Zugl.: Osnabrück, University, Diss., 2006.

University, Diss., 2002--Stuttgart. / Erscheinungsjahr an der Haupttitelstelle: 2002.

Beyond these next four pages lies a product of my travels into the past without ever leaving the present. My experiences with nostalgia are elusive yet frequent, which drove me to attain a greater understanding of this subconscious tendency. Learning that these reminiscent reveries affect humanity at large, I found it odd that the limited literature on the subject includes only speculation regarding its purpose and utility. Consequently, I struggled in my search for a method to accomplish creative research. Quickly discarding the obvious, familiar stimuli summoning recollection, I then grew interested in the unfamiliar stimulating the nostalgic occurrence. With this as a jumping point, I wanted to simultaneously play with the idea of making my contemporary self, whom is so inclined toward the cinematic arts, meet with my past self (accompanied with my childhood interests). Film, already capturing a reality, already representing a representation, I found no better medium to facilitate the expansion of such a concept. My goal was to wipe away the fog on the window to the past and remember what I witnessed. In effect, utilizing this newfound knowledge of nostalgia to create a film of my past that evokes others to ponder their own. This thesis functions to share the memory of my experimental experience.

Zugleich: Diss. Erlangen-Nürnberg, 2008. / Literaturverz.

Die Patellaerstluxation ist eine besonders im jüngeren Lebensalter auftretende Verletzungsform. Bei persistierender Instabilität mit Rezidivereignissen ist die Rekonstruktion des medialen patellofemoralen Ligaments (MPFL) ein etabliertes Operationsverfahren. In dieser Arbeit wurde bei 17 Patienten (Durchschnittsalter 22,65 Jahre) die posturale Stabilität sowie das Kraftverhalten der Oberschenkelmuskulatur im Mittel 400,65 Tage nach Durchführung einer MPFL-Plastik in Form einer klinischen Verlaufsstudie bestimmt. Die Messung der posturalen Instabilität erfolgte im Einbeinstand auf einem Posturomed (Haider Bioswing). Dabei wurde aus der Ruheposition sowie nach Bewegungsimpuls in AP- und ML-Richtung die Wegstrecke der Standplattform aufgezeichnet. Bei allen Testmodi zeigten sich auf der operierten im Vergleich zur Gegenseite leichtgradig bessere Werte (nicht signifikant). Die Kraftdiagnostik erfolgte durch isokinetische Testung der Kniegelenksextensoren bzw. -flexoren im Seitenvergleich mittels Biodex System 3 (Medical Systems) zunächst unter konzentrischen Kontraktionsbedingungen bei 60°/s und 240°/s sowie im Anschluss bei exzentrischer Flexion bei 60°/s Winkelgeschwindigkeit. Im ersten Abschnitt zeigten sich auf der betroffenen Seite in die Knieextension niedrigere Werte als auf der Gegenseite (teilweise signifikant) bei keiner wesentlichen Differenz in die Flexion. Im zweiten Teil erzielten die Probanden im Mittel mit ihrem operierten Bein geringere Werte als mit dem nicht operierten Bein (teilweise signifikant). Zusammenfassend zeigt sich ca. ein Jahr postoperativ kein posturales Defizit jedoch ein Kraftdefizit des Streckapparates der operierten Seite. In der Literatur ist eine postoperative Quadrizepsdsyfunktion nach MPFL-Plastik vielfach beschrieben. Ein möglicher Pathomechanismus ist die arthrogene Muskelinhibition. Die Integration disinhibierender Maßnahmen in herkömmliche Rehabilitationsprogramme stellt einen denkbaren Therapieansatz dar. / Patellar luxation is an injury that affects especially young people. The reconstruction of the medial patellofemoral Ligament (MPFL) is an established surgical procedure in persistent instability with recurrent dislocation. This study examines the postural stability and force behavior of thigh muscles of 17 patients (average age 22.65 years) on average 400.65 days after MPFL reconstruction in a clinical follow-up study. The postural stability was measured with Posturomed (Haider Bioswing) by one-legged stance. Starting from resting position followed by impulse in AP- and ML-direction the distance of the platform was recorded. In all test modes slightly better results were achieved on the operated side compared to the non-operated side (not significant). The strength diagnosis was carried out by isokinetic testing of the knee joint extensors and flexors in a side-by-side comparison using Biodex System 3 (Medical Systems), first under concentric contraction conditions at 60°/s and 240°/s followed by eccentric flexion at 60°/s angular velocity. In the first section of testing the knee extension showed lower values on the affected side than on opposite side (partly significant) with no substantial difference in flexion. In the second section, the test subjects achieved with their operated leg lower values than with the non-operated leg (partly significant). In summary, one year postoperatively there is no postural deficit, but a strength deficit of the Quadriceps muscle on the operated side. Postoperatively dysfunction of the Quadriceps after MPFL reconstruction has been described frequently in the literature. A possible pathomechanism is the arthrogenic muscle inhibition. The integration of disinhibitory modalities into conventional rehabilitation programs is a conceivable therapeutic approach.

Photoreceptors are highly specialized neurons within the eye and the key retinal cells sensing light. They are indispensable for our visual perception and loss of photoreceptors consequently leads to loss of vision, a sense that alone is responsible for more than 30% of the input to our brain. Vision impairment and blindness is a leading cause of disability in the industrialized world and is in many cases ultimately due to a degeneration of the photoreceptors, which cannot be halted or reversed. Retinal degenerative diseases encompass a heterogeneous group of etiologies, mainly caused by various mutations in a plethora of proteins involved in the visual process. Currently, several therapeutic options are being explored, with so far one gene therapy for a rare inherited blinding condition being clinically approved. However, the gene therapy approach requires not only the presence of remaining photoreceptors but the tailoring of the therapy to each individual mutation. An alternative, more generally applicable approach is to restore vision through photoreceptor replacement therapy. As such, research on mouse-to-mouse photoreceptor transplantations has been carried out for many years, though with mixed results. In the last decade, it has however also become possible to generate large quantities of human photoreceptors through retinal organoid technology, allowing to instead transplant human cells. While promising, this field is still in development and principal conditions for successful photoreceptor transplantation have yet to be defined. Here, human-to-mouse photoreceptor transplantations were performed and assessed with the aim to receive insights into retinal cell replacement technology with specific focus on photoreceptor maturation, polarization and functional integration. Using a cone-degeneration host line, large-scale incorporation of human photoreceptor grafts into the murine retina was shown for the first time. It was found that for human photoreceptors, the choice of developmental stage strongly affects incorporation and maturation capacity. Furthermore, the results demonstrate the necessity of adequate graft-host interaction for successful transplant maturation and function, suggesting that photoreceptor replacement strategies might benefit from transplantation in earlier rather than late stages of retinal degeneration. Taken together, this thesis lays important groundwork for the further development of human photoreceptor replacement strategies to treat retinal degenerative disease.:ACKNOWLEDGEMENTS I ABSTRACT III ZUSAMMENFASSUNG V PUBLICATIONS VII TABLE OF CONTENTS IX LIST OF FIGURES XIII LIST OF TABLES XIV GENERAL ABBREVIATIONS XV GENE AND PROTEIN ABBREVIATIONS XVII 1 INTRODUCTION 1 1.1 THE RETINA AND LIGHT PERCEPTION 1 1.1.1 General structure of the eye 1 1.1.2 General structure of the retina 1 1.1.3 General photoreceptor structure 3 1.1.4 Phototransduction 4 1.1.5 Signal transmission to the brain 6 1.1.6 Major differences between rods and cones 7 1.1.7 The role of Müller glia in photoreceptor support and light perception 9 1.2 RETINAL DEGENERATION DISEASES AND TREATMENT OPTIONS 11 1.2.1 Retinal degeneration diseases 11 1.2.2 Therapeutic approaches to treat retinal degeneration diseases 12 1.3 CELL REPLACEMENT AS TREATMENT APPROACH FOR RETINOPATHIES 14 1.3.1 Transplantations of rodent retinal tissue and cells 14 1.3.2 Transplantations of human retinal tissue and cells 17 1.4 AIM OF THIS THESIS 22 2 CHARACTERIZATION OF CRX-MCHERRY HUMAN RETINAL ORGANOIDS AS PHOTORECEPTOR CELL SOURCE 23 2.1 AIMS 23 2.2 CHARACTERIZATION OF CRX-MCHERRY REPORTER-EXPRESSING CELLS 23 2.2.1 Crx-mCherry expression overlaps with endogenous CRX expression and increases over time 23 2.2.2 Crx-mCherry organoids contain an outer and an inner nuclear layer 24 2.2.3 Crx-mCherry+ cells express early and mature rod and cone markers 25 2.2.4 Crx-mCherry+ cells do not express proliferation markers 27 2.3 ENRICHMENT AND CHARACTERIZATION OF CRX-MCHERRY+ DONOR CELLS 28 2.3.1 Enrichment of Crx-mCherry+ cells by FACS 28 2.3.2 Characterization of Crx-mCherry enriched cells by single cell sequencing 29 2.3.3 Characterization of D200 Crx-mCherry-enriched cells by immunocytochemistry 30 2.4 SUMMARY 31 3 TRANSPLANTATION OF HUMAN CRX-MCHERRY+ GRAFTS AGED D100, D200 AND D300 INTO CPFL1 MICE 33 3.1 AIMS 33 3.2 CRX-MCHERRY+ CELLS OF ALL AGES CAN BE TRANSPLANTED AND SURVIVE IN THE MURINE RETINA 33 3.2.1 Human grafts can be identified by RCVRN staining 34 3.2.2 D100 Crx-mCherry+ transplants are larger than D200 and D300 grafts 34 3.2.3 Graft volume increase over time is not due to in vivo proliferation 36 3.3 GRAFT MORPHOLOGY DIFFERS WITH DONOR AGES 37 3.3.1 Human grafts can adopt an intraretinal position 37 3.3.2 Graft positioning changes over time 37 3.3.3 Qualitative differences in graft morphology between donor ages 38 3.4 GRAFT MATURATION 41 3.4.1 D200 but not D100 or D300 grafts develop large quantities of inner segments 41 3.4.2 Inner segment development is associated with close proximity to the host retina 42 3.5 HUMAN IDENTITY OF INTRARETINAL GRAFTS 43 3.5.1 Intraretinal Crx-mCherry+ grafts are largely a result of true morphological incorporation 43 3.5.2 Rare indications of potential human-to-mouse material transfer 45 3.6 SUMMARY 47 4 IN DEPTH CHARACTERIZATION OF TRANSPLANTED D200 CRX-MCHERRY+ CELLS 49 4.1 AIMS 49 4.2 EARLY POST TRANSPLANTATION DYNAMICS IN GRAFT POSITIONING AND GRAFT-HOST INTERACTIONS 49 4.2.1 Intraretinal and proximal D200 grafts interact with the host retina while isolated and distal clusters show only little interaction 49 4.2.2 Incorporation of D200 grafts is first evident at 8 weeks post transplantation 50 4.2.3 Host Müller glia extend processes into the graft before host bipolar cells 51 4.2.4 MG staining in D200 grafts originates from host MG 51 4.3 INCORPORATING D200 GRAFTS POLARIZE AND FORM STRUCTURES OF MATURE PHOTORECEPTORS 53 4.3.1 Grafts and host form an outer limiting membrane (OLM)-like structure 53 4.3.2 Inner segment formation occurs where an OLM is formed 54 4.3.3 Incorporating grafts form outer segment-like structures 55 4.3.4 Incorporating grafts form synaptic structures 57 4.3.5 Transplanted Crx-mCherry+ cells become enriched for cones 58 4.3.6 Higher levels of mature photoreceptor markers in ex vivo compared to in vitro cones 60 4.4 INCORPORATION AND MATURATION CAPACITY DEPEND ON THE HOST ENVIRONMENT 63 4.4.1 Graft morphology and maturation in C57BL/6JRj recipients resembles that in Cpfl1 hosts 63 4.4.2 Graft morphology and maturation in highly degenerated rd1 and tgCR host lines differs strongly from the outcome in models with an ONL 63 4.5 SUMMARY 67 5 FUNCTIONAL ASSESSMENT OF TRANSPLANTED CRX-MCHERRY+ CELLS 69 5.1 AIMS 69 5.2 HIGH-LEVEL FUNCTION 69 5.2.1 Light-Dark Box 69 5.3 TISSUE-LEVEL FUNCTION 71 5.3.1 Multi-electrode array assessment of D200+26w grafts in Cpfl1 mice 71 5.3.2 Isolation of cone-mediated RGC response through photopic light stimulation and L-AP4 addition 71 5.3.3 Graft-containing retinal portions exhibit cone-mediated light responses 72 5.4 SUMMARY 74 6 DISCUSSION AND FUTURE PERSPECTIVES 75 6.1 HUMAN GRAFTS CAN MORPHOLOGICALLY INCORPORATE INTO THE MODERATELY DEGENERATED MOUSE RETINA 75 6.2 INTRARETINAL GRAFTS MOSTLY REPRESENT TRUE INCORPORATION EVENTS, NOT MATERIAL TRANSFER 76 6.3 GRAFT MATURATION DEPENDS ON GRAFT-HOST INTERACTION 77 6.4 ESTABLISHMENT OF GRAFT-HOST INTERACTION AND GRAFT INCORPORATION 78 6.5 D200 CRX-MCHERRY+ CELLS ARE THE PREFERABLE DONOR POPULATION COMPARED TO D100 AND D300 80 6.6 CONES SHOW PREFERENTIAL SURVIVAL POST GRAFTING 81 6.7 FUNCTIONAL ANALYSES OF TRANSPLANTED ANIMALS 82 6.8 FUTURE CLINICAL TRANSLATION 85 6.9 MAJOR CONTRIBUTION TO OTHER WORK 88 7 FINAL CONCLUSION 89 8 MATERIALS AND METHODS 91 8.1 STUDY APPROVAL 91 8.2 MATERIALS 91 8.2.1 Materials and Chemicals 91 8.2.2 Cell Line 92 8.2.3 Mouse Lines 92 8.2.4 Antibodies 93 8.3 METHODS 95 8.3.1 Cell culture 95 8.3.2 Transplantations 96 8.3.3 Functional analyses 98 8.3.4 Immunohistochemistry and Immunocytochemistry 100 8.3.5 Imaging and image processing 103 8.3.6 Statistics 106 8.3.7 Single cell sequencing 107 8.3.8 Bioinformatic analysis 108 9 BIBLIOGRAPHY 111 10 APPENDIX 128 10.1 APPENDIX 1: ERKLÄRUNGEN ZUR ERÖFFNUNG DES PROMOTIONSVERFAHRENS 128 10.2 APPENDIX 2: BESTÄTIGUNG ÜBER EINHALTUNG DER AKTUELLEN GESETZLICHEN VORGABEN 129

Thema der Arbeit – der Ersatz des immateriellen Schadens als ein Mittel der Schutz der immateriellen Gütter. Ersatz des immateriellen Schadens als ein Mittel der Schutz der immateriellen Gütter ist in der Rechtsregelung des Litauens ziemlich neu, deshalb erregt die praktische Anwendug dieses Mittels bei der Schutz der bürgerlichen Rechten ziemlich viele Probleme. Autor der Arbeit analisiert die Konzeption des Ersatzes des immateriellen Schadens als des Mittels der Schutz der immateriellen Gütter, die Stelle des Ersatzes des immateriellen Schadens im System der Mittel der Schutz der immateriellen Gütter und beschäftigt sich mit den einzelnen Problemen der praktischen Anwendung des Ersatzes des immateriellen Schadens. In der vorliegenden Arbeit verhandelt man die Rechtsregelung des Ersatzes des immateriellen Schadens als des Mittels der Schutz der immateriellen Gütter in Litauen und in anderen Rechtsordnungen, basiert man auf die Rechtslehre und die Rechtsprechung. Im ersten Abschnitt der Arbeit beschäftigt sich man mit der Konzeption des Ersatzes des immateriellen Schadens als des Mittels der Schutz der immateriellen Gütter: verhandelt man die historische Entwicklung des immateriellen Schadensersatzes, vergleicht man die Regelung des Ersatzes des immateriellen Schadens in Litauen und in anderen Staaten, analisiert man die Ziele des Ersatzes des immateriellen Schadens. In der zweiten Abschnitt wird die Stelle des Ersatzes des immateriellen Schadens im System der Mittel der... [to full text]

Diss., 2006--Universiẗat Hagen.

Zugleich: Diss. Regensburg, 2008. / Literaturverz.

Eukaryotische DNA ist in Chromatin verpackt, einem Komplex aus DNA und Proteinen. Das ermöglicht Transkriptionsregulation von Genen durch Modulation ihrer Zugänglichkeit. Histone sind evolutionär konservierte Chromatinproteine, die durch posttranslationale Modifikationen (PTMs) modifiziert sind. Das legt nahe, dass deren Primärstruktur und ihre PTMs funktionellen Beschränkungen unterliegen. Genetische Ansätze zur Entschlüsselung von Struktur-Funktions-Beziehungen für Histone waren auf Einzeller und D. melanogaster beschränkt. Das Histon-Ersatz-System in D. melanogaster, bei dem Histontransgene verwendet werden, um endogene Histone zu ersetzen, war nicht für systematische Untersuchung dieser Beziehungen ausgelegt. In meiner Arbeit habe ich die Funktion von Threonin 11 in Histon H3 (H3T11) untersucht, das phosphoryliert werden kann. Ich analysierte zwei Mutationen in H3T11 (H3T11A und H3T11E) und stellte fest, dass beide zur Derepression von Transposons führen. H3T11E hat in Gegenwart von Wildtyp-Histon H3 einen dominanten Phänotyp mit transkriptomweiten Folgen. Dazu gehören Induktion von Immun-Genen und Unterdrückung von mit DNA-Stoffwechsel in Verbindung stehenden Genen. Die Mutationen wurden unter der Prämisse charakterisiert, ein Analyse-Schema für eine große Anzahl von Histon-Ersatz-Stämmen zu entwickeln und Probleme zu identifizieren, die die Analyse beeinträchtigen. Dabei habe ich das Verfahren zur Erzeugung von Histon-Ersatz-Stämmen optimiert. Dazu gehören die optionale Verwendung größerer Histon-Transgene und zuverlässigere Produktion und optimierte Rekombinations-Strategie dieser. Ich habe das Klonierungsverfahren gestrafft und eine Plasmid-Bibliothek erstellt, die es erlaubt, 178 verschiedene mutierte Histon-H3-Transgene zu erzeugen. Mit den Änderungen am Produktionsschema, ist diese Bibliothek eine wertvolle Ressource und wird dazu beitragen, die Funktion von Histon H3 und seiner PTMs während der Entwicklung eines Vielzellers besser zu verstehen. / Eukaryotic DNA is packaged into chromatin, a complex composed of DNA and proteins. This enables transcriptional regulation of genes through modulation of their accessibility. Histones are chromatin proteins, modified by post-translational modifications (PTMs) and their sequences are conserved in evolution. This suggests functional constraints for the primary structure of histones and their PTMs. Genetic approaches to decipher structure-function relationships for histone proteins were restricted to unicellular organisms and D. melanogaster. The histone replacement system in D. melanogaster, which uses histone transgenes to replace endogenous histones, was not adapted for systematic interrogation of such relationships. Here, I investigated the function of threonine 11 in histone H3 (H3T11), which can be phosphorylated. I analyzed two mutations in H3T11 (H3T11A and H3T11E) and found that both lead to de-repression of transposable elements. I also found that H3T11E, has a dominant phenotype in the presence of wildtype histone H3 with transcriptome-wide consequences. These include induction of immune-related genes and repression of genes associated with DNA metabolism. I characterized both mutations under the premise of establishing an analysis scheme suitable for a large set of histone replacement strains and identifying problems that interfere with this analysis. As a consequence, I optimized the procedure to generate histone replacement strains. These include an option to incorporate larger histone transgenes, a more reliable production of transgenes and an optimized strategy to recombine them. I streamlined the cloning procedure and created a plasmid library allowing for the generation of 178 distinct mutant histone H3 transgenes. Together with my amendments to the production scheme, this library provides a valuable resource to the field and will help to better understand the function of histone H3 and its PTMs during the development of a multicellular organism.