreto com o rejeito de carvão, procurou-se ao máximo não alterar o processo desenvolvido na fábrica onde são produzidos os blocos de concreto empregados na pavimentação das vias públicas de Criciúma. Sendo assim, a única alteração imposta às amostras foi a substituição proporcional do agregado miúdo natural, areia lavada, pelo agregado de rejeito de carvão mineral, nas proporções de 25%, 50%, 75% e 100% O lote de estudo contou ainda com a produção de blocos de concreto sem adição de rejeito de carvão, que teve como função servir de base comparativa para as análises de compressão, abrasão e absorção de água. Posteriormente foi realizado um estudo para avaliar a possibilidade de geração, pelos blocos de concreto, de drenagem ácida de mina (DAM), que é a principal fonte de poluição do solo e dos corpos d’agua dos municípios pertencentes à AMREC – Associação dos Municípios da Região Carbonífera. Como resultado, aponta-se a viabilidade de utilização de rejeito de carvão nas proporções de 25% e 50% em substituição ao agregado miúdo natural (areia), criando assim, uma nova perspectiva às mineradoras de carvão da AMREC, que poderão dar um fim nobre ao rejeito de carvão, proporcionando uma produção mais limpa e sustentável, além de poderem contar com uma nova fonte de receita proporcionada pela comercialização do rejeito de carvão produzido na sua principal atividade comercial. / This document presents the results obtained in the study of using raw waste of coal extraction. In this work the concrete block for public roads paving were produced in a factory owned by Criciuma city hall, which is situated at the municipal Penitentiary Santa Augusta. During the concrete samples blocks production with the coal waste, it was tried its best in the way of not changing the process developed at the concrete blocks production site used in the paving of Criciuma´s streets. Thus, the only change that was proportionally imposed in the sample was the replacement of fine natural aggregate, washed sand, for the coal tailings aggregate in the proportions of 25%, 50%, 75% and 100%. The batch study also included the production of concrete blocks without adding coal tailing, which was to form the basis of comparative function for the compression tests, abrasion and water absorption. Afterwards, it was done a study to assess the possibility of generating, by concrete blocks, acid mine drainage (AMD), which is the main source of soil and water pollution in the municipalities belonging from AMREC - Association of Municipalities of the Coal Region. As a result, it points feasibility of using waste coal in the proportions of 25% and 50% to replace the natural aggregate (sand), thus creating a new perspective to AMREC coal mining companies, which may give a noble and to reject coal, providing a cleaner and sustainable production, besides being able to count on a new source of revenue provided by the commercialization of coal waste produced in its main commercial activity.

Objetivos: comparar o telescópio com lente de contato (TLC) com o telescópio convencional (TC) em pacientes com degeneração macular relacionada à idade (DMRI) e baixa visão, com respeito a: acuidade visual (AV), campo de visão (CV), satisfação do paciente com a visão oferecida pelo telescópio (SV), dificuldade de uso do telescópio (DU) e satisfação do paciente com o aspecto estético do telescópio (SE). Materiais e Métodos: Em ensaio clínico randomizado mascarado, foram incluídos 12 pacientes com DMRI e baixa visão, formando 2 grupos de 6 pacientes cada: grupo 1 (uso de TLC) e grupo 2 (uso de TC). Os telescópios usados no estudo foram padronizados para que tivessem a mesma magnificação (2,8x). A AV e o CV foram aferidos antes e durante o uso do telescópio, enquanto a SV, a DU e a SE foram obtidas após a intervenção. Resultados: Não houve diferenças entre os grupos na linha de base. Os dois telescópios demonstraram melhora da AV em relação à linha de base (P=0,002 com o TLC e P<0,001 com o TC) e não houve diferença entre os grupos a este respeito. O CV foi reduzido em 15° com o TLC (P<0.001) e em 54.3° com o TC (P<0.001), em comparação com a linha de base, e tais diferenças também foram significativas entre os grupos durante a intervenção (P<0.001). Os escores de SV foram semelhantes entre os grupos testados. Já os escores de SE, foram maiores com o TLC (P<0.001), assim como os de DU (P=0.003), em relação ao TC. Conclusões: Os dois telescópios promoveram melhora semelhante da AV em pacientes com DMRI e baixa visão, mas o TLC acarretou menor perda de CV em relação ao TC. A SE foi maior com o TLC, mas a DU também foi maior com o TLC, em relação ao TC. / Purpose: To compare the conventional telescope (CT) with the contact lens telescope (CLT) in patients with age-related macular degeneration (AMD) and low-vision, with respect to visual acuity (VA), visual field (VF), patient satisfaction with the vision provided by the telescope (VS), telescope use difficulties (UD) and patient satisfaction with the cosmetic appearance of the telescope (CS). Methods: In a masked randomized clinical trial, 12 patients with AMD and low-vision were enrolled in 2 groups with 6 patients each: group 1 (CLT use) and group 2. (CT use) The telescopes used in this study were standardized to have the same magnification power. (2.8x) Visual field and VA were obtained before and during the telescope use, while VS, UD and CS were obtained after the telescope use. Results: There were no significant differences between groups at baseline. Both groups achieved VA improvement with telescopes compared to baseline (P=0.002 in CLT group and P<0.001 in CT group) and there were no significant differences between groups in this regard. Visual field was reduced by 15° in CLT group (P<0.001) and by 54.3° in CT group (P<0.001) compared to baseline, and VF differences between groups were also significant during telescope use. (P<0.001) Scores observed in both groups were similar in regard to VS. Telescope use difficulties were significant higher in CLT group (P=0.003) as well as CS scores (P<0.001) compared to CT group. Conclusions: Both telescopes provide similar improvement in VA in AMD patients with low-vision, but CLT caused less VF reduction than CT use. Patient satisfaction with the cosmetic appearance of the telescope was higher in CLT group, but UD was also higher in this group compared to CT group.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22323号 / 医博第4564号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 大森 孝一, 教授 横出 正之, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Age-related macular degeneration (AMD) is the leading cause for visual impairment in the elderly. The etiology of AMD remains unclear. Clinical and histopathological studies suggest that photoreceptors play a role in disease progression. Here, we found that photoreceptors of AMD patients show adaptive changes in gene expression, suggestive of a nutrient shortage. To study the effect of these changes, we mimicked the metabolic alteration in mouse photoreceptors, by disruption of the Tuberous Sclerosis Complex (TSC). This led to AMD hallmarks, including the advanced stages of geographic atrophy (GA) and choroidal neovascularization (CNV). Furthermore, we found that disease onset requires the activity of the mammalian target of rapamycin complex 1 (mTORC1). To study the contribution of photoreceptors to disease, we profiled retinal phospholipids as photoreceptors are rich in phospholipids. We found a reduction in two docosahexaenoic acid (DHA)-containing phospholipids. Feeding DHA to mutant mice, alleviated most AMD-associated hallmarks. To study the inflammatory complications seen with current anti-vascular endothelial growth factor (VEGF) treatments for CNV we used rAAV-mediated gene transfer to overexpress an anti-VEGF protein. We found that inhibition of VEGF can promote retinal inflammation. The data suggests that targeting photoreceptor metabolism may provide novel therapies to treat AMD.

Ackmann, Charlotte Osmotische Induktion des Komplementfaktors C9 in retinalen Pigmentepithelzellen Universität Leipzig, Dissertation 98 Seiten, 208 Literaturangaben, 28 Abbildungen, 8 Tabellen Die altersbedingte Makuladegeneration (AMD) ist die häufigste Ursache für Erblindung bei Erwachsenen in den industrialisierten Ländern. Die AMD ist unter anderem eine chronisch entzündliche Erkrankung, bei der die Aktivierung der alternativen Komplementkaskade eine Rolle spielt. Daneben erhöht Bluthochdruck, der auch durch eine salzreiche Ernährung getriggert wird, das Risiko an einer AMD zu erkranken. Untersucht wurde die Genexpression des Komplementfaktors C9 unter verschiedenen pathologischen Bedingungen in humanen retinalen Pigmentepithel (RPE)-Zellen sowie deren Wirkung auf die physiologischen Eigenschaften der Zellen. Gezeigt wird, dass die Expression des C9 Gens in humanen RPE-Zellen spezifisch durch Hyperosmolarität, Hypoxie und oxidativen Stress induziert wird. Die Menge an C9 Protein wurde durch Hyperosmolarität leicht aber signifikant erhöht. Die hyperosmotische Induktion der C9 mRNA ist abhängig von der Aktivierung der Signalproteine p38 MAPK, ERK1/2, JNK, PI3K, sowie der Transkriptionsfaktoren STAT3 und NFAT5 während für die Hypoxie-induzierte C9 mRNA Expression nur eine Beteiligung des Transkriptionsfaktors STAT3 nachgewiesen wurde. Die Aktivierung verschiedener Signalwege durch Hyper-osmolarität und Hypoxie lässt vermuten, dass eine hohe Kochsalzaufnahme auch unter normoxischen Verhältnissen die Eigenschaften RPE-Zellen verändert. Hyperosmolarität hemmt die Proliferation und Migration der RPE-Zellen, während chemische Hypoxie nur die Proliferationsrate verringert. Die Wirkung einer erhöhten extrazellulären NaCl-Konzentration auf die C9 mRNA Expression wird über zwei Mechanismen vermittelt: über die Erhöhung der extrazellulären Osmolarität und über die Veränderung des NaCl-Gradienten über der Plasmamembran. Die NaCl Wirkung über den veränderten NaCl-Gradienten lässt vermuten, dass eine übermäßige Aufnahme von Kochsalz nicht nur über die Erhöhung des Blutdruckes die Pathogenese der AMD stimuliert, sondern dass Kochsalz auch eine direkte stimulierende Wirkung auf RPE-Zellen besitzt. Diese Vermutung könnte erklären, weshalb hoher Blutdruck ein Risikofaktor der AMD ist, aber Medikamente zur Behandlung des Bluthochdruckes das Risiko der AMD nicht verändert.:Inhaltsverzeichnis Bibliographische Beschreibung IV Abkürzungsverzeichnis V 1. EINLEITUNG 1 1.1. Das retinale Pigmentepithel (RPE) 1 1.2. Die altersbedingte Makuladegeneration (AMD) 2 1.2.1. Die atrophe AMD 3 1.2.2. Die exsudative AMD 4 1.2.3. Die Aktivierung des Komplementsystems bei der exsudativen AMD 5 1.3. Risikofaktoren der AMD 7 1.4. Einfluss von Hypertonie auf die AMD 9 1.4.1. Hypertonie 9 1.4.2. Hypertonie als Risikofaktor der AMD 10 1.5. Therapie der AMD 11 2. FRAGESTELLUNG 13 3. MATERIAL UND METHODEN 14 3.1. Arbeitsmaterialien 14 3.1.1. Substanzen 14 3.1.2. Medien 15 3.1.3. Kits 15 3.1.4. Primer 16 3.1.5. Längenstandards 16 3.1.6. Antikörper 16 3.1.7. Rekombinante humane Proteine 17 3.1.8. Selektive Hemmer 17 3.1.9. Geräte 17 3.2. Zellbiologische Methoden 19 3.2.1. Zellkultivierung 19 3.2.2. Zellstimulation 20 3.2.3. Bestimmung der Proliferationsrate 21 3.2.4. Zellmigration 22 3.3. Molekularbiologische Methoden 23 3.3.1. RNA-Präparation 23 3.3.2. RNA Quantifizierung 24 3.3.3. cDNA-Synthese 24 3.3.4. Real-Time PCR 24 3.3.5. Agarose-Gel-Elektrophorese 26 3.3.6. Untersuchungen mit short interfering (si) RNA 27 3.4. Immunologische Methoden 28 3.4.1. Western Blot 28 3.4.1.1. Extraktherstellung aus RPE-Zellen 28 3.4.1.2. Proteinkonzentrationsbestimmung nach Bradford 29 3.4.1.3. SDS-Gelektrophorese 30 3.4.1.4. Western Blot 32 3.5. Statistische Auswertung 33 4. ERGEBNISSE 34 4.1. Regulation der Genexpression von Komplementfaktoren in RPE-Zellen 34 4.1.1. Hyperosmolarität erzeugt durch NaCl 34 4.1.2. Hyperosmolarität erzeugt durch Sucrose 36 4.1.3. Wirkung von Hypoosmolarität und oxidativem Stress 37 4.1.4. Wirkung von Hypoxie 38 4.1.5. Gemeinsame Wirkung von CoCl2 und NaCl 38 4.1.6. Wirkung von Zytokinen 39 4.1.7. Wirkung von Serum- und Gerinnungsfaktoren sowie Glukose 40 4.1.8. Wirkung von Entzündungsmediatoren 40 4.1.9. Wirkung von Matrixmetalloproteinase und Triamcinolon 41 4.2. Wirkung von Hyperosmolarität auf die Transkription und die Stabilität der C9 mRNA 42 4.3. Beteiligung intrazellulärer Signalwege und Transkriptionsfaktoren an der Induktion der C9 mRNA unter Hyperosmolarität und Hypoxie in RPE-Zellen 43 4.3.1. Beteiligung intrazellulärer Signalwege und Transkriptionsfaktoren an der NaCl-induzierten C9 mRNA Expression in RPE-Zellen 43 4.3.2. Hyperosmolare Induktion der C9 mRNA: Einfluss des Transkriptionsfaktors NFAT5 44 4.3.3. Beteiligung intrazellulärer Signalwege und Transkriptionsfaktoren an der Hypoxie-induzierten C9 mRNA Expression bei RPE-Zellen 46 4.4. Aktivierung intrazellulärer Signalproteine durch Hyperosmolarität bzw. Hypoxie 47 4.5. Wirkung von Hyperosmolarität auf das C9 Protein 48 4.6. Wirkung von Hyperosmolarität auf die physiologischen Eigenschaften der RPE- Zellen 50 4.6.1. Wirkung von Hyperosmolarität auf die Zellproliferation 50 4.6.2. Wirkung von Hyperosmolarität auf die Zellmigration 51 4.6.3. Wirkung von Hyperosmolarität auf die Zellvitalität 51 5. DISSKUSION 53 5.1. Wirkung von Hyper-, Hypoosmolarqität, Hypoxie und oxidativem Stress auf die Genexpression von C9 in humanen RPE-Zellen 53 5.3. Wirkung von Hyperosmolarität auf die Stabilität der C9 mRNA 56 5.4. Beteiligung intrazellulärer Signalwege und Transkriptionsfaktoren an der NaCl- bzw. CoCl2-induzierten C9 mRNA Expression in RPE-Zellen 56 5.5. Einfluss des Transkriptionsfaktors NFAT5 auf die hyperosmolare C9 Induktion 58 5.6. Aktivierung intrazellulärer Signalproteine durch Hyperosmolarität bzw. Hypoxie 58 5.7. Wirkung von Hyperosmolarität auf das C9 Protein 59 5.8. Wirkung des Komplementfaktors C9 und der Hyperosmolarität auf die physiologischen Eigenschaften von RPE-Zellen 60 5.8.1. Wirkung auf die Zellproliferation 60 5.8.2. Wirkung auf die Zellmigration 60 5.8.3. Wirkung auf die Zellvitalität 61 5.9. Bedeutung für das Verständnis der Pathogenese der AMD 61 6. ZUSAMMENFASSUNG 63 7. LITERATURVERSZEICHNIS 68 8. TABELLENVERZEICHNIS 85 9. ABBILDUNGSVERZEICHNIS 86 10. ANHANG 88 10.1. Eigenständigkeitserklärung 88 10.2. Lebenslauf 89 10.3. Danksagung 90

La Dégénérescence Maculaire Liée à l’Age (DMLA) affecte actuellement plus d’un million de malades en France et concernera 288 millions de personnes dans le monde en 2040. Cette pathologie du sujet âgé est à l’origine d’un handicap fonctionnel majeur, source de perte d’autonomie et de dépendance. Malgré une forte prévalence, les performances posturales et oculomotrices des sujets atteints de DMLA restent peu étudiées et beaucoup d’interrogations sont à ce jour sans réponse. Ce projet de recherche comporte deux parties pour lesquelles nous avons utilisé différents dispositifs : les plateformes de force TechnoConcept® et Framiral®, le Mobile eyeBRAIN tracker EBT2®, et la tablette connectée Metrisquare®. Dans un premier temps, nous avons examiné l’impact de la DMLA unilatérale versus bilatérale sur le contrôle postural en comparaison avec des sujets sains âgés (Etude I), pour permettre de mettre en évidence le bénéfice de la rééducation posturale chez cette population de malades (Etudes II et III). Dans un second temps, nous avons étudié l’oculomotricité des sujets atteints de DMLA à travers leurs performances de saccades visuellement guidés et de lecture, afin de rééduquer ces mêmes performances via des exercices oculomoteurs et visuo-attentionnels (Etude IV, V et VI). En objectivant les mouvements oculaires ainsi que le déplacement du centre de pression chez les sujets atteints de DMLA uni- et bilatérale, en comparaison avec des sujets sains du même âge, nous avons cherché à confirmer notre hypothèse : le déficit visuel des sujets atteints de DMLA, à l’origine d’une malvoyance, entraine la mise en place de mécanismes adaptatifs qui, grâce à une rééducation adaptée, permettent de réduire les restrictions fonctionnelles posturales et oculomotrices. In fine, ces travaux apportent des solutions pour mieux prévenir et rééduquer ces déficits fonctionnels, grâce à des outils innovants qui permettront d’optimiser la prise en charge. / Nowadays, age-related macular degeneration (AMD) affects more than one million of people in France and will concern 288 million of people in the world in 2040. This pathology impacts elderly and generates a serious functional disability which causes autonomy loss and dependance. Despite the high prevalence, the postural and oculomotor performances of AMD subjects are little studied and there are still lot of questions without answers. This research project is composed by two parts and used different experimental techniques: TechnoConcept® and Framiral® force plateforms, Mobile eyeBRAIN tracker EBT2®, and Metrisquare® digital tablet. Firstly, we examined the impact of unilateral versus bilateral AMD in postural control, compared with healthy elderly (Study I), in order to evidence the benefits of postural training in these groups (Studies II and III). Secondly, we studied the oculomotor pattern in AMD subjects during visually guided saccades and reading tasks, to improve their performance with oculomotor and visuo-attentional exercises (Studies IV, V and VI). By objectifying eye movements as well as the displacement of center of pressure in unilateral and bilateral AMD subjects, compared with healthy age-matched controls, we want to confirm our hypothesis: the visual impairment of AMD subjects, which causes low vision, leads to the implementation of adaptive mechanisms that, through appropriate training, reduce postural and oculomotor functional restrictions.In fine, this research provides specialists with solutions to prevent and train these functional deficits with novator techniques, in order to optimize the coverage.

Bei der Altersabhängigen Makuladegeneration (AMD) handelt es sich um eine Erkrankung des Auges, die die Macula lutea, die Stelle des schärfsten Sehens betrifft. Sie ist verbunden mit der Degeneration von RPE-Zellen, die zur Dystrophie von Photorezeptoren und damit zum Verlust des zentralen Sehvermögens führt. Eine ähnliche Pathophysiologie ist bei der sogenannten Retinalen Pigmentepitheldystrophie (RPED) des Hundes zu beobachten. Die Transplantation von gesunden RPE-Zellen in das betroffene Gebiet stellt eine vielversprechende Therapiemöglichkeit dar. Die Transplantatabstoßung als Kom-plikation schränkt die klinische Anwendung ein. Eine beim Patienten nach Transplantation lebenslang durchgeführte systemische Immunsuppression ist mit erheblichen Nebenwirkungen verbunden. Deshalb bietet die Gentherapie unter Einbezug immunsuppressiver Zytokine wie beispielsweise des Interleukin-10 (IL-10) eine Lösung. In der vorliegenden Arbeit wurde ein selbst konstruierter IL-10-Expressionsvektor (Plasmid pCIneoIL-10) mittels Gentransfer in humane RPE-Zellen in vitro eingebracht. Untersucht wurde die Wirkung des sezernierten IL-10 auf die Proliferation von allogenen T-Lymphozyten mit und ohne allogene Makrophagen als professionelle antigenpräsentierende Zellen (APC). Neben humanen Spender RPE-Zellen (Spender-hRPE-Zellen) wurde eine immortalisierte Permanent-Zelllinie (hTERT-RPE1-Zellen) eingesetzt, deren Hauptvorteil in einer gleichbleibend hohen Wachstumsrate lag. Als transientes Transfektions-system für den Transfer von IL-10-DNA in hRPE-Zellen wurden kationische Lipide gewählt. Drei verschiedene Lipidformulierungen wurden miteinander verglichen und das optimale Transfektionsreagenz:DNA-Verhältnis, mit dem die höchste Transfektionseffizienz erreicht werden konnte, evaluiert. Eine Transfektionseffizienz von 23,3 ± 9,0 % (hTERT-RPE1-Zellen) beziehungsweise 10,3 ± 4,5 % (Spender-hRPE-Zellen) konnte erreicht werden. Die Transfektion hatte weder einen negativen Einfluss auf die Vitalität der hRPE-Zellen, noch wurde der natürliche Zelltod, die Apoptose, erhöht. Die IL-10-mRNA-Expression wurde mittels RT-PCR nachgewiesen. Lediglich bei den transfizierten hRPE-Zellen konnte IL-10-mRNA gefunden werden. Mittels ELISA konnte das IL-10-Protein gemessen werden. Die Sekretion des IL-10 in den Kulturüberstand von transfizierten hRPE-Zellen wurde dafür über einen Zeitraum von 7 Tagen untersucht. Es konnte festgestellt werden, dass die maximale IL-10-Proteinkonzentration bei beiden Zelllinien am Tag 3 mit Werten von 10,3 ± 0,8 ng/ml (hTERT-RPE1-Zellen) und 3,1 ng/ml (Spender-hRPE-Zellen) lag. Es bestand überdies eine positive Korrelation zwischen Transfektionseffizienz und synthetisiertem IL-10. Es wurde außerdem gezeigt, dass durch Stimulation mit dem immunmodulatorischen Zytokin Interferon-gamma (IFN-g) hRPE-Zellen MHC Klasse II-Moleküle vermehrt exprimierten. Damit sind sie ebenso wie die Makrophagen zur Antigenpräsentation fähig. Die Wirkung des von den transfizierten hRPE-Zellen sezernierten IL-10 auf die Proliferation von T-Lymphozyten wurde zwischen Tag 2 und Tag 6 (hTERT-RPE1-Zellen) beziehungsweise zwischen Tag 2 und Tag 4 (Spender-hRPE-Zellen) photometrisch untersucht. Die Proliferation allogener T-Lymphozyten mit beziehungsweise ohne Makrophagen konnte durch das sezernierte IL-10 supprimiert werden. Bei den hTERT-RPE1-Zellen lag ohne die Anwesenheit von professionellen APC am Tag 6 eine signifikante Reduktion der T-Lymphozytenproliferation vor, während bei Kokultivierung mit Makrophagen Signifikanzen am Tag 5 und Tag 6 erkennbar waren. Die immunsuppressive Wirkung von IL-10 konnte mittels Anti-IL-10-Antikörper neutralisiert werden. Damit wurde bewiesen, dass die proliferations-supprimierende Wirkung auf IL-10 zurückzuführen war. Diese Ergebnisse könnten demnach neue Möglichkeiten zur Verhinderung einer Abstoßungsreaktion nach RPE-Zelltransplantation bei Patienten mit AMD eröffnen / Age-related macular degeneration (AMD) is a disease of eyes affecting the macula lutea, the area of the retina with the highest density of retinal pigment epithelial cells (RPE cells). The disease is characterized by degeneration of RPE cells resulting in dystrophy of photoreceptors and finally loss of central vision. Transplantation of healthy RPE cells is a promising possibility for therapy but rejection of the allotransplant limits clinical application. One way to avoid this complications is a systemic immunosuppression of the recipient but this is combined with many side effects. In this thesis a self-constructed IL-10 expression vector (plasmid pCIneoIL-10) has been transferred into human RPE cells in vitro by gene transfer. In addition to human donor RPE cells a permanent RPE cell line (hTERT-RPE1 cells) was employed. Kationic lipids were used as transient transfection system for transfer of pCIneoIL-10 into hRPE cells. Three different lipid formulations and various ratios of transfection reagent:DNA were evaluated for highest transfection efficacy. With the optimized protocols a transfection efficacy of 23,3 ± 9,0 % (hTERT-RPE1 cells) and 10,3 ± 4,5 % (donor hRPE cells) was achieved. A negative influence on the viability of the hRPE cells after transfection was not observed. The IL-10 mRNA expression was analysed by reverse transcription-polymerase chain reaction (RT-PCR). Only in transfected hRPE cells the IL-10 mRNA-amplicon with 383 bp in size was found. Secretion of IL-10 protein in the cell culture supernatants of transfected hRPE cells was investigated using an enzyme-linked immunosorbent assay (ELISA) daily for 7 days. The IL-10 protein concentrations peaked at day 3 with 10,3 ± 0,8 ng/ml (hTERT-RPE1 cells) and 3,1 ng/ml (donor hRPE cells). The amount of secreted IL-10 positively correlated with transfection efficacy. After stimulation with the immunmodulatory cytokine interferon-gamma (IFN-g) the expression of MHC class II molecules on hRPE cells is increasing. Therefore they are able to present antigens similar to macrophages. Hence, the effects of recombinantly expressed IL-10 on the proliferation of allogeneic T lymphocytes were investigated both with and without allogeneic macrophages as professional antigen presenting cells (APC). Proliferation of T lymphocytes has been investigated colorimetrically between day 2 and day 6 (hTERT-RPE1 cells) and day 2 and day 4 (donor hRPE cells) respectively. The proliferation of allogeneic T lymphocytes with and without macrophages could be suppressed by the secreted IL-10. Signifikant reduction of proliferation was observed at day 6 in absence of professional APC (14,1 ± 1,1 % to 100% of untransfected control) and between day 5 (44,1 ± 4,9 %) and day 6 (37,4 ± 6,3%) in the presence of macrophages. It was possible to neutralize the immunosuppressive effect of IL-10 with anti-IL-10 antibodies. Proving that the suppressive effect of T lymphocyte proliferation was caused by IL-10. Thus, the specific IL-10 gene transfer into hRPE cells prior to transplantation may prevent rejection process and could prove a reliable method to help prevent loss of central vision due to AMD.

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