DNA methylation in Dictyostelium discoideum

DNA methyltransferases of type Dnmt2 are a highly conserved protein family with enigmatic function. The aim of this work was to characterize DnmA, the Dnmt2 methyltransferase in Dictyostelium discoideum, and further to investigate its implication in DNA methylation and transcriptional gene silencing. The genome of the social amoeba Dictyostelium encodes DnmA as the sole DNA methyltransferase. The enzyme bears all ten characteristic DNA methyltransferase motifs in its catalytic domain. The DnmA mRNA was found by RT-PCR to be expressed during vegetative growth and down regulated during development. Investigations using fluorescence microscopy showed that both DnmA-myc and DnmA-GFP fusions predominantly localised to the nucleus. The function of DnmA remained initially unclear, but later experiment revealed that the enzyme is an active DNA methyltransferase responsible for all DNA (cytosine) methylation in Dictyostelium. Neither in gel retardation assays, nor by the yeast two hybrid system, clues on the functionality of DnmA could be obtained. However, immunological detection of the methylation mark with an α - 5mC antibody gave initial evidence that the DNA of Dictyostelium was methylated. Furthermore, addition of 5-aza-cytidine as demethylating agent to the Dictyostelium medium and subsequent in vitro incubation of the DNA isolated from these cells with recombinant DnmA showed that the enzyme binds slightly better to this target DNA. In order to investigate further the function of the protein, a gene knock-out for dnmA was generated. The gene was successfully disrupted by homologous recombination, the knock-out strain, however, did not show any obvious phenotype under normal laboratory conditions. To identify specific target sequences for DNA methylation, a microarray analysis was carried out. Setting a threshold of at least 1.5 fold for differences in the strength of gene expression, several such genes in the knock-out strain were chosen for further investigation. Among the up-regulated genes were the ESTs representing the gag and the RT genes respectively of the retrotransposon skipper. In addition Northern blot analysis confirmed the up-regulation of skipper in the DnmA knock-out strain. Bisufite treatment and sequencing of specific DNA stretches from skipper revealed that DnmA is responsible for methylation of mostly asymmetric cytosines. Together with skipper, DIRS-1 retrotransposon was found later also to be methylated but was not present on the microarray. Furthermore, skipper transcription was also up-regulated in strains that had genes disrupted encoding components of the RNA interference pathway. In contrast, DIRS 1 expression was not affected by a loss of DnmA but was strongly increased in the strain that had the RNA directed RNA polymerase gene rrpC disrupted. Strains generated by propagating the usual wild type Ax2 and the DnmA knock-out cells over 16 rounds in development were analyzed for transposon activity. Northern blot analysis revealed activation for skipper expression, but not for DIRS-1. A large number of siRNAs were found to be correspondent to the DIRS-1 sequence, suggesting concerted regulation of DIRS-1 expression by RNAi and DNA methylation. In contrast, no siRNAs corresponding to the standard skipper element were found. The data show that DNA methylation plays a crucial role in epigenetic gene regulation in Dictyostelium and that different, partially overlapping mechanisms control transposon silencing for skipper and DIRS-1. To elucidate the mechanism of targeting the protein to particular genes in the Dictyostelium genome, some more genes which were up-regulated in the DnmA knock-out strain were analyzed by bisulfite sequencing. The chosen genes are involved in the multidrug response in other species, but their function in Dictyostelium is uncertain. Bisulfite data showed that two of these genes were methylated at asymmetrical C-residues in the wild type, but not in DnmA knock-out cells. This suggested that DNA methylation in Dictyostelium is involved not only in transposon regulation but also in transcriptional silencing of specific genes.

Protein-Expressions-Analysen zur Etablierung prädiktiver Biomarker bei der Behandlung fortgeschrittener Tumoren der Mamma und der Prostata

Aktuelle Entwicklungen auf dem Gebiet der zielgerichteten Therapie zur Behandlung maligner Erkrankungen erfordern neuartige Verfahren zur Diagnostik und Selektion geeigneter Patienten. So ist das Ziel der vorliegenden Arbeit die Identifizierung neuer Zielmoleküle, die die Vorhersage eines Therapieerfolges mit targeted drugs ermöglichen. Besondere Aufmerksamkeit gilt dem humanisierten monoklonalen Antikörper Trastuzumab (Herceptin), der zur Therapie Her-2 überexprimierender, metastasierter Mammakarzinome eingesetzt wird. Jüngste Erkenntnisse lassen eine Anwendung dieses Medikamentes in der Behandlung des Hormon-unabhängigen Prostatakarzinoms möglich erscheinen. Therapie-beeinflussende Faktoren werden in der dem Rezeptor nachgeschalteten Signaltransduktion oder Veränderungen des Rezeptors selbst vermutet. Mittels Immunhistochemie wurden die Expressions- und Aktivierungsniveaus verschiedener Proteine der Her-2-assoziierten Signaltransduktion ermittelt; insgesamt wurden 37 molekulare Marker untersucht. In Formalin fixierte und in Paraffin eingebettete korrespondierende Normal- und Tumorgewebe von 118 Mammakarzinom-Patientinnen sowie 78 Patienten mit Prostatakarzinom wurden in TMAs zusammengefasst. Die in Zusammenarbeit mit erfahrenen Pathologen ermittelten Ergebnisse dienten u.a. als Grundlage für zweidimensionales, unsupervised hierarchisches clustering. Ergebnis dieser Analysen war für beide untersuchten Tumorentitäten die Möglichkeit einer Subklassifizierung der untersuchten Populationen nach molekularen Eigenschaften. Hierbei zeigten sich jeweils neue Möglichkeiten zur Anwendung zielgerichteter Therapien, deren Effektivität Inhalt weiterführender Studien sein könnte. Zusätzlich wurden an insgesamt 43 Frischgeweben die möglichen Folgen des sog. shedding untersucht. Western Blot-basierte Untersuchungen zeigten hierbei die Möglichkeit der Selektion von Patienten aufgrund falsch-positiver Befunde in der derzeit als Standard geltenden Diagnostik. Zusätzlich konnte durch Vergleich mit einer Herceptin-sensitiven Zelllinie ein möglicher Zusammenhang eines Therapieerfolges mit dem Phosphorylierungs-/ Aktivierungszustand des Rezeptors ermittelt werden. Fehlende klinische Daten zum Verlauf der Erkrankung und Therapie der untersuchten Patienten lassen keine Aussagen über die tatsächliche Relevanz der ermittelten Befunde zu. Dennoch verdeutlichen die erhaltenen Resultate eindrucksvoll die Komplexität der molekularen Vorgänge, die zu einem Krebsgeschehen führen und damit Auswirkungen auf die Wirksamkeit von targeted drugs haben können. Entwicklungen auf dem Gebiet der zielgerichteten Therapie erfordern Verbesserungen auf dem Gebiet der Diagnostik, die die sichere Selektion geeigneter Patienten erlauben. Die Zukunft der personalisierten, zielgerichteten Behandlung von Tumorerkrankungen wird verstärkt von molekularen Markerprofilen hnlich den hier vorgestellten Daten beeinflusst werden.

Design, implementation and application of a generic framework for integrated regional land-use modeling

Land use is a crucial link between human activities and the natural environment and one of the main driving forces of global environmental change. Large parts of the terrestrial land surface are used for agriculture, forestry, settlements and infrastructure. Given the importance of land use, it is essential to understand the multitude of influential factors and resulting land use patterns. An essential methodology to study and quantify such interactions is provided by the adoption of land-use models. By the application of land-use models, it is possible to analyze the complex structure of linkages and feedbacks and to also determine the relevance of driving forces. Modeling land use and land use changes has a long-term tradition. In particular on the regional scale, a variety of models for different regions and research questions has been created. Modeling capabilities grow with steady advances in computer technology, which on the one hand are driven by increasing computing power on the other hand by new methods in software development, e.g. object- and component-oriented architectures. In this thesis, SITE (Simulation of Terrestrial Environments), a novel framework for integrated regional sland-use modeling, will be introduced and discussed. Particular features of SITE are the notably extended capability to integrate models and the strict separation of application and implementation. These features enable efficient development, test and usage of integrated land-use models. On its system side, SITE provides generic data structures (grid, grid cells, attributes etc.) and takes over the responsibility for their administration. By means of a scripting language (Python) that has been extended by language features specific for land-use modeling, these data structures can be utilized and manipulated by modeling applications. The scripting language interpreter is embedded in SITE. The integration of sub models can be achieved via the scripting language or by usage of a generic interface provided by SITE. Furthermore, functionalities important for land-use modeling like model calibration, model tests and analysis support of simulation results have been integrated into the generic framework. During the implementation of SITE, specific emphasis was laid on expandability, maintainability and usability. Along with the modeling framework a land use model for the analysis of the stability of tropical rainforest margins was developed in the context of the collaborative research project STORMA (SFB 552). In a research area in Central Sulawesi, Indonesia, socio-environmental impacts of land-use changes were examined. SITE was used to simulate land-use dynamics in the historical period of 1981 to 2002. Analogous to that, a scenario that did not consider migration in the population dynamics, was analyzed. For the calculation of crop yields and trace gas emissions, the DAYCENT agro-ecosystem model was integrated. In this case study, it could be shown that land-use changes in the Indonesian research area could mainly be characterized by the expansion of agricultural areas at the expense of natural forest. For this reason, the situation had to be interpreted as unsustainable even though increased agricultural use implied economic improvements and higher farmers' incomes. Due to the importance of model calibration, it was explicitly addressed in the SITE architecture through the introduction of a specific component. The calibration functionality can be used by all SITE applications and enables largely automated model calibration. Calibration in SITE is understood as a process that finds an optimal or at least adequate solution for a set of arbitrarily selectable model parameters with respect to an objective function. In SITE, an objective function typically is a map comparison algorithm capable of comparing a simulation result to a reference map. Several map optimization and map comparison methodologies are available and can be combined. The STORMA land-use model was calibrated using a genetic algorithm for optimization and the figure of merit map comparison measure as objective function. The time period for the calibration ranged from 1981 to 2002. For this period, respective reference land-use maps were compiled. It could be shown, that an efficient automated model calibration with SITE is possible. Nevertheless, the selection of the calibration parameters required detailed knowledge about the underlying land-use model and cannot be automated. In another case study decreases in crop yields and resulting losses in income from coffee cultivation were analyzed and quantified under the assumption of four different deforestation scenarios. For this task, an empirical model, describing the dependence of bee pollination and resulting coffee fruit set from the distance to the closest natural forest, was integrated. Land-use simulations showed, that depending on the magnitude and location of ongoing forest conversion, pollination services are expected to decline continuously. This results in a reduction of coffee yields of up to 18% and a loss of net revenues per hectare of up to 14%. However, the study also showed that ecological and economic values can be preserved if patches of natural vegetation are conservated in the agricultural landscape. -----------------------------------------------------------------------

Influence of the buffer environment on the Relative Biological Effectiveness of carbon ion radiation, investigated by Scanning Force Microscopy and gel electrophoresis

This work focuses on the analysis of the influence of environment on the relative biological effectiveness (RBE) of carbon ions on molecular level. Due to the high relevance of RBE for medical applications, such as tumor therapy, and radiation protection in space, DNA damages have been investigated in order to understand the biological efficiency of heavy ion radiation. The contribution of this study to the radiobiology research consists in the analysis of plasmid DNA damages induced by carbon ion radiation in biochemical buffer environments, as well as in the calculation of the RBE of carbon ions on DNA level by mean of scanning force microscopy (SFM). In order to study the DNA damages, besides the common electrophoresis method, a new approach has been developed by using SFM. The latter method allows direct visualisation and measurement of individual DNA fragments with an accuracy of several nanometres. In addition, comparison of the results obtained by SFM and agarose gel electrophoresis methods has been performed in the present study. Sparsely ionising radiation, such as X-rays, and densely ionising radiation, such as carbon ions, have been used to irradiate plasmid DNA in trishydroxymethylaminomethane (Tris buffer) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES buffer) environments. These buffer environments exhibit different scavenging capacities for hydroxyl radical (HO0), which is produced by ionisation of water and plays the major role in the indirect DNA damage processes. Fragment distributions have been measured by SFM over a large length range, and as expected, a significantly higher degree of DNA damages was observed for increasing dose. Also a higher amount of double-strand breaks (DSBs) was observed after irradiation with carbon ions compared to X-ray irradiation. The results obtained from SFM measurements show that both types of radiation induce multiple fragmentation of the plasmid DNA in the dose range from D = 250 Gy to D = 1500 Gy. Using Tris environments at two different concentrations, a decrease of the relative biological effectiveness with the rise of Tris concentration was observed. This demonstrates the radioprotective behavior of the Tris buffer solution. In contrast, a lower scavenging capacity for all other free radicals and ions, produced by the ionisation of water, was registered in the case of HEPES buffer compared to Tris solution. This is reflected in the higher RBE values deduced from SFM and gel electrophoresis measurements after irradiation of the plasmid DNA in 20 mM HEPES environment compared to 92 mM Tris solution. These results show that HEPES and Tris environments play a major role on preventing the indirect DNA damages induced by ionising radiation and on the relative biological effectiveness of heavy ion radiation. In general, the RBE calculated from the SFM measurements presents higher values compared to gel electrophoresis data, for plasmids irradiated in all environments. Using a large set of data, obtained from the SFM measurements, it was possible to calculate the survive rate over a larger range, from 88% to 98%, while for gel electrophoresis measurements the survive rates have been calculated only for values between 96% and 99%. While the gel electrophoresis measurements provide information only about the percentage of plasmids DNA that suffered a single DSB, SFM can count the small plasmid fragments produced by multiple DSBs induced in a single plasmid. Consequently, SFM generates more detailed information regarding the amount of the induced DSBs compared to gel electrophoresis, and therefore, RBE can be calculated with more accuracy. Thus, SFM has been proven to be a more precise method to characterize on molecular level the DNA damage induced by ionizing radiations.

Biological Photonic Crystals: Diatoms

The aim of this work was to produce a variety of fluorescent diatom cell wall material as a basis for spectroscopic investigations of the influence of the photonic structure on the emission of an incorporated laser dye. This goal was achieved by the method of in vivo-fluorochromation, in which the fluorescence dyes are incorporated by the diatom cells during cell wall formation. Several fluorescent dyes (mostly rhodamines) known as strong laser dyes, were tested for a possible application within this method. The results of this work show that half of the tested rhodamines can be applied for an in vivo-fluorochromation of diatom cells. For a successful incorporation into the diatom cell wall, a relatively low toxicity to diatom cells is necessary. Replacement of the carbon acid function at the carboxyphenyl ring of the rhodamine by a methyl or ethylester function showed to convert a rhodamine of relatively low toxicity to a rhodamine leading to severe lethal effects within the cells. In contrast to their carbon acid forms, which posses a net neutral charge of the molecule, rhodamine esters exhibit a net positive charge. The enhanced toxicological effects seem to be due to an increased accumulation of positive charged rhodamines within the mitochondria, an increased hydrophobicity due to the attachment of an alkyl substituent, an increased retention time of the dyes within the mitochondria and a therefore stronger negative effect on the mitochondrial membrane bound energy processes of the diatom cell. Therefore rhodamines with a positive net charge deriving from a methyl or ethylester function at the carboxy phenyl ring instead of a carbon acid substituent showed not to be suitable for long-term investigations/ biomineralization studies of diatoms. Investigations performed on diatom species of different orders showed that rhodamine 19, rhodamine B, and rhodamine 101 can presumably be successfully applied for in vivo-fluorochromation to all diatom species. The results obtained here can help to find further laser dyes for an in vivo-fluorochromation of diatom cells and therefore for the production of fluorescent nanostructural elements for a detailed optical investigation of the diatom cell wall. First optical measurements performed on in vivo-fluorochromated cell walls did not give any hints concerning the photonic structure of the diatom cell. Cell wall parts with different nanostructural elements were investigated and by comparison of the obtained fluorescence emission spectra, no special features that might derive from photonic structural effects could be observed. Results concerning the concentration dependent shifts within the emission spectra, as well as the decrease of fluorescence intensity of the stained cell wall structures with increasing dye concentration, depict that several effects occurring by interaction of the molecules within the cell wall can have an impact on the technical application of fluorescent cell walls. It can be assumed that the investigation of the photonic crystal behaviour and the possibility to achieve laser action within the diatom cell wall can be hampered by molecular interactions. The results give hints to prevent such obstacles. Comparison of the recent findings and state of the art of in vivo-fluorochromation of diatom cell wall material, make clear that the here presented results are of importance and can offer a considerable contribution to the development and establishment of new biosilification markers, for diatoms as well as for other biosilifying organisms.

A Modeling Analysis of Changing Global Land Use as affected by Changing Demands for Wood Products

The 21st century has brought new challenges for forest management at a time when globalization in world trade is increasing and global climate change is becoming increasingly apparent. In addition to various goods and services like food, feed, timber or biofuels being provided to humans, forest ecosystems are a large store of terrestrial carbon and account for a major part of the carbon exchange between the atmosphere and the land surface. Depending on the stage of the ecosystems and/or management regimes, forests can be either sinks, or sources of carbon. At the global scale, rapid economic development and a growing world population have raised much concern over the use of natural resources, especially forest resources. The challenging question is how can the global demands for forest commodities be satisfied in an increasingly globalised economy, and where could they potentially be produced? For this purpose, wood demand estimates need to be integrated in a framework, which is able to adequately handle the competition for land between major land-use options such as residential land or agricultural land. This thesis is organised in accordance with the requirements to integrate the simulation of forest changes based on wood extraction in an existing framework for global land-use modelling called LandSHIFT. Accordingly, the following neuralgic points for research have been identified: (1) a review of existing global-scale economic forest sector models (2) simulation of global wood production under selected scenarios (3) simulation of global vegetation carbon yields and (4) the implementation of a land-use allocation procedure to simulate the impact of wood extraction on forest land-cover. Modelling the spatial dynamics of forests on the global scale requires two important inputs: (1) simulated long-term wood demand data to determine future roundwood harvests in each country and (2) the changes in the spatial distribution of woody biomass stocks to determine how much of the resource is available to satisfy the simulated wood demands. First, three global timber market models are reviewed and compared in order to select a suitable economic model to generate wood demand scenario data for the forest sector in LandSHIFT. The comparison indicates that the ‘Global Forest Products Model’ (GFPM) is most suitable for obtaining projections on future roundwood harvests for further study with the LandSHIFT forest sector. Accordingly, the GFPM is adapted and applied to simulate wood demands for the global forestry sector conditional on selected scenarios from the Millennium Ecosystem Assessment and the Global Environmental Outlook until 2050. Secondly, the Lund-Potsdam-Jena (LPJ) dynamic global vegetation model is utilized to simulate the change in potential vegetation carbon stocks for the forested locations in LandSHIFT. The LPJ data is used in collaboration with spatially explicit forest inventory data on aboveground biomass to allocate the demands for raw forest products and identify locations of deforestation. Using the previous results as an input, a methodology to simulate the spatial dynamics of forests based on wood extraction is developed within the LandSHIFT framework. The land-use allocation procedure specified in the module translates the country level demands for forest products into woody biomass requirements for forest areas, and allocates these on a five arc minute grid. In a first version, the model assumes only actual conditions through the entire study period and does not explicitly address forest age structure. Although the module is in a very preliminary stage of development, it already captures the effects of important drivers of land-use change like cropland and urban expansion. As a first plausibility test, the module performance is tested under three forest management scenarios. The module succeeds in responding to changing inputs in an expected and consistent manner. The entire methodology is applied in an exemplary scenario analysis for India. A couple of future research priorities need to be addressed, particularly the incorporation of plantation establishments; issue of age structure dynamics; as well as the implementation of a new technology change factor in the GFPM which can allow the specification of substituting raw wood products (especially fuelwood) by other non-wood products.

The Corticiaceae (Basidiomycetes) in Taiwan

Geographically, Taiwan is an Island and situated in the northeast of Asia, on the western side of the Pacific Basin, at the southeast of main China, south of Japan, and north of the Philippines. The main topographic character is the longitudinally oriented mountainous area. More than 200 peaks rise above 3000 m. They departed Taiwan into two lowland areas, an eastern and western plain. Taiwan is departed into subtropical (north area) and tropical zone (south area), which have a warm and humid climate, due to the Tropic of Cancer passing through. The average annual temperature in the lowland amounts to 28°C (7~38°C). The temperate climate also presents in the mountainous areas. The tropical typhoons usually come in summer and bring heavy rain, while the monsoon seasons have an important effect on the regional rainfall distribution. The mean annual rainfall of Taiwan is about 2600 mm (1000~6700 mm); the mountainous areas receive more rain than the lowlands. In Taiwan, according to different temperature and vegetation, the ecological environments were given rise to vertical biotic zonations, and form five major types: highland snowfield, highland meadow, coniferous forest, deciduous forest, and tropical forest. Six National Parks in Taiwan are located in the mountainous areas, in the north, the south, and on Jinmen Island. The National Parks represent about 8.4% of the country area. In this study, the collection sites are situated in Yangmingshan, Shei-Pa, Yushan, and Kenting National Park. Due to the island isolation, the proportions of endemic species are great in Taiwan, which also presents a high biodiversity. There are 4255 species of vascular plants including 1133 endemic. 5936 species in 1276 genera of fungi are hitherto reported in Taiwan. Among them, 233 Corticiaceae species were recorded, over one third (79 species) of them are known only from Taiwan. The first fungal report in Taiwan is about Phytophthora cyperi, published by the Japanese researcher T. Kawakami in 1904. Therefore, the history of research about fungi in Taiwan is more than one hundred years old. An eminent Japanese mycologist K. Sawada made an intensive survey from 1919 to 1959, and reported 2464 fungi species in his eleven volumes of “Descriptive Catalogue of Formosan Fungi”. However, only a few species (21 species in 9 genera) of Corticiaceae were recorded. From 1973, Chen and Lin resumed the study on Corticiaceae, and also some other foreign mycologists contributed for this field after 1980. The German research group lead by Franz Oberwinkler from Tübingen University collected in Taiwan several times. They published a number of new species and new records. Since 1989, S. H. Wu, a Taiwanese mycologist, has published a great amount of reports on corticioid fungi from Taiwan. Corticioid fungi were made up by the large and heterogeneous unnatural family Corticiaceae and other resupinate fungi belonging to other natural families in the Agaricomycetes. Molecular studies have shown that corticioid genera are distributed across all major clades of Agaricomycetes indicating that the corticioid fungi represent a polyphyletic group. They have resupinate fruitbodies and similar habitats. Species are characterized by simple fruitbody, more or less effused, and present smooth, porioid, grandinioid to odontioid hymenial surface. The fruitbodies are differently colored and usually soft to tough. Most of the Corticiaceae species are wood-saprobic organisms and gain the energy from the decomposing of wood-substrate such as cellulose or lignin. Materials for this study were collected by the author and other mycologists in Taiwan during surveys in April and May 1996, and March 2007, using the spring season with its high humidity and warm climate which are optimal conditions for the development of fungi. For assembling, the convenience sampling method was used in this study. This approach was chosen because it enables to detect a high biodiversity in a short time, and also to find species with rare or patchy distribution. The collecting sites from the North to the South include four National Parks and some preserved forests. They cover many different habitats such as low lands and high mountains. Fresh specimens were dried and analysed with a light microscope. 265 specimens belonging to Corticiaceae were studied in this research. Among them, 50 species in 21 genera including 11 new records and 10 new species were described with text and drawing. Four new species are belonging to Hyphodontia (H. sp. nov. 1, H. sp. nov. 2, H. sp. nov. 3, and H. sp. nov. 4), four to Schizopora (Sch. sp. nov. 1, Sch. sp. nov. 2, Sch. sp. nov. 3, and Sch. sp. nov. 4), one in Trechispora (T. sp. nov. 1), and one in Tubulicrinis (T. sp. nov. 1). Species recorded as new are Aleurodiscus amorphus, Botryohypochnus isabellinus, Hyphodontia cineracea, Hyphodontia palmae, Hypochnicium vellereum, Merulius tremellosus, Metulodontia nivea, Paullicorticium ansatum, Phlebia radiata, Phlebiella ardosiaca, and Xylobolus frustulatus. Besides, Botryohypochnus, Merulius, Metulodontia, Paullicorticium, and Xylobolus are also newly recorded genera in Taiwan. The genus Hyphodontia presents the highest diversity with 20 out of 50 species recorded. The second important genus is Hyphoderma, however with only 5 species. This indicates that Hyphodontia and Hyphoderma have a higher ability to develop in variable environments and approximately shows the predominance of these two genera in Taiwanese Corticiaceae. There are 11 new records out of the 50 species recorded, representing 22%. Some species, e.g. Hypochnicium vellereum and Paullicorticium ansatum were in the past recorded only in Europe and North America with cold and temperate climate. The samples of them are for the first time found in the subtropical belt, and display some difference from those of temperate regions. These collections should be molecularly investigated to clarify if they represent the same species of temperate areas. Patchily distributed species, for example Phlebiella ardosiaca, previously known only in Europe, and Hyphodontia palmae collected only in Brazil, were first recorded in different continents. Two possibilities are indicated by these new records: they are worldwide species but very rare to be found, or the Taiwanese specimens are taxonomically different. More survey from other continents and molecular study for these collections should be done in the future to solve this question. The distribution of Corticiaceae in Taiwan presents the variations in the north, central, and south areas and shows the diversity in lowlands and high mountains. The results of this study provide the evidence that the temperate Corticiaceae species displays a wider distribution. Subtropical and tropical taxa probably have also high dispersal capacities, and could possibly be found in the future in neighboring areas such as China, Japan, Korea or South Asia, but this needs further researches. In the total of 50 species, 10 new taxa were described in this study, giving about 20%. Some new species (e.g. Hyphodontia sp. 1, Hyphodontia sp. 2, and Hyphodontia sp. 3) are very similar to known species (Hyphodontia sambuci and Hyphodontia formosana), and the distinctive characters of Schizopora sp. nov. 1 are intermediate between those of Schizopora paradoxa and Hyphodontia flavipora. Thus, these small differences between the new and known species, suggest that the speciation occurred when the fungi migrated into Taiwan, due to the high diversity of environment, and amounts of the endemic plants. Taiwan is an intermediate place for the south (tropical) fungal species to migrate and adapt to north (temperate) regions. The middle and high altitude environments in Taiwan offer good conditions for the fungal speciation and possibly the occurrence of physiological changes to adapt to the temperate climate. Thus Taiwan has an important position for the biogeography of Asia mycobiota. 5936 known species in Taiwan represent about only 20% of the estimated number (24000) of Taiwanese fungal taxa. In this study, the findings (22% new records and 20% new species) indicate that amounts of unknown fungi species are expected in Taiwan. The lack of knowledge indicates that many new species are awaiting description, and fungal survey in Taiwan remains in a Pioneer phase. The last three wide surveys of Corticiaceae researches took place 20 years before this study (Chen & Lin 1977, Lin & Chen 1989, Wu 1990). After previous important contributions, the present taxonomic study comprising 21 genera is the most extensive on Corticiaceae of Taiwan.

Characterization of DnmA, the Dnmt2 homolog in Dictyostelium discoideum

Eukaryotic DNA m5C methyltransferases (MTases) play a major role in many epigenetic regulatory processes like genomic imprinting, X-chromosome inactivation, silencing of transposons and gene expression. Members of the two DNA m5C MTase families, Dnmt1 and Dnmt3, are relatively well studied and many details of their biological functions, biochemical properties as well as interaction partners are known. In contrast, the biological functions of the highly conserved Dnmt2 family, which appear to have non-canonical dual substrate specificity, remain enigmatic despite the efforts of many researchers. The genome of the social amoeba Dictyostelium encodes Dnmt2-homolog, the DnmA, as the only DNA m5C MTase which allowed us to study Dnmt2 function in this organism without interference by the other enzymes. The dnmA gene can be easily disrupted but the knock-out clones did not show obvious phenotypes under normal lab conditions, suggesting that the function of DnmA is not vital for the organism. It appears that the dnmA gene has a low expression profile during vegetative growth and is only 5-fold upregulated during development. Fluorescence microscopy indicated that DnmA-GFP fusions were distributed between both the nucleus and cytoplasm with some enrichment in nuclei. Interestingly, the experiments showed specific dynamics of DnmA-GFP distribution during the cell cycle. The proteins colocalized with DNA in the interphase and were mainly removed from nuclei during mitosis. DnmA functions as an active DNA m5C MTase in vivo and is responsible for weak but detectable DNA methylation of several regions in the Dictyostelium genome. Nevertheless, gel retardation assays showed only slightly higher affinity of the enzyme to dsDNA compared to ssDNA and no specificity towards various sequence contexts, although weak but detectable specificity towards AT-rich sequences was observed. This could be due to intrinsic curvature of such sequences. Furthermore, DnmA did not show denaturant-resistant covalent complexes with dsDNA in vitro, although it could form covalent adducts with ssDNA. Low binding and methyltransfer activity in vitro suggest the necessity of additional factor in DnmA function. Nevertheless, no candidates could be identified in affinity purification experiments with different tagged DnmA fusions. In this respect, it should be noted that tagged DnmA fusion preparations from Dictyostelium showed somewhat higher activity in both covalent adduct formation and methylation assays than DnmA expressed in E.coli. Thus, the presence of co-purified factors cannot be excluded. The low efficiency of complex formation by the recombinant enzyme and the failure to define interacting proteins that could be required for DNA methylation in vivo, brought up the assumption that post-translational modifications could influence target recognition and enzymatic activity. Indeed, sites of phosphorylation, methylation and acetylation were identified within the target recognition domain (TRD) of DnmA by mass spectrometry. For phosphorylation, the combination of MS data and bioinformatic analysis revealed that some of the sites could well be targets for specific kinases in vivo. Preliminary 3D modeling of DnmA protein based on homology with hDNMT2 allowed us to show that several identified phosphorylation sites located on the surface of the molecule, where they would be available for kinases. The presence of modifications almost solely within the TRD domain of DnmA could potentially modulate the mode of its interaction with the target nucleic acids. DnmA was able to form denaturant-resistant covalent intermediates with several Dictyostelium tRNAs, using as a target C38 in the anticodon loop. The formation of complexes not always correlated with the data from methylation assays, and seemed to be dependent on both sequence and structure of the tRNA substrate. The pattern, previously suggested by the Helm group for optimal methyltransferase activity of hDNMT2, appeared to contribute significantly in the formation of covalent adducts but was not the only feature of the substrate required for DnmA and hDNMT2 functions. Both enzymes required Mg2+ to form covalent complexes, which indicated that the specific structure of the target tRNA was indispensable. The dynamics of covalent adduct accumulation was different for DnmA and different tRNAs. Interestingly, the profiles of covalent adduct accumulation for different tRNAs were somewhat similar for DnmA and hDNMT2 enzymes. According to the proposed catalytic mechanism for DNA m5C MTases, the observed denaturant-resistant complexes corresponded to covalent enamine intermediates. The apparent discrepancies in the data from covalent complex formation and methylation assays may be interpreted by the possibility of alternative pathways of the catalytic mechanism, leading not to methylation but to exchange or demethylation reactions. The reversibility of enamine intermediate formation should also be considered. Curiously, native gel retardation assays showed no or little difference in binding affinities of DnmA to different RNA substrates and thus the absence of specificity in the initial enzyme binding. The meaning of the tRNA methylation as well as identification of novel RNA substrates in vivo should be the aim of further experiments.

In vitro characterization of two small nucleolytic ribozymes-the hairpin and the hammerhead ribozymes in their natural context

Hairpin Ribozyme kommen natürlich in den Minussträngen der Satelliten RNAs dreier Pflanzenviren (sTRsV, sArMV and sCYMoV) vor. In dieser Arbeit wurden mit dem Programm Mfold darin mehrere distinkte Sekundärstrukturelemente gefunden, die außerhalb des katalytischen Zentrums der Ribozyme lokalisieren. Verschiedene Varianten der drei Ribozyme wurden hergestellt und die Funktion der beobachteten peripheren Strukturelemente biochemisch untersucht. Die sTRsV Hairpin Ribozyme mit unterschiedlichen Längen in Arm C wiesen ähnliche cis-Spaltungsreaktionen auf, unabhängig von der Anzahl interner bulges in Arm C. Das gleiche Verhalten, jedoch bei schnelleren Spaltungsraten, wurde nach Entfernen der three-way junction, die 3’ von der Spaltstelle in Arm A liegt, beobachtet. Hier hat Arm C demnach keinen Einfluss auf die Katalyse, wogegen ein verlängerter Arm A die Reaktion verlangsamt. Unter den experimentellen Bedingungen war die Rückreaktion in Anwesenheit des natürlichen Arms A nicht messbar. Im Gegensatz dazu zeigten alle Varianten ohne die Arm A Erweiterung Ligationsaktivität, die am höchsten in dem Molekül mit dem längsten Arm C war, und gleichermaßen erniedrigt für zwei Varianten mit kürzerem Arm C. Keine der Reaktionen diverser sArMV Hairpin Ribozyme konnte reproduzierbar analysiert werden. Für das sCYMoV Hairpin Ribozym wurde schließlich in cis-Spaltungsreaktionen eine Zunahme der Geschwindigkeit mit Abnahme der Länge von Arm D beobachtet. Dies war der Fall in Anwesenheit der three-way junction in Arm A, nicht jedoch in ihrer Abwesenheit, wo Varianten mit unterschiedlichen Längen des Arms D ähnliche Spaltungsreaktionen aufwiesen. In Anwesenheit der three-way junction in Arm A war eine Reduzierung der Ligationsgeschwindigkeit zu beobachten, und bei ihrer Abwesenheit stieg diese mit der Länge von Arm D. Dies zeigt, dass sowohl die three-way junction in Arm A, als auch die Länge und Anzahl der bulges in Arm D die Reaktion des Hairpin Ribozyms aus sCYMoV beeinflussen, wobei sich Unterschiede in Vorwärts- und Rückreaktion auf die experimentellen Bedingungen zurückführen lassen. In zwei Serien wurde die zentrale five-way junction dieses Ribozyms durch verschiedene four-way junctions ersetzt. Die kinetischen Parameter der Selbstspaltung waren ähnlich für Varianten ohne Arm E auf, jedoch verlangsamt bei Varianten ohne Arm C. Dies zeigt, dass das sCYMoV Hairpin Ribozym auch um eine four-way junction gebildet werden kann, deren konstituierenden Helices jedoch nicht beliebig sind. In einem zweiten Projekt wurde die Konservierung von Hammerhead Ribozym-motiven, die bereits früher im Genom der Brassicacee A. thaliana gefunden worden waren, exemplarisch an zehn Mitgliedern dieser Familie untersucht. Da deren Genome nicht sequenziert sind, wurde PCR mit Primern angewandt, die für die A. thaliana Motive spezifisch waren. Damit konnten Ribozymmotive in allen untersuchten Brassicaceen außer B. nigra and B. oleracea gefunden werden. Diese gehören zu den sechs Brassica Pflanzen, für die der koreanische Botaniker U 1935 im “triangle of U” die genetische Verwandtschaft beschrieb. Darin ist B. carinata, für die Ribozymmotive gezeigt wurden, die Tochterspezies der Brassica Pflanzen ohne diese Motive. Dieser Widerspruch könnte darauf zurückzuführen sein, dass in der PCR unspezifische Primer genutzt wurden, oder aber die Motive aus B. carinata könnten ein Artefakt aus einer Luft-übertragenen Kontamination sein. Technische Schwierigkeiten in der Durchführung von Southern Blots, mit denen zwischen diesen Möglichkeiten unterschieden werden sollte, haben eine abschließende Antwort verhindert. Nach einer Optimierung der Methode sollte diese aber geeignet sein, diese Frage zu klären.

3D Simultaneous Inversion for Seismic Structure and Local Hypocenters in Germany under Consideration of Seismic Anisotropy in the Upper Mantel

The main task of this work has been to investigate the effects of anisotropy onto the propagation of seismic waves along the Upper Mantle below Germany and adjacent areas. Refraction- and reflexion seismic experiments proved the existence of Upper Mantle anisotropy and its influence onto the propagation of Pn-waves. By the 3D tomographic investigations that have been done here for the crust and the upper mantle, considering the influence of anisotropy, a gap for the investigations in Europe has been closed. These investigations have been done with the SSH-Inversionprogram of Prof. Dr. M. Koch, which is able to compute simultaneously the seismic structure and hypocenters. For the investigation, a dataset has been available with recordings between the years 1975 to 2003 with a total of 60249 P- and 54212 S-phase records of 10028 seismic events. At the beginning, a precise analysis of the residuals (RES, the difference between calculated and observed arrivaltime) has been done which confirmed the existence of anisotropy for Pn-phases. The recognized sinusoidal distribution has been compensated by an extension of the SSH-program by an ellipse with a slow and rectangular fast axis with azimuth to correct the Pn-velocities. The azimuth of the fast axis has been fixed by the application of the simultaneous inversion at 25° - 27° with a variation of the velocities at +- 2.5 about an average value at 8 km/s. This new value differs from the old one at 35°, recognized in the initial residual analysis. This depends on the new computed hypocenters together with the structure. The application of the elliptical correction has resulted in a better fit of the vertical layered 1D-Model, compared to the results of preceding seismological experiments and 1D and 2D investigations. The optimal result of the 1D-inversion has been used as initial starting model for the 3D-inversions to compute the three dimensional picture of the seismic structure of the Crust and Upper Mantle. The simultaneous inversion has showed an optimization of the relocalization of the hypocenters and the reconstruction of the seismic structure in comparison to the geology and tectonic, as described by other investigations. The investigations for the seismic structure and the relocalization have been confirmed by several different tests. First, synthetic traveltime data are computed with an anisotropic variation and inverted with and without anisotropic correction. Further, tests with randomly disturbed hypocenters and traveltime data have been proceeded to verify the influence of the initial values onto the relocalization accuracy and onto the seismic structure and to test for a further improvement by the application of the anisotropic correction. Finally, the results of the work have been applied onto the Waldkirch earthquake in 2004 to compare the isotropic and the anisotropic relocalization with the initial optimal one to verify whether there is some improvement.

Highly Sensitive Sensor Based On Intracavity Laser Absorption Spectroscopy By Means of Relative Intensity Noise

This thesis concerns with the main aspects of medical trace molecules detection by means of intracavity laser absorption spectroscopy (ICLAS), namely with the equirements for highly sensitive, highly selective, low price, and compact size sensor. A novel two modes semiconductor laser sensor is demonstrated. Its operation principle is based on the competition between these two modes. The sensor sensitivity is improved when the sample is placed inside the two modes laser cavity, and the competition between the two modes exists. The effects of the mode competition in ICLAS are discussed theoretically and experimentally. The sensor selectivity is enhanced using external cavity diode laser (ECDL) configuration, where the tuning range only depends on the external cavity configuration. In order to considerably reduce the sensor cost, relative intensity noise (RIN) is chosen for monitoring the intensity ratio of the two modes. RIN is found to be an excellent indicator for the two modes intensity ratio variations which strongly supports the sensor methodology. On the other hand, it has been found that, wavelength tuning has no effect on the RIN spectrum which is very beneficial for the proposed detection principle. In order to use the sensor for medical applications, the absorption line of an anesthetic sample, propofol, is measured. Propofol has been dissolved in various solvents. RIN has been chosen to monitor the sensor response. From the measured spectra, the sensor sensitivity enhancement factor is found to be of the order of 10^(3) times of the conventional laser spectroscopy.

Organische Mikro- und Nanodrähte auf Basis von Perylendiimiden

Diese Arbeit thematisiert die optimierte Darstellung von organischen Mikro- und Nanodrähten, Untersuchungen bezüglich deren molekularen Aufbaus und die anwendungsorientierte Charakterisierung der Eigenschaften. Mikro- und Nanodrähte haben in den letzten Jahren im Zuge der Miniaturisierung von Technologien an weitreichendem Interesse gewonnen. Solche eindimensionalen Strukturen, deren Durchmesser im Bereich weniger zehn Nanometer bis zu einigen wenigen Mikrometern liegt, sind Gegenstand intensiver Forschung. Neben anorganischen Ausgangssubstanzen zur Erzeugung von Mikro- und Nanodrähten haben organische Funktionsmaterialien aufgrund ihrer einfachen und kostengünstigen Verarbeitbarkeit sowie ihrer interessanten elektrischen und optischen Eigenschaften an Bedeutung gewonnen. Eine wichtige Materialklasse ist in diesem Zusammenhang die Verbindungsklasse der n-halbleitenden Perylentetracarbonsäurediimide (kurz Perylendiimide). Dem erfolgreichen Einsatz von eindimensionalen Strukturen als miniaturisierte Bausteine geht die optimierte und kontrollierte Herstellung voraus. Im Rahmen der Doktorarbeit wurde die neue Methode der Drahterzeugung „Trocknen unter Lösungsmittelatmosphäre“ entwickelt, welche auf Selbstassemblierung der Substanzmoleküle aus Lösung basiert und unter dem Einfluss von Lösungsmitteldampf direkt auf einem vorgegebenen Substrat stattfindet. Im Gegensatz zu literaturbekannten Methoden ist kein Transfer der Drähte aus einem Reaktionsgefäß nötig und damit verbundene Beschädigungen der Strukturen werden vermieden. Während herkömmliche Methoden in einer unkontrolliert großen Menge von ineinander verwundenen Drähten resultieren, erlaubt die substratbasierte Technik die Bildung voneinander separierter Einzelfasern und somit beispielsweise den Einsatz in Einzelstrukturbauteilen. Die erhaltenen Fasern sind morphologisch sehr gleichmäßig und weisen bei Längen von bis zu 5 mm bemerkenswert hohe Aspektverhältnisse von über 10000 auf. Darüber hinaus kann durch das direkte Drahtwachstum auf dem Substrat über den Einsatz von vorstrukturierten Oberflächen und Wachstumsmasken gerichtetes, lokal beschränktes Drahtwachstum erzielt werden und damit aktive Kontrolle auf Richtung und Wachstumsbereich der makroskopisch nicht handhabbaren Objekte ausgeübt werden. Um das Drahtwachstum auch hinsichtlich der Materialauswahl, d. h. der eingesetzten Ausgangsmaterialien zur Drahterzeugung und somit der resultierenden Eigenschaften der gebildeten Strukturen aktiv kontrollieren zu können, wird der Einfluss unterschiedlicher Parameter auf die Morphologie der Selbstassemblierungsprodukte am Beispiel unterschiedlicher Derivate betrachtet. So stellt sich zum einen die Art der eingesetzten Lösungsmittel in flüssiger und gasförmiger Phase beim Trocknen unter Lösungsmittelatmosphäre als wichtiger Faktor heraus. Beide Lösungsmittel dienen als Interaktionspartner für die Moleküle des funktionellen Drahtmaterials im Selbstassemblierungsprozess. Spezifische Wechselwirkungen zwischen Perylendiimid-Molekülen untereinander und mit Lösungsmittel-Molekülen bestimmen dabei die äußere Form der erhaltenen Strukturen. Ein weiterer wichtiger Faktor ist die Molekülstruktur des verwendeten funktionellen Perylendiimids. Es wird der Einfluss einer Bay-Substitution bzw. einer unsymmetrischen Imid-Substitution auf die Morphologie der erhaltenen Strukturen herausgestellt. Für das detaillierte Verständnis des Zusammenhanges zwischen Molekülstruktur und nötigen Wachstumsbedingungen für die Bildung von eindimensionalen Strukturen zum einen, aber auch die resultierenden Eigenschaften der erhaltenen Aggregationsprodukte zum anderen, sind Informationen über den molekularen Aufbau von großer Bedeutung. Im Rahmen der Doktorarbeit konnte ein molekular hoch geordneter, kristalliner Aufbau der Drähte nachgewiesen werden. Durch Kombination unterschiedlicher Messmethoden ist es gelungen, die molekulare Anordnung in Strukturen aus einem Spirobifluoren-substituierten Derivat in Form einer verkippten Molekülstapelung entlang der Drahtlängsrichtung zu bestimmen. Um mögliche Anwendungsbereiche der erzeugten Drähte aufzuzeigen, wurden diese hinsichtlich ihrer elektrischen und optischen Eigenschaften analysiert. Neben dem potentiellen Einsatz im Bereich von Filteranwendungen und Sensoren, sind vor allem die halbleitenden und optisch wellenleitenden Eigenschaften hervorzuheben. Es konnten organische Transistoren auf der Basis von Einzeldrähten mit im Vergleich zu Dünnschichtbauteilen erhöhten Ladungsträgerbeweglichkeiten präpariert werden. Darüber hinaus wurden die erzeugten eindimensionalen Strukturen als aktive optische Wellenleiter charakterisiert. Die im Rahmen der Dissertation erarbeiteten Kenntnisse bezüglich der Bildung von eindimensionalen Strukturen durch Selbstassemblierung, des Drahtaufbaus und erster anwendungsorientierter Charakterisierung stellen eine Basis zur Weiterentwicklung solcher miniaturisierter Bausteine für unterschiedlichste Anwendungen dar. Die neu entwickelte Methode des Trocknens unter Lösungsmittelatmosphäre ist nicht auf den Einsatz von Perylendiimiden beschränkt, sondern kann auf andere Substanzklassen ausgeweitet werden. Dies eröffnet breite Möglichkeiten der Materialauswahl und somit der Einsatzmöglichkeiten der erhaltenen Strukturen.

Characterization of the molecular clockwork in the cockroach Rhyparobia maderae

Der Wechsel von Tag und Nacht erzeugt einen regelmäßigen Rhythmus von verschiedenen Umweltreizen, allen voran Licht und Temperatur. Fast jedes bis zum heutigen Tage untersuchte Lebewesen besitzt einen endogenen Mechanismus zur Zeitwahrnehmung, und diese "innere Uhr" befähigt Lebewesen dazu, sich vorausschauend an rhythmische Umwelt-Änderungen anzupassen. Circadiane Rhythmen bestehen auch ohne jegliche äußere Reize und basieren auf einem molekularen Rückkopplungs-Mechanismus, der Rhythmen in Genexpression und Proteinkonzentration von etwa 24 Stunden erzeugt. Obwohl sich die grundsätzlichen Mechanismen und Komponenten dieses molekularen Uhrwerks in allen Insekten ähneln, zeigte sich jedoch immer mehr, dass es im Detail doch wesentliche Unterschiede zwischen verschiedenen Insektengruppen gibt. Während das molekulare Uhrwerk der Fruchtfliege Drosophila melanogaster inzwischen sehr gut untersucht ist, fehlen bei den meisten Insektengruppen immernoch eingehende Untersuchungen. Fast nichts ist über die molekulare Basis von circadianen Rhythmen bei der Schabe Rhyparobia maderae bekannt, obwohl diese Art bereits seit Langem als Modellorganismus in der Chronobiologie dient. Um mit der Forschung am molekularen, circadianen System von R. maderae zu beginnen, wurde die Struktur und das Expressionsprofil der core feedback loop Gene per, tim1 und cry2 analysiert. Mittels degenerierten Primern und RACE konnte das vollständige offene Leseraster (OLR) von rmPer und rmCry2, und ein Teil des rmTim1 OLR kloniert werden. Eine phylogenetische Analyse gruppierte rmPER und rmCRY2 gemeinsam mit den Orthologa hemimetaboler Insekten. Viele bei D. melanogaster funktionell charakterisierte Domänen sind bei diesen Proteinen konserviert, was auf eine ähnliche Funktion in der inneren Uhr von R. maderae hinweist. Mittels quantitativer PCR konnte gezeigt werden, dass die mRNA von rmPer, rmTim1 und rmCry2 in verschiedenen Lichtregimen in der gleichen Phasenlage Tageszeit-abhängig schwankt. Die Phasenlage stellte sich bei unterschiedlichen Photoperioden jeweils relativ zum Beginn der Skotophase ein, mit Maxima in der ersten Hälfte der Nacht. Auch im Dauerdunkel zeigen sich Rhythmen in der rmTim1 und rmCry2 Expression. Die Amplitude der rmPer Expressionsrhythmen war jedoch so gering, dass keine signifikanten Unterschiede zwischen den einzelnen Zeitgeberzeiten (ZT) festgestellt werden konnten. Mittels Laufrad-Assays wurde untersucht wie Kurz- und Langtag Lichtregime die Verhaltensrhythmen beeinflussen. Es konnten nur Unterschiede in der Periodenlänge unter freilaufenden Bedingungen festgestellt werden, wenn höhere Lichtintensitäten (1000lx) zur Synchronisation (entrainment) genutzt wurden. Die Periode des freilaufenden Rhythmus war bei Tieren aus dem Kurztag länger. Die photoperiodische Plastizität zeigte sich also auch auf Verhaltensebene, obwohl höhere Lichtintensitäten notwendig waren um einen Effekt zu beobachten. Basierend auf den Sequenzen der zuvor klonierten OLR wurden gegen rmPER, rmTIM1 und rmCRY2 gerichtete Antikörper hergestellt. Die Antikörper gegen rmPER und rmTIM1 erkannten in western blots sehr wahrscheinlich spezifisch das jeweilige Protein. Zeitreihen von Gehirngewebe-Homogenisaten zeigten keinen offensichtlichen circadianen Rhythmus in der Proteinkonzentration, wahrscheinlich auf Grund einer Oszillation mit niedriger Amplitude. In Immunhistochemischen Färbungen konnte nur mit dem gegen rmPER gerichteten Antikörper aus Kaninchen ein Signal beobachtet werden. Beinahe jede Zelle des Zentralnervensystems war rmPER-immunreaktiv im Zellkern. Es konnten keine Unterschiede zwischen den untersuchten ZTs festgestellt werden, ähnlich wie bei den western blot Zeitreihen. In dieser Studie konnten erstmals molekulare Daten der circadianen Uhr von R. maderae erfasst und dargestellt werden. Die Uhrgene per, tim1 und cry2 werden in dieser Schabenart exprimiert und ihre Domänenstruktur sowie das circadiane Expressionsmuster ähneln dem hypothetischen ursprünglichen Insektenuhrwerk, welches der circadianen Uhr von Vertebraten nahesteht. Das molekulare Uhrwerk von R. maderae kann sich an unterschiedliche Photoperioden anpassen, und diese Anpassungen manifestieren sich im Expressionsprofil der untersuchten Uhrgene ebenso wie im Verhalten.

Development of an ArcGIS extension to model urban climate factors

The possibility to develop automatically running models which can capture some of the most important factors driving the urban climate would be very useful for many planning aspects. With the help of these modulated climate data, the creation of the typically used “Urban Climate Maps” (UCM) will be accelerated and facilitated. This work describes the development of a special ArcGIS software extension, along with two support databases to achieve this functionality. At the present time, lacking comparability between different UCMs and imprecise planning advices going along with the significant technical problems of manually creating conventional maps are central issues. Also inflexibility and static behaviour are reducing the maps’ practicality. From experi-ence, planning processes are formed more productively, namely to implant new planning parameters directly via the existing work surface to map the impact of the data change immediately, if pos-sible. In addition to the direct climate figures, information of other planning areas (like regional characteristics / developments etc.) have to be taken into account to create the UCM as well. Taking all these requirements into consideration, an automated calculation process of urban climate impact parameters will serve to increase the creation of homogenous UCMs efficiently.

Physiological analysis of central and peripheral insect circadian pacemaker neurons

Alle bisher untersuchten Lebewesen besitzen (circadiane) innere Uhren, die eine endogene Perioden-länge von ungefähr 24 Stunden generieren. Eine innere Uhr kann über Zeitgeber mit der Umwelt synchronisiert werden und ermöglicht dem Organismus, rhythmische Umweltveränderungen vorweg zu nehmen. Neben einem zentralen Schrittmacher, der Physiologie und Verhalten des Organismus steuert, gibt es in unterschiedlichen Organen auch periphere Uhren, die die zeitlichen Abläufe in der spezifischen Funktion dieser Organe steuern. In dieser Arbeit sollten zentrale und periphere Schrittmacherneurone von Insekten physiologisch untersucht und verglichen werden. Die Neurone der akzessorischen Medulla (AME) von Rhyparobia maderae dienten als Modellsystem für zentrale Schrittmacher, während olfaktorische Rezeptorneurone (ORNs) von Manduca sexta als Modellsystem für periphere Schrittmacher dienten. Die zentralen Schrittmacherneurone wurden in extrazellulären Ableitungen an der isolierten AME (Netzwerkebene) und in Patch-Clamp Experimenten an primären AME Zellkulturen (Einzelzellebene) untersucht. Auf Netzwerkebene zeigten sich zwei charakteristische Aktivitätsmuster: regelmäßige Aktivität und Wechsel zwischen hoher und niedriger Aktivität (Oszillationen). Es wurde gezeigt, dass Glutamat ein Neurotransmitter der weitverbreiteten inhibitorischen Synapsen der AME ist, und dass in geringem Maße auch exzitatorische Synapsen vorkommen. Das Neuropeptid pigment-dispersing factor (PDF), das von nur wenigen AME Neuronen exprimiert wird und ein wichtiger Kopplungsfaktor im circadianen System ist, führte zu Hemmungen, Aktivierungen oder Oszillationen. Die Effekte waren transient oder langanhaltend und wurden wahrscheinlich durch den sekundären Botenstoff cAMP vermittelt. Ein Zielmolekül von cAMP war vermutlich exchange protein directly activated by cAMP (EPAC). Auf Einzelzellebene wurde gezeigt, dass die meisten AME Neurone depolarisiert waren und deshalb nicht feuerten. Die Analyse von Strom-Spannungs-Kennlinien und pharmakologische Experimente ergaben, dass unterschiedliche Ionenkanäle vorhanden waren (Ca2+, Cl-, K+, Na+ Kanäle sowie nicht-spezifische Kationenkanäle). Starke, bei hohen Spannungen aktivierende Ca2+ Ströme (ICa) könnten eine wichtige Rolle bei Ca2+-abhängiger Neurotransmitter-Ausschüttung, Oszillationen, und Aktionspotentialen spielen. PDF hemmte unterschiedliche Ströme (ICa, IK und INa) und aktivierte nicht-spezifische Kationenströme (Ih). Es wurde angenommen, dass simultane PDF-abhängige Hyper- und Depolarisationen rhythmische Membranpotential-Oszillationen verursachen. Dieser Mechanismus könnte eine Rolle bei PDF-abhängigen Synchronisationen spielen. Die Analyse peripherer Schrittmacherneurone konzentrierte sich auf die Charakterisierung des olfaktorischen Corezeptors von M. sexta (MsexORCO). In anderen Insekten ist ORCO für die Membran-Insertion von olfaktorischen Rezeptoren (ORs) erforderlich. ORCO bildet Komplexe mit den ORs, die in heterologen Expressionssystemen als Ionenkanäle fungieren und Duft-Antworten vermitteln. Es wurde die Hypothese aufgestellt, dass MsexORCO in pheromonsensitiven ORNs in vivo nicht als Teil eines ionotropen Rezeptors sondern als Schrittmacherkanal fungiert, der unterschwellige Membranpotential-Oszillationen generiert. MsexORCO wurde mit vermeintlichen Pheromonrezeptoren in human embryonic kidney (HEK 293) Zellen coexprimiert. Immuncytochemie und Ca2+ Imaging Experimente zeigten sehr schwache Expressionsraten. Trotzdem war es möglich zu zeigen, dass MsexORCO wahrscheinlich ein spontan-aktiver, Ca2+-permeabler Ionenkanal ist, der durch den ORCO-Agonisten VUAA1 und cyclische Nucleotide aktiviert wird. Außerdem wiesen die Experimente darauf hin, dass MsexOR-1 offensichtlich der Bombykal-Rezeptor ist. Eine weitere Charakterisierung von MsexORCO in primären M. sexta ORN Zellkulturen konnte nicht vollendet werden, weil die ORNs nicht signifikant auf ORCO-Agonisten oder -Antagonisten reagierten.