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Unstable environments and habitats changing due to climate change force individuals to either respond by genetic adaptation, phenotypic plasticity or by dispersal to suitable environments. Theodoxus fluviatilis (Linneaus, 1758) is a good study organisms when researching phenotypic plasticity and genetic adaptation as it naturally appears in freshwater (FW) as well as brackish water (BW) and thus inhabits a wide range of environmental salinities (0-18‰). It is a euryhaline snail that can be found in shallow waters with stony ground or on Fucus spp. and has formed regional subgroups. The brackish water and the freshwater subgroups are spatially separated and the species cannot be found in areas inbetween, e.g. estuaries.
The species shows great variability in shell patterning and shell size and there is still debate whether the subgroups are distinguishable by these traits or not. The mitochdrial RNA marker cytochrome c subunit I did not show differences between the subgroups indicating that they must be closely related, but salinity tolerance has been observed to be higher in BW snails. This might be caused by the different protein expression patterns and osmolyte accumulation (measured as ninhydrin-positive substances) observed in this species in previous studies. The exact mechanisms regulating protein expression and osmolyte accumulation, however, are not fully understood yet.
Data collected for this thesis shows differences in shell size and suggests a less strict grouping of FW and BW individuals as shell sizes of one FW site are more similar to BW individuals than the other FW ones. A better salinity tolerance towards high salinities and a higher physiological salinity limit of BW snails was confirmed and extended by demonstrating an expanded tolerance range through slow acclimation to challenging salinities in snails from both subgroups. This was achieved by a shift in the slope of their reaction norms that was much more pronounced in BW snails than FW ones. S3 individuals showed a shift similar to that of BW individuals. The data for the salinity tolerance indicates that the underlying mechanism for these tolerances are a combination of phenotypic plasticity and genetic adaptation. Despite an acclimation and shift in the slope of the reaction norms and therefore an increased tolerance towards high salinities (plasticity) FW individuals from two collection sites were not able to cope with salinities as high as BW individuals (local adaptation). The general ability to mobilise free amino acids (FAA) as organic osmolytes was not the reason for this tolerance difference. Individuals from BW and FW sites were capable of accumulating quantities of FAAs equally well. Proline, alanine and urea were the most important components of the accumulated cocktail of organic osmolytes. Even though the total amount of FAAs accumulated under hyperosmotic conditions was the same in both subgroups, there were differences in the metabolic pathways involved in osmolyte accumulation in the foot muscle. The data indicates that the hydrolysis of storage proteins and the synthesis of proline and alanine are the main processes to avoid detrimental body volume shrinkage in T. fluviatilis. While FW individuals seemed to rely on the degradation of proteins and synthesis of alanine, BW individuals depended on newly synthesising proline and alanine and accumulating urea as a side product of transamination. The accumulation of urea is a new finding in aquatic living snails and has not been reported as a mechanism to avoid cell volume shrinkage in these animals.
Differing protein expression patterns were observed under control conditions across all collection sites. 9 spots showed volume changes in BW snails opposite to those of FW snails from collection sites S1 and S2. For 6 of those spots, S3 individuals showed patterns similar to those of BW individuals and for the remaining 3 they showed patterns similar to those of FW animals. The patterns observed when exposing snails to hypo- or hyperosmotic stress were not conclusive in relation to pinpointing individual spots that show the same pattern in all collection sites, but revealed the heterogeneity of protein expression in snails from the different collection sites and in the process of osmoregulation. It also showed the general tendency of protein reduction when snails where under osmotic stress of either kind (hypo- or hyperosmotic), which supports the hypothesis of storage protein degradation.
The investigation of an ANP-receptor showed two variations of the encoding sequence expressed in T. fluviatilis. S3 individuals as well as BW individuals were found to express one type, while FW individuals, with the exception of one sample expressed the other type. This showed that the FW subgroup of T. fluviatilis seems to be more heterogeneous than the BW subgroup, but also raises the question of the dispersal history of this species. The collected data indicates that T. fluviatilis individuals are firstly capable of surviving the acidity of a duck's gizzard and secondly can tolerate acute salinity changes to 16‰ when introduced into a new environment. Hence, if snails from the FW were to be transported to waters with a salinity of up to 16‰ by man, bird, drifting plants or some other means of transport, they would most likely survive and possibly be able to thrive and spread.
Die akute Pankreatitis ist durch eine vorzeitige intraazinäre Proteasen-Aktivierung gekennzeichnet, wobei diese im Verlauf der Erkrankung durch eine zunehmende Immunantwort mit in das Pankreas infiltrierenden Immunzellen ergänzt wird. Eine besondere Bedeutung hat die intrazelluläre Aktivierung der Serinprotease Trypsinogen, die in Abhängigkeit der lysosomalen Hydrolase Cathepsin B (CTSB) verläuft.
Wir konnten zeigen, dass verschiedene lysosomale Proteine (Cathepsin D (CTSD), Cathepsin C (CTSC)) nach pathologischem Stimulus in das sekretorische Kompartiment (Zymogengranula) umverteilt werden. Cathepsin D ist in der Lage, das Schlüsselenzym Cathepsin B zu aktivieren, indem es das Pro-Enzym zu aktivem Enzym spaltet. Der Ort dieser proteolytischen Aktivierung sind die sekretorischen Vesikel. Eine pharmakologisch induzierte Permeabilisierung der Lysosomen mit nachfolgendem Ausbleiben der Umverteilung der Enzyme in das sekretorische Kompartiment zeigte, dass die vorzeitige Zymogen-Aktivierung in der Frühphase der Pankreatitis erhalten geblieben ist und unabhängig vom Lysosom verläuft. Eine CTSB-Abhängigkeit bleibt jedoch bestehen. Ein Fehlen von CTSD in den Azinuszellen führt zu einem nur transient milderen Verlauf der akuten Pankreatitis, wie anhand von CTSDf/f/p48Cre/+ Mäusen demonstiert werden konnte, die einen Pankreas-spezifischen CTSD Knockout besitzen. Ein anhaltend milderer Verlauf der Pankreatitis fand sich in CTSD-/- Mäusen, der auf eine verminderte Sekretion pro-inflammatorischer Zytokine in Immunzellen zurückzuführen ist. Auch bei Defizienz von CTSC war der Schweregrad der akuten Pankreatitis milder, wie in CTSC-/- Mäusen experimentell demonstiert werden konnte. Ursächlich hierfür ist vor allem ein reduziertes Einwandern neutrophiler Granulozyten in das Pankreas und in die extrapankreatischen Organe (Lunge), die auf eine geringere Aktivität der Serinprotease Neutrophilen Elastase und verminderte Spaltung des Zell-Kontakt Moleküls E-Cadherin beruhen. Umgekehrt beeinflusste das Fehlen von CTSC in den Azinuszellen nicht die vorzeitige Proteasen-Aktivierung.
Unsere Arbeit unterstreicht die Bedeutung lysosomaler Enzyme in der akuten Pankreatitis und zeigt, dass diese Enzyme maßgeblichen Einfluss auf die Funktion von Immunzellen haben, die den Verlauf der Erkrankung wesentlich mitbestimmen. Unsere Arbeit zeigt außerdem, dass der primäre Ort der intrazellulären und vorzeitigen Proteasen-Aktivierung alleinig im sekretorischen Kompartiment stattfindet und nicht von einer Fusion mit dem lysosomalen Kompartiment abhängig ist.