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In der folgenden Arbeit werden Eigenschaften von Verzweigungsprozessen in zufälliger Umgebung (engl. branching processes in random environment, kurz BPREs) untersucht. Das Modell geht auf Smith (1969) und Athreya (1971) zurück. Ein BPRE ist ein einfaches mathematisches Modell für die Entwicklung einer Population von apomiktischen (d.h. sich ungeschlechtlich fortpflanzenden) Individuen in diskreter Zeit, wobei die Umgebungsbedingungen einen Einfluß auf den Fortpflanzungserfolg der Individuen haben. Dabei wird angenommen, dass die Umgebungsbedingungen in den einzelnen Generationen zufällig sind, und zwar unabhängig und identisch verteilt von Generation zu Generation. Man denke z.B. an eine Population von Pflanzen mit einem einjährigen Zyklus, die in jedem Jahr anderen Witterungsbedingungen ausgesetzt sind, wobei angenommen wird, dass diese sich unabhängig und identisch verteilt ändern. In Kapitel 1 wird eines der wichtigsten Hilfsmittel zur Beschreibung von BPREs, die sogenannte zugehörige Irrfahrt, eingeführt und die Klassifizierung von BPREs beschrieben. In Kapitel 2 werden bekannte Resultate, insbesondere zu kritischen, schwach subkritischen und stark subkritischen Verzweigungsprozessen, wiederholt. In Kapitel 3 wird der sogenannte intermediär subkritische Fall behandelt. Mithilfe von funktionalen Grenzwertsätzen für bedingte Irrfahrten wird die genaue Asymptotik der Überlebenswahrscheinlichkeit des Prozesses, die bereits in Vatutin (2004) bewiesen wurde, unter etwas allgemeineren Voraussetzungen gezeigt. Anschließend wird untersucht, wie häufig der Prozess, bedingt auf Überleben, nur noch aus einem Individuum besteht. Im letzten Teil des Kapitels wird ein funktionaler Grenzwertsatz für die zugehörige Irrfahrt, bedingt aufs Überleben des Prozesses, gezeigt. Diese konvergiert, richtig skaliert, gegen einen Levy-Prozess, der darauf bedingt ist, sein Minimum am Ende anzunehmen. In Kapitel 4 werden große Abweichungen von BPREs untersucht. Die Ratenfunktion des BPRE wird sowohl für den Fall mindestens geometrisch schnell abfallender Tails, als auch für den Fall von Nachkommenverteilungen mit schweren Tails bestimmt. Wie sich herausstellt, hängt die Ratenfunktion von der Ratenfunktion der zugehörigen Irrfahrt, der exponentiellen Abfallrate der Überlebenswahrscheinlichkeit sowie, bei Nachkommenverteilungen mit schweren Tails, auch von den Tails derselben ab. In der Ratenfunktion spiegeln sich die wahrscheinlichsten Wege, um Ereignisse der großen Abweichungen zu realisieren, wider, was in Kapitel 4.3 beschrieben wird. In Kapitel 4.4 wird im speziellen Fall von Nachkommenverteilungen mit gebrochen-linearer Erzeugendenfunktion die Ratenfunktion für Ereignisse bestimmt, bei denen ein superkritischer BPRE überlebt, aber klein im Vergleich zum Erwartungswert bleibt. In Kapitel 4.5 werden die großen Abweichungen, bedingt auf die Umgebung untersucht (engl. quenched). In diesem Fall können unwahrscheinliche Ereignisse nur über den Verzweigungsmechanismus und nicht mehr über eine außergewöhnliche Umgebung realisiert werden. Zum Abschluss der Dissertation werden Verzweigungsprozesse in zufälliger Umgebung, bedingt auf Überle-ben, simuliert. Dazu wird eine Konstruktion nach Geiger (1999) angewendet. Diese erlaubt es, Galton-Watson Bäume in variierender Umgebung, bedingt auf Überleben, entlang einer Ahnenlinie zu konstruieren. Der Fall geometrischer Nachkommenverteilungen, auf den wir uns in Kapitel 5 beschränken, erlaubt die explizite Berechnung der benötigten Verteilungen. Als Anwendung des Grenzwertsatzes aus Kapitel 3.1 können nun intermediär subkritische Verzweigungsprozesse, bedingt auf Überleben, wie folgt simuliert werden: Zunächst wird die Umgebung zufällig bestimmt, und zwar als Irrfahrt, bedingt darauf ihr Minimum am Ende anzunehmen. Anschließend wird, der Geiger-Konstruktion folgend, ein Verzweigungsprozess in dieser Umgebung, bedingt auf Überleben, simuliert. Zum Abschluss wird in einem kurzen Ausblick auf aktuelle Forschung verwiesen. Im Anhang befinden sich einige technische Resultate.
Proliferation and apoptosis are fundamental cellular processes that are important for the development and homeostasis of multi-cellular organisms. Deregulation of these processes plays an important role in tumor formation. Often, genes that control homeostasis by regulating proliferation and apoptosis are mutated or improperly expressed in tumors. In this project, the physiological and pathological functions of FUSE Binding Protein 1 (FBP1) were studied to elucidate the involvement of this gene in the context of embryonic development and tumorigenesis. Two reasons led to the hypothesis that FBP1 might be relevant in this context. FBP1 was isolated in the group of PD Dr. Martin Zörnig using a functional yeast survival screen for the identification of anti-apoptotic genes involved in tumorigenesis, and the anti-apoptotic function of FBP1 was confirmed in the human colon carcinoma cell line RKO. In addition, FBP1 had been published to function as a transcriptional regulator that activates expression of the proto-oncogene c-myc. This gene stimulates cell proliferation and is overexpressed in many tumors. Analysis of FBP1 expression by immunhistochemistry in normal and tumor tissue samples revealed frequent and significant overexpression of FBP1 in Hepatocellular Carcinoma (HCC). To study the functional relevance of FBP1 activity for this tumor type, apoptosis and proliferation of the HCC cell line Hep3B were studied in dependence of FBP1 expression. Downregulation of FBP1 by lentiviral expression of FBP1-specific short hairpin RNA (shRNA) reduced proliferation and increased sensitivity to apoptosis. Subcutaneous injection of FBP1-deficient Hep3B cells into immunodeficient NOD/SCID mice demonstrated that tumor growth was strongly decreased in comparison to control cells. mRNA expression studies by quantitative real time PCR showed reduced mRNA levels of the pro-apoptotic genes Bik, Noxa, TRAIL and TNF-􀀁 in the absence of FBP1. In addition, the cell cycle inhibitors p21 and p15 were repressed by FBP1 while Cyclin D2 expression was decreased in the absence of FBP1. Surprisingly, expression of c-myc was not altered by FBP1 downregulation, indicating a different mechanism of c-myc regulation in HCC cells. These results demonstrate that overexpression of FBP1 inhibits apoptosis and stimulates proliferation in HCC cells by regulating the transcription of relevant target genes. Therefore, FBP1 might represent a promising therapeutic target for the treatment of HCC. For analysis of the physiological function of FBP1, a gene trap mouse model was established. In these mice, the gene trap vector pT1􀀂geo is inserted in intron 19 of the FBP1 locus, leading to the expression of a fusion protein consisting of a truncated FBP1 (lacking the last 62 amino acids), 􀀁-Galactosidase and Neomycin Phosphotransferase. Luciferase reporter assays demonstrated that the fusion protein was not capable of activating the c-myc promoter and even showed a dominant negative effect. Thus, this gene trap mouse serves as a functional FBP1 knockout model. Phenotyping of the FBP1 gene trap mice showed that homozygous mutation of FBP1 resulted in embryonic lethality at late stages of embryonic development (E15.5-E16.5). Heterozygous mice were viable, but born at lower frequencies, indicating a gene dosage- or a dominant negative effect of the FBP1 fusion protein. The cellular effects of FBP1 inactivation were tested in mouse embryonic fibroblasts isolated from FBP1 gene trap mice. While proliferation was reduced in the absence of wildtype FBP1, apoptosis was not affected. Expression analysis showed that in homozygous MEFs p15 and p21 transcripts were upregulated, while decreased cmyc mRNA levels were measured. Closer inspection of homozygous gene trap embryos revealed an anemic phenotype that appeared most pronounced around embryonic day 15.5. Analysis of fetal livers, the main site of hematopoiesis at this stage of development, showed a strongly reduced total cell number in homozygous embryos. Evaluation of the different hematopoietic cell lineages did not reveal significant changes in particular differentiated cell types. Instead, all cell lineages seemed to be affected equally by FBP1 inactivation. In contrast, analysis of hematopoietic progenitor cell populations showed an increased percentage of multipotent progenitor cells (MPPs) and a strongly reduced number of long-term hematopoietic stem cells (LT-HSCs). Functional analysis of MPPs by in vitro colony formation assays demonstrated that the FBP1-mutant cells possess a normal colony formation potential while their expansion capacity was reduced. Competitive transplantation of lineage negative fetal liver cells into irradiated recipient mice resulted in reduced engraftment of liverderived progenitor cells from homozygous FBP1 gene trap mice. However, stable engraftment was observed over a period of 12 weeks, demonstrating that the FBP1-deficient LT-HSCs are in principle capable of long-term repopulation. These results demonstrate that FBP1 exerts an essential function during definitive hematopoiesis. It can be speculated that FBP1 influences proliferation, apoptosis and possibly also stem cell self-renewal through the regulation of specific target genes within the hematopoietic progenitor cells. Alternatively, extrinsic effects caused by the absence of FBP1 activity could impair the function of the progenitor cells.
Tumor hypoxia and nutrient starvation are common phenomena in cancerous tissue. Cells that resist this hostile environment are selected for a more aggressive phenotype, usually accompanied by therapy resistance. The hypoxia inducible factors HIF-1a and HIF-2a play a key role in the adaptive homeostatic responses to these challenging conditions inducing a number of target genes that are involved in the regulation of a variety of cellular processes such as angiogenesis, proliferation, metabolism, self-renewal and cell death/cycle arrest. Thus, the HIF pathway encompasses opposing adaptive responses on tumor growthgrowth promoting abilities on the one hand and growth inhibiting on the other. A recent study in our lab uncovered that this switch between cell death and cell survival critically depends on HIF-2a protein levels. Since PHDs (HIF prolyl hydroxylases) are the main regulators of HIF protein abundance and hypoxia drives the malignant phenotype of tumors, we wanted to characterize HIF regulatory functions of PHDs under hypoxic conditions. Our intention was to reveal the importance of PHD contribution to the opposing functions of HIFs under hypoxia. Characterization of PHD1-4 mRNA and protein expression levels under normoxic and hypoxic conditions in glioblastoma cell lines led to the identification of PHD2 and PHD3 as hypoxia inducible PHD isoforms and highlighted their predominant function under hypoxia. Mechanistically, we demonstrated that HIF mediates the hypoxic induction of PHD2 and 3 within a negative feedback loop, promoting its own degradation during prolonged hypoxia. The functional impact of PHD2 and 3 abundance on cell viability under hypoxic conditions was analyzed by disrupting PHD2 and PHD3 function either through a siRNA mediated approach or by application of the PHD inhibitor DMOG. These experiments uncovered that PHD2 and 3 are protective under hypoxic conditions and that PHD inhibition expedites cell death. Combined HIF and PHD suppression under hypoxic conditions abrogated this increased susceptibility to cell death, clearly showing that PHD2 and 3 act in a negative feedback regulatory loop to limit the HIF response under prolonged hypoxia. With respect to possible future therapeutical applications we co-treated cells with a PHD inhibitor and pro-apoptotic agents staurosporine or TRAIL. Co-challenging tumor cells even potentiated the cell death response, indicating a more widespread protective function of PHD. Taken together PHD2 and 3 protect tumor cells from cell death induction, functioning in a negative feedback regulatory loop to constrain the HIF dependent cell death responses under hypoxia. Interestingly, however, when assessing the role of PHD2 and PHD3 in in vivo tumor growth using an intracranial tumor model, we identified an exclusive tumor suppressor function for PHD3. Loss of PHD3 function enhanced tumor growth whereas increased PHD3 expression diminished the tumor burden. The accelerated tumor growth following PHD3 loss could be attributed to a decrease in the induction of apoptosis and an increase in proliferation. Tumor cells are frequently exposed to temporary and spatial depletion of nutrients. Interestingly, PHD3 loss conferred a growth advantage under growth factor deprivation. The growth regulatory function of PHD3 was isoform specific, HIF independent and importantly, did not require the hydroxylase function of PHD3. Previous reports have uncovered a regulatory function of the PHD system in NF-kB signaling. However, our results demonstrated that NF- kB signaling remained unaffected by alteration in the PHD3 status of the cell. Additionally, the PHD3 tumor suppressor function proved to be independent of two putative PHD3 downstream effectors, ATF4 and KIF1Bb. Mechanistically, PHD3 suppression reduced EGFR internalization, enhancing the amount of EGFR expressed on the cell surface. We further showed that the impaired EGFR internalization during PHD3 loss resulted in receptor hyperactivation under stimulated and growth factor deprived conditions. Importantly, PHD3 physcially associated with the EGFR complex as evidenced by co-immunoprecpitation. Consequently, this extended EGFR activation in PHD3 deficient cells resulted in enhanced downstream activation of EGFR signaling and increased proliferation. Consistent with the interpretation that PHD3 loss is beneficial for tumor growth, we found PHD3 promoter methylation in glioblastoma cell lines, hinting at a epigenetic mechanism to finetune PHD3 expression on top of the hypoxic driven gene regulation. Finally, we demonstrated that PHD3 tumor suppressor function is not restricted to glioblastomas since PHD3 suppression in lung adenocarcinoma accelerated subcutaneous tumor growth. With these findings, we expand the knowledge of PHD3 action from its oxygen sensing role to a regulatory function in growth factor signaling. This clearly discriminates PHD3 from the other isoforms and supports the exclusive tumor suppressor function in glioblastoma. Taken together our results identify a complex role of PHD signaling in cancer and delineate HIF dependent and HIF independent functions of the PHD system. We think that the HIF dependent protective effect of PHD2 and 3 and the HIF independent PHD3 tumor suppressor function are not mutually exclusive, but might be activated according to the heterogeneous intra-tumoral conditions. However, PHD3 hydroxylase activity is dispensable for its HIFindependent tumor suppressor function in glioma. This uncouples PHD3 function from co-factor and co-substrate requirements and allows it to act over a broader physiological range, since its influence on cellular processes is not constrained by the availability of rate limiting factors. It might explain, why the enzymatic independent functions of PHD3 predominate in vivo. Thus, therapeutic modulation of the PHD system to inhibit tumor growth has to be based on these contrasting functions of the PHD system. However, their differential dependence on the hydroxylase activity may facilitate a therapeutic strategy to specifically inhibit or promote the protective versus suppressive functions of the PHD system.
Biodegradation and elimination of industrial wastewater in the context of whole effluent assessment
(2010)
The focus of this thesis is on the assessment of the degradability of indirectly discharged wastewater in municipal treatment plants and on assessing indirectly discharged effluents by coupling the Zahn-Wellens test with effect-based bioassays. With this approach persistent toxicity of an indirectly discharged effluent can be detected and attributed to the respective emission source. In the first study 8 wastewater samples from different industrial sectors were analysed according to the “Whole-Effluent Assessment“ (WEA) approach developed by OSPAR. In another study this concept has been applied with 20 wastewater samples each from paper manufacturing and metal surface treating industry. In the first study generally low to moderate ecotoxic effects of wastewater samples have been determined. One textile wastewater sample was mutagenic in the Ames test and genotoxic in the umu test. The source of these effects could not be identified. After treatment in the Zahn-Wellens test the mutagenicity in the Ames test was eliminated completely while in the umu test genotoxicity could still be observed. Another wastewater sample from chemical industry was mutagenic in the Ames test. The mutagenicity with this wastewater sample was investigated by additional chemical analysis and backtracking. A nitro-aromatic compound (2-methoxy-4-nitroaniline) used for batchwise azo dye synthesis and its transformation products are the probable cause for the mutagenic effects analysed. Testing the mother liquor from dye production confirmed that this partial wastewater stream was mutagenic in the Ames test. The wasteweater samples from paper manufacturing industry of the second study were not toxic or genotoxic in the acute Daphnia test, fish egg test and umu test. In the luminescent bacteria test, moderate toxicity was observed. Wastewater of four paper mills demonstrated elevated or high algae toxicity, which was in line with the results of the Lemna test, which mostly was less sensitive than the algae test. The colouration of the wastewater samples in the visible band did not correlate with algae toxicity and thus is not considered as its primary origin. The algae toxicity in wastewater of the respective paper factory could also not be explained with the thermomechanically produced groundwood pulp (TMP) partial stream. Presumably other raw materials such as biocides might be the source of algae toxicity. In the algae test, often flat dose–response relationships and growth promotion at higher dilution factors have been observed, indicating that several effects are overlapping. The wastewater samples from the printed circuit board and electroplating industries (all indirectly discharged) were biologically pre-treated for 7 days in the Zahn–Wellens test before ecotoxicity testing. Thus, persistent toxicity could be discriminated from non-persistent toxicity caused, e.g. by ammonium or readily biodegradable compounds. With respect to the metal concentrations, all samples were not heavily polluted. The maximum conductivity of the samples was 43,700 micro S cm -1 and indicates that salts might contribute to the overall toxicity. Half of the wastewater samples proved to be biologically well treatable in the Zahn–Wellens test with COD elimination above 80%, whilst the others were insufficiently biodegraded (COD elimination 28–74%). After the pre-treatment in the Zahn–Wellens test, wastewater samples from four companies were extremely ecotoxic especially to algae. Three wastewater samples were genotoxic in the umu test. Applying the rules for salt correction to the test results following the German Wastewater Ordinance, only a small part of toxicity could be attributed to salts. In one factory, the origin of ecotoxicity has been attributed to the organosulphide dimethyldithiocarbamate (DMDTC) used as a water treatment chemical for metal precipitation. The assumption, based on rough calculation of input of the organosulphide into the wastewater, was confirmed in practice by testing its ecotoxicity at the corresponding dilution ratio after pre-treatment in the Zahn–Wellens test. The results show that bioassays are a suitable tool for assessing the ecotoxicological relevance of these complex organic mixtures. The combination of the Zahn–Wellens test followed by the performance of ecotoxicity tests turned out to be a cost-efficient suitable instrument for the evaluation of indirect dischargers and considers the requirements of the IPPC Directive.
Acute myeloid/lymphoid leukemia is a fatal hematological malignancy characterized by accumulation of nonfunctional, immature blasts, which interferes with the production of normal blood cells. Activating mutations of receptor tyrosine kinases are common genetic lesions in leukemia. FLT3-ITD is a frequent activating mutation found in AML patients, leading to uncontrolled proliferation of leukemic blasts. FLT3-ITD directly activates STAT5, leading to the induction of STAT5 target gene expression like PIM kinases and SOCS genes. STAT5 and PIM kinases have been shown to play a crucial role in the FLT3-ITD mediated transformation. On the other hand, the role of SOCS proteins in FLT3-ITD mediated transformation has not been studied to date. SOCS proteins are part of a negative feedback mechanism that controls Jak kinases downstream of cytokine receptors. One of the SOCS family members, SOCS1 has been reported to suppress oncogenecity of several activating kinases implicated in hematologic malignancies. In this thesis the role of these SOCS proteins in FLT3-ITD mediated transformation (in vitro) and leukemogenesis (in vivo) is systematically explored. Expression of FLT3-ITD in cell lines of myeloid (32D) and lymphoid (Ba/F3) origin, led to CIS, SOCS1 and SOCS2 expression. FLT3-ITD expression in primary murine bone marrow stem/progenitor cells led to a 59 fold induction of SOCS1 expression. Furthermore, FLT3-ITD positive AML cell lines (MV4-11, MOLM-13) show kinase dependent CIS, SOCS1, and SOCS3 expression. Importantly SOCS1 is highly expressed in AML patients with FLT3-ITD compared to healthy individuals. SOCS1 protein was expressed in FLT3-ITD transduced murine bone marrow stem cells and SOCS1 expression was abolished with kinase inhibition in MOLM-13 cell line. In conclusion, SOCS1 was highly regulated by FLT3-ITD in myeloid, lymphoid cell lines, in bone marrow stem/progenitors and in AML patient samples. SOCS1 co-expression did not affect FLT3-ITD mediated signaling and proliferation, but abolished IL-3 mediated proliferation and protected 32D cells from interferon-α and interferon-γ mediated growth inhibition. FLT3-ITD expressing 32D cells showed diminished STAT1 activation in response to interferons (α and γ). Alone, SOCS1 strongly inhibited cytokine induced colony formation of bone marrow stem and progenitors, but not FLT3-ITD induced colony formation. Most importantly, in the presence of growth inhibitory interferon-γ, SOCS1 co-expression with FLT3-ITD led to increased colony formation compared to FLT3-ITD alone. Taken together, FLT3-ITD induced and exogenously expressed SOCS1, shielded cells from external cytokines, signals, while not affecting FLT3-ITD induced proliferation/signaling. In further experiments the in vivo effects of SOCS1 were studied in a bone marrow transplantation model. SOCS1 bone marrow transplants were unable to engraft/proliferate in mice. FLT3-ITD was shown to induce a myeloproliferative disease. Both control (empty vector), SOCS1 transplanted mice were normal and did not show any disease phenotype. FLT3-ITD alone and SOCS1 co-expressing FLT3-ITD developed either myeloproliferative disease or acute lymphoblastic leukemia with equal distribution. SOCS1 co-expression with FLT3-ITD led to a decreased latency. Mice transplanted with FLT3-ITD alone and SOCS1 co-expressing FLT3-ITD displayed enlarged spleens, liver and hypercellular bone marrow indicating infiltration of leukemic cells. Mice were also anemic and showed decreased platelet counts. Importantly SOCS1 co-expression particularly shortened the latency of myeloproliferative disease but not of acute lymphoblastic leukemia. In summary, in the context of FLT3-ITD, SOCS1 acts as a ‘conditional oncogene’ and cooperates with FLT3-ITD in the development of myeloproliferative disease. With these data we propose the following model: FLT3-ITD induces SOCS gene expression, which shields cells against proliferation and differentiation signals from cytokines, while not affecting FLT3-ITD mediated proliferative signals. This leaves cells under the dictate of FLT3-ITD thereby contributing to leukemogenesis. Similar to FLT3-ITD, BCR/ABL (P190) (an oncogenic fusion kinase often found in acute lymphoblastic leukemia) induces SOCS gene expression in K562 and long-term cultured cells from patients with acute lymphoblastic leukemia. SOCS1 co-expression does not affect BCR/ABL mediated proliferation while abrogating IL-3 mediated proliferation. These findings suggest that SOCS proteins may play a general co-operative role in the context of oncogenes which aberrantly activate STAT3/5 independently of JAK kinases. This study reveals a novel molecular mechanism of FLT3-ITD mediated leukemogenesis and suggests SOCS genes as potential therapeutic targets.
The comparison of persons is pervasive in social judgement and human decision making and yet its neural substrate is poorly explored. Using functional magnetic resonance imaging we investigated the brain activities of participants comparing other persons with each other (other vs. other comparison - OOC) and with themselves (self vs. other comparison - SOC) as regards psychological (intelligence) and physical (height) characteristics. We found that the comparison of these two person characteristics differ in their neural activation patterns in the OOC as well as in the SOC with higher activity increases for intelligence than height comparison in several areas in medial frontal and orbitofrontal cortex and posterior cingulate cortex suggesting that their activation scales with the demand on person comparison. The person comparison network strikingly overlaps the one commonly described for the classic theory of mind tasks. We interpret this overlap as indicating perspective taking common to person comparison and theory of mind. Furthermore, we suggest that the neural differences between the SOC and the OOC especially in the dorsal part of the medial frontal cortex rely on the different degree of the self involved in the two types of comparisons. The results additionally suggest that the decision directions of self-relevant comparisons, especially in the intelligence comparison of the SOC, resulted in differences in the activation of the medial frontal cortex, which also relies on differences in the reward anticipation and self-relatedness of these decisions.
Wastewater treatment plants (WWTPs) do not eliminate micropollutants completely and are thus important point sources for these substances. In particular, concerns about en-docrine disrupting compounds in WWTP effluents give rise to the implementation of advanced treatment steps for the elimination of trace organic contaminants. The present study investigated ozonation (O3) and activated carbon treatment (AC) at two WWTPs. For an ecotoxicological assessment at WWTP Regensdorf, conventionally treated wastewater, wastewater after ozonation, and ozonated wastewater after sand filtration were evaluated in parallel via the fish early life stage toxicity test (FELST) using rainbow trout (Oncorhynchus mykiss). Additionally, a comparative toxicity evalu-ation of ozonated and activated carbon treated effluents was performed at the pilot scale treatment plant in Neuss (WWTP Neuss). For this purpose, four invertebrate tests and one higher plant toxicity test were selected to assess potential biological effects on or-ganisms [Lemna minor growth inhibition test, chironomid toxicity test with Chironomus riparius, Lumbriculus variegatus toxicity test, comet assay with haemolymph of the zebra mussel (Dreissena polymorpha), reproduction test with Potamopyrgus antipo-darum]. All in vivo assays were performed on site at the treatment plants in flow-through test systems. Furthermore, the present study investigated the effects of ozona-tion and activated carbon treatment on endocrine activities [estrogenicity, anti-estrogenicity, androgenicity, anti-androgenicity, aryl-hydrocarbon receptor (AhR) agonistic activity] with yeast based bioassays using solid phase extracted water samples. To evaluate the removal of in vitro non-specific toxicity, a cytotoxicity assay using a rat cell line was applied. The FELST at WWTP Regensdorf revealed a considerable developmental retardation of test organisms exposed to ozonated WW. This was accompanied by a significant decrease in body weight and length compared to reference water, to the conventionally treated WW, and to the ozonated water after sand filtration. Hence sand filtration obvi-ously prevents from adverse ecotoxicological effects of ozonation. An additional test – starting with yolk-sac larvae – resulted in a significant reduction of vitellogenin levels in fish exposed to ozonated wastewater compared to fish reared in conventionally treat-ed wastewater. This demonstrates the effective removal of estrogenic activity by ozonation. At WWTP Neuss, the reproduction test with the mudsnail P. antipodarum exhibited a decreased reproductive output after advanced treatment compared to conventional treatment. This indicates an effective estrogenicity removal by ozonation and activated carbon treatment and is confirmed by results of the yeast estrogen screen with a reduc-tion of in vitro estrogenic activity by > 75%. The L. variegatus test revealed a signifi-cantly enhanced toxicity after ozonation compared to conventional treatment, whereas this effect was reduced following subsequent sand filtration. When ozonation was applied, a significantly increased genotoxicity was observed, detected with the comet assay using haemolymph of the zebra mussel. Again, this effect was removed by subsequent sand filtration to the level of conventional treatment. Activated carbon treatment even resulted in a significant reduction of genotoxicity. At both treatment plants, adverse effects after ozonation may have been a result of the formation of toxic oxidation by-products. However, sand filtration reduced toxication effects, indicating that these oxidation by-products are readily degradable or adsorbable. The results point out that, in any case, ozonation should not be applied without subsequent biologically active post treatment appropriate for oxidation by-products removal (e.g. sand filtration). However, only activated carbon achieved a toxicity reduction compared to the conventional treated wastewater. Thus, it cannot be excluded that po-tential beneficial effects due to ozonation might be masked by residual toxic oxidation by-products passing the sand filter or ozonation is not as effective in toxicity removal as PAC treatment. The yeast based assays with solid phase extracted samples revealed an effective endo-crine activity removal during ozonation and activated carbon filtration (estrogenicity: 77 – 99%, anti-androgenicity: 63 – 96%, AhR agonistic activity: 79 – 82%). The cyto-toxicity assay exhibited a 32% removal of non-specific toxicity after ozonation com-pared to conventional treatment. Ozonation in combination with sand filtration reduced cytotoxic effects by 49%, indicating that sand filtration contributes to the removal of toxicants. Activated carbon treatment was the most effective technology for cytotoxici-ty removal (61%). Sample evaporation reduced cytotoxic effects by 52% (after activated carbon treatment) to 73% (after ozonation), demonstrating that volatile substances contribute considerably to toxic effects, particularly after ozone treatment. These results confirm an effective removal or transformation of toxicants with receptor mediated mode of action and non-specific toxicants during both investigated treatment steps. However, due to the limited extractability, polar ozonation by-products were neglected for toxicity analysis, and hence non-specific toxicity after O3 is underestimated. In the long run, only on-site comparisons at WW receiving water bodies (e.g. communi-ty analysis of fish, macroinvertebrates, plants, microorganisms) – before and after up-grading WWTPs – allow drawing environmentally relevant conclusions regarding bene-fits and risks of advanced WW treatment methods. Conclusively, the benefits and possible negative impacts have to be carefully evaluated to prove that not more environmental impact will be induced than removed by advanced treatment technologies as each additional treatment requires considerable amounts of energy, resources, and infrastructure facilities. Accordingly, comprehensive sustainable approaches for pollution prevention and wastewater treatment (e.g. source control and source separation) are preferable compared to end-of-pipe treatment systems.
The TTL is the transition layer between the tropical troposphere and stratosphere, and is the main region where tropospheric air enters the stratosphere. In this thesis different transport processes are studied by using in situ measurements of tracers. Long-lived tracers were measured with the High Altitude Gas Analyzer (HAGAR) on board the M55 Geophysica aircraft. The instrument was developed by the University of Frankfurt and measures the long-lived tracers CO2, N2O, CFC-12, CFC-11, H-1211, SF6, CH4 and H2 with two gas chromatographic channels and a CO2 sensor (LICOR). The measurements are supported by CO and O3 measurements of other instruments. Two campaigns were conducted to obtain measurements in the TTL: SCOUT-O3 (November/December 2005 in Darwin, Australia) and AMMA-SCOUT-O3 (August 2006 in Ouagadougou, Burkina Faso). After a general introduction of the thesis in chapters one and two, the third chapter describes the findings during this last campaign. Five local flights are analyzed to study the different transport processes that occur in the tropical tropopause layer above West-Africa: deep convection up to the level of main convective outflow, vertical mixing after overshooting of air in deep convection, horizontal inmixing from the extratropical lower stratosphere, and horizontal transport across the subtropical barrier. Main findings are that the TTL over West-Africa is mostly influenced by remote convection. The subtropical barrier is not a strong barrier but more a region of transition between the extratropical and the tropical stratosphere. Chapter 4 presents the results obtained during the SCOUT-O3 campaign. From the eight local flights the last four flights (051129, 051130a, 051130b, 051205) show enhanced values of ozone, CO and CO2 between 355 and 380 K potential temperature in comparison with the first four flights (051116, 051119, 051123, 051125). Horizontal inmixing from the extra-tropical stratosphere and influence of the local convective system Hector cannot explain the enhanced values of the two flights on 30 November Therefore, other possible explanations for these enhanced CO, CO2 and ozone levels are proposed. The first explanation is vertical mixing in the vicinity of the jet stream. However, the jet cannot explain the differences between the flights on 30 November and the flights on 29 November and 5 December. Another possible explanation is influence of polluted boundary layer air masses from the Indonesian region. Especially air sampled during the flights on November 30 crossed large parts of northern Indonesia between 8 and 10 days before the measurements. Convective uplift of biomass burning and other pollution plumes can transport CO and ozone precursors into the upper troposphere, where they can significantly enhance the ozone production. The last chapter deals with the vertical ascent rate in the TTL and uses measurements of both the SCOUT-O3 and AMMA-SCOUT-O3 campaign as well as data from previous aircraft campaigns (TROCCINOX and APE-THESEO). Time scales and residence times for mean vertical transport in the background TTL are estimated for different seasons and over different geographic regions using in situ observations of CO2 and long-lived tracers. The vertical transport time scales are constrained using the seasonal variation of CO2 in the tropical troposphere as a “tracer clock” for vertical ascent. Two methods are applied to calculate the residence time in the layer between 360 and 390 K potential temperature. The first method uses the slope of the CO2 index, the second method fits the CO2 index directly to the measurements assuming a constant ascent rate. The first method yields residence times for Australia,West Africa, and Brazil of the same order, 35-45 days to 380 K and 50 days to 390 K (where no value can be derived for Australia as the slope is changing approximately one month before the campaign). For APE-THESEO, the method does not yield reasonable results. The best estimates using the second method show moderate residence times between 360 and 390 K of 60±25 days SCOUT-O3 (NH autumn) and 43±8 days for AMMA/SCOUT-O3 (NH summer). These results agree well with the results calculated using the first method. For APE-THESEO and TROCCINOX the best fits yield shorter residence times of 23±7 and 40±10 days, respectively, both during winter. These results correspond well to the expectations based on the seasonal variation of the Brewer-Dobson circulation.
Feral cats (Felis catus), introduced into Australia with European settlers in the 19th century, colonized the entire Australian continent in less than 100 years, including the Australian arid zone which covers more than 70% of the continent. Feral cats are responsible for the decline and extinction of a number of native species and the failure of a number of reintroduction attempts, especially in the arid zone. Many ecological studies on feral cats have been conducted on home range size and movement patterns in different environments, abundance and diet, with the aim of gaining a better understanding about their successful invasion of the Australian continent. There are no physiological studies on the feral cat to date. However, there is evidence that there is a strong interrelation between physiology and abiotic factors such as climate. Thus, distribution, habitat, and dispersal of species can not fully be understood without background knowledge of physiology. This PhD aims to contribute to a better understanding of three physiological parameters: metabolism, body mass and body temperature patterns. These parameters may possibly identify physiological adaptation to different climate zones, seasonal conditions and island isolation.
To date it is not clear at which stage of differentiation mature T cell leukaemia/lymphoma is initiated. Previous studies in our group showed that mature T cells are relatively resistant to transformation. We wanted to further investigate the transformation potential of NPM-ALK, p21SNFT and the viral oncoprotein Tax on mature T cells. First, we analyzed the effects on T cell growth in vitro after transducing human T cell lines with gammaretroviral vectors encoding these genes. No growth or proliferation promoting effect of all three genes was observed. In the second part of the project, we transduced murine, mature T cells and/or haematopoietic stem cells (HPCs/HSCs) and transplanted these cells into Rag-1 deficient recipients. All mice transplanted with NPM-ALK transduced monoclonal mature T cells (OT-1) developed leukaemia/lymphoma. In contrast, only few NPM-ALK transduced polyclonal T cell and HPC/HSC transplanted mice developed leukaemia/lymphoma. From the p21SNFT group, only two mice transplanted with transduced OT-1 T cells developed leukaemia/lymphoma, which showed high eGFP and interestingly CD19 expression. No malignancies were observed in Tax transplanted animals so far. Furthermore, the recipients do not show any eGFP marking in the periphery. In conclusion, our results show that compared to polyclonal T cells, monoclonal T cells are transformable after gammaretroviral transfer of NPM-ALK and p21SNFT.
This thesis is dedicated to the study of fluctuation and correlation observables of hadronic equilibrium systems. The statistical hadronization model of high energy physics, in its ideal, i.e. non-interacting, gas approximation will be investigated in different ensemble formulations. The hypothesis of thermal and chemical equilibrium in high energy interaction will be tested against qualitative and quantitative predictions.
Die Maillard-Reaktion findet während der Lagerung und thermischen Verarbeitung von Lebensmitteln zwischen den darin enthaltenen Proteinen und reduzierenden Kohlehydraten statt. Als Ergebnis der Reaktion entstehen sogenannte advanced glycation end products (AGEs), Protein-Derivate mit Glykierungs-Strukturen. Da Lebensmittel vor dem Verzehr häufig erhitzt werden, ist der Einfluss von AGEs auf die Pathogenese von Nahrungsmittelallergien von großem Interesse. Die Maillard-Reaktion könnte zur Bildung von neuen, für die Pathogenese der Nahrungsmittelallergie relevanten, Immunepitopen beitragen. Das Ziel dieser Arbeit war es, den Einfluss der Maillard-Reaktion auf die T-Zell-Immunogenität, die Antigenität und die von beiden Eigenschaften abhängige Allergenität von Nahrungsmittelallergenen zu untersuchen. Zunächst wurde der Einfluss der Maillard-Reaktion auf die T-Zell-Immunogenität von Ovalbumin (OVA), einem Allergen des Hühnereiweißes, untersucht. Dafür wurde glykiertes OVA (AGE-OVA) hergestellt indem das Protein zusammen mit Glukose erhitzt wurde. In dieser Arbeit konnte zum ersten Mal gezeigt werden, dass ein AGE-Derivat eines Lebensmittelallergens eine höhere T-Zellen-Immunogenität besitzt, als sein natives Gegenstück. Die Aktivierung und Proliferation von CD4+ T-Zellen durch AGE-OVA wurde in vitro durch Co-Kultivierung der T-Zellen mit dendritischen Zellen (DZ) untersucht. DZ sind professionelle Antigen- präsentierende Zellen, welche im Pathomechanismus der Allergie eine wichtige Rolle spielen. Im Vergleich zu nativen OVA und OVA welches ohne Glukose erhitzt wurde, führte die Stimulierung mit AGE-OVA zu einer deutlich erhöhten Aktivierung von OVA-spezifischen CD4+ T-Zellen. Damit DZ T-Zellen aktivieren können, muss das Allergen zunächst durch die DZ aufgenommen werden. In der vorliegenden Arbeit wurde gezeigt, dass die Aufnahme von AGE-OVA wesentlich höher war als die der Kontrollen. Außerdem konnte der scavenger receptor class A type I and II (SR-AI/II) als einer der hauptverantwortlichen Rezeptoren für die Aufnahme von AGE-OVA identifiziert werden. Zusammenfassend lässt sich aus den Ergebnissen dieser Arbeit die Hypothese aufstellen, dass die Glykierung von OVA eine erhöhte Assoziation des Allergens mit SR-AI/II ermöglicht, welche zu einer verstärkten Aufnahme des Allergens durch die DZ führt. Dadurch können mehr Peptide des Allergens an MHC II gebunden und auf der Zelloberfläche präsentiert werden. Das wiederum führt zur beobachteten stärkeren OVA-spezifischen CD4+ T-Zell-Aktivierung durch AGE-OVA. Als nächstes wurde die T-Zell-Immunogenität und Antigenität von AGE-OVA in vivo in einem Mausmodel untersucht. Es zeigte sich, dass AGE-OVA auch in vivo im Vergleich zu den nicht glykierten OVA-Formen eine erhöhte T-Zell-Immunogenität besitzt. Des weiteren führte die Immunisierung mit AGE-OVA zu einer erhöhten Produktion von IgE-Antikörpern. Somit wurde in dieser Arbeit gezeigt, dass AGE-OVA in vivo nicht nur eine erhöhte CD4+ T-Zell-Immunogenität besitzt, sondern auch eine höhere Antigenität hat als natives und ohne Glukose erhitztes OVA. Diese Ergebnisse harmonieren gut miteinander da CD4+ T-Zellen eine zentrale Rolle in der Aktivierung von B-Zellen und der IgE-Produktion durch selbige Zellen spielen. IgE-Antikörper besitzen eine essentielle Funktion beim Auslösen der klinischen Symptomatik der Allergie. Zusammenfassend lässt deshalb sagen, dass die Maillard-Reaktion die Allergenität von OVA erhöhen könnte. Zum Schluss wurden noch die immunstimulatorischen Eigenschaften des Erdnussallergens (AGE)-Ara h 2 untersucht. Da Erdnüsse häufig ernsthafte allergische Reaktionen hervorrufen und selten roh verzehrt werden, war es vom großen Interesse den Einfluss der Maillard-Reaktion auf Immunogenität und Antigenität von rekombinanten Ara h 2 (rAra h 2) zu untersuchen. Es zeigte sich, dass die Glykierung von rAra h 2 durch die Maillard-Reaktion die T-Zellen-Immunogenität, als auch die Antigenität des Allergens reduziert. Abschließend lässt sich sagen, dass die Maillard-Reaktion die allergenen Eigenschaften von Lebensmittelallergenen erheblich beeinflusst indem es die T-Zell-Immunogenität des Allergens verändert. Die Mechanismen welche die T-Zell-Immunogenität beeinflussen wurden hier näher untersucht. Wenn die Glykierung nicht die Bindung der T-Zellen- und/oder B-Zellen-Rezeptoren inhibiert, wird die Allergen-spezifische CD4+ T-Zell-Aktivierung und die davon abhängige IgE-Produktion dadurch erhöht, dass das glykierte Allergen durch DZ verstärkt über SR-AI/II aufgenommen wird. Die vorliegende Arbeit liefert wertvolle Information über die Allergenität von Proteinen die durch die Maillard-Reaktion modifiziert wurden and trägt dazu bei die Mechanismen von Nahrungsmittelallergien besser zu verstehen.
This work reports on the study of the projectile x-ray emission in relativistic ion-atom collisions. Excitation of K-shell in He-like uranium ions, electron capture into H-like uranium ions and Simultaneous ionization and excitation of initially He-like uranium ions have been studied using the experimental storage ring at GSI. Information about the population of the excited states for the H- and He-like uranium ions, can be obtained by measuring the angular distribution of the decay radiation. Since the Ly_alpha2 transition is isotropic, the intensities of the Ly_alpha1 and K_alpha transitions were normalized to the Ly_alpha2 line. For the K_alpha1 and K_alpha2 transitions originating from the excitation of the He-like uranium ions, no alignment was observed. In contrast, the Ly_alpha1 radiation from the simultaneous ionization-excitation process of the He-like uranium ions shows a clear alignment. It is shown that the alignment of Ly_alpha1 was obtained by the Alignment parameter A_20. The experimental value leads to the inclusion of a magnetic term in the interaction potential. It is interesting to note that in the case of the Ly_alpha1 emission the small M2 contribution added coherently to the E1 transition amplitudes enhances the anisotropy. The capture process of target electrons into the highly-charged heavy ions was studied using H-like uranium ions at an incident energy of 220 MeV/u, impinging on N2 gas-target. It was shown that, the strongly aligned electrons captured in 2p3/2 level will couple with the available 1s1/2 electron which shows no initial directional preference. The magnetic sub-state population of the 2p3/2 electron will be redistributed according to the coupling rules to the magnetic sub-states of the relevant two-electron states. Consequently, the 1^P1 and 3^P2 states are corresponding to the the strongly aligned 2p3/2 state. This leads to the large anisotropy in the corresponding individual ground state transitions contributing to the K_alpha1 emission. Due to the fact that the 1^P1 --> 1^S0 and 3^P2 --> 1^S0 transitions are experimentally not resolved, a more detailed analysis of the angular dependence of the K_alpha1 radiation is required. From the K_alpha1/K_alpha2 ratio, the current results show that the incoherent addition of the E1 and M2 transition components yield to an almost isotropic emission of the total K_alpha1. In contrast to the radiative electron capture, the experimental results for the K-shell single excitation of He-like uranium ions indicate that only the 1^P1 level contributes to the K_alpha1 transition. For this case, the anisotropy parameter beta_20 was found to be -0.20 + 0.03 which is similar to that one calculated for pure E1 transition. This work also reports on the study of a two-electron process: the simultaneous ionization and excitation occurring in relativistic collisions of heavy highly-charged ions with gaseous targets. The investigation was performed on He-like uranium ions impinging upon xenon gas-target at an incident energy of 220 MeV/u. The measurements have been performed at the ESR gas-target using atomic xenon with a typical area density of 10^12 particles/cm^2. In contrast to the solid state target, the use of gas target offers the advantage of clear separation of the one step two-electron process due to the fact that the probability of two consecutive collision in such thin targets is negligible and the double step processes can be excluded. During the process of simultaneous ionization and excitation in He-like uranium ions, one of the ground-state electrons is promoted into the continuum and the other into the L-subshell states of the projectile. To select this process, the Lyman-series radiation has been measured at various observation angles in coincidence with up-charged projectiles (U^91+). From the yields of the Ly_alpha1 and Ly_alpha2 projectile radiation, the relative cross section for the process of simultaneous ionization and excitation was directly determined. The angle dependent measurement of the radiation yields provide information about the angular distributions of the emitted radiation and permits the determination of the alignment parameter A_{20}. This parameter gives information on the level population and the collision impact parameter. The present results (b^exp = 810 fm) show that the simultaneous ionization and excitation is a process which occurs at small impact parameter.
Within the present study the occurrence and fate of the organophosphorus flame retardants and plasticizers tris(2-chloroethyl) phosphate (TCEP), tris(2-chloro-1-methylethyl) phosphate (TCPP), tris(1,3-dichloro-2-propyl) phosphate (TDCP), tris(2-butoxyethyl) phosphate (TBEP), tri-iso-butyl phosphate (TiBP), and tri-n-butyl phosphate (TnBP) in precipitation, lake water, surface runoff and groundwater from urban and remote areas in Germany was investigated between June 2007 and October 2009. 255 samples of precipitation, 210 samples of lentic surface water and 72 samples of groundwater were analyzed for the six organophosphates (OPs) by solid phase extraction followed by gas chromatography-mass spectrometry. The research focused on aspects concerning (1) the atmospheric washout of OPs by precipitation, (2) the temporal variation of OP concentrations in precipitation and in lentic surface waters as well as (3) the pollution of groundwater by OPs. The results of the study emphasize the importance of precipitation as an all-season entry-pathway for OPs in the aquatic environment, particularly in densely populated urban environments with high traffic volume and abundant usage of flame-protected products. No seasonal trends were observed for all analytes in precipitation at the urban sampling site. TCPP dominated in all precipitation and storm water holding tank (SWHT) water samples with maximum levels exceeding 1 µg/L. An accumulation of OPs deposited in SWHTs was observed with concentrations often exceeding those observed in wet precipitation. Median concentrations of TCPP (880 ng/L), TDCP (13 ng/L), and TBEP (77 ng/L) at the urban SWHT were more than twice as high as those measured at the urban precipitation sampling site (403 ng/L, 5 ng/L, 21 ng/L) located close to the SWHT. OP levels in more remote lakes were often below or close to the limits of quantitation (LOQ). Nevertheless, TCPP was the substance with the highest median concentration in rural volcanic lakes (7–18 ng/L) indicating an atmospheric transport of the compound. At urban lakes the median OP concentrations were in the range of 23–61 ng/L (TCEP), 85–126 ng/L (TCPP), <LOQ–53 ng/L (TBEP), 8–10 ng/L (TiBP), and 17–32 ng/L (TnBP). In laboratory experiments, TBEP, TiBP, and TnBP were photochemically degraded in spiked lake water samples upon exposure to sunlight. In the SWHT a seasonal trend with decreasing concentrations in summer/autumn was evident for TiBP and TnBP but not for the chlorinated OPs. The decreasing concentrations can be explained by in-lake photodegradation. Results have also shown that the occurrence of OPs in groundwater is depending on the anthropogenic impact during groundwater recharge/natural replenishment. Infiltration of precipitation was found to be no important entry-pathway for OPs into aquifers at rural sites. Highest OP concentrations (>0.1 µg/L) were determined in groundwater polluted by percolating leachate from contaminated sites or groundwater recharged via bank filtration of OP-loaded recipients. Concentrations of TCEP, TCPP, TiBP and TnBP in groundwater decreased rapidly (89–97%) during bank filtration with increasing distance from the recipient due to adsorption processes and/or biotransformation. Although TCEP and TCPP are stable within the aquifer, they are not suitable as conservative organic tracers in groundwater.
The HADES (High Acceptance DiElectron Spectrometer) is an experimental
apparatus installed at the heavy-ion synchrotron SIS-18 at GSI, Darmstadt.
The main physics motivation of the HADES experiment is the measurement
of e+e− pairs in the invariant-mass range up to 1 GeV/c2 in heavy-ion collisions
as well as in pion and proton-induced reactions.
The HADES physics program is focused on in-medium properties of the light
vector mesons ρ(770), ω(783) and φ(1020), which decay with a small branching
ratio into dileptons. Dileptons are penetrating probes which allow to study
the in-medium properties of hadrons. However, in heavy-ion collisions, the
measurement of such lepton pairs is difficult because they are rare and have a
very large combinatorial background.
Recently, HADES has been upgraded with new detectors and new electronics
in order to handle higher intensity beams and reactions with heavy nuclei up
to Au.
HADES will continue for a few more years its rich physics program at its
current place at SIS-18 and then move to the upcoming international Facility
for Antiproton and Ion Research (FAIR) accelerator complex. In this context
the physics results presented in this work are important prerequisites for the investigation
of in-medium vector meson properties in p + A and A+A collisions.
This work consists of five chapters. The first chapter introduces the physics
motivation and a review of recent physics results. In the second chapter, the
HADES spectrometer is described and its sub-detectors are presented. Chapter
three deals with the issue of lepton identification and the reconstruction of
the dielectron spectra in p + p collisions is presented. Here, two reactions
are characterized: inclusive and exclusive dilepton production reactions. From
the spectra obtained, the corresponding cross sections are presented with the
respective statistical and systematical errors. A comparison with theoretical
models is included as well. Conclusions are given in chapter four.
The final part of this work is dedicated to the HADES upgrade, whose goal
is among others the achievement of a reliable and fast data acquisition of the
Multiwire Drift Chambers (MDCs). Chapter five presents my contribution to
this successful project during the three years of my stay at GSI.
G-protein coupled receptors (GPCRs) are the key players in signal perception and transduction and one of the currently most important class of drug targets. An example of high pharmacological relevance is the human endothelin (ET) system comprising two rhodopsin-like GPCRs, the endothelin A (ETA) and the endothelin B (ETB) receptor. Both receptors are major modulators in cardiovascular regulation and show striking diversities in biological responses affecting vasoconstriction and blood pressure regulation as well as many other physiological processes. Numerous disorders are associated with ET dysfunction and ET antagonism is considered an efficient treatment of diseases like heart failure, hypertension, diabetes, artherosclerosis and even cancer. This study exemplifies strategies and approaches for the preparative scale synthesis of GPCRs in individual cell-free (CF) systems based on E. coli, a newly emerging and promising technique for the production of even very difficult membrane proteins. The preparation of high quality samples in sufficient amounts is still a major bottleneck for the structural determination of the ET receptors. Heterologous overexpression has been a challenge now for decades but extensive studies with conventional cell-based systems had only limited success. A central milestone of this study was the development of efficient preparative scale expression protocols of the ETA receptor in qualities sufficient for structural analysis by using individual CF systems. Newly designed optimization strategies, the implementation of a variety of CF expression modes and the development of specific quality control assays finally resulted in the production of several milligrams of ETA receptor per one millilitre of reaction mixture. The versatility of CF expression was extensively used to modulate GPCR sample quality by modification of the solubilization environment with detergents and lipids in a variety of combinations at different stages of the production process. Downstream processing procedures of CF synthesized GPCRs were systematically optimized and sample properties were analysed with respect to homogeneity, protein stability and receptor ligand binding competence. Evaluation was accomplished by an array of complementary and specifically modified techniques. Depending on its hydrophobic environment, CF production of the ETA receptor resulted in non-aggregated, monodisperse forms with sufficient long-term stability and high degrees of secondary structure thermostability. The obtained results document the CF production of the ETA receptor in two different modes as an example of a class A GPCR in ligand-binding competent and non-aggregated form in quantities sufficient for structural approaches. The presented strategy could serve as basic guideline for the production of related receptors in similar systems.
Der L-Carnitin/gamma-Butyrobetain Antiporter CaiT ist ein Mitglied der Betain/Carnitin/Cholin Transporter (BCCT) Familie. Sekundärtransporter der BCCT Familie transportieren Substrate, die eine positiv-geladene quartäre Ammoniumgruppe besitzen. CaiT besteht aus 504 Amiosäuren und besitzt ein moleculares Gewicht von etwa 56 kDa. In Enterobakterien wie Escherichia coli, Proteus mirabilis und Salmonella typhimurium wird die Expression des caiTABCDE Operons unter anaeroben Bedingungen induziert. Unter diesen Bedinungen ist CaiT der Haupttransporter des Betain-Derivates L-Carnitin. In Enterobakterien wird L-Carnitin unter anaeroben Bedingungen aufgenommen und dehydratisiert wobei Crotonobetain ensteht. Crotonobetain wird anschließend zum Endprodukt gamma-Butyrobetain reduziert. Gamma-Butyrobetain ist das Gegensubstrat, das aus der Zelle hinaustransportiert wird, wenn L-Carnitin in die Zelle aufgenommen wird. Der Austauschmechanismus von LCarnitin gegen gamma-Butyrobetain geschieht ohne das Vorhandensein eines elektrochemischen Gradients, d.h. CaiT ist sowohl H+- als auch Na+-unabhängig. Ein Ziel dieser Arbeit war es die drei-dimensionale (3D) Struktur von CaiT mittels Röntgenstrukturanalyse zu lösen. Weiterhin sollten mit Hilfe der 3D-Struktur und funktionellen Studien detailiertere Erkenntnisse über den kationenunabhängigen Antiportmechanismus von CaiT ermittelt werden. Im Rahmen dieser Arbeit wurden die 3D-Röntgenkristallstrukturen von drei CaiT-Homologen der Enterobakterien P. mirabilis (PmCaiT), E. coli (EcCaiT) und S. typhimurium (StCaiT) mittels molekularem Ersatz (engl.: molecular replacement, MR) mit einem Alanin-Model des CaiT verwandten Na+/Glycinbetain Symporters BetP gelöst. PmCaiT konnte mit einer Auflösung von 2.3 Å gelöst werden. Das Protein kristallisierte in der Kristallraumgruppe H3, mit drei Molekülen in der asymmetrischen Einheit (engl.: asymmetric unit, AU). Die drei PmCaiT-Moleküle ordneten sich innerhalb der AU um eine kristallographische dreifach Symmetrieachse an. EcCaiT wurde mittels MR mit einem Alanin-Model von PmCaiT bei einer Auflösung von 3.5 Å gelöst. EcCaiT kristallisierte in der Kristallraumgruppe P32, ebenfalls mit drei Molekülen in der AU, jedoch ohne kristallographische Symmetry. Während der Verfeinerung des EcCaiT-Models wurde eine strenge dreifache nichtkristallographische Symmetry (engl.: non-crystallographic symmetry, NCS) angewandt. StCaiT, das ebenfalls mittels MR mit einem Alanin-Model von PmCaiT, aber bei einer Auflösung von 4.0 Å gelöst wurde, kristallisierte in der Kristallraumgruppe P65, ebenfalls mit drei StCaiT-Molekülen in der AU, ohne kristallographische Symmetry. Bei der Verfeinerung des StCaiT-Modells wurde wie bei EcCaiT eine strenge NCS angewandt. Da die Auflösung von 4.0 Å bei StCaiT zu niedrig ist um detailierte moleculare Erkenntnisse zu gewinnen, wurden Protein- sowie Substratinteraktionen nur an den Strukturen von PmCaiT und EcCaiT analysiert. Alle drei CaiT-Homologe weisen jedoch einen ähnlichen strukturellen Aufbau auf. In der Röntgenkristallstruktur bildet CaiT ein symmetrisches Trimer, das über ionische und polare Wechselwirkungen zwischen den Protomeren stabilisiert wird. Der trimere Oligomerisierungszustand von CaiT in Detergenzlösung sowie in zweidimensionalen Lipidmembrankristallen wurde bereits in früheren Arbeiten gezeigt. Jedes der drei CaiT-Protomere besteht aus zwölf Transmembranhelices (TMH), die N- und C-terminalen Domänen des Proteins befinden sich auf der cytoplasmatischen Seite. Zehn der TMH bilden zwei invertierte Wiederholungseinheiten aus jeweils fünf TMH. Die erste Einheit besteht aus den TMH 3 – 7, die invertierte zweite Einheit besteht aus den TMH 8 – 12. Beide Wiederholungseinheiten sind strukturell nahezu identisch und lassen sich fast vollständig übereinanderlegen, jedoch weisen die Aminosäuren der beiden Einheiten keine signifikante Sequenzidentität auf. Die ersten beiden Helices der Wiederholungseinheiten, die TMH 3 – 4 und die TMH 8 – 9, bilden ein antiparalleles vier-Helix-Bündel, in dem in CaiT zwei Substratbindestellen lokalisiert sind. Eine derartige Transporterarchitektur wurde erstmals in der Struktur des Na+/Alanin Symporters LeuTAa des thermophilen Bakteriums Aquifex aeolicus gezeigt. Bislang wurden, inklusive CaiT, sieben Sekundärtransporterstrukturen gelöst, die diese LeuT-Transporterarchitektur aufweisen. Ungewöhnlich dabei ist, dass diese sieben Sekundärtransporter fünf verschiedenen Transporterfamilien angehören und eine Verwandschaft auf Basis der Aminosäuren nicht zu finden ist. Da jedoch die tertiäre Struktur dieser Tansporter konserviert ist, kann davon ausgegangen werden, dass sie alle von einem Urprotein entstanden sind, welches zunächst aus fünf TMH bestanden haben muss. Im Laufe der Evolution hat sich das Urgen des Urproteins zunächst dupliziert und die weitere Evolution hat zwar die Aminosäuresequenz verändert und den Umweltbedingungen angepasst, jedoch ist die tertiäre Struktur erhalten geblieben. Da sich die tertiäre Struktur der sieben Sekundärtransporter so stark ähnelt, ist zu vermuten, dass auch der Transportmechanismus ähnlich, jedoch nicht identisch ist. Nach dem strukturellen Aufbau der Transporter, der Lage der Substratbindestellen in den jeweiligen Transportern und der Tatsache, dass es sich bei diesen Proteinen um Membranproteine handelt, wurde ein Transportmechanismus aufgestellt, in dem die Bindestelle des zu transportierende Substrats alternierend zu beiden Seiten der Membran zugänglich ist, ohne jedoch jemals den Substratweg innerhalb des Proteins vollständig zu öffnen. Dieser Mechanismus wurde als “alternating access mechanism” beschrieben. Anhand der unterschiedlichen Zustände, in denen einige der Transporter kristallisierten, kann abgeleitet werden, welche Konformationsänderungen erforderlich sind um das Substrat von einer Seiter der Membran auf die andere zu transportieren. Bisher kristallisierten einzelne der sechs Transporter in der nach außen gerichteten offenen Form, der nach außen gerichteten Form, in der die Substratbindestelle jedoch nicht mehr zugänglich ist, in einer Form, die keine Öffnungspräferenz der Substratbindestelle zu einer Seite der Membran hat und in der nach innen gerichteten Form, in der die Substratbindestelle jedoch nicht geöffnet ist. CaiT kristallisierte in der noch fehlenden Konformation, der nach innen gerichteten Form, in der die Substratbindestelle zugänglich ist. Mit dieser noch fehlenend Konformation kann der Transportzyklus des “alternating access mechanism” vollständig beschrieben werden. Alle drei CaiT-Homologe kristallisierten in der nach innen gerichteten, offenen Konformation. Im Gegensatz zur EcCaiT-Struktur kristallisierte PmCaiT in der substratungebundenen Form. In der StCaiT-Struktur konnte aufgrund der niedrigen Auflösung kein Substrat nachgewiesen werden. In der EcCaiT-Struktur sind zwei gamma-Butyrobetain-Moleküle gebunden. Das erste Molekül wurde in der zentralen Substratbindestelle, der sogenannten Tryptophan-Box bestehend aus vier Tryptophanen, im Zentrum des Protein lokalisiert. Das zweite gamma-Butyrobetain-Molekül wurde in einer Vertiefung an der extrazellulären Proteinoberfläche gefunden. Beide Substrate werden hauptsächlich über Kation-Pi-Interaktionen zwischen der positiv geladenen quatären Ammoniumgruppe des Substrats und des Pi-Elektronensystems der Tryptophane in den jeweiligen Bindestellen gebunden. Eine besondere Eigenschaft von CaiT ist der H+- bzw. Na+-unabhängige Substrattransport. Die CaiT-Struktur erklärt warum kein zusätzliches Kation benötigt wird um Substrat zu binden oder zu transportieren. In der EcCaiT-Struktur ist eine wichtige polare nicht-bindende Interaktion zwischen der Carboxylgruppe des gamma-Butyrobetains und dem Schwefelatom eines Methionins in der zentrale Bindestelle zu erkennen. Dieses Methionin ist konserviert in den prokaryotischen CaiTs und in den Na+-unabhängigen eukaryotischen L-Carnitin Transportern (OCTN), jedoch ist es nicht konserviert im Na+-abhängigen verwandten Glycinbetain Transporter BetP. In BetP ist diese Position des Methionins durch ein Valin ersetzt. Die Mutation des Methionins in CaiT zu Valin ermöglicht zwar immernoch die H+- bzw. Na+-unabhängige Bindung des Substrates durch die Tryptophan-Box, jedoch ist der Substrattransport nahezu vollständig zerstört. Eine derart wichtige Substratkoordinierende Funktion des Schwefelatoms eines Methionins wurde bisher nicht beschrieben. Eine weitere Stelle, die in H+- bzw. Na+-abhängigen Transporter mit H+ bzw. Na+ besetzt ist, ist in CaiT von einem positiv geladenen Arginin eingenommen. Eine positive Ladung an dieser Stelle stabilisiert den Bereich im Protein in der Nähe der zentralen Substratbindestelle. Die Mutation des Arginins zu Glutamat in CaiT erzielt eine vollständige Inaktivierung des Substrattansports. Durch Zugabe von Na+ im Transportansatz kann die Substrattransportaktivität der Glutamat-Mutante jedoch teilweise zurückerlangt werden. Diese eben beschriebenen Aminosäurereste in den beiden Stellen des Proteins erklären die Kationenunabhängigkeit von CaiT. Die Aktivierung des Antiportmechanismus in CaiT wurde mit Hilfe von Bindungsstudien an rekonstituiertem Protein ermittelt. Diese Messungen ergaben für das Wildtypprotein ein sigmoidales Substratbindungsverhalten, was auf ein positiv-kooperatives Bindungsverhalten hindeutet. Die beiden Substratbindestellen im Protein sowie die beiden unterschiedlichen Substrate, L-Carnitin und gamma-Butyrobetain, lassen auf einen heterotropen positiv-kooperativen Bindungs- und einen allosterisch regulierten Transportmechanismus schließen. Bei diesem Mechanismus erhöht die Bindung eines Substrats in der regulatorischen Bindestelle durch induzierte Konformationsänderungen die Affinität eines anderen Substrats in einer weiteren Substratbindestelle. Die regulatorische Bindestelle in CaiT befindet sich an der extrazellulären Proteinoberfläche. Eine Schwächung der Substrataffinität in dieser Bindestelle durch Einführung einer Mutation, verstärkt das sigmoidale Substratbindungsverhalten und hat einen negativen Einfluss auf den Substrattransport. Durch die in dieser Arbeit gelösten 3D-Röntgenkristallstrukturen der zwei CaiT-Homologen, PmCaiT und EcCaiT, sowie den durchgeführten funktionellen Studien sowohl an Wildtypprotein wie auch an Mutanten konnte ein L-Carnitin/gamma-Butyrobetain Antiport-Mechanismus für CaiT vorzuschlagen werden.
This thesis consists of four chapters. Each chapter covers a topic in international macroeconomics and monetary policy. The first chapter investigates the impact of unexpected monetary policy shocks on exchange rates in a multi-country econometric model. The second chapter examines the linkage between macroeconomic fundamentals and exchange rates through the monetary policy expectation channel. The third chapter focuses on the international transmission of bank and corporate distress. The last chapter unfolds the interest rate channel of monetary policy transmission in-an emerging economy-China, where regulations and market forces co-exist in this transmission.
This thesis investigates the development of early cognition in infancy using neural network models. Fundamental events in visual perception such as caused motion, occlusion, object permanence, tracking of moving objects behind occluders, object unity perception and sequence learning are modeled in a unifying computational framework while staying close to experimental data in developmental psychology of infancy. In the first project, the development of causality and occlusion perception in infancy is modeled using a simple, three-layered, recurrent network trained with error backpropagation to predict future inputs (Elman network). The model unifies two infant studies on causality and occlusion perception. Subsequently, in the second project, the established framework is extended to a larger prediction network that models the development of object unity, object permanence and occlusion perception in infancy. It is shown that these different phenomena can be unified into a single theoretical framework thereby explaining experimental data from 14 infant studies. The framework shows that these developmental phenomena can be explained by accurately representing and predicting statistical regularities in the visual environment. The models assume (1) different neuronal populations processing different motion directions of visual stimuli in the visual cortex of the newborn infant which are supported by neuroscientific evidence and (2) available learning algorithms that are guided by the goal of predicting future events. Specifically, the models demonstrate that no innate force notions, motion analysis modules, common motion detectors, specific perceptual rules or abilities to "reason" about entities which have been widely postulated in the developmental literature are necessary for the explanation of the discussed phenomena. Since the prediction of future events turned out to be fruitful for theoretical explanation of various developmental phenomena and a guideline for learning in infancy, the third model addresses the development of visual expectations themselves. A self-organising, fully recurrent neural network model that forms internal representations of input sequences and maps them onto eye movements is proposed. The reinforcement learning architecture (RLA) of the model learns to perform anticipatory eye movements as observed in a range of infant studies. The model suggests that the goal of maximizing the looking time at interesting stimuli guides infants' looking behavior thereby explaining the occurrence and development of anticipatory eye movements and reaction times. In contrast to classical neural network modelling approaches in the developmental literature, the model uses local learning rules and contains several biologically plausible elements like excitatory and inhibitory spiking neurons, spike-timing dependent plasticity (STDP), intrinsic plasticity (IP) and synaptic scaling. It is also novel from the technical point of view as it uses a dynamic recurrent reservoir shaped by various plasticity mechanisms and combines it with reinforcement learning. The model accounts for twelve experimental studies and predicts among others anticipatory behavior for arbitrary sequences and facilitated reacquisition of already learned sequences. All models emphasize the development of the perception of the discussed phenomena thereby addressing the questions of how and why this developmental change takes place - questions that are difficult to be assessed experimentally. Despite the diversity of the discussed phenomena all three projects rely on the same principle: the prediction of future events. This principle suggests that cognitive development in infancy may largely be guided by building internal models and representations of the visual environment and using those models to predict its future development.
This dissertation connects two independent fields of theoretical neuroscience: on the one hand, the self-organization of topographic connectivity patterns, and on the other hand, invariant object recognition, that is the recognition of objects independently of their various possible retinal representations (for example due to translations or scalings). The topographic representation is used in the presented approach, as a coordinate system, which then allows for the implementation of invariance transformations. Hence this study shows, that it is possible that the brain self-organizes before birth, so that it is able to invariantly recognize objects immediately after birth. Besides the core hypothesis that links prenatal work with object recognition, advancements in both fields themselves are also presented. In the beginning of the thesis, a novel analytically solvable probabilistic generative model for topographic maps is introduced. And at the end of the thesis, a model that integrates classical feature-based ideas with the normalization-based approach is presented. This bilinear model makes use of sparseness as well as slowness to implement "optimal" topographic representations. It is therefore a good candidate for hierarchical processing in the brain and for future research.
Clinical application of transcranial Doppler for detection of cerebral emboli during cardiac surgery
(2010)
Objective: Neurologic injury is one of the most damaging complications for cardiac surgery. How to decrease neurologic impairment by improving perioperative monitoring remains a challenge for both cardiac surgeons and anesthetists. For this reason, transcranial doppler (TCD) has been widely used in cerebral monitoring during cardiac surgery. In this study, two experiments of clinical application of TCD for detection of cerebral emboli during cardiac surgery were to be done. One was “Solid and gaseous cerebral emboli during valvular surgery are significantly reduced with axillary artery cannulation”. The other was “Do intraoperative cerebral embolic signals differ between valvular surgery (VS) and CABG”. Methods: In experiment one, 20 valve and combined procedures with aortic cannulation (AoC group) were compared to 18 procedures with axillary cannulation (AxC group) in a prospective non-randomized study. In experiment two, 18 VS patients and 18 CABG patients were matched by extracorporeal circulation (ECC) time retrospectively. Intraoperative monitoring of both middle cerebral arteries was performed with TCD discriminating between solid and gaseous embolic signals (ES). Results: In experiment one, the AxC group had less solid ES than the AoC group (38±22 vs 55±25, P<0.05), but no significant difference was found in gaseous (501±271 vs 538±333, P>0.05) and total (539 ± 279 vs 593 ± 350, P>0.05) ES. The AxC group had less solid ES during arterial cannulation (2.1±1.5 vs 6.6±3.6, P<0.05) and during aortic cross-clamp time (4.4 ±3.1 vs 10.2 ± 5.1, P<0.05) than the AoC group. During ECC, gaseous ES was not significantly different between groups (398±210 vs 448±291, P>0.05). However, AxC showed less gaseous ES (85±68 vs 187±148, P<0.05) and less gaseous ES per minute (1.8±1.5 vs 4.5±3.2, P<0.05) during weaning off extracorporeal circulation than the AoC group. No significant difference in gaseous ES (313±163 vs 261±189, P>0.05) and gaseous ES per minute (3.1±2.2 vs 2.8±2.2, P>0.05) was found between groups from bypass start to aortic declamping. No neurologic complications occurred. In experiment two, no significant difference was found in solid (38±20 vs 40±26, P>0.05) or gaseous (457±263 vs 412±157, P>0.05) ES between the VS and CABG group during the whole recording time. During ECC, solid ES (20±10 vs 24±19, P>0.05) and gaseous ES (368±230 vs 317±157, P>0.05) were comparable between groups. Specifically, during weaning off ECC, the VS group had more gaseous ES/min (5.6±3.6 vs 3.1±1.2, P<0.05) than the CABG group. But this difference in gaseous ES/min was not significant during the period from bypass start to aortic declamping (2.5±1.8 vs 3.0±1.8, P>0.05). Conclusion: Cerebral embolization does occur during cardiac surgery. Through these two experiments, we demonstrated the feasibility and importance of clinical application of transcranial doppler for detection of cerebral emboli during cardiac surgery. Due to the diversity in clinical application of TCD, it is impossible to compare the number of ES between different research centers. More unified standards should be drawn in order to make wider clinical application possible. Up till now, no robust evidence shows the correlation between intraoperative ES and postoperative neurological impairment. The research on intraoperative ES and postoperative neurological impairment should rely on a complete concept.
In this thesis, the structure of the C-terminal domain of presenilin-1, the catalytic component of the y-secretase complex, is investigated by NMR spectroscopy. The ysecretase complex has a definitive role in the pathogenic development of Alzheimer's disease, in that it mediates the cleavage of aprecursor to create the amyloid ß peptide. Aggregates of amyloid ß which form amyloid plaques are the most overt clinieal feature observed in the post-mortem brains of Alzheimer's patient. In addition, many of the mutations found in the aggressive early onset familial Alzheimer's disease have been linked to presenilin-1, highlighting its importance in disease progression and deeming it an important target for investigation. One of the greatest challenges for the structural investigation of the y-secretase components is their low expression yields in cell-based systems. We therefore applied continuous-exchange cell-free expression to obtain sufficient amounts of protein for our structural studies. An added benefit of the cell-free expression system is the freedom to incorporate any desired combination of stable-isotope labels directly into sampies. We were therefore able to develop a labeling scheme which targets the amino acid composition of transmembrane a-helices, allowing us to simplify an assignment procedure whieh tends to be cumbersome and diffieult for most a-helical transmembrane proteins. The y-secretase complex is a member of the intramembrane cleaving proteases which, as their name implies, cleave their transmembrane substrates within the bilayer. Single particle analysis of the y-secretase (1) as weil as crystal structures of rhomboid (2) and S2P (3) have revealed the presence of hydrophilie po res within the membrane where catalysis occurs. In light of evidence that certain elements of CTF reside in close proximity or even contribute to the formation of the hydrophilic pore, we chose to study the structure of CTF in mieelles, whieh may be better suited to accommodate CTF in isolation as compared with solid membranes in the absence of the other y-secretase components. The structure of CTF was solved to 1.7 A (backbone r.m.s.d) and revealed the presence of unusual features, including a partially membrane-spanning helix which situates the catalytic asparte at its N-terminus in what would be the center of the membrane where catalysis is proposed to occur, as weil as a severely kinked helix which is partially embedded beneath the surface of the membrane (P6). Interestingly, similar features have been observed in the crystal structure of the GlpG rhomboid. In addition, a soluble helix was found in the long N-terminal loop of CTF which until now has been described as unstructured. The first part of the thesis is designed to provide an introduction to Alzheimer's disease, the role of y-secretase and its presenilin-l catalytic component in disease progression, as weil as cell-free expression and liquid-state NMR techniques involved in the structural investigation of membrane proteins. In chapter 2, the reader is familiarized with the history, the clinical manifestation, and biochemical features of Alzheimer's disease. The chapter goes further to describe the role of the y-secretase complex and its individual components in disease progression and substrate processing. Chapter 3 focuses more specifically on presenilin-l in the context of the newly emerging class of intramembrane proteases. In chapter 4, attention is shifted to the cell-free expression system with special focus on the expression of membrane proteins, and chapter 5 explores the various liquid-state NMR techniques that were required for the characterization of CTF. The second part of the thesis is cumulative and contains original research, method, and review articles that were produced during the course of study. Chapter 6 explores the various techniques and innovations used to study membrane proteins using continuous exchange cell-free expression coupled with NMR spectroscopy. In chapter 7, a new technique, transmembrane segment targeted labeling, is described as a tool that facilitates the backbone assignment of transmembrane proteins which display severe overlap in NMR spectra. Chapter 8 presents the novel NMR structure of the C-terminal fragment of presenilin-l solved in SOS micelles.
Therapy of hemorrhagic shock with following resuscitation-induced liver injury : in vivo study
(2010)
Shock resulting from life-threatening blood-loss (hemorrhagic shock) represents the most frequent injury pattern after a traumatic insult. Hemorrhagic shock induces inflammatory changes, characterized by highly complex pathophysiological pathways often resulting in death. In this study, we establish an experimental in vivo model of H/R in rats and study the mechanisms which determine the hepatic injury after H/R. Furthermore, we show that hemorrhagic shock with following resuscitation is accompanied with release of systemic and local pro-inflammatory mediators, increased infiltration of hepatic neutrophils in the liver, increased oxidative and nitrosative stress, enhanced cell death of both types, apoptosis and necrosis, conspicuous cytoskeletal rearrangements, loss of hepatic integrity and finally high general mortality rates, up to 80%. In addition, the effects of two potential therapeutic interventions to prevent the H/R induced liver injury are explored in a model of H/R in rats. First, the role of JNK and its inhibition by D-JNKI-1 in preservation of hepatic integrity following H/R was analyzed. Second, we investigated the potential of simvastatin to prevent the disturbed inflammatory response and hepatic injury after H/R. The effects of both therapeutic interventions were studied by looking at several inflammatory parameters, markers of oxidative and nitrosative stress, cytoskeleton integrity, microcirculatory parameters, underlying signaling cascades, liver damage and mortality. Highly specific blockade of JNK with the potent, inhibitory peptide D-JNKI-1 revealed the crucial role of the JNK signaling pathway in the H/R induced pathophysiology and strong protective effects of DJNKI- 1 in H/R induced liver injury, when the peptide was applied before and even after hemorrhagic shock. The other therapeutic intervention tested in this study was the use of simvastatin which also revealed protective effects after H/R and even a remarkable improvement in survival after H/R. We show that H/R induced release of pro-inflammatory cytokines, hepatic PMNL infiltration, increased oxidative and nitrosative stress, apoptosis and necrosis can be diminished by treatment with D-JNKI-1 but also with simvastatin in vivo. Furthermore, simvastatin reduces H/R induced cytoskelatal rearrangements, loss of liver integrity and the mortality rate after H/R. The key pathway which underlies these beneficial effects of simvastatin is the Rho kinase pathway. Identification of both mechanisms as well as the effectiveness of both substances provide new insights in the close interaction between hypoxia and the immune system and present a promising basis for the anti-inflammatory, hepatoprotective treatment after H/R.
The Alborz Mountains are forming a ~100 km wide, E-W trending mountain chain where individual summits are up to 5000 m in elevation. The Alborz Mountains range are part of the Alpine orogen and are straddling a 2000 km wide area S of the Caspian Sea. The rocks of the Alborz Mountains consist of Neogen sediments, which are affected by folding and faulting. In the western part of the Alborz Mountains the folds and faults are trending NW-SE, whereas in the eastern part they are trending NE-SW. GPS data confirm N-S shortening including dextral strike-slip along ESE-WNW trending faults, and sinistral strike-slip along ENE-WSW trending faults. The present thesis is focusing on the active Garmsar salt nappe, the fragmented roof of which is pierced by rock salt which extruded near the front of the Alborz Mountains Range. During the past 5 m.y. the front of the Alborz chain migrated towards SSW on top of the salt of the Garmsar basin. The salt was squeezed towards SSW and took place at the Great Kavir. The extruded salt is forming the Eyvanekey plateau between the cities of Eyvanekey and Garmsar. Both the Garmsar salt nappe and the Eyvanekey plateau are dextrally displaced for ca. 9 km along the Zirab-Garmsar fault. Structural analyses of the Garmsar salt nappe indicate three different groups of joints which are trending perpendicular and parallel to the local mechanical anisotropy. The folds of the study area are congruent (type 2 and 3 after Ramsay) resulting from viscose inhomogeneous flow. InSAR-Investigations suggest the Alborz Mountains to be lifted up by ca. 1 cm/a, while horizontal shortening is active at a rate of 8 ±2 mm/a. These values are consistent with GPS data. Based on nine „Advanced Synthetic Aperture Radar“ (ASAR) scenarios, produced by the ENVISAT satellite of the European space agency between 2003 and 2006, we used interferograms to map the displacement via 22 increments during 2 – 18 months. The results suggest that the topographic height of the surface of the salt is changing at a rate which is controlled by the season. The displacement ranges from subsidence at -40 to -50 mm/a to uplift of 20 mm/a. In order to investigate the time-dependent deformation with high spatial resolution, we used algorithms which are based on data of small base lines (SBAS). The resulting interferometric SAR time series analyses also suggest that the study area is largely subsiding at a rate that is controlled by the seasons. The map with the averaged LOS deformation velocities, on the other hand, suggests the subsidence to increase from the upper part of the salt nappe towards deeper topographic 5 levels of the agricultural lowlands. The major part of subsidence is probably caused by the annual rainfall which results in subrosion of salt. The spatial changes in the subsidence rate are probably controlled by the distribution of fountains, mining activity at the margin of the salt glacier, and faults and fractures inside the salt. Striking seasonal imprints are obvious along the agricultural areas which are surrounding the Garmsar salt nappe. These areas are rapidly subsiding in summer and spring when groundwater is used for irrigations. The maximum rate of subsidence (40-50 mm/a) is located E and W of the Eyvanekey plateau, where large areas are irrigated. The maximum displacement is 20 mm/a in the farmland and 5 mm/a in the center of the salt nappe. Depth estimates using Euler deconvolution method for gravimetric and magnetic data suggest the salt to extrude from a depth less than ca. 2000 m. The gravity field of the study area is characterized by strong anomalies in the SW and weak anomalies in the NE. A considerable negative anomaly in the N indicates that the northern part subsided, whereas the southern part was lifted up. The seismic data show three major horizons inside the Miocene sediments: the Lower Red Formation, the Qom Formation, and the Upper Red Formation. The western part of the study area seems to be free from salt domes. The layers of the upper part of the Qom Formation show thinning along the NE and NW trending faults. In some areas the seismic reflectors indicate steep faults close the saddle of the folds. NE-SW-, NW-SE and E-Wtrending faults prevail. Analogue experiments have been carried out to extend our knowledge about the evolution of the Garmsar salt dome. We used a scaled model (34 cm * 25 cm * 2.5 cm) that was shortened perpendicular to its long side. The wedge shape of the Alborz Mountains was simulated by a wedge consisting of Styrofoam. Rock salt was simulated using Polydimethylsiloxan (PDMS), a linear viscous material with a viscosity of 2.3*104 Pa s and a density of 0.96 g/cm3 at room temperature. Other sediments were modeled using dry quartz sand. The experimental results can be used to simulate the structural evolution of the study area: The Alborz deformation front was emplaced on top of the salt rocks in the Garmsar area while migrating towards SSW. A salt basin and a salt extrusion have also been produced in the model. Cross sections through the wedge shaped analogue model indicate N- and S-dipping reverse faults, which are in line with the wedge shape of the Alborz chain. Moreover, ENE-WSW trending sinistral and ESE-WNW trending dextral strike-slip faults led to N-S shortening during the Miocene. Structural marker horizons, 6 which have been turned into Z-folds on the western fold limbs and to S-folds on the eastern fold limbs, are comparable with the folds of the study area. Solving the problem of waste is one of the central tasks of environmental protection. It is becoming increasingly difficult to find suitable sites that are acceptable to the public. Salt and salt formations have relevant properties to be utilizing as a repository for each kind of waste. The favorable properties make rock salt highly suitable as a host rock, in particular for nonradioactive and radioactive wastes. The Qom and Garmsar basins are the nearest salt diapirs to the Tehran province, and there are suitable repositories for waste disposal. Based on surface and subsurface data, the Garmsar salt diapir has been investigated as a case example for its suitability as a host and repository for various types of waste. The data used are based on field studies, interferometry, and geophysical investigations. The results of this study suggest the deep bedded salt of the Garmsar Salt Basin to be an appropriate host for the deposition of industrial waste. Rock salt of surficial layers or domes, on the other hand, is not regarded as an appropriate candidate for waste disposal.
Iron uptake is an essential process in all Gram-negative bacteria including cyanobacteria and therefore different transport systems evolved during evolution. In cyanobacteria, however, the iron demand is higher than in proteobacteria due to the function of iron as cofactor in e.g. photosynthesis and nitrogen fixation. Most of the transport systems depend on outer membrane localized TonB-dependent transporters (TBDTs), a periplasma-facing TonB protein and a plasma membrane localized machinery (ExbBD). So far, iron chelators (siderophores), oligosaccharides and polypeptides have been identified as substrates of TBDTs. However, in proteobacteria TonB-dependent outer membrane transporter represent a well-explored subject whereas for cyanobacteria almost nothing is known about possible TonB-dependent uptake systems for iron or other substrates. The heterocyst-forming filamentous cyanobacterium Anabaena sp. PCC 7120 is known to secrete the siderophore schizokinen, but its transport system has remained unidentified. For Anabaena sp. PCC 7120 22 genes were identified as putative TBDTs covering almost all known TBDT subclasses. This is a high number of TBDTs compared to other cyanobacteria. The expression of the 22 putative TBDTs individually depends on the presence of iron, copper or nitrogen. The atypical dependence of TBDT gene expression on different nutrition points to a yet unknown regulatory mechanism. In addition, the hypothesis of the absence of TonB in Anabaena sp. PCC 7120 was clarified by the identification of an according sequence, all5036. Inspection of the genome of Anabaena sp. PCC 7120 shows that only one gene encoding a putative TonB-dependent iron transporter, namely alr0397, is positioned close to genes encoding enzymes involved in the biosynthesis of a hydroxamate siderophore. The expression of alr0397 was elevated under iron-limited conditions. Inactivation of this gene caused a moderate phenotype of iron starvation in the mutant cells. The characterization of the mutant strain showed that Alr0397 is a TonB-dependent schizokinen transporter (SchT) of the outer membrane and that alr0397 expression and schizokinen production are regulated by the iron homeostasis of the cell. Additional two genes of Anabaena sp. PCC 7120 involved in this process were identified. SchE encoded by all4025 is a putative cytoplasmic membrane-localized transporter involved in TolC-dependent siderophore secretion. The mutation of schE resulted in an enhanced sensitivity to high metal concentrations and in drastically reduction of secretion of hydroxamate-type siderophores. IacT coded by all4026 is a predicted outer membrane-localized TonB-dependent iron transporter. Inactivation of iacT resulted in reduced sensitivity to elevated iron and copper levels, whereas decoupling the expression from putative regulation by exchange of the promoter resulted in sensitization against tested metals. Further analysis showed that iron and copper effects are synergistic because decrease of iron induced a significant decrease of copper levels in the iacT insertion mutant but an increase of those levels in Anabaena sp. PCC 7120 where expression of all4026 is under the trc-promoter. In consequence, the results unravel a link between iron and copper homeostasis.
Leukotrienes (LTs) are pro-inflammatory lipid mediators that belong to the group of eicosanoids, which are oxygenated metabolites of one common precursor, the aracidonic acid (AA). This polyunsaturated fatty acid is esterified at the sn-2 position of cellular membrane phospholipids and can be released by cytosolic phospholipase A2 alpha (cPLA2alpha) enzymatic deacylation. AA can be converted into LTs by the catalytic reaction of 5-lipoxygenase (5-LO). Enzymatic activation of cPLA2alpha as well as of 5-LO is regulated by similar determinants. In response to cellular stimuli that elevate the intracellular Ca2+ level and/or activate MAP kinase pathways, cPLA2alpha and 5-LO comigrate from a soluble cell compartment (mainly the cytosol) to the nuclear membrane, where AA is released und converted into LTs. LTs play a significant role in promoting inflammatory reactions and immune processes. They have been shown to be released from leukocytes in response to bacterial and viral infections and substantially contribute to an effective immune reaction for host defense. Innate immune pathogen recognition is mediated to a substantial part by the Toll-like receptor (TLR) family. So far, 10 human TLR subtypes have been identified, all of which detect distinct highly conserved microbial structures and trigger the induction of signaling pathways that lead to the expression of numerous immune and inflammatory genes. TLR signaling culminates in the activation NF-kappaB and/or MAP kinases, which as well are known to be involved in the regulation of cellular LT biosynthesis. In this regard, it seemed conceivable that the release of LTs might be regulated by TLR activation. Present studies were undertaken in order to verify and characterize a possible influence of TLR activation on the LT biosynthesis, and furthermore to identify the involved signaling pathways and underlying mechanisms. First experiments revealed that pre-incubation of differentiated Mono Mac 6 (MM6) cells with a TLR4 ligand, a TLR5 ligand, as well as with different TLR2 ligands led to an about 2-fold enhancement of Ca2+ ionophore induced LT biosynthesis. Ligands of other TLR subtypes did not show any influence. These observations could also be confirmed in primary human monocytes stimulated with ionophore or fMLP. With focus on TLR2 ligands, further studies were carried out to characterize the observed enhancement of LT biosynthesis in MM6 cells. It was demonstrated that the extent of LT formation was dependent on the ligand concentration used, but was also dependent on the duration of pre-incubation. Ligand pre-incubation of 15 minutes was optimal to maximally enhance LT formation and further prolongation of pre-incubation decreased LT formation again. Moreover, simultaneous addition of TLR2 ligands with ionophore did also not enhance LT formation. These results indicated that TLR2 ligands seemed to prime human monocytes for an enhanced response upon ionophore stimulation, but did not act as costimuli, which per se were not capable of directly stimulating the biosynthesis of LTs. To analyze the underlying mechanism, the impact of TLR2 ligands on the two key enzymes of the LT biosynthesis pathway, cPLA2alpha and 5-LO, was investigated. In this regard, 5-LO could not been shown to be positively regulated by TLR ligand priming. Neither a direct stimulation, nor an enhancement of 5-LO activity by TLR ligands was detectable in MM6 cells. Similarly, TLR2 ligands did also not enhance ionophore induced 5-LO translocation to the nuclear membrane. However, it was shown that TLR2 ligands enhanced ionophore induced release of AA in MM6 cells, which occurred with a similar time course as LT formation, displaying a maximum at 10 minutes of pre-incubation. A direct stimulation of AA release, however, could not been detected. Inhibitor studies revealed cPLA2alpha to be essential for AA release in TLR2 ligand primed, ionophore stimulated MM6 cells, but also sPLA2 was found to be involved. However, the priming effect of TLR2 ligands was mediated exclusively by cPLA2alpha. Western Blot analyses revealed that p38 MAP kinase, as well as ERK1/2, are activated in MM6 cells in response to TLR2 ligands, and also Ser-505 phosphorylation of cPLA2alpha was detected, which is known to be mediated by MAP kinases and to increase cPLA2alpha activity in vitro. Maximal cPLA2alpha phosphorylation occurred after 5-10 minutes of TLR2 ligand incubation, slightly preceding maximal AA release at 10 minutes and maximal LT formation at 15 minutes of priming. The combined use of a specific p38 MAPK inhibitor with an inhibitor of the ERK1/2 signaling pathway resulted in a complete prevention of cPLA2alpha phosphorylation and TLR2 ligand mediated enhancement of AA release. Thus, both MAPK pathways seem to play a role for TLR2 ligand mediated priming effects on the release of AA. An impact of other kinases such as Mnk-1 and CamKII, which can also regulate cPLA2alpha by phosphorylation, was excluded. Finally, an anti-hTLR2 antibody significantly reduced enhanced AA release, confirming the priming effects to be dependent on TLR2 activation. In summary, it was concluded that the increase of LT biosynthesis by TLR2 ligand priming is considerably due to an enhanced cellular AA supply, which arises from a MAPK mediated phosphorylation and up-regulation of cPLA2alpha. TLR dependent enhancement of LT biosynthesis represents an interesting link between activation of innate immune receptors and the rapid formation of pro-inflammatory lipid mediators. On the one hand, this support the role of LTs in host defence and infectious diseases, but may also be relevant in pathophysiological processes, which involve TLRs as well as LTs, as it has been shown for the pathogenesis of atherosclerosis or allergic diseases.
Within this thesis, an experimental study of the photo double ionization (PDI) and the simultaneous ionization-excitation is performed for lithium in different initial states Li (1s22l) (l = s, p). The excess energy of the linearly polarized VUV-light is between 4 and 12 eV above the PDI-threshold. Three forefront technologies are combined: a magneto-optical trap (MOT) for lithium generating an ultra-cold and, by means of optical pumping, a state-prepared target; a reaction microscope (ReMi), enabling the momentum resolved detection of all reaction fragments with high-resolution and the free-electron laser in Hamburg (FLASH), providing an unprecedented brilliant photon beam at favourable time structure to access small cross sections. Close to threshold the total as well as differential PDI cross sections are observed to critically depend on the excitation level and the symmetry of the initial state. For the excited state Li (1s22p) the PDI dynamics strongly depends on the alignment of the 2p-orbital with respect to the VUV-light polarization and, thus, from the population of the magnetic substates (mp = 0, ±1). This alignment sensitivity decreases for increasing excess energy and is completely absent for ionization-excitation. Time-dependent close-coupling calculations are able to reproduce the experimental total cross sections with deviations of at most 30%. All the experimental observations can be consistently understood in terms of the long range electron correlation among the continuum electrons which gives rise to their preferential back-to-back emission. This alignment effect, which is observed here for the first time, allows controlling the PDI dynamics through a purely geometrical modification of the target initial state without changing its internal energy.
Transmissible spongiform encephalopathies (TSEs) are rare but fatal neurodegenerative diseases affecting human and animals. The prion protein which is the causative agent, according to “protein-only” hypothesis misfold in to rogue amyloid conformer. Despite several years of studies, the atomic structural details of the rogue conformers have not been clearly understood. This study focused on developing an in-vitro conversion method, which allows us to monitor the transition from unfolded state of prion protein to fibril state. In order to reach maximal unfolded state, we have used 8 M urea as chemical denaturant, pH 2 and prion fragment 90-230 as the model. It has been demonstrated earlier that acidic pH and mild denaturant induce the fibril formation. The mechanism underlying the structural transition from monomeric state to polymeric form is largely unknown. We have confirmed by EM and AFM that fibrils are formed in our conditions, which resemble to naturally occurring fibrils in morphologies observed. The agitation accelerates the rate of fibril formation and, which allow us to do time-resolved NMR on these preparations. The conformational flexibility is inherent to amyloid fibrils and has been observed in our preparations. We aimed to map the important segment of prion protein, which forms the rigid core in its fibrillar structured form. Our time-resolved NMR studies allowed us to monitor the changes happening from unfolded state to fibrillar state. Analysis of data identified the segment between residues 145 to 223 forming the rigid core in these fibrils, which correspond to β strand 2, helix 2 and major part of helix 3 of native prion monomeric structure. Most of the point mutations which are associated with hereditary prion disease are part of rigid core, which undergo a refolding on fibril formation. The C-terminal residues from 224 to 230 displayed peak shifting and therefore, indicate the adaptation to a fibril specific conformation. The major part of N-terminal 90-144 segment, remains dynamic, which can be understood by their accessibility to amyloid specific antibodies. This provides novel structural insight to the amyloid formation from unfolded state of prion protein fragment 90-230, which represents the proteinase-K resistant part naturally occurring prions. Earlier studies have established the core to 160-220 where hydrogen-deuterium exchange mass spectrometry or site-directed spin labeling EPR spectroscopy was used for analysis. Those studies have been initiated from either native-like or partially unfolded state of recombinant prion protein, and therefore, it is quite striking to find out that fibrils initiated from unfolded monomeric state share the same “amyloid core”. This structural insight has important implications for understanding the molecular basis of prion propagation.
Despite sensible guidelines for the use of opioid analgesics, respiratory depression remains a significant risk with a possibility of fatal outcomes. Clinicians need to find a balance of analgesia with manageable respiratory effects. The ampakine CX717 (Cortex Pharmaceuticals, Irvine, CA, USA), an allosteric enhancer of glutamate-stimulated AMPA receptor activation, has been shown to counteract opioid-induced respiratory depression in rats while preserving opioid-induced analgesia. Adopting a translational approach, we orally administered 1500 mg of CX717 to 16 male healthy volunteers in a placebo controlled double-blind study. Starting 100 min after CX717 or placebo intake, alfentanil was administered by computerized intravenous infusion targeting a plateau of effective alfentanil plasma concentrations of 100 ng/ml. One hour after start of opioid infusion, its effects were antagonized by intravenous injection of 1.6 mg of the classical opioid antidote naloxone. Respiration was quantified prior to drug administration (baseline), during alfentanil infusion and after naloxone administration by (i) counting the spontaneous respiratory frequency at rest and (ii) by employing hypercapnic challenge with CO2 rebreathing that assessed the expiratory volume at a carbon dioxide concentration in the breathable air of 55% (VE55). Pain was quantified at the same time points, immediately after assessment of respiratory parameters, by (i) measuring the tolerance to electrical stimuli (5 Hz sine increased by 0.2 mA/s from 0 to 20 mA and applied via two gold electrodes placed on the medial and lateral side of the mid-phalanx of the right middle finger) and (ii) by measuring the tolerance to heat (increased by 0.3°C/s from 32 to 52.5°C applied to a 3 x 3 cm2 skin area of the left volar forearm, after sensitization with 0.15 g capsaicin cream 0.1%). CX717 was tolerated by all subjects without side effects that would have required medical intervention. We observed that CX717 was approximately as effective as naloxone in reversing the opioid induced reduction of the respiratory frequency. Despite the presence of high plasma alfentanil concentrations, the respiratory frequency decreased only by 8.9 ± 22.4% when CX717 was pre-administered, which was comparable to the 7.0 ± 19.3% decrease observed after administration of naloxone. In contrast, after placebo pre-administration the respiratory rate decreased by 30.0 ± 21.3% (p=0.0054 for CX717 versus placebo). In agreement with this, periods of a very low respiratory frequency of <= 4 min-1 under alfentanil alone were shortened by ampakine pre-dosing by 52.9% (p=0.0182 for CX717 versus placebo). Furthermore, VE55 was decreased during alfentanil infusion by 55.9 ± 16.7% under placebo preadministration but only by 46.0 ± 18.1% under CX717 pre-administration (p=0.017 for CX717 versus placebo). Most importantly, in contrast to naloxone, CX717 had no effect on opioid induced analgesia. Alfentanil increased the pain tolerance to electrical stimuli by 68.7 ± 59.5% with placebo pre-administration. With CX717 pre-administration, the increase of the electrical pain tolerance was similar (54.6 ± 56.7%, p=0.1 for CX717 versus placebo). Similarly, alfentanil increased the heat pain tolerance threshold by 24.6 ± 10.0% with placebo pre-administration. Ampakine co-administration had also no effect on the increase of the heat pain tolerance of the capsaicin-sensitized skin (23.1 ± 8.3%, p=0.46 for CX717 versus placebo). The results of this study allow us to draw the conclusion, that opioid induced ventilatory depression can be selectively antagonized in humans by co-administering an ampakine. This is the first successful translation of a selective antagonism of opioidinduced respiratory depression from animal research into application in humans. Ampakines, namely CX717, thus are the first selective antidote for opioid-induced respiratory depression without loss of analgesia, available for the use in humans.
This dissertation consists of three essays, which study the relation between stock prices and the macroeconomy using vector autoregressions (VARs). The first essay focuses on the link between stock prices and the current account. I find that stock markets provide a channel, in addition to the traditional exchange rate channel, through which external balance for a country with a current account imbalance can be restored. The second essay explores the transmission of U.S. stock price shocks to real activity and prices in G-7 countries. I achieve identification by imposing a small number of sign restrictions on impulse responses, while controlling for monetary policy, business cycle and government spending shocks. The results suggest that stock price movements are important for fluctuations in G-7 real activity and prices, but do not qualify as demand side business cycle shocks. The third essay investigates the impact of monetary and technology shocks on the stock market. I find an important role for technology shocks, but not monetary shocks, in explaining variations in real stock prices. The identification method is flexible enough to study the effects of technology news shocks. The responses are consistent with the idea that news on technology improvements have an immediate impact on stock prices.
Type 1 diabetes (T1D) is a chronic T cell-mediated autoimmune disorder that results in the destruction of insulin-producing pancreatic ß cells leading to life-long dependence on exogenous insulin. Attraction, activation and transmigration of inflammatory cells to the site of ß-cell injury depend on two major molecular interactions. First, interactions between chemokines and their receptors expressed on leukocytes result in the recruitment of circulating inflammatory cells to the site of injury. In this context, it has been demonstrated in various studies that the interaction of the chemokine CXCL10 with its receptor CXCR3 expressed on circulating cells plays a key role in the development of T1D. Second, once arrived at the site of inflammation adhesion molecules promote the extravasation of arrested cells through the endothelial cell layer to penetrate the site of injury. Here, the junctional adhesion molecule (JAM) JAM-C expressed on endothelial cells is involved in the process of leukocyte diabedesis. It was recently demonstrated that blocking of JAM-C efficiently attenuated cerulein-induced pancreatitis in mice. In my thesis I studied the influence of the CXCL10/CXCR3 interaction on the one hand, and of the adhesion molecule JAM-C on the other hand, on trafficking and transmigration of antigen-specific, autoaggressive T cells in the RIP-LCMV mouse model. RIP-LCMV mice express the glycoprotein (GP) or the nucleoprotein (NP) of the lymphocytic choriomeningitis virus (LCMV) as a target autoantigen specifically in the ß cells of the islets of Langerhans and turn diabetic after LCMV-infection. In my first project I found that pharmacologic blockade of CXCR3 during development of virus-induced T1D results in a significant delay but not in an abrogation of overt disease. However, neither the frequency nor the migratory properties of islet-specific T cells was significantly changed during CXCR3 blockade. In the second project I was able to demonstrate that JAM-C was upregulated around the islets in RIP-LCMV mice after LCMV infection and its expression correlated with islet infiltration and functional ß-cell impairment. Blockade with a neutralizing anti-JAM-C antibody slightly reduced T1D incidence, whereas overexpression of JAM-C on endothelial cells did not accelerate virus-induced diabetes. In summary, our data suggest that both CXCR3 as well as JAM-C are involved in trafficking and transmigration of antigen-specific autoaggressive T cells to the islets of Langerhans. However, the detection of only a moderate influence on the onset of clinical disease during CXCR3 or JAM-C blockade reflects the complex pathogenesis of T1D and indicates that several different inflammatory factors need to be neutralized in order to achieve a stable and persistent protection from disease.
In this retrospective study, case records of clinical forensic examinations and respective investigation records of the police and the public prosecutor’s (state attorney) office along with the resulting verdicts were examined in terms of type and site of injury found and extent of agreement or discrepancy between the story given by the accused party and the medical conclusions drawn from the injury pattern. Particular attention was focussed on the relevance of the expert opinion for the legal assessment through case-specific analysis of the respective verdicts. A total of 118 cases originating from the scope of the Institute of Forensic Medicine, Goethe-University Frankfurt/Main (2002 – 2005) were examined. These included bodily injury, child abuse, sexual compulsion, self-mutilation and injury patterns of individuals under suspicion of attempted or completed manslaughter/homicide. As compared to former studies, the results of this analysis were additionally correlated with the investigation records of the public prosecutor’s office (state attorney) to elucidate the importance of the forensic findings for police investigation and legal evaluation. The forensic examination involved 19 accused and 99 victims. As for the gender distribution of the victims, 51 females and 48 males were encountered. Slight female preponderance was seen in cases of sexual compulsion. The group of accused individuals consisted of 16 males and 3 females. Injuries due to blunt force impact, in particular hematomas involving skull and trunk, dominated as diagnostic findings in cases of bodily injury, sexual offenses and child abuse. In cases with suspected self-mutilation and in examinations of accused perpetrators of manslaughter/homicide scratches and lacerations prevailed. Correlating injury patterns and police inquiries, conclusions drawn from medical findings and results of police investigations were in good agreement in 46 % of the cases, but showed major discrepancies in another 25 %. In the remaining 29 % of the cases, the injury pattern did not allow for a definite expert opinion on the mode of infliction. Nevertheless, a detailed documentation of the medical findings proved to be of substantial value for police investigations. 39 % of the cases resulted in a final verdict, whilst in 59 % of the cases the charge was dismissed. Especially in the ladder forensic expert opinion was of considerable importance, since forensic assessment of injuries could either not be attributed to a certain perpetrator or contributed to the exoneration of the accused. In 2 % the judicial assessment was not available. In 82 % of the cases of child abuse the proceedings were stopped, e.g. since maltreatment could not be assigned to a particular perpetrator. In these cases, it became obvious, that forensic examination and assessment alone does not suffice, but has to be embedded in police investigations to achieve optimal results. Medical conclusions by forensic experts were – almost without any exception – considered in legal assessment and differentiatedly taken into account when weighing the sentence, thus reflecting the objectivity and neutrality of the medical assessment. In synopsis, albeit evidential value of forensic examination is assessed to be high optimal clarification of a case requires integration into the complete spectrum of investigations performed in a case.
A basic introduction to RFQs has been given in the first part of this thesis. The principle and the main ideas of the RFQ have been described and a small summary of different resonator concepts has been given. Two different strategies of designing RFQs have been introduced. The analytic description of the electric fields inside the quadrupole channel has been derived and the limitation of these approaches were shown. The main work of this thesis was the implementation and analysis of a Multigrid Poisson solver to describe the potential and electric field of RFQs which are needed to simulate the particle dynamics accurately. The main two ingredients of a Multigrid Poisson solver are the ability of a Gauß-Seidel iteration method to smooth the error of an approximation within a few iteration steps and the coarse grid principle. The smoothing corresponds to a damping of the high frequency components of the error. After the smoothing, the error term can well be approximated on a coarser grid in which the low frequency components of the error on the fine grid are converted to high frequency errors on the coarse grid which can be damped further with the same Gauß-Seidel method. After implementation, the multigrid Poisson solver was analyzed using two different type of test problems: with and without a charge density. After illustrating the results of the multigrid Poisson solver, a comparison to the field of the old multipole expansion method was made. The multipole expansion method is an accurate representation of the field within the minimum aperture, as limited by cylindrical symmetry. Within these limitations the multigrid Poisson solver and the multipole expansion method agree well. Beyond the limitation the two method give different fields. It was shown that particles leave the region in which the multipole expansion method gives correct fields and that the transmission is affected therefrom as well as the single particle dynamic. The multigridPoisson solver also gives a more realistic description of the field in the beginning of the RFQ, because it takes the tank wall into account, and this effect is shown as well. Closing the analysis of the external field, the transmission and fraction of accelerated particles of the set of 12 RFQs for the two different methods were shown. For RFQs with small apertures and big modulations the two different method give different values for the transmission due to the limitation of the multipole expansion method. The internal space charge fields without images was analyzed at the level of single particle dynamic and compared to the well known SCHEFF routine from LANL, showing major differences for the analyzed particle. For comparing influences on the transmissions of the set of 12 RFQs a third space charge routine (PICNIC) was considered as well. The basic shape of the transmission curve was the same independent of space charge routines, but the absolute values differ a little from routine to routine, with SCHEFF about 2% lower than the other routines. The multigrid Poisson solver and PICNIC agree quite well (less than 1%), but PICNIC has an extremely long running time. The major advantage of the multigrid Poisson solver in calculating space charge effects compared to the other two routines used here is that the Poisson solver can take the effect of image charges on the electrodes into account by just changing the boundaries to have the shape of the vanes whereas all other settings remain unchanged. It was demonstrated that the effect of image charges on the vanes on the space charge field is very big in the region close to the electrodes. Particles in that region will see a stronger transversely defocusing force than without images. The result is that the transmission decreases by as much as 10% which is considerably more than determined by other (inexact) routines before. This is an important result, because knowing about the big effect of image charges on the electrodes it allows it to taken into account while designing the RFQ to increase the performance of the machine. It is also an important factor in resolving the traditional difference observed between the transmission of actual RFQs and the transmission predicted by earlier simulations. In the last chapter of this thesis some experimental work on the MAFF (Munich Accelerator for Fission Fragments) IH-RFQ is described. The machine was assembled in Frankfurt and a beam test stand was built. The shunt impedance of the structure was measured using different techniques, the output energy of the structure were measured and finally its transmission was determined and compared to the beam dynamics simulations of the RFQ. Unfortunately, the transmission measurements were done without exact knowledge of the beam’s emittance. So the comparison to the simulation is somewhat rough, but with a reasonable guess of the emittance a good comparison between the measurement and simulation was obtained.
The power to dissociate : molecular function of the twin-ATPase ABCE1 in archaeal ribosome recycling
(2010)
Relational data exchange deals with translating relational data according to a given specification. This problem is one of the many tasks that arise in data integration, for example, in data restructuring, in ETL (Extract-Transform-Load) processes used for updating data warehouses, or in data exchange between different, possibly independently created, applications. Systems for relational data exchange exist for several decades now. Motivated by their experiences with one of those systems, Fagin, Kolaitis, Miller, and Popa (2003) studied fundamental and algorithmic issues arising in relational data exchange. One of these issues is how to answer queries that are posed against the target schema (i.e., against the result of the data exchange) so that the answers are consistent with the source data. For monotonic queries, the certain answers semantics proposed by Fagin, Kolaitis, Miller, and Popa (2003) is appropriate. For many non-monotonic queries, however, the certain answers semantics was shown to yield counter-intuitive results. This thesis deals with computing the certain answers for monotonic queries on the one hand, and on the other hand, it deals with the issue of which semantics are appropriate for answering non-monotonic queries, and how hard it is to evaluate non-monotonic queries under these semantics. As shown by Fagin, Kolaitis, Miller, and Popa (2003), computing the certain answers for unions of conjunctive queries - a subclass of the monotonic queries - basically reduces to computing universal solutions, provided the data transformation is specified by a set of tgds (tuple-generating dependencies) and egds (equality-generating dependencies). If M is such a specification and S is a source database, then T is called a solution for S under M if T is a possible result of translating S according to M. Intuitively, universal solutions are most general solutions. Since the above-mentioned work by Fagin, Kolaitis, Miller, and Popa it was unknown whether it is decidable if a source database has a universal solution under a given data exchange specification. In this thesis, we show that this problem is undecidable. More precisely, we construct a specification M that consists of tgds only so that it is undecidable whether a given source database has a universal solution under M. From the proof it also follows that it is undecidable whether the chase procedure - by which universal models can be obtained - terminates on a given source database and the set of tgds in M. The above results in particular strengthen results of Deutsch, Nash, and Remmel (2008). Concerning the issue of which semantics are appropriate for answering non-monotonic queries, we study several semantics for answering such queries. All of these semantics are based on the closed world assumption (CWA). First, the CWA-semantics of Libkin (2006) are extended so that they can be applied to specifications consisting of tgds and egds. The key is to extend the concept of CWA-solution, on which the CWA-semantics are based. CWA-solutions are characterized as universal solutions that are derivable from the source database using a suitably controlled version of the chase procedure. In particular, if CWA-solutions exist, then there is a minimal CWA-solution that is unique up to isomorphism: the core of the universal solutions introduced by Fagin, Kolaitis, and Popa (2003). We show that evaluation of a query under some of the CWA-semantics reduces to computing the certain answers to the query on the minimal CWA-solution. The CWA-semantics resolve some the known problems with answering non-monotonic queries. There are, however, two natural properties that are not possessed by the CWA-semantics. On the one hand, queries may be answered differently with respect to data exchange specifications that are logically equivalent. On the other hand, there are queries whose answer under the CWA-semantics intuitively contradicts the information derivable from the source database and the data exchange specification. To find an alternative semantics, we first test several CWA-based semantics from the area of deductive databases for their suitability regarding non-monotonic query answering in relational data exchange. More precisely, we focus on the CWA-semantics by Reiter (1978), the GCWA-semantics (Minker 1982), the EGCWA-semantics (Yahya, Henschen 1985) and the PWS-semantics (Chan 1993). It turns out that these semantics are either too weak or too strong, or do not possess the desired properties. Finally, based on the GCWA-semantics we develop the GCWA*-semantics which intuitively possesses the desired properties. For monotonic queries, some of the CWA-semantics as well as the GCWA*-semantics coincide with the certain answers semantics, that is, results obtained for the certain answers semantics carry over to those semantics. When studying the complexity of evaluating non-monotonic queries under the above-mentioned semantics, we focus on the data complexity, that is, the complexity when the data exchange specification and the query are fixed. We show that in many cases, evaluating non-monotonic queries is hard: co-NP- or NP-complete, or even undecidable. For example, evaluating conjunctive queries with at least one negative literal under simple specifications may be co-NP-hard. Notice, however, that this result only says that there is such a query and such a specification for which the problem is hard, but not that the problem is hard for all such queries and specifications. On the other hand, we identify a broad class of queries - the class of universal queries - which can be evaluated in polynomial time under the GCWA*-semantics, provided the data exchange specification is suitably restricted. More precisely, we show that universal queries can be evaluated on the core of the universal solutions, independent of the source database and the specification.
Summary: Information and communication is critical to the successful management of infectious diseases because an effective communication strategy prevents the surge of anxious patients who have not been genuinely exposed to the pathogen ('low risk patients') affecting medical infrastructures (1) and the future transmission of the infectious agent (2). Surge of low risk patients: The arrival of large numbers of low risk patients at hospitals following an infectious diseases emergency would be problematic for three main reasons. First, it would complicate the situation at hospitals receiving exposed patients, delaying the treatment of the acutely ill, creating difficulties of crowd control and tying up medical resources. Second, for the low risk patients themselves, attending hospital following an infectious disease emergency might increase their risk of exposure to the agent in question. Third, the needs of low risk patients may be poorly attended to at hospitals which are already overstretched dealing with medical casualties. Future transmission: Obtaining early information about symptoms and isolating infected patients is the most effective strategy to interrupt the chain of infection in the public in the absence of specific prophylaxis or treatment. Particularly at the beginning of an outbreak, these nonpharmaceutical interventions play an important role in enabling the early detection of signs or symptoms and in encouraging passengers to adopt appropriate preventive behaviour in order to limit the spread of the disease. This thesis includes two papers dealing with this problem: The first part is a systemic literature review of information needs following an infectious disease emergency (Anthrax, SARS, Pneumonic Plague). The key question was: what are the information needs of the public during an infectious disease emergency? The second part is an empirical investigation of information needs and communication strategies at the airport during the early stage of the Influenza Pandemic. The key question here was: what communication strategies help to meet the information needs and to enable the public to behave appropriately and responsibly? Conclusions: Evidence from the anthrax attacks in the United States suggested that a surge of low risk patients is by no means inevitable. Data from the SARS outbreak illustrated that if hospitals are seen as sources of contagion, many patients with non-bioterrorism related health care needs may delay seeking help. Finally, the events surrounding the Pneumonic Plague outbreak of 1994 in Surat, India, highlighted the need for the public to be kept adequately informed about an incident to avoid creating rumours. Clear, consistent and credible information is key to the successful management of infectious disease outbreaks. The results of the empirical investigation suggested that the desire for information is a reflection of current anxiety and does not mirror the objective scientific assessment of exposure. The airport study showed that perceived information needs were directly related to anxiety – the least anxious did not require any further information, the most anxious reported significant information needs concerning medical treatment, public health management and the assessment of the ongoing situation – irrespective of their actual exposure. A communication strategy only focussing on the 'real' exposed individuals neglects the information needs of those worrying about having contracted the virus and seeking medical attendance. Effective communication strategies should enable the general public to detect early signs or symptoms and provide them with behaviour advice to prevent the further transmission of the infectious agent. These include the provision of clear information about the incident, the symptoms and what to do to prevent the further transmission, detailed and regularly updated information in various media formats (telephone, internet, etc.) and rapid triage at hospital entrances to guide patients to the appropriate medical infrastructures. Relevance: These research findings could contribute to a shift in the organisational and communicative approach responding to infectious diseases outbreaks and could be considered relevant for future risk communication and policy decision making.
The aim of this work is to develop an effective equation of state for QCD, having the correct asymptotic degrees of freedom, to be used as input for dynamical studies of heavy ion collisions. We present an approach for modeling an EoS that respects the symmetries underlying QCD, and includes the correct asymptotic degrees of freedom, i.e. quarks and gluons at high temperature and hadrons in the low-temperature limit. We achieve this by including quarks degrees of freedom and the thermal contribution of the Polyakov loop in a hadronic chiral sigma-omega model. The hadronic part of the model is a nonlinear realization of an sigma-omega model. As the fundamental symmetries of QCD should also be present in its hadronic states such an approach is widely used to describe hadron properties below and around Tc. The quarks are introduced as thermal quasi particles, coupling to the Polyakov loop, while the dynamics of the Polyakov loop are controlled by a potential term which is fitted to reproduce pure gauge lattice data. In this model the sigma field serves a the order parameter for chiral restoration and the Polyakov loop as order parameter for deconfinement. The hadrons are suppressed at high densities by excluded volume corrections. As a next step, we introduce our new HQ model equation of state in a microscopic+macroscopic hybrid approach to heavy ion collisions. This hybrid approach is based on the Ultra-relativistic Quantum Molecular Dynamics (UrQMD) transport approach with an intermediate hydrodynamical evolution for the hot and dense stage of the collision. The present implementation allows to compare pure microscopic transport calculations with hydrodynamic calculations using exactly the same initial conditions and freeze-out procedure. The effects of the change in the underlying dynamics - ideal fluid dynamics vs. non-equilibrium transport theory - are explored. The final pion and proton multiplicities are lower in the hybrid model calculation due to the isentropic hydrodynamic expansion while the yields for strange particles are enhanced due to the local equilibrium in the hydrodynamic evolution. The elliptic and directed flow are shown to be not sensitive to changes in the EoS while the smaller mean free path in the hydrodynamic evolution reflects directly in higher flow results which are consistent with the experimental data. This finding indicates qualitatively that physical mechanisms like viscosity and other non equilibrium effects play an essentially more important role than the EoS when bulk observables like flow are investigated. In the last chapter, results for the thermal production of MEMOs in nucleus-nucleus collisions from a combined micro+macro approach are presented. Multiplicities, rapidity and transverse momentum spectra are predicted for Pb+Pb interaction at different beam energies. The presented excitation functions for various MEMO multiplicities show a clear maximum at the upper FAIR energy regime making this facility the ideal place to study the production of these exotic forms of multistrange objects.
The Opisthobranchia comprise highly specialized marine gastropods and have therefore been subject to diverse investigations covering various biological disciplines. However, a robust phylogeny of these gastropods is still lacking and several subclades have only been rarely studied. Furthermore, crucial aspects for the evolution of Opisthobranchia have not been comparatively analysed. Therefore, the aim of the present thesis is to gain new insights into the phylogeny of the Opisthobranchia with special focus on certain critical groups (Pleurobranchomorpha, Acteonoidea) and to assess several crucial features of the evolution of the investigated clades. The combination of four different gene markers (18S rDNA, 28S rDNA, 16S rDNA and CO1) and modern molecular systematic analysis tools were used to construct phylogenetic hypotheses focussing on Opisthobranchia as a whole as well as Pleurobranchomorpha and Acteonoidea in more detail. Intriguing new aspects of phylogeny and evolution of Opisthobranchia were revealed. First of all, monophyly of Opisthobranchia is definitely rejected based on the present data, while monophyly of Euthyneura (comprising Opisthobranchia and Pulmonata) is supported. Monophyly of opisthobranch subclades is confirmed for Nudipleura (as well as its constituting groups Nudibranchia and Pleurobranchomorpha), Umbraculida, Pteropoda (as well as subclades Thecosomata and Gymnosomata) and Acochlidiacea, for Cephalaspidea (if Runcinacea is regarded as a separate clade) and for Sacoglossa (if Cylindrobulla is accepted as an Oxynoacea). Aplysiomorpha are rendered paraphyletic due to the position of Akera bullata, but this result needs further investigation and should be considered with caution. The Nudipleura are found as the first single offshoot of the Euthyneura implying an early evolutionary separation of the last common ancestor of this clade. The remaining taxa form two main clades, one comprising the opisthobranch subgroups Umbraculida, Cephalaspidea, Aplysiomorpha and Pteropoda, while the other contains the pulmonate taxa and the opisthobranch Sacoglossa and Acochlidiacea. The interrelationships within these clades remain largely unresolved due to low statistical support values. However, a possible sister group relationship of Acochlidiacea and Eupulmonata receives statistical support. Opisthobranchia display various highly specific adaptations to diverse food sources. However, evolution of these specialized traits has never been assessed at an analytical level. The current thesis reconstructs the evolution of dietary preferences with novel methodologies based on the newly proposed phylogenetic hypothesis. Reconstruction of dietary evolution revealed herbivory as the ancestral condition in Euthyneura implying that carnivory evolved at least five times independently in the diverse lineages. The first comprehensive molecular phylogenetic hypothesis of the Pleurobranchomorpha could not reveal monophyly of the two main subclades Pleurobranchaeidae and Pleurobranchidae. This is due to the position of a single taxon (Euselenops luniceps) which is assigned to the Pleurobranchaeidae based on morphology but clusters within Pleurobranchidae in the current hypothesis. Furthermore, the tribe Berthellini and the genus Berthella are rendered paraphyletic by the current analyses. The results of molecular systematic analyses were used to reconstruct historical biogeography of Pleurobranchomorpha. Four different methodological approaches were applied yielding ambiguous results for Pleurobranchomorpha. However, the Pleurobranchidae comprising about 80% of the extant Pleurobranchomorpha most probably derived from an Antarctic origin. Dating of the phylogenetic tree via molecular clock methods yielded divergence of Pleurobranchidae into the Antarctic Tomthompsonia antarctica and the remaining species in Early Oligocene. Afterwards the latter underwent rapid radiation during Oligocene and Early Miocene. This divergence event coincides with two major geological events in the Antarctic region. On the one hand, the onset of glaciation and on the other hand the opening of the Drake Passage with concurrent formation of an Antarctic circumpolar current (ACC). I suppose that these sudden and dramatic changes in climate and palaeogeography probably accounted for migration of the last common ancestor of Pleurobranchidae (besides Tomthompsonia) into warmer regions via the Drake Passage to the Western Atlantic and Eastern Pacific and via the South Tasman Rise to the Indo-West Pacific. Furthermore, the ACC may have triggered larval dispersal to the Eastern Atlantic. The phylogenetic position of Acteonoidea has been a matter of debate for decades and they have long been considered as basal opisthobranchs. Results of the present thesis rather support placement in “Lower Heterobranchia” as sister group of Rissoelloidea. The current division of Acteonoidea into three families has never been investigated by means of phylogenetic methods. Thus, this thesis provides the first comprehensive investigation of this clade challenging present division into three families. The results rather support division into two main clades with the monogeneric Bullinidae clustering within Aplustridae doubting its separate status. Additionally, Rictaxis punctocaelatus which has been assigned to Acteonidae clusters basal to Aplustridae rendering Acteonidae paraphyletic. Since information on morphology of R. punctocaelatus was lacking until now, I conducted the first detailed investigation on morphology and histology of this species in order to reassess the unexpected molecular systematic placement. Character tracing analyses revealed similarities with both acteonoidean families implying an intermediate position of this species which might be assigned to a separate family in the future. Furthermore, the common features of Acteonidae and Rictaxis (massive shell, small foot, anterior mantle cavity opening, and absence of oral gland) are possibly plesiomorphic for the whole Acteonoidea. In summary, the results of the present thesis provide valuable novel insights into the phylogeny and evolution of the Opisthobranchia by employing state-of-the-art approaches of molecular systematics and evolutionary reconstruction. Thus, diverse hypotheses on opisthobranch phylogeny and evolution were either supported or rejected as well as novel hypotheses proposed which offer the basis for further research on these extraordinary gastropods.
In order to fully understand the new state of matter formed in heavy ion collisions, it is vital to isolate the always present final state hadronic contributions within the primary Quark-Gluon Plasma (QGP) experimental signatures. Previously, the hadronic contributions were determined using the properties of the known mesons and baryons. However, according to Hagedorn, hadrons should follow an exponential mass spectrum, which the known hadrons follow only up to masses of M = 2 GeV. Beyond this point the mass spectrum is flat, which indicates that there are "missing" hadrons, that could potentially contribute significantly to experimental observables. In this thesis I investigate the influence of these "missing" Hagedorn states on various experimental signatures of QGP. Strangeness enhancement is considered a signal for QGP because hadronic interactions (even including multi-mesonic reactions) underpredict the hadronic yields (especially for strange particles) at the Relativistic Heavy Ion Collider, RHIC. One can conclude that the time scales to produce the required amount of hadronic yields are too long to allow for the hadrons to reach chemical equilibrium within the lifetime of a cooling hadronic fireball. Because gluon fusion can quickly produce strange quarks, it has been suggested that the hadrons are born into chemical equilibrium following the Quantum Chromodynamics (QCD) phase transition. However, we show here that the missing Hagedorn states provide extra degrees of freedom that can contribute to fast chemical equilibration times for a hadron gas. We develop a dynamical scheme in which possible Hagedorn states contribute to fast chemical equilibration times of X X pairs (where X = p, K, Lambda, or Omega) inside a hadron gas and just below the critical temperature. Within this scheme, we use master equations and derive various analytical estimates for the chemical equilibration times. Applying a Bjorken picture to the expanding fireball, the hadrons can, indeed, quickly chemically equilibrate for both an initial overpopulation or underpopulation of Hagedorn resonances. We compare the thermodynamic properties of our model to recent lattice results and find that for both critical temperatures, Tc = 176 MeV and Tc = 196 MeV, the hadrons can reach chemical equilibrium on very short time scales. Furthermore the ratios p/pi, K/pi , Lambda/pi, and Omega/pi match experimental values well in our dynamical scenario. The effects of the "missing" Hagedorn states are not limited to the chemical equilibration time. Many believe that the new state of matter formed at RHIC is the closet to a perfect fluid found in nature, which implies that it has a small shear viscosity to entropy density ratio close to the bound derived using the uncertainty principle. Our hadron resonance gas model, including the additional Hagedorn states, is used to obtain an upper bound on the shear viscosity to entropy density ratio, eta/s, of hadronic matter near Tc that is close to 1/(4pi). Furthermore, the large trace anomaly and the small speed of sound near Tc computed within this model agree well with recent lattice calculations. We also comment on the behavior of the bulk viscosity to entropy density ratio of hadronic matter close to the phase transition, which qualitatively has a different behavior close to Tc than a hadron gas model with only the known resonances. We show how the measured particle ratios can be used to provide non-trivial information about Tc of the QCD phase transition. This is obtained by including the effects of highly massive Hagedorn resonances on statistical models, which are generally used to describe hadronic yields. The inclusion of the "missing" Hagedorn states creates a dependence of the thermal fits on the Hagedorn temperature, TH , and leads to a slight overall improvement of thermal fits. We find that for Au+Au collisions at RHIC at sqrt{sN N} = 200 GeV the best square fit measure, chi^2 , occurs at TH = Tc = 176 MeV and produces a chemical freeze-out temperature of 172.6 MeV and a baryon chemical potential of 39.7 MeV.
The single unit doctrine proposes that each one of our percepts and sensations is represented by the activity of specialized high-level cells in the brain. A common criticism applied to this proposal is the one referred to as the "combinatorial problem". We are constantly confronted with unlimited combinations of elements and features, and yet we face no problem in recognizing patterns and objects present in visual scenes. Are there enough neurons in the brain to singly code for each one of our percepts? Or is it the case that perceptions are represented by the distributed activity of different neuronal ensembles? We lack a general theory capable of explaining how distributed information can be efficiently integrated into single percepts. The working hypothesis here is that distributed neuronal ensembles signal relations present in the stimulus by selectively synchronizing their spiking responses. Synchronization is generally associated with oscillatory activity in the brain. Gamma oscillations in particular have been linked to various integrative processes in the visual system. Studies in anesthetized animals have shown a conspicuous increase in power for the gamma frequency band (30 to 60 Hz) in response to visual stimuli. Recently, these observations have been extended to behavioral studies which addressed the role of gamma activity in cognitive processes demanding selective attention. The initial motivation for carrying out this work was to test if the binding-by-synchronization (BBS) hypothesis serves as a neuronal mechanism for perceptual grouping in the visual system. To this aim we used single and superimposed grating stimuli. Superimposed gratings (plaids) are bi-stable stimuli capable of eliciting different percepts depending on their physical characteristics. In this way, plaids can be perceived either as a single moving surface (pattern plaids), or as two segregated surfaces drifting in different directions (component plaids). While testing the BBS hypothesis, we performed various experiments which addressed the role of both stimulus and cortical architecture on the properties of gamma oscillations in the primary visual cortex (V1) of monkeys. Additionally, we investigated whether gamma activity could also be modulated by allocating attention in time. Finally, we report on gamma-phase shifts in area V1, and how they depend on the level of neuronal activation. ...
Atherosclerosis is accompanied by infiltration of macrophages to the intima of blood vessels. There they engulf oxLDL (oxidized low-density lipoproteins) and differentiate to foam cells. These cells are known as major promoters of atherosclerosis progression. In initial experiments I could demonstrate that foam cell formation caused a severe loss in the ability to produce IFNA (interferon A) in response to stimulation with the bacterial cell wall component LPS (lipopolysaccharide). Since IFNA is discussed to have anti-atherosclerotic potential and has the capability to induce immune tolerance, its inhibition in foam cells might promote the atherosclerotic process. For this reason the aim of my PhD project was to clarify the underlying molecular mechanisms that attenuate LPS-induced IFNA expression in foam cells. LPS activates TLR4 (Toll-like receptor 4) in macrophages. Downstream this receptor two distinct signaling pathways are activated, namely a MyD88 (myeloid differentiation primary response gene 88)-dependent and a TRIF (TIR-domain-containing adapter-inducing IFNA)-dependent one. Foam cell formation targeted the TRIF-dependent TLR4 signaling pathway, as seen by loss of IRF3 activation and IFNA expression inhibition, whereas MyD88-initiated NFBB (nuclear factor 'B-light-chain-enhancer' of activated B-cells) activation and subsequent TNF@ (tumor necrosis factor @) expression remained unaltered. The TRIF signaling cascade results in transactivation of the transcription factor IRF3 (interferon regulatory factor 3), the main activator of IFNA expression. This event demands IRF3 phosphorylation by TBK1 (TANK-binding kinase 1), whereas TBK1 needs to be recruited to TRAF3 (TNF receptor associated factor 3) by the scaffold protein TANK (TRAF family member-associated NFBB activator) for its activation. This work allowed to propose the following scheme: OxLDL utilizes SR-A1 (scavenger receptor A1) to activate IRAK4 (interleukin-1 receptor-associated kinase 4), IRAK1 and Pellino3. Active IRAK1 and Pellino3 associate with TRAF3 and Pellino3 promotes mono-ubiquitination of the adaptor molecule TANK. Mono-ubiquitination of TANK interrupts TBK1 recruitment to TRAF3 and thereby abrogates phosphorylation and transactivation of IRF3 as well as subsequent expression of IFNA. In this study I provide evidence for a negative regulatory role of Pellino3 for TRIF-dependent TLR4 signaling. This expands the current knowledge of the interplay between pathways downstream scavenger and Toll-like receptors. Due to the multifaceted roles of TLR4 signaling in pathology, the new TRIF-signaling inhibitor Pellino3 might be of importance as therapeutical target for disease intervention.
In this work we study compact stars, i.e. neutron stars, as cosmic laboratories for the nuclear matter. With a mass of around 1 - 3 solar masses and a radius of around 10km, compact stars are very dense and, besides nucleons, can contain exotic matter such as hyperons or quark matter. The KaoS collaboration studied nuclear matter for densities up to 2-3 times saturation density by analysing kaon multiplicities from Au+Au and C+C collisions. The results show that nuclear matter in the corresponding density region is very compressible, with a compressibility of <200MeV. For such soft nuclear equations of state the maximum masses of neutron stars are ca. 1.8 - 1.9 solar masses, whereas the central densities are higher than 5 times nuclear saturation density and therefore point towards a possible phase transition to quark matter. If quark matter would be present in the interior of neutron stars, so-called hybrid stars, it could be produced already during their birth in supernova explosions. To study this we implement a quark matter phase transition in a hadronic equation of state which is used in supernova simulations. Supernova simulations of low and intermediate mass progenitors and two different bag constants show a collapse of the proto neutron star due to the softening of the equations of state in the quark-hadron mixed phase. The stiffening of the equation of state for pure quark matter halts the collapse and leads to the production of a second shock wave. The second shock wave is energetic enough to lead to an explosion of the star and produces a neutrino burst when passing the neutrinospheres. Furthermore, first studies of the longtime cooling of hybrid stars show, that colour superconductivity can significantly influence the cooling behaviour of hybrid stars, if all quarks form Cooper Pairs. For the so-called CSL phase (colour-spin locking) with pairing energies of several MeV, the cooling of the quark phase is suppressed and the hybrid star appears as a pure hadronic star.
This dissertation introduces in chapter 1 a new comparative approach to model-based research and policy analysis by constructing an archive of business cycle models. It includes many well-known models used in academia and at policy institutions. A computational platform is created that allows straightforward comparisons of models’ implications for monetary and fiscal stabilization policies. Chapter 2 applies business cycle models to forecasting. Several New Keynesian models are estimated on historical U.S. data vintages and forecasts are computed for the five most recent recessions. The extent of forecast heterogeneity for models and professional forecasts is analysed. Chapter 3 extends the forecasting analysis to a long sample and to the evaluation of density forecasts. Weighted forecasts are computed using a variety of weighting schemes. The accuracy of forecasts is evaluated and compared to professional forecasts and forecasts from nonstructural time series methods. Chapter 4 adds a new feature to existing business cycle models. Specifically, a medium-scale New Keynesian model is constructed that allows for strategic complementarities in price-setting. The role of trade integration for monetary policy transmission is explored. A new dimension of the exchange rate channel is highlighted by which monetary policy directly impacts domestic inflation. Chapter 5 tests whether simple symmetric monetary policy rules used in most business cycle models are a sufficient description of reality. I use quantile regressions to estimate policy parameters and find asymmetric reactions to inflation, the output gap and past interest rates.
Direct photon emission from heavy-ion collisions has been calculated and compared to available experimental data. Three different models have been combined to extract direct photons from different environments in a heavy-ion collision: Thermal photons from partonic and hadronic matter have been extracted from relativistic, non-viscous 3+1-dimensional hydrodynamic calculations. Thermal and non-thermal photons from hadronic interactions have been calculated from relativistic transport theory. The impact of different physics assumptions about the thermalized matter has been studied. In pure transport calculations, a viscous hadron gas is present. This is juxtaposed with ideal gases of hadrons with vacuum properties, hadrons which undergo a chiral and deconfinement phase transition and with a system that has a strong first-order phase transition to a deconfined ideal gas of quarks and gluons in the hybrid model calculations with the various Equations of State. The models used for the determination of photons from both hydrodynamic and transport calculations have been elucidated and their numerical properties tested. The origin of direct photons, itemised by emission stage, emission time, channel and baryon number density, has been investigated for various systems, as have the transverse momentum spectra and elliptic flow patterns of direct photons. The differences of photon emission rates from a thermalized transport box and the hadronic photon emission rates that are used in hydrodynamic calculations are found to be very similar, as are the spectra from calculations of heavy-ion collisions with transport model and hybrid model with hadronic Equation of State. Taking into account the full (vacuum) spectral function of the rho-meson decreases the direct photon emission by approximately 10% at low photon transverse momentum. The numerical investigations show that the parameter with the largest impact on the direct photon spectra is the time at which the hydrodynamic description is started. Its variation shows deviations of one to two orders of magnitude. In the regime that can be considered physical, however, the variation is less than a factor of 3. Other parameters change the direct photon yield by up to approximately 20%. In all systems that have been considered -- heavy-ion collisions at E_lab = 35 AGeV and 158 AGeV, (s_NN)**1/2 = 62.4 GeV, 130 GeV and 200 GeV -- thermal emission from a system with partonic degrees of freedom is greatly enhanced over that from hadronic systems, while the difference between the direct photon yields from a viscous and a non-viscous hadronic system (transport vs. hydrodynamics) is found to be very small. Predictions for direct photon emission in central U+U-collisions at 35 AGeV have been made. Since non-soft photon sources are very much suppressed at this energy, experimental results should very easily be able to distinguish between a medium that is entirely hadronic and a system that undergoes a phase transition from partonic to hadronic matter. In the case of lead-lead collisions at 158 AGeV, the situation is not so clear. In central collisions, the complete direct photon spectra including prompt photons seem to favour hadronic emission sources, while the partonic calculations only slightly overpredict the data. In peripheral collisions at the same energy, the hadronic contribution is more than one order of magnitude smaller than the prompt photon contribution, which fits the available experimental data. A similar picture presents itself at higher energies. At RHIC energies, however, the difference between transport calculations and hadronic hybrid model calculations is largest. Hybrid model calculations with partonic degrees of freedom can describe the experimental results in gold-gold collisions at 200 GeV. The elliptic flow component of direct photon emission is found to be consistently positive at small transverse momenta. This means that the initial photon emission from a non-flowing medium does not completely overshine the emission patterns from later stages. High-pt photons dominantly come from the beginning of a heavy-ion collision and therefore do not carry the directed information of an evolving medium.
One of the earliest and most striking observations made about HIV is the extensive genetic variation that the virus has within individual hosts, particularly in the hypervariable regions of the env gene which is divided into 5 variable regions (V1-V5) and 5 more constant (C1-C5) regions. HIV evolves at any time over the course of an individual’s infection and infected individuals harbours a population of genetically related but non-identical viruses that are under constant change and ready to adapt to changes in their environment. These genetically heterogeneous populations of closely related genomes are called quasispecies [65]. Tuberculosis or tubercle forming disease is an acute and/or chronic bacterial infection that primarily attacks the lungs, but which may also affect the kidneys, bones, lymph nodes, and brain. The disease is caused by Mycobacterium tuberculosis (MTB), a slow growing rod-shaped, acid fast bacterium. It is transmitted from person to person through inhalation of bacteria-carrying air droplets. Worldwide, one person out of three is infected with Mycobacterium tuberculosis – two billion people in total. TB currently holds the seventh place in the global ranking of causes of death [73]. In 2008, there were an estimated 9.4 (range, 8.9–9.9 million) million incident cases (equivalent to 139 cases per 100 000 population) of TB globally [75]. A complex biological interplay occurs between M. tuberculosis and HIV in coinfected host that results in the worsening of both pathologies. HIV promotes progression of M. tuberculosis either by endogenous reactivation or exogenous reinfection [77, 78] and, the course of HIV-1 infection is accelerated subsequent to the development of TB [80]. Active TB is associated with an increase in intra-patient HIV-1 diversity both systemically and at the infected lung sites [64,122]. The sustainability or reversal of the HIV-1 quasispecies heterogeneity after TB treatment is not known. Tetanus toxoid vaccinated HIV-1 infected patients developed a transient increase in HIV-1 heterogeneity which was reversed after few weeks [121]. Emergence of a heterogeneous HIV-1 population within a patient may be one of the mechanisms to escape strong immune or drug pressure [65,128]. The existence of better fitting and/or immune escape HIV-variants can lead to an increase in HIV-1 replication [129,130]. It might be that TB favourably selected HIV-1 variants which are sources for consistent HIV-1 replication. Understanding the mechanisms underlying the impacts of TB on HIV-1 is essential for the development of effective measures to reduce TB related morbidity and mortality in HIV-1 infected individuals. In the present study we studied whether the increase in HIV-1 quasispecies diversity during active TB is reversed or preserved throughout the course of antituberculous chemotherapy. For this purpose Two time point HIV-1 quasispecies were evaluated by comparing HIV-1 infected patients with active tuberculosis (HIV-1/TB) and HIV-1 infected patients without tuberculosis (HIV-1/non TB). Plasma samples were obtained from the Frankfurt HIV cohort and HIV-1 RNA was isolated. C2V5 env was amplified by PCR and molecular cloning was performed. Eight to twenty five clones were sequenced from each patient. Various phylogenetic analyses were performed including tree inferences, intra-patient viral diversity and divergence, selective pressure, co-receptor usage prediction and two time point identity of quasispecies comparison using Mantel’s test. We found out from this study that: 1) Active TB sustains HIV-1 quasispecies diversity for longer period 2. Active TB increases the rate of HIV-1 divergence 3) TB might slow down evolution of X4 variants And we concluded that active TB has an impact on HIV-1 viral diversity and divergence over time. The influence of active TB on longitudinal evolution of HIV- 1 may be predominant for R5 viruses. The use of CCR5-coreceptor inhibitors for HIV-1/TB patients as therapeutic approach needs further investigation.
This dissertation is devoted to the study of thermodynamics for quantum gauge theories.The poor convergence of quantum field theory at finite temperature has been the main obstacle in the practical applications of thermal QCD for decades. In this dissertation I apply hard-thermal-loop perturbation theory, which is a gauge-invariant reorganization of the conventional perturbative expansion for quantum gauge theories to the thermodynamics of QED and Yang-Mills theory to three-loop order. For the Abelian case, I present a calculation of the free energy of a hot gas of electrons and photons by expanding in a power series in mD/T, mf /T and e2, where mD and mf are the photon and electron thermal masses, respectively, and e is the coupling constant.I demonstrate that the hard-thermal-loop perturbation reorganization improves the convergence of the successive approximations to the QED free energy at large coupling, e ~ 2. For the non-Abelian case, I present a calculation of the free energy of a hot gas of gluons by expanding in a power series in mD/T and g2, where mD is the gluon thermal mass and g is the coupling constant. I show that at three-loop order hard-thermal-loop perturbation theory is compatible with lattice results for the pressure, energy density, and entropy down to temperatures T ~ 2 - 3 Tc. The results suggest that HTLpt provides a systematic framework that can be used to calculate static and dynamic quantities for temperatures relevant at LHC.
Employing NMR spectroscopy, it is not only possible to calculate the three dimensional structures of single proteins, but also to study dynamics and conformational changes of protein-complexes. In fact that is an important aspect, since the protein function depends on dynamics and interactions with other molecules. Therefore the study of protein-protein interactions is of highest importance for a better understanding of biological processes. Based on NMR methods, in this thesis we were able to determine protein-protein interactions within the enterobacterial Rcs signalling complex which is regulated via a phosphorelay. Originally identified as regulator of capsule synthesis, the Rcs phosphorelay is now considered to be implicated in stress response caused by disturbances in the peptidoglycan layer. Beyond that the Rcs system is involved in multiplex transcriptional networks including cell division, motility, biofilm formation and virulence. Because of such global nature and its extraordinary structural organisation involving membrane integrated sensor proteins (RcsC, RcsD), coactivators (RcsF, RcsA) and a transcription factor (RcsB), the Rcs system is one of the most remarkable phosphorelays in the family of enterobacteriacaea. During the complex phosphotransfer the histidine phosphotransferase (HPt) domain of the intermediary RcsD protein mediates the phosphotransfer between RcsC and RcsB, and probably modulates the phosphorylation state of the response regulator RcsB. Therefore the present work has been focused on the interface between RcsD and RcsB in more detail. In the first part of the thesis a new domain within the RcsD protein has been identified and structurally analysed by liquid NMR spectroscopy. RcsD is an inner membrane bound hybrid sensor like-kinase composed of a periplasmic sensor domain and a cytoplasmic portion. The cytoplasmic part contains the histidine like-kinase (HK) domain and the histidine phosphotransferase (HPt) domain. By analysis of the secondary structure in more detail, it was shown here that the two domains are intermitted by an additional 13.3 kDa domain. Corresponding to the position of the ABL (α−β−loop) domain of RcsC, located C-terminal to the RcsC-HK domain, the new identified domain was named RcsD-ABL. The central structural element of RcsD-ABL is a β-sheet composed of six strands with a β1−β2−β3−β4−β6−β5 topology and surrounded by two α-helices α1 and α2. In the second part of the thesis, RcsD-ABL is identified as a binding domain for the response regulator RcsB by NMR titration experiments. Such a binding domain for a response regulator has so far only been described for the histidine kinase CheA. In reportergene assays with β-galactosidase and ONPG as substrate it was shown that overexpression of RcsD-ABL in high amounts inhibited binding of RcsB to its target promoter. The β-galactosidase activity was reduced by 80 % with respect to cells carrying no plasmid encoding RcsD-ABL. The mapping of the binding interface was successfully achieved by chemical shift perturbations, a fast mapping protocol and selective labelling. It was shown that the interaction between RcsD-ABL and RcsB takes place via a binding interface comprising mainly the two α-helices of RcsD-ABL and the α-helices α7, α8 and α10 in the effector domain of RcsB. In the third part of the thesis, the interaction of RcsB with RcsD-ABL was related to that with RcsD-HPt. Using NMR titration experiments and ITC measurements, a comparison of the binding constants (Kd) of RcsB interacting either with the isolated RcsD-ABL (2 PM) or the isolated RcsDHPt domain (40 PM) revealed a higher affinity of RcsD-ABL to RcsB. A conjugate of RcsD-ABL-HPt interacting with RcsB decreased the Kd in the one-site fitting mode to 10 PM. However, the two-site fitting mode applied for RcsD-ABL-HPt/RcsB interaction resulted in a Kd (RcsD-ABL) of 2 PM and a Kd (RcsD-HPt) of 8 PM, indicating that RcsD-ABL enhances the binding of RcsD-HPt to RcsB. In the last part of the thesis, it was partly possible together with the data obtained from NMR titration experiments, PRE measurements and a HADDOCK protocol to develop a geometrical model for the interaction of RcsD with RcsB. In this model the receiver domain of RcsB interacts with the RcsD-HPt domain and the RcsB effector domain interacts with the RcsD-ABL domain. These results lead to surprising insights on the regulation of phosphorelays, since normally the effector domain binds to DNA. Here the effector domain is recognized by the newly identified RcsD-ABL domain. Prospectively, further investigations of phosphorylation affects and mutational studies will be of great interest.
Genes coding for membrane proteins make up 25%-30% of the genome in most organisms. Membrane proteins play an important role in cell functioning and their importance is enhanced by the fact that a large number of drugs are targeted at membrane proteins. Paradoxically, experimentally determined structures of membrane protein correspond to only about 1.7% of protein structures deposited in the protein data bank (PDB). This is largely due to the fact that membrane proteins are difficult to deal with owing to their amphipathic nature. The low abundance of membrane proteins in native tissue makes heterologous overexpression of these genes a necessity. This thesis work aimed at heterologous production of several secondary active transporter proteins for structural and functional characterizations and establishing alternative strategies to overcome the obstacles associated with heterologous overproduction. Four members of the heavy metal transporting cation diffusion facilitator (CDF) family from S. typhimurium and A. aeolicus were heterologously overproduced in E. coli and functionally characterized by an in vivo complementation assay using the zinc transport deficient E. coli GG48 strain. Out of these four, Aq_2073 from A. aeolicus was produced in large scale with substantial yield and purity sufficient to carry out structural studies. After extensive stability studies with different detergents, pHs and temperatures, the protein was subjected to 3D and 2D crystallization trials. Several C- terminal truncated constructs were made and the simultaneous crystallization screenings were carried out. These resulted in initial needle like crystals in 3D crystallization trials or optimum sized vesicles with crystalline patches in 2D crystallization trials but no obvious crystal. The protein showed significant increase in melting temperature in the presence of cadmium, when tested by differential scanning calorimetry. Another transporter, STM3880 of the potassium uptake permease (KUP) family from S. typhimurium, was heterologously overproduced in E. coli, purified by affinity chromatography, reconstituted into artificial liposome and functionally characterized by solid supported membrane based electrophysiology. In order to establish alternative expression strategies, continuous exchange cell free expression (CECF) of proteins from four different families was carried out. This method found to be aptly complementing the cell-based production approach. Targets from resistance to homoserine/threonine (RhtB) family not expressing in vivo could be expressed and purified using CECF. STM1781 of the sulfate permease (SulP) family was expressed, purified and characterized for stability while the cell-based production resulted in extensive degradation. PF0780 of multidrug/oligosaccharidyllipid/polysaccharide flippase (MOP) family was also purified to homogeneity and the stability was comparable to in vivo produced protein. Moreover, the effect of maltose binding protein (MBP) fusion at N-terminus on production and membrane integration was tested with three selected targets. The analysis revealed decreased yields in the presence of MBP if the protein had both termini in the cytoplasm. This work succeed in heterologously overproducing and establishing purification protocols for several secondary active transporters aiming at structural and functional characterization in a structural genomics framework. It also showed that integration of alternative strategies, like employing both cell-based and cell-free heterologous expression systems, expands the overall expression space coverage and in turn increases the chance of success of a structural genomics styled project.
Succinate:quinone oxidoreductases (SQORs) are integral membrane protein complexes, which couple the two-electron oxidation of succinate to fumarate (succinate → fumarate + 2H+ + 2e-) to the two-electron reduction of quinone to quinol (quinone + 2H+ + 2e- → quinol) as well as catalyzing the opposite reaction, the reduction of fumarate by quinol. In mitochondria and some aerobic bacteria, succinate:ubiquinone reductase, also known as complex II of the aerobic respiratory chain or as succinate dehydrogenase from the tricarboxylic acid (TCA or Krebs) cycle, catalyzes the oxidation of succinate by ubiquinone, which is mildly exergonic under standart conditions and not directly associated with energy storage in the form of a transmembrane electrochemical proton potential (Δp). Gram-positive bacteria do not contain ubiquinone but rather menaquinone, a quinone with significantly lower oxidation-reduction (“redox”) midpoint potential. In these cases, the catalyzed oxidation of succinate by quinone is endergonic under standard conditions. Consequently, these bacteria face a thermodynamic problem in supporting the catalysis of this reaction in vivo. Based on experimental evidence obtained on whole cells and purified membranes, it had previously been proposed that the SQR from Gram-positive bacteria supports this reaction at the expense of the protonmotive force, Δp. Nonetheless, it has been argued that the observed Δp dependence is not associated specifically with the activity of SQR because the occurrence of artifacts in experiments with bacterial membranes and whole cells can not be fully excluded. Clearly, definitive insight into the mechanism of catalysis of this intriguing reaction required a corresponding functional characterization of an isolated, membranebound SQR from a Gram-positive bacterium. The first aim of the present work addresses the question if the general feasibility of the energetically uphill electron transfer from succinate to menaquinone is associated specifically to a single enzyme complex, the SQR. The prerequisite to achieve this goal was stable preparation of this enzyme.
The nicotinamide-adenine-dinucleotide (NADH):ubiquinone oxidoreductase (complex I) from the strictly aerobic yeast Y. lipolytica contains at least 26 “accessory” subunits however the significance of most of them remains unknown. The aim of this study was to characterize the role of three accessory subunits of complex I, recently identified: two mitochondrial acyl carrier proteins, ACPM1 and ACPM2 and a sulfurtransferase (st1) subunit. ACPMs are small (approx. 10 kDa) acidic proteins that are homologous to the corresponding central components of prokaryotic fatty acid synthase complexes. Genomic deletions of the two genes ACPM1 and ACPM2 resulted in strains that were not viable or retained only trace amounts of assembled mitochondrial complex I, respectively, as assessed using two-dimensional blue native/sodium dodecyl sulfate polyacrylamide gel electrophoresis (BN/SDS) PAGE. This suggested different functions for the two proteins that despite high similarity could not be complemented by the respective other homolog still expressed in the deletion strains. To test whether complex I was affected by deletion of the ACPM2 gene, its activities in mitochondrial membranes were measured. Consequently, specific inhibitor sensitive dNADH: decylubiquinone (DBQ) oxidoreductase activity was lost completely and a strong decrease in dNADH: hexa-ammine-ruthenium (HAR) oxidoreductase activity was measured. Remarkably, the same phenotypes were observed if just the conserved serine carrying the phosphopantethein moiety was exchanged with alanine. Although this suggested a functional link to the lipid metabolism of mitochondria, using HPLC chromatography no changes in the lipid composition of the organelles were found. Proteomic analysis revealed that both ACPMs were tightly bound to purified mitochondrial complex I. Western blot analysis revealed that the affinity tagged ACPM1 and ACPM2 proteins were exclusively detectable in mitochondrial membranes but not in the mitochondrial matrix as reported for other organisms. Hence it has been concluded that the ACPMs can serve all their possible functions in mitochondrial lipid metabolism and complex I assembly and stabilization as subunits bound to complex I. A protein exhibiting rhodanese (thiosulfate:cyanide sulfurtransferase) activity was found to be associated with homogenous preparation of complex I. From a rhodanese deletion strain, functional complex I that lacked the additional protein but was fully assembled and displayed no functional defects or changes in EPR signature was purified. In contrast to previous suggestions, this indicated that the sulfurtransferase associated with Y. lipolytica complex I is not required for assembly of its iron–sulfur clusters.
Functional and structural characterization of Aquifex aeolicus sulfide:quinone oxidoreductase
(2010)
This work presents the first complete structure of the membrane protein sulfide:quinone oxidoreductase (SQR), obtained by X-ray crystallography. Its description is complemented by the results of biochemical and functional experiments. SQRs are ubiquitous flavoprotein disulfide reductases (FDRs), present in all domains of life, including in humans. Their physiological role extends from sulfide detoxification to sulfide-dependent respiration and photosynthesis (in archaea and bacteria), to heavy metal tolerance (in yeast) and possibly to sulfide signalling (in higher eukaryotes). Until now understanding the function of SQRs was difficult because of the poor level of sequence conservation in this enzyme family, the limited functional characterization available and the absence of any structural data. SQR was identified in the native membranes of the hyperthermophilic bacterium Aquifex aeolicus by peptide mass fingerprinting (PMF) and by a spectrophotometric activity assay. The protein was solubilized in the detergent dodecyl-beta-D-maltoside (DDM) and purified to homogeneity in a functionally active state. It binds one FAD molecule per protein monomer and FAD is its only cofactor. Its structure was determined in the “as-purified”, substrate-bound and inhibitor-bound forms at resolutions of 2.3, 2.0 and 2.9 Å, respectively. It is composed of two Rossmann-fold domains and of one membrane-attachment region. Despite the overall monomeric architecture being similar to that of FDRs, the structure reveals properties that had not been observed in FDRs until now and that have strong implications for the SQR catalytic mechanism. Surprisingly, A. aeolicus SQR is trimeric in the crystal structure and in solution, as determined by density-matched analytical ultracentrifugation, cross-linking and single particle electron microscopy. The trimer creates an appropriate surface for binding lipids and thus ensures that SQR exclusively reduces hydrophobic quinones. SQR inserts to a depth of about 12 Å into the membrane as an integral monotopic membrane protein. The interaction is mediated by an amphipathic helix-turn-helix tripodal motif and two lipid clamps. A channel in the membrane-binding domain extends towards the si-side of FAD and represents the quinone-binding site. The quinone ring is sandwiched between the conserved amino acids Phe 385 and Ile 346 and is possibly protonated upon reduction via Glu 318, Lys 382 and/or neighboring solvent molecules. Sulfide polymerization occurs on the re-side of FAD, where the highly conserved Cys 156 and Cys 347 appear to be covalently bound to the putative product of the reaction, a polysulfur chain which takes the form of an S8 ring in some monomers. Finally, the structure shows that FAD is covalently connected to the protein in an unprecedented way, via a putative disulfide bridge between the 8-methyl group of the isoalloxazine moiety and Cys 124. The high resolution insight into the protein and all unexpected structural observations presented in this work suggest that the catalytic mechanism of SQRs is significantly different from that of FDRs. In agreement with the structural and functional data, two reaction schemes are proposed for A. aeolicus SQR. They both provide a detailed description of how sulfide and quinones reach and bind the active site, how electrons are transferred from sulfide to quinone via FAD and how the elongating polysulfur product is attached to the polypeptide and is finally released. The two hypotheses differ in defining the structure of the covalent protein-FAD intermediate that forms during the reaction cycle and whose identity still remains experimentally undetermined. Remarkably, the structure of the active site and the FAD-binding mode of A. aeolicus SQR are not conserved in another SQR structure which also became available recently, that of the archaeon Acidianus ambivalens. The variability in SQRs suggests that not all of these enzymes follow the same catalytic mechanism, despite having been considered homologous. Consequently, the currently available but contradictory sequence-based classifications of the SQR family were revised. A structure-based alignment calculated on the increasing number of available sequences allowed to define new SQR groups and their characteristic sequence fingerprints in agreement with the reported structural and functional data. In conclusion, the results obtained in this work offer for the first time a detailed look into the intriguing but complicated reactions catalysed by SQRs and provide a stimulus for further genetic, biochemical and structural investigation.
Gene therapy is a promising therapeutic strategy that emerged from the attractive idea of targeting therapy at the molecular level. For many patients who suffer from genetic and acquired diseases that cannot be effectively treated by conventional treatment approaches gene therapy remains a huge hope of cure in spite of the hurdles regarding efficacy and safety that need to be overcome. The development of efficient gene transfer vehicles, mainly retroviral vectors, led to the first successful gene therapy trial, to treat patients suffering from X-linked severe combined immunodeficiency syndrome (X-SCID) using gene modified stem cells (Hacein-Bey-Abina, Le Deist et al. 2002). Despite the success of this trial, it revealed the danger of retroviral insertional mutagenesis as a major adverse event of gene therapy using gene-modified stem cells (Hacein-Bey-Abina, von Kalle et al. 2003). In contrast to stem cells, T cells are relatively resistant to insertional mutagenesis and transformation even after transduction with potent oncogenes using retroviral vectors (Newrzela, Cornils et al. 2008). However, mature T cells can self-renew, proliferate and survive for long periods. These criteria are supposed to render T cells prone to transformation. Therefore, the questions of mature T cells transformability and the control mechanism limiting their transformation are still elusive.
Drug toxicity and viral resistance limit long-term efficacy of antiviral drug treatment for HIV
infection. Thus, alternative therapies need to be explored. Previously, group of “Prof. von Laer”
tested the infusion of T lymphocytes transduced with a retroviral vector (M87o) that expresses an
HIV entry inhibitory peptide (maC46). Gene-modified autologous T cells were infused into 10
HIV-infected patients with advanced disease and multidrug resistant virus during antiretroviral
combination therapy. T cell infusions were tolerated well with no severe side effects. A
significant increase of CD4 counts was observed post infusion. At the end of the one-year
follow-up, the CD4 counts of all patients were still around or above baseline. Gene-modified
cells could be detected in peripheral blood, lymph nodes and bone marrow throughout the oneyear
follow-up, whereby marking levels correlated with the cell dose. No significant changes of
viral load were observed during the first four months. Four of the seven patients that changed
their antiviral drug regimen thereafter responded with a significant decline in plasma viral load.
In conclusion, the transfer of gene-modified cells was safe, led to sustained levels of gene
marking and may improve immune competence in HIV-infected patients with advanced disease
and multidrug resistant virus. However, the low level of gene marking and the lack of substantial
long-term in vivo accumulation of gene-protected cells observed in this trial clearly demonstrate
the requirement for new vectors with new strategy.
In this thesis self‐inactivating lentiviral vectors harboring internal promoters and RNA elements
were therefore evaluated for their potential use in a clinical gene‐therapy trial. The results from
this work provide the basis for the selection of a suitable candidate vector for extensive
preclinical testing. Apart from being capable of transducing non‐dividing cells, lentiviral vectors
incorporate a number of additional features that are of potential value for gene therapeutic
applications. These include a larger packaging capacity, higher titers than γ‐retroviral vectors
and, most importantly, a reduced risk of deregulating cellular genes due to its natural integration
profile. The use of internal promoters to drive expression of the therapeutic transgene maC46
should further improve the safety profile of these new‐generation vectors, while an additional
artificial splice acceptor (SA) into the 5‟UTR of the transgene over all elevate transgene
expression. The rationale for this is that hematopoietic stem and progenitor cells will be
Summary
98
protected from enhancer‐mediated transactivation effects and also from potential side effects due
to the aberrant expression of maC46 while at the same time the full clinical benefit for the
patients is maintained.
In order to find a suitable candidate for preclinical studies, two candidate therapeutic vectors
harboring different regulatory elements were selected based on results from pilot experiments.
The internal promoters used to drive expression of codon optimized maC46 were the PGK
promoter and MPSV promoter. This work focuses on the transgene expression levels in
lymphoid cells and antiviral activity. The issues of long term expression, propensity to
methylation mediated silencing of the promoters, and genotoxicity were also touched. In a first
step the performance of different vectors was evaluated in the human T cell lines. Based on
promising data from ex vivo human peripheral blood mononuclear cells, the vector carrying the
MPSV promoter along with intron were selected for in vivo transplantation experiments.
In summary, the ex vivo data suggested the long term survival of lentiviral gene modified cells,
along with maintained expression of introduced genes. It was observed that the expression of
these constructs depends strongly on the activation and differentiation status of the targeted T
cells. This regulation was not linked to any specific promotor. In vivo study shows that maC46
can be introduced into murine multiple hematopoietic lineages via lentiviral vector and expressed
at high levels in their mulilineage progeny, without altering the hematopoiesis. There was no
sign of any kind of hematopoietic or lymphoid malignancies. Although gene-modified
lymphocytes persisted in-vivo, the downregulation of transgene expression was consistent with
the ex-vivo observation. In contrast to that the T cells transplanted group showed delayed
engraftment of donor cells and there was no expression of C46 in blood and lymphatic organs. .
In conclusion, when considering HIV gene therapy focusing CD4+ T cells, potential problems of
T cell activation status as related to the desired clinical effect must be addressed. These results
might open the way for a gene therapy targeting mainly or exclusively activated T cells and
could be exploited for immunostimulatory as well as suppressive approaches.
The pathophysiology of schizophrenia is still poorly understood. Investigating the neurophysiological correlates of cognitive dysfunction with functional neuroimaging techniques such as electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) is widely considered to be a possible solution for this problem. Working memory impairment is one of the most prominent cognitive impairments found in schizophrenia. Working memory can be divided into a number of component processes, encoding, maintenance and retrieval. They appear to be differentially affected in schizophrenia, but little is known about the neurophysiological disturbances which contribute to deficits in these component processes. The aim of this dissertation was to elucidate the neurophysiological underpinnings of the component processes of working memory and their disturbance in schizophrenia. In the first study the the neurophysiological substrates of visual working memory capacity limitations were investigated during encoding, maintenance and retrieval in 12 healthy subjects using event-related fMRI. Subjects had to encode up to four abstract visual shapes and maintain them in working memory for 12 seconds. Afterwards a test stimulus was presented, which matched one of the previously shown shapes in fifty percent of the trials. A bilateral inverted U-shape pattern of BOLD activity with increasing memory load in areas closely linked with selective attention, i.e. the frontal eye fields and areas around the intraparietal sulcus, was observed already during encoding. The increase of the number of stored items from memory load three to memory load four in these regions was negatively correlated with the increase of BOLD activity from memory load three to memory load four. These results point to a crucial role of attentional processes for the limited capacity of working memory. In the second study, the contribution of early perceptual processing deficits during encoding and retrieval to working memory dysfunction was investigated in 17 patients with schizophrenia and 17 healthy control subjects using EEG and event-related fMRI. A slightly modified version of the working memory task used in the fist study was employed. Participants only had to encode and maintain up to three items. In patients the amplitude of the P1 event-related potential was significantly reduced already during encoding in all memory load conditions. Similarly, BOLD activity in early visual areas known to generate the P1 was significantly reduced in patients. In controls, a stronger P1 amplitude increase with increasing memory load predicted better performance. These findings indicate that in addition to later memory related processing stages early visual processing is disturbed in schizophrenia and contributes to working memory dysfunction by impairing the encoding of information. In the third study, which was based on the same data set as the second study, cortical activity and functional connectivity in 17 patients with schizophrenia and 17 to healthy control subjects during the working memory encoding, maintenance and retrieval was investigated using event-related fMRI. Patients had reduced working memory capacity. During encoding activation in the left ventrolateral prefrontal cortex and extrastriate visual cortex was reduced in patients but positively correlated with working memory capacity in controls. During early maintenance patients switched from hyper- to hypoactivation with increasing memory load in a fronto-parietal network which included left dorsolateral prefrontal cortex. During retrieval right ventrolateral prefrontal hyperactivation was correlated with encoding-related hypoactivation of left ventrolateral prefrontal cortex in patients. Cortical dysfunction in patients during encoding and retrieval was accompanied by abnormal functional connectivity between fronto-parietal and visual areas. These findings indicate a primary encoding deficit in patients caused by a dysfunction of prefrontal and visual areas. The findings of these studies suggest that isolating the component processes of working memory leads to more specific markers of cortical dysfunction in schizophrenia, which had been obscured in previous studies. This approach may help to identify more reliable biomarkers and endophenotypes of schizophrenia.
Current theories of schizophrenia suggest that the pathophysiology of the disorder may be the result of a deficit in the coordination of neural activity within and between areas of the brain, which may lead to impairments in basic cognitive functions such as contextual disambiguation and dynamic grouping (Phillips and Silverstein, 2003). This notion has been supported by recent studies showing that patients with schizophrenia are characterized by reduced synchronous, oscillatory activity in the gamma-frequency band during sensory processing (Spencer et al. 2003, Green et al. 2003, Wynn et al. 2005). However, it is currently unclear to what extent high-frequency gamma-band oscillations (> 60 Hz) contribute to impaired neural synchronization as research has so far focussed on gamma-band oscillations between 30 and 60 Hz. In addition, it is not known whether deficits in high-frequency oscillations are already present at the onset of the disorder and to what extent reductions may be related to the confounding influence of antipsychotic medication. Finally, the neural generators underlying impairments in synchronous oscillatory activity in schizophrenia have not been investigated yet. To address these questions, we recorded MEG activity during a visual closure task (Mooney faces task) in medicated chronic schizophrenia patients, drug-naive first-episode schizophrenia patients and healthy controls. MEG data were analysed for spectral power between 25 and 150 Hz, and beamforming techniques were used to localize the sources of oscillatory gamma-band activity. In healthy controls, we observed that the processing of Mooney faces was associated with sustained high-frequency gamma-band activity (> 60 Hz). A time-resolved analysis of the neural generators underlying perceptual closure revealed a network of distributed sources in occipito-temporal, parietal and frontal regions, which were differentially activated during specific time intervals. In chronic schizophrenia patients, we found a pronounced reduction of high-frequency gamma-band oscillatory activity that was accompanied by an impairment in perceptual organization and involved reduced source power in various brain regions associated with perceptual closure. First-episode patients were also characterized by a deficit in high-frequency gamma-band activity and reductions of source power in multiple areas; these impairments, however, were less pronounced than in chronic patients. Regarding behavioral performance, first-episode patients were not impaired in their ability to detect Mooney faces, but exhibited a loss in specificity of face detection. In conclusion, our results suggest that schizophrenia is associated with a widespread reduction in high-frequency oscillations that indicate local network abnormalities. These dysfunctions are independent of medication status and already present at illness onset, suggesting a possible progressive deficit during the course of the disorder.
Plasticity supports the remarkable adaptability and robustness of cortical processing. It allows the brain to learn and remember patterns in the sensory world, to refine motor control, to predict and obtain reward, or to recover function after injury. Behind this great flexibility hide a range of plasticity mechanisms, affecting different aspects of neuronal communication. However, little is known about the precise computational roles of some of these mechanisms. Here, we show that the interaction between spike-timing dependent plasticity (STDP), intrinsic plasticity and synaptic scaling enables neurons to learn efficient representations of their inputs. In the context of reward-dependent learning, the same mechanisms allow a neural network to solve a working memory task. Moreover, although we make no any apriori assumptions on the encoding used for representing inputs, the network activity resembles that of brain regions known to be associated with working memory, suggesting that reward-dependent learning may be a central force in working memory development. Lastly, we investigated some of the clinical implications of synaptic scaling and showed that, paradoxically, there are situations in which the very mechanisms that normally are required to preserve the balance of the system, may act as a destabilizing factor and lead to seizures. Our model offers a novel explanation for the increased incidence of seizures following chronic inflammation.
The goal of this project is to develop a framework for a cell that takes in consideration its internal structure, using an agent-based approach. In this framework, a cell was simulated as many sub-particles interacting to each other. This sub-particles can, in principle, represent any internal structure from the cell (organelles, etc). In the model discussed here, two types of sub-particles were used: membrane sub-particles and cytosolic elements. A kinetic and dynamic Delaunay triangulation was used in order to define the neighborhood relations between the sub-particles. However, it was soon noted that the relations defined by the Delaunay triangulation were not suitable to define the interactions between membrane sub-particles. The cell membrane is a lipid bilayer, and does not present any long range interactions between their sub-particles. This means that the membrane particles should not be able to interact in a long range. Instead, their interactions should be confined to the two-dimensional surface supposedly formed by the membrane. A method to select, from the original three-dimensional triangulations, connections restricted to the two-dimensional surface formed by the cell membrane was then developed. The algorithm uses as starting point the three-dimensional Delaunay triangulation involving both internal and membrane sub-particles. From this triangulation, only the subset of connections between membrane sub-particles was considered. Since the cell is full of internal particles, the collection of the membrane particles' connections will resemble the surface to be obtained, even though it will still have many connections that do not belong to the restricted triangulation on the surface. This "thick surface" was called a quasi-surface. The following step was to refine the quasi-surface, cutting out some of the connections so that the ones left made a proper surface triangulation with the membrane points. For that, the quasi-surface was separated in clusters. Clusters are defined as areas on the quasi-surface that are not yet properly triangulated on a two-dimensional surface. Each of the clusters was then re-triangulated independently, using re-triangulation methods also developed during this work. The interactions between cytosolic elements was given by a Lennard-Jones potential, as well as the interactions between cytosolic elements and membrane particles. Between only membrane particles, the interactions were given by an elastic interaction. For each particle, the equation of motion was written. The algorithm chosen to solve the equations of motion was the Verlet algorithm. Since the cytosol can be approximated as a gel, it is reasonable to suppose that the sub-cellular particles are moving in an overdamped environment. Therefore, an overdamped approximation was used for all interactions. Additionally, an adaptive algorithm was used in order to define the size of the time step used in each interaction. After the method to re-triangulate the membrane points was implemented, the time needed to re-triangulate a single cluster was studied, followed by an analysis on how the time needed to re-triangulate each point in a cluster varied with the cluster size. The frequency of appearance for each cluster size was also compared, as this information is necessary to guarantee that the total time needed by to re-triangulate a cell is convergent. At last, the total time spent re-triangulating a surface was plotted, as well as a scaling for the total re-triangulation time with the variation. Even though there is still a lot to be done, the work presented here is an important step on the way to the main goal of this project: to create an agent-based framework that not only allows the simulation of any sub-cellular structure of interest but also provides meaningful interaction relations to particles belonging to the cell membrane.
Apoptotic cell (AC)-derived factors alter the physiology of macrophages (M Phi s) towards a regulatory phenotype that is characterized by enhanced production of anti-inflammatory mediators, an attenuated pro-inflammatory cytokine profile and reduced nitric oxide (NO) formation. Impaired NO production in response to ACs or AC-conditioned medium (CM) is facilitated by arginase II (ARG II) expression, which competes with inducible NO synthase for L-arginine. In this study, I investigated the signaling pathway that allowed CM to upregulate ARG II in M Phi s. A sphingolipid, further identified as sphingosine-1-phosphate (S1P), was required but authentic S1P alone only produced small effects. S1P acted synergistically with a so far unidentified factor to elicit high ARG II expression. S1P signaled through S1P receptor 2 (S1P2), since the S1P2-antagonist JTE013 and siRNA knock-down of S1P2 prevented ARG II upregulation. Further, inhibition and knock-down of extracellular signal-regulated kinase 5 (ERK5) attenuated CM-mediated ARG II protein induction. Exploring ERK5-dependent transcriptional regulation, promoter deletion and luciferase reporter analysis of the murine ARG II promoter (mpARG II) suggested the involvement of cyclic adenosine monophosphate (cAMP) responsive element binding protein (CREB). This was confirmed by EMSA analysis and decoyoligonucleotides scavenging CREB, thereby preventing it from activating target genes and thus, blocking ARG II expression. I concluded that AC-derived S1P binds to S1P2 and acts synergistically with other factors to activate ERK5 and concomitantly CREB. This signaling cascade shapes an anti-inflammatory M Phi phenotype by ARG II induction. Further investigations of ERK5-dependent CREB activation suggested an indirect mechanism implying that ERK5 inhibited phosphodiesterase 4 (PDE4) and thus, prevented hydrolysis of cAMP. Since S1P-dependent ERK5 activation presumably inhibited PDE4, subsequent cAMP accumulation led to enhanced PKA activity and CREB-mediated transcription. The unidentified factor(s) besides S1P probably provoked the required elevation of cAMP production in M Phi s. Indeed, pharmacological inhibition of cAMP-producing adenylyl cyclase with SQ22536 as well as cAMP-dependent protein kinase A (PKA) with KT5720 suggested cAMP to be involved in CM-mediated ARG II up-regulation. Furthermore, forskolin-dependent activation of adenyly cyclase and simultaneous rolipram-mediated inhibition of PDE4 mimicked CM-induced ARG II expression. Considering these findings, I propose that one or several unidentified factors in CM provoke cAMP production in M Phi s. In parallel, AC-derived S1P activates ERK5, which inhibits PDE4-dependent cAMP hydrolysis, further raising intracellular cAMP levels. Thus, unrestricted continuous cAMP signaling via PKA/CREB, results in a time-dependent and sustained ARG II induction.
One of the key functions of blood vessels is to transport nutrients and oxygen to distant tissues and organs in the body. When blood supply is insufficient, new vessels form to meet the metabolic tissue demands and to re-establish cellular homeostasis. Expansion of the vascular network through sprouting angiogenesis requires the specification of ECs into leading (sprouting) tip and following (non-sprouting) stalk cells. Attracted by guidance cues tip cells dynamically extend and retract filopodia to navigate the nascent vessel sprout, whereas trailing stalk cells proliferate to form the extending vascular tube. All of these processes are under the control of environmental signals (e.g. hypoxia, metabolism) and numerous cytokines and peptide growth factors. The Dll4/Notch pathway coordinates several critical steps of angiogenic blood vessel growth. Even subtle alterations in Notch activity can profoundly influence endothelial cell behavior and blood vessel formation, yet little is known about the intrinsic regulation and dynamics of Notch signaling in endothelial cells. In addition, it remains an open question, how different growth factor signals impinging on sprouting ECs are coordinated with local environmental cues originating from nutrient-deprived, hypoxic tissue to achieve a balanced endothelial cell response. Acetylation of lysines is a critical posttranslational modification of histones, which acts as an important regulatory mechanism to control chromatin structure and gene transcription. In addition to histones, several non-histone proteins are targeted for acetylation reversible acetylation is emerging as a fundamental regulatory mechanism to control protein function, interaction and stability. Previous studies from our group identified the NAD+-dependent deacetylase SIRT1 as a key regulator of blood vessel growth controlling endothelial angiogenic responses. These studies revealed that SIRT1 is highly expressed in the vascular endothelium during blood vessel development, where it controls the angiogenic activity of endothelial cells. Moreover, in this work SIRT1 has been shown to control the activity of key regulators of cardiovascular homeostasis such as eNOS, Foxo1 and p53. The present study describes that SIRT1 antagonizes Notch signaling by deacetylating the Notch intracellular domain (NICD). We showed that loss of SIRT1 enhances DLL4-induced endothelial Notch responses as assessed by different luciferase responsive elements as well as transcriptional analysis of Notch endogenous target genes activation. Conversely, SIRT1 gain of function by overexpression of pharmacological activation decreases induction of Notch targets in response to DLL4 stimulation. We also showed that the NICD can be directly acetylated by PC AF and p300 and that SIRT1 promotes deacetylation of NICD. We have identified 14 lysines that are targeted for acetylation and their mutation abolishes the effects of SIRT1 of Notch responses. Furthermore, over-expression or activation of SIRT1 significantly reduces the levels of NICD protein. Moreover, SIRT1-mediated NICD degradation can be reversed by blockade of the proteasome suggesting a mechanism resulting from ubiquitin-mediated proteolysis. Indeed, we have shown that SIRT1 knockdown or pharmacological inhibition decreased NICD ubiquitination. We propose a novel molecular mechanism of modulation of the amplitude and duration of Notch responses in which acetylation increases NICD stability and therefore permanence at the promoters, while SIRT1, by inducing NICD degradation through its deacetylation, shortens Notch responses. In order to evaluate the physiological relevance of our findings we used different models in which the Notch functions during blood vessel formation have been extensively characterized. First, retinal angiogenesis in mice lacking SIRT1 activity shows decreased branching and reduced endothelial proliferation, similar to what happens after Notch gain of function mutations. ECs from these mice exhibit increased expression of Notch target genes. Second, these results were reproducible during intersomitic vessel growth in sirt1-deficient zebrafish. In both models, the defects could be partially rescued by inhibition of Notch activation. Third, we used an in vitro model of vessel sprouting from differentiating embryonic bodies in response to VEGF in a collagen matrix. Our results showed that Sirt1-deficient cells shows impaired sprouting which correlated with increased NICD levels. In addition, when in competition with wild-type cells in this assay, Sirt1-deficient cells are more prone to occupy the stalk cell position. Taken together, our study identifies reversible acetylation of NICD as a novel molecular mechanism to adapt the dynamics of Notch signaling and suggest that SIRT1 acts as a rheostat to fine-tune endothelial Notch responses. The NAD+-dependent feature of SIRT1 activity possibly links endothelial Notch responses to environmental cues and metabolic changes during nutrient deprivation in ischemic environments or upon other cellular stresses.
Event-by-event fluctuations of the particle yield ratios in heavy-ion collisions at 20 - 158 AGeV
(2010)
Non-statistical event-by-event fluctuations are considered as an important signal for the critical endpoint of the QCD phase diagram. Event-by-event fluctuations of different observables are thus investigated in detail in current experiments but are also an important observable to be studied at the future CBM experiment at FAIR. In this work we present the energy and centrality dependence of event-by-event fluctuations of particle yield ratios measured by the NA49 experiment in Pb+Pb collisions at 20 - 158 AGeV. Systematic studies of the influence of the dE/dx resolution on the particle identification and the centrality bin size were performed. Results can be compared to event-by-event fluctuations measured by NA49 for different observables such as <pt> or the mean charged particle multiplicity. Main results of these studies are an increase of absolute value of the dynamical particle ratio fluctuations with decreasing centrality for all considered ratios, saturation of the K/Pi and K/p ratio fluctuations for peripheral Pb + Pb collisions at 158A GeV and scaling of the energy and centrality dependences of the p/Pi ratio fluctuations with NpNPi. The measured energy and centrality dependences of the K/Pi and K/p ratio fluctuations scale with NK in a different way. The saturation of the mentioned ratios fluctuations was attributed to the development of pronounced spike at zero in the eventwise ratio distributions, which, as was shown by Monte Carlo simulations, influence the measured fluctuations in the very peripheral Pb + Pb collisions at 158A GeV. In future, the CBM experiment at FAIR will investigate the intermediate region of the QCD phase diagram in great detail searching for the first order phase transition line and the expected critical endpoint. It is therefore important to closely investigate its sensitivity towards particle ratio fluctuations in Au+Au collisions at 10-45 AGeV beam energy. Detailed simulation studies will be presented. As an outcome of these feasibility studies we conclude that the CBM experiment will be able to provide high quality data on the subject of the event-by-event flucutations of particle yield ratios with sensitivity in the order of 1%.
The role of gamma oscillatory activity in magnetoencephalogram for auditory memory processing
(2010)
Recent studies have suggested an important role of cortical gamma oscillatory activity (30-100 Hz) as a correlate of encoding, maintaining and retrieving auditory, visual or tactile information in and from memory. It was shown that these cortical stimulus representations were modulated by attention processes. Gamma-band activity (GBA) occurred as an induced response peaking at approximately 200-300 ms after stimulus presentation. Induced cortical responses appear as non-phase-locked activity and are assumed to reflect active cortical processing rather than passive perception. Induced GBA peaking 200-300 ms after stimulus presentation has been assumed to reflect differences between experimental conditions containing various stimuli. By contrast, the relationship between specific oscillatory signals and the representation of individual stimuli has remained unclear. The present study aimed at the identification of such stimulus-specific gamma-band components. We used magnetoencephalography (MEG) to assess gamma activity during an auditory spatial delayed matching-to-sample task. 28 healthy adults were assigned to one of two groups R and L who were presented with only right- or left-lateralized sounds, respectively. Two sample stimuli S1 with lateralization angles of either 15° or 45° deviation from the midsagittal plane were used in each group. Participants had to memorize the lateralization angle of S1 and compare it to a second lateralized sound S2 presented after an 800-ms delay phase. S2 either had the same or a different lateralization angle as S1. After the presentation of S2, subjects had to indicate whether S1 and S2 matched or not. Statistical probability mapping was applied to the signals at sensor level to identify spectral amplitude differences between 15° and 45° stimuli. We found distinct gamma-band components reflecting each sample stimulus with center frequencies ranging between 59 and 72 Hz in different sensors over parieto-occipital cortex contralateral to the side of stimulation. These oscillations showed maximal spectral amplitudes during the middle 200-300 ms of the delay phase and decreased again towards its end. Additionally, we investigated correlations between the activation strength of the gamma-band components and memory task performance. The magnitude of differentiation between oscillatory components representing 'preferred' and 'nonpreferred' stimuli during the final 100 ms of the delay phase correlated positively with task performance. These findings suggest that the observed gamma-band components reflect the activity of neuronal networks tuned to specific auditory spatial stimulus features. The activation of these networks seems to contribute to the maintenance of task-relevant information in short-term memory.
Top-down and bottom-up approaches are the general methods used to analyse proteomic samples today, however, the bottom-up approach has been dominant in the last decade. Establishing a bottom-up method involves not only the choice of adequate instruments and the optimisation of the experimental parameters, but also choosing the right experimental conditions and sample preparation steps. LC-ESI MS/MS has widely been used in this field due to its advanced automation. The primary objective of the present study was to establish a sensitive high-throughput nLC-MALDI MS/MS method for the identification and characterisation of proteins in biological samples. The method establishment included optimisation and validation of parameters such as the capillaries in the HPLC systems, gradient slopes, column temperature, spotting frequencies or the MS and MS/MS acquisition methods. The optimisation was performed using two HPLC-systems (Agilent 1100 series and Proxeon Easy nLC system), three spotters and the 4800 MALDI-TOF/TOF analyzer. Furthermore, samples preparation protocols were modified to fit to the established nLCMALDI- TOF/TOF-platform. The potentials of this method was demonstrated by the successful analysis of complex protein samples isolated from lipid particles, pre-adipocytes/adipocytes tissues, membrane proteins and proteins pulled-down from protein-proteins interaction studies. Despite the small amount of proteins in the lipid particles or oil bodies, and the challenges encountered in studying such proteins, 41(6 novel + 14 mammal specific + 21 visceral specific) proteins were added to the already existing proteins of the secretome of human subcutaneous (pre)adipocytes and 6 novel proteins localised in the yeast lipid particles. Protein-protein interaction studies present another area of application. Here the analytical challenges are mostly due to the loss of binding partner upon sample clean-up and to differentiate from non-specific background. Novel interaction partners for AF4•MLL and AF4 protein complex were identified. Furthermore, a novel sample protocol for the analysis of membrane proteins, based on the less specific protease, elastase, was established. Compared to trypsin, a higher sequence coverage and higher coverage of the transmembrane domains were achieved. The use of this enzyme in proteomics has been limited because of its non specific cleavage. However, from the results obtained in these studies, elastase was found to cleave preferentially at the C-terminal site of the amino acids AVLIST. The advantage of the established protocol over conventional protocols is that the same enzyme can be used for shaving of the soluble dormains of intact proteins in membranes and the digestion of the hydrophobic domain after solubilisation. Furthermore, the solvents used are compatible with the nLC-MALDI method setup. In addition, it was also shown that for less specific enzymes, a higher mass accuracy is required to reduce the rate of false positive identifications, since current search engines are not perfectly adapted for these types of enzymes. A brief statistical analysis of the MS/MS data obtained from the LC-MALDI TOF/TOF system showed that for less specific enzymes, under high-energy collision conditions, approximately 43 % of the fragment ions could not be matched to the known y- b type ions and their resultant internal fragments. This limitation greatly influenced the search results. However, this limitation can be overcome by modifying the N-terminal amino acids with basic moieties such as TMT. The use of elastase as a digestion enzyme in proteomic workflow further increased the complexity of the sample. Therefore, orthogonal multidimensional separation was necessary. Offgel-IEF was used as the separation technique for the first dimension. Here peptides are separated according to the pI. However, the acquired samples could not be loaded to the nLC due to the high viscosity of the concentrated samples when using the standard protocol. In order to achieve compatibility of the Offgel-IEF to the nLC-MALDI-TOF/TOF-platform, the separation protocol of the Offgel-IEF was modified by omitting the glycerol, which was the cause of the viscous solution. The novel glycerol free protocol is advantageous over the conventional method because the samples could directly be picked-up and loaded onto the pre-column without resulting in an increase in back pressure or a subsequent pre-column clogging. The glycerol free protocol was then assessed using purple membrane and membrane fraction of C. glutamicum. The results obtained were comparable to those applied in published reports. Therefore, the absence of glycerol did not affect the separation efficiency of the Offgel-IEF. In addition the applicability of elastase and the glycerol free Offgel-IEF for quantitation of membrane proteins was assessed. Most of the unique peptides identified were in the acidic region and 85 % were focused only into one fraction and approximately 95 % in only two fractions. These results are in accordance with previously published results (Lengqvist et al., 2007). When compared with theoretical digests of the proteins identified in this study, it can be concluded that basic moiety (TMT) on the peptide backbone, did not affect the separation efficiency of the Offgel-IEF. In an applied study, changes in the protein content of yeast strain grown in two different media were relatively quantified. For example, prominent proteins, such as the hexose tranporter proteins responsible for transporting glucose accross the membrane, were successfully quantified. Last but not least, the nLC-MALDI-TOF/TOF platform also served as a basis for the development of a high-throughput method for the identification of protein phosphorylation. The establishment of such a method using MALDI has been challenging due to the lack of sensitive matrices, such as CHCA for non-modified peptides, which exhibit a homogenous crystallisation and thus yield stable signal intensity over a long period of time in an automated setup. The first step of this method was the establishment of a matrix/matrix mixture with better crystal morphology and higher analyte signal intensity than the matrix of choice, i.e. DHB. From MS and MS/MS measurements of standard phosphopeptides, a combination of FCCA and CHAC in a 3:1 ratio and 3 mM NH4H2PO4 facilitated high analyte signal intensities and good fragmentation behaviour. Combining a custom-packed biphasic column for the enrichment of phosphopeptides, the applicability of the matrix mixture was assessed in anautomated phosphopeptide analysis using standard phosphopeptides spiked to a 20-fold excess BSA digest. These analyses showed that this method is reproducibile and both flow throughs can be analysed. Applying the method to the analysis of 2 standard phosphoproteins, alpha/beta-casein, and a leukemia related protein, ENL, 13 phosphopeptides from both alpha/beta-Casein and 13 phosphopeptides with 6 phosphorylation sites from the ENL were identified. As a general conclusion, it can be stated that the nLC-MALDI-TOF/TOF method established here in various modifications for different analytical purposes is a robust platform for proteomic analyses.
Over the last years there has been an increasing interest in the involvement of the MVA-pathway and of members of the small GTPases, in the development and progression of AD. Earlier investigations mainly focused on the role of cholesterol in disease pathology. This research was supported by retrospective cohort studies, initially showing beneficial effects of the long-term intake of cholesterol lowering statins, on the incidence of the development of sporadic AD. However, in more recent literature increasing attention has been paid to the isoprenoids, FPP and GGPP, due to their crucial role in the post-translational modifications of members of the superfamily of small GTPases. In AD, these proteins were amongst others shown to be involved in mechanisms affecting APP processing, ROS generation and synaptic plasticity. A major factor impeding the clarification of the role of the MVA-pathway intermediates in these mechanisms was the lack of a sensitive and accurate method to determine FPP and GGPP levels in brain tissue. Hence, a state of the art HPLC-FLD method for the quantification of the isoprenoids FPP and GGPP in brain tissue was successfully developed. After the introduction of a double clean-up step from complex brain matrix samples and the synthesis of an appropriate IS (DNP), the method was fully validated according to the latest FDA guideline for bioanalytical method validation. Furthermore, this method was transferred to a faster and more sensitive, state of the art UHPLC-MS/MS application. Additionally, the method was shown to be applicable for mouse brain tissue and data was generated from an in vivo mouse simvastatin study and for different mouse models. According to the aims of the thesis, the current work describes for the first time absolute isoprenoid concentrations in human frontal cortex white and grey matter. Furthermore, this is the first report of isoprenoid levels in the frontal cortex of human AD brains. Further results were shown from mouse brains originating from different mouse models, including the Thy-1 APP mouse model mimicking AD pathology in terms of Aβ formation or C57Bl/6 mice at different ages. AD prevalence can be clearly correlated with increasing age. Therefore, three different generations of mice were investigated. The study demonstrated constant isoprenoid and cholesterol levels in the first half of their life followed by a significant increase of FPP and GGPP in the second half (between 12 and 24 month of age). Cholesterol levels were also elevated in the aged group, but again the effect was less pronounced than shown for the isoprenoids. These results lead to the tentative conclusion that cerebral isoprenoid levels are elevated during aging and that this accumulation is amplified during AD leading to accelerated neuronal dysfunction. In a different mouse study, using the C57Bl/6 mice, in vivo drug intervention with the HMG-CoA reductase inhibitor simvastatin revealed strong inhibition of the rate limiting step of the mevalonate/isoprenoid/cholesterol pathway and resulted in the first report of significantly reduced FPP and GGPP levels in brain tissue of statin treated mice. These results open for the first time the possibility to monitor drug effects on cerebral isoprenoid levels and correlate these data with a modulation of APP processing, which was shown by our group in previous studies. Interestingly, apart from the isoprenoid reduction following statin treatment the reduction of brain cholesterol was also significant but to a lesser extent. These findings support the notion that isoprenoid levels are more susceptible to statin treatment than cholesterol levels. Furthermore, this suggests a strong cellular dependence on FPP and GGPP, as the pool seems to be easily depleted, which finally could lead to cell death. The first investigations of farnesylated Ras and geranylgeranylated Rac protein levels by means of immuno-blotting, substantiated the notion of a decreased abundance of prenylated small GTPases under statin influence as a consequence of reduced isoprenoid levels. These findings demonstrate for the first time a correlation of FPP and GGPP levels with the abundance of small GTPases. These findings together with the results from the AD study prove that isoprenoid levels are not strictly subject to the same regulation as cholesterol levels. To further understand the physiological regulation in the cell, in vitro experiments with different inhibitors of the mevalonate/isoprenoid/cholesterol pathway were conducted. These results confirmed the isoprenoid and cholesterol reducing effects of statin treatment as observed in the aforementioned in vivo mouse study. Interestingly, cholesterol synthesis inhibition targeted after FPP as the branch point, led to significantly elevated FPP levels. FTase inhibition led to significantly reduced FPP levels, whereas inhibition of the GGTase I did not show a significant change of either isoprenoid levels.
Hallin and Mancini’s seminal work Comparing Media Systems: Three Models of Media and
Politics has generated great interest and enthusiasm among media scholars to advance comparative
studies by applying the four dimensions to analyze media performance in different countries. Media
scholars agree that the four variables suggested by the two authors, i.e. the structure of media
markets, political parallelism, role of the state, and professionalization of journalism, provide a
good theoretical framework for the analysis of relationship between political and media system.
Their models for comparing media systems are based on a ‘most similar’ strategy,
analysing media and journalism only in stable Western democracies (i.e. Western European and
North American nations), and the purpose of the research presented in this paper was to develop
the model to include other parts of the world as well.
The most recent attempts to integrate East Central European media systems into the Hallin
and Mancini model, the conclusion being that the East Central European media share most
similarities with the Polarized Pluralist model. This conclusion follows not only Hallin and
Mancini, but also Splichal. The researcher in his earlier works argued that the changes in post-
Soviet media systems could be best explained by referring to the concept of Italianization - the
media are under strong state control, the degree of mass media partisanship is strong, low level of
journalistic professionalism, commercialization.
In fact, out of the three models only two (the Liberal and the Democratic Corporatist
model) are models in any strict sense, whereas the third - Polarized Pluralism - is better defined
as the lack of a model: the Liberal and Democratic Corporatist model are both built on a
consensus around core values, whereas the key feature of the Polarized Pluralism model is that
there is no consensus and no core values. De Albuquerque introduced other variables that also
would be highly relevant to the comparative analysis of media systems, but that have no place in
the Hallin & Mancini framework, the most important one being whether the political system is
presidential or parliamentary. For example, it has been demonstrated that media in presidential systems are more likely to focus on individual politicians and the administrative aspects of
government, as well as acting as an intermediary between different branches of government, than
are media in parliamentary systems.
Scholars dealing with the East Central Europe (and elsewhere) are too interested in fitting
their respective nations to one of the three models, rather than focusing on the variables and on the
comparative dimension. The scholars focus on the variables and on the comparative dimension: it
is strucking that their conclusions are precisely that a strict modeling approach (i.e. trying to fit
any given nation into the three-system model) is not enough if we want to understand media
system differences properly.
Hallin and Mancini (2004: 305) write that “The Democratic Corporatist Model, we suspect,
will have particularly strong relevance for the analysis of those parts of Eastern and Central
Europe that share much of the same historical development, like Poland, Hungary, the Czech
Republic, and the Baltic States”. At the same time, however, they suspect that scholars working on
the East Central European media will find much that is relevant in their analysis of the
Mediterranean region.
The recent attempts integrate East Central European media systems into the Hallin and
Mancini model, the conclusion being that the East Central European media share most similarities
with the Polarized Pluralist model. This conclusion follows not only Hallin and Mancini, but also
Splichal. The researcher in his earlier works argued that the changes in post-Soviet media systems
could be best explaind by referring to the concept of Italianization - including the role of
clientelism, the strong role of the state, the role of the media as an instrument of political struggle,
and a low level of journalistic professionalism.
The Polarized Pluralist model all too often seems to be the default model – what is really
gained, analytically, by saying that post-Communist countries are all basically Polarized Pluralist
media system when they are different in many ways. This question needs further elaboration.
Instead of fitting the Italianization model into East Central Europe, scholars should start working on their own model, introducing other variables, that would allow them to investigate the
media in the region adequately.
Der Neocortex der Säugetiere weist charakteristische Schichtungen auf, und jede dieser Schichten enthält verschiedene Typen von Neuronen, die in stereotypen Mustern angeordnet sind. Die Ausbildung dieser geschichteten Struktur ist nur dann möglich, wenn korrekte Migration von Neuronen von proliferativen Zonen zu deren Endpositionen stattfindet. Die exakte Migration und Schichtung wird von Mutationen beeinflusst, die entweder die migratorische Fähigkeit der Neuronen beeinträchtigen, oder deren Fähigkeit, die Position zu erkennen, an der sie die Wanderung beenden sollten (Gupta et al., 2002, Rice et al., 2001, Walsh et al., 2000). In den letzten Jahren wurde das extrazelluläre Protein Reelin als wichtiger Faktor bekannt, der sich auf mehrere Schritte der neuronalen Migration und Schichtung in der Großhirnrinde auswirkt (zusammengefasst in (Tissir et al., 2003). Das sekretierte Glykoprotein Reelin kontrolliert die Migration der Neuronen durch die Bindung an zwei Lipoproteinrezeptoren, den Very-low-density lipoprotein Rezeptor (VLDLR) und den Apolipoprotein E Rezeptor 2 (ApoER2) (D'Arcangelo et al., 1999). Die Bindung von Reelin an ApoER2 und VLDLR ruft die Phosphorylierung von Disabled-1 (Dab1) (D'Arcangelo et al., 1999, Howell et al., 1997), einem Adapterprotein, das an die intrazelluläre Domäne der Rezeptoren bindet, hervor, indem sie Kinasen der Src-Familie (SFKs) aktiviert (Arnaud et al., 2003, Bock et al., 2003a). Außer der Bedeutung des Reelin-Signalwegs für die korrekte Entwicklung des Nervensystems und dem Wissen, dass die Unterbrechung dieses Signalwegs zu verschiedenen neurologischen Krankheiten wie Epilepsie, Schizophrenie und der Alzheimerkrankheit führt (Costa et al., 2002, Botella-Lopez et al., 2006, Herz et al., 2006), ist die molekulare Grundlage der Aktivierung dieses Signalwegs an der Zellmembran noch kaum charakterisiert. Da VLDLR und ApoER2 keine intrinsische Kinaseaktivität besitzen, wurde die Existenz eines Korezeptors für mindestens eine Dekade vermutet, und die genaue Natur dieses Korezeptors ist unbekannt. EphrinBs, Transmembranliganden für Eph-Rezeptoren, besitzen die Fähigkeit zur Signalgebung, die für synaptische Plastizität und Angiogenese durch Sprossung erforderlich ist, indem sie die Aktivität anderer Transmembranrezeptoren wie AMPAR beziehungsweise VEGFR2 beeinflussen (Sawamiphak et al., 2010b, Segura et al., 2007, Essmann et al., 2008). Darüber hinaus führt die Stimulation von cortikalen Neuronen in Kultur mit löslichen EphB-Rezeptoren zur Rekrutierung und Aktivierung von SFKs in Membranpatches, in denen sich ephrinB-Liganden befinden (Palmer et al., 2002). Deshalb nehmen wir an, dass ephrinB in vivo funktionell mit dem Reelin-Signalweg verbunden sein könnte. Der Fokus dieser Arbeit liegt darin, zu zeigen, dass das neuronale Wegweisermolekül ephrinB einen entscheidenden Korezeptor für die Reelin-Signalgebung während der Entwicklung geschichteter Strukturen im Gehirn darstellt. Um zu erforschen, ob ephrinB und die Reelin-Signalgebung in vivo genetisch interagieren, wurden zuerst Mäuse mit Compound-Mutationen hergestellt, die eine Nullmutation im Gen für ephrinB3 tragen und heterozygot für Reelin sind (rl/+; b3-/-). Reeler ist eine autosomal rezessive Mutation der Maus, die, wenn sie heterozygot auftritt, keinen offenkundigen Phänotyp aufweist (Caviness et al., 1972, Caviness et al., 1978). Wir zeigen, dass ephrinBs genetisch mit Reelin interagieren, da Mäuse mit Compound-Mutationen (rl/+; b3 -/-) und ephrinB1-, B2- und B3-Dreifach-Knockouts die verschiedenen Defekte in der Entwicklung phänokopieren, die im Neocortex, Hippocampus und Cerebellum der reeler-Mäuse beobachtet wurden. Eines der Kennzeichen des reeler-Phänotyps ist die gestörte Schichtung der Großhirnrinde mit einer Marginalzone (MZ), die eine äußerst große Zahl an Zellen enthält (Caviness, 1982). Sowohl die Compound-Mäuse als auch die Triple-ephrinB1B2B3-knockouts zeigten eine Zunahme der Zellzahl in der MZ. Um die cortikalen Defekte detailliert zu charakterisieren, wurde die Verteilung von postmitotischen migrierenden Neuronen im Cortex von rl/+; b3-/- Compound-Mäusen mit Hilfe von unterschiedlichen schichtenspezifischen Markern für früh (Tbr1) (Hevner et al., 2001) und spät entstandene (SatB2 and Brn1) (Britanova et al., 2008, McEvilly et al., 2002) Neuronen, analysiert . Unsere Untersuchungen ließen die veränderte cortikale Schichtung in den rl/+; b3-/- Compound-Mäusen erkennen. So befanden sich früh entstandene Neuronen in den oberen cortikalen Schichten und spät entstandene in den unteren cortikalen Schichten, was für eine outside-in-Schichtung spricht, wie man sie von reeler kennt. Interessanterweise ist eine der frühesten strukturellen Abnormalitäten, die man im reeler-Cortex erkennen kann, die Unfähigkeit, die Preplate, die reich an extrazellulärer Matrix ist, in die Marginalzone und die Subplate aufzuspalten (Sheppard et al., 1997). Zum Zeitpunkt E17.5 zeigten rl/+; b3-/- Compound-Mäuse eine beachtliche Anhäufung von Chondroitin-Sulfat-Proteoglykan (CSPG), einer Komponente der extrazellulären Matrix, im gesamten Neocortex mit einer ungeteilten Schicht an der Oberfläche, welche übermäßig viel CSPG enthielt und somit die abnorme Teilung der Preplate der reeler-Maus nachahmte. Um zu bestätigen, dass die beobachteten Effekte auf die Schichtung des Cortex der rl/+; b3-/- Compound-Mäuse als Folge der Beeinträchtigung der neuronalen Migration auftritt, wurden zusätzlich BrdU-Puls-Experimente durchgeführt. BrdU wird in sich teilende Vorläuferzellen eingebaut und spiegelt deshalb das migratorische Verhalten von neu entstandenen Neuronen zum Zeitpunkt der Injektion wieder. Schwangeren Weibchen wurde BrdU zu den Zeitpunkten E12.5, E15.5 und E17.5 injiziert und die Gehirne wurden am postnatalen Tag 20 ausgewertet. Die Verteilung der mit BrdU gekennzeichneten Neuronen zu verschiedenen Zeitpunkten der Entwicklung in der Großhirnrinde bestätigte unsere Untersuchungen, die mit Hilfe der schichtspezifischen Marker durchgeführt worden waren. Deshalb deuten unsere Ergebnisse an, dass die beobachteten Defekte in der Schichtung des Cortex tatsächlich eine Folge von beeinträchtigter neuronaler Migration sind. Es wurde beobachtet, dass auch geschichtete Strukturen im Hippocampus in den rl/+; b3-/- Compound-Mäusen verändert sind, was für einen Crosstalk zwischen ephrinB3 und Reelin auch während der Entwicklung des Hippocampus spricht. Die CA1-Region des Hippocampus zeigte eine lockere Verbindung der pyramidalen Zellschichten, welche zu einer signifikanten Erhöhung der Dicke dieser Region und zu einer Einwanderung von Pyramidalzellen in das Stratum oriens führte. Darüber hinaus haben die Anomalien in den dendritischen Verzweigungen von Pyramidalneuronen der CA1-Region, die in Richtung der Reelin-produzierenden Cajal-Retzius-Zellen im stratum locunosum moleculare projizieren, in den rl/+; b3-/- Compound-Mäusen eine auffallende Ähnlichkeit mit denen, die in reeler-Mutanten beobachtet wurden. Reelin fungiert auch als Differenzierungsfaktor und Positionierungssignal für radiale Gliazellen, die positiv für glial fibrillary acidic protein (GFAP) sind und ein Gerüst für die korrekte Migration von neu entstandenen Granularzellen, die auf das Netzwerk der Granularzellen im Gyrus dentatus zuwandern (Forster et al., 2002) bilden. In rl/+; b3-/- Compound-Mäusen ist dieses Gerüst aus radialen Gliazellen schwerwiegend beeinträchtigt, was ebenfalls zu einer lockeren Organisation der Granularzellen im Gyrus dentatus führt. Die Ataxie in reeler-Mäusen ist das Ergebnis einer schwerwiegenden Fehlorganisation im Cerebellum dieser Mutanten (Tissir et al., 2003). Interessanterweise wurden nur milde Defekte in den Granularzellen, die sich in der internen Granularschicht des Cerebellums von rl/+; b3-/- Compound-Mäusen angesammelt haben, und keine Defekte in der Migration und der Verzweigung der Purkinjezellschicht, festgestellt. Stattdessen ist ephrinB2 in den Purkinjezellen des Cerebellums stark exprimiert (Liebl et al., 2003) und obwohl keine bedeutenden Defekte der Migration dieser Zellen festgestellt wurden, zeigte die Untersuchung der Verzweigung der Purkinjezellen in b2-/- Mäusen eindeutige Defekte, die bereits in einfachen ephrinB2-Mutanten auftraten. Bedeutend ist, dass die Defekte in der Verzweigung bei rl/+; b2-/- Compound-Mäusen signifikant verstärkt waren, was darauf hindeutet, dass der Reelin-Signalweg im Cerebellum spezifisch ephrinB2 benötigt. Um Einblicke in den Mechanismus zu erhalten, wie ephrinB-Liganden den Crosstalk mit Reelin durchführen, um die korrekte Positionierung von Neuronen in den geschichteten Strukturen des Gehirns zu kontrollieren, wurde als nächstes die biochemische Interaktion dieser beiden Signalwege untersucht. In einer gerichteten proteomischen Untersuchung mit Hilfe der Tandem affinity purification-mass spectometry-Methode (Angrand et al., 2006) von Proteinen aus eine Neuroblastom-Zelllinie, die ephrinB binden, wurde Reelin als ein Protein, das mutmaßlich mit ephrinB interagiert, identifiziert. Zunächst bestätigten wir die Fähigkeit von Reelin, mit ephrinBs zu assoziieren mit Ko-Immunpräzipitation beider endogener Proteine aus Gehirnlysaten. Das extrazelluläre Protein Reelin zeigte eine starke Bindung an die extrazelluläre Domäne von ephrinB3 und auch von ephrinB2, was andeutet, dass beide ephrin-Liganden die Funktionen von Reelin in vivo beeinflussen könnten. Die Stimulierung von cortikalen Neuronen mit Reelin führt zu einer effektiven Tyrosin-Phosphorylierung des Adapters Dab1. Da die Stimulation von cortikalen Neuronen mit einer löslichen, vorgeclusterten Form von EphB-Rezeptoren zur Rekrutierung und Aktivierung von Src-Kinasen in ephrinB-Clustern führt (Palmer et al., 2002), nehmen wir an, dass ephrinBs Src-Kinasen in VLDLR- und ApoER2-Rezeptor-Clustern rekrutieren und aktivieren könnten. Aktivierte Src-Kinasen phosphorylieren dann wiederum das Adapterprotein Dab1, das an VLDLR und ApoER2 gebunden ist und initiieren die weitere Signalgebung. In Übereinstimmung damit ko-immunpräzipitiert phosphoryliertes Dab1 zum Zeitpunkt E16.5 mit ephrinBs, während die neuronale Migration und die Schichtung des Cortex stattfindet. Darüber hinaus konnten wir beobachten, dass ephrinB3, das durch EphB3-Fc aktiviert wurde, sowohl Reelin, als auch ApoER2 und VLDLR in ephrinB3-Membranpatches in cortikalen Neuronen anhäuft. Die Aktivierung von ephrinB-Liganden durch Stimulation von cortikalen Neuronen mit EphB3-Fc führt zur Rekrutierung und Phosphorylierung von Dab1 in ephrinB-Clustern. Als nächstes befassten wir uns mit der Notwendigkeit von der durch ephrinB vermittelten Rekrutierung und Aktivierung von Src-Kinasen für den Reelin-Signalweg, indem wir Loss-of-function-Studien sowohl in cortikalen Neuronen in Kultur als auch in vivo in Mäusen durchführten. Cortikale Neuronen, die aus ephrinB3- und ephrinB2-Knockouts isoliert wurden, zeigten eine signifikante Beeinträchtigung der durch Reelin vermittelten Phosphorylierung von Dab1 und die Phosphorylierungslevels von Dab1 in ephrinB3 Mausmutanten waren stark verringert, was andeutet, dass ephrinBs Korezeptoren, die notwendig für einwandfreie Signalgebung durch Reelin sind, darstellen. Um die Bedeutung von ephrinBs für die Kontrolle der Funktion von Reelin zu untersuchen, arrangierten wir eine Reihe von Rescue-Experimenten sowohl in Neuronenkulturen als auch während der neuronalen Migration im Cortex in vivo. Aus reeler-Mäusen isolierte cortikale Neuronen zeigten die erwartet verringerte Phosphorylierung von Dab1, die rückgängig gemacht werden konnte, indem die Neuronen mit exogenem Reelin stimuliert wurden. Noch bedeutender ist die Tatsache, dass die Phosphorylierung von Dab1 durch die alleinige Aktivierung von ephrinBs mit EphB wiederhergestellt werden konnte, was die Bedeutung der ephrinBs als Korezeptoren für die Aktivierung des Signalwegs über die Rezeptoren für Reelin, VLDLR und ApoER2, wiederspiegelt. Um die Rolle von ephrinBs als Korezeptoren für den Reelin-Signalweg während der neuronalen Migration in der Großhirnrinde zu unterstreichen, setzten wir ähnliche Rescue-Experimente in organotypischen Schnittkulturen an. In den Schnitten von reeler-Mäusen und Wildtyp-Wurfgeschwistern wurde die Migration von Neuronen, die durch Fc als Kontrolle und EphB3-Fc stimuliert wurde, nach drei Tagen in Kultur untersucht. Die reeler-Schnitte zeigten den typischen reeler-Phänotyp in der Großhirnrinde. In Übereinstimmung mit der Annahme einer wirksamen Regulation des Reelin-Signalwegs war die Aktivierung von eprhinB mit EphB-Rezeptoren in der Lage, die migratorischen Defekte in reeler-Schnitten aufzuheben. Zusammengefasst identifizieren unsere Ergebnisse ephrinBs als Korezeptoren für den Reelin-Signalweg, die für die Funktion von Reelin in der neuronalen Migration während der Entwicklung der geschichteten Strukturen der Großhirnrinde, dem Hippocampus und dem Cerebellum notwendig sind. Unsere genetischen Analysen von ephrinB-Mutanten zeigen gemeinsam mit starken biochemischen Untersuchungen, dass ephrinBs in vivo für zahlreiche Aktivitäten von Reelin erforderlich sind.
This work investigated the applicability of global pairwise sequence alignment to the detection of functional analogues in virtual screening. This variant of sequence comparison was developed for the identification of homologue proteins based on amino acid or nucleotide sequences. Because of the significant differences between biopolymers and small molecules several aspects of this approach for sequence comparison had to be adapted. All proposed concepts were implemented as the ‘Pharmacophore Alignment Search Tool’ (PhAST) and evaluated in retrospective experiments on the COBRA dataset in version 6.1. The aim to identify functional analogues raised the necessity for identification and classification of functional properties in molecular structures. This was realized by fragment-based atom-typing, where one out of nine functional properties was assigned to each non-hydrogen atom in a structure. These properties were pre-assigned to atoms in the fragments. Whenever a fragment matched a substructure in a molecule, the assigned properties were transferred from fragment atoms to structure atoms. Each functional property was represented by exactly one symbol. Unlike amino acid or nucleotide sequences, small drug-like molecules contain branches and cycles. This was a major obstacle in the application of sequence alignment to virtual screening, since this technique can only be applied to linear sequences of symbols. The best linearization technique was shown to be Minimum Volume Embedding. To the best of knowledge, this work represents the first application of dimensionality reduction to graph linearization. Sequence alignment relies on a scoring system that rates symbol equivalences (matches) and differences (mismatches) based on functional properties that correspond to rated symbols. Existing scoring schemes are applicable only to amino acids and nucleotides. In this work, scoring schemes for functional properties in drug-like molecules were developed based on property frequencies and isofunctionality judged from chemical experience, pairwise sequence alignments, pairwise kernel-based assignments and stochastic optimization. The scoring system based on property frequencies and isofunctionality proved to be the most powerful (measured in enrichment capability). All developed scoring systems performed superior compared to simple scoring approaches that rate matches and mismatches uniformly. The frameworks proposed for score calculations can be used to guide modifications to the atom-typing in promising directions. The scoring system was further modified to allow for emphasis on particular symbols in a sequence. It was proven that the application of weights to symbols that correspond to key interaction points important to receptor-ligand-interaction significantly improves screening capabilities of PhAST. It was demonstrated that the systematic application of weights to all sequence positions in retrospective experiments can be used for pharmacophore elucidation. A scoring system based on structural instead of functional similarity was investigated and found to be suitable for similarity searches in shape-constrained datasets. Three methods for similarity assessment based on alignments were evaluated: Sequence identity, alignment score and significance. PhAST achieved significantly higher enrichment with alignment scores compared to sequence identity. p-values as significance estimates were calculated in a combination of Marcov Chain Monte Carlo Simulation and Importance Sampling. p-values were adapted to library size in a Bonferroni correction, yielding E-values. A significance threshold of an E-value of 1*10-5 was proposed for the application in prospective screenings. PhAST was compared to state-of-the-art methods for virtual screening. The unweighted version was shown to exhibit comparable enrichment capabilities. Compound rankings obtained with PhAST were proven to be complementary to those of other methods. The application to three-dimensional instead of two-dimensional molecular representations resulted in altered compound rankings without increased enrichment. PhAST was employed in two prospective applications. A screening for non-nucleoside analogue inhibitors of bacterial thymidin kinase yielded a hit with a distinct structural framework but only weak activity. The search for drugs not member of the NSAID (non-steroidal anti-inflammatory drug) class as modulators of gamma-secretase resulted in a potent modulator with clear structural distiction from the reference compound. The calculation of significance estimates, emphasizing on key interactions, the pharmacophore elucidation capabilities and the unique compound rannkings set PhAST apart from other screening techniques.
Development of a computational method for reaction-driven de novo design of druglike compounds
(2010)
A new method for computer-based de novo design of drug candidate structures is proposed. DOGS (Design of Genuine Structures) features a ligand-based strategy to suggest new molecular structures. The quality of designed compounds is assessed by a graph kernel method measuring the distance of designed molecules to a known reference ligand. Two graph representations of molecules (molecular graph and reduced graph) are implemented to feature different levels of abstraction from the molecular structure. A fully deterministic construction procedure explicitly designed to facilitate synthesizability of proposed structures is realized: DOGS uses readily available synthesis building blocks and established reaction schemes to assemble new molecules. This approach enables the software to propose not only the final compounds, but also to give suggestions for synthesis routes to generate them at the bench. The set of synthesis schemes comprises about 83 chemical reactions. Special focus was put on ring closure reactions forming drug-like substructures. The library of building blocks consists of about 25,000 readily available synthesis building blocks. DOGS builds up new structures in a stepwise process. Each virtual synthesis step adds a fragment to the growing molecule until a stop criterion (upper threshold for molecular mass or number of synthesis steps) is fulfilled. In a theoretical evaluation, a set of ~1,800 molecules proposed by DOGS is analyzed for critical properties of de novo designed compounds. The software is able to suggest drug-like molecules (79% violate less than two of Lipinski’s ‘rule of five’). In addition, a trained classifier for drug-likeness assigns a score >0.8 to 51% of the designed molecules (with 1.0 being the top score). In addition, most of the DOGS molecules are deemed to be synthesizable by a retro-synthesis descriptor (77% of molecules score in the top 10% of the decriptor’s value range). Calculated logP(o/w) values of constructed molecules resemble a unimodal distribution centred close to the mean of logP(o/w) values calculated for the reference compounds. A structural analysis of selected designs reveals that DOGS is capable of constructing molecules reflecting the overall topological arrangement of pharmacophoric features found in the reference ligands. At the same time, the DOGS designs represent innovative compounds being structurally distinct from the references. Synthesis routes for these examples are short and seem feasible in most cases. Some reaction steps might need modification by using protecting groups to avoid unwanted side reactions. Plausible bioisosters for known privileged fragments addressing the S1 pocket of trypsin were proposed by DOGS in a case study. Three of them can be found in known trypsin inhibitors as S1-adressing side chains. The software was also tested in two prospective case studies to design bioactive compounds. DOGS was applied to design ligands for human gamma-secretase and human histamine receptor subtype 4 (hH4R). Two selected designs for gamma-secretase were readily synthesizable as suggested by the software in one-step reactions. Both compounds represent inverse modulators of the target molecule. In a second case study, a ligand candidate selected for hH4R was synthesized exactly following the three-step synthesis plan suggested by DOGS. This compound showed low activity on the target structure. The concept of DOGS is able to deliver synthesizable and bioactive compounds. Suggested synthesis plans of selected compounds were readily pursuable. DOGS can therefore serve as a valuable idea generator for the design of new pharmacological active compounds.
Until now, the NW Indian Ocean was sparsely covered with coral proxy records, and records from the Maldives Archipelago do not exist. The first such coral proxy record from the central Maldives is presented in this study. It originates from a massive Porites lutea (Quoy and Gaimard, 1833) colony that was sampled March 2007 in the lagoon of Rasdhoo Atoll (4°N/ 73°W), which is located in the central Maldives. The record spans a period of 90 yrs and reaches back to 1917 AD with monthly to bimonthly resolution. This study investigates temporal variations of the skeletal stable oxygen (delta18O) and carbon (delta13C) isotopes, the strontium-to-calcium (Sr/Ca), and the annual extension-rates, and their relationship to historical climate variations 1917-2007. Annual extension-rates show an increase over the 20th century, and are correlated with instrumental sea surface temperatures (SST). The interannual variation of the extension-rates within 2.5-4 years is driven by the El Niño-Southern Oscillation (ENSO). The amount of skeletal extension during the summer months is triggered by variations in the strength of the SW monsoon. Interannual and decadal variability in monsoon current activity (18-19 yrs) and rainfall over India are an expression of the summer monsoon strength. This is the reason why a statistical link between coral extension-rates and precipitation over India can be established. This implies that annual extension-rates in corals can be used as a new proxy for Indian monsoon variability on decadal resolution. The delta18O record exhibits the 20th century warming trend that is influenced by the effect of monsoon-induced cooling. delta18O also reveals interannual ENSO triggered variability, which is due to ENSO-forced variations in SST and sea surface salinity (SSS). A decadal variation at 12-14 yrs cannot be linked to SST variations in the NW Indian Ocean, but with decadal variations of SSS. They could be caused by ENSO- forced variations of the monsoon currents during the mature phase of ENSO teleconnections in the Indian Ocean in boreal winter. The Sr/Ca record does not indicate a significant warming, in spite of the observed SST rise at the sampling site. Changes in seawater Sr/Ca cannot be excluded. Nevertheless, interannual ENSO forcing is still evident. Evidence for the Pacific Decadal Oscillation (PDO) is found during 1917-1955. Afterwards, the Sr/Ca data indicate the disappearance of PDO forcing. By the combination of Sr/Ca and delta18O it is possible to detect ~80% of historical El Niño and La Niña events at the sample site. This study confirms the notion that interannual to multi-decadal climate fluctuations in the Pacific play a crucial role for climate variability in the Indian Ocean.
Fas Ligand (FasL; CD95L; CD178; TNSF6) is a 40 kDa glycosylated type II transmembrane protein with 279 aa in mice and 281 aa in humans that belongs to the tumor necrosis factor (TNF) family. The extracellular domain (ECD) harbors a TNF homology domain, the receptor binding site, a motif for self assembly and trimerization, and several putative N-glycosylation and a metalloprotease cleavage site/s. The cytoplasmic tail of FasL is the longest of all TNFL family members and contains several conserved signaling motifs, such as a putative tandem Casein kinase I phosphorylation site, a unique proline-rich domain (PRD) and phosphorylatable tyrosine residues (Y7 in mice; Y7, Y9, Y13 in human). The FasL/Fas system is renowned for the potent induction of apoptosis in the receptor-bearing cell and is especially important for immune system functions. It is involved in the killing of target cells by natural killer (NK) and cytotoxic T cells, in the (self) elimination of effector cells following the proliferative phase of an immune response (activation-induced cell death; AICD), in the maintenance of immuneprivileged sites and in the induction and maintenance of peripheral tolerance. Owing to its potent pro-apoptotic signaling capacity and important functions, FasL expression and activity are tightly regulated at transcriptional and posttranscriptional levels and restricted to few cell types, such as immune effector cells and cells of immune-privileged sites. In contrast, Fas is expressed in a variety of tissues including lymphoid tissues, liver, heart, kidney, pancreas, brain and ovary. In addition to its pro-apoptotic function, the FasL/Fas system can also elicit nonapoptotic signals in the receptor-expressing cell. Among others, Fas-signaling exerts co-stimulatory functions in the immune system, e.g. by promoting survival, activation and proliferation of T cells. Besides the capacity to deliver a signal into receptor-bearing cells (‘forward signal’), FasL can receive and transmit signals into the ligand-expressing cell. This phenomenon has been described for several TNF family ligands and is known as ‘reverse signaling’. The first evidence for the existence of reverse signaling into FasL-bearing cells stems from two studies that demonstrated either co-stimulation of murine CD8+ T cell lines by FasL cross-linking or inhibition of activation-induced proliferation of murine CD4+ T cells. In both cases, the observed changes of proliferative behaviour critically depended on the presence of a signaling-competent FasL. Almost certainly, the FasL ICD is functionally involved in signal-transmission: (i) The ICD is highly conserved across species and harbors several signaling motifs, most notably a unique PRD. (ii) Numerous proteins have been identified which interact with the FasL PRD via their SH3 or WW domains and regulate various aspects of FasL biology, such as FasL sorting, storage, cell surface expression and the linkage of FasL to intracellular signaling pathways. (iii) Post-translational modifications of the ICD have been implicated in the sorting of FasL to vesicles and the FasL-dependent activation of Nuclear factor of activated T cells (NFAT). (iv) Proteolytic processing of FasL liberates the ICD and allows its translocation into the nucleus where it might influence gene transcription. (v) It could be shown that overexpression of the FasL ICD is sufficient to initiate reverse signaling upon concomitant T cell receptor (TCR) stimulation and ICD cross-linking. Conflicting data on the consequences of FasL reverse signaling exist, and costimulatory as well as inhibitory functions have been reported. These discrepancies probably reflect the use of artificial experimental systems. Neither the precise molecular mechanism underlying FasL reverse signaling, nor its physiological relevance have been addressed at the endogenous protein level in vivo. Therefore, a ‘knockout/knockin’ mouse model in which wildtype FasL was replaced with a deletion mutant lacking the intracellular portion (FasL Delta Intra) was established in the group of PD Dr. Martin Zörnig. In the present study, FasL Delta Intra mice were phenotypically characterized and were employed to investigate the physiological consequences of FasL reverse signaling at the molecular and cellular level. To ensure that FasL Delta Intra mice represent a suitable model to study the consequences of FasL reverse signaling, we demonstrated that activated lymphocytes from homozygous FasL Delta Intra or wildtype mice express comparable amounts of (truncated) FasL at the cell surface. The truncated protein retains the capacity to induce apoptosis in Fas receptor-positive target cells, as co-culture assays with FasL-expressing activated lymphocytes and Fas-sensitive target cells showed. Additionally, systematic screening of unchallenged mice did not reveal any phenotypic abnormalities. Notably, signs of a lymphoproliferative autoimmune disease associated with FasL-deficiency could not be detected. As several reports have implicated FasL reverse signaling in the regulation of T cell expansion and activation, proliferation of lymphocytes isolated from FasL Delta Intra and wildtype mice in response to antigen receptor stimulation was investigated. Using CFSE dilution assays it could be demonstrated that the proliferative response of CD4+ T cells, CD8+ T cells and of B cells was enhanced in the absence of the FasL ICD. Interestingly, this effect was most pronounced in B cells and could only be detected in CD4+ T cells after depletion of CD4+CD25+ regulatory T cells. To our Summary knowledge, this is the first time that FasL reverse signaling has been demonstrated in B cells. In a series of experiments, the activation of several pathways that are known to play important roles in signal-transmission initiated upon antigen receptor triggering was assessed. As a molecular correlate for the observed enhancement of activation-induced proliferation, Extracellular signal regulated kinase (ERK1/2) phosphorylation was significantly increased in FasL Delta Intra mice following antigen receptor crosslinking. Surprisingly, B cell stimulation lead to a comparable extent of activating phosphorylations on S38 in c-Raf and S218/S222 in MEK1/2 in cells isolated from wildtype and FasL Delta Intra mice, indicating that Mitogen activated protein kinases (MAPKs) upstream of ERK1/2 (Raf-1 and MEK1/2) apparently do not contribute to the differential regulation of ERK1/2. Experiments in which activation-induced Akt phosphorylation (S473) was quantified also did not suggest a participation of Phosphoinositol specific kinase 3 (PI3K)/Akt signals in this process. Instead, further characterization of the upstream pathway revealed an involvement of Phospholipase C gamma (PLC gamma) and Protein kinase C (PKC) signals in FasL-dependent ERK1/2- regulation. Previous studies in our group revealed a Notch-like processing of FasL, resulting in the transcriptional regulation of a reporter gene. Furthermore, an interaction of the FasL ICD with the transcription factor Lymphoid-enhancer binding factor-1 (Lef-1) that affected Lef-1-dependent reporter gene transcription could be demonstrated. Therefore, a molecular analysis of activated lymphocytes was performed to identify FasL reverse signaling target genes. The differential expression of promising candidates was verified by quantitative real-time PCR (qRT-PCR), which showed that the transcription of genes associated with lymphocyte proliferation and activation was increased in FasL Delta Intra mice compared to wildtype mice. Interestingly, an extensive regulation of Lef-1-dependent Wnt/beta-Catenin signalingrelated genes was found. Lef-1 mRNA (RT-PCR) and protein (intracellular FACS staining) could be detected in mature B cells, suggesting the possibility of FasL ICD-mediated inhibition of Lef-1-dependent gene expression in these cells, initiated by Notch-like processing of FasL. To investigate the consequences of FasL reverse signaling in vivo, a potential participation of the FasL ICD in the regulation of immune responses upon various challenges was analyzed. In experiments in which thymocyte proliferation or the expansion of antigen-specific T cells following a challenge with the superantigen Staphylococcus enterotoxin B (SEB), with Lymphocytic choriomeningitis virus (LCMV) or with Listeria monocytogenes were investigated, comparable results were obtained with wildtype and FasL Delta Intra mice. Likewise, the recruitment of neutrophils in a thioglycollate-induced model of peritonitis was not affected by deletion of the FasL ICD. These findings might reflect regulatory mechanisms operating in vivo, such as control exerted by regulatory T cells. Along these lines, proliferative differences in CD4+ T cells could only be detected ex vivo after depletion of CD4+CD25+ regulatory T cells. Furthermore, several in vitro studies indicate that retrograde FasL signals can be observed under conditions of suboptimal lymphocyte stimulation, but not when the TCR is optimally stimulated. Therefore, the potent initiation of antigen receptor signaling by stimuli like SEB or LCMV might have masked inhibitory FasL reverse signaling in these experiments. In agreement with the observed hyperactivation of lymphocytes in the absence of the ICD ex vivo, the increase in germinal center B cells (GCs) following immunization with the hapten 3-hydroxy 4-nitrophenylacetyl (NP) and the number of antibody-secreting PCs was significantly higher in FasL Delta Intra mice. The larger quantity of PCs correlated with increased titers of NP-binding, i.e. antigen-specific, IgM and IgG1 antibodies in the serum of FasL Delta Intra mice after immunization. These data suggest that FasL reverse signaling exerts immunmodulatory functions. Supporting this notion, a model of Ovalbumin-induced allergic airway inflammation revealed an involvement of retrograde FasL-signals in the recruitment of immune effector cells into the lung and in the activation of T cells following exposure of mice to Ovalbumin. Together, our ex vivo and in vivo findings based on endogenous FasL protein levels demonstrate that FasL ICD-mediated reverse signaling is a negative modulator of certain immune responses. It is tempting to speculate that FasL reverse signaling might be a fine-tuning mechanism to prevent autoimmune diseases, a theory which will be tested in adequate mouse models in the future.
The glycine receptor (GlyR) is the major inhibitory neurotransmitter receptor in spinal cord and brainstem. Heteropentameric GlyRs are clustered and anchored at inhibitory postsynaptic sites by the binding of the large intracellular loop between transmembrane domains 3 and 4 of the GlyRbeta subunit (GlyRbeta-loop) to the cytoplasmic scaffolding protein gephyrin. GlyRs are also cotransported with gephyrin along microtubules in the anterograde and retrograde direction due to the binding of gephyrin to microtubule-associated motor proteins. Additionally, GlyRs undergo lateral diffusion in the plasma membrane from extrasynaptic to synaptic sites and vice versa. Since its discovery, gephyrin has remained for many years the only binding partner interacting directly with the GlyRbeta subunit. In an attempt to elucidate further mechanisms involved in GlyR function and regulation at inhibitory postsynaptic sites, a proteomic screen for putative binding partners to the GlyRbeta loop was performed. Three proteins were identified as putative interactors. In this thesis, the interaction between these putative binding proteins and the GlyRbeta subunit was analyzed and characterized. Binding studies with glutathione-S-transferase fusion proteins revealed that all putative binding proteins, Syndapin (Sdp), Vacuolar Protein Sorting 35 (Vps35) and Neurobeachin (Nbea), interact specifically with the GlyRbeta loop. The Sdp family of proteins are F-BAR and SH3 domain containing proteins. Inmmunocytochemical experiments showed that SdpI as well as the isoforms SdpII-S and SdpIIL colocalize with the full-length GlyRbeta subunit in a mammalian cell expression system. In cultured spinal cord neurons, a partial colocalization of endogenous SdpI with several excitatory and inhibitory synaptic markers was demonstrated. Mapping experiments using deletion mutants narrowed the SdpI binding site down to 22 amino acids. Peptide competition experiments confirmed the specificity of the interaction between SdpI and this sequence of the GlyRbeta subunit. Point mutation analysis revealed a SH3-proline rich domain dependent interaction between SdpI and the GlyRbeta subunit, respectively. In addition, binding studies in mammalian cells showed that both splice variants of SdpII as well as SdpI interact with the GlyR scaffolding protein gephyrin. Although the SdpI and gephyrin binding sites do not overlap, protein competition studies revealed that interaction of the E-domain of gephyrin with the GlyRbeta loop interferes with SdpI binding. Since SdpI is a dynamin binding protein involved in vesicle endocytosis and recycling pathways, a possible function of SdpI in the regulation of GlyR synaptic distribution was investigated. Co-immunoprecipitation experiments confirmed a SdpI-GlyR association in the vesicle-enriched fraction of rat spinal cord tissue. Immunocytochemical studies of SdpI knock out mice showed that the clustering and distribution of GlyRs in the brain stem is unchanged. However, acute down-regulation of SdpI in rat spinal cord neurons by viral shRNA expression led to a reduction in the number and size of GlyR clusters, an effect that could be rescued upon shRNA-resistant SdpI overexpression. Further immunocytochemical analysis of the localization of gephyrin, the gamma2 subunit of the type A gamma-aminobutyric acid receptor (GABAARgamma2 subunit) and the vesicular inhibitory amino acid transporter (VIAAT) under SdpI knock-down conditions showed that both the number and average size of the gamma2-subunit containing GABAA receptor clusters were significantly reduced in spinal cord neurons. In contrast to GlyR and GABAARgamma2 immunoreactivity, the number and average size of gephyrin and VIAAT clusters were barely reduced upon SdpI downregulation. These results suggest that SdpI has a role in GlyR trafficking that can be compensated by other syndapin isoforms or other trafficking pathways. Furthermore, SdpI might be required for the clusters of GlyRs and gamma2-subunit containing GABAARs in spinal cord and brainstem. Vps35 is the core protein of the retromer complex, which mediates the endosome to Golgi apparatus retrieval of different types of receptors in mammals and yeast. Here, protein-protein interaction assays revealed for the first time that Vps35 interacts directly with the GlyRbeta loop as well as with gephyrin. The generation of specific Vps35 antibodies allowed to determine the distribution of this protein in the central nervous system. Immunocytochemical analyses revealed the presence of Vps35 in the somata and neurites of spinal cord neurons, suggesting a possible interaction of Vps35 with the GlyR under physiological conditions. Nbea is a BEACH domain containing, neuron-specific protein. Binding studies revealed a direct interaction between two regions of Nbea and the GlyRbeta loop. Immunocytochemical experiments confirmed a somatic and synaptic distribution of Nbea in primary cultures. In spinal cord neurons, a partial colocalization of Nbea with excitatory and inhibitory synaptic markers suggests a possible interaction of Nbea with the GlyR at inhibitory synaptic sites.
The analysis of biomolecular macrocomplexes requires certain preconditions to be fulfilled. The preparation of biomolecular samples usually results in low yields. Due to this constraint of low availability any method should provide a sufficient sensitivity to cope with typical sample amounts. Biomolecules also often show a reduced stability, i.e. a propensity for fragmentation upon ionisation, which requires reasonable soft methods for the investigation. Furthermore macromolecular complexes usually are composed by means of non-covalent interactions presenting additional demands on the softness. This holds true for specific complexes like protein-ligand or DNA double strand binding. For the formation of non-covalent, specific complexes the biomolecules’ native structure and environment are a basic prerequisite and hence crucial. Therefore it is desirable during analysis to keep the biomolecules in a native environment to preserve their structure and weak interactions. One suitable method for analysing biomolecules is mass spectrometry. Mass spectrometry is capable of high throughput screening as well as determining masses with high accuracy and high sensitivity. Especially since the availability of MALDI-MS and ESI-MS mass spectrometry evolved to a versatile tool to investigate biomolecular complexes. Both, MALDI- and ESI-MS are sufficiently soft methods to observe fragile biomolecules. Yet both methods have their advantages and disadvantages. During the recent years an alternative mass spectrometric approach has been developed in our group, termed LILBID-MS (Laser Induced Liquid Bead Ionisation/Desorption). In LILBID microdroplets of aqueous solution containing buffer, salt and further additives among the analyte molecules are injected into vacuum and irradiated one-by-one by mid-IR laser pulses. The absorption of the energy by the water leads to a rapid ablation of the preformed analyte ions. LILBID is highly tolerant for the addition of salts and detergents allowing to study biomolecular complexes in a native environment. As LILBID-MS is soft enough to avoid fragmentation, specific non-covalent complexes can be analysed directly from their native environment by this method. In addition dissociation can be induced on demand by increasing the laser intensity which allows for the study of subunit compositions. A further prominent property of LILBID is the possibility to study hydrophobic membrane proteins due to the tolerated use of detergents. During the course of this work, several instrumental improvements mostly concerning ion focussing and beam steering were introduced. Together with refinements of different modes of measurement the result is a significantly improved signal-to-noise ratio as well as a further improvement in sensitivity. In addition the accessible m/z range for a given flight time has been vastly increased. The new possibilities that LILBID now offers for the study of biomolecular complexes were investigated. The ability to detect specific binding in LILBID-MS was investigated by means of nucleic acids and their interaction with proteins. It could be shown that the stability of a 16bp dsDNA corresponds to that in solution phase regarding the dependency on concentration and type of the salts used. In addition a competitive experiment with the well-known transcription factor p50 was used to demonstrate the detection of sequence-specific binding with LILBID. The improved sensitivity allowed to detect single stranded DNA at nanomolar concentrations and even the 2686bp plasmid pUC19 could be easily detected without fragmentation using a concentration of only 80nM. In case of the transcription factor p63 the mass spectrometric analysis could help to identify a new model of activation and inhibition. For the first time known quarternary structures of membrane proteins like the light-driven proton pump bacteriorhodopsin and the potassium channel KcsA could be detected with mass spectrometry. For the light-driven proton pump proteorhodopsin the type and the concentration of the used detergents significantly influenced the stability of this protein as well as the preferred quarternary structure.
In my dissertation I study the transmission of monetary and fiscal policy in New Keynesian DSGE models. In the first chapter we revisit the exchange rate channel in a two-country model of the U.S. and a panel of industrialized countries to analyse how monetary policy transmission in the U.S. changes if it becomes more trade integrated. We find that more openness lowers the sacrifice ratio, although the effect is quantitatively small and depends on the pricing of the firms. In the second chapter we simulate the impact of the U.S. fiscal stimulus package in 2009 on GDP. We find that the government spendingmultiplier is well below 1. The finding is robust to including rule-of-thumb consumers and simulating the stimulus in the recent recession. In the third chapter we collect the fiscal stimulus measures in the eleven biggest countries of the euro area. Then we do a robustness study by simulating the european package in five different models of the euro area. The macroeconomic models vary in terms of backward-looking decision making of the agents and openness. Our findings provide no support for a Keynesian multiplier. Instead they suggest that additional government spending will reduce private spending for consumption and investment purposes. If government spending faces an implementation lag, the initial effect on GDP may even be negative. In the fourth chapter I estimate a DSGE model for Germany and compute forecasts for the debt-to-GDP ratio. I find that the expected economic recovery will lead to a decrease in Germany’s indebtedness in the medium-term given that policy makers stick to the fiscal policy rules.
Project I: The progression of rod and cone degeneration in retinally degenerate (rd) mice ultimately results in a complete loss of photoreceptors and blindness. The inner retinal neurons survive and several recent studies using genetically targeted, light activated channels have made these neurons intrinsically light sensitive. We crossbred a transgenic mouse line expressing channelrhodopsin2 (ChR2) under the control of the Thy1 promoter with the Pde6b(rd1) mouse, a model for retinal degeneration (rd1/rd1). Approximately 30-40% of the ganglion cells of the offspring expressed ChR2. Extracellular recordings from ChR2-expressing ganglion cells in degenerated retinas revealed their intrinsic light sensitivity which was approximately 7 log U less sensitive than the scotopic threshold and approximately 2 log U less sensitive than photopic responses of normal mice. All ChR2-expressing ganglion cells were excited at light ON. The visual performance of rd1/rd1 mice and ChR2 rd1/rd1 mice was compared. Behavioral tests showed that both mouse strains had a pupil light reflex and they were able to discriminate light fields from dark fields in the visual water task. Cortical activity maps were recorded with optical imaging. The ChR2rd1/rd1 mice did not show a better visual performance than rd1/rd1 mice. In both strains the residual vision was correlated with the density of cones surviving in the peripheral retina. The expression of ChR2 under the control of the Thy1 promoter in retinal ganglion cells does not rescue vision. Project II: Lentiviral vectors are becoming the vector of choice for transgene delivery into cells due to their ability to infect non- dividing cells and stably integrate the gene into the genome of the host. Two different viral vector systems, namely HIV-1 and SIV and three different viral vectors PLECYT, PHRCMVChR2 of HIV-1 family and PBjChR2 of SIV were used in this study. The efficiency of the vectors was analyzed by applying them onto the retinal explants in culture and checking the transgene expression. The transgene in the PLECYT lentiviral vector was driven by the EF1A promoter. Upon administration of 5.2 X 106 infectious units of PLECYT viral vector suspension onto the retinal explant resulted in the transduction of retinal ganglion cells. Very few other retinal neurons were found transduced. In the case of PHRCMVChR2, approximately 5 X 105 TU/ml of the vector was used and resulted in the transduction of different neuronal subtypes. Many amacrine cells, ganglion cells and Müller cells were found expressing the transgene. For PBjChR2, 5.6 X104 TU/ml was used which resulted in Müller cell- specific transduction. Very few or no other retinal neurons were found transduced. This study demonstrates the transduction efficiency of different viral vectors on the retinal neurons in vitro. An interesting observation on these viral vectors is their altered tropism. The glycoprotein of the virus is critical for determining their tropism and in this study, all the viral vectors generated were pseudotyped with VSVG, which confers a broad non-specific spectrum of infection. However, analyzing the transgene expression, the viral vectors differ from one another and show remarkable difference in their transduction pattern. To list a few factors that might possibly responsible for the drastic transduction difference exerted by the viral vectors include; 1. Promoters used to drive the transgene expression. 2. HIV or SIV component of the vector in combination with the promoter 3. Titre of the vector used and 4. Other factors like pH and serum used in the study. Therefore optimizing the viral vectors and generating high titers would increase the efficiency and cell-type specific expression of the transgene.
Suicide genes have been broadly used in gene therapy. They can serve as safety tools for conditional elimination of infused cells or for directed tumor therapy. To date, the Herpes simplex virus thymidine kinase/ ganciclovir (HSVtk/GCV) system is the most prominent and the most widely used suicidegene/prodrug combination. Despite its promising performance, the system displays limitations, which include relatively slow killing kinetics and toxicity of the prodrug GCV. Consequently, several groups have either developed new suicide-gene/prodrug combinations or attempted to improve the established HSVtk/GCV suicide system. The present study also aimed towards optimization of the HSVtk/GCV system. To do so, a novel, codon-optimized point mutant (A168H) of HSVtk was developed. The novel mutant was named TK.007. It was extensively tested for its efficiency in two relevant settings: (1) control of severe graft-versus-host disease (GvHD) after adoptive immunotherapy with Tlymphocytes, and (2) direct elimination of targeted tumor cells. TK.007 was compared to the broadly used wild-type, splice-corrected scHSVtk and to a codon-optimized HSVtk (coHSVtk) not bearing the above point mutation. (1) For experiments related to the adoptive immunotherapy approach, HSVtkvariants were expressed from a γ-retroviral MP71 vector as a fusion construct with the selection and marker gene tCD34. Expression levels for TK.007 in transduced lymphoid and myeloid cell lines were significantly higher at initial transduction and over a 12 week period compared to the commonly used scHSVtk and coHSVtk indicating reduced toxicity of TK.007. Killing kinetics of transduced cell lines (PM1 and K562) and primary human T cells were significantly faster for TK.007 in comparison to scHSVtk and coHSVtk in vitro. In vivo-functionality of TK.007 was assessed in an allogeneic transplantation model. T cells derived from C57BL/6J.Ly5.1 donor mice were transduced with MP71 vectors expressing scHSVtk or TK.007. Transduced cells were selected and transplanted into Balb/c Rag2-/- γ-/- immune-deficient recipient mice. Acute, severe GvHD occurred and was effectively abrogated in all mice transplanted with TK.007- transduced T cells, and in five out of six mice transplanted with scHSVtk-transduced cells. In a slightly modified quantitative allogeneic transplantation mouse model, significantly faster and more efficient in vivo killing was demonstrated for TK.007 as compared to scHSVtk, especially at low doses of GCV. (2) In order to assess TK.007 functionality in cells derived from solid tumors, HSVtk-variants were expressed from lentiviral gene ontology (LeGO) vectors in combination with an eGFP/neo-opt selection cassette. Transduced and selected tumor cell lines that derived from several tissues were eliminated at significantly lower GCV doses and to higher extents when transduced with TK.007 compared to scHSVtk. Moreover, a significantly stronger bystander effect of TK.007 was demonstrated. The superior in vitro efficiency of TK.007 was confirmed in an in vivo subcutaneous xenograft mouse model for glioblastoma in NOD/SCID mice. Mice transplanted with TK.007 transduced cells stayed tumor-free after treatment with different GCV-doses. On the contrary, mice of the scHSVtk group either demonstrated only transiently reduced tumor growth in the low-dose GCV group (10 mg/kg) compared to the control groups or suffered from relatively fast relapses after initial tumor shrinking in the standarddose (50 mg/kg) GCV group. As a result, all mice in the scHSVtk group died from vigorous tumor growth. In summary, in two different applications for suicide gene therapy the present study has demonstrated superior functional performance of the novel suicide gene TK.007 as compared to the broadly used wild-type scHSVtk. Differences became particularly pronounced at low doses of GCV. It can be concluded that the new TK.007-gene represents a promising alternative to the commonly used scHSVtk for gene therapeutic applications.
EGFL7 regulates adult neural stem cell maintenance and differentiation by inhibition of Notch1
(2010)
In neurobiology the preexisting dogma on the unchangeability of the adult mammalian brain and its inability to give rise to new neurons has been challenged since the early nineties. Generally, it is now accepted that neurogenesis persists in adults. Progress in developmental and stem cell biology in recent years led to an increasing interest in regeneration-based treatment strategies for damaged tissue of the central nervous system. Thus, the enhancement of the endogenous potential of the brain to repair itself is potentially a feasible therapeutic strategy to treat various types of brain damage. Therefore, it is of great interest to understand the molecular mechanism that regulate adult neurogenesis. One of the prominent pathways suggested to be involved here is the Notch signaling cascade. Previously, it has been shown that various components of Notch signaling are expressed in the stem cell niche of the adult subventricular zone (SVZ) in vivo. Interestingly, a recent study demonstrated that the self-renewal potential of adult neural stem cells (NSCs) isolated from the SVZ depend on Notch signaling in vitro.
Recently, we identified a novel non-canonical Notch ligand termed epidermal growth factor-like domain 7 (EGFL7), which was originally described as a protein secreted by endothelial cells and functionally implicated in cellular responses of the vascular system such as cell migration and blood vessel formation. We were able to show that secreted EGFL7 binds to a region in Notch that is involved in ligand-mediated receptor activation, thus acting as an antagonist of Notch signaling.
The present study identifies neurons of the human and murine brain as a novel source of EGFL7, which suggests functions of EGFL7 in the neural system. Expression analyses by quantitative RT-PCR (qRT-PCR) revealed EGFL7 is down regulated in the adult SVZ, which suggests that endogenous EGFL7 may act as a Notch modulator of NSCs. We assessed the expression of Notch pathway components in adult NSCs isolated from the SVZ of adult mice and demonstrated that inhibition of Notch activity by the γ-secretase inhibitor DAPT reduced the self-renewal potential of NSCs. Accordingly, adenoviral-mediated expression of EGFL7 in vitro decreased Notch-specific signaling and reduced proliferation and self-renewal of NSCs. Conversely, activation of Notch by a constitutive active form of Notch (NICD) rescued the EGFL7-mediated reduction of NSC self-renewal verifying that this effect was directly linked to Notch signaling. Congruent to the reduced proliferation rate measured in vitro, induced expression of EGFL7 in vivo significantly reduced the number of Ki-67 positive cells within the SVZ upon cerebroventricular injection of EGFL7 adenovirus.
Expression analyses in the developing brain showed single EGFL7-positive cells within the marginal zone of the neocortex as measured by in situ hybridization. These cells might be Cajal-Retzius cells, specialized neurons, which specifically express Reelin, which is a protein of the extracellular matrix known to control neuronal migration and differentiation. Interstingly, we could show that Reelin and EGFL7 are expressed in a subtype of neurons of the adult mouse cortex. This implied an interaction of both proteins and was verified by co-immunoprecipitation assays, suggesting an additional role for EGFL7 in neuronal maintenance. QRT-PCR based expression analyses in vitro comparing differentiated and non-differentiated NSCs displayed an increase in EGFL7 expression during the differentiation process, which was paralled by reduced Notch signaling. NSCs differentiated on coverslips coated with EGFL7 differentiated into all three cell types - neurons, oligodendrocytes and astrocytes. EGFL7 favored the formation of neurons as compared to control comparable to the effect of the Notch-inhibitor DAPT. Furthermore, additional oligodendrocytes were formed. These cells displayed a mature morphology with distinct sprouts and branches in contrast to the small and round oligodendrocytes that formed on control coverslips, which resembled us of precursor cells. Neurons and oligodendrocytes were formed at the expense of astrocytes. Congruently to the effect observed in vitro, adenoviral-based expression of EGFL7 in the SVZ yielded a slight induction of neuronal differentiation in vivo. Taken together, these results show for the first time a previously unrecognized role for EGFL7 in the brain by modulation of the Notch pathway in adult NSCs, which changes the proliferation and differentiation potential of adult NSCs in vitro and in vivo.
At present, there is a huge lag between the artificial and the biological information processing systems in terms of their capability to learn. This lag could be certainly reduced by gaining more insight into the higher functions of the brain like learning and memory. For instance, primate visual cortex is thought to provide the long-term memory for the visual objects acquired by experience. The visual cortex handles effortlessly arbitrary complex objects by decomposing them rapidly into constituent components of much lower complexity along hierarchically organized visual pathways. How this processing architecture self-organizes into a memory domain that employs such compositional object representation by learning from experience remains to a large extent a riddle. The study presented here approaches this question by proposing a functional model of a self-organizing hierarchical memory network. The model is based on hypothetical neuronal mechanisms involved in cortical processing and adaptation. The network architecture comprises two consecutive layers of distributed, recurrently interconnected modules. Each module is identified with a localized cortical cluster of fine-scale excitatory subnetworks. A single module performs competitive unsupervised learning on the incoming afferent signals to form a suitable representation of the locally accessible input space. The network employs an operating scheme where ongoing processing is made of discrete successive fragments termed decision cycles, presumably identifiable with the fast gamma rhythms observed in the cortex. The cycles are synchronized across the distributed modules that produce highly sparse activity within each cycle by instantiating a local winner-take-all-like operation. Equipped with adaptive mechanisms of bidirectional synaptic plasticity and homeostatic activity regulation, the network is exposed to natural face images of different persons. The images are presented incrementally one per cycle to the lower network layer as a set of Gabor filter responses extracted from local facial landmarks. The images are presented without any person identity labels. In the course of unsupervised learning, the network creates simultaneously vocabularies of reusable local face appearance elements, captures relations between the elements by linking associatively those parts that encode the same face identity, develops the higher-order identity symbols for the memorized compositions and projects this information back onto the vocabularies in generative manner. This learning corresponds to the simultaneous formation of bottom-up, lateral and top-down synaptic connectivity within and between the network layers. In the mature connectivity state, the network holds thus full compositional description of the experienced faces in form of sparse memory traces that reside in the feed-forward and recurrent connectivity. Due to the generative nature of the established representation, the network is able to recreate the full compositional description of a memorized face in terms of all its constituent parts given only its higher-order identity symbol or a subset of its parts. In the test phase, the network successfully proves its ability to recognize identity and gender of the persons from alternative face views not shown before. An intriguing feature of the emerging memory network is its ability to self-generate activity spontaneously in absence of the external stimuli. In this sleep-like off-line mode, the network shows a self-sustaining replay of the memory content formed during the previous learning. Remarkably, the recognition performance is tremendously boosted after this off-line memory reprocessing. The performance boost is articulated stronger on those face views that deviate more from the original view shown during the learning. This indicates that the off-line memory reprocessing during the sleep-like state specifically improves the generalization capability of the memory network. The positive effect turns out to be surprisingly independent of synapse-specific plasticity, relying completely on the synapse-unspecific, homeostatic activity regulation across the memory network. The developed network demonstrates thus functionality not shown by any previous neuronal modeling approach. It forms and maintains a memory domain for compositional, generative object representation in unsupervised manner through experience with natural visual images, using both on- ("wake") and off-line ("sleep") learning regimes. This functionality offers a promising departure point for further studies, aiming for deeper insight into the learning mechanisms employed by the brain and their consequent implementation in the artificial adaptive systems for solving complex tasks not tractable so far.
Statistical physics of power flows on networks with a high share of fluctuating renewable generation
(2010)
Renewable energy sources will play an important role in future generation of electrical energy. This is due to the fact that fossil fuel reserves are limited and because of the waste caused by conventional electricity generation. The most important sources of renewable energy, wind and solar irradiation, exhibit strong temporal fluctuations. This poses new problems for the security of supply. Further, the power flows become a stochastic character so that new methods are required to predict flows within an electrical grid. The main focus of this work is the description of power flows in a electrical transmission network with a high share of renewable generation of electrical energy. To define an appropriate model, it is important to understand the general set-up of a stable system with fluctuating generation. Therefore, generation time series of solar and wind power are compared to load time series for whole Europe and the required balancing or storage capacities analyzed. With these insights, a simple model is proposed to study the power flows. An approximation to the full power flow equations is used and evaluated with Monte-Carlo simulations. Further, approximations to the distributions of power flows along the links are analytically derived. Finally, the results are compared to the power flows calculated from the generation and load data.
In nature, society and technology many disordered systems exist, that show emergent behaviour, where the interactions of numerous microscopic agents result in macroscopic, systemic properties, that may not be present on the microscopic scale. Examples include phase transitions in magnetism and percolation, for example in porous unordered media, biological, and social systems. Also technological systems that are explicitly designed to function without central control instances, like their prime example the Internet, or virtual networks, like the World Wide Web, which is defined by the hyperlinks from one web page to another, exhibit emergent properties. The study of the common network characteristics found in previously seemingly unrelated fields of science and the urge to explain their emergence, form a scientific field in its own right, the science of complex networks. In this field, methodologies from physics, leading to simplification and generalization by abstraction, help to shift the focus from the implementation's details on the microscopic level to the macroscopic, coarse grained system level. By describing the macroscopic properties that emerge from microscopic interactions, statistical physics, in particular stochastic and computational methods, has proven to be a valuable tool in the investigation of such systems. The mathematical framework for the description of networks is graph theory, in hindsight founded by Euler in 1736 and an active area of research since then. In recent years, applied graph theory flourished through the advent of large scale data sets, made accessible by the use of computers. A paradigm for microscopic interactions among entities that locally optimize their behaviour to increase their own benefit is game theory, the mathematical framework of decision finding. With first applications in economics e.g. Neumann (1944), game theory is an approved field of mathematics. However, game theoretic behaviour is also found in natural systems, e.g. populations of the bacterium Escherichia coli, as described by Kerr (2002). In the present work, a combination of graph theory and game theory is used to model the interactions of selfish agents that form networks. Following brief introductions to graph theory and game theory, the present work approaches the interplay of local self-organizing rules with network properties and topology from three perspectives. To investigate the dynamics of topology reshaping, coupling of the so called iterated prisoners' dilemma (IPD) to the network structure is proposed and studied in Chapter 4. In dependence of a free parameter in the payoff matrix, the reorganization dynamics result in various emergent network structures. The resulting topologies exhibit an increase in performance, measured by a variance of closeness, of a factor 1.2 to 1.9, depending in the chosen free parameter. Presented in Chapter 5, the second approach puts the focus on a static network structure and studies the cooperativity of the system, measured by the fixation probability. Heterogeneous strategies to distribute incentives for cooperation among the players are proposed. These strategies allow to enhance the cooperative behaviour, while requiring fewer total investments. Putting the emphasis on communication networks in Chapters 6 and 7, the third approach investigates the use of routing metrics to increase the performance of data packet transport networks. Algorithms for the iterative determination of such metrics are demonstrated and investigated. The most successful of these algorithms, the hybrid metric, is able to increase the throughput capacity of a network by a factor of 7. During the investigation of the iterative weight assignments a simple, static weight assignment, the so called logKiKj metric, is found. In contrast to the algorithmic metrics, it results in vanishing computational costs, yet it is able to increase the performance by a factor of 5.
In the present work, the problem of protein folding is addressed from the point of view of equilibrium thermodynamics. The conformation of a globular protein in solution at common temperatures is quite complicated without any geometrical symmetry, but it is an ordered state in the sense of its biological activity. This complicated conformation of a single protein molecule is destroyed upon increasing the temperature or by the addition of appropriate chemical agents, as is revealed by the loss of its activity and change of the physical properties, and so on. Once the complicated native structures having biological activity are lost, it would be natural to suppose that the native structure could hardly be restored. Nevertheless, pioneers, such as Anson and Mirsky, recognized as early as in 1925 that this was not always the case. If one defines the folded and unfolded states of a protein as two distinct phases of a system, then under the variation of temperature the system is transformed from one phase state into another and vice versa. The process of protein folding is accompanied by the release or absorption of a certain amount of energy, corresponding to the first-oder-type phase transitions in the bulk. Knowing the partition function of the system one can evaluate its energy and heat capacity under different temperatures. This task was performed in this work. The results of the developed statistical mechanics model were compared with the results of molecular dynamic simulations of alanine poylpeptides. In particular, the dependencies on temperature of the total energy of the system and heat capacity were compared for alanine polypeptides consisting of 21, 30, 40, 50 and 100 amino acids. The good correspondence of the results of the theoretical model with the results of molecular dynamics simulations allowed to validate the assumptions made about the system and to establish the accuracy range of the theory. In order to perform the comparison of the results of theoretical model and the molecular dynamics simulations it is necessary to perform the efficient analysis of the results of molecular dynamics simulations. This task was also addressed in the present work. In particular, different ways to obtain dependence of the heat capacity on temperature from molecular dynamics simulations are discussed and the most efficient one is proposed. The present thesis reports the result of molecular dynamic simulations for not only alanine polypeptides by also for valine and leucine polypeptides. In valine and leucine polypeptides, it is also possible to observe the helix↔random coil transitions with the increase of temperature. The current thesis presents a work that starts with the investigation of the fundamental degrees of freedom in polypeptides that are responsible for the conformational transitions. Then this knowledge is applied for the statistical mechanics description of helix↔coil transitions in polypeptides. Finally, the theoretical formalism is generalized for the case of proteins in water environment and the comparison of the results of the statistical mechanics model with the experimental measurements of the heat capacity on temperature dependencies for two globular proteins is performed. The presented formalism is based on fundamental physical properties of the system and provides the possibility to describe the folding↔unfolding transitions quantitatively. The combination of these two facts is the major novelty of the presented approach in comparison to the existing ones. The “transparent” physical nature of the formalism provides a possibility to further apply it to a large variety of systems and processes. For instance, it can be used for investigation of the influence of the mutations in the proteins on their stability. This task is of primary importance for design of novel proteins and drug delivering molecules in medicine. It can provide further insights into the problem of protein aggregation and formation of amyloids. The problem of protein aggregation is closely associated with various illnesses such as Alzheimer and mad cow disease. With certain modifications, the presented theoretical method can be applied to the description of the protein crystallization process, which is important for the determination of the structure of proteins with X-Rays. There many other possible applications of the ideas described in the thesis. For instance, the similar formalism can be developed for the description of melting and unzipping of DNA, growth of nanotubes, formation of fullerenes, etc.
Nanocarbon structures, such as fullerenes and nanotubes, have generated considerable interest and research, due to their unique properties and potential applications. In this thesis, we present a study of the phase transition properties of nanocarbon clusters,in particular, we pay special consideration to fullerenes. The work presented in this thesis is largely theoretical and computational in nature, employing as a tool, molecular dynamics simulations to probe the dynamic stability of fullerenes and associated nanocarbon structures such as graphenes and nanotubes.
Purpose of the Study: The purpose of the current study was to evaluate the role of radiofrequency (RF) and microwave (MW) ablation in the treatment of pulmonary neoplasms. Materials and Methods: From March 2004 to January 2009, 164 patients (92 males, 72 females; mean age 59.7 years, SD: 10.2) underwent computed tomography (CT)-guided percutaneous RFA of pulmonary malignancies. RFA was performed on 248 lung lesions (20 primary lesions and 228 metastatic lesions) in 248 sessions (one lesion per session). Tumors were pathologically proven and were classified as primary lung neoplasms in 20 patients (non-small cell lung cancer) and as metastatic lung neoplasms in 144 patients. RFA was performed using: a) CelonProSurge bipolar internally cooled applicator b) RITA®StarburstTMXL. From December 2007 to October 2009, 80 patients (30 males, 50 females; mean age 59.7 years, range: 48-68, SD: 6.4) underwent computed tomography (CT) guided percutaneous MW ablation of pulmonary metastases from variable histopathological primaries. MW was performed on 130 lung lesions in 130 sessions (one lesion per session) using Valleylab TM system. Results: The overall success rate of RFA was 67.7% (168/248 lesions), with overall failure rate either due to tumor residue or recurrence on follow up in 32.3% (80/248) with mean time to tumor progress was 5.6 months SD: 2.99 (Range:1-18 months). Complete successful ablation was achieved in patients treated by MWA in 73.1% (95/130 lesions), with failure rate either due to tumor residue or recurrence on follow up in 26.9% (35/130) with mean time to tumor progress 6 months SD: 2.83 (Range:1-12months). Correlation of the histopathological type of the lesion and the end result of ablation therapy revealed insignificant correlation in both RFA and MWA (p > 0.1). The preablation tumor size was one of the most significant factors that determined the end result of ablation. In RFA successful tumor ablation was significant statistically for lesions with maximal axial diameter up to 2.5 cm (110/140) in comparison to lesions of more than 2.5 cm in maximal axial diameter (58/108) (Fisher’s exact test: p < 0.0001). While in MW ablated lesions successful tumor ablation was significant statistically for lesions with maximal axial diameter up to 3 cm (90/110) in comparison to lesions of more than 3 cm in maximal axial diameter (5/20) (Fisher’s exact test: p < 0.001). The location of the lesion was another important factor that determined the end result of ablation. In both RFA and MWA successful ablation was significantly more correlated to peripheral lesions (RFA: 120/160, 80% / MWA: 80/100, 80%) than centrally located lesions (RFA: 48/88, 50%; MWA: 15/30, 50%) (Fisher’s Exact Test: p > 0.001). For successfully RFA ablated cases mean preablation tumor volumes 1.9 cc SD: 0.9 (range: 0.3 - 4.25 cc) while for failed cases the mean tumor volume was 3.7 SD: 2.4 (range: 0.8 – 6.8cc). For successfully MW ablated cases the mean preablation tumor volume: 2.4 cc SD: 2.2 (range: 0.25-8.2 cc) while for failed cases the mean tumor volume was 3.5 SD: 2.6 (range: 0.3 – 7.1 cc). In RFA the survival rates at 12, 24 and 36 months were 90%, 78% and 68% respectively while in MWA treated patients the survival rate within 12 months follow up period was 96% while at 20 month the survival rate was 77%. Complications associated with the ablation therapy were: a) procedure related mortality: 0.4% (1/248) in RFA due to massive pulmonary hemorrhage versus 0% (0/130) in MWA, b) pneumothorax: 11.3% (28/240) in RFA versus 8.5% (11/130) in MWA, c) pulmonary Hemorrhage: 17.7% (44 of 248 sessions) of which one patient had massive uncontrolled bleeding and immediate death versus 6.2% (8/130) in MWA, d) pleural effusion: 3.2 % (8 of 248 sessions) in RFA versus 3.8 % (6/130) in MWA, e) hemoptysis: 4% (10/248) in RFA versus 4.6% (6/130) in MWA ranging from mild tinged sputum to frank bleeding, f) infection: 0.4% (1/248) in RFA, versus 0% in MWA, and g) post ablation pain: 10% (25/248) in RFA versus 9.2% (12/130) in MWA. Pain was generally adequately controlled by analgesics. Conclusion: Radiofrequency and microwave ablation are effective minimally invasive tools and may be safely applied for management of lung malignancy. The success of ablation therapy is significantly correlated to the preablation tumor size, volume and tumor location.
2.1. Background & purpose The recent introduction of new technical innovations such as CT perfusion (CTP) and dual energy CT (DECT) increases the diagnostic abilities of CT for imaging of the head and neck (H&N). The aim of this work was to evaluate the role of CTP and DECT in head and neck imaging. The first part tests whether CTP can differentiate between malignant H&N tumors and surrounding muscle, and discusses the impact of arterial input selection and tumor region of interest (ROI) on CTP of H&N cancer. The second part of the study evaluates radiation dose and image quality of DECT of the H&N. Finally the use of DE derived weighted averaging to improve lesion delineation and image quality is discussed. 2.2. Patients and methods CT perfusion Retrospective analysis of CTP was done for a total number of 55 cases of H&N tumors. Perfusion parameters were calculated for 33 cases of squamous cell carcinoma (SCC) and compared to those of muscles. CTP parameters of 50 cases of H&N tumors calculated using different arterial input functions were compared. CTP was calculated for 28 SCC cases using the single dynamic CT section that shows maximal tumor dimension compared to using average values obtained from all tumor-containing dynamic CT sections. Dual energy CT of head and neck This prospective part of the study was further divided into 2 parts. In the first part 32 consecutive patients underwent DECT of the H&N and were compared to a standard single energy CT (SE) control group. Radiation doses were compared. Weighted-average images from raw data of the 2 DE tubes (weighting factor 0.3 from 80 kVp and 0.7 from 140 KVp) were compared to SE images. Image noise was compared at 5 anatomic levels. Two blinded readers compared subjective overall image quality on a 5-point grading scale. In the second part 35 proved SCC cases underwent DECT of the neck. Pure 140 kVp and 80 kVp image datasets as well as weighted-average images from raw data of the 2 DE tubes at weighting factors 0.3, 0.6, 0.8 (30%, 60% and 80% from 80 kVp raw data respectively) were reconstructed. Objective image noise, contrast to noise ratio (CNR) and subjective image quality were compared between the 5 image datasets. Results CT perfusion Tumor perfusion parameters were significantly higher than those of muscle (p <0.05). Significant high correlation with no significant differences between the means (p >0.05) were observed between perfusion parameters obtained using internal carotid artery (ICA) versus external carotid artery (ECA) and ipsilateral versus contralateral ICA. High correlation was observed between perfusion parameters calculated using one section with maximal tumor dimension and the average of multiple sections. Differences between the means were non significant, p values>0.05. The 95% limits of agreement between repeated measurements using average of multiple sections were slightly narrower for blood volume and permeability than those of repeated measurements using one section. Dual energy CT of head and neck CTDIvol was 12% lower with DE than SECT (p<0.0001). There were no significant differences in objective noise between DECT and SECT at any of the anatomic levels (p >0.05). There were no significant differences between DE- and SECT in attenuation measurements, all p values >0.05. No significant differences in subjective image quality scores were observed between DE- and SECT at any of the 5 anatomic levels (p >0.05). At weighting factor 0.6 the lesion CNR was significantly higher than at weighting factor 0.3 and at pure 140 kVp image dataset (p< 0.0001); while non significantly lower than at weighting factor 0.8 and pure 80 kVp (p=1.00). The 0.6 weighting factor was rated the best at subjective image quality and lesion delineation. 2.4. Conclusion In conclusion; this study demonstrated the ability of CTP to differentiate SCC from surrounding muscle tissue. The choice of arterial input selection has no significant impact on quantitative CTP of H&N tumors. CTP of SCC calculated from one section with maximal tumor dimensions and the average values from multiple sections are not significantly different. The second part of the study showed that DE scanning can be routinely used for H&N imaging; preserving high diagnostic image quality even when the radiation dose was lowered by 12%. Average weighting of DE raw data, with a weighting factor 0.6, results in significant improvement in both tumor delineation and image quality.
Aim: To study the changes in leiomyoma volume following uterine artery embolization (UAE) and to correlate these changes with the initial leiomyoma volume and location within the uterus and to evaluate the impact of preprocedural prediction of the best tube angle obliquity for visualization of the uterine artery origin using 3D-reconstructed contrast-enhanced MR angiography (CE-MRA) on the radiation dose, fluoroscopy time and contrast medium volume used during UAE. Materials and Methods: The study was performed in two parts. The first part was retrospectively done on 28 patients (age range: 37-57 years, mean: 48 years, SD: 4.81) in whom UAE was performed. All leiomyomas in all patients were evaluated. In total, 84 leiomyomas were evaluated. MRI studies were performed before, 3 months and 1 year after UAE. The volumes and location of each leiomyoma in each patient were evaluated in consensus by two radiologists. The second part included 40 consecutive patients (age range: 37-56 years, mean: 46 years, SD: 4.49) and was done in a controlled prospective/retrospective manner. In 20 sample patients (prospective part) pre-procedural prediction of the best tube angle obliquity was predicted using 3D-reconstructed CE-MRA and provided to the interventionalist. 3D-reconstruction was done using Inspace application. The radiation dose, fluoroscopy time and contrast medium volume for those patients were compared with the data of the last 20 procedures (control) performed by the same interventionalist (retrospective part). Results: For the first part the mean pre-embolization volume was 51.6 cm3 range:0.72-371.1cm3, SD=79.3). At 3-month follow-up 83 (98.8%) leiomyomas showed a mean volume reduction of 52.62% (range: 12.79–96.67%, SD=21.85) and 1 leiomyoma (1.2%) increased in volume. At 1-year follow-up 5 (6%) leiomyomas were not detectable, 72 (85.7%) showed a further mean of 20.5% (range: 2.52–58.72%, SD=11.92) volume reduction compared to the 3-month follow-up volume and 7 (8.3%) leiomyomas increased in volume. A statistically significant (p=0.026 at 3-month, p=0.0046 at 1-year) difference in percentage of volume change was observed based on leiomyoma location; submucous leiomyomas showed the largest volume reduction. The initial leiomyoma volume showed a weak negative correlation (Spearman's correlation-coefficient =-0.35 at 3m and -0.36 at 1y) with the leiomyoma volume change. For the second part the tube angle prediction resulted in a significant reduction of the radiation dose utilized (p<0.001), fluoroscopy time (p=0.002) and contrast medium volume (p<0.001) for the sample patients when compared with the control patients. The overall radiation dose was reduced from a mean of 11044 μGym2 to a mean of 4172.5 μGym2, fluoroscopy time was reduced from a mean of 15.45 minutes to 8.81 minutes and contrast medium volume was reduced from a mean of 135 ml to 75 ml. Conclusion: UAE results in significant leiomyoma volume reduction at 3-month and 1- year follow-up. The leiomyoma location plays an important role in volume changes while the initial leiomyoma volume plays a minor role. Pre-procedural prediction of the best tube angle obliquity for visualization of the origin of the uterine artery using 3D-reconstructed CE-MRA results in a significant reduction of the radiation dose, fluoroscopy time and contrast medium volume used during UAE.
Philosophy is essentially dialectical. One gets into a dialectic by a puzzle, an aporia in thought and understanding. The point of philosophizing is not (necessarily) to get hold of the ultimate, objective, and immutably correct answers. The point is rather to come to be able to see one’s way out of the aporia, and to understand how one got into it in the first place.
The essay that follows – which I am submitting as my dissertation – is dialectical in two senses. As a piece of philosophizing, the essay is guided by a problem, the problem of understanding how laws of nature are possible, and how it is possible for us to know them. The movement of thought generated by attempts to get out of the problem then yields some ideas that do not stay in the original context in which the problem was felt to exist. Two important ones are, first, perceptual experience is not the only ultimate source of warrant we have for empirical knowledge claims, and second, perceptual experience is not the only epistemically significant experience we can have. Both are consequences of the idea that the mastery of skills is a form of interaction with nature that provides epistemic warrant for nomological claims. I shall leave it to the epilogue to examine how this view of skills contrasts with the ways skills are ordinarily thought of in philosophy and the implication of it for empiricism.
The other sense in which the essay is dialectical is more interesting, and it has to do with the way in which I approach the problem that got me into started, namely, by paying special attention to the dialectic exchange between the realists and the antirealists about the laws of nature. Antirealism about the lawfulness of nature has experienced something like a post-Humean revival since the publication of van Fraassen’s The Scientific Image. Most, including me, have strong realist intuitions about nomological “connections” in nature. Philosophical positions that are strongly counterintuitive have mostly not ended well in history. So it becomes something of a puzzle why antirealism about laws of nature manages to enjoy popularity every now and then.
ABCB9 is a peptide transporter belonging to the ATP-binding cassette (ABC) transporter subfamily B. Due to its high sequence identity to the transporter associated with antigen processing (TAP) the protein was named TAP-like (TAPL). The primary aim of this PhD thesis was the functional characterization of the TAPL transport complex. Despite the lack of TAPL function in the classical MHC class I pathway an involvement of TAPL in antigen presentation was still suggested. Apart from the crucial role of TAP for peptide delivery into the ER, TAP-independent translocation pathways in professional antigen presenting cells (pAPC) have been proposed, but not identified so far. Remarkably, TAPL mRNA and protein expression is strongly induced during differentiation of monocytes to immature and mature dendritic cells. This result was confirmed in the promonocytic cell line THP-1, which was used as a model system for monocyte to macrophage differentiation. By using quantitative immunofluorescence microscopy and subcellular fractionation, TAPL was detected in the lysosomal compartment co-localizing with the lysosome associated membrane protein 2 (LAMP-2) thus excluding the ER-localization formerly reported. Furthermore, by in vitro assays, a TAPL-specific and ATPdependent translocation of peptides into isolated lysosomes was demonstrated. Hence, TAPL is a candidate mediating peptide transport in alternative antigen presentation pathways in pAPCs. The presence of an extra N-terminal transmembrane domain (TMD0) lacking sequence homology to any known protein distinguishes TAPL from most other ABC transporters of its subfamily. By dissecting the TAPL translocation complex into its four putative transmembrane helices containing TMD0 and the core complex, distinct functions to the core complex and TMD0 were assigned. The core-TAPL complex composed of six predicted transmembrane helices and the nucleotide-binding domain (NBD) was expressed transiently in HeLa or stably in Raji cells. Crude membranes containing core-TAPL showed the same peptide transport activity as wt-TAPL demonstrating that the six core helices and the NBD are sufficient for peptide transport. This result also shows that the core transport complex is correctly targeted to and assembled in the membrane. Strikingly, in contrast to the wt transporter, the core complex localizes only partially to lysosomes and is mistargeted to the plasma membrane as observed by immunofluorescence microscopy and confirmed biochemically by cell surface biotinylation. Thus, a crucial role for TMD0 in proper subcellular targeting can be postulated. The vast majority of biological processes are mediated by protein complexes, hence characterization of such protein-protein-interactions is essential for understanding protein function on the cellular level. To identify interaction partners of TAPL, the transporter was isolated by tandem affinity purification. By tandem mass spectrometry the membrane proteins LAMP-1 and LAMP-2 were deciphered as specific proteins interacting with wt-TAPL. Notably, core-TAPL lacks these interactions indicating a role for TMD0 in recruiting other proteins. These results were verified for endogenous TAPL by co-immunoprecipitation. Using cells deficient in LAMP-1 and/or in LAMP-2 an escort function for the LAMP proteins was excluded. Very importantly, the physiological function of the LAMP-1and LAMP-2 interaction with TAPL is an increase in stability, since in their absence half-life of TAPL is drastically reduced.
Magnetic characteristics of metal organic low-dimensional quantum spin systems at low temperatures
(2010)
In dieser Arbeit wurden neue Klassen von niedrigdimensionalen metallisch-organischen Materialien untersucht, die es ermöglichen interessante quantenkritische Phänomene (quantum critical phenomena, QCP) wie die Bose-Einstein-Kondensation (Bose-Einstein condensation, BEC) der magnetischen Anregung in gekoppelten Spin-Dimer-Systemen, den Berezinskii-Kosterlitz-Thouless Übergang (Berezinskii-Kosterlitz-Thouless transition, BKT) und die Divergenz des magnetokalorischen Effekts (magnetocaloric effect, MCE) in Quanten-Spinsystemen beim Anlegen eines magnetischen Feldes zu beobachten. Die Niedrigdimensionalität der untersuchten Systeme war sowohl für die theoretische Beschreibung, als auch für die experimentelle Beobachtung der Phänomene von großer Bedeutung. Aus theoretischer Sicht eröffnet die Beschäftigung mit diesen Systemen die Möglichkeit, einfache Modelle zu entwickeln, die exakt lösbar sind und erlaubt somit ein qualitatives Verständnis der magnetischen Phänomene. Von experimenteller Seite ist es von größtem Interesse, dass durch das Zusammenspiel von Niedrigdimensionalität, konkurrierenden Wechselwirkungen und starker Quantenfluktuation exotische und aufregende magnetische Phänomene (quantenkritische Phänomene) entstehen, die mit verschiedenen experimentellen Methoden untersucht werden können. Um die intrinsischen Eigenschaften der quantenkritischen Phänomene zu verstehen ist es wichtig, die Phänomene an einfachen und gut kontrollierbaren niedrigdimensionalen Modellsystemen wie ein- oder zweidimensionalen Systemen zu untersuchen. ...
Paleoecology is the study of organismal interactions with the environment in the geological past. Organisms are influenced in their distribution and abundance by abiotic factors such as temperature and precipitation. A change in these factors, for example by major climatic shifts, would then affect the communities of organisms. Studying this hypothesized causal link between climatic and faunal change is especially interesting for the Plio-Pleistocene of East Africa due to the fact that our own ancestors also inhabited these regions. Both the Turkana basin in Kenya and the Lake Albert region in Uganda offer unique opportunities to investigate these paleoecological issues. Their late Miocene through Pleistocene deposits provide a very good record of climatic, vegetation and faunal change in East Africa (Pickford et al. 1993, Leakey et al. 1995, 1998, McDougall & Feibel 2003, Wynn 2004). This study focuses on the mammal family Bovidae as they are good indicator of vegetation and environment (e.g. Vrba 1980, 1995, Shipman & Harris 1988, Bobe & Eck 2001, Bobe & Behrensmeyer 2004, Bobe et al. 2007). Bovidae are quite species-rich and inhabit a wide range of habitats from tropical rain forests to deserts which predicates their array of morphological adaptations (ecovariables) to these environments. Diet is the ecovariable that is most to climate and thus habitat change. Therefore, the fossil Bovidae are especially suitable for reconstructing past environments. The objective of this thesis is to test the hypothesis that, from the late Miocene through the Holocene, Africa has experienced an overall increase in aridity and concomitant pulses of habitat change. The hypothesis predicts that increasing aridity causes a likewise growth in the abundance of taxa adapted to open arid environments. In particular, an increase in bovid grazers should be observed in combination with a decrease of bovid browsers. To test this hypothesis, I examine the fossil bovid communities from each stratigraphic member of Lake Turkana (Lothagam, Kanapoi, West Turkana and Koobi Fora) and Lake Albert (Nkondo-Kaiso region) and through a taxonomic and a functional perspective reconstruct the paleoenvironments and -climates from approximately 8 to 0.6 Ma. This study is the first to use taxonomic and ecomorphological data together to reconstruct the paleoenvironments of the Turkana basin and the Nkondo-Kaiso region of Lake Albert. In a first analysis, mesowear, as introduced by Fortelius & Solounias (2000), is used to gather information about the diet of bovids. As a result of my preliminary investigations on upper vs. lower molars of recent species, the sample of fossil bovid specimens from the Turkana basin and Lake Albert were found to be unsuitable to reveal a meaningful diet reconstruction. Therefore, the bovids are assigned to diet categories based on literature. For each member of the time period from 8.0 to 0.6 Ma, I provide a detailed characterization of the bovid fauna in terms of α- and β- diversity both on tribe and diet level based on presence-absence as well as for the Turkana basin on abundance data. Statistical comparisons between the fossil bovid communities and those in modern protected areas with known vegetation and climatic conditions have yielded modern analogues for each stratigraphic member. Following that I provide paleoclimatic conditions such as assumed mean annual temperature for each member. Based on abundance of diet categories in the bovid communities, the paleoclimate of the Turkana basin was in general cooler and considerably more humid during the late Miocene to the Pleistocene than today. The mean annual temperature at Lothagam is assumed as 22.2 °C, the annual precipitation as 685 mm for 8.0 – 6.54 Ma and 4.9 – 3.4 Ma. The intervening time period is characterized by a slightly lower mean annual temperature and precipitation (20.3 °C, 583 mm). From 4.17 to 4.07 Ma Kanapoi faced 21.3 °C and 592 mm rainfall. In the eastern part of the basin the climate was warmer and more humid (3.4 – 2.68 Ma: 26.2, 961 mm; 2.68 – 1.3 Ma: 27.1 °C, 935 mm) from 3.4 to 1.3 Ma than in the preceeding eras. In the western part, the climate became warmer and more humid ~500,000 years later and was more variable than that in the eastern basin. From 2.94 to 2.52 Ma the mean annual temperature was 26.2 °C and the annual precipitation 961 mm. Between 2.34 and 1.6 Ma the climate again cooled and became drier as before 2.94 Ma. A second shift to higher temperature and precipitation occurred after 1.6 Ma (27.1 °C, 935 mm) lasted until 1.34 Ma. The results of the bovid community analyses do not support the hypothesis of increasing aridity in Eastern Africa during the late Mio- to Pleistocene. Instead, the results show that the bovid communities differed much over time and on a relatively small spatial scale. Regional paleovegetation and paleoclimate exhibit fluctuations through the studied time period at western Turkana and differences between the western and eastern part of the Turkana basin. This is indicative of a patchy habitat distribution both on temporal and spatial levels. Increased climate variability predicts an increase in landscape complexity as proposed by the ‘variability selection hypothesis’ (Potts 1998a+b). Therefore, this thesis research supports the hypothesis of increased landscape complexity on the spatial level. This study has important implications for future research. First, an analysis based on ecovariable characteristics such as diet may be preferred to a taxonomic analysis. Second, abundance data should be used for an ecovariable analysis because the results then provide more precise information on the paleovegetation and –climate than just the presence of these adaptations in the faunal community. Lastly, as this study is based on one mammal family, further studies on other mammal groups should be conducted to increase the database of exploited resource by the entire faunal community. Most significantly this study provides a basis for new interpretations of faunal community distributions. It also raises the question whether small scale spatial community variability is also to be expected at other fossil sites. If so then this methodology has important implications for reconstructions of paleovegetation and paleoclimate.
In this thesis, we study the properties of excitations in the systems of interacting fermions. These excitations can be bosonic such as collective modes which we handle in the first part of this thesis or fermionic like quasi particles and quasi holes. One of the important points, to investigate the excitations is their damping which corresponds to their life-time in the system. This thesis consists of two parts, where in both parts, we use the field-theoretical methods to examine the problem.
The political, the urban, and the cosmopolitan : the 1970s generation in Romanian-German poetry
(2010)
This study is an introduction to the body of work produced by the German poets who were born during or after World War II in Romania and whose almost simultaneous debut lies in the relatively liberal period 1965 – 1971. Helped onto the Romanian-German literary scene by a propitious environment and informed by the socialist ideology they were born “into,” the poets born between 1942 and 1955 formed a remarkable generation unit which sought to significantly renew German-language literature in Romania. Rejecting identification with the insulary Romanian-German communities, the young poets strove to create a socially and politically relevant verse expressing an urban and cosmopolitan attitude. The growing nationalist rhetoric and isolationist stance of Romania's regime and the material and psychological hardships endured by its population through the 1970s and 80s forced the generation to revise its incipient enthusiasm for Romanian socialism. Increasingly, the poets' work came to depict the threatened existence of the German minority and the harsh general living conditions in Romania and to provide an alternative to the absurd official proclamations of a “golden age” under Ceauşescu, despite the poetry's growing reliance on obscuring literary techniques. The emigration of most of the generation members in the mid to late 1980s brought about the eventual unravelling of the generation unit and marks the end of my study. By following the evolution of three themes – social and political engagement, the German minority, and the urban environment – which define the poets as a generation throughout their literary careers in Romania, the analysis illuminates not only the generation's development from identification with Romanian socialism and rejection of the German minority to criticism of the country's policies and a renewed interest in the fate of the German community but also the changing possibilities and limits of literary expression under communism. In addition to providing an introduction to the body of work created by the 1970s generation in Romania, the study also expands the understanding of German literature in the 20th century by providing new material on literature written under totalitarianism and of intercultural German literature.
Planning problems, like real-world planning and scheduling problems, are complex tasks. As an efficient strategy for handing such problems is the ‘divide and conquer’ strategy has been identified. Each sub problem is then solved independently. Typically the sub problems are solved in a linear way. This approach enables the generation of sub-optimal plans for a number of real world problems. Today, this approach is widely accepted and has been established e.g. in the organizational structure of companies. But existing interdependencies between the sub problems are not sufficiently regarded, as each problem are solved sequentially and no feedback information is given. The field of coordination has been covered by a number of academic fields, like the distributed artificial intelligence, economics or game theory. An important result is, that there exist no method that leads to optimal results in any given coordination problem. Consequently, a suitable coordination mechanism has to be identified for each single coordination problem. Up to now, there exists no process for the selection of a coordination mechanism, neither in the engineering of distributed systems nor in agent oriented software engineering. Within the scope of this work the ECo process is presented, that address exactly this selection problem. The Eco process contains the following five steps. • Modeling of the coordination problem • Defining the coordination requirements • Selection / Design of the coordination mechanism • Implementation • Evaluation Each of these steps is detailed in the thesis. The modeling has to be done to enable a systemic analysis of the coordination problem. Coordination mechanisms have to respect the given situation and the context in which the coordination has to be done. The requirements imposed by the context of the coordination problem are formalized in the coordination requirements. The selection process is driven by these coordination requirements. Using the requirements as a distinction for the selection of a coordination mechanism is a central aspect of this thesis. Additionally these requirements can be used for documentation of design decisions. Therefore, it is reasonable to annotate the coordination mechanisms with the coordination requirements they fulfill and fail to ease the selection process, for a given situation. For that reason we present a new classification scheme for coordination methods within this thesis that classifies existing coordination methods according to a set of criteria that has been identified as important for the distinction between different coordination methods. The implementation phase of the ECo process is supported by the CoPS process and CoPS framework that has been developed within this thesis, as well. The CoPS process structures the design making that has to be done during the implementation phase. The CoPS framework provides a set of basic features software agents need for realizing the selected coordination method. Within the CoPS process techniques are presented for the design and implementation of conversations between agents that can be applied not only within the context of the coordination of planning systems, but for multiagent systems in general. The ECo-CoPS approach has been successfully validated in two case studies from the logistic domain.
Lattice simulation of a center symmetric three dimensional effective theory for SU(2) Yang-Mills
(2010)
We present lattice simulations of a center symmetric dimensionally reduced effective field theory for SU(2) Yang Mills which employ thermal Wilson lines and three-dimensional magnetic fields as fundamental degrees of freedom. The action is composed of a gauge invariant kinetic term, spatial gauge fields and a potential for the Wilson line which includes a "fuzzy" bag term to generate non-perturbative fluctuations between Z(2) degenerate ground states. The model is studied in the limit where the gauge fields are set to zero as well as the full model with gauge fields. We confirm that, at moderately weak coupling, the "fuzzy" bag term leads to eigenvalue repulsion in a finite region above the deconfining phase transition which shrinks in the extreme weak-coupling limit. A non-trivial Z(N) symmetric vacuum arises in the confined phase. The effective potential for the Polyakov loop in the theory with gauge fields is extracted from the simulations including all modes of the loop as well as for cooled configurations where the hard modes have been averaged out. The former is found to exhibit a non-analytic contribution while the latter can be described by a mean-field like ansatz with quadratic and quartic terms, plus a Vandermonde potential which depends upon the location within the phase diagram. Other results include the exact location of the phase boundary in the plane spanned by the coupling parameters, correlation lengths of several operators in the magnetic and electric sectors and the spatial string tension. We also present results from simulations of the full 4D Yang-Mills theory and attempt to make a qualitative comparison to the 3D effective theory.
Dessins d'enfants (children's drawings) may be defined as hypermaps, i.e. as bipartite graphs embedded in compact Riemann surfaces. They are very important objects in order to describe the surface of the embedding as an algebraic curve. Knowing the combinatorial properties of the dessin may, in fact, help us determining defining equations or the field of definition of the surface. This task is easier if the automorphism group of the dessin is "large". In this thesis we consider a special type of dessins, so-called Wada dessins, for which the underlying graph illustrates the incidence structure of points and of hyperplanes of projective spaces. We determine under which conditions they have a large orientation-preserving automorphism group. We show that applying algebraic operations called "mock" Wilson operations to the underlying graph we may obtain new dessins. We study the automorphism group of the new dessins and we show that the dessins we started with are coverings of the new ones.
European pea crabs - taxonomy, morphology, and host-ecology (Crustacea: Brachyura: Pinnotheridae)
(2010)
Pinnotherids are small crabs symbiotic to a variety of invertebrates. The European species infest bivalves and sea squirts. Their way of life is parasitic and poses a threat to commercially exploited bivalves. While juveniles of both sexes still look very similar - being agile swimmers and partially free living - a metamorphosis takes place in the female after mating and results in a conspicuous sexual dimorphism. Thereafter, the female settles in its host definitely and is morphologically strongly adapted to the parasitic life phase. A very high reproductive output was demonstrated among several pea crab species infesting bivalves. Despite from that, hardly any information is present in the literature on the pinnotherids’ reproductive biology and the underlying morphology.
Due to their cryptic way of life, the sexual dimorphism, and the different morphotypes of the female, the taxonomy of the Pinnotheridae is a serious challenge. Two widely accepted species are recognized on European coasts: Pinnotheres pisum and Nepinnotheres pinnotheres. Pinnotheres pectunculi was so far only known from the bivalve Glycymeris glycymeris in its type locality Roscoff (France), while Pinnotheres ascidicola and Pinnotheres marioni were described as living exclusively in ascidians without careful comparison with the previously described species. In order to produce standardized comparative descriptions, pea crabs were collected and studied from different hosts and localities in the Northeast Atlantic and in the Mediterranean. Nepinnotheres pinnotheres and Pinnotheres pisum were redescribed with consideration to characters of female and male. According to our morphological analysis, Pinnotheres ascidicola and Pinnotheres marioni are junior synonyms of Nepinnotheres pinnotheres, whereas the status of Pinnotheres pectunculi as a valid species was ascertained. Important characters are the mouthparts, the male gonopods, and especially chelipeds that showed consistent characteristics among different crab stages of both sexes.
Based on our sampling, we estimated the host-range of the European species. Nepinnotheres pinnotheres lives in ascidians and in the pen shell Pinna nobilis. Pinnotheres pisum infests numerous bivalve species - Pinna nobilis included. For Pinnotheres pectunculi novel host records are presented, all from the bivalve family Veneridae. Furthermore, feeding of the Pinnotheres-species was observed. They use a setae comb ventrally on the claw to brush mucus (and the accumulated food particles) from the bivalve gills. Feeding strategies and host-ecology will be thoroughly discussed in consideration to other Pinnotheridae.
We investigated the reproductive systems of European pinnotherids by histological methods, scanning and transmission electron microscopy, and confocal laser scanning microscopy.
The Eubrachyura have internal fertilization: paired vaginas enlarge into storage structures, the spermathecae, which are connected to the ovaries by oviducts. Sperm is stored until the oocytes are mature and transported into the spermathecae, where fertilization takes place. In the investigated pinnotherids, the vagina is of the ‘concave pattern’. Musculature is attached alongside flexible parts of the vagina-wall to control the dimension of its lumen. The genital opening is closed by a muscular mobile operculum.
The spermatheca can be divided into two distinct regions by function and morphology. The ventral part includes the connection with vagina and oviduct and is regarded as the zone where fertilization takes place. It is lined with cuticle except where the oviduct enters the spermatheca by the ‘holocrine transfer tissue’. At ovulation, the oocytes have to pass through this multi-layered glandular epithelium, which has a holocrine mode secretion. The dorsal part of the spermatheca is lined by a highly secretory apocrine glandular epithelium, which was to date only found in fiddler crabs of the genus Uca.
The male internal reproductive system consists of paired testes and corresponding vasa deferentia. The sperm morphology of pinnotherids conforms to other thoracotremes, with slight differences between Nepinnotheres pinnotheres and Pinnotheres pisum. Spermatozoa become enveloped into spermatophores in the secretory proximal vas deferens. The medial vas deferens is strongly enlarged and stores spermatophores embedded in seminal plasma. The distal vas deferens holds tubular appendices, which extend into the ventral cephalothorax and slightly into the pleon. These appendices produce and store vast quantities of seminal plasma. The copulatory system of the Brachyura is formed by paired penes and two pairs of gonopods, which function in sperm transfer. In pinnotherids, the long first gonopods transfers the sperm mass to the female. It holds the ejaculatory canal inside, which opens proximally and distally. The second gonopod is solid, short and conical. During copulation, the penis and the second gonopod are inserted into the base of the tubular first gonopod. The second gonopod functions in the transport of the sperm mass inside the ejaculatory canal towards its distal opening. The specific shape of the second gonopod is strongly adapted for a sealing of the tubular first gonopod with longitudinal cuticle foldings that interlock inside the first gonopod. The presented results are discussed concerning their function in reproduction and in respect of the systematic account.
The role of secretion in sperm transfer, storage and fertilization among the Brachyura is still under debate. It is notable that structure and function of secretion are more complex in pinnotherids and probably more efficient than in other brachyuran crabs, which will be discussed, in view of the parasitic way of life and the high fecundity of pinnotherids.
In the adult mammalian central nervous system, two defined neurogenic regions retain the capacity to generate new neurons throughout adulthood, namely the subependymal zone (SEZ) at the lateral ventricles and the subgranular layer of the hippocampus (SGL). Adult neurogenesis consists of a whole set of events including proliferation, fate specification, migration, survival and finally synaptic integration of newly born neurons. Each of these events is controlled by the interplay of numerous factors. In this study two signalling systems were analysed with regard to their functional role in adult neurogenesis in vivo, namely the purinergic system and the growth factor EGF. Neither short- nor long-term application of the P2Y receptor agonists UTP and ADPβS and the P2Y receptor antagonist suramin into the lateral ventricle of adult mice altered cell responses as compared to vehicle controls in vivo. In contrast, analysis of the expansion rates of cultured neural stem cells (NSCs) from knockout mice revealed a strong increase in the number of NSCs from NTPDase2-/- mice, whereas cell numbers of NSCs from P2Y1-/- and P2Y2-/- mice were significantly reduced in comparison to wildtype levels. Notably, in vivo proliferation rates were potently elevated in the SGL and the SEZ of NTPDase2-deficient mice. However, in vivo proliferation in both neurogenic niches of the single receptor knockout mice P2Y1-/- and P2Y2-/- and P2Y1-/- P2Y2-/-double-knockout mice did not differ significantly from the wildtype. In mice lacking the P2Y2 receptor the survival of newly born neurons in the hippocampal granule cell layer was significantly increased. These data provide the first line of evidence that purinergic signalling is involved in the control of neural stem cells behaviour not only in vitro but also in vivo. In order to further characterise the role of epidermal growth factor (EGF) in adult neurogenesis, transit amplifying precursors (TAPs) and type B astrocytes were identified as EGF-responsive cell populations following ventricular EGF injection, whereas ependymal cells, neuroblasts and NG2-positive cells did not or only to a minor extent respond to EGF injection. These EGF-responsive cell populations were found on both, the septal as well as striatal lateral ventricle walls. Long-term ventricular EGF infusion for 6d, 1. increased cell proliferation of both ventricle walls revealing a gradient along the rostro-caudal axis, 2. altered the balance between neuronal and macroglial cell fates to generate oligodendrocyte precursors and 3. lead to an entire remodelling of the classical architecture of the SEZ.
Fuer die schlechte Prognose von Glioblastompatienten mit einer ueberlebenszeit von 9-15 Monaten (Norden and Wen, 2006) ist vor allem die hohe Invasivitaet dieser Tumore verantwortlich. Nach operativer Entfernung des Haupttumors entstehen aus den verbleibenden invadierten Zellen sekundaere Tumore, die sich mitunter ueber weite Bereiche des Hirns verteilen. Des Weitern sind die hochinvasiven Tumorzellen oft resistent gegen Chemo- und Strahlentherapie (Drappatz et al., 2009; Lefranc et al., 2005). In Maustumormodellen und Pateinten konnte zudem gezeigt werden, dass die neuartige antiangiogenetische Therapie zwar das Tumorwachstum verringert, jedoch die Invasivitaet stark erhoeht. (Norden et al., 2008; Ebos et al., 2009; Paez-Ribes et al., 2009). Ueber die Mechanismen die diese hohen Invasivitaet induzieren, ist bislang nur sehr wenig bekannt. Die durch Reduktion von Blutgefaessen steigende Hypoxie des Tumors foerdert die Expression von Matrix-Metalloproteinasen (MMPs). Dies fuehrt zum Abbau der extrazelluaeren Matrix des umgebenden gesunden Gewebes und beguenstigt dadurch die Tumorzellinvasion (Indelicato et al., 2010; Miyazaki et al., 2008; Shyu et al., 2007). Die Umformung des Aktinzytoskeletts und damit die Mobilitaet von Zellen wird vorwiegend durch ein akkurates Zusammenspeil der Rho GTPasen Rac, Rho und Cdc42, kontrolliert (Ridley et al., 2003). Fuer die Organisation von Axonen im Nervensystem und fuer die Blut- und Lymphgefaessbildung wurde gezeigt, dass die Interaktion der Eph-Rezeptortyrosinkinasen und Ihrer Ephrin-Liganden Signalwege induziert, die in die Regulation dieses Zusammenspiels involviert sind (Egea and Klein, 2007; Makinen et al., 2005; Palmer et al., 2002; Sawamiphak et al., 2010). Des Weiteren zeigt die Analyse der Genloci von Eph-Rezeptoren und Ephrinen in verschieden Hirntumoren eine gehaeufte Deletionen des Ephrin-B2-Gens. Die Quantifizierung von Ephrin-B2 mRNA in diesen Tumoren hat ausserdem ergeben, dass mit zunehmender Malignitaet die Expression von Ephrin-B2 sinkt. Aus diesen Gruenden wurden die Untersuchungen in dieser Arbeit auf die Rolle von Ephrin-B2 anhaengigen Signalwegen in der Glioblastomzellinvasion konzentriert. In einem modifiziertem Boyden-Chamber-Assay konnte gezeigt werden, dass das Ephrin-B2 induzierte EphB4 forward signaling und EphB4 induzierte Ephrin-B2 reverse signaling die Invasivitaet der human Glioblastomzelllinien LN-229, G55 und SNB-19 reduziert. In einem Maustumormodel konnte weiterhin gezeigt werden, dass Ephrin-B2 Knock-Out (KO) Astrozytomzellen, im Vergleich zu Wild-Typ (WT) Zellen, Tumore mit einem groesseren Volumen und einer erhoehten Invasivitaet bilden. Da die Expressionslevel fuer die Ephrin-B2 bindenden Rezeptoren EphA4, EphB1 EphB3 und EphB6 auch im adulten Hirn hoch sind (Hafner et al., 2004), weisen diese in vitro und in vivo Ergebnisse auf eine Tumorsupressorfunktion von Ephrin-B2 hin, die durch repulsive Effekte des Ephrin-B2 reverse signaling vermittelte werden koennten. Dies geht mit Erkenntnissen ueber kolorektale Tumore einher (Batlle et al., 2005). Die in einem Sphaeroid-Invasionsassay mit einer EphB-Rezeptoren freien Umgebung beobachtete verminderte Invasion von Ephrin-B2 WT deutet auf eine zusaetzliche invasionsblockierende Rolle der Ephrin-B2-Eph-Rezeptor Interaktion zwischen benachbarten Tumorzellen hin, wie sie auch in Brusttumoren gefunden wurde (Noren et al., 2006). Es scheint als sei Tumorprogression und Invasion erst moeglich, nachdem die Expression von Ephrin-B2 vermindert wurde. Es konnte weiterhin gezeigt werden, dass in hypoxischen Glioblastomzellen die Ephrin-B2 Expression durch die direkte Bindung des den Transkriptionsfaktors ZEB2 an den Ephrin-B2 Promoter reprimiert wird. In einem Weiteren Maustumormodel konnte gezeigt werden, dass die Blockierung der ZEB2 Expression mittels shRNA und die damit einhergehenden Inhibition der hypoxie induzierten Ephrin-B2 Repression das Wachstum und die Invasivitaet von Glioblastomen verringert. Zusaetzlich wurde gezeigt, dass der Verlust von ZEB2 ausreicht, die durch antiangiogenetische Therapie induzierte stark erhoehte Invasivitaet zu vermeiden. Die in dieser Arbeit gewonnen Erkenntnisse fuehren zu folgendem Modelmechanismus. In kleinen normoxischen Tumoren koennen repulsive Effekte des Ephrin-B2 reverse signalings und EphB forward signalings zwischen Tumorzellen und Zellen des umgebenden Gewebes die Ausbreitung und Invasion des Tumors unterdruecken. Zusaetzlich koennte das Ephrin-B2 induzierte EphB forward signaling zwischen benachbarten Tumorzellen die Mobilitaet der Tumorzellen wie in Brusttumoren inhibieren. Beim Erreichen einer bestimmten Tumorgroesse tritt Hypoxie auf, wodurch HIF-1alpha stabilisiert wird. Dies fuehrt dann zur ZEB2 Expression und leitet die Repression von Ephrin-B2 ein, was wiederum zur erhoehten Tumorzellemobilitaet und im Zusammenspiel mit MMPs zu Invasion fuehren kann. Gleichzeitig werden durch den HIF-induzierten VEGF-Gradienten neue Blutgefaesse rekrutiert. Damit wird der hypoxie-induzierten Invasivitaet entgegengewirkt. Wird mittels antiangiogenetischer Behandlung versucht Tumorprogression entgegenzuwirken, resultiert daraus eine erneut gesteigerte Hypoxie, die dann durch die ZEB2 vermittelte Repression von Ephrin-B2 wieder eine erhoehte Invasivitaet induzieren kann. Das Blockieren der ZEB2 Expression kann dieser durch antiangiogenetischen Behandlung induzierten Invasivitaet entgegenwirken.
LmrA is a member of the ATP Binding Cassette (ABC) transporter family of membrane proteins and a structural and functional homologue of P-glycoprotein1, 2. ABC-transporters share a common architecture of two transmembrane domains and two nucleotide binding domains. The NBDs are highly conserved in this transporter family whereas the TMDs are highly diverse3. The TMDs recognize the substrate and the NBDs bind and hydrolyze ATP and thus contribute the energy for substrate translocation. ABC transporters as a protein family transport a high number of substrates including peptides, nutrients, ions, bile acids, lipids and other lipophilic compounds. LmrA is a multidrug transporter that recognizes a number of hydrophobic substrates including fluorescent dyes and antibiotics1, 4-6. LmrA is a native protein of the gram-positive bacterium Lactococcus lactis. In this thesis, L. lactis was used as a homologous expression host for the preparation of LmrA for a variety of experiments. Wildtype LmrA as well as a number of cysteine mutants were successfully expressed in L. lactis, purified and subsequently characterized by a variety of biochemical assays (Chapter 4). LmrA can be expressed to very high amounts in L. lactis. The purification and reconstitution were optimized for the requirements of solid-state NMR experiments in this thesis. For the first time, an ABC transporter has been reconstituted in synthetic lipids to a ratio of up to 1:150 (mol/mol). LmrA was shown to be active under magic angle spinning conditions with these reconstitution ratios. By taking advantage of the slower ATP hydrolysis by LmrA ΔK388 (lysine deletion in the Walker A motif), a real-time 31P solid-state NMR ATPase assay was established (Chapter 5). This assay allowed, for the first time, the investigation of all phosphor nuclei during the ATP hydrolysis cycle of a membrane protein simultaneously and in real time7. This assay has been successfully adapted to investigate both ATP hydrolysis and substrate phosphorylation of diacylglycerol kinase (together with S. Wollschlag) and ATP hydrolysis at high temperatures of the thermophilic ABC transporter ABC1 from Thermos thermophilus (together with A. Zutz). In the course of this thesis, the gene for LmrA has been cloned into expression vectors suitable for Escherichia coli and the heterologous expression of LmrA was established (Chapter 4). The functionality of the heterologously expressed protein has been investigated and compared to L. lactis LmrA. In these experiments, LmrA was shown to yield a distinct multidrug resistance phenotype in its E. coli host and to show secondary active multidrug transport in the absence of ATP and presence of a proton gradient [Hellmich et al, in prep] (Chapter 4). Previously, it had been shown that LmrA acts as a seconadary active transporter when the NBDs are truncated8. The overexpression in minimal and defined medium and the purification of LmrA from E. coli have been optimized. Isotope labeling for ssNMR has been established and the first multinuclear ssNMR experiments have been carried out on a functional ABC transporter (Chapter 8). ABC transporters couple two cycles: upon ATP binding, the NBDs dimerize, hydrolyze the ATP, subsequently release Pi and ADP and finally dissociate. During this cycle, conformational changes are relayed to the TMDs which utilize the energy from ATP binding and/or hydrolysis to translocate the respective substrate. The prehydrolysis state can be trapped by beryllium fluoride, whereas the post-hydrolysis state of this cycle can be trapped by vanadate9-12. Trapping protocols for these reagents were successfully established for LmrA in this thesis (Chapter 4). This allowed for the investigation of different catalytic states by both ssNMR and EPR. A general 19F labeling protocol for membrane proteins has been established in the course of this thesis and successfully applied to proteorhodopsin (together with N. Pfleger)13 and LmrA (chapter 6). Single cysteine mutants of LmrA that line out the dimer interface have been labeled with a fluorine label for ssNMR. In the apo state, the 19F labeling indicates highly flexible transmembrane domains, a finding that is supported by 13C ssNMR and EPR measurements. The addition of drugs has a different effect on different positions within the LmrA dimer, therefore indicating that different drugs are recognized at a different position within the protein. For P-glycoprotein and LmrA it has been previously shown by biochemical methods that different drug binding sites co-exist. For a 19F label attached at position 314 (LmrA E314C), the spectra showed two distinct peaks with similar populations. This could hint towards a structural asymmetry within the LmrA dimer that might also be reflected in the alternating ATP hydrolysis at the NBDs. E314 has been specifically implicated with drug transport. Thus, structural asymmetry at this position might be functionally relevant for guiding a substrate through the transporter. Structural asymmetry within a homodimeric ABC transporter has also been shown for BtuCD, the E. coli vitamin B12 importer14. In addition, the conserved glutamates in EmrE, a small multidrug resistance protein, were shown to be asymmetric in the drug bound state15. Both, uniformly 13C/15N labeled as well as selectively amino acid type labeled LmrA has been investigated in different conformational states. Interestingly, significant dynamic changes in the b-sheet regions of LmrA (confined to the NBDs) were observed in the pre-hydrolysis (beryllium fluoride) and transition state (vanadate trapped) state. These were interpreted as the transition from a domain in fast conformational exchange in the apo state to one of intermediate exchange in the nucleotide bound state. A significant change in NBD mobility upon nucleotide binding was previously also shown with 2H ssNMR on LmrA16. By EPR it was shown that LmrA in both the vanadate and BeFx trapped states displays a significantly higher rigidity and therefore defined distances, whereas the apo state resembled a “floppy” protein with no preferred distance distribution. This concurs with data obtained from 19F ssNMR with fluorine labeled single-cysteine mutants. Here, in agreement with the EPR data, a higher label (and possibly) protein mobility was observed in the apo state displaying rather broad line widths. Upon trapping with vanadate, the line widths of the majority of fluorine-labeled mutants decreased due to an enhanced protein rigidity and a more homogenous environment of the fluorine labels. A similar observation was made when increasing the temperature that can be explained due to higher protein flexibility at increased temperatures. Solution NMR was employed to investigate the isolated soluble NBD of LmrA (Chapter 9). First 2D and 3D spectra were successfully obtained and could be utilized for a preliminary assignment of a significant fraction of residues. Additionally, binding of ATP and ADP in absence and presence of magnesium was investigated. Finally, the effects of peptides emulating the coupling helices of the full-length transporter on the soluble NBD were investigated. Strikingly, binding of one of these peptides only occurred in the presence of nucleotides (whereas the other showed no binding at all) hinting towards a tightly coupled regulation of the NBD and TMD during the substrate translocation/ATP hydrolysis cycle based on nucleotide binding.
Vibronic (vibrational-electronic) transition is one of the fundamental processes in molecular physics. Indeed, vibronic transition is essential both in radiative and nonradiative photophysical or photochemical properties of molecules such as absorption, emission, Raman scattering, circular dichroism, electron transfer, internal conversion, etc. A detailed understanding of these transitions in varying systems, especially for (large) biomolecules, is thus of particular interest. Describing vibronic transitions in polyatomic systems with hundreds of atoms is, however, a difficult task due to the large number of coupled degrees of freedom. Even within the relatively crude harmonic approximation, such as for Born-Oppenheimer harmonic potential energy surfaces, the brute-force evaluation of Franck-Condon intensity profiles in a time-independent sum-over-states approach is prohibitive for complex systems owing to the vast number of multi-dimensional Franck-Condon integrals. The main goal of this thesis is to describe a variety of molecular vibronic transitions, with special focus on the development of approaches that are applicable to extended molecular systems. We use various representations of Fermi’s golden rule in frequency, time and phase spaces via coherent states to reduce the computational complexity. Although each representation has benefits and shortcomings in its evaluation, they complement each other. Peak assignment of a spectrum can be made directly after calculation in the frequency domain but this sum-over-states route is usually slow. In contrast, computation is considerably faster in the time domain with Fourier transformation but the peak assignment is not directly available. The representation in phase space does not immediately provide physically-meaningful quantities but it can link frequency and time domains. This has been applied to, herein, for example (non-Condon) absorption spectra of benzene and electron transfer of bacteriochlorophyll in the photosynthetic reaction center at finite temperature. This work is a significant step in the treatment of vibronic structure, allowing for the accurate and efficient treatment of complex systems, and provides a new analysis tool for molecular science.
The display of foreign polypeptides and proteins on the surface of viruses or cells provides an important tool for the engineering of biomolecules and the analysis of their interactions with binding partners. The most extensively used display platform is the coat protein of the filamentous bacteriophage (Smith, 1985). Phage display libraries have often been selected for polypeptides, e.g. single chain (sc) antibodies that bind to a protein of interest, but in vivo selection could only be demonstrated for peptides so far. An alternative display platform is the retrovirus murine leukemia virus (MLV). Here, polypeptides are displayed at the N-terminus of the viral envelope glycoprotein. Proof of principle for this platform was demonstrated for protease substrate libraries, which can be selected through coupling proteolytic activation with viral infectivity (Buchholz et al., 1998). Selection of the library CX4A on living cells resulted in viruses with more than three orders of magnitude improved spreading efficiency through tumor cells (Hartl et al., 2005). Also scAb libraries have recently been displayed and selected using retroviruses (Urban et al., 2005). The library scFvlibxMo displays the repertoire of phage display preselected sc antibodies for laminin-1 binding. The retrovirus based selection process resulted in laminin-specific sc antibodies with improved expression levels in mammalian cells.
This thesis describes the in vivo (i.e. in mouse tumor models) selection of the C-X4-A and scFvlibxMo for tumor homing upon systemic delivery.
For selection of the protease substrate library C-X4-A a subcutaneous tumor was induced in SCID mice followed by three systemic injections of the library. The selection process was monitored over a period of 34 days. After the incubation period mice were sacrificed and virus load in organs and tumor determined. PCR analysis after 34 days showed that virus from the library had preferentially infected the tumor. Sequence analysis showed the selection of protease substrates with the most prominent one with a frequency of over 65%. The four most prominent protease substrate variants where reconstituted into the original viral backbone for further investigation (C-SK-A, C-HI-A, C-HM-A and C-HS-A). Interestingly, these viruses exhibited a reduced spreading capacity in vitro on HT1080 cells as compared to the C-AK-A virus, which had previously been selected on HT1080 cells. When assayed for tumor homing, however, viruses C-HI-A and C-HS-A had clearly improved in comparison to C-AK-A. Tumor tissue had been infected at rates of over 55% while virus load of extratumoral organs was very low (infection rates <0.7 for C-HS-A and <0.02 for C-HI-A). Tumor targeting capacity had thus been improved over 10-fold by the in vivo selection of the C-X4-A library.
The experimental set up for the in vivo selection of the scFvlibxMo library was performed according to that of the C-X4-A library. Fingerprint analysis of the selected viruses that infected tumor tissue resulted in the identification of seven antibody variants showing unique CDR3 sequences. Two prominent clones (M49T-A and M49T-B) were cloned back into the MoMLV genome for further analysis of the reconstituted viruses. While variant B bound laminin-1 efficiently, variant A was unable to do so, although it was selected at highest frequency (76%). Both reconstituted viruses were equally well infectious and spread through HT1080rec1 cells at a similar efficiency as MoMLV. In an in vivo competition experiment the selected viruses clearly out-competed a laminin-1 binding reference virus L36xMo for tumor homing. To understand the molecular driving forces behind the in vivo selection process the epitope of the selected scFv M49T-A was identified using a phage peptide library approach. In silico analysis led to the identification of a small group of possible antigens, including tenascin, fibronectin and collagen.
The data described in this thesis demonstrate that the retrovirus display platform is capable of allowing the in vivo selection of protease substrates and scFvs. Notably, the replication competence of the system introduced an additional level of complexity to the library. The performed in vivo selections significantly enhanced tumor tropism. Selective infection of tumor cells combined with transfer of anti-tumoral genes is an attractive strategy for cancer therapy being in focus of current research. The viruses selected in this thesis build prime candidates for targeted retrovirus based tumor therapy.