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Institute
- Graduate School of Life Sciences (32)
- Theodor-Boveri-Institut für Biowissenschaften (32)
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Spreading drug resistances among Gram-negative pathogens and the paucity of new agents on the antibacterial drug market against these tenacious bacteria create a pressing need for the development of new antibiotics. The bacterial fatty acid biosynthesis pathway FAS-II, especially the enoyl-ACP reductase catalyzing the last step of the elongation cycle, is an established drug target against tuberculosis but has not been extensively exploited for drug design against other bacterial pathogens. In this thesis the enoyl-ACP reductases of the Gram-negative biothreat organisms Burkholderia pseudomallei and Yersinia pestis were targeted in a structure-based drug design approach. The structure of the most recently identified enoyl-ACP isoenzyme FabV was characterized by X-ray crystallography and could be determined in three different states. FabV from B. pseudomallei was obtained in the apo-form of the enzyme, whereas FabV from Y. pestis was characterized in a binary complex with the cofactor NADH as well as in a ternary complex with NADH and the triclosan-based 2-pyridone inhibitors PT172 and PT173. Analysis of the FabV structure revealed the typical fold of the short chain dehydrogenase/reductase superfamily with the NADH-binding Rossmann fold and a substrate-binding pocket with a conserved active site geometry compared to the related isoenzyme FabI. Additional structural elements of FabV are located around the active site. The monomeric form of the enzyme is thereby stabilized and the substrate-binding loop is kept in a closed, helical conformation. The ternary complexes of FabV exhibited a similar inhibitor-binding mode as observed for triclosan inhibition in FabI and point to a potential substrate-binding mechanism. B. pseudomallei possesses FabI as an additional enoyl-ACP reductase isoenzyme, which was structurally characterized in the apo form and in ternary complexes with NAD+ and the diphenyl ether inhibitors triclosan, PT02, PT12 or PT404 as well as the 4-pyridone inhibitor PT155. The structural data of the ternary enoyl-ACP reductases complexes of B. pseudomallei and Y. pestis hold the promise for the possibility to develop antibacterials targeting FabV or even both isoenzymes, FabI and FabV, based on the triclosan scaffold.
In summary, structure-activity relationships in peptide and dendrimer carriers modified with different organometal complexes were studied on a human breast cancer cell line. Variation of the organometal cargo and carrier can significantly influence their biological properties and might open the way to new approaches in chemotherapy. Furthermore, the incorporation of complexes with different C≡O vibrational signatures in a model peptide was explored to examine information encoding in biomolecules in a barcoding strategy for potential imaging applications. In particular for the latter, additional stable metal-carbonyl markers need to be prepared in future work to expand the pool of vibrational labels available.
Hey1, Hey2 and HeyL are downstream effectors of the Notch signalling pathway. Hey genes play decisive roles during embryonic development for example in cardiovascular development. However, the precise transcriptional programmes and genes, which are affected by each single Hey gene, are still poorly understood. One drawback for the analysis of Hey1, Hey2 or HeyL single gene function is that these genes are co-expressed in many tissues and share a high degree of functional redundancy. Thus, it was necessary to establish a system, which is either devoid of Hey expression, or just comprises one single Hey gene family member. For this, Hey1(fl/fl)/Hey2(-/-)/HeyL(-/-)- as well as Hey-triple- knock out (KO)-ES cells (embryonic stem cells) were generated in this work, because ES cells and their differentiation as EBs (embryoid bodies) represent a valuable tool for the in vitro analysis of embryonic developmental processes. After the establishment of Hey1(fl/fl)/Hey2(-/-)/HeyL(-/-)- and Hey-triple- KO-ES cells, it could be seen by ALP staining and pluripotency marker expression that loss of Hey expression did not affect ES cell pluripotency features. Thus, these ES cells represent bona fide ES cells and could be further used for the differentiation as EBs. Here, differences in gene expression between Hey1(fl/fl)/Hey2(-/-)/HeyL(-/-)- and Hey-triple- KO-ES cells (after the loss of Hey1) could be observed in realtime-RT-PCR analysis for the endodermal marker AFP as well as for neural and myogenic markers in d10 EBs. However, the establishment of inducible Hey1, Hey2 or HeyL ES cell lines will be essential to confirm these findings and to search for novel Hey target genes. To get further insight into the mode of Hey action, the analysis of Hey interaction partners is necessary. One such binding partner, the Bre protein, has previously been found in a yeast-two-hybrid screen. Bre has been described to be a member of two distinct complexes (i.e. the nuclear BRCA1-A complex with a function in DNA damage response and the cytoplasmic BRISC complex), to directly interact with the TNF-receptor and Fas and to interfere with apoptotic signalling. The Hey-Bre interaction could be further corroborated in this work; yet, it was not possible to narrow down the interaction site of Bre with Hey1. It rather seems that non-overlapping parts of the Bre protein may bind to Hey. This interaction may be direct– pointing to more than one interaction site inside the Bre protein – or via a common binding partner such as the endogenous Bre protein itself. Besides the interaction studies, functional assays were performed for a more detailed characterisation of Hey1 and Bre interaction. Here, it could be shown that Hey1 over-expression did not have any influence on Bre sub-cellular localisation. Interestingly, it could be demonstrated that Bre positively interfered with Hey1 repressive function in luciferase assays at three of four promoters analysed. Moreover, interaction with Bre seems to lead to a stabilisation of Hey1. As Bre has been described to modulate the E3-ligase activity intrinsic to the BRCC complex it was analysed whether Bre over-expression results in an ubiquitination of Hey1. Yet, this could not be observed in the present work. Furthermore, an interaction of Bre with ubiquitinated proteins could not be demonstrated in an ubiquitin binding assay. To obtain a better insight into Bre function, Bre LacZ gene trap-ES cells and animals were generated. However, realtime-RT-analyses revealed that these cells and mice did not show a loss of Bre expression on mRNA level indicating that insertion mutagenesis did not occur as expected. However, embryos derived from these mice could nevertheless be used for the detection of tissues with Bre expression by β-galactosidase staining. Bre deficiency on mRNA levels was only achieved after the deletion of the floxed exon 3 resulting in the generation of Bre del-mice. Bre del-mice were fertile and without any obvious phenotype and they were used for the generation of Bre del- and wt-MEFs (murine embryonic fibroblasts). Characterisation of these cells showed that proliferation was not affected after loss of Bre (neither under normal nor under stress conditions). However, loss of Bre notably resulted in a reduction in the BRCA1 DNA damage response, in a slightly increased sensitivity towards apoptosis induction by FasL treatment and in an increase in the K63-poly-ubiquitin content in Bre del-cytoplasmic fractions, probably linked to a change in the BRISC de-ubiquitinase activity. Even though these results have the same tendencies as observed in former studies, the effects in the present work are less striking. Further studies as well as intercrossing of Bre del- to Hey KO-animals will be necessary to further understand the functional relevance of Hey and Bre interaction.
Ants of the species Camponotus floridanus live in huge colonies composed of genetically identical or closely related animals, which should predispose them to an increased vulnerability towards infection by pathogens (Cremer et al. 2007). Therefore the question is how ants (or social insects in general) can nevertheless efficiently combat infections. In order to investigate the immune response of the ant C. floridanus, the present study initially focused on the identification of possible immune factors, encoded by the ant´s genome. By using the method “suppression subtractive hybridization” as well as by Illumnia sequencing technology, several immune-related genes could be identified. Among these were genes encoding proteins involved in pathogen recognition, signal transduction, antimicrobial activity, or general stress response. In accordance with the ant´s genome sequence (Bonasio et al. 2010), only three antimicrobial peptide (AMP) genes could be identified in C. floridanus. The gene and cDNA sequences of these AMPs were established and their expression was shown to be induced by microbial challenge. Two different defensin genes (type 1 and 2) were characterized. A detailed characterization of the mRNA and gene sequence of the other AMP, a hymenoptaecin, revealed a special repeat structure. The C. floridanus hymenoptaecin has a signal and a pro-sequence followed by a hymenoptaecin-like domain and six directly repeated hymenoptaecin domains (HDs). Since each HD is flanked by two known processing sites, proteolytic processing of the precursor protein may generate several mature AMPs. Bioinformatical analyses revealed the presence of hymenoptaecin genes with similar multipeptide precursor structure in genomes of other ant species suggesting an evolutionary conserved important role of this gene in ant immunity. C. floridanus ants harbor the obligate intracellular bacterium, Blochmannia floridanus, in specialized cells (so-called bacteriocytes), which are intercalated between midgut cells as well as in ovaries of females (Blochmann 1882; Sauer et al. 2002; Schröder et al. 1996). Ant hosts face the problem that on the one hand they have to maintain the beneficial symbiotic bacteria and on the other hand they need to raise an immune response against harmful pathogenic bacteria during an infection. It was investigated, if endosymbionts are actually detected by the host immune system. Injection of B. floridanus induced an immune response of its host C. floridanus, which was comparable to the one towards pathogens. This means that, despite the evolutionary established cooperation of the endosymbionts and their hosts, these bacteria are still recognized as „non-self“ by the host immune system. This finding led to the question, if the ant immune system might be involved in regulation of the endosymbiont number in the midgut tissue in order to avoid their uncontrolled replication. During the holometabolous life cycle of the ant hosts the distribution of bacteriocytes and of Blochmannia endosymbionts is remarkably dynamic and peaks in late pupal stages, in which the entire midgut is transformed into a symbiotic organ (Stoll et al. 2010). It was hypothesized that hosts could regulate the number of endosymbionts present in their tissues via the innate immune system. A quantitative gene expression analysis of assumed symbiosis-relevant candidate genes revealed distinct expression patterns of some genes according to developmental stage and tissue. Moreover, the immune gene expression in response to bacterial challenge was investigated in the pupal stage. By an artificial immune-challenge of pupae it was confirmed that in fact the immune response of the endosymbiont-bearing midgut tissue differs from that of other body parts. The data support a key role for amidase peptidoglycan recognition proteins (PGRPs), especially PGRP-LB, in endosymbiont tolerance and suggest an involvement of the lysosomal system in control of Blochmannia endosymbionts. In sum, this thesis provides a first description of the immune response of the ant C. floridanus. A comprehensive set of immune-relevant genes was determined. Especially, the identification and molecular characterization of the hymenoptaecin gene delivered new insights into the immune competence of ants in general. Moreover, first indications could be gathered for the involvement of the immune system in controlling the endosymbiont B. floridanus.
Recent development of proteomic approaches and generation of large-scale proteomic datasets calls for new methods for biological interpretation of the obtained results. Systems biological approaches such as integrated network analysis and functional module search have become an essential part of proteomic investigation. Proteomics is especially applied in anucleate cells such as platelets. The underlying molecular mechanisms of platelet activation and their pharmacological modulation are of immense importance for clinical research. Advances in platelet proteomics have provided a large amount of proteomic data, which has not yet been comprehensively investigated in a systems biological perspective. To this end, I assembled platelet specific data from proteomic and transcriptomic studies by detailed manual curation and worked on the generation of a comprehensive human platelet repository for systems biological analysis of platelets in the functional context of integrated networks (PlateletWeb) (http:/PlateletWeb.bioapps.biozentrum.uni-wuerzburg.de). I also added platelet-specific experimentally validated phosphorylation data and generated kinase predictions for 80% of the newly identified platelet phosphosites. The combination of drug, disease and pathway information with phosphorylation and interaction data makes this database the first integrative platelet platform available for platelet research. PlateletWeb contains more than 5000 platelet proteins, which can also be analyzed and visualized in a network context, allowing identification of all major signaling modules involved in platelet activation and inhibition. Using the wealth of integrated data I performed a series of platelet-specific analyses regarding the platelet proteome, pathways, drug targets and novel platelet phosphorylation events involved in crucial signaling events. I analyzed the statistical enrichment of known pathways for platelet proteins and identified endocytosis as a highly represented pathway in platelets. Further results revealed that highly connected platelet proteins are more often targeted by drugs. Using integrated network analysis offered by PlateletWeb, I analyzed the crucial activation signaling pathway of adenosine diphosphate (ADP), visualizing how the signal flow from receptors to effectors is maintained. My work on integrin inside-out signaling was also based on the integrated network approach and examined new platelet-specific phosphorylation sites and their regulation using kinase predictions. I generated hypothesis on integrin signaling, by investigating the regulation of Ser269 phosphorylation site on the docking protein 1 (DOK1). This phosphorylation site may influence the inhibiting effect of DOK1 on integrin a2bb3. Extending the integrated network approach to further cell lines, I used the assembled human interactome information for the analysis of functional modules in cellular networks. The investigation was performed with a previously developed module detection algorithm, which finds maximum-scoring subgraphs in transcriptomic datasets by using assigned values to the network nodes. We extended the algorithm to qualitative proteomic datasets and enhanced the module search by adding functional information to the network edges to concentrate the solution onto modules with high functional similarity. I performed a series of analyses to validate its performance in small-sized (virus-infected gastric cells) and medium-sized networks (human lymphocytes). In both cases the algorithm extracted characteristic modules of sample proteins with high functional similarity. The functional module search is especially useful in site-specific phosphoproteomic datasets, where kinase regulation of the detected sites is often sparse or lacking. Therefore, I used the module detection algorithm in quantitative phosphoproteomic datasets. In a platelet phosphorylation dataset, I presented a pipeline for network analysis of detected phosphorylation sites. In a second approach, the functional module detecting algorithm was used on a phosphoproteome network of human embryonic stem cells, in which nodes represented the maximally changing phosphorylation sites in the experiment. Additional kinases from the human phosphoproteome in PlateletWeb were included to the network to investigate the regulation of the signal flow. Results indicated important phosphorylation sites and their upstream kinases and explained changes observed in embryonic stem cells during differentiation. This work presents novel approaches for integrated network analysis in cells and introduces for the first time a systematic biological investigation of the human platelet proteome based on the platelet-specific knowledge base PlateletWeb. The extended methods for optimized functional module detection offer an invaluable tool for exploring proteomic datasets and covering gaps in complex large-scale data analysis. By combining exact module detection approaches with functional information data between interacting proteins, characteristic functional modules with high functional resemblance can be extracted from complex datasets, thereby focusing on important changes in the observed networks.
The mechanisms that enable cells to regulate their gene expression and thus their metabolism, proliferation or cellular behaviour are not only important to understand the basic biology of a living cell, but are also of crucial interest in cancerogenesis. Highly interwoven and tightly regulated pathways are the basis of a robust but also flexible regulatory network. Interference with these pathways can be either causative for tumorigenesis or can modify its outcome. The receptor tyrosine kinase (RTK) and RAS dependent pathways leading to AKT or ERK1/2 activation are of particular interest in melanoma. These signaling modules are commonly activated by different mutations that can be found in various pathway components like NRAS, BRAF or PTEN. The first part of this work deals with the diverse and versatile functions of the ERK1/2 pathway feedbackregulator MKP2 in different cellular, melanoma relevant settings. In addition, a functional role of the AP1-complex member FOSL1, an ERK1/2 transcriptional target being implicated in the regulation of proliferation, is demonstrated. Secondly, aspects of direct pharmacological inhibition of the ERK1/2 pathway with regard to the induction of apoptosis have been analysed. Due to the high frequency of melanoma related mutations occurring in the RAS/RAF/MEK/ERK pathway (e.g. NRASQ61K, BRAFV600E), inhibition of this signaling cascade is deemed to be a promising therapeutic strategy for the treatment of malignant melanoma. However, although in clinical trials mono-therapeutic treatment with MEK- or RAF inhibitors was successful in the short run, it failed to show satisfactory long-lasting effects. Hence, combination therapies using a MAPK pathway inhibitor and an additional therapy are currently under investigation. I was able to demonstrate that inhibition of MEK using the highly specific inhibitor PD184352 can have a protective effect on melanoma cells with regard to their susceptibility towards the apoptosis inducing agent cisplatin. Single application of cisplatin led to strong DNA damage and the induction of caspase-dependent apoptosis. Additional administration of the MEK inhibitor, however, strongly reduced the apoptosis inducing effect of cisplatin in several melanoma cell lines, These cells displayed an increased activation of the serine/threonine kinase AKT after MEK inhibition. This AKT activation concomitantly led to the phosphorylation of FOXO transcription factors, attenuating the cisplatin induced expression of the BH3-only protein PUMA. PUMA in turn was important to mediate the apoptosis machinery after cisplatin treatment. My results also indicate a participation of RTKs, in particular EGFR, in mediating MEK inhibitor induced activation of AKT. These results demonstrate that inhibition of the RAS/RAF/MEK/ERK signaling pathway in melanoma cell lines does not necessilary have favourable effects in a cytotoxic co-treatment situation. Instead, it can even enhance melanoma survival under pro-apoptotic conditions.
Systems biology looks for emergent system effects from large scale assemblies of molecules and data, for instance in the human platelets. However, the computational efforts in all steps before such insights are possible can hardly be under estimated. In practice this involves numerous programming tasks, the establishment of new database systems but as well their maintenance, curation and data validation. Furthermore, network insights are only possible if strong algorithms decipher the interactions, decoding the hidden system effects. This thesis and my work are all about these challenges. To answer this requirement, an integrated platelet network, PlateletWeb, was assembled from different sources and further analyzed for signaling in a systems biological manner including multilevel data integration and visualization. PlateletWeb is an integrated network database and was established by combining the data from recent platelet proteome and transcriptome (SAGE) studies. The information on protein-protein interactions and kinase-substrate relationships extracted from bioinformatical databases as well as published literature were added to this resource. Moreover, the mass spectrometry-based platelet phosphoproteome was combined with site-specific phosphorylation/ dephosphorylation information and then enhanced with data from Phosphosite and complemented by bioinformatical sequence analysis for site-specific kinase predictions. The number of catalogued platelet proteins was increased by over 80% as compared to the previous version. The integration of annotations on kinases, protein domains, transmembrane regions, Gene Ontology, disease associations and drug targets provides ample functional tools for platelet signaling analysis. The PlateletWeb resource provides a novel systems biological workbench for the analysis of platelet signaling in the functional context of protein networks. By comprehensive exploration, over 15000 phosphorylation sites were found, out of which 2500 have the corresponding kinase associations. The network motifs were also investigated in this anucleate cell and characterize signaling modules based on integrated information on phosphorylation and protein-protein interactions. Furthermore, many algorithmic approaches have been introduced, including an exact approach (heinz) based on integer linear programming. At the same time, the concept of semantic similarities between two genes using Gene Ontology (GO) annotations has become an important basis for many analytical approaches in bioinformatics. Assuming that a higher number of semantically similar gene functional annotations reflect biologically more relevant interactions, an edge score was devised for functional network analysis. Bringing these two approaches together, the edge score, based on the GO similarity, and the node score, based on the expression of the proteins in the analyzed cell type (e.g. data from proteomic studies), the functional module as a maximum-scoring sub network in large protein-protein interaction networks was identified. This method was applied to various proteome datasets (different types of blood cells, embryonic stem cells) to identify protein modules that functionally characterize the respective cell type. This scalable method allows a smooth integration of data from various sources and retrieves biologically relevant signaling modules.
Alveolar echinococcosis (AE) is a severe and life-threatening disease caused by the metacestode larva of the fox-tapeworm Echinococcus multilocularis. Parasite entry into the host evokes an early and potentially parasiticidal Th1 immune response that is gradually replaced by a permissive Th2 response. An immunoregulatory environment has also been reported in the host as the disease progresses. As a result of immunomodulation, E. multilocularis larvae persist in the host for decades without being expelled, and thus almost act like a perfect transplant. Very little is currently known on the molecular basis of the host immunomodulation by E. multilocularis. In this work, in vitro cultivation systems were used to assess the influence of metabolites released by the parasite larvae (E/S products) on host immune effector cells. E/S products of cultivated larvae that respresent the early (primary cells) and chronic (metacestode vesicles) phase of AE induced apoptosis and tolerogenic properties (poor responsiveness to LPS stimulation) in host dendritic cells (DC) whereas those of control larvae (protoscoleces) failed to do so. These findings show that the early infective stage of E. multilocularis induces tolerogenicity in host DC, which is most probably important for generating an immunosuppressive environment at an infection phase in which the parasite is highly vulnerable to host attacks. Interestingly, metacestode E/S products promoted the conversion of naïve CD4+ T-cells into Foxp3+ regulatory T-cells in vitro, whereas primary cell and protoscolex E/S products failed to do it. Since Foxp3+ regulatory T-cells are generally known to mediate immunosuppression, the present finding indicates that Foxp3+ regulatory T-cells, expanded by E/S products of the metacestode larva, could play a role in the parasite-driven immunomodulation of the host observed during AE. Furthermore, a substantial increase in number and frequency of suppressive Foxp3+ regulatory T-cells could be observed within peritoneal exudates of mice following intraperitoneal injection of E. multilocularis metacestodes, indicating that Foxp3+ regulatory T-cells could also play an important role in E. multilocularis-driven immunomodulation in vivo. Interestingly, a parasite activin ortholog, EmACT, secreted by metacestodes, was shown to expand host regulatory T-cells in a TGF-β-dependent manner, similarly to mammalian activin A. This observation indicated that E. multilocularis utilizes evolutionarily conserved TGF-β superfamily ligands, like EmACT, to expand host regulatory T-cells. Taken together, the present findings suggest EmACT, a parasite activin secreted by the metacestode and capable of expanding host regulatory T-cells, as an important player in the host immunomodulation by E. multilocularis larvae. Another parasite factor EmTIP, homologous to mammalian T-cell immunomodulatory protein (TIP) was characterized in this work. EmTIP could be detected in the secretions of the parasite primary cells and localized to the intercellular space within the parasite larvae. EmTIP blockade inhibited the proliferation of E. multilocularis primary cells and the formation of metacestode vesicles indicating a major role for parasite development. Furthermore, EmTIP evoked a strong release of IFN-γ by CD4+ T-cells hence suggesting that the secretion of this factor as a result of its role in parasite development could “secondarily” induce a potentially protective Th1 response. In conclusion, this work identified two molecules, EmACT and EmTIP, with high immunomodulatory potential that are released by E. multilocularis larvae. The data presented do provide insights into the mechanisms of parasite-driven host immunomodulation during AE that are highly relevant for the development of anti-parasitic immune therapies.
Dementia, or any form of degenerative cognitive decline, is one of the major problems in present, and even more will be in future medicine. With Alzheimer's disease (AD) being the most prevalent, Vascular Dementia is the second most entity of dementing processes in the elderly. As diagnostic criteria are still imprecise and in many cases do not embrace early stages of the disease, recent studies have proposed more detailed classifications of the newly created condition Vascular Cognitive Impairment (VCI). Of all conditions subsumed under this term, subcortical small-vessel alterations are the most common cause for cognitive decline. The diagnosis of dementia / cognitive impairment is presently often made in late stages of the disease, when therapeutical options are poor. Thus, early detection of changes of the subcortical small vessels is desirable, when there is still time to identify and aggressively treat risk factors and underlying conditions like diabetes, hyper- or hypotension, and hyperlipidemia. This study aimed to evaluate whether cTT correlates to cognitive dysfunction, i.e. if cTT is fit as an early diagnostic tool for VCI. The study cohort included 38 patients from the Neurological Clinic of the Würzburg University hospital admitted due to diagnoses other than dementia or stroke. As a result of this study it turned out that cTT is certainly capable of fulfilling the task to easily and effectively detect and evaluate possible microvascular lesions of the brain with respect to the actual clinical relevance for the patient. When compared to the other proposed diagnostic tools, neuropsychological testing and MRI, the advantages of cTT are obvious: its measurement is a low-cost and quick procedure which would spare both patients and examiners a long neuropsychological exam or complement it. cTT is safe to assess as the only possible risks derive from the use of the contrast agent, which are rare and easily manageable. It has also proven to be more accurate in showing the extent of cognitive impairment than MRI. Finally, it is widely available. The only prerequisite is an ultrasound machine capable of transcranial color-coded duplex sonography. No cost-intensive procedures like MRI are needed. So, with neuropsychological testing remaining the gold standard, cTT here proved to be a reliable alternative which is more time- and cost-effective than MRI.
Parasitic helminths share a large degree of common genetic heritage with their various hosts. This includes cell-cell-communication mechanisms mediated by small peptide cytokines and lipophilic/steroid hormones. These cytokines are candidate molecules for host-parasite cross-communication in helminth diseases. In this work the function of two evolutionary conserved signaling pathways in the model cestode Echinococcus multilocularis has been studied. First, signaling mechanisms mediated through fibroblast growth factors (FGF) and their cognate receptors (FGFR) which influence a multitude of biological functions, like homeostasis and differentiation, were studied. I herein investigated the role of EmFR which is the only FGFR homolog in E. multilocularis. Functional analyses using the Xenopus oocyte expression system clearly indicate that EmFR can sense both acidic and basic FGF of human origin, resulting in an activation of the EmFR tyrosine kinase domain. In vitro experiments demonstrate that mammalian FGF significantly stimulates proliferation and development of E. multilocularis metacestode vesicles and primary cells. Furthermore, DNA synthesis and the parasite’s Erk-like MAPK cascade module was stimulated in the presence of exogenously added mammalian FGF. By using the FGFR inhibitor BIBF1120 the activity of EmFR in the Xenopus oocyte system was effectively blocked. Addition of BIBF1120 to in vitro cultivated Echinococcus larval material led to detrimental effects concerning the generation of metacestode vesicles from parasite stem cells, the proliferation and survival of metacestode vesicles, and the dedifferentiation of protoscoleces towards the metacestode. In conclusion, these data demonstrate the presence of a functional EmFR-mediated signaling pathway in E. multilocularis that is able to interact with host-derived cytokines and that plays an important role in larval parasite development. Secondly, the role of nuclear hormone receptor (NHR) signaling was addressed. Lipophilic and steroid hormone signaling contributes to the regulation of metazoan development. By means of in silico analyses I demonstrate that E. multilocularis expresses a set of 17 NHRs that broadly overlaps with that of the related flatworms Schistosoma mansoni and S. japonicum, but also contains several NHR encoding genes that are unique to this parasite. One of these, EmNHR1, is homolog to the DAF-12/HR-96 subfamily of NHRs which regulate cholesterol homeostasis in metazoans. Modified yeast-two hybrid analyses revealed that host serum contains a ligand which induces homodimerization of the EmNHR1 ligand-binding domain. Also, a HNF4-like homolog, EmHNF4, was characterized. Human HNF4 plays an important role in liver development. RT-PCR experiments showed that both isoforms of the EmHNF4 encoding gene are expressed stage-dependently suggesting distinct functions of the two isoforms in the parasite. Moreover, specific regulatory mechanisms on the convergence of NHR signaling and TGF-β/BMP signaling pathways in E. multilocularis have been identified. On the one hand, EmNHR1 directly interacted with the EmSmadC and on the other hand EmHNF4b interacted with EmSmadD, EmSmadE which are all downstream signaling components of the TGF-β/BMP signaling pathway. This suggests cross-communication in order to regulate target gene expression. With these results, further studies on the role of NHR signaling in the cestode will be facilitated. Also, the first serum-free in vitro cultivation system for E. multilocularis was established using PanserinTM401 as medium. Serum-free co-cultivation with RH-feeder cells and an axenic cultivation method have been established. With the help of this serum-free cultivation system investigations on the role of specific peptide hormones, like FGFs, or lipophilic/steroid hormones, like cholesterol, for the development of helminths will be much easier.
Non-small cell lung cancer (NSCLC) is the deadliest form of lung cancer and has a poor prognosis due to its high rate of metastasis. Notably, metastasis is one of the leading causes of death among cancer patients. Despite the clinical importance, the cellular and molecular mechanisms that govern the initiation, establishment and progression of metastasis remain unclear. Moreover, knowledge gained on metastatic process was largely based on cultured or in vitro manipulated cells that were reintroduced into immune-compromised recipient mice. In the present study, a spontaneous metastasis mouse model for NSCLC was generated with a heritable fluorescent tag (DsRed) driven by CAG (combination of cytomegalovirus early enhancing element and chicken beta actin) promoter in alveolar type II cells (SpC-rtTA/TetO-Cre/LSL-DsRed). This approach is essential, keeping in mind the reprogramming nature of Myc oncogene (Rapp et al, 2009). Such genetic lineage tracing approach not only allowed us to monitor molecular and cellular changes during development of primary tumor but also led us to identify the different stages of secondary tumor development in distant organs. Upon combined expression of oncogenic C Raf-BXB and c-Myc (MYC-BXB-DsRed) in lung alveolar type II epithelial cells, macroscopic lung tumors arose comprising of both cuboidal and columnal cellular features. C Raf-BXB induced tumors (CRAF-DsRed) exhibit cuboidal morphology and is non-metastatic whereas Myc-BXB induced lung tumors (Myc-BXB-DsRed) present cuboidal-columnar cellular features and is able to undergo metastasis mainly in liver. Surprisingly, cystic lesions which were negative for SpC (Surfactant protein C) and CCSP (Clara cell secretory protein), strongly expressed DsRed proteins indicating its origin from lung alveolar type II cells. Moreover, early lung progenitor markers such as GATA4 (GATA-binding protein 4) and TTF1 (Thyroid Transcription Factor 1) were still expressed in these early cystic lesions suggesting metastasis as a faulty recapitulation of ontogeny (Rapp et al, 2008). Interestingly, mixed cystic lesions and metastatic tumors contained DsRed and SpC positive cells. These results demonstrate secondary tumor progression from cystic, mixed cystic to malignant transformation. Our results shed tremendous light on reprogramming of metastasizing cells during secondary tumor development. Moreover, such fluorescent tagged metastatic mice model can also be used to track the migration ability of metastatic cancer cell to different organs and its potential to differentiate into other cell types such as blood vessel or stromal cell within the primary tumor.
The “Akọ na Uche” (Wisdom and Justifiability) of Preemptive-strike in Self-defense and Alternative Conflict Resolutions is an ethical examine on man’s inherent right of self-defense, not only as a right that is innate, but also as an individual’s or a nation’s right enshrined in, and guaranteed by the Charter provisions of the United Nations.
Stemming from the painful experience of the First and Second World Wars, nations wishing never again to engage one another in such full scale wars of destruction, met in San Francisco, California, accepted the formation of a new organization, the United Nations, to replace the League of Nations considered as ineffectual. The participating nations articulated a set of guiding principles in the form of rules, rights and responsibilities endorsed by all the early member-nations on June 26, 1945, but effective from October 24, same year. This is the birth of the United Nations Charter.
With the endorsement of the Charter, all member-nations assumed the responsibility of making the world a better place, peaceful and secure for humanity. They vowed never again to engage in unethical wars, they accepted to respect and foster human rights, to fight poverty, to spread democracy and to promote more healthy and robust international relations through a more vibrant cooperation and aggressive diplomacy. The Charter also reaffirmed the intrinsic right of self-defense of the victim of an armed attack, which sometimes has been utilized as well as exploited.
Non-coding RNAs constitute a major class of regulators involved in bacterial gene expression. A group of riboregulators of heterogeneous size and shape referred to as small regulatory RNAs (sRNAs) control trans- or cis-encoded genes through direct base-pairing with their mRNAs. Although mostly inhibiting their target mRNAs, several sRNAs also induce gene expression. An important co-factor for sRNA activity is the RNA chaperone, Hfq, which is able to rearrange intramolecular secondary structures and to promote annealing of complementary RNA sequences. In addition, Hfq protects unpaired RNA from degradation by ribonucleases and thus increases sRNA stability. Co-immunoprecipitation of RNA with the Hfq protein, and further experimental as well as bioinformatical studies performed over the last decade suggested the presence of more than 150 different sRNAs in various Enterobacteria including Escherichia coli and Salmonellae. So-called core sRNAs are considered to fulfill central cellular activities as deduced from their high degree of conservation among different species. Approximately 25 core sRNAs have been implicated in gene regulation under a variety of environmental responses. However, for the majority of sRNAs, both the riboregulators’ individual biological roles as well as modes of action remain to be elucidated. The current study aimed to define the cellular functions of the two highly conserved, Hfq-dependent sRNAs, SdsR and RydC, in the model pathogen Salmonella Typhimurium. SdsR had been known as one of the most abundant sRNAs during stationary growth phase in E. coli. Examination of the conservation patterns in the sdsR promoter region in combination with classic genetic analyses revealed SdsR as the first sRNA under direct transcriptional control of the alternative σ factor σS. In Salmonella, over-expression of SdsR down-regulates the synthesis of the major porin OmpD, and the interaction site in the ompD mRNA coding sequence was mapped by a 3'RACE-based approach. At the post-transcriptional level, expression of ompD is controlled by three additional sRNAs, but SdsR plays a specific role in porin regulation during the stringent response. Similarly, RydC, the second sRNA adressed in this study, was initially discovered in E. coli but appeared to be conserved in many related γ-proteobacteria. An interesting aspect of this Hfq-dependent sRNAs is its secondary structure involving a pseudo-knot configuration, while the 5’ end remains single stranded. A transcriptomic approach combining RydC pulse-expression and scoring of global mRNA changes on microarrays was employed to identify the targets of this sRNA. RydC specifically activated expression of the longer of two versions of the cfa mRNA encoding for the phospholipid-modifying enzyme cyclopropane fatty acid synthase. Employing its conserved single-stranded 5' end, RydC acts as a positive regulator and masks a recognition site of the endoribonuclease, RNase E, in the cfa leader.
The objective of this thesis focuses on the development of strategies for precise control of perylene bisimide (PBI) self-assembly and the in-depth elucidation of structural and optical features of discrete PBI aggregates by means of NMR and UV/Vis spectroscopy. The strategy for discrete dimer formation of PBIs is based on delicate steric control that distinguishes the two facets of the central perylene surface. The strategy applied in this thesis for accessing discrete PBI quadruple and further oligomeric stacks relies on backbone-directed PBI self-assembly. For this purpose, two tweezer-like PBI dyads bearing the respective rigid backbones, diphenylacetylene (DPA) and diphenylbutydiyne (DPB), were synthesized. The distinct aggregation behavior of these structurally similar PBI dyads can be ascribed to the intramolecular distance between the two PBI chromophores imparted by the DPA and DPB spacers.
In initial experiments, the well characterized VACV strain GLV-1h68 and three wild-type LIVP isolates were utilized to analyze gene expression in a pair of autologous human melanoma cell lines (888-MEL and 1936 MEL) after infection. Microarray analyses, followed by sequential statistical approaches, characterized human genes whose transcription is affected specifically by VACV infection. In accordance with the literature, those genes were involved in broad cellular functions, such as cell death, protein synthesis and folding, as well as DNA replication, recombination, and repair. In parallel to host gene expression, viral gene expression was evaluated with help of customized VACV array platforms to get better insight over the interplay between VACV and its host. Our main focus was to compare host and viral early events, since virus genome replication occurs early after infection. We observed that viral transcripts segregated in a characteristic time-specific pattern, consistent with the three temporal expression classes of VACV genes, including a group of genes which could be classified as early-stage genes. In this work, comparison of VACV early replication and respective early gene transcription led to the identification of seven viral genes whose expression correlated strictly with replication. We considered the early expression of those seven genes to be representative for VACV replication and we therefore referred to them as viral replication indicators (VRIs). To explore the relationship between host cell transcription and viral replication, we correlated viral (VRI) and human early gene expression. Correlation analysis revealed a subset of 114 human transcripts whose early expression tightly correlated with early VRI expression and thus early viral replication. These 114 human molecules represented an involvement in broad cellular functions. We found at least six out of 114 correlates to be involved in protein ubiquitination or proteasomal function. Another molecule of interest was the serine-threonine protein kinase WNK lysine-deficient protein kinase 1 (WNK1). We discovered that WNK1 features differences on several molecular biological levels associated with permissiveness to VACV infection. In addition to that, a set of human genes was identified with possible predictive value for viral replication in an independent dataset. A further objective of this work was to explore baseline molecular biological variances associated with permissiveness which could help identifying cellular components that contribute to the formation of a permissive phenotype. Therefore, in a subsequent approach, we screened a set of 15 melanoma cell lines (15-MEL) regarding their permissiveness to GLV-1h68, evaluated by GFP expression levels, and classified the top four and lowest four cell lines into high and low permissive group, respectively. Baseline gene transcriptional data, comparing low and highly permissive group, suggest that differences between the two groups are at least in part due to variances in global cellular functions, such as cell cycle, cell growth and proliferation, as well as cell death and survival. We also observed differences in the ubiquitination pathway, which is consistent with our previous results and underlines the importance of this pathway in VACV replication and permissiveness. Moreover, baseline microRNA (miRNA) expression between low and highly permissive group was considered to provide valuable information regarding virus-host co-existence. In our data set, we identified six miRNAs that featured varying baseline expression between low and highly permissive group. Finally, copy number variations (CNVs) between low and highly permissive group were evaluated. In this study, when investigating differences in the chromosomal aberration patterns between low and highly permissive group, we observed frequent segmental amplifications within the low permissive group, whereas the same regions were mostly unchanged in the high group. Taken together, our results highlight a probable correlation between viral replication, early gene expression, and the respective host response and thus a possible involvement of human host factors in viral early replication. Furthermore, we revealed the importance of cellular baseline composition for permissiveness to VACV infection on different molecular biological levels, including mRNA expression, miRNA expression, as well as copy number variations. The characterization of human target genes that influence viral replication could help answering the question of host cell response to oncolytic virotherapy and provide important information for the development of novel recombinant vaccinia viruses with improved features to enhance replication rate and hence trigger therapeutic outcome.
γ-Aminobutyric acid type A receptors (GABAARs) and glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the central nervous system. For proper synaptic function their precise localization and exact concentration within the neuronal surface membrane is essential. These properties are mediated by scaffolding proteins which directly contact the large intracellular loops of the receptors and tether them to cytoskeletal elements of the neuronal cells. In my thesis I deciphered the molecular details of several underlying protein-protein interactions, namely the interaction of a subset of GABAAR and GlyR subunits with the scaffolding proteins gephyrin, radixin and collybistin. I determined short linear motifs within the large intracellular loops of the receptors that directly engage in subunit specific scaffold protein interactions. My quantitative binding studies revealed that gephyrins E domain primarily recognizes the GABAAR α1 (Kd = 17 M) and α3 (Kd = 5 M) subunits, in contrast, the SH3 domain of collybistin mainly interacts with the GABAAR α2 subunit (Kd = 1 µM), while the FERM domain of radixin tightly binds to the GABAAR α5 subunit (Kd = 8 µM). My work additionally demonstrated that this simple relationship is complicated by (i) missing or (ii) overlapping binding specificities between the scaffold proteins and the receptor subunits. Moreover, this thesis addressed the possibility of (iii) posttranslational negative regulation as well as amplification generated by (iv) avidity effects as summarized below. (i) First, using biochemical methods I mapped the radixin-GABAAR α5 interaction in detail. My structural analysis and competition assays suggest that radixin mediates the receptor subunit binding via a universal binding site within the F3 subdomain of its FERM domain. This binding site is formed by an α-helix that offers a large hydrophobic pocket, which accepts a variety of different hydrophobic residues adopting different conformations, and a β-strand that readily engages in peptide backbone interactions. Not surprisingly, this binding site has been implicated in a wide variety of different scaffold interactions, thus emphasizing the importance of the essential FERM activation mechanism described earlier and suggesting additional pathways to allow tight regulation of this interaction. (ii) Next, I analyzed in detail the process of gephyrin-mediated GABAAR clustering. My X-ray crystallographic studies and binding assays revealed that gephyrin mediates binding of the GABAAR α1, α2 and α3 subunit via a universal binding site that also mediates the interactions with the GlyR β subunit. Using structure-guided mutagenesis I identified key residues within gephyrin and the receptor subunits that act as major contributors to the overall binding strength. Namely, two conserved aromatic residues within the N-terminal half of the receptor binding region engage in crucial hydrophobic interactions with gephyrin. Accordingly, J. Mukherjee from the group of our collaborator Steven J. Moss verified a substantial decrease in GABAAR cluster number and size in primary hippocampal neurons upon exchange of these residues within the GABAAR α2 subunit. Extension of my studies to collybistin (CB) revealed an overlapping but reciprocal subunit preference for this protein in comparison to gephyrin. The GABAAR α3 subunit exclusively binds gephyrin, in contrast the GABAAR α1 subunit mainly targets gephyrin (Kd = 17 µM) but additionally displays a moderate affinity (Kd ≈ 400 µM) towards the SH3 domain of CB. The GABAAR α2 subunit binds tightly to the SH3 domain of CB (Kd = 1 µM) and additionally displays a weak gephyrin affinity (Kd ≈ 500 µM). Notably, I could exclude the possibility of synergistic effects between gephyrins E domain, the SH3 domain of CB and the GABAAR α2 subunit. Instead, I found that the GABAAR α2 subunit binds gephyrin and CB in a mutually exclusive manner. These results suggest that CBs role in receptor clustering is solely determined by competing binding events of its constituting domains. Namely, the intra-molecular association between the PH/DH domain and the SH3 domain within CB competes with different inter-molecular interactions of CB: GABAAR α2 binding to the SH3 domain, PIP2 binding to the PH domain and gephyrin presumably binding to the PH and DH domain of CB. (iii) Interestingly, the receptor motifs, which have been mapped in my thesis to directly interact with the scaffold proteins, were shown in earlier studies to be posttranslationally modified in vivo. In particular, the GABAAR α1 and GlyR β subunits have been implicated as targets of the ERK/MAPK and PKC phosphorylation-pathways, respectively, while the GABAAR α5 subunit motif was shown to be ubiquitinated. In this dissertation, I analyzed Thr348, a possible ERK phosphorylation site within GABAAR α1. My binding assays verified a severe reduction of the direct gephyrin binding strength upon introduction of the respective phosphomimetic residue. The relevance of this in vitro result was highlighted by J. Mukherjee who confirmed a significant reduction in GABAAR cluster number and size upon introduction of the same mutation. The ERK/MAPK pathway is therefore a promising candidate for regulation of GABAergic transmission. (iv) In vivo, gephyrin presumably forms a multivalent scaffold, which is based on the self-association of its G (GephG) and E domains (GephE). Given the multimeric nature of gephyrin and the pentameric receptor architecture, I tested the possibility of avidity in the clustering of inhibitory neurotransmitter receptors. Cocrystallization of selected minimum peptides with GephE and their crystal structure analyses enabled me to define a receptor-derived peptide that offers a maximized gephyrin affinity. The structure of the GephE-GlyR receptor complex reveals two receptor-binding sites in close spatial vicinity (15 Å). I therefore designed bivalent peptides that enable to target both GephE sites at the same time and, as expected, a variety of biophysical methods verified an avidity-potentiated and unmatched high gephyrin affinity for these bidentate compounds. Notably, I could extend the dimerization approach to low affinity gephyrin ligands, namely short GABAAR-derived peptides that could not be studied using conventional monomeric ligands. Additionally, I verified that this compound specifically targets GephEs receptor binding site, and that it thereby inhibits its receptor binding activity. Further development of this molecule may offer the possibility to specifically analyze the effect of uncoupling the gephyrin-receptor interaction in cell culture-based assays, without altering protein function or expression level that accompanies conventional methods such as protein knock-out, RNA interference or the usage of antibodies.
Cancer-related anemia is prevalent in cancer patients. Anemia negatively affects normal mental and physical function capacity with common symptoms s like fatigue, headache, or depression. Human erythropoietin (hEPO), a glycoprotein hormone regulating red blood cell formation, is approved for the treatment of cancer-related anemia. It has shown benefits in correcting anemia, and subsequently improving health-related quality of life and/or enhancing radio-, and chemotherapy. Several recent clinical trials have suggested that recombinant hEPO (rhEPO) may promote tumor growth that raises the questions concerning the safety of using rhEPO for cancer treatment. However in others, such effects were not indicated. As of today, the direct functional effect of rhEPO in tumor models remains controversial and needs to be further analyzed. Based on the GLV-1h68 backbone, the hEPO-expressing recombinant VACV strains (EPO-VACVs) GLV-1h210, GLV-1h211, GLV-1h212 and GLV-1h213 were generated by replacing the lacZ expression cassette at the J2R locus with hEPO under the control of different vaccinia promoters p7.5, pSE, pSEL, pSL, respectively. Also, GLV-1h209 was generated, which is similar to GLV-1h210 but expresses a mutated non-functinal EPO (R103A). The EPO-VACV strains were characterized for their oncolytic efficacy in lung (A549) cancer cells in culture and tumor xenografts. Concomitantly, the effects of locally expressed hEPO in tumors on virus replication, host immune infiltration, tumor vascularization and tumor growth were also evaluated. As expected, EPO-VACVs enhanced red blood cell (RBC) formation in xenograft model. The number of RBCs and hemoglobin (Hb) levels were significantly increased in EPO-VACVs-treated mice compared to GLV-1h68-treated or untreated control mice. However, the mean size of RBC or Hb content per RBC remained normal. Furthermore, over-expression of hEPO did not significantly affect numbers of lymphocytes, monocytes, leucocytes or platelets in the peripheral blood stream. The expression of hEPO in colonized tumors of mice treated with EPO-VACVs was demonstrated by immunohistological staining. Interestingly, there were 9 - 10 hEPO isoforms detected either in tumors, cells, or supernatant, while 3-4 basic isoforms were missing in blood serum, where only six hEPO isoforms were found. Tumor-bearing mice after treatment with EPO-VACVs showed enhanced tumor regression compared to GLV-1h68. The virus titers in tumors in EPO-VACVs-treated mice were 3-4 fold higher compared to GLV-1h68-treated mice. Nevertheless, no significant difference in virus titers among EPO-VACVs was found. The blood vessels in tumors were significantly enlarged while the blood vessel density remained unchanged compared to the GLV-1h68 treated mice, indicating that hEPO did not affect endothelial cell proliferation in this model. Meanwhile, rhEPO (Epoetin alfa) alone or in combination with GLV-1h68 did not show any signs of enhanced tumor growth when compared to untreated controls and GLV-1h68 groups, while doses used were clinical relevant (500 U/kg). These findings suggested that hEPO did not promote angiogenesis or tumor growth in the A549 tumor xenograft model. Human EPO has been reported to function as an immune modulator. In this study, however, we did not find any involvement of hEPO in immune cytokine and chemokine expression or innate immune cell infiltration (leucocytes, B cells, macrophages and dendritic cells) into infected tumors. The degree of immune infiltration and cytokine expression was directly correlated to the number of virus particles. Increased virus replication, led to more recruited immune cells and secreted cytokines/chemokines. It was proposed that tumor regression was at least partially mediated through activation of innate immune mechanisms. In conclusion, the novel EPO-VACVs were shown to significantly increase the number of RBCs, Hb levels, and virus replication in tumors as well as to enhance tumor regression in the A549 tumor xenograft model. Moreover, locally expressed hEPO did not promote tumor angiogenesis, tumor growth, and immune infiltration but was shown to causing enlarged tumoral microvessels which facilitated virus spreading. It is conceivable that in a possible clinical application, anemic cancer patients could benefit from the EPO-VACVs, where they could serve as “wellness pills” to decrease anemic symptoms, while simultaneously destroying tumors.
Previous studies by our group revealed that chronic low grade inflammation implicating phagocytosing macrophages is a highly relevant mechanism in the pathogenesis of Charcot-Marie-Tooth disease. The lack of CSF-1, the primary regulator of macrophage function and survival, led to a robust and persistent amelioration of the phenotype in two authentic mouse models of CMT. Moreover, a close contact between CSF-1 producing fibroblasts and endoneurial macrophages carrying CSF-1R has been confirmed in nerve biopsies of CMT patients, further supporting the clinical significance of this pathway. In the current study we treated 3 distinct mouse models of CMT1: the PMP22tg mice as a model for CMT1A, the P0+/- mice as a model for CMT1B and the Cx32def mice as a model for CMT1X, with a CSF-1R specific kinase (c-FMS) inhibitor (800-1200 mg PLX5622/ kg chow) according to different treatment regimes mimicking an ideal early onset treatment, a late onset treatment and the withdrawal of the drug. Using the above mentioned doses of PLX5622, we documented a dramatic decrease in macrophage numbers in the PNS of all 3 myelin mutants, except for the quadriceps nerve of Cx32def mice. Fibroblast numbers remained unchanged in treated animals. Surprisingly, in spite of the decrease in the number of detrimental macrophages we could not detect an unequivocal phenotypic improvement. CMAP amplitudes were reduced in both wild type and myelin mutant mice treated with CSF-1R inhibitor in comparison to untreated littermates. Corresponding to the electrophysiological findings, the axon number and the percentage of large diameter axons were reduced in the quadriceps nerve of treated P0+/- and Cx32def mice. By contrast we observed a higher number of fully myelinated axons, in parallel with a decrease in the percentage of demyelinated (and hypermyelinated in PMP22tg mice) fibers in the ventral roots of P0+/- mice treated with CSF-1R inhibitor from 3 months up to 6 months of age and PMP22tg animals treated from 9 months up to 15 months of age. Our results indicate that CSF-1R inhibitor has the potential to improve the demyelinating phenotype of at least two models of CMT1. Nevertheless, further studies are necessary (for example with lower doses of the inhibitor) to minimize or even eliminate the putative neurotoxic effect we observed with high dose treatment conditions.
Glioblastoma multiforme (GBM) represents the most aggressive form of malignant brain tumors and remains a therapeutically challenge. Intense research in the field has lead to the testing of oncolytic viruses to improve tumor control. Currently, a variety of different oncolytic viruses are being evaluated for their ability to be used in anti-cancer therapy and a few have entered clinical trials. Vaccinia virus, is one of the viruses being studied. GLV-1h68, an oncolytic vaccinia virus engineered by Genelux Corporation, was constructed by insertion of three gene cassettes, RUC-GFP fusion, β-galactosidase and β- glucuronidase into the genome of the LIVP strain. Since focal tumor radiotherapy is a mainstay for cancer treatment, including glioma therapy, it is of clinical relevance to assess how systemically administered oncolytic vaccinia virus could be combined with targeted ionizing radiation for therapeutic gain. In this work we show how focal ionizing radiation (IR) can be combined with multiple systemically delivered oncolytic vaccinia virus strains in murine models of human U-87 glioma. After initial experiments which confirmed that ionizing radiation does not damage viral DNA or alter viral tropism, animal studies were carried out to analyze the interaction of vaccinia virus and ionizing radiation in the in vivo setting. We found that irradiation of the tumor target, prior to systemic administration of oncolytic vaccinia virus GLV-1h68, increased viral replication within the U-87 xenografts as measured by viral reporter gene expression and viral titers. Importantly, while GLV-1h68 alone had minimal effect on U-87 tumor growth delay, IR enhanced GLV-1h68 replication, which translated to increased tumor growth delay and mouse survival in subcutaneous and orthotopic U-87 glioma murine models compared to monotherapy with IR or GLV-1h68. The ability of IR to enhance vaccinia replication was not restricted to the multi-mutated GLV-1h68, but was also seen with the less attenuated oncolytic vaccinia, LIVP 1.1.1. We have demonstrated that in animals treated with combination of ionizing radiation and LIVP 1.1.1 a strong pro-inflammatory tissue response was induced. When IR was given in a more clinically relevant fractionated scheme, we found oncolytic vaccinia virus replication also increased. This indicates that vaccinia virus could be incorporated into either larger hypo-fraction or more conventionally fractionated radiotherapy schemes. The ability of focal IR to mediate selective replication of systemically injected oncolytic vaccinia was demonstrated in a bilateral glioma model. In mice with bilateral U-87 tumors in both hindlimbs, systemically administered oncolytic vaccinia replicated preferentially in the focally irradiated tumor compared to the shielded non- irradiated tumor in the same mouse We demonstrated that tumor control could be further improved when fractionated focal ionizing radiation was combined with a vaccinia virus caring an anti-angiogenic payload targeting vascular endothelial growth factor (VEGF). Our studies showed that following ionizing radiation expression of VEGF is upregulated in U-87 glioma cells in culture. We further showed a concentration dependent increase in radioresistance of human endothelial cells in presence of VEGF. Interestingly, we found effects of vascular endothelial growth factor on endothelial cells were reversible by adding purified GLAF-1 to the cells. GLAF-1 is a single- chain antibody targeting human and murine VEGF and is expressed by oncolytic vaccinia virus GLV-109. In U-87 glioma xenograft murine models the combination of fractionated ionizing radiation with GLV-1h164, a vaccinia virus also targeting VEGF, resulted in the best volumetric tumor response and a drastic decrease in vascular endothelial growth factor. Histological analysis of embedded tumor sections 14 days after viral administration confirmed that blocking VEGF translated into a decrease in vessel number to 30% of vessel number found in control tumors in animals treated with GLV-164 and fractionated IR which was lower than for all other treatment groups. Our experiments with GLV-1h164 and fractionated radiotherapy have shown that in addition to ionizing radiation and viral induced tumor cell destruction we were able to effectively target the tumor vasculature. This was achieved by enhanced viral replication translating in increased levels of GLAF-2 disrupting tumor vessels as well as the radiosensitization of tumor vasculature to IR by blocking VEGF. Our preclinical results have important clinical implications of how focal radiotherapy can be combined with systemic oncolytic viral administration for highly aggressive, locally advanced tumors with the potential, by using a vaccinia virus targeting human vascular endothelial growth factor, to further increase tumor radiation sensitivity by engaging the vascular component in addition to cancer cells.
Fanconi anemia (FA) is an autosomal recessive or X-chromosomal inherited disorder, which is not only phenotypically but also genotypically very heterogeneous. While its hallmark feature is progressive bone marrow failure, many yet not all patients suffer additionally from typical congenital malformations like radial ray defects and growth retardation. In young adulthood the cumulative risk for developing hematological or other malignancies is compared to the general population several hundred-fold increased. The underlying molecular defect is the deficiency of DNA interstrand crosslink (ICL) repair. ICLs are deleterious lesions, which interfere with crucial cellular processes like transcription and replication and thereby can lead to malignant transformation, premature senescence or cell death. To overcome this threat evolution developed a highly complex network of interacting DNA repair pathways, which is conserved completely only in vertebrates. The so called FA/BRCA DNA damage response pathway is able to recognize ICLs on stalled replication forks and promotes their repair through homologous recombination (HR). Today we know 15 FA genes (FANCA, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O and -P) whose products are involved in this pathway. Although more than 80% of FA patients carry biallelic mutations in either FANCA, FANCC or FANCG, there are still some who cannot be assigned to any of the known complementation groups. This work aimed to indentify the di¬sease causing mutations in a cohort of those unassigned patients. Initial screens of the candidate genes FAN1, MHF1 and MHF2 did not reveal any pathogenic alterations. Moreover, FAN1 could be excluded as FA candidate gene because patients carrying a homozygous microdeletion including the FAN1 locus did not show a phenotype comparable to FA patients. In the case of MHF1 and MHF2 the reason for the negative screening result is not clear. Mutation carriers might be rare or, regarding the diverse and also FA pathway independent protein functions, phenotypically not comparable to FA patients. Nevertheless, this study contri¬buted to the identification and characterization of the most recent members of the FA pathway - RAD51C (FANCO), SLX4 (FANCP) and XPF (FANCQ). FANCO is one of the RAD51 paralogs and is involved in crucial steps of HR. But since the only reported FA-O patient has so far not developed any hematological anomalies, FANCO is tentatively designated as gene underlying an FA-like disorder. In contrast, patients carrying biallelic mutations in FANCP do not only show hematological anomalies, but as well congenital malformations typical for FA. The distinct role of FANCP in the FA pathway could not be determined, but it is most likely the coordination of structure-specific nucleases during ICL excision. One of these nucleases is the heterodimer XPF/ERCC1. XPF is probably disease causing in the complementation group FA-Q and is the first FA gene, which was identified by Next Generation Sequencing (NGS). Extraordinarily is that mutations in this gene had previously been reported to cause two other disorders, xeroderma pigmentosum and segmental progeria. Despite some overlaps, it was shown that the divergent phenotypes could clearly be distinguished and are caused by distinct functional defects of XPF. Additionally, this work aimed to improve and accelerate the genotyping process of FA patients in general. Therefore, classical approaches should be complemented or fully replaced by approa¬ches using NGS. Massively parallel sequencing of the whole exome proved to be most appro¬priate and the establishment of an FA-specific analysis pipeline facilitated improved molecular diagnostics by combining complementation group assignment and mutation analysis in one step. Consequently two NGS studies revealed the pathogenic defect in several previously unassigned FA patients and thereby added another patient to one of the most recent subtypes, FA-P. In summary, this work contributed not only to further completion of the FA/BRCA DNA repair network by adding three novel genes, it also showed that classical molecular approaches for re¬search as well as for diagnostics could be replaced by NGS.
Interleukin-6 (IL-6), oncostatin M (OSM), leukaemia inhibitory factor (LIF) and cardiotrophin-1 (CT-1) are members of the IL-6-type cytokine family that is characterised by sharing the common receptor subunit gp130. While the involvement of these polypeptides in cell differentiation, cell survival, proliferation, apoptosis, inflammation, haematopoiesis, immune response and acute phase reaction has already been demonstrated, the description of their role in development and progression of cardiac hypertrophy is still rather limited. A model has been postulated that declares the transient expression of IL-6-type cytokines as protective, while a continuous cardiac secretion of these proteins seems to be rather harmful for the heart. Within the first part of the study (results 4.1, 4.2 and 4.3) it was shown that OSM induces hypertrophy of primary neonatal rat cardiomyocytes (NRCM), just as its related cytokines LIF, CT-1 and hIL-6/hsIL-6R (hsIL-6R, human soluble IL-6 receptor). Regarding the hypertrophic potentials the LIFR/gp130 utilising cytokines (hLIF, hOSM and hCT-1) are stronger inducers than the OSMR/gp130 utilising mOSM. Human IL-6/hsIL-6R which signals via a gp130 homodimer has the weakest hypertrophic effect. The thorough analysis of typical signalling pathways initiated by IL-6-type cytokines revealed that STAT3 phosphorylation at Y705 seems to be the most important hypertrophy promoting pathway. In addition and in contrast to published work, we clearly demonstrate that classical IL-6 signalling (upon pure IL-6 treatment) has no hypertrophic effect on cardiomyocytes, because they lack sufficient amounts of the membrane-bound IL-6R. This is also true for neonatal rat cardiac fibroblasts (NRCFB). Since these cells can also influence cardiac hypertrophy, signalling pathways and target genes were additionally examined in NRCFB in response to OSM, LIF and IL-6/sIL-6R. One of the key findings of this thesis is the selective change in expression of cytokines and receptors of the IL-6 family in both cell types upon IL-6-type cytokine stimulation. A striking difference between NRCM and NRCFB is the fact that the target gene induction in NRCM is of similar duration upon mOSM and hIL-6/hsIL-6R treatment, while hIL-6/hsIL-6R is capable of promoting the induction of OSMR and IL-6 significantly longer in NRCFB. By searching for transcription factors or intermediate cytokines which could be responsible for this difference, a strong correlation between increased Il6 transcription and amount of mRNA levels for C/EBPβ and C/EBPδ was observed in response to IL-6/sIL-6R stimulation. Interestingly, mOSM also mediates the induction of C/EBPβ and δ, but the initiation is significantly less efficient than in response to IL-6/sIL-6R. Therefore, we assume that mOSM stimulation fails to reach threshold values required for a prolonged IL-6 secretion. Since we additionally observe a slight IL-6R mRNA upregulation in NRCFB, we assume that the combination of IL-6, LIF, C/EBPβ, C/EBPδ and IL-6R expression might be responsible for the observed different kinetics with which IL-6 and OSM stimulate NRCFB. In addition to the aforementioned proteins, members of the renin-angiotensin system seem to support the IL-6-type cytokine mediated hypertrophy. Since it has already been shown that angiotensin II vice versa induces IL-6 expression in NRCM and NRCFB, this enhanced expression of AT1α and ACE could be of crucial interest for the hypertrophy supporting phenotype. The second part of the presented work dealt with the characterisation of the receptor complexes of rat OSM. The central question of this analysis was, whether rOSM, just like mOSM, only binds the type II (OSMR/gp130) receptor complex or is able to utilise the type II and type I (LIFR/gp130) receptor complex. Using different experimental approaches (knock-down of the OSMR expression by RNA interference, blocking of the LIFR by LIF-05, an antagonistic LIF variant, and generation of stably transfected Ba/F3 cells expressing the newly cloned rat OSMR/gp130 or LIFR/gp130 receptor complex) we can clearly show that rat OSM surprisingly utilises both, the type I and type II receptor complex. Therefore it closely mimics the human situation. Furthermore, rOSM displays cross-species activities and stimulates cells of human as well as murine origin. Its signaling capacities closely mimic those of human OSM in cell types of different origin in the way that strong activation of the JAK/STAT, the MAP kinase as well as the PI3K/Akt pathways can be observed. Therefore, the results obtained in the last section of this thesis clearly suggest that rat disease models would allow evaluation of the relevance of OSM for human biology much better than murine models.
Effects of stem cell transcription factor-expressing vaccinia viruses in oncolytic virotherapy
(2012)
Cancer remains the second leading cause of death in the industrialized. The data from many different studies investigating the nature of cancer-initiating cells coined the description ‘cancer stem cells’ and has major implications on conventional cancer therapy. Thus, to improve the outcome of cancer treatment and to lower negative side effects, the development of novel therapeutic regimens is indispensable. It has been demonstrated in many preclinical studies that oncolytic virotherapy using vaccinia virus may provide a powerful and well-tolerable new tool in cancer therapy which is currently investigated in several clinical trials (Phase I & II) as stand-alone treatment or in combination with conventional cancer therapy. Cancer-initiating cells and stem cells share a variety of characteristics like the ability to self-renew, differentiation potential, quiescence, drug and radiation resistance, activation and inhibition of similar signaling pathways as well as expression of cell surface markers and stem cell-related genes. In this work, two new recombinant vaccinia viruses expressing the transcription factors Nanog (GLV-1h205) and Oct4 (GLV-1h208) were engineered to provide deeper insight of these stem cell master regulators in their significance of cancer-initiation and their impact on oncolytic virotherapy. Both viruses were analyzed for their replication potential in A549 and PC-3 human cancer cells. Marker gene expression was assessed by RT-PCR, SDS-PAGE and Western blotting, ELISA or immunocytochemistry.Furthermore, the effect of GLV-1h205 infection on the cell cycle in A549 cells was analyzed. Next, the effects of virus-mediated expression of stem cell transcription factors on therapeutic efficacy and survival rates in A549 xenograft mouse models was analyzed. A non-functional Nanog mutant-expressing virus strain (GLV-1h321) was engineered to analyze whether the observed therapeutic benefits were promoter- or payload-driven. Furthermore, this study analyzed the potential of GLV-1h68 to infect, replicate in, and lyse colorectal cancer cell lines to study whether oncolytic vaccinia viruses can be potential new and less invasive treatment regimens for late stage colorectal cancer. Marker gene expression was assessed by fluorescence microscopy and FACS. The transcription factor Klf4 is highly expressed in quiescent, terminally differentiated cells in the colonic epithelium whereas it is dramatically downregulated in colon cancers. Klf4 expression leads to cell growth arrest and inhibits Wnt signaling by binding to beta-catenin. To further improve the treatment of colorectal cancers, new recombinant vaccinia viruses (GLV-1h290-292) mediating the expression of differing amounts of the tumor suppressor Klf4 by using different promoter strengths were engineered. Initial characterization of recombinant vaccinia viruses expressing Klf4 by replication assay, cell viability assay, SDS-PAGE and Western blotting, immuncytochemistry and analysis of protein functionality by qPCR and ELISA analysis for cellular beta-catenin expression, demonstrated promoter strength-dependent expression of and impact of Klf4. To further boost the effects of tumor suppressor Klf4, a vaccinia virus strain expressing Klf4 with a C-terminal fusion of the TAT transduction domain (GLV-1h391) was engineered. Treatment of HT-29 non-responder tumors in vivo with GLV-1h291 and GLV-1h391 led to significant tumor growth inhibition and improved overall survival compared to GLV-1h68. This makes the Klf4-TAT expressing GLV-1h391 a promising candidate for the treatment of colorectal cancer in man.
The objective of this prospective observational controlled study was to evaluate potential effects and dose-response relationship of LT4 administration on BMD, parameters of bone and muscle strength, and biochemical variables of calcium homoeostasis and bone turnover.Ninety-seven men and pre-menopausal women after near total thyroidectomy and ¹³¹I remnant ablation for well-differentiated thyroid carcinoma or after strumectomy for non-toxic goitre were stratified by degree of TSH suppression and by gender in three subgroups: 28 men and 46 women on LT4 suppressive treatment and 23 women on LT4 replacement therapy. Patients were matched for age, gender and BMI to 89 healthy controls with a negative history of thyroid disease. Patients and controls were followed and studied for a mean time of 1.1±0.2 years. Peripheral volumetric total and trabecular BMD as well as bone strength (pQCT) were determined at the ultra-distal radius. Central areal BMD (DXA) was measured at the lumbar spine, left and right femoral neck as well as left and right total hip. Maximum grip strength (dynamometer) of the non-dominant forearm and serum markers of calcium and bone metabolism were assessed. BMD at the axial skeleton and muscle strength were not impaired by LT4 medication irrespective of gender, underlying diagnosis or treatment regimen. By contrast, a general trend of inversely affected total and trabecular BMD and of decreased bone strength was detected at the ultra-distal radius. Only in women on LT4 suppressive treatment, loss of total BMD at the ultra-distal radius reached a level of high significance. In women on LT4 replacement therapy, a significant decline of maximum grip strength appeared in comparison with female controls, while appendicular total and trabecular BMD as well as bone strength remained unchanged and did not differ from respective controls. In men on LT4 suppressive treatment, greater reduction of bone strength as compared to female thyroid cancer patients was marginally significant. Calcium balance was stable and serum concentrations of bone metabolism markers levelled off or rather decreased contradicting (high turnover) bone loss. The study did not reveal any dose-related differential influence of LT4 administration either on primary or secondary study endpoints in female patients. A gender-related difference of bone strength in response to LT4 suppressive treatment might not be excluded, as male thyroid cancer patients showed greater decline of bone strength despite unaffected peripheral BMD and muscle strength. In conclusion, there was only little evidence of adverse LT4 effects. For the most part, LT4 administration irrespective of degree of TSH suppression was not associated with low or accelerated loss of BMD at the peripheral and central skeleton and loss of bone and muscle strength, a finding also confirmed biochemically. The ultra-distal radius as a non-weight bearing skeletal site might be at risk for BMD reduction. According to the results, pre-menopausal women on LT4 suppressive therapy might be at risk of bone loss. The more complex approach of this study also took into account biomechanical qualities of bone material as well as structural and geometrical characteristics of bone architecture implying a causal muscle-bone interrelationship.
This dissertation provides both empirically and theoretically new insights into the economic effects of housing and housing finance within NK DSGE models. Chapter 1 studies the drivers of the recent housing cycle in Ireland by developing and estimating a two-country NK DSGE model of the European Economic and Monetary Union (EMU). It finds that housing preference (demand) and technology shocks are the most important drivers of real house prices and real residential investment. In particular, housing preference shocks account for about 87% of the variation in real house prices and explain about 60% of the variation in real residential investment. A robustness analysis finally shows that a good part of the variation of the estimated housing preference shocks can be explained by unmodeled demand factors that have been considered in the empirical literature as important determinants of Irish house prices. Chapter 2 deals with the implications of cross-country mortgage market heterogeneity for the EMU. The chapter shows that a change in cross-country institutional characteristics of mortgage markets, such as the loan-to-value (LTV) ratio, is likely to be an important driver of an asymmetric development in the housing market and real economic activity of member states. Chapter 3 asks whether monetary policy shocks can trigger boom-bust periods in house prices and create persistent business cycles. The chapter addresses this question by implementing behavioral expectations into an otherwise standard NK DSGE model with housing and a collateral constraint. Key to the approach in chapter 3 is that agents form heterogeneous and biased expectations on future real house prices. Model simulations and impulse response functions suggest that these assumptions have strong implications for the transmission of monetary policy shocks. It is shown that monetary policy shocks might trigger pronounced waves of optimism, respectively, pessimism that drive house prices and the broader economy, all in a self-reinforcing fashion. The chapter shows that in an environment in which behavioral mechanisms play a role an augmented Taylor rule that incorporates house prices is superior, because it limits the scope of self-fulfilling waves of optimism and pessimism to arise. Chapter 4 challenges the view that the observed negative correlation between the Federal Funds rate and the interest rate implied by consumption Euler equations is systematically linked to monetary policy. Using a Monte Carlo experiment based on an estimated NK DSGE model, this chapter shows that risk premium shocks have the capability to drive a wedge between the interest rate targeted by the central bank and the implied Euler equation interest rate, so that the correlation between actual and implied rates is negative. Chapter 4 concludes by arguing that the implementation of collateral constraints tied to housing values is a promising way to strengthen the empirical performance of consumption Euler equations.
In the context of this dissertation very long ranged exciton diffusion lengths (LD) were simulated for perylene-based materials under ideal conditions. This leads to the conclusion that the short LD values in existing materials result from an extrinsic and intrinsic immobilization. The latter, which is a specific material property, is based on a relaxation of the exciton into self-trapping states. An in-depth understanding of the atomistic processes defining self-trapping is essential to developing materials with long LD in the future, in which intrinsic immobilization is prevented. For the development of such a mechanistic understanding it is crucial that a clear relationship between molecular structure and LD is available. This is given by single crystals of diindeno perylene (DIP) and α-perylene tetracarboxylic anhydride (α-PTCDA). An extraordinary large LD of 90 nm was measured for the first one, while the latter possesses only 22 nm. Part of this thesis was to deliver reasons for this discrepancy. Only self-trapping comes into question to explain the different LD values. One reason for the different self-trapping in DIP and α-PTCDA could lie in the electronic structure. However, it was possible to demonstrate that a wide range of perylene-based materials possess no significant differences in their electronic structures. Consequently, such differences can be neglected for the explanation of immobilization mechanisms for the exciton. A further possible explanation could be polarization effects in the crystal, which influences the electronic structure of perylene based materials differently. Especially their influence on charge transfer (CT) states, which are located above the optically bright Frenkel state, was in question because such states could be stabilized by a polarizable surrounding. A significant influence of polarization effects on all considered states were excluded by using a polarizable continuum model. Hence, the small LD values in α-PTCDA are an evidence for self-trapping, which produces a crystal structure built up by π-stacks, while the one of DIP is of herringbone type. Since polarization effects can be neglected, is the dimer only via steric restrictions influenced by the crystal. Hence, a method describing self-trapping has to consider such effects, so that a mechanical embedding QM/MM approach is sufficient. Now, potential energy surfaces were calculated, on which wave packet dynamics were subsequently performed. In this way, atomistic mechanisms for the immobilization of excitons were described for the first time in organic materials. Self-trapping was studied in crystals of α-PTCDA by potential energy surfaces, which map an intermolecular shift motion of the dimer in the crystal. An immobilization of excitons occurs within 500 fs, which results from an irreversible energy loss together with a local deformation of the crystal lattice. This prevents a further transport of the exciton. In the case of DIP, this immobilization does not proceed due to high barriers. These barriers result from the herringbone type packing motif in the DIP crystal. This discrepancy in the dynamics explains the different LD values in DIP and α-PTCDA. In a further example, an exciton immobilization was found in helical π-aggregates of perylene tetracarboxylic bisimide (PBI) molecules. Self-trapping is caused by a relaxation mechanism, in which the exciton is transferred by asymmetric vibrations of the aggregate from the bright to a dark Frenkel state within 200 fs, whereby the transition is mediated by a CT state. However, the CT state is almost non-populated during the whole mechanism so that its participation could not yet be proven experimentally. This entire procedure is solely possible in helical aggregates, because only for such structures is there a CT state located next to the bright Frenkel state. At the final Frenkel state a torsional motion around the π-stacking axis is possible so that the loss in energy and the local rearrangement of the aggregate structure occurs, which means a self-trapping of the exciton. This mechanism is in perfect agreement with all available experimental data. These insights allow the conclusion that in future materials for organic solar cells an irreversible and ultrafast deformation of aggregates after photo-absorption must be avoided. Only in this way long LD values can be achieved and exciton self-trapping can be prevented. However, small LD values are always predicted in helical aggregates of perylene-based materials, because exciton immobilization occurs already due to small molecular motions. For this reason such aggregates are inappropriate for the use in organic solar cells. Long LD values are expected for aggregate structures with long intermolecular shifts or molecules with bulky substituents.
Precise control of progression through mitosis is essential to maintain genomic stability and to prevent aneuploidy. The DREAM complex is an important regulator of mitotic gene expression. Depletion of Lin9, one core-subunit of DREAM, leads to reduced expression of G2/M genes and impaired proliferation. In conditional mouse knockout cells (MEFs) Lin9 deletion causes defects in mitosis and cytokinesis and cells undergo premature senescence in order to prevent further proliferation. In this work it could be shown that the senescence phenotype in Lin9 knockout MEFs is independently mediated by the two tumor suppressor pathways p53-p21 and p16-pRB. Studies using the conditional Lin9 knockout mouse model demonstrated an important function of Lin9 in the regulation of mitotic gene expression and proliferation in vivo. Deletion of Lin9 caused reduced proliferation in the intestinal crypts resulting in atrophy of the intestinal epithelium and in rapid death of the animals. In the second part of this work, the pathways leading to p53 mediated G1 arrest after failed cytokinesis were analyzed by using a chemical inhibitor of the mitotic kinase Aurora B. In a high throughput siRNA screen the MAP kinase MAP3K4 was identified as an upstream activator of p53. It could be shown that MAP3K4 activates the downstream stress kinase p38b to induce the p53 mediated cell cycle arrest of tetraploid cells. p38b was required for the transcriptional activation of the p53 target gene p21 in response to Aurora B inhibition. In contrast, phosphorylation, stabilization and recruitment of p53 to the p21 promoter occured independently of p38 signaling. Partial inhibition of Aurora B demonstrated that chromosome missegregation also activates the MAP3K4-p38-p53 pathway, suggesting that subtle defects in mitosis are sufficient for inducing this stress signaling pathway. Although p38 was required for the G1 cell cycle arrest after mitotic failures, long-term co-inhibition of p38 and Aurora B resulted in reduced proliferation probably due to increased apoptosis. Presumably, MAP3K4-p38-p53 signaling is a common pathway that is activated after errors in mitosis or cytokinesis to arrest cells in G1 and to prevent chromosomal instability.
Stimulatory or superagonistic (SA) CD28-specific monoclonal antibodies (mAbs) are potent polyclonal activators of regulatory T cells and have proven highly effective as treatment in a wide range of rodent models for autoimmune and inflammatory diseases. In these models, a preferential activation of regulatory T cells was observed by in vivo administration of CD28SA. In stark contrast, human volunteers receiving TGN1412, a humanized CD28-specific mAb, experienced a life-threatening cytokine release syndrome during the first-in-man trial. Preclinical tests employing human peripheral blood mononuclear cells (PBMC) failed to announce the rapid cytokine release measured in the human volunteers in response to TGN1412. The aim of this thesis project was to find an explanation of why standard PBMC assays failed to predict the unexpected TGN1412-induced "cytokine storm" observed in human volunteers. CD28 superagonists can activate T cells without T cell receptor (TCR) ligation. They do depend, however, on “tonic” TCR signals received by MHC scanning, signals that they amplify. PBMC do not receive these signals in the circulation. Short-term in vitro preculture of human PBMC at a high cell density (HDC) resulted in massive cytokine release during subsequent TGN1412 stimulation. Restoration of reactivity was cell-contact dependent, associated with TCR polarization and tyrosine-phosphorylation, and blocked by HLA-specific mAb. In HDC, both CD4 T cells and monocytes functionally mature in a mutually dependent fashion. However, only CD4 memory T-cells proliferate upon TGN1412 stimulation, and were identified as the main source of pro-inflammatory cytokines. Importantly, responses to other T-cell activating agents were also enhanced if PBMC were first allowed to interact under tissue-like conditions. A new in vitro protocol is provided that returns circulating T-cells to a tissue-like status where they respond to TGN1412 stimulation, and it might represent a more reliable preclinical in vitro test for both activating and inhibitory immunomodulatory drugs. Finally, the surprising observation was made that the IgG1 “sibling” of TGN1412, which is of the poorly Fc receptor-binding IgG4 isotype, has a much lower stimulatory activity. We could exclude steric hindrance as an explanation and provide evidence for removal of TGN1112 from the T-cell surface by trans-endocytosis.
Animals need to evaluate their experiences in order to cope with new situations they encounter. This requires the ability of learning and memory. Drosophila melanogaster lends itself as an animal model for such research because elaborate genetic techniques are available. Drosphila larva even saves cellular redundancy in parts of its nervous system. My Thesis has two parts dealing with associative olfactory learning in larval Drosophila. Firstly, I tackle the question of odour processing in respect to odour quality and intensity. Secondly, by focusing on the evolutionarily conserved presynaptic protein Synapsin, olfactory learning on the cellular and molecular level is investigated. Part I.1. provides a behaviour-based estimate of odour similarity in larval Drosophila by using four recognition-type experiments to result in a combined, task-independent estimate of perceived difference between odour-pairs. A further comparison of these combined perceived differences to published calculations of physico-chemical difference reveals a weak correlation between perceptual and physico-chemical similarity. Part I.2. focuses on how odour intensity is interpreted in the process of olfactory learning in larval Drosophila. First, the dose-effect curves of learnability across odour intensities are described in order to choose odour intensities such that larvae are trained at intermediate odour intensity, but tested for retention either with that trained intermediate odour intensity, or with respectively HIGHer or LOWer intensities. A specificity of retention for the trained intensity is observed for all the odours used. Such intensity specificity of learning adds to appreciate the richness in 'content' of olfactory memory traces, and to define the demands on computational models of associative olfactory memory trace formation. In part II.1. of the thesis, the cellular site and molecular mode of Synapsin function is investigated- an evolutionarily conserved, presynaptic vesicular phosphoprotein. On the cellular level, the study shows a Synapsin-dependent memory trace in the mushroom bodies, a third-order “cortical” brain region of the insects; on the molecular level, Synapsin engages as a downstream element of the AC-cAMP-PKA signalling cascade.
Platelet activation and aggregation are essential to limit posttraumatic blood loss at sites of vascular injury, but also contribute to arterial thrombosis, leading to myocardial infarction and stroke. Thrombus formation is the result of well-defined molecular events, including agonist-induced elevation of intracellular calcium ([Ca2+]i) and series of cytoskeletal rearrangements. With the help of genetically modified mice, the work presented in this thesis identified novel mechanisms underlying the process of platelet activation in hemostasis and thrombosis. Store-operated calcium entry (SOCE) through Orai1 was previously shown to be the main Ca2+ influx pathway in murine platelets. The residual Ca2+ entry in the Orai1 deficient platelets suggested a role for additional non-store-operated Ca2+ (non-SOC) and receptor operated Ca2+ entry (ROCE) in maintaining platelet calcium homeostasis. Canonical transient receptor potential channel 6 (TRPC6), which is expressed in both human and murine platelets, has been attributed to be involved in SOCE as well as in diacylglycerol (DAG)-triggered ROCE. In the first part of the study, the function of TRPC6 in platelet Ca2+ signaling and activation was analyzed by using the TRPC6 knockout mice. In vitro agonist induced Ca2+ responses and in vivo platelet function were unaltered in Trpc6-/- mice. However, Trpc6-/- mice displayed a completely abolished DAG mediated Ca2+-influx but a normal SOCE. These findings identified TRPC6 as the major DAG operated ROC channel in murine platelets, but DAG mediated ROCE has no major functional relevance for hemostasis and thrombosis. In the second part of the thesis, the involvement of the PDLIM family member CLP36 in the signaling pathway of the major platelet collagen receptor glycoprotein (GP) VI was investigated. The GPVI/FcR-chain complex initiates platelet activation through a series of tyrosine phosphorylation events downstream of the FcR-chain-associated immunoreceptor tyrosine-based activation motif (ITAM). GPVI signaling has to be tightly regulated to prevent uncontrolled intravascular platelet activation, but the underlying mechanisms are not fully understood. The present study reports the adaptor protein CLP36 as a major inhibitor of GPVI-ITAM signaling in platelets. Platelets from mice expressing a truncated form of CLP36, (Clp36ΔLIM) and platelets from mice lacking the entire protein (Clp36-/-) displayed profound hyper-activation in response to GPVI-specific agonists, whereas GPCR signaling pathways remained unaffected. These alterations translated into accelerated thrombus formation and enhanced pro-coagulant activity of Clp36ΔLIM platelets and a pro-thrombotic phenotype in vivo. These studies revealed an unexpected inhibitory function of CLP36 in GPVI-ITAM signaling and established it as a key regulator of arterial thrombosis.
Is behaviour response or action? In this Thesis I study this question regarding a rather simple organism, the larva of the fruit fly Drosophila melanogaster. Despite its numerically simple brain and limited behavioural repertoire, it is nevertheless capable to accomplish surprisingly complex tasks. After association of an odour and a rewarding or punishing reinforcement signal, the learnt odour is able to retrieve the formed memory trace. However, the activated memory trace is not automatically turned into learned behaviour: Appetitive memory traces are behaviourally expressed only in absence of the rewarding tastant whereas aversive memory traces are behaviourally expressed in the presence of the punishing tastant. The ‘decision’ whether to behaviourally express a memory trace or not relies on a quantitive comparison between memory trace and current situation: only if the memory trace (after odour-sugar training) predicts a stronger sugar reward than currently present, animals show appetitive conditioned behaviour. Learned appetitive behaviour is best seen as active search for food – being pointless in the presence of (enough) food. Learned aversive behaviour, in turn, can be seen as escape from a punishment – being pointless in absence of punishment. Importantly, appetitive and aversive memory traces can be formed and retrieved independent from each other but also can, under appriate circumstances, summate to jointly organise conditioned behaviour. In contrast to learned behaviour, innate olfactory behaviour is not influenced by gustatory processing and vice versa. Thus, innate olfactory and gustatory behaviour is rather rigid and reflexive in nature, being executed almost regardless of other environmental cues. I suggest a behavioural circuit-model of chemosensory behaviour and the ‘decision’ process whether to behaviourally express a memory trace or not. This model reflects known components of the larval chemobehavioural circuit and provides clear hypotheses about the kinds of architecture to look for in the currently unknown parts of this circuit. The second chapter deals with gustatory perception and processing (especially of bitter substances). Quinine, the bitter tastant in tonic water and bitter lemon, is aversive for larvae, suppresses feeding behaviour and can act as aversive reinforcer in learning experiments. However, all three examined behaviours differ in their dose-effect dynamics, suggesting different molecular and cellular processing streams at some level. Innate choice behaviour, thought to be relatively reflexive and hard-wired, nevertheless can be influenced by the gustatory context. That is, attraction toward sweet tastants is decreased in presence of bitter tastants. The extent of this inhibitory effect depends on the concentration of both sweet and bitter tastant. Importantly, sweet tastants differ in their sensitivity to bitter interference, indicating a stimulus-specific mechanism. The molecular and cellular processes underlying the inhibitory effect of bitter tastants are unknown, but the behavioural results presented here provide a framework to further investigate interactions of gustatory processing streams.
Applications in various research areas such as signal processing, quantum computing, and computer vision, can be described as constrained optimization tasks on certain subsets of tensor products of vector spaces. In this work, we make use of techniques from Riemannian geometry and analyze optimization tasks on subsets of so-called simple tensors which can be equipped with a differentiable structure. In particular, we introduce a generalized Rayleigh-quotient function on the tensor product of Grassmannians and on the tensor product of Lagrange- Grassmannians. Its optimization enables a unified approach to well-known tasks from different areas of numerical linear algebra, such as: best low-rank approximations of tensors (data compression), computing geometric measures of entanglement (quantum computing) and subspace clustering (image processing). We perform a thorough analysis on the critical points of the generalized Rayleigh-quotient and develop intrinsic numerical methods for its optimization. Explicitly, using the techniques from Riemannian optimization, we present two type of algorithms: a Newton-like and a conjugated gradient algorithm. Their performance is analysed and compared with established methods from the literature.
The superfamily of G protein-coupled receptors (GPCR) regulates numerous physiological and pathophysiological processes. Hence GPCRs are of significant interest for pharmacological therapy. Embedded into cytoplasmic membranes, GPCRs represent the core of large signaling complexes, which are critical for transduction of exogenous stimuli towards activation of downstream signaling pathways. As a member of the GPCR family B, the parathyroid hormone receptor (PTHR) activates adenylyl cyclases, phospholipases C β as well as mitogen-activated protein kinase-dependent signaling pathways, thereby mediating endocrine and paracrine effects of parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), respectively. This regulates, calcium homeostasis, bone metabolism and bone development. Paradoxically, PTH is able to induce both catabolic and anabolic bone metabolism. The anabolic effect of PTH is successfully applied in the therapy of severe osteoporosis. Domination of anabolic or catabolic bone-metabolism is entailed by temporal and cell-type specific determinants. The molecular bases are presumably differential arrangements of adaptor proteins within large signaling complexes that may lead to differential activation of signaling pathways, thereby regulating physiological effects. The molecular mechanisms are largely unclear; thus, there is significant interest in revealing a better understanding of PTHR-related adaptor proteins. To identify novel adaptor proteins which direct PTHR signaling pathways, a proteomic screening approach was developed. In this screening, vav2, a guanine-nucleotide exchange factor (GEF) for small GTPases which regulates cytoskeleton reorganization, was found to interact with intracellular domains of PTHR. Evidence is provided that vav2 impairs PTH-mediated phospholipase C β (PLCβ) signaling pathways by competitive interactions with G protein αq subunits. Vice versa, PTH was shown to regulate phosphorylation and subsequent GEF activity of vav2. These findings may thus shed new light on the molecular mechanisms underlying the effects of PTH on bone metabolism by PLC-signaling, cell migration and cytoskeleton organization. In addition to the understanding of intracellular molecular signaling processes, screening for ligands is a fundamental and demanding prerequisite for modern drug development. To this end, ligand binding assays represent a fundamental technique. As a substitution for expensive and potentially harmful radioligand binding, fluorescence-based ligand-binding assays for PTHR were developed in this work. Based on time-resolved fluorescence, several assay variants were established to facilitate drug development for the PTHR.
Human society understands itself as a place where man enjoys an authentic human existence. In the society therefore, man should have all the basic necessities of life. The universal destination of all the created goods as God intends it, is for the good use of man in order to live a fulfilled human life. In so far as, man is the source, the focus and the end of all economic and social life, this must be respected. In order to enhance his dignity as the imago Dei , man has natural right to all the created goods. However, there are tools that make this situation possible. One of such tools is governance which is the means of organising the created things in the society for the common good. The ultimate goal and purpose of the society is the good of man. If the goods are not brought to this use, there exist socio-ethical problems. There are so many socio-ethical ills in the world today, because of the denial of the created good to some people by the organisers of the society. The social ills include armed robbery, corruption of all sorts, official lies and deceit. These constitute a complex social problem as regards their justification in the society. Subsequently, of all these social ills in the world today, corruption is the most devastating. Most people acknowledge this fact, but some others see it only as a means of survival, especially in countries that seem to appreciate or favour rigid capitalism, whereby wealth is power. Corruption is a common phenomenon in human society, and it is as old as the human society. Otive Igbuzor confirms it thus, “Corruption is as old as the society and cuts across nations, cultures, races and classes of people.” Corruption knows no bound. At any rate, corrupt practices vary from country to country and even within a country it also varies from section to section in its intensity and propensity. However, the manner with which it is manifesting itself presently accounts for its recent addition to the dictionary of the most global social problems. Unfortunately, nobody admits that he is corrupt and yet corruption as a social problem is widespread. In this light, Lightowler Brain observes that, “Worldwide corruption is by its nature generally hidden, but nevertheless insidious and as potentially dangerous to society.” It affects the society adversely, and therefore leads to misery of some members of the society. In this line, Peter Eigen argues, “Corruption is a daunting obstacle to sustainable development, and results in a major loss of public funds needed for basic necessities of life, both in developed and developing countries.” These are the devastating effects of corruption. The word corruption provokes an active discussion and condemnation among all classes of people in the society. It can be situated and discussed in any discipline but more especially in Social Ethics because this discipline deals with the interaction of human beings in the society. Problems in life are divided into private problems and social problems. Julius O. Onwuka, differentiates the two thus, “When a situation affects members of the society adversely leading to misery, regret, despair, it becomes a social problem. On the contrary, when a problem is suffered by an individual alone, without other members of the society being affected, this is a personal problem.” From Onwuka, I understand that, social problem is more problematic than personal problem, because it concerns more than one person. Social problems are seen in the societies of human beings where the basic necessities of life are denied some people by corrupt organisers of the society.
Melanoma is the most aggressive skin cancer with very limited treatment options. Upon appearance of metastases chemotherapeutics are used to either kill or slow down the growth of cancer cells by inducing apoptosis or senescence, respectively. With melanomas originating from melanocytes, it is vital to elucidate the mechanisms that distinguish senescence induction from proliferation and tumourigenicity. Xmrk (Xiphophorus melanoma receptor kinase), the fish orthologue of the human epidermal growth factor receptor (EGFR), causes highly aggressive melanoma in fish. Using an inducible variant, HERmrk, I showed that high receptor levels result in melanocyte senescence, whereas low and medium expression allows for cell proliferation and tumourigenicity. Mechanistically, HERmrk leads to increased reactive oxygen species (ROS) levels, which trigger a DNA damage response. Consequently, multinucleated, senescent cells develop by both endomitosis and fusion. Furthermore, oncogenic N‐RAS (N-‐RAS61K) induces a similar multinucleated phenotype in melanocytes. In addition, I found that both overexpression of C‐MYC and the knockdown of miz‐1 (Myc‐interacting zinc finger protein 1) diminished HERmrk‐induced senescence entry. C‐MYC prevent ROS induction, DNA damage and senescence, while acting synergistically with HERmrk in conveying tumourigenic features to melanocytes. Further analyses identified cystathionase (CTH) as a novel target gene of Myc and Miz-1 crucial for senescence prevention. CTH encodes an enzyme involved in the synthesis of cysteine from methionine, thereby allowing for increased ROS detoxification. Even though senescence was thought to be irreversible and hence tumour protective, I demonstrated that prolonged expression of the melanoma oncogene N‐RAS61K in pigment cells overcomes initial OIS by triggering the emergence of tumour‐initiating, mononucleated stem‐like cells from multinucleated senescent cells. This progeny is dedifferentiated, highly proliferative, anoikis‐resistant and induces fast‐growing, metastatic tumours upon transplantation into nude mice. Our data demonstrate that induction of OIS is not only a cellular failsafe mechanism, but also carries the potential to provide a source for highly aggressive, tumour‐initiating cells.
Argumentation and proof have played a fundamental role in mathematics education in recent years. The author of this dissertation would like to investigate the development of the proving process within a dynamic geometry system in order to support tertiary students understanding the proving process. The strengths of this dynamic system stimulate students to formulate conjectures and produce arguments during the proving process. Through empirical research, we classified different levels of proving and proposed a methodological model for proving. This methodological model makes a contribution to improve students’ levels of proving and develop their dynamic visual thinking. We used Toulmin model of argumentation as a theoretical model to analyze the relationship between argumentation and proof. This research also offers some possible explanation so as to why students have cognitive difficulties in constructing proofs and provides mathematics educators with a deeper understanding on the proving process within a dynamic geometry system.
This thesis on the “Impacts of extreme hydro-meteorological events on electricity generation and possible adaptation measures – a GIS-based approach for corporate risk management and enhanced climate mitigation concepts in Germany” presents an identification of hydro-meteorological extreme events in Germany and their effects on electricity generating units, i.e. on conventional thermal and nuclear power plants as well as on installations of the renewable energies of hydropower, wind energy and photovoltaic installations. In addition, adaptation measures and strategies are named that help power plant operators to prepare for a changing climate. Due to the different requirements of large facility operators and local planners and owners of renewable energies, the work contains the two approaches of corporate risk management and climate mitigation concepts. A changing climate not only consists of a shift in mean values of weather parameters such as global and regional air temperature and precipitation, but may also result in more frequent and more severe single events such as extreme precipitation, tornadoes and thunderstorms. In two case studies, these findings are implemented into an adjusted general risk management structure. This is enhanced by the use of Geographical Information Systems (GIS) to accomplish a localisation of events and infrastructure. The first example gives insight into the consequences of ice throw from wind turbines and how climate mitigation concepts can act as a framework for an adapted, sustainable energy planning. The second example on the other hand highlights a GIS-based flood risk management for thermal power plants and the benefits of an adjusted corporate risk management cycle. The described approach leads to an integrated management of extreme hydro-meteorological events at power plant site respectively district level by combining two cycles of site-related and local planning in addition to GIS-based analyses. This is demonstrated as an example by the comparison of two districts in Germany. The practical outcome is a comprehensive support for decision-making processes.
Two-Dimensional Electron Systems at Surfaces — Spin-Orbit Interaction and Electronic Correlations
(2012)
This thesis addresses three different realizations of a truly two-dimensional electron system (2DES), established at the surface of elemental semiconductors, i.e., Pt/Si(111), Au/Ge(111), and Sn/Si(111). Characteristic features of atomic structures at surfaces have been studied using scanning tunneling microscopy and low energy electron diffraction with special emphasis on Pt deposition onto Si(111). Topographic inspection reveals that Pt atoms agglomerate as trimers, which represent the structural building block of phase-slip domains. Surprisingly, each trimer is rotated by 30° with respect to the substrate, which results in an unexpected symmetry breaking. In turn, this represents a unique example of a chiral structure at a semiconductor surface, and marks Pt/Si(111) as a promising candidate for catalytic processes at the atomic scale. Spin-orbit interactions (SOIs) play a significant role at surfaces involving heavy adatoms. As a result, a lift of the spin degeneracy in the electronic states, termed as Rashba effect, may be observed. A candidate system to exhibit such physics is Au/Ge(111). Its large hexagonal Fermi sheet is suggested to be spin-split by calculations within the density functional theory. Experimental clarification is obtained by exploiting the unique capabilities of three-dimensional spin detection in spin- and angle-resolved photoelectron spectroscopy. Besides verification of the spin splitting, the in-plane components of the spin are shown to possess helical character, while also a prominent rotation out of this plane is observed along straight sections of the Fermi surface. Surprisingly and for the first time in a 2DES, additional in-plane rotations of the spin are revealed close to high symmetry directions. This complex spin pattern must originate from crystalline anisotropies, and it is best described by augmenting the original Rashba model with higher order Dresselhaus-like SOI terms. The alternative use of group-IV adatoms at a significantly reduced coverage drastically changes the basic properties of a 2DES. Electron localization is strongly enhanced, and the ground state characteristics will be dominated by correlation effects then. Sn/Si(111) is scrutinized with this regard. It serves as an ideal realization of a triangular lattice, that inherently suffers from spin frustration. Consequently, long-range magnetic order is prohibited, and the ground state is assumed to be either a spiral antiferromagnetic (AFM) insulator or a spin liquid. Here, the single-particle spectral function is utilized as a fundamental quantity to address the complex interplay of geometric frustration and electronic correlations. In particular, this is achieved by combining the complementary strengths of ab initio local density approximation (LDA) calculations, state-of-the-art angle-resolved photoelectron spectroscopy, and the sophisticated many-body LDA+DCA. In this way, the evolution of a shadow band and a band backfolding incompatible with a spiral AFM order are unveiled. Moreover, beyond nearest-neighbor hopping processes are crucial here, and the spectral features must be attributed to a collinear AFM ground state, contrary to common expectation for a frustrated spin lattice.
The superconducting properties of complex materials like the recently discovered iron-pnictides or strontium-ruthenate are often governed by multi-orbital effects. In order to unravel the superconductivity of those materials, we develop a multi-orbital implementation of the functional renormalization group and study the pairing states of several characteristic material systems. Starting with the iron-pnictides, we find competing spin-fluctuation channels that become attractive if the superconducting gap changes sign between the nested portions of the Fermi surface. Depending on material details like doping or pnictogen height, these spin fluctuations then give rise to $s_{\pm}$-wave pairing with or without gap nodes and, in some cases, also change the symmetry to $d$-wave. Near the transition from nodal $s_{\pm}$-wave to $d$-wave pairing, we predict the occurrence of a time-reversal symmetry-broken $(s+id)$-pairing state which avoids gap nodes and is therefore energetically favored. We further study the electronic instabilities of doped graphene, another fascinating material which has recently become accessible and which can effectively be regarded as multi-orbital system. Here, the hexagonal lattice structure assures the degeneracy of two $d$-wave pairing channels, and the system then realizes a chiral $(d+id)$-pairing state in a wide doping range around van-Hove filling. In addition, we also find spin-triplet pairing as well as an exotic spin-density wave phase which both become leading if the long-ranged hopping or interaction parameters are slightly modified, for example, by choosing different substrate materials. Finally, we consider the superconducting state of strontium-ruthenate, a possible candidate for chiral spin-triplet pairing with fascinating properties like the existence of half-quantum vortices obeying non-Abelian statistics. Using a microscopic three orbital description including spin-orbit coupling, we demonstrate that ferromagnetic fluctuations are still sufficient to induce this $\bs{\hat{z}}(p_x\pm ip_y)$-pairing state. The resulting superconducting gap reveals strong anisotropies on the $d_{xy}$-dominated Fermi-surface pocket and nearly vanishes on the other remaining two pockets.
The "Large Hadron Collider" (LHC) is currently the most powerful particle accelerator. It provides particle collisions at a center of mass energy in the Tera-electronvolt range, which had never been reached in a laboratory before. Thereby a new era in high energy particle physics has began. Now it is possible to test one of the most precise theories in physics, the Standard Model of particle physics, at these high energies. The purpose is particularly served by four large experiments installed at the LHC, namely "A Toroidal LHC ApparatuS" (ATLAS), the "Compact-Muon-Solenoid" (CMS), the "Large Hadron Collider beauty" (LHCb) and "A Large Ion Collider Experiment" (ALICE). Besides exploring the high energy behavior of the well-established portions of the Standard Model, one of the main objectives is to find the Higgs boson included in the model, but not discovered by any preceding effort. It is of tremendous importance since fermions and heavy electroweak gauge bosons acquire mass because of this boson. Although the success of the Standard Model in describing nature is already undisputed, there are some flaws due to observations inexplicable within this theory only. Therefore searches for physics beyond the Standard Model are promoted at the LHC experiments as well. In order to achieve the defined goals, crucial aspects are firstly precise measurements, to verify Standard Model predictions in detail, and secondly an evaluation of as much information as accessible by the detectors, to recognize new phenomena as soon as possible for subsequent optimizations. Both challenges are only possible with a superior understanding of the detectors. An inevitable contribution to attain this knowledge is a realistic simulation, partially requiring new implementation techniques to describe the very complex instrumentation. The research presented here is performed under the patronage of the ATLAS collaboration with a special focus on measurements done with muon spectrometer. Thus a first central issue is the performance of the spectrometer in terms of physics objects that are recognized by the device, the compatibility of data and the existing simulation as well as its improvement and finally the extension of the acceptance region. Once the excellent behavior and comprehension of the muon spectrometer is demonstrated, a second part addresses one physics use case of reconstructed muons. The electroweak force is part of the Standard Model and causes the interaction of heavy electroweak gauge bosons with fermions as well as their self-interaction. In proton-proton collisions such gauge bosons are produced. However, they decay immediately into a pair of fermions. In case of the Z boson, which is one of the gauge bosons, oppositely charged fermions of the same generation, including muons, emerge. The various decay modes are determined precisely at particle accelerators other than the LHC. However, the associated production of two Z bosons is measured less exactly at those facilities because of a very low cross section. The corresponding results acquired with the ATLAS experiment exceed all previous measurements in terms of statistics and accuracy. They are reported in this thesis as obtained from the observation of events with four charged leptons. The enhancement of the signal yield based on the extension of the muon spectrometer acceptance is especially emphasized as well as alternative methods to estimate background events. Furthermore, the impact on the probing of couplings of three Z bosons and intersection with the search for the Standard Model Higgs boson are pointed out.
The present thesis is devoted to the spectroscopic study of rare earth manganites RMnO3 (R = Gd, Dy, Tb, Eu(1 - x)Y(x)) in the submillimeter frequency range. A dynamic manifestation of a strong magnetoelectric coupling in these systems is the existence of electromagnons - spin waves excited by the electric component of the electromagnetic wave. The exact analytical solution of the Landau-Lifshitz equations obtained for cycloidal antiferromagnets builds the bridge between the inelastic neutron scattering and the optical experiments. A semi-quantitative agreement is achieved between the theory and the results by these two experimental techniques. Two suggested mechanisms of the magnetoelectric coupling, the inverse Dzyaloshinskii-Moriya (IDM) interaction and the symmetric Heisenberg exchange (HE) striction, are introduced in a perturbative manner. The qualitative conclusions regarding both static and dynamic electric properties are also in agreement with the experiment. GdMnO3 is the system in which the electromagnons were first detected at low frequencies. Far infrared measurements in GdMnO3 presented here have confirmed the existence of a second high frequency electromagnon at 75 reciprocal centimeter. The detection of an additional mode suggests the existence of at least short range ferroelectric order. Such order has not been observed in static experiments so far. The electromagnons in Eu(1 - x)Y(x)MnO3 helped to clarify the role of the rare earth magnetism. As the Y(3+) ions are diamagnetic and Eu(3+) ions possess Van Vleck paramagnetism only, it is the Mn subsystem that is primarily responsible for the magnetoelectric properties of rare earth manganites. The electromagnons in DyMnO3 and TbMnO3 do not change their excitation conditions upon the flop of the spin cycloid in external magnetic fields. This fact still lacks consistent theoretical explanation. Detailed measurements on TbMnO3 of different orientations have allowed to prove the existence of the IDM electromagnon. The study of DyMnO3 in external magnetic fields has shown that, depending on the Dy ordering, the electromagnons and static electric polarization can be either enhanced or suppressed. Thus, the magnetic order of rare earth moments still plays an important role. As a general result of the present work, the IDM interaction is capable to describe the static electric polarization and the weak electro-active excitation in the high-field phase of TbMnO3. The HE model is successful in explaining the high frequency electromagnon, including its excitation conditions and the spectral weight. However, both models are still unable to describe the energy and the spectral weight of the low frequency electromagnon. Further theoretical and experimental efforts are required in this direction.
The Mediterranean area reveals a strong vulnerability to future climate change due to a high exposure to projected impacts and a low capacity for adaptation highlighting the need for robust regional or local climate change projections, especially for extreme events strongly affecting the Mediterranean environment. The prevailing study investigates two major topics of the Mediterranean climate variability: the analysis of dynamical downscaling of present-day and future temperature and precipitation means and extremes from global to regional scale and the comprehensive investigation of temperature and rainfall extremes including the estimation of uncertainties and the comparison of different statistical methods for precipitation extremes. For these investigations, several observational datasets of CRU, E-OBS and original stations are used as well as ensemble simulations of the regional climate model REMO driven by the coupled global general circulation model ECHAM5/MPI-OM and applying future greenhouse gas (GHG) emission and land degradation scenarios.
This dissertation focuses on selected novels written by contemporary indigenous authors from Aotearoa/New Zealand and examines the fictional imagination of the human body as a medium of cultural identity and memory. The novels discussed are Keri Hulme’s »The Bone People« (1984), »Nights in the Gardens of Spain« (1995) and »The Uncle’s Story« (2000) by Witi Ihimaera as well as James George’s »Hummingbird« (2003). In order to further decolonisation processes and to come to terms with the colonial past and the complexity of present realities, the fictional works position the human body as an active entity in the negotiation of specific cultural epistemologies. This project explores the narrative translation of corporeality that is used to locate alternative concepts of identity and cultural memory. Taking into account indigenous perspectives, this thesis makes use of the current theoretical approaches presented by pragmatism and affect theory in order to analyse the investment of the novels in feeling and the reciprocal relationship between text and corporeality depicted by the narratives. On the one hand, the novels aim to undermine oppressive and marginalising categories by placing particular emphasis on »sensuous gaps« in the text. On the other hand, the narratives intend to construct alternative identities and evoke specific aspects of indigenous histories and knowledge by imagining the human body in terms of »sensuous inscription«. The novels portray individuals who act from a place in-between different cultures, and articulate a desire to dissolve polarities and emphasise individual and cultural transformation as a formative element in the creation of complex identities and new perspectives.
Acute graft-versus-host disease (aGvHD) is an immune syndrome associated with allogeneic hematopoietic cell transplantation (allo-HCT) that is mediated by alloreactive donor T cells attacking the gastrointestinal tract, liver, and skin of the host. Early diagnosis remains problematic and to date mainly relies on clinical symptoms and histopathology. Previously, different groups demonstrated that in order to cause aGvHD, alloreactive T cells require the expression of appropriate homing receptors to efficiently migrate from their priming sites to their target tissues. Therefore, the development of a predictive test based on the homing receptor expression profile of peripheral blood T cells seems attractive to identify patients at risk before the onset of aGvHD. The aim of this study was to analyze migrating alloreactive donor T cell kinetics in the peripheral blood early after allo-HCT in a murine model across minor histocompatibility antigens (miHAg) followed by a precise characterization of the homing receptor expression profile of migrating donor lymphocytes in order to identify suitable predictive markers. Combining daily bioluminescence imaging (BLI) and flow cytometry (FC) allowed defining two weeks of massive alloreactive donor T cell migration before clinical aGvHD symptoms became apparent. Peripheral blood donor T lymphocytes highly up-regulated the homing markers α4β7 integrin, and P- and E-selectin-ligand at peak time points of cell migration. The combination with the activation markers CD25 and CD69 and low expression levels of L-selectin allowed alloreactive donor T cell definition. Based on this migration phase we postulated a potential diagnostic window to precisely identify alloreactive donor T cells upon their homing receptor expression profile. Consequently, targeted pre-emptive treatment with rapamycin starting at the earliest detection time point of alloreactive donor T cells in the peripheral blood (day+6) significantly prolonged survival of treated mice. Based on this data, we propose a potential diagnostic window for alloreactive cell detection based on their homing receptor expression profile for a timely and effective therapeutic intervention before the clinical manifestation of aGvHD.
Attention-deficit/hyperactivity disorder (ADHD) is the most frequent psychiatric disorder in children and adolescents and is often treated with methylphenidate (MPH), resulting in MPH exposure in more than 1% of all children in many countries. A 2005 report on cytogenetic effects in peripheral lymphocytes from 12 ADHD children treated for 3 months with MPH raised questions about its genetic toxicity and potential carcinogenicity. A healthy control group (23 individuals), a chronically MPH-treated (>12 months) group (21 patients), and a drug naïve group of ADHD-affected children (26 patients), which was analyzed again after 3 months (17 patients) and 6 months (11 patients), provided samples for analysis of micronucleated lymphocytes. No significant alteration in genomic damage as seen as micronucleus frequency in peripheral lypmphocytes was detected after MPH treatment. No indication for genomic damage induced by MPH was obtained in this study. Ongoing studies in the USA, as well as continuation of recently published epidemiological cancer incidence analysis should provide additional reassurance for MPH-treated ADHD patients.
Protection of healthy tissues from infection with systemically administered vaccinia virus strains
(2012)
Oncolytic virotherapy using recombinant vaccinia virus strains is a promising approach for the treatment of cancer. To further improve the safety of oncolytic vaccinia viruses, the cellular microRNA machinery can be applied as the host’s own security mechanism to avoid unwanted viral replication in healthy tissues. MicroRNAs are a class of small single-stranded RNAs which due to their ability to mediate post-transcriptional gene-silencing, play a crucial role in almost every regulatory process in cellular metabolism. Different cancers display unique microRNA expression patterns, showing significant up- or downregulation of endogenously expressed microRNAs. Furthermore, the behavior of cancer cells can be altered by either adding microRNAs known to inhibit cancer cell spread and proliferation or suppressing cancer promoting microRNAs (oncomirs) making microRNAs promising targets for cancer gene therapy. The cell’s own RNAi machinery can also be utilized to control viral replication due to the virus dependence on the host cell replication machinery, a process controlled by microRNAs. GLV-1h68 is a replication-competent recombinant oncolytic vaccinia virus constructed and generated by Genelux Corp., San Diego, CA, USA which carries insertions of three reporter gene cassettes for detection and attenuation purposes and is currently being evaluated for cancer treatment in clinical trials. Though there are hardly any side effects found in GLV-1h68 mediated oncolytic therapy an increased tropism for replication exclusively in cancer cells is desirable. Therefore it was investigated whether or not further cancer cell specificity of a recombinant vaccinia virus strain could be obtained without compromising its oncolytic activity using microRNA interference. Let-7a is a well characterized microRNA known to be expressed in high levels in healthy tissues and strongly downregulated in most cancers. To control vaccinia virus replication rates, four copies of the mature human microRNA let-7a target sequence were cloned behind the stop codon in the 3’end of the vaccinia virus D4R gene, using a GLV-1h68 derivative, GLV-1h190, as parental strain yielding the new recombinant virus strain GLV-1h250. The D4R gene belongs to the group of early transcribed vaccinia genes and encodes an essential enzyme, uracil DNA glycosylase, which catalyzes the removal of uracil residues from double-stranded DNA. A defect in D4R prevents vaccinia virus from entering into the intermediate and late phase of replication, leading to an aborted virus replication. After expression of the microRNA target sequence from the vaccinia virus genome, the endogenously expressed microRNA-let-7a should recognize its target structure within the viral mRNA transcript, thereby binding and degrading the viral mRNA which should lead to a strong inhibition of the virus replication in healthy cells. GLV-1h250 replication rates in cancerous A549 lung adenocarcinoma cells, which show a strong down-regulation of microRNA let-7a, was comparable to the replication rates of its parental strain GLV-1h190 and the control strain GLV-1h68. In contrast, GLV-1h250 displayed a 10-fold decrease in viral replication in non-cancerous ERC cells when compared to GLV-1h190 and GLV-1h68. In A549 tumor bearing nude mice GLV-1h250 replicated exclusively in the tumorous tissue and resulted in efficient tumor regression without adverse effects leading to the conclusion that GLV-1h250 replicates preferentially in cancerous cells and tissues, which display low endogenous let-7a expression levels.
This thesis deals with the chaotic dynamics of nonlinear networks consisting of semiconductor lasers which have time-delayed self-feedbacks or mutual couplings. These semiconductor lasers are simulated numerically by the Lang-Kobayashi equations. The central issue is how the chaoticity of the lasers, measured by the maximal Lyapunov exponent, changes when the delay time is changed. It is analysed how this change of chaoticity with increasing delay time depends on the reflectivity of the mirror for the self-feedback or the strength of the mutal coupling, respectively. The consequences of the different types of chaos for the effect of chaos synchronization of mutually coupled semiconductor lasers are deduced and discussed. At the beginning of this thesis, the master stability formalism for the stability analysis of nonlinear networks with delay is explained. After the description of the Lang-Kobayashi equations and their linearizations as a model for the numerical simulation of semiconductor lasers with time-delayed couplings, the artificial sub-Lyapunov exponent $\lambda_{0}$ is introduced. It is explained how the sign of the sub-Lyapunov exponent can be determined by experiments. The notions of "strong chaos" and "weak chaos" are introduced and distinguished by their different scaling properties of the maximal Lyapunov exponent with the delay time. The sign of the sub-Lyapunov exponent $\lambda_{0}$ is shown to determine the occurence of strong or weak chaos. The transition sequence "weak to strong chaos and back to weak chaos" upon monotonically increasing the coupling strength $\sigma$ of a single laser's self-feedback is shown for numerical calculations of the Lang-Kobayashi equations. At the transition between strong and weak chaos, the sub-Lyapunov exponent vanishes, $\lambda_{0}=0$, resulting in a special scaling behaviour of the maximal Lyapunov exponent with the delay time. Transitions between strong and weak chaos by changing $\sigma$ can also be found for the Rössler and Lorenz dynamics. The connection between the sub-Lyapunov exponent and the time-dependent eigenvalues of the Jacobian for the internal laser dynamics is analysed. Counterintuitively, the difference between strong and weak chaos is not directly visible from the trajectory although the difference of the trajectories induces the transitions between the two types of chaos. In addition, it is shown that a linear measure like the auto-correlation function cannot unambiguously reveal the difference between strong and weak chaos either. Although the auto-correlations after one delay time are significantly higher for weak chaos than for strong chaos, it is not possible to detect a qualitative difference. If two time-scale separated self-feedbacks are present, the shorter feedback has to be taken into account for the definition of a new sub-Lyapunov exponent $\lambda_{0,s}$, which in this case determines the occurence of strong or weak chaos. If the two self-feedbacks have comparable delay times, the sub-Lyapunov exponent $\lambda_{0}$ remains the criterion for strong or weak chaos. It is shown that the sub-Lyapunov exponent scales with the square root of the effective pump current $\sqrt{p-1}$, both in its magnitude and in the position of the critical coupling strengths. For networks with several distinct sub-Lyapunov exponents, it is shown that the maximal sub-Lyapunov exponent of the network determines whether the network's maximal Lyapunov exponent scales strongly or weakly with increasing delay time. As a consequence, complete synchronization of a network is excluded for arbitrary networks which contain at least one strongly chaotic laser. Furthermore, it is demonstrated that the sub-Lyapunov exponent of a driven laser depends on the number of the incoherently superimposed inputs from unsynchronized input lasers. For networks of delay-coupled lasers operating in weak chaos, the condition $|\gamma_{2}|<\mathrm{e}^{-\lambda_{\mathrm{m}}\,\tau}$ for stable chaos synchronization is deduced using the master stability formalism. Hence, synchronization of any network depends only on the properties of a single laser with self-feedback and the eigenvalue gap of the coupling matrix. The characteristics of the master stability function for the Lang-Kobayashi dynamics is described, and consequently, the master stability function is refined to allow for precise practical prediction of synchronization. The prediction of synchronization with the master stability function is demonstrated for bidirectional and unidirectional networks. Furthermore, the master stability function is extended for two distinct delay times. Finally, symmetries and resonances for certain values of the ratio of the delay times are shown for the master stability function of the Lang-Kobyashi equations.
Multi-Wavelength Observations of the high-peaked BL Lacertae objects 1ES 1011+496 and 1ES 2344+514
(2012)
BL Lacertae objects belong to the most luminous sources in the Universe. They represent a subclass of active galactic nuclei with a spectrum that is dominated by non-thermal emission, extending from radio wavelengths to tera electronvolt (TeV) energies. The emission is strongly variable on time scales of years down to minutes, and arises from relativistic jets pointing at small angles to the line of sight of the observer, which is the reason for naming them “blazars”. Blazars are the dominant extragalactic source class in the radio, microwave and gamma-ray regime, are prime candidates for the origin of the Cosmic Rays and excellent laboratories to study black hole and jet physics as well as relativistic effects. Despite more than 20 years of observational efforts, the physical mechanisms driving their emission are not yet fully understood. So far, studies of their broad-band continuum emission were mostly concentrated on bright, flaring states. However, for a better understanding of the central engine powering the jets, the bias from flux-limited observations of the past must be overcome and their long-term average continuum spectral energy distributions (SEDs) must be determined. This work presents the first simultaneous multi-wavelength campaigns from the radio to the TeV regime of two high-frequency peaked BL Lacertae objects known to emit at TeV energies. The first source, 1ES 1011+496, was observed between February and May 2008, the second one, 1ES 2344+514, between September 2008 and February 2009. The extensive observational campaigns were organised independently from an external trigger for the presence of a flaring state. Since the duty cycle of major flux outbursts is known to be rather low, the campaigns were expected to yield SEDs representative of the long-term average emission. Central for this thesis is the analysis of data obtained with the MAGIC Cherenkov telescope, measuring energy spectra and light curves from ~0.1 to ~10 TeV. For the remaining instruments, observation time was proposed and additional data was organised by collaboration with the instrument teams by the author of this work. Such data was obtained mostly in a fully reduced state. Individual light curves are investigated as well as combined in a search for inter-band correlations. The data of both sources reveal a notable lack of a correlation between the emission at radio and optical wavelengths, indicating that the radio and short-wavelength emission arise in different regions of the jet. Quasi-simultaneous SEDs of two different flux states are observationally determined and described by a one-zone as well as a self-consistent two-zone synchrotron self-Compton model. First approaches to model the SEDs by means of a Chi2 minimisation technique are briefly discussed. The SEDs and the resulting model parameters, characterising the physical conditions in the emission regions, are compared to archival data. Though the models can describe the data well, for 1ES 1011+496 the model parameters indicate that in addition to the synchrotron and inverse-Compton emission of relativistic electrons, emission due to accelerated protons seems to be required. The SEDs of 1ES 2344+514 reveal one of the lowest activity states ever detected from the source. Despite that, the model parameters are not indicative of a distinct quiescent state, which may be caused by the degeneracy of the different parameters in one-zone models. Moreover, indications accumulate that the radiation can not be attributed to a single emission region. The results disfavour some of the current blazar classification schemes and the so-called “blazar sequence”, emphasising the need for a more realistic explanation of the systematics of the blazar SEDs in terms of fundamental parameters.
Both specific stimulus valence and unspecific processing dynamics can influence evaluative responses. Eight experiments investigated their respective influence on evaluative judgments in the domain of stereotyping. Valence of stereotypic information and consistency-driven fluency were manipulated in an impression formation paradigm. When information about the to-be-evaluated target person was strongly valenced, no effects of consistency-driven fluency were observed. Higher cognitive processes, valence of inconsistent attributes, processing priority of category information, and impression formation instructions were ruled out as possible factors responsible for the non-occurrence of fluency effects. However, consistency-driven fluency did influence the evaluative judgment, if the information about a target person was not strongly valenced. It is therefore concluded that both stimulus valence and consistency-driven processing fluency play a role in evaluative judgments in the domain of stereotyping. The respective impact of stimulus valence is much stronger than the impact of unspecific processing dynamics, however. Implications for fluency research and the applied field of stereotype change are discussed.
In this study, a double-donor concept is used to improve the performance of thermally evaporated merocyanine(s)/C60 bulk heterojunction (BHJ) solar cells. It is shown that the co-evaporation of two merocyanine dyes with absorption bands at ~ 500 nm (SW dye) and ~ 650 nm (LW dye), respectively, together with C60 fullerene results in an improvement of open-circuit voltage (VOC), short-circuit current (JSC) as well as total power conversion efficiency (PCE) compared to the best single-donor cell. The enhancement of JSC is attributed to a higher photon harvesting efficiency of the mixed-donor devices due to a better spectral coverage.
Upon synthesis, nascent polypeptide chains are subject to major rearrangements of their side chains to obtain an energetically more favorable conformation in a process called folding. About one third of all cellular proteins pass through the secretory pathway and undergo oxidative folding in the endoplasmic reticulum (ER). During oxidative folding, the conformational rearrangements are accompanied by the formation of disulfide bonds – covalent bonds between cysteine side chains that form upon oxidation. Protein disulfide isomerase (PDI) assists in the folding of substrates by catalyzing the oxidation of pairs of cysteine residues and the isomerization of disulfide bonds as well as by acting as chaperones. In addition to PDI itself, a family of related ER-resident proteins has formed. All PDI family members share the thioredoxin fold in at least one of their domains and exhibit a subset of the PDI activities. Despite many studies, the role of most PDI family members remains unclear. The project presented in this thesis was aimed to establish tools for the biochemical characterization of single members of the PDI family and their role in the folding process. A combination of fluorescence based assays was developed to selectively study single functions of PDI family members and relate their properties of either catalysis of oxidation or catalysis of isomerization or chaperone activity to the rest of the protein family. A binding assay using isothermal titration calorimetry (ITC) was established to complement the activity assays. Using ITC we could show for the first time that members of the PDI family can distinguish between folded and unfolded proteins selectively binding the latter. The unique information provided by this method also revealed a two-site binding of unfolded proteins by PDI itself. In addition to the functional characterization, experiments were conducted to further investigate the oligomeric state of PDI. We could show that the equilibrium between structurally different states of PDI is heavily influenced by the redox state of the protein and its environment. This new data could help to further our understanding of the interplay between oxidases like PDI and their regenerative enzymes like Ero1, which may be governed by structural changes in response to the change in redox status. Another structural approach was the screening of all investigated PDI family members for suitable crystallization conditions. As a result of this screening we could obtain protein crystals of human ERp27 and were able to solve the structure of this protein with X-ray crystallography. The structure gives insight into the mechanisms of substrate binding domains within the PDI family and helps to understand the interaction of ERp27 with the redox active ERp57. In collaboration with the group of Heike Hermanns we could further show the physiological importance of this interaction under oxidative stress. In conclusion, the project presented in this thesis provides novel tools for an extensive analysis of the activities of single PDI family members as well as a useful set of methods to characterize novel oxidoreductases and chaperones. The initial results obtained with the our novel methods are very promising. At the same time, the structural approach of this project could successfully solve the structure of a PDI family member and give information about the interplay within the PDI family.