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Characterization of tolerogenic rat bone marrow-derived dendritic cells and regulatory T cells
(2010)
Tolerogenic dendritic cells (DC) and regulatory T (Treg) cells are able to prevent destructive immune responses. There is reason to hope that it may soon be possible to use DC and Treg cells to suppress immune responses antigen-specific, not only after transplantation, but also in the case of autoimmunity and allergy. At the moment, the generation of such cell types is very time-consuming and not suitable for clinical routine. In addition, it is not yet fully understood how these cells elicit a desired protective immune response in vivo and how the risks of an excessive immune suppression can be managed. The rat is one of the most important animal models in biomedical research. It is therefore surprising that tolerogenic DC and Treg cells in particular have not been more thoroughly investigated in this model. Thus, the aim of the present study was to systematically characterize these immune cells and investigate their impact on the immune system. Tolerogenic DC were generated from bone marrow precursors cultured with GM-CSF and IL-4 (= IL-4 DC). The proportion of naturally occurring Treg cells with a CD4posCD25posFoxp3pos phenotype comprises approximately 5-8% of the peripheral CD4pos T cells. The characterization of IL-4 DC revealed an up to 26-fold reduced expression of surface molecules such as MHC class II molecules, CD80, CD86, ICAM-1 and CD25 in comparison to mature splenic DC (S-DC). This low expression did not change when the cells where stimulated with different maturation-inducing signals such as replating, LPS, TNF- α and CD40L. Thus, these cells possess a robust phenotype resistant to maturation-inducing stimuli. IL-4 DC take up antigen via endocytosis and are not able to activate naïve T cells or to restimulate antigen-specific T cells. Furthermore, they are able to inhibit and prolongate mature S-DC induced T cell proliferation as well as mature S-DC induced restimulation of antigen-specific T cells, respectively. Thereby, the T cell proliferation was reduced up to 95%. This strong inhibitory effect was mediated within 24 hours in association with a reduced cytokine production (IL-2 about 49% and IFN-γ about 92%). The inhibitory properties of IL-4 DC don´t seem to be caused exclusively by the reduced expression of co-stimulatory molecules. In this study, the detection of the inhibitory molecules PD-L1 and PD-L2 on IL-4 DC suggests they have an impact on mediating inhibitory signals to the T cells. In addition, a suppressive effect of soluble factors was shown. The supernatant of one million IL-4 DC, collected after a 24 hour culture, suppressed mature S-DC induced proliferation of naïve T cells by about 90%. TGF-β, which was detected in the supernatant (up to 300 pg/ml), appears to be the causing soluble factor for this immune inhibition. By contrast, the supernatants of mature S-DC, which did not inhibit the activation of T cells, showed a TGF-β concentration of only about 100 pg/ml. The cytotoxic nitric oxide does not contribute to the IL-4 DC-mediated inhibition of T cell proliferation. The NO synthase inhibitor NMMA reduced the amount of NO by about 50%, but the decreased NO levels did not influence T cell proliferation. Indeed, IL-4 DC are not able to induce T cell proliferation, but this doesn´t mean that there is no change on the molecular level. For instance, T cells co-cultured with IL-4 DC during a first culture are not able to proliferate in the presence of mature S-DC during a second culture. This anergic-like state, however, could be abolished by adding exogenous IL-2. In addition, T cells co-cultured with IL-4 DC are able to inhibit the activation of naïve T cells. Naïve and activated T cells were not able to inhibit the mature S-DC induced T cell proliferation. This observation suggests the induction of Treg cells and was investigated in more detail. Indeed, flow cytometric analysis showed a 1.6-fold expansion of CD4posCD25posFoxp3pos T cells from naturally occurring Treg cells in the presence of IL-4 DC. Thereby, the expansion of CD4posCD25posFoxp3pos T cells occurs independently of the maturation state of DC. Both immature IL-4 DC as well as mature S-DC were able to expand the percentage of naturally occurring Treg cells. However, Treg cells pre-incubated with mature S-DC demonstrated a diminished inhibitory effect compared to Treg cells pre-incubated with IL-4 DC. Treg cells pre-incubated with IL-4 DC were able to inhibit the activation of naïve T cells. In this study it was shown that the regulatory potential of DC cannot be deduced solely by their phenotype or maturation state. Other factors, such as functional properties, need to taken into consideration, too. The induction of Treg cells with suppressive properties induced by in vitro generated tolerogenic IL-4 DC might provide an important mechanism for the maintenance of peripheral tolerance. However, for clinical application further investigation is necessary, not only to understand the interactions between tolerogenic DC and Treg cells, but also to investigate the impact of the transfer of a larger quantity of regulatory cells on the immune system of the recipient.
Resin, a sticky sap emitting terpenoids and other volatiles, is produced by various plant species to seal wounds and protect themselves against herbivores and microbes. Among several other insects, bees have evolved the surprising ability to handle the repellent plant sap and use it to construct and defend their nests. Whereas the collection of pollen and nectar has been intensively studied in bees, resin collection has received only little attention. The aim of this dissertation was to better understand how the physiological and chemical properties of resin and resin-derived compounds (terpenes) affect the ecology of stingless bees. I therefore asked why, where and how stingless bees of Borneo (seven study-species), Australia (eight) and Costa Rica (27) collect and process plant resins, addressing the importance of a largely neglected resource not only for building and defensive properties, but also for the bees’ chemical diversity. Stingless bees are highly opportunistic resin foragers with all species collecting resin from a similar set of tree species. They locate and/or recognize resin sources on the basis of several volatile mono- and sesquiterpenes. I found that different bee species and even colonies significantly varied in the amount of resin collected. Predator attack (e.g., by ants) had the strongest affect on resin intake, whereas manual nest destruction only slightly increased the number of resin foragers. Resin is used to build, maintain and defend nests, but also as source for chemical compounds (terpenes) which stingless bees include in their surface profiles (chemical profiles). They directly transfer resin-derived compounds to their body surfaces (cuticular terpenes), but only include a subset (8 %) of the large number (>> 1000) of terpenes found in tree resins. This phenomenon can only be explained by a hitherto unknown ability to filter environmentally derived compounds which results in species-specific terpene profiles and thus in an increased chemical heterogeneity among species. Moreover, due to the addition of resin-derived substances the diversity of compounds on the bees’ body surfaces by far exceeds the chemical diversity of profiles in other hymenopterans. Because stingless bees filter but do not modify resin-derived compounds, species from Borneo, Australia and Costa Rica all resemble the characteristic resin of typical trees in their regions of origin. This chemical similarity reveals a strong correlation between the diversity of tree resins and the diversity of cuticular terpenes among stingless bees in a given habitat. Because different tree species are found in different tropical regions, the chemical composition of tree resins varies between tropical regions as does the composition of cuticular terpenes in bee species from these regions. Cuticular terpenes are however most common among stingless from Borneo, with 100 % of species studied having resin-derived terpenes in their chemical profiles. They are least common in Costa Rica, with only 40 % of species having terpenes. Likewise, resin collection was found to be highest in Tetragonilla collina colonies of Borneo where occasionally up to 90 % of foragers collected resin. By contrast, resin collection was only performed by 10 % of foragers of a given colony in Australia and by a maximum of 40 % in Costa Rica. The dominance of resin and resin-derived compounds in the chemical ecology of bees from Borneo may mirror the dominance of a particular Southeast Asian tree family: the highly resinous dipterocarps. Such a correlation between the chemistry of bees and the chemistry of tree resins therefore underlines the close relationship between stingless bees and the trees of their habitat. Cuticular terpenes are assumed to protect bees against predators and/or microbes. Sesquiterpenes, a specific group of terpenes, most vary between species and impair inter-specific aggression by reducing aggressive behavior in species without sesquiterpenes, thereby providing a novel mechanism to achieve interspecific tolerance among insects. Reduced interspecific aggression may also be an important factor enabling the non-aggressive aggregation of nests from stingless bee colonies of up to four different species, because such aggregations frequently comprise both species with and species without sesquiterpenes. Given its various functions, resin represents a highly important resource for stingless bees which directly affects their chemical ecology, defensive properties and inter-specific communication. It remains to be investigated how the bees influence the resin-derived terpene profiles on their body surface and in their nests, particularly how they manage to exclude entire groups of terpenes. Whether bees actually need a high diversity of different resin sources and therefore tree species to maintain the homeostasis of their colonies or whether they would do equally well with a limited amount of resin sources available, should also be addressed in future studies. Answers to this question will directly impair bee and forest management in (sub)tropical regions.
The aim of this project was to investigate whether reflex-like innate facial reactions to tastes and odors are altered in patients with eating disorders. Qualitatively different tastes and odors have been found to elicit specific facial expressions in newborns. This specificity in newborns is characterized by positive facial reactions in response to pleasant stimuli and by negative facial reactions in response to unpleasant stimuli. It is, however, unclear, whether these specific facial displays remain stable during ontogeny (1). Despite the fact that several studies had shown that taste-and odor-elicited facial reactions remain quite stable across a human’s life-span, the specificity of research questions, as well as different research methods, allow only limited comparisons between studies. Moreover, the gustofacial response patterns might be altered in pathological eating behavior (2). To date, however, the question of whether dysfunctional eating behavior might alter facial activity in response to tastes and odors has not been addressed. Furthermore, changes in facial activity might be linked to deficient inhibitory facial control (3). To investigate these three research questions, facial reactions in response to tastes and odors were assessed. Facial reactions were analyzed using the Facial Action Coding System (FACS, Ekman & Friesen, 1978; Ekman, Friesen, & Hager, 2002) and electromyography.
Integrating neurobiological markers of depression: an fMRI-based pattern classification approach
(2010)
While depressive disorders are, to date, diagnosed based on behavioral symptoms and course of illness, the interest in neurobiological markers of psychiatric disorders has grown substantially in recent years. However, current classification approaches are mainly based on data from a single biomarker, making it difficult to predict diseases such as depression which are characterized by a complex pattern of symptoms. Accordingly, none of the previously investigated single biomarkers has shown sufficient predictive power for practical application. In this work, we therefore propose an algorithm which integrates neuroimaging data associated with multiple, symptom-related neural processes relevant in depression to improve classification accuracy. First, we identified the core-symptoms of depression from standard classification systems. Then, we designed and conducted three experimental paradigms probing psychological processes known to be related to these symptoms using functional Magnetic Resonance Imaging. In order to integrate the resulting 12 high-dimensional biomarkers, we developed a multi-source pattern recognition algorithm based on a combination of Gaussian Process Classifiers and decision trees. Applying this approach to a group of 30 healthy controls and 30 depressive in-patients who were on a variety of medications and displayed varying degrees of symptom-severity allowed for high-accuracy single-subject classification. Specifically, integrating biomarkers yielded an accuracy of 83% while the best of the 12 single biomarkers alone classified a significantly lower number of subjects (72%) correctly. Thus, integrated biomarker-based classification of a heterogeneous, real-life sample resulted in accuracy comparable to the highest ever achieved in previous single biomarker research. Furthermore, investigation of the final prediction model revealed that neural activation during the processing of neutral facial expressions, large rewards, and safety cues is most relevant for over-all classification. We conclude that combining brain activation related to the core-symptoms of depression using the multi-source pattern classification approach developed in this work substantially increases classification accuracy while providing a sparse relational biomarker-model for future prediction.
Leaf-cutting ants have a highly developed thermal sense which the insects use to regulate the own body temperature and also to optimize brood and fungus development. Apart from the already described temperature guided behaviors inside the nest it is unknown to what extent the ants may use their thermal sense outside the nest. As part of the present thesis, the question was addressed whether leaf-cutting ants (Atta vollenweideri) are able to learn the position of a warm object as landmark for orientation during foraging. Using absolute conditioning, it was shown that ten training trials are sufficient to elicit the association be-tween food reward and the temperature stimulus. In the test situation (without reward) a significantly higher amount of ants preferred the heated site compared to the unheated con-trol. Importantly, thermal radiation alone was sufficient to establish the learned association and served as orientation cue during the test situation (chapter IV). Based on the experi-mental design used in the previous chapter, the localization of thermosensitive neurons, which detect the underlying thermal stimuli, is restricted to the head or the antennae of the ants. The antennal sensillum coeloconicum is a potential candidate to detect the thermal stimuli during the orientation behavior. In chapter V the sensillum coeloconicum of Atta vollenweideri was investigated concerning its gross morphology, fine-structure and the phy-siology of the associated thermosensitive neuron. The sensillum is predominantly located on the apical antennal segment (antennal tip) where around 12 sensilla are clustered, and it has a peg-in-pit morphology with a double walled, multiporous peg. The sensory peg is deeply embedded in a cuticular pit, connected to the environment only by a tiny aperture. The sen-sillum houses three receptor neurons of which one is thermosensitive whereas the sensory modality of the other two neurons remains to be shown. Upon stimulation with a drop in temperature, the thermosensitve neuron responds with a phasic-tonic increase in neuronal activity (cold-sensitive neuron) and shows rapid adaptation to prolonged stimulation. In ad-dition, it is shown that thermal radiation is an effective stimulus for the thermosensitive neuron. This is the first evidence that sensilla coeloconica play an important role during the thermal orientation behavior described in chapter IV. During the test situation of the classic-al conditioning paradigm, the ants showed rapid antennal movements, indicating that they scan their environment in order to detect the heated object. Rapid antennal movements will result in rapid discontinuities of thermal radiation that re-quire thermosensitive neurons with outstanding sensitivity and high temporal resolution. In Chapter VI the question was addressed whether the thermosensitive neuron of the sensilla coeloconica fulfils these preconditions. Extracellular recordings revealed that the neuron is extremely sensitive to temperature transients and that, due to the response dynamics, an estimated stimulus frequency of up to 5 Hz can be resolved by the neuron. Already a tem-perature increase of only 0.005 °C leads to a pronounced response of the thermosensitive neuron. Through sensory adaptation, the sensitivity to temperature transients is maintained over a wide range of ambient temperatures. The discovered extreme sensitivity, the high temporal resolution and the pronounced adaptation abilities are further evidence support-ing the idea that sensilla coeloconica receive information of the thermal environment, which the ants may use for orientation. In order to understand how the ants use their thermal environment for orientation, it is ne-cessary to know where and how thermal information is processed in their central nervous system. In Chapter VII the question is addressed where in the brain the thermal information, specifically received by the thermosensitive neuron of sensilla coeloconica, is represented. By selectively staining single sensilla coeloconica, the axons of the receptor neurons could be tracked into the antennal lobe of Atta vollenweideri workers. Each of the three axons termi-nated in a single functional unit (glomerulus) of the antennal lobe. Two of the innervated glomeruli were adjacent to each other and are located lateral, while the third one was clear-ly separate and located medial in the antennal lobe. Using two-photon Ca2+ imaging of an-tennal lobe projection neurons, the general representation of thermal information in the antennal lobe was studied. In 11 investigated antennal lobes up to six different glomeruli responded to temperature stimulation in a single specimen. Both, warm- and cold-sensitive glomeruli could be identified. All thermosensitive glomeruli were located in the medial half of the antennal lobe. Based on the correlative evidence of the general representation of thermal information and the results from the single sensilla stainings, it is assumed that thermal information received by sensilla coeloconica is processed in the medial of the three target glomeruli. This part of the thesis shows the important role of the antennal lobe in temperature processing and links one specific thermosensitive neuron to its target region (a single glomerulus). In chapter V it was shown that the sensilla coeloconica are clustered at the antennal tip and have an extraordinary peg-in-pit morphology. In the last chapter of this thesis (Chapter VIII) the question is addressed whether the morphology of the sensilla coeloconica predicts the receptive field of the thermosensitive neuron during the detection of thermal radiation. The sensory pegs of all sensilla coeloconica in the apical cluster have a similar orientation, which was not constraint by the shape of the antennal tip where the cluster is located. This finding indicates that the sensilla coeloconica function as a single unit. Finally the hypothesis was tested whether a single sensillum could be direction sensitive to thermal radiation based on its eye-catching morphology. By stimulating the thermosensitive neuron from various angles around the sensillum this indeed could be shown. This is the last and most significant evi-dence that the sensilla coeloconica may be adapted to detect spatially distributed heated objects in the environment during the thermal landmark orientation of ants.
Characterization of allosteric mechanisms on the M2 and M4 mACh receptor using the FRET-technique
(2010)
Allosteric modulators have been proposed as promising new compounds to modify protein function. Allosteric binding sites have been discovered for several G-protein-coupled receptors, including M1-5 muscarinic receptors. Since these receptors play a pivotal role in the regulation of a plethora of organ functions, it is particularly important to investigate the mechanisms of allosteric modulation. To study molecular mechanisms of allosteric modulation in the M2 muscarinic receptor, a new FRET-based sensor was designed. CFP fused to the C-terminus of the receptor and a small fluorescent compound FlAsH, which labels a specific binding sequence in the third intracellular loop, were used as donor and acceptor fluorophores, respectively. The first part of the study was to design a functional FRET receptor sensor. After several optimization steps the constructs FLAG-M2-sl3-FlAsH-GSGEG-CFP and HA-FLAG-M2-sl3-FlAsH-GSGEG-CFP were generated which showed good cell-surface expression, robust changes in FRET and the ability to deliver reproducible data. The second part of this thesis sought to elucidate the mechanisms of the allosteric ligand binding and their effects on the receptor conformation. The described modifications, which were introduced in the wild type M2 mAChR to create the FRET sensor can alter receptor functionality and influence receptor expression. Radioligand binding studies revealed that the used transfection method provided sufficient receptor expression but, unfortunately, about 60 % of the FLAG-M2-sl3-FlAsH-GSGEG-CFP receptor remains in the cytosol. However, this was sufficient to perform FRET experiments. Patch clamp GIRK-measurements with acetylcholine evinced that the new M2-sensor was able to activate Gi-proteins. Also, radioligand-binding assays with the second construct HA-FLAG-M2-sl3-FlAsH-GSGEG-CFP showed ligand affinity comparable to the wildtype receptor. Furthermore inhibition of forskolin-stimulated cAMP production was indistinguishable from the behaviour of the wildtype receptor. According to that, the full functionality of both receptor constructs could be confirmed. FRET measurements with the full muscarinic receptor agonists carbachol and acetylcholine confirmed that the FLAG-M2-sl3-FlAsH-GSGEG-CFP receptor construct showed rapid changes in FRET upon addition of both ligands, which were concentration-dependent. Concentration response curves and the resulting EC50 values of both agonists were similar to those already published in literature. In addition, the orthosteric antagonists atropine and methoctramine inhibited the FRET changes induced by the agonists. This inhibition was significantly faster than the washout kinetics, pointing to an active displacement of the agonists by the antagonists. Allosteric ligands gallamine, tacrine and dimethyl-W84 did not alter receptor conformation when added without an orthosteric ligand. However, when applied in addition to muscarinic agonists, all three substances inhibited the FRET-signal. The extent of this inhibition was dependent on the used concentration of the allosteric ligands. These results reveal that conformational changes brought about by allosteric ligands can be measured with the FRET technique. Furthermore real-time FRET-based kinetic measurements could be performed in living cells and showed that the allosteric ligands gallamine and dimethyl-W84 alter receptor conformation significantly faster than the antagonists atropine and methoctramine. This data indicate that allosteric ligands actively induce the conformational changes in the receptor.
Studies on platelet cytoskeletal dynamics and receptor regulation in genetically modified mice
(2009)
Platelets are produced by bone marrow megakaryocytes in a process involving actin dynamics. Actin-depolymerizing factor (ADF) and cofilin are actin-binding proteins that act as key regulators in actin turnover by promoting filament severing and depolymerization. The overall significance of ADF/cofilin function and actin turnover in platelet formation is presently unclear. In the first part of this thesis, platelet formation and function were studied in mice constitutively lacking ADF and/or mice with a conditional deficiency (Cre/loxP) in n-cofilin. To delete cofilin exclusively in megakaryocytes and platelets, cofilinfl/fl mice were crossed with PF4 (platelet factor 4)-Cre mice. While a single-deficiency in ADF or n-cofilin resulted in no or only a minor platelet formation defect, respectively, a double-deficiency in ADF and n-cofilin led to an almost complete loss of platelets. Bone marrow megakaryocytes of ADF/n-cofilin-deficient mice showed defective platelet zone formation. Interestingly, in vitro and ex vivo megakaryocyte differentiation revealed reduced proplatelet formation and absence of platelet-forming swellings. These data establish that ADF and n-cofilin have redundant but essential roles in the terminal step of platelet formation in vitro and in vivo. In the second part of the thesis, mechanisms underlying cellular regulation of the major platelet collagen receptor, glycoprotein VI (GPVI), were studied. GPVI mediates platelet activation on exposed subendothelial collagens at sites of vascular injury, and thereby contributes to normal hemostasis but also to occlusion of diseased vessels in the setting of myocardial infarction or stroke. Thus, GPVI is an attractive target for anti-thrombotic therapy, particularly because previous studies have shown that anti-GPVI antibodies induce irreversible down-regulation of the receptor in circulating platelets by internalization and ectodomain shedding. Metalloproteinases of the ADAM (a disintegrin and metalloproteinase domain) family are suspected to mediate this ectodomain shedding, but in vivo evidence for this is lacking. To study the mechanism of GPVI regulation in vivo, two mouse lines, Gp6 knock-out and Adam10fl/fl, PF4-Cre mice, were generated and in addition low TACE (TNFalpha converting enzyme) mice were analyzed. It was shown that GPVI can be cleaved in vitro by ADAM10 or TACE depending on the shedding-inducing signaling pathway. Moreover, GPVI was down-regulated in vivo upon antibody injection in ADAM10-deficient and low TACE mice suggesting that either both or an additional metalloproteinase is involved in GPVI regulation in vivo.
Die Regulation des Tonus glatter Muskelzellen wird entscheidend von den beiden antagonistisch wirkenden second messengern cAMP und Ca2+ beeinflusst. Ein Ziel dieser Arbeit war herauszufinden, ob diese beiden Botenstoffe auch direkten Einfluss aufeinander haben können und welche Enzyme in diesem Fall an den Prozessen beteiligt sind. cAMP-Signale in intakten Zellen konnten wir in Echtzeit mit Hilfe des FRET-basierten cAMP-Sensors Epac1-camps beobachten; Ca2+-Signale durch Markieren der Zellen mit Fura-2. Anstiege der intrazellulären Ca2+-Konzentration in VSMCs wurden durch Aktivierung von endogen exprimierten, Gq-gekoppelten P2Y6-Rezeptoren mit Uridindiphosphat (UDP) ausgelöst. Durch eine zusätzliche in-vitro Kalibrierung des Epac1-camps konnten darüber hinaus absolute cAMP-Konzentrationen in einzelnen lebenden Zellen berechnet werden. Während ein Anstieg der Ca2+-Konzentration auf nicht vorstimulierte VSMCs keinen signifikante Einfluss auf die intrazellulären cAMP-Konzentrationen hatte, bewirkte die Aktivierung der purinergen Rezeptoren einen deutlichen Rückgang der intrazellulären cAMP-Konzentration in mit Isoproterenol vorstimulierten VSMCs. Dieser Effekt konnte sowohl durch die Komplexierung von Ca2+ mit BAPTA-AM als auch durch die Überexpression der Ca2+-insensitiven AC4 antagonisiert werden. Adenylatcyclase-Aktivitäts-Assays in VSMC-Membranen zeigten ebenfalls einen Rückgang der Cyclaseaktivität nach Zugabe von 2 und 5 μM freiem Ca2+. Die Hemmung der einzigen Ca2+-regulierbaren PDE1 mit dem selektiven PDE1-Inhibitor 8-Methoxymethyl-IBMX (8-MM-IBMX) hatte im Gegensatz dazu keinen Einfluss auf die durch UDP verursachte Änderung der cAMP-Konzentration in vorstimulierten VSMCs. Schließlich bewirkte die Herunterregulation der Ca2+-inhibierbaren AC5 und 6 mit siRNA einen signifikante Hemmung des durch UDP verursachten Effekts. Fasst man alle diese Ergebnisse zusammen, so lässt sich folgende Schlussfolgerung ziehen: Der durch purinerge Stimulation verursachte Rückgang der cAMP-Konzentration in mit Isoproterenol vorstimulierten VSMCs wird durch eine Hemmung der Ca2+-hemmbaren AC5 und 6 vermittelt. Dadurch sind zwei für die Regulation des Tonus wichtige Signalwege in VSMCs miteinander verbunden, die sich somit gegenseitig entscheidend beeinflussen können. Ein weiterer Bestandteil dieser Arbeit war die Entwicklung eines transgenen Mausmodells, das glattmuskelspezifisch den cAMP-Sensor Epac1-camps exprimiert. Mit Hilfe eines solchen Tiermodells könnten in Zukunft cAMP-Änderungen in intakten Geweben und vielleicht sogar in lebenden Tieren beobachtet werden. Durch Anwendung des Cre-loxP-Rekombinationssystems gelang es eine glatt¬muskelspezifische, für den Epac1-camps transgene Mauslinie zu generieren. Mit isolierten VSMCs dieser Tiere konnten bereits erste FRET-Messungen durchgeführt und agonistinduzierte cAMP-Änderungen beobachtet werden.
Der Parathormonrezeptor Typ 1 (PTHR) ist ein G-Protein-gekoppelter Rezeptor der Gruppe 2 und wichtigster Regulator des Kalziumstoffwechsels. Im ersten Teil der Arbeit wurde eine neuartige posttranslationale Modifikation des PTHR in Form einer proteolytischen Spaltung der Ektodomäne identifiziert, charakterisiert und deren Regulation beschrieben. Nach langanhaltender Stimulation des Rezeptors mit Agonisten – aber nicht mit Antagonisten – wurde eine Massen- und Mengenzunahme des Rezeptorproteins beobachtet. Es konnte gezeigt werden, dass der Rezeptor unter basalen Bedingungen einer Spaltung unterliegt. Der Massenunterschied entsteht durch die proteolytische Spaltung der Ektodomäne des PTHR, was nachfolgend die Stabilität des Rezeptors beeinträchtigt. Die Spaltung erfolgte innerhalb einer unstrukturierten Schleife der Ektodomäne, welche die Bereiche für die Ligandenbindung miteinander verbindet. Hierbei handelt es sich um eine Region, die im Vergleich zu anderen Gruppe 2-Rezeptoren spezifisch für den PTHR ist. Das durch die Spaltung entstandene N-terminale Fragment bleibt durch eine Disulfidbrücke mit dem Transmembranteil des Rezeptors verbunden. Durch Versuche mit verschiedenen Proteaseinhibitoren konnte die verantwortliche Protease der Familie der zinkabhängigen extrazellulären Proteasen zugeordnet werden. Diese Ergebnisse beschreiben einen Mechanismus wie die Homoöstase des PTHR reguliert sein könnte. In einem zweiten Abschnitt wurde die Interaktion der Adapterproteine NHERF1 und beta-Arrestin2 mit dem PTHR untersucht. Beide Proteine interagierten unabhängig mit dem Rezeptor, wobei NHERF1 über eine PDZ-Domäne konstitutiv an den C-Terminus des Rezeptors bindet. beta-Arrestin2 hingegen bindet nach Aktivierung des Rezeptors und führt zur Desensitisierung des Rezeptors. Mittels biochemischer und mikroskopischer Methoden konnte gezeigt werden, dass beide Proteine gemeinsam einen ternären Komplex mit dem PTHR bilden, welcher durch die direkte Interaktion zwischen NHERF1 und beta-Arrestin2 vermittelt wird. Dies hat zur Folge, dass beta-Arrestin im basalen Zustand durch NHERF1 an den Rezeptor gekoppelt wird. Durch Analyse der Assoziationskinetik mittels Fluoreszenz-Resonanz-Energietransfer-Messungen zeigte sich, dass diese Kopplung zu einer zweifach erhöhten Rekrutierungsgeschwindigkeit von beta-Arrestin2 an den PTHR führt. Somit stellt unterstützt NHERF1 die beta-Arrestin2-vermittelte Desensitisierung des PTHR.
An increase in cytosolic Ca2+ levels ([Ca2+]i) is a key event that occurs downstream of many signaling cascades in response to an external stimulus and regulates a wide range of cellular processes, including platelet activation. Eukaryotic cells increase their basal [Ca2+]i allowing extracellular Ca2+ influx into the cell, which involves different mechanisms. Store-operated Ca2+ entry (SOCE) is considered the main mechanism of extracellular Ca2+ influx in electrically non-excitable cells and platelets, and comprises an initial Ca2+ depletion from intracellular Ca2+ stores prior to activation of extracellular Ca2+ influx. Although the close relation between Ca2+ release from intracellular stores and extracellular Ca2+ influx was clear, the nature of the signal that linked both events remained elusive until 2005, when Stromal Interaction Molecule 1 (STIM1) was identified as an endoplasmic reticulum (ER) Ca2+ sensor essential for inositol (1,4,5)-trisphosphate (IP3)-mediated SOCE in vitro. However, the function of its homologue STIM2 in Ca2+ homeostasis was in general unknown. Therefore, mice lacking STIM2 (Stim2-/-) were generated in this work to study initially STIM2 function in platelets and in cells of the immune system. Stim2-/- mice developed normally in size and weight to adulthood and were fertile. However, for unknown reasons, they started to die spontaneously at the age of 8 weeks. Unexpectedly, Stim2-/- mice did not show relevant differences in platelets, revealing that STIM2 function is not essential in these cells. However, STIM2 seems to be involved in mammary gland development during pregnancy and is essential for mammary gland function during lactation. CD4+ T cells lacking STIM2 showed decreased SOCE. Our data suggest that STIM2 has a very specific function in the immune system and is involved in Experimental Autoimmune Encephalomyelitis (EAE) at early stages of the disease progression. Stim2-/- neurons were also defective in SOCE. Surprisingly, our results evidenced that STIM2 participates in mechanisms of neuronal damage after ischemic events in brain. This is the first time that the involvement of SOCE in ischemic neuronal damage has been reported. This finding may serve as a basis for the development of novel neuroprotective agents for the treatment of ischemic stroke, and possibly other neurodegenerative disorders in which disturbances in cellular Ca2+ homeostasis are considered a major pathophysiological component.