Cyclic-3',5 '-adenosine monophosphate (cAMP) is an ancient signaling molecule that is found in a variety of species from prokaryotes to eukaryotes and translates extra-cellular stimuli into tightly controlled intra-cellular responses. The two major mammalian cAMP sensors are protein kinase A (PKA), for the phosphorylation of the downstream effectors, and the exchange protein directly activated by cAMP (Epac ), for the guanine nucleotide exchange in the small GTPase Rap proteins. In this study, we investigated the intra-molecular cAMP dependent allosteric network of Epac cyclic nucleotide binding domain (CBD) via solution NMR spectroscopy. Epac proteins have been shown to employ an auto-inhibition strategy in the control of the equilibrium between the active and the inactive states. In the absence of cAMP, the periphery of the Rap recognition site is masked via an ionic interface provided by the N-terminus of the CBD. Binding of cAMP at the distal Phosphate Binding Cassette (PBC), results in weakening of this interface. Here we show that the cAMP binding signal is propagated to the sites important in Epac activation, i.e. the ionic interface, via two key allosteric spots within the CBD. We have also determined the dynamics as a key carrier of cAMP effects to the region forming the ionic interface (ionic latch). Hence entropic enhancements emerged as a key effector in the cAMP mediated ionic latch weakening. We have also provided initial evidence of a negative allosteric contribution from the C-terminal Hinge-Lid region (CHLR) on the cAMP induced Epac activation. In addition to these findings, we also observed critical differences in the mode of cAMP recognition and inter-subdomain communication between the Epac and PKA. A detailed understanding of these two ubiquitous systems, will aid in the development of agonists and antagonists that are relevant in the drug lead development for related diseases, such as Alzheimer's and diabetes. / Thesis / Master of Science (MSc)

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Signaling molecules are critically important to regulate cellular processes. Therefore, their incorporation into engineered biomaterials is indispensable for the applications in tissue engineering and regenerative medicine. In particular, the functionalization of highly hydrated polymer networks, so-called hydrogels, with the signaling molecules, has been quite beneficial to provide multiple cell-instructive signals. Following this strategy, the incorporation of sulfated glycosaminoglycans (GAGs) into such polymer networks offers unprecedented options to control the administration of signaling molecules via electrostatic interactions. Moreover, mathematical models can be instrumental in designing materials to tune the transport and adjust the local concentration of the signaling molecules to precisely modulate cell fate decisions. Accordingly, this study aims to systematically investigate the impact of different binary poly(ethylene glycol)-glycosaminoglycan hydrogel networks on the transport of signaling molecules by developing and applying mathematical modeling in combination with experimental approaches. The gained knowledge was then applied to modulate the bioactivities of pro-angiogenic growths factor within the binary hydrogel and rationally design a new class of cytocompatible GAG-based materials for the controlled administration of pro-angiogenic growth factors. Firstly, systematic studies on the mobility of signaling molecules within GAG-based polymer networks revealed differential effects of hydrogel network parameters such as mesh size, GAG content, and the sulfation pattern of the GAG building block on the transport of these signaling molecules. Secondly, the effect of the GAG content of the hydrogel and the sulfation pattern of the GAG building block on the bioactivity of hydrogel administrated vascular endothelial growth factor (VEGF) have been analyzed. Since VEGF is a GAG-affine protein that plays a major role in angiogenesis, its ability to promote vascular morphogenesis has been investigated. The simulation and experimental results demonstrated the determining impact of the availability of free (unbound) VEGF as well as the presence of GAGs with a specific sulfation pattern within the polymer network on the formation of the endothelial capillary network within the hydrogel. Finally, a rational design strategy has been applied to extend a GAG-hydrogel platform to allow for a far-reaching control of its cell instructive properties. The resulting materials are independently tunable over a broad range for their mechanical properties and GAG content. The GAG content of the hydrogel matrices, in particular, was shown to modulate the transport of pro-angiogenic growth factors most. Moreover, the hydrogel also supports endothelial vascular morphogenesis. In conclusion, the in here followed approach of combining experimental results and mathematical modeling for predicting the transport of signaling molecules and the rational design concept for customizing GAG-based hydrogel networks provide the fundamentals to precisely modulate cell fate decisions within GAG-based biohybrid polymer networks rationalizing their application for tissue engineering and regenerative medicine

Two isoforms of sphingosine kinase (SphK) catalyze the formation of sphingosine-1-phosphate (SIP). Whereas, SphKl stimulates cell growth and survival, it was found that when overexpressed in mouse NIH 3T3 fibroblasts SphK2 enhances caspase-dependent apoptosis in response to serum deprivation, independently of S1P receptors. Sequence analysis revealed that SphK2 contains a 9 amino acid motif similar to that present in BH3-only proteins. Studies showed that the BH3-only domain, catalytic activity, endoplasmic reticulum (ER) stress, and uptake of calcium by the mitochondria may all contribute to the apoptotic effects of overexpressed SphK2 in NIH 3T3 cells. Further studies in human carcinoma cells showed that overexpression of SphK2 increased the expression of the cyclin dependent kinase (cdk) inhibitor p21, but interestingly had no effect on p53 or its phosphorylation. Correspondingly, downregulation of endogenous SphK2 with small interfering RNA (siRNA) targeted to unique mRNA sequences decreased basal and doxorubicin-induced expression of p21 without affecting p53. In addition, downregulation of SphK2 decreased G2/M arrest in response to doxorubicin. Surprisingly however, siSphK2 markedly enhanced apoptosis induced by doxorubicin in MCF7 and HCT-116 cells. This result raises the question of how overexpression of SphK2 decreases cell growth and enhances apoptosis while its downregulation sensitizes cells to apoptosis. A partial answer may come from the possibility that when SphK2 is overexpressed it does not always have the same subcellular distribution as the endogenous protein. It may also be possible that proteolysis of overexpressed SphK2 might induce apoptosis due to liberation of its BH3 peptide domain, which does not occur at the levels at which endogenous SphK2 is expressed. Collectively, these results demonstrate that endogenous SphK2 is important for p53-independent induction of p21 expression by doxorubicin and suggest that SphK2 expression may influence the balance between cytostasis and apoptosis.

Saccharosetransporter sind Membranproteine, die von der SUT-Genfamilie kodiert werden. In Solanaceen wurden Subfamilien: SUT1, SUT2, und SUT4 identifiziert. Die Funktion von SUT4 wurde bisher nur fragmentarisch geklärt. Mittels real time PCR wurde eine Organ- und Entwicklungs-spezifische SUT4-Expression in Wildtyp Kartoffeln (Solanum tuberosum ssp. tuberosum) der Varietät Désirée gezeigt. Die Expression von SUT4, SUT2 und SUT1 zeigt eine Tagesrhythmik. Mit Hilfe der RNA Interferenz-Technik wurden transgene Pflanzen mit einer reduzierten SUT4-Expression hergestellt. StSUT4-RNAi Kartoffeln zeigten: reduzierte Stängelelongation, frühere Blühinduktion, frühzeitige Knollenbildung, Veränderungen des Kohlenhydratprofils in den Source- und Sink-Organen, sowie in den Phloemexudaten. Eine vermehrte Zuckertranslokation von Source-zu–Sink wurde postuliert. Bestimmte Aspekte des StSUT4-RNAi Phänotyps wurden früher bereits für Pflanzen mit Veränderungen der Gibberellin-Antwort, sowie für Pflanzen mit deregulierter photoperiodischen Signaltransduktion beschrieben. In den StSUT4-RNAi Blättern wurden verringerte Transkriptmengen eines GA-Biosynthese-Enzyms (GA20ox1) und eine erhöhte PhyB-mRNA-Akkumulation festgestellt. Der Versuch der Komplementation des StSUT4-RNAi Phänotyps durch GA3-Behandlung war nicht erfolgreich. Es wurde gezeigt, dass es unter Beschattung zu einer erhöhten SUT4-mRNA-Akkumulation kommt. In den StSUT4-RNAi Pflanzen wurden Veränderungen der Expression photoperiodisch regulierter Gene beobachtet. Eine Rolle des SUT4 als ein Integrator der pflanzlichen Antwort auf Licht, Hormone und Zucker wurde postuliert. / Sucrose transporters are membrane proteins, which are encoded by the SUT gene family. In Solanaceen the SUT1, SUT2, and SUT4 subfamily have been identified. So far, the function of the sucrose transporters belonging to the SUT4 subfamily is only poorly understood. The expression of SUT4 in wild type of potato, Solanum tuberosum ssp. tuberosum var. Désirée analyzed via real time PCR shows to be sink and development specific. The expression of SUT4, SUT2 und SUT1 follow a diurnal rhythm. An RNA interference approach was used for the generation of transgenic plants with reduced SUT4 expression. The transgenic potato plants show a reduced internode elongation, early flowering, early tuberization, changes of the carbohydrate content in source as well as in sink organs of these plants, together with changes in phloem efflux from leaves. Increased translocation rate of soluble sugars was postulated. Particular aspects of the StSUT4-RNAi phenotype were already described for plants with changes in the gibberellin response or changes in the photoperiodic signaling pathway. In the StSUT4-RNAi leaves was observed a reduction of the transcript level of the GA biosynthetic enzyme GA20ox1 and increased accumulation of PhyB transcripts. However, the complementation of the StSUT4-RNAi phenotype by GA3 treatment was not successful. Under shade conditions (or under far red light enrichement), the StSUT4 transcripts accumulated to higher levels. In the StSUT4-RNAi plants were observed changes in the expression of genes involved in the photoperiodic pathway. An integrative function of SUT4 in the coordination of the light signalling, the hormone signalling and the sugar signalling pathways of higher plants was postulated.