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With the advent of highthroughput sequencing (HTS), profiling immunoglobulin (IG) repertoires has become an essential part of immunological research. The dissection of IG repertoires promises to transform our understanding of the adaptive immune system dynamics. Advances in sequencing technology now also allow the use of the Ion Torrent Personal Genome Machine (PGM) to cover the full length of IG mRNA transcripts. The applications of this benchtop scale HTS platform range from identification of new therapeutic antibodies to the deconvolution of malignant B cell tumors. In the context of this thesis, the usability of the PGM is assessed to investigate the IG heavy chain (IGH) repertoires of animal models. First, an innovate bioinformatics approach is presented to identify antigendriven IGH sequences from bulk sequenced bone marrow samples of transgenic humanized rats, expressing a human IG repertoire (OmniRatTM). We show, that these rats mount a convergent IGH CDR3 response towards measles virus hemagglutinin protein and tetanus toxoid, with high similarity to human counterparts. In the future, databases could contain all IGH CDR3 sequences with known specificity to mine IG repertoire datasets for past antigen exposures, ultimately reconstructing the immunological history of an individual. Second, a unique molecular identifier (UID) based HTS approach and network property analysis is used to characterize the CLLlike CD5+ B cell expansion of A20BKO mice overexpressing a natural short splice variant of the CYLD gene (A20BKOsCYLDBOE). We could determine, that in these mice, overexpression of sCYLD leads to unmutated subvariant of CLL (UCLL). Furthermore, we found that this short splice variant is also seen in human CLL patients highlighting it as important target for future investigations. Third, the UID based HTS approach is improved by adapting it to the PGM sequencing technology and applying a custommade data processing pipeline including the ImMunoGeneTics (IMGT) database error detection. Like this, we were able to obtain correct IGH sequences with over 99.5% confidence and correct CDR3 sequences with over 99.9% confidence. Taken together, the results, protocols and sample processing strategies described in this thesis will improve the usability of animal models and the Ion Torrent PGM HTS platform in the field if IG repertoire research.
Besides well-known positive aspects of conservation tillage combined with mulching, a drawback may be the survival of phytopathogenic fungi like Fusarium species on plant residues. This may endanger the health of the following crop by increasing the infection risk for specific plant diseases. In infected plant organs, these pathogens are able to produce mycotoxins like deoxynivalenol (DON). Mycotoxins like DON persist during storage, are heat resistant and of major concern for human and animal health after consumption of contaminated food and feed, respectively. Among fungivorous soil organisms, there are representatives of the soil fauna which are obviously antagonistic to a Fusarium infection and the contamination with mycotoxins. Earthworms (Lumbricus terrestris), collembolans (Folsomia candida) and nematodes (Aphelenchoides saprophilus) provide a wide range of ecosystem services including the stimulation of decomposition processes which may result in the regulation of plant pathogens and the degradation of environmental contaminants. Several investigations under laboratory conditions and in the field were conducted to test the following hypotheses: (1) Fusarium-infected and DON-contaminated wheat straw provides a more attractive food substrate than non-infected control straw (2) the introduced soil fauna reduce the biomass of F. culmorum and the content of DON in infected wheat straw under laboratory and field conditions (3) the species interaction of the introduced soil fauna enhances the degradation of Fusarium biomass and DON concentration in wheat straw; (4) the degradation efficiency of soil fauna is affected by soil texture. The results of the present thesis pointed out that the degradation performance of the introduced soil fauna must be considered as an important contribution to the biological control of plant diseases and environmental pollutants. As in particular L. terrestris revealed to be the driver of the degradation process, earthworms contribute to a sustainable control of fungal pathogens like Fusarium and its mycotoxins in wheat straw, thus reducing the risk of plant diseases and environmental pollution as ecosystem services.
Exposure to fine and ultra-fine environmental particles is still a problem of concern in many industrialized parts of the world and the intensified use of nanotechnology may further increase exposure to small particles. Since many years air pollution is recognized as a critical problem in western countries, which led to rigorous regulation of air quality and the introduction of strict guidelines. However, the upper thresholds for particulates in ambient air recommended by the world health organization are often exceeded several times in newly industrialized countries. Such high levels of air pollution have the potential to induce adverse effects on human health. The response triggered by air pollutants is not limited to local effects of the respiratory system but is often systemic, resulting in endothelial dysfunction or atherosclerotic malady. The link between air pollution and cardiovascular disease is now accepted by the scientific community but the underlying mechanisms responsible for the pro-atherogenic potential still need to be unraveled in detail. Based on the results from in- vivo and in vitro studies the production of reactive oxygen species due to exposure to particles is the most important mechanism to explain the observed adverse effects. However, the doses that were applied in many in vivo and in vitro studies are far beyond the range of what humans are exposed to and there is the need for more realistic exposure studies. Complex in vitro coculture systems may be valuable tools to study particle-induced processes and to extrapolate effects of particles on the lung. One of the objectives of this PhD thesis was the establishment and further improvement of a complex coculture system initially described by Alfaro-Moreno et al. [1]. The system is composed of an alveolar type-II cell line (A549), differentiated macrophage-like cells (THP-1), mast cells (HMC-1) and endothelial cells (EA.hy 926), seeded in a 3D-orientation on a microporous membrane to mimic the cell response of the alveolar surface in vitro in conjunction with native aerosol exposure (VitrocellTM chamber). The tetraculture system was carefully characterized to ensure its performance and repeatability of results. The spatial distribution of the cells in the tetraculture was analyzed by confocal laser scanning microscopy (CLSM), showing a confluent layer of endothelial and epithelial cells on both sides of the Transwellâ„¢. Macrophage-like cells and mast cells can be found on top of the epithelial cells. The latter cells formed colonies under submerged conditions, which disappeared at the air-liquid-interface (ALI). The VitrocellTM aerosol exposure system was not significantly influencing the viability. Using this system, cells were exposed to an aerosol of 50 nm SiO2-Rhodamine nanoparticles (NPs) in PBS. The distribution of the NPs in the tetraculture after exposure was evaluated by CLSM. Fluorescence from internalized particles was detected in CD11b-positive THP-1 cells only. Furthermore, all cell lines were found to be able to respond to xenobiotic model compounds, such as benzo[a]pyrene (B[a]P) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with the upregulation of CYP1 mRNA. With this tetraculture system the response of the endothelial part of the alveolar barrier was studied in- vitro in a still realistic exposure scenario representing the conditions for a polluted situation without direct exposure of endothelial cells. After exposure to diesel exhaust particulate matter (DEPM) the expression of different anti-oxidant target genes and inflammatory genes such as NAD(P)H dehydrogenase quinone 1 (NQO1), superoxide dismutase 1 (SOD1) and heme oxygenase 1 (HMOX1), as well as the nuclear translocation nuclear factor erythroid-derived 2 (Nrf2) was evaluated. In addition, the potential of DEPM to induce the upregulation of CYP1A1 mRNA in the endothelium was analyzed. DEPM exposure led not to an upregulation of the anti-oxidant or inflammatory target genes, but to clear nuclear translocation of Nrf2. The endothelial cells responded to the DEPM treatment also with the upregulation of CYP1A1 mRNA and nuclear translocation of the aryl hydrocarbon receptor (AhR). Overall, DEPM triggered a response in the endothelial cells after indirect exposure of the tetraculture system to low doses of DEPM, underlining the sensitivity of ALI exposure systems. The use of the tetraculture together with the native aerosol exposure equipment may finally lead to a more realistic judgment regarding the hazard of new compounds and/or new nano-scaled materials in the future. For the first time, it was possible to study the response of the endothelial cells of the alveolar barrier in vitro in a realistic exposure scenario avoiding direct exposure of endothelial cells to high amounts of particulates.
Climate change and habitat fragmentation modify the natural habitat of many wetland biota and lead to new compositions of biodiversity in these ecosystems. While the direct effects of climate are often well known, indirect effects due to biotic interactions remain poorly understood. The water meadow grasshopper, Chorthippus montanus, is a univoltine habitat specialist, which is adapted to permanently moist habitats. Land use change and drainage led to highly fragmented populations of this generally flightless species. In large parts of the Palaearctic Ch. montanus occurs sympatrically with its widespread congener, the meadow grasshopper Chorthippus parallelus. Due to their close relationship and their similar songs, hybridization is likely to occur in syntopic populations. Such a species pair of a habitat specialist and a habitat generalist represents an ideal model system to examine the role of ongoing climate change and an accumulation of extreme climatic events on the life history strategies, population dynamics and inter-specific interactions. In Chapter I a laboratory experiment was conducted to identify the impact of environmental factors on intra-specific life-history traits of Ch. montanus. Like other Orthoptera species, Ch. montanus follows a converse temperature size rule. In line with the dimorphic niche hypothesis, which states that sexual size dimorphism evolved in response to the different sexual reproductive roles, both sexes showed different responses to increasing density at lower temperatures. Males attained smaller body sizes at high densities, whereas females had a prolonged development time. This is the first evidence for a sex-specific phenotypic plasticity in Ch. montanus. Females benefit from the prolonged development as their reproductive success depends on the size and number of egg clutches they may produce. By contrast, the reproductive success of males depends on the chance to fertilize virgin females, which increases with faster development. This may become a disadvantage for Ch. montanus as an intraspecific phenology shift may increase hybridization risk with the sibling species. Despite the widespread assumption that hybridization between two sympatric species is rare due to complete reproductive barriers, the genetic analyses of 16 populations (Chapter II) provided evidence for wide prevalence of hybridization between both species in the wild. As no complete admixture was found in the examined population, it is assumed that hybridization only occurs in ecotones between wetlands and drier parts. Reproductive barriers (habitat isolation, behavior, phenology) seem to prevent the genetic swamping of Ch. montanus populations. Although a behavioral experiment showed that mate choice presents an important reproductive barrier between both species, the experiment also revealed that reproductive barriers could be altered by environmental change (e.g. increasing heterospecific frequency). Chapter III analyzes the impact of extreme climatic events on population dynamics and interspecific hybridization. A mark-recapture analysis combined with weather records over five years provides evidence that the embryonic development in Ch. montanus is vulnerable to extreme climatic events. Strong population declines in Ch. montanus lead to a disequilibrium between Ch. montanus and Ch. parallelus populations and increases the risk of hybridization. The highest hybridization risk was found in the first weeks of a season, when both species had an overlapping phenology. Furthermore, hybrids were generally localized at the edge of the Ch. montanus distribution with higher heterospecific encounter probabilities. The hybridization rate reached up to 19.6%. The genetic analyses in Chapter II and III show that hybridization differentially affects specialists and generalists. While generalists may benefit from hybridization by an increasing genetic diversity, such a positive correlation was not found for Ch. montanus. The results underline the importance of reproductive barriers for the co-existence of these sympatric species. However, climate change and other anthropogenic disturbances alter reproductive barriers and promote hybridization, which may threaten small populations by genetic displacement. As anthropogenic hybridization is recognized as a major threat to biodiversity, it should be considered in environmental law and policy. In Chapter IV the role of hybrids and hybridization in three levels of law and the historical backgrounds of hybrids becoming a part of legal instruments is analyzed. Due to legal uncertainties and the complexity of this topic a legal assessment of hybrids is challenging and argues for species-specific approaches. Nonetheless, existing legal norms provide a suitable basis, but need to be specified. Finally, this chapter discusses different opportunities for the management of hybrids and hybridization in a conservation perspective and their necessity.
Um die weiterhin ansteigende Weltbevölkerung auch zukünftig mit Nahrungsmitteln und anderen wichtigen Rohstoffen zu versorgen, bedarf es einer immer größeren Nutzungsausweitung der Landwirtschaft auf natürliche Flächen. Im Jahre 2009 wurde ein Drittel der Landoberfläche der Erde bereits landwirtschaftlich genutzt. In jüngster Zeit kommt der Landwirtschaft zusätzlich eine wichtige Bedeutung als Energielieferant zu. Ein weiter steigender Flächenbedarf ist die Konsequenz dieser Entwicklung. Dies führt zum Verlust von Habitaten und somit zu einer starken Fragmentierung der Landschaft. Die direkten und indirekten Auswirkungen dieser Entwicklung auf den weltweiten Biodiversitätsrückgang werden mittlerweile als eine der Hauptursachen für den Rückgang vieler Arten anerkannt. Auch der Gesetzgeber hat dieses Problem erkannt und versucht dem entgegenzuwirken. In der vorliegenden Arbeit wird durch einen multiplen Methodenansatz der Einfluss der Habitatfragmentierung auf die Konnektivität der Populationen von zwei unterschiedlich mobilen Insektenarten untersucht. Als Modelarten wurden eine wenig mobile Heuschreckenart (Chorthippus montanus) und ein hoch mobiler Tagfalter (Brenthis ino) ausgewählt, welche beide an feuchte Grünländer gebunden sind. Für C. montanus wurde gezeigt, dass die Art nur ein sehr eingeschränktes Mobilitätspotenzial besitzt und stark an ihr Habitat gebunden ist. Daher ist eine Durchquerung von ungeeigneten Lebensräumen als äußerst unwahrscheinlich zu erachten. Eine Studie zur Mobilität und dem Verhalten von Brenthis ino belegt eine starke Philopatrie für eine nicht territoriale Insektenart. Zusätzlich zu den Fang-Wiederfang-Untersuchungen im Freiland wurden populations- und landschaftsgenetische Analysen durchgeführt. Die populationsgenetischen Analysen zeigen eine starke Isolation der Populationen von C. montanus, während zwischen den untersuchten Populationen von B. ino eine gute Konnektivität besteht. Mittels der landschaftsgenetischen Analyse wird gezeigt, dass Wälder und bebaute Flächen für beide Arten substanzielle Genefluss-Barrieren darstellen. Basierend auf einer GIS gestützten Analyse wird dargelegt, dass eine lokal ausreichende Vernetzung von Grünländern in Rheinland-Pfalz durch den Biotopverbund existiert. Die Berücksichtigung der Konnektivität bei der Auswahl und Ausgestaltung der Instrumente zur rechtlichen Sicherung und Umsetzung des Biotopverbundes (-§ 21 BNatSchG) bergen großes Potenzial zum Schutz von Grünländern. Ferner wird gezeigt, dass die Landschaftsplanung zwar ein geeignetes Instrument zur Erarbeitung des Biotopverbundes ist, aber kein Instrument zur rechtlichen Sicherung desselbigen im Sinne des -§ 21 Abs. 4 BNatSchG sein kann.
Geographic ranges of species and their determinants are of great interest in the field of biogeography and are often studied in terms of the species" ecological niches. In this context, the range of a species is defined by the accessibility of an area, abiotic factors and biotic interactions, which affect a species" distributions with different intensities across spatial scales. Parapatry describes a distributional pattern in which the ranges of two species meet along sharp range limits with narrow contact zones. Such parapatric range limits are determined by changing abiotic conditions along sharp environmental gradients or can result from interspecific resource competition. However, it has been shown that often the interplay of abiotic conditions and species interactions determine parapatry. The geographic ranges of the land salamanders, Salamandra salamandra and S. atra, narrowly overlap in the European Alps with only few syntopic localities and to date, the cause of parapatry is unknown. The goal of this thesis was thus to identify the importance of abiotic and biotic factors for their parapatric range limits at different spatial scales. On a broad spatial scale, the role of climate for the parapatric range limits of the species was investigated within three contact zones in Switzerland. Climatic conditions at species" records were analysed and species distribution modelling techniques were used to explore the species" climatic niches and to quantify the interspecific niche overlap. Furthermore, it was tested whether the parapatric range limit coincides with a strong climatic gradient. The results revealed distinct niches for the species as well as the presence of strong climatic gradients which could explain the parapatric range limits of the species. Yet, there was a moderate interspecific niche overlap in all contact zones indicating that the species may co-occur and interact with each other in areas where they both find adequate conditions. Comparison among contact zones revealed geographic variation in the species" niches as well as in the climatic conditions at their records suggesting that the species can occur in a much wider range of conditions than they actually do. These findings imply that climate represents a main factor for the species" parapatric range limits. Yet, interspecific niche overlap and the geographic variation provide indirect evidence that interspecific interaction may also affect their spatial distribution. To test whether competition restricts the species" ranges on the habitat scale and to understand local syntopic co-occurrence of the salamanders within their contact zones, site-occupancy modelling was used. This approach allowed to find the habitat predictors that best explain the species" local distribution. While the slope of the site positively affected the occupancy probability of S. salamandra, no tested predictor explained that of S. atra. Also, there was no effect of the occurrence of one species on the occupancy probability of the other providing no evidence for competition. Should competition occur, it does not lead to spatial segregation of the species on this scale. Because biotic interactions most significantly affect the ranges of species on small spatial scales, the microhabitat conditions at locations of the species within syntopic contact zones were compared and a null model analysis was applied to determine their niche overlap. Resource selection probability function models were used to assess those attributes that affect the species" habitat selections. The results revealed species-specific microhabitat preferences related to leaf litter cover, tree number and that the species were active at different temperatures as well as times of the day. The high degree of diurnal activity of S. atra may be due to its preference of forest floor microhabitats that long remain suitable during daytime. Besides, there was a great niche overlap for shelters indicating that the species may compete for this resource. Differential habitat selection and the use of the available shelters at different times of the day may minimize species interactions and allow their local co-occurrence within contact zones. To identify whether the potential infection with the pathogenic chytrid fungus could serve as an alternative biotic explanation for the range margins of S. atra, several populations throughout its range were screened for infection. Since the occurrence of this pathogen was detected mostly at lower altitudes of the Alps, it may confine the range of S. atra to higher elevations. Because chytrid was not detected in any of the samples, the pathogen unlikely plays a role in determining its range limits. Overall, these findings underline the complexity of mechanisms that determine the range margins of parapatric species and provide an important basis for subsequent studies regarding the determinants of the parapatric distribution of the two salamander species.
Comparing the results of the phylogeographies of the four species included in this thesis, some accordances have been found, even though certain patterns are only represented in one or two species. In all cases, the findings of the studied species strongly support the existence of forests or forest-like ecosystems beyond the classic forest refugia in the Mediterranean areas (Iberian, Apennine and Balkan peninsulas) during glacial times. However, evidence of glacial refugial areas in Southeastern Europe, especially the Balkans, have been found in this study as well. The analysed populations of Aposeris foetida, Melampyrum sylvaticum and Erebia euryale showed high genetic diversity values and mostly higher private fragments in this area, which is a strong indicator for centres of glacial survival during Würm and, regarding the results of M. sylvaticum, even during the Riss ice age. Three of the analysed species (A. foetida, M. sylvaticum and Colias palaeno) supported a second main glacial refuge area located along the Northern Alps. Again, high genetic diversity values and the uniqueness of the populations living in this region today prove the importance of this area as a glacial centre of survival. Those results confirm several recently published studies on forest species and strongly indicate the persistence of forest-like structures or even forests during the ice ages along the foothills of the Northern Alps. Additionally, the persistence of C. palaeno in this area furthermore supports the existence of peatlands north of the Alps, at least during the last glacial. The results of M. sylvaticum and E. euryale further indicate the vicinity of the Tatra Mountains as core areas for glacial survival. However, the genetic patterns found for E. euryale are ambiguous. Due to an intermediate position of two genetic lineages (originating in the Eastern Alps and Southeastern Europe), the Tatras could also reflect a postglacial mixture zone of those lineages. Moreover, the glacial and postglacial importance of this area for woodland species was accentuated, supporting other phylogeographic studies published. Besides the congruities among the results of the study species, some unique patterns and therefore further potential glacial refugia have also been illuminated in this thesis. For instance, the calcicole species, A. foetida, most probably had further survival area at both sides of the Dinaric Alps, supported by high genetic diversity values and a high number of private fragments found in Croatian populations. Furthermore, the surroundings of the German Uplands and the margin of the Southern Alps provided suitable conditions for glacial survival for M. sylvaticum, while the Eastern and Southeastern Alpine region most probably sheltered the Large Ringlet E. euryale during ice ages. Additionally, this butterfly species survived at least the glaciation along the foothills of the Massif Central, whose present populations showed a unique genetic lineage and their genetic diversity values have been measurably higher than in other populations for this species. Finally, a large and continuous Würm distribution is highly likely south of the Fennoscandian glaciers in Central Europe for C. palaeno, which might indicate extended peatland areas during Würm glacial. With all the patterns found in this study, the understanding of glacial persistence of forest, respectively forest-like structures and peatlands during Würm or even Riss glacial in Europe could be advanced. The congruencies among the analysed woodland and bog species illustrate the importance and location of extra-Mediterranean refugia for European mountain forests and the glacial presence of Central European peatlands. Thus, already postulated theories could be supported and further pieces of the overall puzzle could be added. The varieties of the different survival centres once more clarified that further phylogeographic studies on mountain forest of different habitat requirements and especially peatland species have to be implemented to get a clearer picture of the glacial history of these habitats.
Global change, i.e. climate and land use changes, severely impact natural ecosystems at different scales. Poikilothermic animals as butterflies, amphibians and reptiles have proven to be useful indicators for global change impacts as their phenology, spatial distribution, individual fitness and survival strongly depend on external environmental factors. In this aspect, phenological changes in terms of advanced flight or breeding periods, immigrations of foreign species, range shifts concomitant with temperature increases and even local population declines have been observed in both species groups. However, to date much attention has been paid to global change impacts on the species or population level and analyses concerning entire ecosystems are scarce. Applying a novel statistical modelling algorithm we assessed future changes in the extent and composition of terrestrial ecoregions as classified by the World Wide Fund for Nature (WWF). They are defined as coarse-scale conservation units containing exceptional assemblages of species and ecological dynamics. Our results demonstrate dramatic geographical changes in the extent and location of these ecoregions across all continents and even imply a repriorisation of conservation efforts to cope with future climate change impacts on biodiversity. On the local scale, climate change impacts become unequivocal. Comparing historical to contemporary butterfly assemblages on vineyard fallows of the Trier Region, a significant decline in butterfly richness, but also a severe depletion in trait diversity was observed. Comparisons of community temperature indices reveal a striking shift in community composition leading to a replacement of sedentary and monophagous habitat specialists by ubiquitous species. Similar changes have been observed in nature reserves in the Saar-Mosel-area. Monitoring data reveal strong losses of species diversity and remarkable shifts of community compositions at the expense of habitat specialists. Besides climatic variability, these findings are largely attributed to changes in habitat structures, mostly due to eutrophication and monotonisation. Management activities are unlikely to counterbalance these effects, thus severely questioning current conservation strategies. Most dramatic global change impacts are suspected on closely associated species and disruptions of biotic interactions are often hold responsible for species declines. A strong host-parasite association has developed in Myrmica ants and Maculinea butterflies, the later crucially depending on specific host ants for their larval survival. Applying environmental niche models we determined considerable niche dynamics in the observed parasite-host relation with a pronounced niche plasticity in the butterfly species adapting to previous evasive niche shifts in their host ants. Moreover, the new emergence of species continuously expanding their northernmost range borders concomitant with global warming like the Short-tailed blue (Cupido argiades) is attributed to climate change. However, species distribution models predict a severe habitat loss and shifts of potentially suitable habitats of this species towards north-eastern Europe and higher altitudes under several IPCC scenarios making the presence of this species in the Trier region a contemporary phenomenon. Species distribution models have emerged as powerful tools to predict species distributions over spatial and temporal scales. However, not only the presence of a species, but also its abundance have significant implications for species conservation. The ability to deduce spatial abundance patterns from environmental suitability might more efficiently guide field surveys or monitoring programs over large geographical areas saving time and money. Although the application of species distribution models to deduce vertebrate abundances is well recognized, our results indicate that this method is not an adequate approach to predict invertebrate abundances. Structural and ecological factors as well as climatic patterns acting at the microscale are key drivers of invertebrate occurrence and abundances limiting conclusions drawn from modeling approaches. Population declines should be interpreted with care as in butterflies and amphibians various reasons are debated. Both species groups are acknowledged to be highly susceptible to land use changes and variations in landscape structure. Moreover, climate and land use are not independently operating factors. The combined impact of both is demonstrated in our study linking climate-driven changes in amphibian phenologies to temporal advanced applications of pesticides and fertilizers. Both environmental factors already represent severe threats to amphibians when standing alone, but linking their combined impacts may result in an potentiated risk for amphibian populations. As all amphibians and numerous butterfly species are legally protected under the Federal Nature Conservation Act, intensifications of agricultural land use in large parts of Germany as well as new agrarian practices (including genetically manipulated plants accompanied by new herbicide technologies) might severely challenge regional conservation activities in the future.
Cortisol exhibits typical ultradian and circadian rhythm and disturbances in its secretory pattern have been described in stress-related pathology. The aim of this thesis was to dissect the underlying structure of cortisol pulsatility and to develop tools to investigate the effects of this pulsatility on immune cell trafficking and the responsiveness of the neuroendocrine system and GR target genes to stress. Deconvolution modeling was set up as a tool for investigation of the pulsatile secretion underlying the ultradian cortisol rhythm. This further allowed us to investigate the role of the single cortisol pulses on the immune cell trafficking and the role of induced cortisol pulses on the kinetics of expression of GR target genes. The development of these three tools, would allow to induce and investigate in future the significance of single cortisol pulses for health and disease.
In this thesis, in order to shed light on the biological function of the membrane-bound Glucocorticoid Receptor (mGR), proteomic changes induced by 15 min in vivo acute stress and by short in vitro activation of the mGR were analyzed in T-lymphocytes. The numerous overlaps between the two datasets suggest that the mGR mediates physiologically relevant actions and participates in the early stress response, triggering rapid early priming events that pave the way for the slower genomic GC activities. In addition, a new commercially available method with suitable sensitivity to detect the human mGR is reported and the transcriptional origin of this protein investigated. Our results indicates that specific GR-transcripts, containing exon 1C and 1D, are associated with the expression of this membrane isoform.