It is well known that ultraviolet (UV) B (280-315 nm) and blue light (400-500 nm) radiation can produce phototoxic lesions in the neural retina and the retinal pigment epithelium (RPE). In the first section of this thesis, bovine lens cells (epithelium and superficial cortical fibre cell) and human retinal pigment epithelial (ARPE-19) cells were used to characterize in vitro changes following oxidative stress with UVB radiation in ocular lens optics and cellular function in terms of mitochondrial dynamics. In the second part, human retinal pigment epithelial (ARPE-19) cells and in vitro bioassays were used together to develop an in vitro approach for UV radiation-induced retinal toxicology research. In the third chapter, the in vitro approach developed above was used with intraocular lens (IOL) materials to evaluate the UV radiation blocking efficiency of commercially available IOL’s. Lastly, narrowband blue light irradiation and in vitro assays were used to determine more precisely the wavelengths of blue light responsible for photochemical lesions of the retina as an effort to contribute to future IOL designs. The results from mitochondrial dynamics of lens cells and RPE cells show significant decreases in mitochondrial movement after UVB irradiation in a dose dependent manner. Results obtained from four in vitro assays (Alamar blue assay, confocal microscopy for mitochondrial distribution and nucleic acids damage, phagocytotic activity assay) for evaluating the UVB-induced damage in ARPE-19 show significant decreases in cell viability as well as phagocytotic activity of RPE cells after UVB radiation. In addition, the results show that UV radiation can also induce the degradation of DNA/RNA and mitochondria of RPE cells in a dose dependent manner. The results of the UV blocking efficiency test of commercially available IOL materials show very effective UV blocking ability, allowing no cellular damage at all, in comparison to an IOL uncovered control cell. The results of three different wavelengths of blue light exposure show that only 400 nm blue light radiation can cause significant damage to RPE cells, while 420 and 435.8 nm blue light radiation cause no cellular damage at all. In conclusion, UVB and blue light radiation can cause phototoxic damage to the retinal pigment epithelium as a result of oxidative stress, and in vitro bioassays used for this research may offer a sensitive, and meaningful biomarker approach, not only for evaluating RPE function after oxidative and chemical stress, but also for evaluating IOL effectiveness.

It is well known that ultraviolet (UV) B (280-315 nm) and blue light (400-500 nm) radiation can produce phototoxic lesions in the neural retina and the retinal pigment epithelium (RPE). In the first section of this thesis, bovine lens cells (epithelium and superficial cortical fibre cell) and human retinal pigment epithelial (ARPE-19) cells were used to characterize in vitro changes following oxidative stress with UVB radiation in ocular lens optics and cellular function in terms of mitochondrial dynamics. In the second part, human retinal pigment epithelial (ARPE-19) cells and in vitro bioassays were used together to develop an in vitro approach for UV radiation-induced retinal toxicology research. In the third chapter, the in vitro approach developed above was used with intraocular lens (IOL) materials to evaluate the UV radiation blocking efficiency of commercially available IOL’s. Lastly, narrowband blue light irradiation and in vitro assays were used to determine more precisely the wavelengths of blue light responsible for photochemical lesions of the retina as an effort to contribute to future IOL designs. The results from mitochondrial dynamics of lens cells and RPE cells show significant decreases in mitochondrial movement after UVB irradiation in a dose dependent manner. Results obtained from four in vitro assays (Alamar blue assay, confocal microscopy for mitochondrial distribution and nucleic acids damage, phagocytotic activity assay) for evaluating the UVB-induced damage in ARPE-19 show significant decreases in cell viability as well as phagocytotic activity of RPE cells after UVB radiation. In addition, the results show that UV radiation can also induce the degradation of DNA/RNA and mitochondria of RPE cells in a dose dependent manner. The results of the UV blocking efficiency test of commercially available IOL materials show very effective UV blocking ability, allowing no cellular damage at all, in comparison to an IOL uncovered control cell. The results of three different wavelengths of blue light exposure show that only 400 nm blue light radiation can cause significant damage to RPE cells, while 420 and 435.8 nm blue light radiation cause no cellular damage at all. In conclusion, UVB and blue light radiation can cause phototoxic damage to the retinal pigment epithelium as a result of oxidative stress, and in vitro bioassays used for this research may offer a sensitive, and meaningful biomarker approach, not only for evaluating RPE function after oxidative and chemical stress, but also for evaluating IOL effectiveness.

Hintergrund: Die Funktion von Immunzellen hängt von einer konstanten und ausreichenden Energieversorgung ab, die über die OXPHOS in den Mitochondrien und die Glykolyse im Zytosol realisiert wird. Die wichtigsten Substrate dafür sind Sauerstoff und Glukose. Fragestellung: Bei schweren Erkrankungen oder in Entzündungsgebieten ist die zelluläre Energieversorgung stark beeinträchtigt, weil in der Mikroumgebung der Zelle Sauerstoff und Nährstoffe inadäquat bereitgestellt werden. Ziel war herauszufinden, ob und wie humane Immunzellen ihre Lebensfähigkeit und funktionellen Aktivitäten unter solchen Umständen aufrechterhalten. Methoden: Humane CD4+ T-Zellen und CD14+ Monozyten wurden durch MACS aus peripherem Blut gesunder Spender isoliert. Die Sauerstoffverbrauchsmessung mittels Clark-Elektrode war Maß der oxidativen Energiebildung, die mit Myxothiazol und Glukoseentzug gehemmt wurde. Die CD3/CD28-stimulierte T-Zell-Proliferation wurde durchflußzytometrisch mittels CFDA SE analysiert. Basierend auf dem Paraformaldehyd-Saponin-Prozedere wurde die Zytokinsynthese ebenfalls am FACS bewertet, nachdem die T-Zellen in Anwesenheit von Brefeldin A mit PMA/Ionomycin stimuliert wurden. Mit einem käuflichen Testsystem (FACS-Technik) wurde die monozytäre Phagozytose untersucht. Die HIF-1alpha-Expression wurde nach PMA-Ionomycin-Stimulation von Myxothiazol-behandelten T-Zellen auf mRNA- und Proteinebene gemessen. Ergebnisse: Bei Glukoseanwesenheit waren alle untersuchten Immunfunktionen trotz vollständig gehemmter OXPHOS unbeeinträchtigt. Erst bei gleichzeitigem Glukoseentzug, der per sé Proliferation und Phagozytose signifikant beeinträchtigte, waren sie signifikant vermindert. Es wird vermutet, daß T-Zellen die Energieverluste mit einem überschießenden Effekt ihres Sauerstoffverbrauchs und stark angetriebener Glykolyse kompensieren. HIF-1alpha ist dabei nicht entscheidend für die Umschaltung auf anaerobe Energiesynthese. Schlußfolgerung: Die Daten quantifizieren die Energieanforderungen der funktionellen Aktivität in hochgereinigten humanen Immunzellfraktionen. Es wurde nachgewiesen, daß sich Immunzellen unerwartet lange an eine massiv beeinträchtigte Energetik adaptieren können und ihre spezifischen Funktionen aufrechterhalten. / Background: The function of immune cells is dependent upon a constant and adequate supply of energy. Energy is formed via OXPHOS in the mitochondria and via cytosolic glycolysis. Oxygen and glucose are the main substrates for energy synthesis. Objective: In severe diseases or in inflamed areas cellular energy supply is significantly impaired due to inadequate supply of cellular microenvironment with oxygen and nutrients. The aim of this study was to answer the question, whether and how human immune cells maintain viability and functional activity under these circumstances. Methods: Human CD4+ T cells and CD14+ monocytes were isolated by MACS from peripheral blood of healthy donors. The extent of oxidative energy formation was determined via measurement of oxygen consumption using a Clark type electrode. Energy production was restricted in glucose-free cell culture medium and by gradually inhibited OXPHOS using myxothiazol. T cell proliferation was flow-cytometrically analysed using CFDA SE after stimulation with CD3 and CD28 antibodies. Cytokine synthesis was assessed by flow-cytometrical immunofluorescence and the paraformaldehyde-saponin procedure after stimulation of T cells with PMA/ionomycin in the presence of brefeldin A. Phagocytosis of monocytes was measured using a commercial test system (FACS technique). HIF-1alpha expression was assessed by semiquantitative PCR and immunoblot after the stimulation of myxothiazol treated T cells with PMA/ionomycin. Results: In glucose-containing medium all investigated immune functions were unaffected even under complete suppression of OXPHOS. Only when OXPHOS and glycolysis were simultaneously and almost completely suppressed a significant decrease was found. Glucose deprivation per se caused both a significantly reduced proliferation and phagocytosis. It is supposed, that T cells are able to compensate for an energy deficit by an excess of oxygen consumption and strongly induced glycolysis. However, HIF-1alpha was found to be not crucial for switching to anaerobic energy synthesis. Conclusion: These data quantify the energy requirement of functional activity in highly purified human immune cell fractions. An unexpectedly high adaptive potential of immune cells to maintain specific functions even under massively impaired energetic conditions could be demonstrated.