DNA methylation, through 5-methyl- and 5-hydroxymethylcytosine (5mC and 5hmC) is considered to be one of the principal interfaces between the genome and our environment and it helps explain phenotypic variations in human populations. Initial reports of large differences in methylation level in genomic regulatory regions, coupled with clear gene expression data in both imprinted genes and malignant diseases provided easily dissected molecular mechanisms for switching genes on or off. However, a more subtle process is becoming evident, where small (<10%) changes to intermediate methylation levels were associated with complex disease phenotypes. This has resulted in two clear methylation paradigms. The latter "subtle change" paradigm is rapidly becoming the epigenetic hallmark of complex disease phenotypes, although we were currently hampered by a lack of data addressing the true biological significance and meaning of these small differences. The initial expectation of rapidly identifying mechanisms linking environmental exposure to a disease phenotype led to numerous observational/association studies being performed. Although this expectation remains unmet, there is now a growing body of literature on specific genes, suggesting wide ranging transcriptional and translational consequences of such subtle methylation changes. Data from the glucocorticoid receptor (NR3C1) has shown that a complex interplay between DNA methylation, extensive 5"UTR splicing and microvariability gives rise to the overall level and relative distribution of total and N-terminal protein isoforms generated. Additionally, the presence of multiple AUG translation initiation codons throughout the complete, processed, mRNA enables translation variability, hereby enhancing the translational isoforms and the resulting protein isoform diversity; providing a clear link between small changes in DNA methylation and significant changes in protein isoforms and cellular locations. Methylation changes in the NR3C1 CpG island, alters the NR3C1 transcription and eventually protein isoforms in the tissues, resulting in subtle but visible physiological variability. Implying external environmental stimuli act through subtle methylation changes, with transcriptional microvariability as the underlying mechanism, to fine-tune the total NR3C1 protein levels. The ubiquitous distribution of genes with similar structure as NR3C1, combined with an increasing number of studies linking subtle methylation changes in specific genes with wide ranging transcriptional and translational consequences, suggested a more genome-wide spread of subtle DNA methylation changes and transcription variability. The subtle methylation paradigm and the biological relevance of such changes were supported by two epigenetic animal models, which linked small methylation changes to either psychopathological or immunological effects. The first model, rats subjected to maternal deprivation, showed long term behavioural and stress response changes. A second model, exposing mice to early life infection with H1N1, illustrated long-term immunological effects. Both models displayed subtle changes within the methylome. Suggesting/Indicating that early life adversity and early life viral infection "programmed" the CNS and innate immune response respectively, via subtle DNA methylation changes genome-wide. The research presented in this thesis investigated the ever-growing roles of DNA methylation; the physiological and functional relevance of subtle small DNA methylation changes genome-wide, in particular for the CNS (MD model) and the immune system (early life viral infection model) ; and the evidence available, particularly from the glucocorticoid of the cascade of events initiated by such subtle methylation changes, as well as addressing the underlying question as to what represents a genuine biologically significant difference in methylation.

Cortisol is one of the key substances released during stress to restore homeostasis. Our knowledge of the impact of this glucocorticoid on cognition and behavior in humans is, however, still limited. Two modes of action of cortisol are known, a rapid, nongenomic and a slow, genomic mode. Both mechanisms appear to be involved in mediating the various effects of stress on cognition. Here, three experiments are presented that investigated fast and slow effects of cortisol on several functions of the human brain. The first experiment investigated the interaction between insulin and slow, genomic cortisol effects on resting regional cerebral blood flow (rCBF) in 48 young men. A bilateral, locally distinct increase in rCBF in the insular cortex was observed 37 to 58 minutes after intranasal insulin admission. Cortisol did not influence rCBF, neither alone nor in interaction with insulin. This finding suggests that cortisol does not influence resting cerebral blood flow within a genomic timeframe. The second experiment examined fast cortisol effects on memory retrieval. 40 participants (20 of them female) learned associations between neutral male faces and social descriptions and were tested for recall one week later. Cortisol administered intravenously 8 minutes before retrieval influenced recall performance in an inverted U-shaped dose-response relationship. This study demonstrates a rapid, presumably nongenomic cortisol effect on memory retrieval in humans. The third experiment studied rapid cortisol effects on early multisensory integration. 24 male participants were tested twice in a focused cross-modal choice reaction time paradigm, once after cortisol and once after placebo infusion. Cortisol acutely enhanced the integration of visual targets and startling auditory distractors, when both stimuli appeared in the same sensory hemi-field. The rapidity of effect onset strongly suggests that cortisol changes multisensory integration by a nongenomic mechanism. The work presented in this thesis highlights the essential role of cortisol as a fast acting agent during the stress response. Both the second and the third experiment provide new evidence of nongenomic cortisol effects on human cognition and behavior. Future studies should continue to investigate the impact of rapid cortisol effects on the functioning of the human brain.

During pregnancy every eighth woman is treated with glucocorticoids. Glucocorticoids inhibit cell division but are assumed to accelerate the differentiation of cells. In this review animal models for the development of the human fetal and neonatal hypothalamic-pituitary-adrenal (HPA) axis are investigated. It is possible to show that during pregnancy in humans, as in most of the here-investigated animal models, a stress hyporesponsive period (SHRP) is present. In this period, the fetus is facing reduced glucocorticoid concentrations, by low or absent fetal glucocorticoid synthesis and by reduced exposure to maternal glucocorticoids. During that phase, sensitive maturational processes in the brain are assumed, which could be inhibited by high glucocorticoid concentrations. In the SHRP, species-specific maximal brain growth spurt and neurogenesis of the somatosensory cortex take place. The latter is critical for the development of social and communication skills and the secure attachment of mother and child. Glucocorticoid treatment during pregnancy needs to be further investigated especially during this vulnerable SHRP. The hypothalamus and the pituitary stimulate the adrenal glucocorticoid production. On the other hand, glucocorticoids can inhibit the synthesis of corticotropin-releasing hormone (CRH) in the hypothalamus and of adrenocorticotropic hormone (ACTH) in the pituitary. Alterations in this negative feedback are assumed among others in the development of fibromyalgia, diabetes and factors of the metabolic syndrome. In this work it is shown that the fetal cortisol surge at the end of gestation is at least partially due to reduced glucocorticoid negative feedback. It is also assumed that androgens are involved in the control of fetal glucocorticoid synthesis. Glucocorticoids seem to prevent masculinization of the female fetus by androgens during the sexual gonadal development. In this work a negative interaction of glucocorticoids and androgens is detectable.

α2-adrenerge Rezeptoren sind entscheidende Strukturen für den Ablauf einer physiologischen Stressreaktion. Verschiedene Ursachen sind bekannt, welche die Funktionalität/Effektivität der vorwiegend inhibitorischen α2-adrenergen Wirkung einschränken. Darunter fallen frühe und lebenszeitliche Stressbelastung, genetische Faktoren sowie pharmakologische Einflüsse. Ziel der vorliegenden Dissertation war die Identifikation und Charakterisierung von kognitiven und zentralnervös gesteuerten kardiovaskulären Parametern, welche durch α2-adrenerge Rezeptoren in besonderem Maße beeinflusst werden. Weiterhin sollte anhand pharmakologischer Modelle eine Methode entwickelt werden, um diese Beeinflussbarkeit quantitativ zu beschreiben. In einem komplexen pharmakologischen Versuchsplan wurde die Aktivität der α2-adrenergen Rezeptoren durch jeweils fünf Dosisstufen Dexmedetomidin (α2-adrenerger Agonist) und Yohimbin (α2-adrenerger Antagonist) manipuliert. In einem placebokontrollierten einfach-blinden Design wurde die Konzentrations-Wirkungs-Beziehung bzw. die Dosis-Wirkungs-Beziehung zwischen der Medikation und kognitiven sowie zentralnervös gesteuerten kardiovaskulären Parametern ermittelt. Zudem wurden die Effekte von α2-adrenergem Agonismus und Antagonismus auf die akustische Startlereaktion sowie auf die Plasmakonzentration von Noradrenalin (NA) und DHPG erfasst. Mittels linearer pharmakodynamischer Modellierung sollte anschließend die maximale Spannweite der α2-adrenerg vermittelten Effekte vorhergesagt werden, um so Aussagen über die pharmakologische Beeinflussbarkeit der Rezeptoren treffen zu können. Es zeigten sich deutliche Effekte von α2-adrenergem Agonismus und Antagonismus auf einfache Reaktionszeiten und kardiovaskuläre Parameter. Insbesondere sympathisch vermittelte Funktionen waren durch die pharmakologische Manipulation beeinflusst, ebenso wie die Magnitude der Blinzelreaktion auf einen akustischen Schreckreiz. Die Substanzen hatten zudem deutliche Einflüsse auf die Plasmakonzentration von NA und DHPG. Der besondere Erfolg dieser Arbeit liegt in der systematischen Quantifizierung der pharmakologischen Beeinflussung zentralnervöser Parameter durch α2-adrenergen Agonismus und Antagonismus. Es konnte gezeigt werden, dass die pharmakodynamische Modellierbarkeit zentralnervöser Parameter Aufschluss über potenzielle Gruppen-unterschiede in der Funktionalität/Effektivität α2-adrenerger Mechanismen geben kann.

The allergic contact dermatitis (ACD) to small molecular weight compounds is a common inflammatory skin reaction. ACD is restricted to industrialized countries, has an enormous sociomedical and socioeconomic impact. About 2,800 compounds from the six million chemicals known in our environment are believed to have allergic, and to a lesser degree also contact-sensitizing or immunogenic properties causing allergic contact dermatitis. ACD results from T cell responses to harmless, low molecular weight chemicals (haptens) applied to the skin. Haptens are not directly recognized by the cells of the immune system. They need to be presented by subsets of antigen presenting cells to the cells of the immune system. In this regard, epidermal Langerhans cells (LC) and the cells into which they mature (dendritic cells) are believed to play a pivotal role in the sensitization process for ACD. LC are able to bind the haptens, internalize them, and present them to naive T cells and induce thereby the development of effector T cells. They are so-called professional antigen presenting cells. This process is initiated and maintained by the release of several mediators, which are released by various cells after their contact with the haptens. One of the first proteins secreted into the environment is interleukin (IL)-1ß. This cytokine is produced and secreted minutes after an antigen enters the cell. It is commonly believed that the large amounts of this protein and other cytokines such as granulocyte-colony stimulation factor (GM-CSF) and tumor necrosis factor alpha (TNF-) needed for the initiation and activation of ACD are coming first from other cells residing in the skin, e.g., keratinocytes, monocytes and macrophages. These cytokines provide the danger signals needed for the activation of the Langerhans cell (LC), which then produce via a positive feedback loop various cytokines themselves. In addition, other proteins such as chemokines influence the generation of danger signals, migration, homing of T cells in the local lymph nodes as well as the recruitment of T cells into the skin. Thus, a small molecular compounds or hapten needs to be able to induce danger signals in order to become immunogenic. In this study, we investigated whether para-phenylenediamine (PPD), an arylamine and common contact allergen, is able to induce danger signals and likely provide the signals needed for an initiation of an immune response[162, 163]. PPD is used as an antioxidant, an ingredient of hair dyes, intermediate of dyestuff, and PPD is found in chemicals used for photographic processing. But up to date, it has not been clearly demonstrated if PPD itself is a sensitizing agent. Thus, this study aimed on the potential of PPD to provide the danger signals by studying IL-1β, TNF-, and monocyte chemoattractant proteins (MCP-1) in human monocytes, peripheral blood mononuclear cells (PBMC) from healthy volunteers, and also in two human monocyte cell lines namely U937, and THP-1. This study found that PPD decreased dose- and time-dependently the expression and release of three relevant mediators involved in the generation of danger signals. Namely, PPD reduced the mRNA and protein levels for IL-1ß, TNF-, and MCP-1 in primary human monocytes from various donors. These findings were extended and validated by investigations using the cell line U937. The data were highly specific for PPD, and no such results were gained for its known auto oxidation product called Bandrowski- base or for meta-phenylenediamine (MPD), and ortho-phenylenediamine (OPD). Therefore, we can speculate that this effect is likely to be dependent on the para-substitution. Based on these results we conclude that PPD itself is not able to mount a cascade for the induction of danger signals. It should be mentioned that it is still possible that PPD induces danger signals for sensitization by other unknown processes. Therefore, more research is still needed focusing on this subject especially in professional antigen presenting cells in order to solve the still open question whether PPD itself sensitizes naive T cells or if PPD is solely an allergen. Independently we found unexpectedly that PPD as well as other haptens such as 2, 4-Dinitrochlorobenzene, nickelsulfate, as well as some terpenoide increased clearly the expression of CC chemokin receptor 2 (CCR2), the receptor for the chemokine MCP-1. Up to date, the main importance for the CCR2 receptor comes from results demonstrating that CCR2 is critical for the migration of monocytes after encounter with bacterial lipopolysaccharides. Under these circumstances the receptor disappears from the cell surface and is down regulated. An up regulation of CCR2 has not been reported for haptens, and deserves further investigations.