Alguns estudos sugerem que as pequenas vias aéreas têm um papel importante na fisiopatologia da lesão pulmonar aguda/ síndrome do desconforto respiratório agudo (LPA/SDRA). O epitélio respiratório que reveste as vias aéreas é capaz de liberar mediadores inflamatórios e está relacionado ainda com a produção de surfactante nas vias aéreas. Até o presente momento, existem poucos estudos que avaliaram se estas funções do epitélio que reveste as pequenas vias aéreas encontram-se alteradas na SDRA. No presente estudo, nós mensuramos a expressão da proteína de surfactante (PS) A e PS-B, a expressão de citocinas inflamatórias interleucina (IL)-6 e IL-8, e um índice de apoptose do epitélio que reveste as pequenas vias aéreas de pacientes com SDRA que foram submetidos a autópsia e comparamos estes resultados com os de indivíduos controle. Foram incluídos no estudo pulmões de autópsia de 31 pacientes com SDRA (PaO2/FiO2200, 45±14 anos, 16 homens) e 11 controles (52±16 anos, 7 homens). A expressão de IL-6, IL-8, PS-A e PS-B no epitélio das pequenas vias aéreas (diâmetro2.0mm) foi verificada através de reações de imunohistoquímica e análise de imagem. O índice de apoptose epitelial das vias aéreas foi avaliado através do método de TUNEL e da expressão de FAS/FASL. Avaliou-se ainda a densidade de células inflamatórias positivas para IL-6 e IL-8 na parede das pequenas vias aéreas. As vias aéreas dos pacientes com SDRA apresentaram maior expressão epitelial de IL-8 (p=0,006) e maior densidade de células inflamatórias expressando IL-6 (p=0,004) e IL-8 (p<0,001) quando comparadas com o grupo controle. Não houve diferenças na expressão epitelial de PS-A e PS-B ou no índice de apoptose epitelial entre os grupos SDRA e controle. Nossos resultados mostram que as pequenas vias aéreas participam da inflamação pulmonar de pacientes com SDRA, caracterizada pelo aumento na expressão de interleucinas próinflamatórias tanto em células inflamatórias da parede da via aérea quanto no epitélio. Nossos resultados sugerem ainda que a apoptose não é um mecanismo importante de morte de células epiteliais das vias aéreas de pacientes com SDRA / Recent studies suggest a role for distal airway injury in the pathophysiology of human ALI/ARDS. The epithelium lining the airways modulates airway function secreting a large number of molecules such as surfactant components and inflammatory mediators. So far, there is little information on how these secretory functions of the small airways are altered in ARDS. In the present study we assessed the airway expression of surfactant protein (SP) A and SP B, the expression of inflammatory cytokines IL-6 and IL-8, and an index of airway epithelial apoptosis of patients with ARDS submitted to autopsy and compared the results with those of control subjects. We studied autopsy lungs of 31 ARDS patients (PaO2/FiO2200, 45±14 years, 16 males) and 11 controls (52±16 years, 7 males). Using immunohistochemistry and image analysis, we quantified the expression of IL-6, IL-8 and SP-A and SP-B in the epithelium of small airways (diameter2.0mm). Airway epithelial apoptosis index was obtained with the TUNEL assay and FAS/FASL expression. We also quantified the density of inflammatory cells expressing IL-6 and IL-8 within the small airway walls. ARDS airways showed an increase in the epithelial expression of IL-8 (p=0.006) and an increased density of inflammatory cells expressing IL-6 (p=0.004) and IL-8 (p<0.001) when compared to controls. There were no differences in SP-A and SP-B epithelium expression or in epithelial apoptosis index between ARDS and controls. Our results show that the distal airways are involved in ARDS lung inflammation with higher expression of pro-inflammatory interleukins in both airway epithelial and inflammatory cells. Our results also suggest that apoptosis is not a major mechanism of airway epithelial cell death in ARDS

Retinopathy of prematurity, a disease characterised by aberrant retinal vascular development in premature neonates, is a leading cause of blindness and visual impairment in childhood. This work sought to examine differences in the susceptibility of inbred rat strains to oxygen-induced retinopathy, a model of human retinopathy of prematurity. The overriding aim was to identify genetic factors in rats that might be generalisable to humans. Newborn rats of six different strains were exposed to alternating cycles of hyperoxia and relative hypoxia for fourteen days. Rats were removed to room air and killed for analysis immediately, to assess oxygen-induced retinal vascular attenuation, or four days later to evaluate the extent of hypoxia-induced vasoproliferation. Whole flat-mounted retinae were stained with fluorophore conjugated isolectin GS-IB4, and measurement of vascular area was conducted using fluorescence microscopy and video-image analysis. A hierarchy of susceptibility to the inhibitory effects of cyclic hyperoxia and relative hypoxia on postnatal retinal vascularization was identified for the rat strains studied. Susceptibility to vascular attenuation was predictive of the subsequent risk of vascular morphological abnormalities. Cross-breeding experiments between susceptible and resistant strains demonstrated that the susceptible phenotype was dominantly inherited in an autosomal fashion. These studies confirmed an association between ocular pigmentation and retinopathy risk, however the finding of differential susceptibility amongst albino rat strains implicated factors in addition to those associated with ocular pigmentation. Quantitative real-time reverse transcription-polymerase chain reaction was used to compare the retinal expression of angiogenic factor genes in susceptible and resistant strains with the aim of identifying a genetic basis for the strain difference. Eight angiogenic factor genes were selected for study: vascular endothelial growth factor (VEGF); VEGF receptor 2; angiopoietin 2; Tie2; pigment epithelium-derived factor; erythropoietin; cyclooxygenase-2 and insulin-like growth factor-1. The most notable difference between strains was the expression of vascular endothelial growth factor (VEGF) during the cyclic hyperoxia exposure period - higher VEGF expression was associated with relative resistance to retinopathy. Other differences in retinal angiogenic factor gene expression between strains, such as higher expression of VEGF receptor 2 and angiopoietin 2 in resistant strains, appeared to be secondary to those in VEGF. Following cyclic hyperoxia, the expression pattern of angiogenic factor genes changed - messenger RNA levels of hypoxia-induced genes, including VEGF, VEGF receptor 2, angiopoietin 2 and erythropoietin, were significantly higher in those strains with larger avascular areas, than in those strains that were relatively resistant to retinopathy. These findings provide firm evidence for hereditary risk factors for oxygen-induced retinopathy in the rat. Differences in the regulatory effects of oxygen on VEGF expression appear to be central to the risk of retinopathy. The potential relevance of these hereditary factors is discussed in the context of the human disease.

<p>Over the past decade, the development of macromolecular drugs based on peptides, proteins and nucleic acids has increased the interest in microparticulate drug delivery, i.e., the delivery of drug systems in the nanometer and micrometer ranges. However, little is known so far about the effect that microparticulate systems have on various tissues after administration. Additionally, the knowledge of mechanisms responsible for the uptake and transport of microparticles across the human intestine is incomplete and requires further investigation to improve both the safety profiles and the efficiency of these drug delivery systems.</p><p>This thesis is comprised of two parts. The first one investigates gene expression responses obtained from DNA arrays in local and distal tissues after microparticulate drug delivery. The second part focuses on the mechanisms responsible for the transport of microparticles across epithelial cells lining the intestine.</p><p>The results presented in the first part demonstrated that gene expression analysis offers a detailed picture of the tissue responses after intramuscular or pulmonary administration of microparticulate drug delivery systems compared to the traditional techniques used for such evaluations. In addition, DNA arrays provided a useful and sensitive tool for the initial characterization and evaluation of both local and distal tissue responses, making it possible to distinguish between gene expression patterns related to each studied delivery system.</p><p>The results presented in the second part demonstrated that the surface properties of the microparticle were important for the extent of transport across an <i>in vitro</i> model of the follicle-associated epithelium (FAE), comprised of intestinal epithelial cells specialized in particle transport (M cells). Another important finding was that the enteropathogen bacterium, <i>Yersinia pseudotuberculosis</i>, induced microparticle transport across the normal intestinal epithelium, represented by Caco-2 cells and excised human ileal tissue. This transport was most probably mediated by an increased capacity for macropinocytosis in the epithelial cells.</p>

Over the past decade, the development of macromolecular drugs based on peptides, proteins and nucleic acids has increased the interest in microparticulate drug delivery, i.e., the delivery of drug systems in the nanometer and micrometer ranges. However, little is known so far about the effect that microparticulate systems have on various tissues after administration. Additionally, the knowledge of mechanisms responsible for the uptake and transport of microparticles across the human intestine is incomplete and requires further investigation to improve both the safety profiles and the efficiency of these drug delivery systems. This thesis is comprised of two parts. The first one investigates gene expression responses obtained from DNA arrays in local and distal tissues after microparticulate drug delivery. The second part focuses on the mechanisms responsible for the transport of microparticles across epithelial cells lining the intestine. The results presented in the first part demonstrated that gene expression analysis offers a detailed picture of the tissue responses after intramuscular or pulmonary administration of microparticulate drug delivery systems compared to the traditional techniques used for such evaluations. In addition, DNA arrays provided a useful and sensitive tool for the initial characterization and evaluation of both local and distal tissue responses, making it possible to distinguish between gene expression patterns related to each studied delivery system. The results presented in the second part demonstrated that the surface properties of the microparticle were important for the extent of transport across an in vitro model of the follicle-associated epithelium (FAE), comprised of intestinal epithelial cells specialized in particle transport (M cells). Another important finding was that the enteropathogen bacterium, Yersinia pseudotuberculosis, induced microparticle transport across the normal intestinal epithelium, represented by Caco-2 cells and excised human ileal tissue. This transport was most probably mediated by an increased capacity for macropinocytosis in the epithelial cells.

Crumbs is a transmembrane protein and apical determinant in Drosophila epithelial cells. Its cytoplasmic tail contains a PDZ and a FERM domain-binding site through which Crumbs interacts with the FERM proteins Yurt, Moesin and Expanded. Recent evidence suggests that Crumbs can also interact with the uncharacterised FERM proteins CG34347 and Cdep. The main objective of my thesis was to generate mutations in CG34347 and Cdep to facilitate the functional analysis of these genes. I generated a mutation for Cdep that remains to be characterised and two mutant lines for CG34347; one lacking the first exon and one lacking the entire gene, using a FRT-based recombination strategy. Both CG34347 mutants cause severe ovarian defects. The most consistent defect is a multilayering of the interfollicular stalk. These defects are also observed when Notch, Hippo, Wingless and Hedgehog signalling pathways are overactive in ovaries suggesting that CG34347 participates in one of those pathways.

Crumbs is a transmembrane protein and apical determinant in Drosophila epithelial cells. Its cytoplasmic tail contains a PDZ and a FERM domain-binding site through which Crumbs interacts with the FERM proteins Yurt, Moesin and Expanded. Recent evidence suggests that Crumbs can also interact with the uncharacterised FERM proteins CG34347 and Cdep. The main objective of my thesis was to generate mutations in CG34347 and Cdep to facilitate the functional analysis of these genes. I generated a mutation for Cdep that remains to be characterised and two mutant lines for CG34347; one lacking the first exon and one lacking the entire gene, using a FRT-based recombination strategy. Both CG34347 mutants cause severe ovarian defects. The most consistent defect is a multilayering of the interfollicular stalk. These defects are also observed when Notch, Hippo, Wingless and Hedgehog signalling pathways are overactive in ovaries suggesting that CG34347 participates in one of those pathways.

Tesi realitzada a l'Institut d'Investigacions Biomèdiques de Barcelona (IIBB-CSIC IDIBAPS) / Les patologies renals s’han convertit en una problemàtica a nivell mundial, en l’actualitat poden afectar a una de cada 9 persones al llarg de la seva vida i porten associats elevats costos econòmics. Malgrat els últims avenços científics i millores en el tractament d’aquestes patologies, el transplantament renal i la diàlisi continuen sent les dues úniques opcions terapèutiques efectives en el tractament de la insuficiencia renal. La regeneració de l’epiteli renal és determinant en la recuperació del pacient ja que determina que es pugui restablir la funcionalitat d’aquest òrgan. L’obtenció de precursors renals a partir de cèl•lules mare que puguin integrar-se i regenerar el ronyó lesionat s’ha convertit en una àrea de recerca biomèdica molt important. L’objecte d’estudi d’aquesta tesi va ser conèixer els mecanismes involucrats en la diferenciació de les cèl•lules mare embrionàries (ESCs) i les cèl•lules mare adiposes (ASCS) cap al llinatge epitelial renal ja que aquestes cèl•lules poden ser una potencial font d’aquests precursors renals amb capacitat regenerativa. Recentment s’ha descobert que els miRNAs, que tenen la funció de regular l’expressió gènica en l’etapa post-transcripcional, són essencials en la diferenciació de les cèl•lules mare i s’han trobat miRNAs específics que regulen la diferenciació a un llinatge cel•lular en concret. En aquest sentit, el nostre objectiu general en aquest treball va ser estudiar el paper dels miRNAs en la diferenciació de cèl•lules mare a progenitors epitelials renals. Per això, primer de tot es van realitzar experiments de diferenciació en que els cossos embrionaris (EBs) induïts a partir de les cèl•lules mare embrionàries (ESCs) es van cultivar amb un medi de cultiu suplementat amb all-trans-retinoic acid (ATRA) i activina A, i les cèl•lules mare adiposes (ASCs) amb un medi amb una concentració fisiológica de glucosa suplementat amb ATRA. Amb aquests protocols de diferenciació, es van obtenir cèl•lules amb característiques de progenitors renals. Els EBs cultivats amb el protocol de diferenciació expressaven els marcadors de l’organogènesi renal inicial (Pax2, WT1, Wnt4, Notch2 i Wnt9b). Les ASCs cultivades amb ATRA varen canviar la seva morfologia a una morfologia epitelial i van expressar marcadors tant de l’organogènesi renal inicial (Pax2, WT1, Wnt4, Six2 i Megalina) com els marcadors epitelials (Citoqueratina 18, E-caderina). Un anàlisis de miRNAs va demostrar que la família de miRNAs let-7 es sobreexpressava durant la diferenciació dels EBs i que el miRNA let-7e més concretament era essencial en l’expressió dels marcadors Pax2, Wt1i Wnt4 ja que la seva silenciació disminuïa l’expressió d’aquests gens. En les ASCs, en canvi, es va demostrar que el miRNA let-7e també augmentava en la diferenciació i que a més tenia característiques d’inductor de la diferenciació, essent essencial en l’expressió tant dels marcadors de l’organogènesi renal (Pax2, Wt1, Wnt4, Megalina) com del marcador epitelial CK18. Profunditzant en el paper del miRNA let-7e en la diferenciació, es va demostrar amb experiments de Western blot, que el miRNA let-7e modula els nivells de β-catenina a través d’un mecanisme que implica la inhibició de la PKCβ i la conseqüent disminució en la fosforilació de la GSK3β (Ser-9) i que això és imprescindible per la correcta diferenciació de les ESCs. Per altra banda, en les ASCs es va demostrar utilitzant l’assaig del gen reporter de la luciderasa, que el miRNA let-7e inhibeix directament l’expressió de la metal•loproteinasa 9 i que d’aquesta manera modula la diferenciació de les ASCs al llinatge epitelial renal. / Role of miRNA in stem cell differentiation Renal diseases have become a worldwide problem that can affect one in nine people throughout their life. Despite recent scientific advances, kidney transplantation and dialysis are still the only two effective therapeutic options in renal failure. The regeneration of the epithelium is critical for patient recovery as it determines the restoration of the kidney functionality. Cell precursors obtained from renal stem cells that can regenerate and integrate the injured kidney have become an important research area. The aim of our study was to determine the mechanisms involved in the differentiation of embryonic stem cells (ESCs) and adipose stem cells (ASCs) to renal epithelial lineage as these cells can be a source of these renal precursors with regenerative potential. It was recently discovered that miRNAs, which have the function of regulating gene expression in the post-transcriptional level, are essential in the differentiation of stem cells. In this sense, our main objective was to study the role of miRNAs in stem cells differentiation to renal epithelial progenitors. For this purpose, We have carried out experiments of stem cells differentiation. Embryoid bodies (EBs) from ESCs were cultured with activin A and ATRA and ASCs where cultured in a medium with physiological concentration of glucose supplemented with ATRA, obtaining progenitor cells with renal epithelial characteristics. EBs expressed the early kidney organogenesis markers (Pax2, WT1, Wnt4, Notch2 Wnt9b) and ASCs changed their morphology to a more epithelial one and expressed both markers of kidney organogenesis (Pax2, WT1, Wnt4, SIX2 i Megalin) and epithelial markers (cytokeratin-18 and E-cadherin). Furthermore, miRNA analysis and the subsequent overexpression and silencing of the miRNA let-7e in stem cells, demonstrates that this miRNA has characteristics of differentiation inductor and that is essential in the expression of both kidney organogenesis markers and epithelial markers. Furthermore, the ESCs, let-7e miRNA modulates β-catenin levels through a mechanism involving inhibition of PKCβ and the consequent decrease in the phosphorylation of GSK3 (Ser-9). Moreover, it was demonstrated using the luciferase assay, that the miRNA let-7e directly inhibits expression of gelatinase B (MMP9) in ASCs and thereby modulates its renal epithelial differentiation.

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.

Influence of stress factors on tunneling nanotubes in cultivated human retinal pigment epithelial cells (ARPE-19). The eye as one of the most important sense organs of the human body is exposed to visible light radiation and other stress factors every day. Especially the retina (of the eye) is a sensible tissue for oxidative damage (Wu et al., 2006). The retinal pigment epithelium (RPE) is an important layer of the retina, which forms the outer layer and phagocytises the shed disc membranes of the photoreceptor outer segments. Furthermore, the RPE is involved in the maintenance of the visual cycle and regulates the retinal balance (Bok, 1993). To maintain those functions, a steady communication between the RPE-cells and the adjacent neighbour cells is necessary. Tunneling nanotubes (TNTs) build a newly discovered variety of cell communication and thus establish intercellular signal transduction and transport different cell components including pathogens (Rustom et al., 2004; Onfelt et al., 2006; Sherer und Mothes, 2008; Veranic et al., 2008). The formation of TNTs in the neuron-like pheochromocytoma cell line PC12 was first reported by Rustom et al in 2004. In the following years a growing number of cell types containing TNTs were described. For example a lot of TNT-reports were found between immune cells (Onfelt et al., 2004; Sowinski et al., 2008). Chinnery et al. first described TNTs in vivo in 2008. Here they found TNTs between dendritic cells in the cornea of the mouse. An important characteristic of TNTs is that they do not attach to the substratum. They contain F-actin as a characteristic feature of there structure (Rustom et al., 2004). Our study group detected the formation of TNTs between ARPE-19-cells, a human retinal pigment epithelial cell line. They contain F-actin, but no microtubules. Further it was observed an exchange of electrical signals, small molecules and even the transfer of organelles between cells via TNTs (see publication Wittig et al., 2012). It is often described in the literature, that TNTs are very sensitive against stress factors, like prolonged light excitation, mechanical and chemical stress, which then can result in rupture of the TNTs (Rustom et al., 2004; Koyanagi et al., 2005; Gurke et al., 2008a; Pontes et al., 2008; Sowinski et al., 2008; Domhan et al., 2011; Wang und Gerdes, 2012). Up to now it is widely unclear how pathological conditions influences TNTs. There are several studies, which report an induction but also an inhibition of TNT-formation by different factors. The reaction of cell-cell-interactions between RPE cells on stress factors is not jet analysed. So our motivation was, to analyse the influence of different stress factors on the number, the morphology and formation of TNTs. ARPE-19-cells were treated with blue light, with a wavelength of 470 and 405 nm, with 3000 μM glyoxal, with 200 μM H2O2, with medium without serum as well as with cytochalasin-D and latrunculin-B. With the help of differential interference contrast (DIC) microscopy the formed TNTs were counted and the morphology was evaluated. A 24 hours cultivation of untreated ARPE-19 cells resulted in 15 TNTs per 100 cells on average. After excitation of the ARPE-19-cells with blue light 470 and 405 nm the number of TNTs decreased 50 % and 28,5 % accordingly in comparison to untreated cells (100 %). Furthermore, the cell culture, which was treated with glyoxal and H2O2 resulted in a reduction of 17,5 % and 53 % TNTs in comparison to the untreated cell culture. Cells which were cultured with serum free medium had an decreased TNT-number of 56.8 % in comparison with serum containing medium. TNTs of untreated ARPE-19-cells have a diameter from 50 to 300 nm (Wittig et al., 2012). Every TNTs, which were formed under named stress factors had the same diameter like untreated cells. In this study an average TNT length of 23 +/- 16 μm was measured between cells without treatment. This correlated with the TNT-lengths of cells which excitated with blue light 405 and 470 nm with 26 +/- 13 μm and 24 +/- 14 μm. In contrast the TNT-lenghts of cells treated with glyoxal and H2O2 with 16 +/- 11 μm and 15 +/- 13 μm were less and from cells cultured without serum with 34 +/- 20 μm were above the average length of TNTs of untreated cells. TNTs of ARPE-19-cells without treatment and TNTs which were treated with stress factors contained F-actin but no microtubules. Depolymerisation of F-actin, induced by addition of cytochalasin-D or latrunculin-B, led to disappearance of TNTs. This is an evidence for the importance of F-actin as an essential component of TNTs between ARPE-19-cells. Under the influence of blue light excitation the TNTs formed as good as untreated cells after contact of migrating cells. Reason for the reduced TNT-formation under stress factors could be explained by the generation of oxidative stress due to reactive oxygen species (ROS). ROS induced under blue light- or glyoxal-treatment as well as H2O2 could influence cell function by inactivation of cell-mediated proteins or induction of F-actin oxidation with subsequent destruction of the actin-network and inhibition of the actin-polymerisation (Chen, 1993; Ballinger et al., 1999; Thornalley et al., 1999; Valen et al., 1999; Dalle-Donne et al., 2002; Nilsson et al., 2003; Shangari und O'Brien, 2004; Zhu et al., 2005; Knels et al. 2008; Roehlecke et al., 2009). The reduced actin-polymerisation as well as the disruption of the TNTs due to changes at the actin-cytoskeleton and at the membranes could explain the reduced TNT-formation (Valen et al., 1999; Dalle-Donne et al., 2002; Reber et al., 2002; Zhu et al., 2005; Knels et al., 2008). The inhibition of the cell growth under oxidative stress conditions and under nutritional deficiency by serum free medium could lead to a reduced TNT-formation too. In this study we found a reduction of TNT-number between ARPE-19-cells under different stress conditions. It is possible, that TNTs are formed between RPE- and photoreceptor-cells in vivo, where they can exchange useful or recyclable materials between cells (Wang et al., 2011; Wittig et al., 2012). Disruption of TNTs by reactive oxygen species could cause a decreased exchange of informations. It is possible, that the cells, RPE- as well as photoreceptor-cells, die due to a deficiency of nutrients. This could be another reason in the formation of age related macular degeneration, which shows a destruction of RPE-cells and secondary of the photoreceptorcells. / Das Auge ist als eines der wichtigsten Sinnesorgane des Menschen täglich sichtbarer Lichtstrahlung und weiteren Stressfaktoren ausgesetzt. Die Netzhaut des Auges ist besonders empfindlich für oxidative Schäden (Wu et al., 2006). Eine bedeutende Schicht der Netzhaut im Auge stellt das retinale Pigmentepithel (RPE) dar, welches die äußere Schicht der Retina bildet und täglich die abgeworfenen Photorezeptoraußensegmentscheiben phagozytiert. Zudem ist das RPE wesentlich am visuellen Prozess sowie der Aufrechterhaltung des retinalen Gleichgewichts beteiligt (Bok, 1993). Um diese Funktionen zu gewährleisten, ist eine ständige Kommunikation zwischen den RPEZellen sowie zu angrenzenden Nachbarzellen innerhalb der Netzhaut notwendig. So ist über Tunneling Nanotubes (TNTs), als neu entdeckte Kommunikationsform, ein interzellulärer Transport von Signalen und verschiedensten Zellkomponenten, aber auch von Pathogenen, möglich (Rustom et al., 2004; Onfelt et al., 2006; Sherer und Mothes, 2008; Veranic et al., 2008). Erstmals 2004 beschrieben Rustom et al. die Bildung von TNTs zwischen Rattennierenzellen in vitro. In den folgenden Jahren kam es zu einer Vielzahl weiterer TNT-Entdeckungen zwischen verschiedensten Zellen in vitro. So findet man zum Beispiel vermehrt TNTBeschreibungen zwischen Immunzellen (Onfelt et al., 2004; Sowinski et al., 2008). Ein erster Nachweis an TNTs in vivo erfolgte 2008 durch die Arbeitsgruppe Chinnery et al.. Hierbei fand man TNTs zwischen dendritischen Zellen in der Mauscornea. Ein wichtiges Merkmal von TNTs ist, dass sie sich als frei im Medium schwebende interzelluläre Verbindungen darstellen, ohne Kontakt zum Substrat zu haben. TNTs sind im Wesentlichen als stabilisierendes Hauptstrukturmerkmal aus Aktin aufgebaut (Rustom et al., 2004). In unserer Arbeitsgruppe wurde die Bildung von TNTs zwischen ARPE-19-Zellen, einer humanen Pigmentepithelzelllinie, entdeckt. Neben dem strukturellen Aufbau aus Aktin, konnte ein Austausch von elektrischen Signalen sowie molekularen Stoffen und der Transport von Organellen (Mitochondrien) durch TNTs zwischen ARPE-19-Zellen nachgewiesen werden (siehe Publikation Wittig et al., 2012). Wie schon mehrfach in der Literatur beschrieben, reagieren TNTs sehr sensibel auf Stressfaktoren, so zum Beispiel auf längere Lichtreizung, mechanischen und chemischen Stress, was jeweils zur Ruptur der Strukturen führen kann (Rustom et al., 2004; Koyanagi et al., 2005; Gurke et al., 2008; Pontes et al., 2008; Sowinski et al., 2008; Domhan et al., 2011; Wang und Gerdes, 2012). Weitgehend unklar ist bisher der Einfluss von pathologischen Bedingungen auf die TNTs. Es gibt mehrere Studien, in denen durch verschiedenste Faktoren über eine Induktion, aber auch über eine Hemmung der TNT-Bildung berichtet wurde. Die Reaktion von Zell-Zell-Interaktionen zwischen RPE-Zellen auf Stressfaktoren wurde bisher in wissenschaftlichen Arbeiten nicht untersucht. Dies nahmen wir zum Anlass, den Einfluss von unterschiedlichen Stressfaktoren auf die Anzahl von TNTs, ihre Morphologie und Bildung zu untersuchen. Es erfolgte eine Behandlung der ARPE-19-Zellen mit Blaulicht in den Wellenlängen 405 und 470 nm, mit 3000 μM Glyoxal, mit 200 μM H2O2, mit serumfreiem Medium sowie mit Cytochalasin D und Latrunculin B. Die gebildeten TNTs wurden anschließend mit Hilfe der Lichtmikroskopie ausgezählt sowie deren Morphologie beurteilt. So bildeten unbehandelte ARPE-19-Zellen nach 24 Stunden Kultivierung im Durchschnitt 15 TNTs pro 100 Zellen aus. Nach 24stündiger Bestrahlung der ARPE-19-Zellen mit Blaulicht 470 nm und 405 nm fiel die TNT-Anzahl auf 50 % und 28,5 % im Vergleich zu unbehandelten Zellen (100 %). Weiterhin fanden sich in den Glyoxal- und H2O2-behandelten Kulturschalen 17,5 % und 53 % TNTs verglichen mit der unbehandelten Zellkultur. In der serumfreien Kulturschale verringerten sich die TNTs 24 Stunden nach Ausplattierung der Zellen auf 56,8 % im Vergleich zu in Medium mit Serum kultivierten Zellen. TNTs unbehandelter ARPE-19-Zellen besitzen einen Durchmesser von 50 bis 300 nm (Wittig et al., 2012). Alle unter oben genannten Stressfaktoren gebildeten TNTs befanden sich in Hinblick auf ihren Durchmesser im Bereich der TNTs unbehandelter Zellen. Bei TNTs unbehandelter Zellen wurde in dieser Arbeit eine durchschnittliche Länge von 23 +/- 16 μm gemessen. Dies entsprach dem TNT-Längendurchschnitt von mit Blaulicht 405 nm und 470 nm bestrahlter ARPE-19-Zellen mit 26 +/- 13 μm und mit 24 +/- 14 μm. Unter Glyoxal und H2O2 gebildete TNTs lagen im Gegensatz dazu mit 16 +/- 11 μm und 15 +/- 13 μm unterhalb und unter serumfreier Kultivierung mit 34 +/- 20 μm über dem TNTLängendurchschnitt unbehandelter Zellen. Alle TNTs, sowohl unbehandelter als auch mit Stressfaktoren behandelter ARPE-19-Zellen, sind aus Aktin aufgebaut. Jedoch ließ sich kein Tubulin nachweisen. Nach Zugabe von Aktinpolymerisationshemmern waren keine TNTs nachweisbar, was beweist, dass F-Aktin essentieller Bestandteil von TNTs zwischen ARPE-19-Zellen ist. Unter dem Einfluss von Blaulicht 470 und 405 nm bildeten sich die TNTs, wie auch bei unbehandelten Zellen, durch ein Zusammentreffen der Zellen mit anschließendem Auseinandergleiten. Die Ursache für die verminderte Bildung an TNTs unter verschiedenen Stressfaktoren könnte in der Entstehung von oxidativem Stress durch die Ausbildung von reaktiven Sauerstoffspezies (ROS) begründet sein. So können zum Beispiel die unter Blaulicht- und Glyoxalexposition entstehenden ROS sowie H2O2, als eine Hauptform der ROS, die Zellfunktion durch Inaktivierung zellulärer Proteine beeinflussen sowie eine direkte Oxidation an Aktin hervorrufen mit folglicher Aktinnetzwerkzerstörung und Hemmung der Aktinpolymerisation (Chen, 1993; Ballinger et al., 1999; Thornalley et al., 1999; Valen et al., 1999; Dalle-Donne et al., 2002; Nilsson et al., 2003; Shangari und O'Brien, 2004; Zhu et al., 2005; Knels, Worm et al. 2008; Roehlecke et al., 2009). Die verminderte Aktinpolymerisation, aber auch die Zerreißungen der TNTs durch Veränderungen am Aktinzytoskelett sowie an den Membranen könnten zu einer verringerten TNT-Bildung führen (Valen et al., 1999; Dalle-Donne et al., 2002; Reber et al., 2002; Zhu et al., 2005; Knels et al., 2008). Auch eine Hemmung des Zellwachstums unter oxidativen Stressbedingungen sowie unter Nährstoffmangel durch Serumentzug könnte mit einer verminderten TNT-Bildung einhergehen. Wir haben in unserer Untersuchung gezeigt, dass es durch verschiedene Stresseinflüsse zu einer Reduktion der TNTs zwischen ARPE-19-Zellen kommt. Es ist denkbar, dass solche TNTs in vivo zwischen RPE- und Photorezeptorzellen ausgebildet werden, wo sie nützliches oder recycelbares Material zwischen Zellen austauschen (Wang et al., 2011; Wittig et al., 2012). Bei Zerstörung der TNTs durch zum Beispiel oxidative Faktoren könnte es zu einer Verringerung des Informationsaustausches kommen. Es ist möglich, dass durch die Minderversorgung die Zellen absterben, sowohl RPE- als auch Photorezeptorzellen. Dies könnte ein weiterer möglicher Ursachenansatz in der Entstehung der altersabhängigen Makuladegeneration sein, welche als Erkrankungserscheinung den Untergang der RPEZellen und damit sekundär der Photorezeptorzellen aufweist.