The human skin provides us with an excellent protective barrier and possesses a remarkable ability of constant renewal. Basal cell carcinoma is the most common type of skin cancer. The aim of this project was to verify results from an earlier study investigating the molecular differences between basal cell carcinoma (BCC) and basal cells of normal human epidermis. In that study microdissection of cell populations from BCC and basal cells of normal epidermis respectively was performed in five cases of confirmed BCC. Following RNA extraction and amplification, a gene expression analysis was performed using a 46 k human cDNA microarray. Comparison of expression profiles showed a differential expression of approximately 300 genes in BCC. An upregulation of signaling pathways previously known to be of importance in BCC development could be observed, as well as a downregulation of differentiation markers, MHC class II molecules, and proteins active in scavenging of oxygen radicals. We wanted to confirm these findings for a number of selected genes, using real time PCR. The focal point of this project was microdissection of cells from BCC and subsequent isolation of RNA. Microdissection based methods offer a possibility of selecting well defined cell populations for further analysis by using a focused laser beam. Initially tests in order to optimize the method were also performed, concerning the dehydration process and choice of slides used in microdissection. Isolation of RNA may, as we experienced, be associated with problems due to destruction of RNA by degrading enzymes.

The mammalian skin provides a necessary barrier between the organism and the environment, defending against loss of water and solutes, preventing the invasion of pathogens as well as protecting against chemical and physical assault. Claudin (Cldn)-based Tight Junctions (TJs) are the main functional part of the skin barrier. In particular, Cldn6 through its cytoplasmic tail has been shown to be important for barrier function. In other to further investigate the role of the Cldn6 tail in TJ-function, we developed Cldn6 mouse mutants carrying varying truncations of the Cldn6 tail. Both of these mice present with epidermal differentiation perturbations and delayed barrier function that is repaired later in life. These studies support the importance of the tail portion of the Cldn molecules in epidermal differentiation and barrier function. In addition, both of these mouse models are useful for the study of barrier function in preterm infants and in aging, with the hope of developing novel therapeutics for the alleviation of barrier dysfunction. Cdx is a family of homeodomain (HD) transcription factors (TFs) essential for many key developmental processes. In particular, Cdx2 is important for the establishment and maintenance of posterior identity in the developing endoderm. In spite of this, only a few Cdx targets in the developing endoderm have been discovered. In addition, the interplay between Cdx and its targets within the endoderm is poorly understood. In this study, we show that the forkhead box transcription factor, Foxa2 is a Cdx2 target. We also show that Foxa2 and Cdx2 physically and genetically interact to regulate a subset of genes that are implicated in endodermal development. These studies help to further our understanding of endoderm biology with the goal of developing new strategies to diagnose and treat diseases associated with defective endoderm development.

Mucociliary epithelia are found in the human airways and act as the first line of defence against inhaled foreign agents. Mucus traps potentially damaging particles and the cilia transport the mucus away from the airways to remove the threat. Modelling mucociliary epithelia for research purposes is challenging. This is because the airways are enclosed and are thus difficult to study directly. Instead, tissue is extracted or in vitro techniques are employed. Whilst these systems are useful, there is a need for accessible in vivo models to complement them. In this thesis I assess a new model system for studying mucociliary epithelia. This system is the larval epidermis of the amphibian, Xenopus tropicalis. Its epidermis comprises multi-ciliated cells that beat in a polarised direction reminiscent of those found in the human airways. It is also proposed to have a number of other cell types including mucus-secreting cells, but very little is known about them. The epidermis is open and accessible to manipulation meaning that it has great potential to be used in the study of mucociliary epithelia in live, native conditions. Such a system would be a valuable addition to the current models employed. However, the epidermis has not been thoroughly characterized before so its utility as a model system remains speculative.To develop and evaluate this new model, I fully characterize the epidermis, showing that it has five distinguishable cell types. This includes a population of cells called ionocytes that are shown to be essential for the health and function of the epidermis. I also test for the presence of mucins, the structural component of mucus, secreted from the epidermis in order to evaluate the proposal that mucus-secreting cells are present in the epidermis. A mucin-like protein called otogelin is identified. After characterizing the epidermal cell types, I compare them to the human mucociliary epithelium and consider potential applications and future perspectives for this model.

Das Tragen von Schutzhandschuhen gilt als wichtigste Maßnahme zum Schutz der Haut vor beruflichen Risiken. Bei langen Tragezeiten können luft- und wasserdampfundurchlässige (impermeable) Schutzhandschuhe nachteilige Wirkungen entfalten und zu irritativen Hautschäden führen. Besonders negativ wirkt dieser Effekt bei bereits bestehenden Dermatosen, da eine impermeable Abdeckung der Haut die Hautregeneration hemmt und weitere Schädigungen hervorruft. In der vorliegenden Arbeit wurde untersucht, wie eine Abdeckung mit semipermeablen Materialien im Vergleich zu impermeablen Materialien auf die Regeneration der Haut wirkt. Bei einem Kollektiv von 25 Probanden wurden experimentell induzierte, irritative Hautschäden mit impermeablen und semipermeablen Materialien abgedeckt. Die Regeneration der Haut wurde mittels nichtinvasiver, hautphysiologischer Messungen erfasst. Zudem wurde der Einfluss der Affektregulation (Handlungs- vs. Lageorientierung) auf den Heilungsverlauf der Haut untersucht. Die Ergebnisse der Untersuchung zeigten, dass die Heilungsprozesse der Haut unter semipermeablem Material nicht beeinträchtigt wurden und zum Teil sogar besser verliefen als an Teststellen, die nicht abgedeckt waren. Impermeable Materialien verzögerten hingegen die Regeneration der Haut. Als weiteres Ergebnis konnte gezeigt werden, dass die Regeneration der Haut bei Handlungsorientierten nach Misserfolg rascher verlief als bei Lageorientierten nach Misserfolg.

Es gibt Hinweise darauf, dass der weibliche Zyklus in unterschiedlicher Weise die Barrierehomöostase beeinflusst. Zudem wurden Assoziationen zwischen ausgeprägtem psychischen Stress und der Barrierehomöostase dokumentiert sowie die beeinflussende Wirkung des Serumcortisols diskutiert. In dieser Arbeit wurde getrennt voneinander untersucht, ob die Irritabilität der epidermalen Barriere nach chemischer und physikalischer Irritation vom Menstruationszyklus bzw. vom "alltäglich" wahrgenommenen psychischen Wohlbefinden oder vom ausgeschütteten Cortisol abhängig ist. Methode: Die Barrierefunktion wurde in verschiedenen Zyklusphasen (späte Follikel-, späte Lutealphase) hautphysiologisch erfasst; die Hautreaktion visuell beobachtet. Zur Irritation wurden Natriumlaurylsulfat, Natronlauge, Isopropylalkohol, Pyramidenarray und Tapestripping eingesetzt. Die Studie wurde an hautgesunden Probandinnen mit regelmäßigem Zyklus durchgeführt, die keine hormonellen Kontrazeptiva verwendeten. Das psychische Befinden wurde mittels Fragebogen dokumentiert, die Cortisolkonzentration im Speichel erfasst. Ergebnis: Es fanden sich nach 20-minütiger NaOH-Exposition in der Lutealphase hoch signifikant höhere TEWL-Werte. Bei den anderen Irritationen fanden sich keine signifikanten Unterschiede. Der Vergleich des Zeitpunktes des höheren vs. des niedrigeren psychische Wohlbefindens sowie der Vergleich der Zeitpunkte der höheren vs. der niedrigeren Cortisolwerte ergab keine relevanten Unterschiede. Schlussfolgerung: Die Ergebnisse liefern Hinweise darauf, dass bei einigen angewendeten Irritationsverfahren eine mit dem Zyklus assoziierte Variation der Irritabilität existiert, wobei die Reaktion des Hautorgans auf den schädigenden Einfluss in der lutealen Phase stärker als in der Follikelphase ist. Dagegen scheint eine Assoziation zwischen dem "alltäglichen" psychischen Befinden bzw. dem Speichelcortisol und der Irritabilität bzw. der Regeneration nicht gegeben.

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Yes / The maintenance of a proper nuclear architecture and 3D organization of the genes, enhancer elements and transcription machinery plays an essential role in tissue development and regeneration. Here we show that in the developing skin, epidermal progenitor cells of mice lacking p63 transcription factor display alterations in the nuclear shape accompanied by marked decrease in expression of several nuclear envelop-associated components (Lamin B1, Lamin A/C, SUN1, Nesprin-3, Plectin) compared to controls. Furthermore, ChIP-qPCR assay showed enrichment of p63 on Sun1, Syne3 and Plec promoters, suggesting them as p63 targets. Alterations in the nuclei shape and expression of nuclear envelope-associated proteins were accompanied by altered distribution patterns of the repressive histone marks H3K27me3, H3K9me3 and heterochromatin protein 1- alpha in p63-null keratinocytes. These changes were also accompanied by downregulation of the transcriptional activity and relocation of the keratinocyte-specific gene loci away from the sites of active transcription towards the heterochromatin-enriched repressive nuclear compartments in p63-null cells. These data demonstrate functional links between the nuclear envelope organization, chromatin architecture and gene expression in keratinocytes and suggest nuclear envelope-associated genes as important targets mediating p63-regulated gene expression programme in the epidermis.

No / The nucleus of epidermal keratinocytes (KCs) is a complex and highly compartmentalized organelle, whose structure is markedly changed during terminal differentiation and transition of the genome from a transcriptionally active state seen in the basal and spinous epidermal cells to a fully inactive state in the keratinized cells of the cornified layer. Here, using multicolor confocal microscopy, followed by computational image analysis and mathematical modeling, we demonstrate that in normal mouse footpad epidermis, transition of KCs from basal epidermal layer to the granular layer is accompanied by marked differences in nuclear architecture and microenvironment including the following: (i) decrease in the nuclear volume; (ii) decrease in expression of the markers of transcriptionally active chromatin; (iii) internalization and decrease in the number of nucleoli; (iv) increase in the number of pericentromeric heterochromatic clusters; and (v) increase in the frequency of associations between the pericentromeric clusters, chromosomal territory 3, and nucleoli. These data suggest a role for nucleoli and pericentromeric heterochromatin clusters as organizers of nuclear microenvironment required for proper execution of gene expression programs in differentiating KCs, and provide important background information for further analyses of alterations in the topological genome organization seen in pathological skin conditions, including disorders of epidermal differentiation and epidermal tumors.