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- De Gruyter (22)
This thesis presents the results of experimental investigations of the vertical and lateral properties of polyelectrolyte multilayer films (PEMs) adsorbed on a solid support. PEMs are a new class of organic thin films based on self-assembly layer-by-layer (LbL) processes of oppositely charged polyelectrolytes (charged polymers). The LbL assembly technique allows precise control of film thickness within a few nanometers and makes PEM systems especially interesting for technical applications. Thin films are prepared by alternating exposure of a hydrophilic substrate to solutions of oppositely charged polyelectrolytes. In this work, synthetic polycation poly (allylamine hydrochloride) (PAH) and polyanion poly (styrene sulfonate) (PSS) have been used. Range and amplitude of the electrostatic force during PEM build-up, has been shielded by use of high salt concentration in the deposition solution. As a foundation of any theory, role of non-elecrostatic (secondary) forces is explored. Four complementary methods have been combined to investigate the properties and composition of PEMs. X-ray reflectivity is sensitive to electron density gradients, and therefore provides information about film thickness, average electron density and interfacial roughness between materials of different electron densities (like PEM and air). Neutrons are the unique probe that is sensitive to the internal order of the multilayers (scattering length density variation) due to selective deuteration of the layers (PSS replaced by PSS_d). Therefore neutron reflectivity at V6 beamline, at the research reactor BER II, Helmholtz Centre for Materials and Energy (former Hahn-Meitner-Institute), was used in this work. Ultraviolet-visible (UV-Vis) light induces the characteristic absorption peak of polyelectrolytes and metallic nanoparticles, therefore with UV-Vis absorption spectroscopy is possible to probe the aggregation of metallic nanoparticles embedded into PEM by measuring their absorption spectra (imaginary part of the refraction index). Atomic force microscopy (AFM) allows to observe lateral structures at nano-level and to obtain surface topology of the films. Application of only small forces (pN) is achieved by use of a intermittent contact (tapping) mode in air. Summarizing the main results, the unambiguous parametrization of the investigated system for neutron reflectivity measurements enables to obtain detailed information about internal interfaces. New approach for polyelectrolyte multilayer architectures consisting of thick protonated and deuterated blocks can be used in order to distinguish different zones of the thin film growth which can be described as precursor and core zones. Thus, almost no bound water is found in precursor layers at 0% relative humidity, which suggests that water is mobile and the precursor layer is not in the glassy state like in the central zone of the PEM. Swelling behaviour of the PEMs (reversibility of the swelling) can be understood in terms of equilibrium reactions. Explored influence of temperature and type of salt used during preparation contributes to a better understanding of the formation of PEMs. The dependence of the film thickness on preparation temperature, concentration and the type of salt can be described by the hydrophobic nature of the effect. Experimental observations demonstrate that it is possible to decrease both the range and the amplitude of the electrostatic force by using an ion concentration of at least 0.1 mol/L in the solution. The role of secondary interactions such as hydrophobic attraction of the chains that can overcome electrostatic repulsion and become the major contributing factor for the layer formation and resulting structures is emphasized.
This work proposes and experimentally evaluates a new method and the first prototypic design of an apparatus for the objective assessment of peripheral dynamic stereovision. Peripheral dynamic stereovision is understood as the ability to perceive, recognize and observe moving objects in the visual periphery without sacrificing foveal fixation, i.e. shifting the line of sight (e.g. moving the eyes or the head) and loosing attention to the scene of interest. The presence of stereopsis on top further enhances the perceptive quality by adding the third dimension with judgment of depth and estimation of relative spatial positions as central clues for orientation, coordinative interaction and navigation in space. Based on findings that moving stereoscopic contours can induce distinctive optokinetic reactions, a panoramic stimulus pattern was rotated round subjects’ heads. Designed after the Frisby Stereotest, solely the binocular parallax resulting from the plate thickness carries stereoscopic information, excluding all other depth clues. A specially fitted goggle frame assured correct binocular alignment of the subjects adding objectivity. In first experiments, voluntary reactions (pushing a buzzer button) and involuntarily triggered ocular responses have been registered. The Performance Level (PL) and Confidence Ratio (CR) were introduced as benchmarks for voluntary reactions. Despite larger spreads, the PL averaged at 60 % while an elevated CR confirmed a low error rate of 16 % and thus high overall credibility. Poor performance of available recording hard- and software, in many cases, rendered the analysis of involuntary ocular reactions less exhaustive. It was however observable that objects that appear in the periphery of the visual field triggered the onset of nystagmoid search and tracking mechanisms. Finally, mean peripheral locations of subjects’ active reactions have been established at 30 degrees. The outcome of this pilot study in principle confirmed basic feasibility, conceptual validity and practical applicability of this novel method. Prospective fields of application with raised demands on peripheral dynamic stereopsis have been identified and critically assessed. The applicative possibilities and exclusive advantages of this test combining the assessment of stereopsis and dynamic visual field testing have not been matched by solutions published so far. Before however commercialization should be aimed at, design-related issues, including the implementation of electrophysiological ocular measurements, need to be addressed first to lead the post-prototype development to higher diagnostic expressiveness and reliability.
To uncover the genetic structure of Populus euphratica forests along the Tarim River in Xinjiang, China, a PCR set of eight microsatellite markers was established. 18 primer pairs originally developed for P. tremuloides and P. trichocarpa were screened for amplification in P. euphratica. The eight most variable loci were selected for further genotyping experiments. Subsequently, two multiplex PCR assays, each containing four loci, were set up and optimized. Three populations containing altogether 436 trees were used to characterize the selected loci. The set was found to be moderately polymorphic (mean expected heterozygosity = 0.57). The resolution was sufficient to discriminate even siblings with high confidence (PID = 1.81x10-5). Cumulative exclusion probabilities were 0.89 (single parent), 0.98 (paternity), and 1.00 (parent pair) and proved the set’s suitability for parentage analysis. Practical and theoretical analysis of consequences of genotyping errors in this semi-clonal plant showed that the vast majority of errors (62.1%) lead to division of identical genotypes. Merging of different genotypes was found to be a very rare case (0.4%). This always leads to an overestimation of genotypes. A similarity threshold of one allele difference between two genotypes to be regarded as being identical lead to an underestimation of clonal richness and genotype number of one per cent compared to an overestimation of more than 20 per cent without such a threshold. Allowing a certain amount of variation is therefore expected to reflect the clonal structure better than an analysis that considers exact matches only. Using a combination of morphological and molecular analyses, a first study demonstrated that root suckers are clearly distinguished from seedlings in their root architecture. Root suckering starts when trees are 10–15 years old and bridges distances of up to 40 m at a time. Root suckers depend on their parent tree for at least five years and are expected to have a higher mortality than generatively grown trees. Molecular analysis of old growth stands revealed a highly variable proportion of clonal growth between different stands. In the study area, the proportion of clonality decreases with distance to the main river bed (R = 0.31 at the site closest to the main river, R = 0.97 at the site farthest away from the river). An analysis of the history of river movements at different sites indicates a dependency of clonal growth on the frequency of ground water replenishment by the yearly floods. Genetic differentiation among the stands in the study area is low (FST = 0.055), and isolation by distance was not detectable (P = 0.058). Also, the river does not function as a vector for directed gene flow in downstream direction (P > 0.11). The forests are therefore considered to be one large panmictic metapopulation with unrestricted gene flow. Clonal growth does not lead to higher final stand densities (P = 0.99) and is obviously not of crucial importance for stand survival. Furthermore, analysis of vitality measures and size differences indicate that root suckers are in disadvantage both in vitality and in survival rate compared to seedlings. In this light, a possible function of clonal growth as a luxury strategy to enhance a genetic individual’s reproduction success under good site conditions can be discussed. The genetic structure of the (meta)population bears direct implications for management and conversation of the Tugai forest in Xinjiang. Due to the low degree of differentiation and the unhindered gene flow even small, fragmented, or isolated populations have conservational value, thereby clearly answering the SLOSS question (a single large or several small protected areas) in the latter sense. More than that, non-clonal stands with the highest amount of genotypic diversity can be easily identified on satellite and aerial images. Selection of such stands for conservation is therefore possible without expensive and time-consuming molecular analyses.
The objectives of the present work are to relate the spatial distribution of benthic macrofauna in the Baltic Sea to patterns in environmental variables describing near-bottom hydrographical conditions and sediment characteristics, analyzing the data for two various spatial extents. The first case study is devoted to an exploratory statistical description of the prevailing ecological structure within the limited area attached to the region of the Mecklenburg Bight. Key environmental descriptors of spatial distribution of macrofaunal communities were disclosed within the area of investigation: water depth, regarded as a proxy for other environmental factors, and total organic content. Distinct benthic assemblages that are discriminated by particular species (Hydrobia ulvae–Scoloplos armiger, Lagis koreni–Mysella bidentata and Capitella capitata–Halicryptus spinulosus) were defined. Each assemblage is related to different spatial subarea and is characterized by a certain variability of environmental factors. This study represented the basis for the predictive modelling of species distribution in the selected investigation area, which constituted the next part of the investigation. Species-specific models predicting the probability of occurrence relative to environmental and sedimentological characteristics were developed for 29 representative macrofaunal species using a logistic regression modelling approach. Subsequently, the technique for a predictive modelling of species distributions in response to abiotic parameters based on single-factor logistic regression models, utilizing Akaike’s information criterion (AIC) and Akaike weights for multimodel inference, was used. Thus, probabilities of occurrence for selected exemplary species (Arctica islandica, Hediste diversicolor, Pygospio elegans, Tubificoides benedii and Scoloplos armiger) were modelled and mapped. Finally, the investigation proceeded on a large spatial scale. The discriminating ability of such factors as salinity, bathymetry, and sediment characteristics (considered only generally due to the lack of more detailed data) to explain the occurrence of typical macrozoobenthic species on the Baltic Sea-wide extend was tested. Full coverage macrofauna distribution maps, though being increasingly demanded, are generally lacking, with information being merely restricted to point observations. In contrast to spatial interpolation, periled by presence of short distance changes in community structure and dependence of the result on density of the samples, predictive habitat suitability modelling allows to objectively produce distribution maps at a level of detail limited only by the availability and resolution of the environmental data. Various literature sources and available databases were analyzed in respect to the information on macrozoobenthos distribution in the Baltic Sea, resulting in the compilation of an extensive list of taxa and an inventory dataset on species distribution for the whole Baltic Sea. The study demonstrates the need to analyze species’ relationships in gradient systems such as the Baltic Sea and provides a basis for a tool to predict natural and anthropogenic forced changes in species distribution.
Kurze Inhaltszusammenfassung in einer weiteren Sprache (Englisch): Muong Lay is one of the important social and economic areas in Northwestern Vietnam. Landslides occur frequently in the area and seriously affect local livelihoods and living conditions. Therefore, the problem of landslide hazard and mitigation for a sustainable development of this area is significant. The spatial analysis of landslide hazard assessment in the mountainous regions in Muong Lay is important to address this development challenge. This study focuses on the application of GIS and RS to landslide hazard assessment, especially for support of GIS modeling to landslide hazard susceptibility for Muong Lay area. By using Remote sensing with LandSat TM image and aerial photos of scale 1:50,000 and using statistical models with GIS-software’s ENVI3.4, ILWIS3.0, PCI9.0 and ARC/GIS9.1, the study tries to evaluate and estimate the landslide in relation with naturally different elements of natural conditions such as geology, geomorphology, geology-engineering, tectonics, hydrology, rainfall, etc… Especial, the study firstly aims to produce the causal factor maps by verifying digital data. These factors then will be applied in a methodology based on statistical methods such as: “bivariate statistical analysis” and “multivariate statistical analysis” approach to calculate the susceptibility level of each class of each factor to landslide. The integration of Geographic Information System (GIS) and Remote Rensing (RS)for landslide hazard zonation and assessment is a valid approach. In these researches various methods for image integration and information extraction have been analysed and evaluated in detail.
Summary Cyanobacteria are a diverse and ancient group of photosynthetic prokaryotic organisms that can inhabit a wide range of environments including extreme conditions such as hot springs, desert soils and the Antarctic. They are abundant producers of natural products well recognized for their bioactivity and utility in drug discovery and biotechnology applications. Novel intracellular and extracellular compounds from various cultured and field cyanobacteria with diverse biological activities and a wide range of chemical classes have considerable potential for development of pharmaceuticals and other biomedical applications. However, cyanobacteria are still viewed as unexplored source of potential drugs. Especially the collections of cyanobacterial strains from South East Asia where biodiversity is high are still largely unexplored. Thus, we investigated twelve soil cyanobacterial strains isolated from soil samples collected from rice, cotton, and coffee fields in Dak Lak province of Vietnam and one marine strain, Lyngbya majuscula collected from Khanh Hoa province of Vietnam for the search for new compounds with antimicrobial and cytotoxic activities. From the 12 soil cyanobacterial strains, 48 extracts prepared with n-hexane, methanol, and water for biomasses and ethyl acetate for growth media were screened for antibacterial activity against Gram-positive bacteria (Bacillus subtilis ATCC 6051 and Staphylococcus aureus ATCC 6538) and Gram-negative bacteria (Escherichia coli ATCC 11229, Pseudomonas aeruginosa ATCC 27853). Of 48 extracts, 47.92% and 45.83% showed activity against Bacillus subtilis and Staphylococcus aureus, respectively, while 22.92% and 6.25% exhibited activity against Escherichia coli and Pseudomonas aeruginosa, respectively. All investigated cyanobacteria (12/12) showed antibacterial activity to at least one of the test organisms applied. Among the active extracts, extracts obtained from 5 cyanobacterial strains, Westiellopsis sp. VN, Calothrix javanica, Scytonema ocellatum, Anabaena sp. and Nostoc sp. showed the highest strength and range of antibacterial activity and therefore were selected for chemical investigation with an emphasis on the isolation and structure elucidation of antimicrobial compounds. Bioassay-guided fractionation of the methanol extract prepared from biomass of Westiellopsis sp. VN by silica gel chromatography, followed by sephadex LH-20 chromatography and reversed-phase HPLC led to isolation and identification of 6 compounds as ambiguine D isonitrile, ambiguine B isonitrile, dechloro-ambiguine B isonitrile, fischerellin A, hydroxy-eicosatetraenoic acid and methoxy-nonadecadienoic acid. Identification of these active compounds was established by direct comparison of our spectroscopic data, including 1H NMR and HR-ESI-MS with those reported in the literature. All these compounds showed biological activity. The identification of fatty acids and other volatile components by GS-MS in the active MeOH fraction obtained from EtOAc extract of growth medium was done before commencing further fractionation processes. Culture optimization of Westiellopsis sp.VN showed that NaNO3 deficiency increased accumulation of antimicrobial compounds. Biosynthesis of antimicrobial compounds increased over cultivation time resulting in increased diameter of inhibition zone of the methanol extract towards the end of the 7-to 8- week growth period, but the most clear inhibition zone of this extract was detected after cultivation time of 8 weeks. Bioassay-guided fractionation of the methanol extract prepared from biomass of either Calothrix javanica by C18 chromatography followed by reversed-phase HPLC or Scytonema ocellatum by C18 chromatography followed by silica gel chromatography and reversed-phase HPLC led to isolation and structure elucidation of new cyclic peptide named daklakapeptin. Structure of daklakapeptin was elucidated by exhaustive 1D (1H) and 2D (COSY, TOCSY, NOESY, HMQC, HMBC) NMR spectroscopy in combination with HR-ESI-MS. Daklakapeptin was found to have totally 12 residues including 6 proteinogenic amino acids (Pro, Tyr, Ile, Leu, Gln, Thr), 4 complexes (X,Y,T,Z) and the methyl derivative of Ile. The exact sequence of daklakapeptin is shown in following figure with X: (CH3)2CHCH2CH2CH(NH-)CH2CO-, Y:(CH3)2CHCH(OH)CH(NH-)CO-, T: HOCH2CH2CH(NH-)CO-, Z: HOCH2CHOHCH(NH-)CO- This new cyclic peptide exhibited antibacterial activity against Staphylococcus aureus with diameter of inhibition zone of 12.5 mm in concentration of 200 mg/disc. Further test for activity to other bacteria and for cytotoxic activity are in progress. Using reversed-phase HPLC to separate compounds in the crude ethyl acetate extract obtained from culture medium of Anabaena sp. led to isolation and structure elucidation of flourensadiol. The structure of flourensadiol was established using an extensive array of 1D (1H, 13C, DEPT-135) and 2D (HMQC, COSY, HMBC) NMR and HR-ESI-MS experiments. Flourensadiol was isolated previously from the common western shrub Flourensia cernua. However, only MS, IR, and proton NMR data but no reports on biological activity were available. In this study, we report the complete NMR data of flourensadiol for the first time. Flourensadiol was found to be very strong antibacterial active against Escherichia coli with diameter of inhibition zone of 20.0 mm in concentration of 200 mg/disc. Further test for activity to other bacteria and cytotoxic activity are in progress. Bioassay-guided fractionation of the methanol extract from biomass of Nostoc sp. by silica gel chromatography followed by C18 chromatography and reversed phase HPLC led to isolation of the active fraction NsF2 which exhibited antibacterial activity against Staphylococcus aureus with diameter of inhibition zone of 10.0 mm in concentration of 500 mg/disc. The low resolution ESI-MS of fraction NsF2 showed signal at m/z 426 [M+H]+. The NMR and MS characterization of compounds in fraction NsF2 is in progress. Bioassay-guided fractionation of the methanol extract prepared from biomass of marine cyanobacterium Lyngbya majuscula collected from Khanh Hoa province of Vietnam by various chromatographic methods (CC, PTLC, HPLC) afforded 3 cytotoxic compounds anhydrodebromoaplysiatoxin, debromoaplysiatoxin, and anhydroaplysiatoxin. Identification of these cytotoxic compounds was established by direct comparison of our spectroscopic data, including (1H, 13C) NMR and HR-ESI-MS with those reported in the literature. In our study, debromoaplysiatoxin and anhydroaplysiatoxin exhibited cytotoxic activity against bladder cancer cell line 5637 with IC50 of 86 ng/ml and 40 ng/ml, respectively but anhydrodebromoaplysiatoxin was not yet tested for cytotoxic activity. The identification of fatty acids by GS-MS technique in the n-hexane extract obtained from biomass of this marine cyanobacterium was undertaken before commencing further fractionation processes. The presented results prove that soil cyanobacteria are a promising source to yield chemical and pharmaceutical interesting compounds.
Influence of single amino acid polymorphisms on the in vitro convertibility of goat prion protein
(2010)
Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders which include, among others, scrapie and bovine spongiform encephalopathy (BSE). The causative agent is composed mainly of a misfolded isoform of a cellular prion protein (PrPC), denoted prion protein scrapie (PrPSc). Genetically determined PrPC polymorphisms can modulate the convertibility of PrPC to PrPSc and thus lead to prolonged TSE incubation times or even complete resistance of the animal. In sheep, such polymorphisms are located at codons 136, 154 and 171. Several disease-associated amino acid polymorphisms also exist in caprine PrPC. However, due to their large number and the limited number of goats carrying them, it is difficult to assess their specific impact on TSE susceptibility in vivo. The susceptibility can be simulated in vitro by a cell-free conversion assay, in which the conversion efficiency of recombinant PrPC is determined. In this study, twelve caprine PrPC variants (M112T, M137I, L141F, I142M, H143R, N146S, N146D, R151H, R211Q, Q215R, Q222K and wild-type PrPC (denoted INRQ) were produced by using PCR mutagenesis amplification and expressed in E. coli M15 cells and purified on Ni-NTA agarose columns. The renatured PrPC variants had molecular masses of approx. 23 kDa and the expected conformation as determined by CD spectroscopy. These variants were then subjected to a cell-free conversion assay using different BSE and scrapie strains. Cross species (mouse and goat) cell-free conversion studies were performed and specific monoclonal antibodies were used to discriminate the exogenous PrPSc molecules used to seed the reaction and newly converted PrPres. The studies with the mouse-adapted strain Me7 revealed that polymorphisms M137I, H143R and L141F did not influence the conversion of PrPC in a significant manner. However, the reduced conversion rate of the variant I142M (harbouring a methionine at position 142 instead of isoleucine) correlated with longer scrapie incubation times in goats with this polymorphism. The polymorphisms M112T, R151H and Q211R showed also reduced conversion rates in comparison to INRQ, an effect that related well to reduced scrapie susceptibility of such goats in vivo. Polymorphisms N146S, N146D and Q222K were to date extremely rarely found in scrapie affected goats. It was intriguing to see that these amino acid substitutions also abolished the in vitro conversion efficiency completely as did the Q215R polymorphism, which had not yet been associated with scrapie resistance in vivo. Results of cell free conversion studies with mouse adapted BSE prions (BSE/Bl6 strain) correlated well with the results obtained with Me7, although the results with BSE/Bl6 showed more variation. Again it was possible to observe a reduction in the conversion with I142M, R151H and R211Q and no or almost no conversion with N146S, N146D and Q222K and with Q215R respectively. In subsequent experiments, caprine PrPC variants were directly biotinylated so that goat or sheep scrapie as well as cattle, sheep or goat BSE derived PrPSc could be used. In these assays I142M, H143R and R211Q clearly reduced the conversion of PrPC with ovine and caprine scrapie isolates, whereas R151H did not influence the conversion efficiency of biotin-tagged PrPC. Conversion with scrapie isolates showed a marked reduction or no conversion in the case of N146S and N146D which correlated again with the Me7 data and the in vivo observations. In the case of bovine BSE isolates, the cell-free conversion mimicked the species barrier observed in vivo. BSE material from cattle barely converted any caprine PrPC variant into PrPres, whereas BSE from sheep converted all variants including the resistance-associated N146S and N146D, suggesting that the resistance is also prion strain specific. A marked reduction in the conversion rate was also observed with I142M and, less pronounced, with H143R and R211Q corroborating the protective role of these polymorphisms against TSEs. When co-incubated, resistance-associated variants N146D, N146S and Q222K produced a dominant negative effect on the conversion of the susceptible wild-type PrPC genotype (INRQ). In a similar way, the incubation of I142M and H143R also reduced the amount of PrPres in a mixture with INRQ. In conclusion, the cell-free conversion assay results show that the caprine PrP polymorphisms M112T, I142M, R143H, N146S, N146D, R151H, R215Q and Q222K correlated clearly with the in vivo susceptibilities of the goats carrying these polymorphisms. Apart from practical implications, like the possibility of breeding TSE resistant goats, these data indicate that scrapie resistance is modulated by thermodynamic changes affecting PrPC-PrPSc interactions and the formation of conversion intermediates.
The present study was aimed at associating further genes to selected types of laminopathies applying a functional candidate gene approach. Additionally, genotype/phenotype correlations in defined laminopathies were investigated to extend the clinical spectrum and considering practical aspects of molecular genetic analysis in laminopathies. Primary and secondary laminopathies are rare genetic disorders caused by mutations in genes encoding proteins of the nuclear lamina or proteins interacting with the nuclear lamina. So far at least 14 distinct disease phenotypes of primary laminopathies have been found mostly caused by pleiotropic lamin A/C ( LMNA) mutations. Secondary laminopathies can be caused by mutations in other than lamin genes including emerin (STA), lamin associated protein-2 (LAP2) and ZMPSTE24 (ZMPSTE2).
Wadi Wurayah area is located in the north of Fujairah Emirates between the towns of Khor Bidiyah Fakkan and Oman on the Gulf Coast Line in Fujairah Emirates, United Arab Emirates. It lies within a priority World Wide Fund for Nature ( WWF) Global 200 Ecoregions ( ecoregion 127, Arabian Highland Woodlands and Shrublands ), a rich diversity of sheltering rare and endangered mountainous and freshwater habitats and species , and providing opportunities for the revival and sustenance of local livelihoods. However, as most of the United Arab Emirates and the region , the area is undergoing dramatic changes linked to economic diversification and promotion of tourism. The United Arab Emirates in 1999 approved the programmed of work from the UN convention of Biological Diversity ( CBD). This momentum must be used wants it or disappear. In a first move , the United Arab Emirates established the federal Environment Agency ( FEA) that produced the Environmental Law of 1999 with the role to encourage each to Emirates assess its land and coastal / marine resources, formulate plans for establishing protected areas , upgrade those that may already exist , and help implement the environment law. In early 2006, UAE created its first Ministry of Environment and Water ( MEW ), which was before the Ministry of Agriculture and Fisheries. Environmental issues and a greater role of the civil society are now higher on the agenda of the United Arab Emirates government , partner of the Emirates Wildlife Society ( EWS ), the World Wide Fund for Nature ( WWF) - UAE Project Office is the only international conservation NGOs operating in the UAE that plays a pioneering role in partnering with local- governmental institutions to establish win- win solutions. The United Arab Emirates is making tremendous efforts in diversification is the development of tourism. Too often tourism mass , With all of its negative environmental consequences is privileged. However, the more traditional Emirates are seeking alternatives that would preserve their environment and respect the traditional lifestyles of the local communities. This study aims to help a sustainable biosphere reserve integrating oneself local traditional and lifestyle with the conservation of biodiversity and habitat inimitable by providing a model of economical incentives unique to the region . In order to further the implementation of the Wadi Wurayah Biosphere Reserve, this study will: • Implement a set of carefully - targeted actions in Wadi Wurayah and its hinterland and therefore Fujairah Emirates. • Work to demonstrate the feasibility and viability of combining environmental protection in a sensitive area with the preservation of traditional activities. • Support the capacity building of key national and local authorities and selected partners in the Emirates of Fujairah and the UAE So that they have the awareness and skills to fully realize the aims of the study . To set out and develop options for sustainable natural resource management in the proposed Wurayah Biosphere Reserve , one of the UAE as examples of marginal dry lands , building on environmental information system was the best choice using Geographic information systems (GIS ) as a tool. This has been classified to there steps of work: Field Survey and Analysis Lab Office work. As a first step, this study used to survey this area in the light of the work done by the EWSWWF and the Fujairah Municipality, to evaluate the potential and the feasibility of the creation of a Biosphere Reserve. The traditional field survey has been carried out in three batches between January 2007 and January , 2009 for sample collection using specially tailored database forms that suit the properties and nature of the variables measured, and the database . Design The information obtained from field survey included the Landscape and their local classification and distribution , local habitats , water catchments areas , local rangeland systems and indigenous agro -ecological zones. This information in addition to the laboratory analysis has then be transformed into GIS format, and overlaid with the base maps of the study area in order to produce a georeferenced maps. Various types of maps required according the selected works related to area of study have been used as an input data for the GIS system An integrated management methodology / approach has been proposed associated with the plan of work throughout the forthcoming years. The plan of work is designed to be as consistent as possible with that of the concept of the UNESCO 's Man and Biosphere Program.
Tunable Diode Laser Absorption Spectroscopy in the mid InfraRed spectral range (IR-TDLAS) has been applied to investigate the behaviour of CF, CF2 and C2F4 species produced in pulsed CF4/H2 capacitively coupled radio frequency plasmas (13.56 MHz CCP). This experimental technique was shown to be suitable for temporally resolved measurements of the absolute number density of the target molecules in the studied fluorocarbon discharges. The temporal resolution of about 20…40 ms typically achieved in the standard data acquisition mode (“stream mode”) was sufficient for the real-time measurements of CF2 and C2F4, but not of CF whose kinetics was observed to be much faster. Therefore, a more sophisticated approach (“burst mode”) providing a temporal resolution of 0.94 ms was established and successfully applied to CF density measurements. In order to enable the TDLAS measurements of the target species, preliminary investigations on their spectroscopic data had been carried out. In particular, pure C2F4 has been produced in laboratory by means of vacuum thermal decomposition (pyrolysis) of polytetrafluoroethylene and used as a reference gas. Therefore, an absorption structure consisting of several overlapping C2F4 lines around 1337.11 cm-1 was selected and carefully calibrated, which provided the first absolute measurements of the species by means of the applied experimental technique. The absolute number density traces measured for CF, CF2 and C2F4 in the studied pulsed plasmas were then analysed, in which two differential balance equations were proposed for each of the species to describe their behaviour during both “plasma on” and “plasma off” phases. Analytical solutions of the balance equations were used to fit the experimental data and hence to deduce important information on the kinetics of the studied molecules. In particular, during the “plasma off” phase, the self-recombination of CF2 (CF2 + CF2 (+M) → C2F4 (+M)) was found to be dominant in the kinetics of the radical, but of minor importance for C2F4 production. A rapid consumption of CF observed within 7…25 ms after switching off the plasma was explained mainly by volume reaction with other species (most likely with CF3), whereas diffusion of the radical towards the reactor walls followed by sticking on the surfaces was found to contribute only at relatively low pressures (<10 Pa). Under certain discharge conditions, measured CF density traces exhibited significant overshoots in 50…150 ms after the plasma ignition, which had not been known from literature before. The electron impact fragmentation of C2F4 was shown to be essential for CF production at the beginning of the “plasma on” phase and therefore for formation of the observed CF density overshoots. Finally, the broad band FTIR spectroscopy was applied in order to better characterize the gas phase composition of the studied plasmas. Thus, absorption bands of CF4, C2F4, C2F6, C3F8, CHF3 and HF stable molecules were detected in the FTIR spectra recorded between 400 and 4000 cm-1. The spectra were then successfully deconvolved and the absolute concentration of the detected species was estimated. In particular, the absolute number density of C2F4 obtained from the FTIR measurements was in a good agreement with that achieved by means of the IR-TDLAS technique. The work was supported by the German Research Foundation (DFG) within the framework of the Collaborative Research Centre Transregio 24 “Fundamentals of Complex Plasmas” (SFB/TRR24, project section B5).
In the present work, a time- and radial-dependent fluid model has been developed to describe the glow-to-arc transition of the positive column in the course of constriction. The self-consistent model comprises the particle balance equations for the relevant species, the balance equation of the mean electron energy and the heavy particle temperature in the plasma, the Poisson equation for the space-charge potential, and a current balance determining the axial electric field. The model adopts the nonlocal moment method, i.e., the system of the balance equations resulting from the moments of the radially dependent Boltzmann equation is solved. The electron transport and rate coefficients are adapted as functions of the mean energy of the electrons, the gas temperature and the ionization degree. The model is applied to a description of the constriction of the dc positive column in argon, for a wide range of pressures and applied currents. Pronounced nonlocal features of the mean electron energy balance are found and their influence on the constricted argon positive column is analyzed. Different assumptions concerning the electron velocity distribution function (EVDF) have been considered in the present model. The assumption of a Maxwellian distribution for the electrons was found to be inappropriate, while the assumption of a Druyvesteyn distribution for the electrons was found to be suitable for describing qualitatively the glow-to-arc transition. However, the standard model using the EVDF obtained from the solution of the steady-state, spatially homogeneous electron Boltzmann equation including electron-electron collisions allows to describe the constriction effect and provides best agreement with experimental data and other available modelling results. The fluid model has also been used to study a medium-pressure pulsed positive column in xenon at conditions of the contracted discharge. The simulation results provide a detailed insight in the physical mechanisms of xenon discharges in pulsed mode. The stepwise ionization of the excited atoms, the conversion of the atomic ions into molecular ions as well as the dissociative recombination of the molecular ions are found to be the most important processes for the pulsed positive column in xenon plasmas at conditions of the contracted discharge. The comparison of the model predictions with experimental results generally shows good agreement. In particular, the model predictions are suitable for qualitative reproduction of the significant increase of low-lying atomic levels densities as well as of the higher and of the relaxed lowest vibrational states of the Xe2* excimers in the afterglow phase of the pulse.
Bacillus licheniformis is one of the most important hosts used in the biotechnological industry for the production of technical enzymes, antibiotics and a number of biochemicals. Although this bacterium has been used for a long time as an expression host, only little information on expression systems of this host is available. An expression system could be controlled by a cell density signal, a specific chemical inducer or a thermal shift. A limiting substrate such as glucose or phosphate limitation is suggested to use as the signal for the induction of an expression system. When B. licheniformis cells are subjected to nutrient limitation conditions, numerous genes involved in the metabolism of alternative nutrient sources are induced in order to keep cell survival. Therefore, the main topic of this study was to identify and investigate the regulation of genes or operons which are strongly induced in B. licheniformis cells grown under nutrient limitation conditions in order to apply for the construction of potential new expression systems. The research includes studies on the regulation of genes which are responsible for the acetoin and 2,3-butanediol utilization in B. licheniformis cells grown under glucose limitation conditions. Furthermore, we also analyzed the regulation of phytase gene expression as well as investigated the function of a putative ribonuclease expressed in B. licheniformis under phosphate limitation conditions. From this study, it was shown that in B. licheniformis, the utilization of acetoin and 2,3-butanediol was mainly mediated by enzymes encoded by the acoABCL operon. The transcription of this operon was regulated by sigma L transcription factor and was induced by acetoin. The acuABC operon was suggested to play as an indirect regulatory role for the acetoin utilization in B. licheniformis. This operon was controlled by a typical sigma A dependent promoter, however, acetoin was not an inducer for its expression. Furthermore, the regulation of phytase gene expression was suggested to be controlled by PhoPR-two component systems. The results showed that phytate, which is the substrate of phytase enzyme, was not an inducer for the expression of phy gene. However, growth experiments revealed that phytate served as a good alternative phosphate source for the growth of B. licheniformis cells under these conditions. Finally, the inactivation of BLi03719 gene, coding for a putative ribonuclease, resulted in an increase of the total RNA concentration of B. licheniformis cells grown in phosphate limited medium. However, the mutation did not affect the expression of the heterologous reporter gene. Therefore, it could be speculated that the putative ribonuclease BLi03719 plays a role in ribosomal RNA degradation under these conditions.
The aim of this study was to invistigate the effect of the oral health component of a general health promoting program implemented in primary schools of Greifswald city and east Pomerania region. Methods: This program was part of an innovative multidisciplinary general health promoting program, a longitudinal collaborative project based on health competence concept. Seven hundred and forty students with an age range 9-12 years (mean 10.34, SD±0.56, 48% females) were recruited from the fifth grade students of different 18 primary schools. The schools were randomly allocated into two groups. Oral health education was provided to the teachers in the intervention schools and then they conveyed it to their students, while no additional measures were conducted in the control schools. School dental examinations as well as questionnaires for the students and their parents were conducted at baseline and after one and half year of the program. Results: A significant correlation between caries increment and intervention/control group was reported, with a 35% higher risk in the control group. High socio-economic status has a significant highly protective effect in the intervention program with a reduction in incidence risk ratio of 94% (p < 0.001). In the low socio-economic status no preventive effect could found. The association between overweight/obese students and caries increment was border line significance (IRR 1.37, p = 0.055). The intervention program left a protective effective on the students who reported; lower tooth-brushing frequency, do not take care of their teeth, and do not consider sound and healthy teeth. Conclusion: The implemented program was effective in improving dental health, especially among students with high socio-economic status, but failed to achieve an effect in the low socio-economic group. The program was successful in maintaining a good dental health status among students who lack for the essential oral health competence items at the beginning. Social inequalities are an important issue which was partly tackled with a competence-based health promoting program, therefore, additional 76 compulsory preventive measures, such as daily or weekly tooth-brushing at school, should be seriously considered as behavior-centered approach.
Eight hundred and fifty two students with an age range 9-13 years (mean 10.34, SD±0.56, 48% females) were recruited from the fifth grade students of different 19 primary schools in Greifswald and East Pomerania. In conjunction with the compulsory dental community examination, additional data were collected with two questionnaires for the children and their parents. Newly generated items were taken from the children’s questionnaire to form short scales for oral health-related knowledge, behaviour, attitudes. Parents’ questionnaire contains questions on socio-economic status (SES) and child’s health. The response rate was 93.2%; 78.8% for children; parents, respectively. Results: The distribution of DMFT values was highly polarized with most of the children (71%) exhibiting no carious defects, fillings or missing teeth in the permanent dentition with a mean of 0.6 ±1.2. There was a significant correlation between DMFT and social class levels (rs=-0.19, p=0.001) with mean DMFT values of 0.9 ± 1.3, 0.6 ±1.1 and 0.4 ± 0.9 for the low, medium and higher social strata, respectively. There was a clear correlation between the dental attitude and dental behaviour (rs=0.32, p=0.003). However, correlations between knowledge vs. attitude and knowledge vs. behaviour were loose. A statistically significant correlation between DMFT and dental behaviour was found (rs=-0.15, p=0.003). It should be noted that children with higher self-esteem were found to have significantly higher dental awareness scores (rs=0.19, p=0.001). General health was a significant predictor for caries incidence (rs=0.08, p=0.01). The frequency of drinking lemonade or ice tea and eating salty snacks (chips, nuts) showed clear correlations with the DMFT (rs=0.17 and 0.13, p<0.01). Prolonged daily TV watching was associated directly with DMFT values (rs=0.13, p=0.001). A significant correlation was found between caries and smoking, even after adjusting for age (rs=0.1, p=0.002). Smoking children had a significantly higher DMFT rate than children who were not smokers with a mean DMFT of 0.9 ±1.5 vs 0.6 ±1.2 (p=0.004). Interestingly, each of prolonged TV watching, more lemonade drinking and smoking were correlated directly with the low socioeconomic status (Spearman correlation coefficient of 0.13, 0.2 and 0.17, respectively, p<0.05). Regarding malocclusion, 64% the subjects had at least one type of anomaly. Crowding and maxillary overjet represented the major proportion 28% and 23%, respectively. Males exhibited significantly higher increased overbite scores than females p=0.04. Whereas the prevalence of crowding was more common in females than males (p=0.05). Amazingly, more malocclusion was registered in children with caries-free primary teeth when compared to children with carious primary teeth (p=0.05). No significant differences in the mean of dmft or DMFT value were found between normal and non normal occlusion (p>0.05). Undergoing to orthodontic treatment was associated with significantly higher dental awareness scores (p=0.003). No correlation between socio-economic status and malocclusion was registered. Conclusion: This thesis confirms the decline and polarisation of dental caries. Dental behaviour was mostly independent of dental knowledge, but depended on dental attitude. Higher scores in dental behaviour resulted in lower DMFT scores; possibly, oral health promotion should strengthen attitude and actual behaviour instead of knowledge. Higher scores in self-esteem and general health connected with lower caries incidence and higher score in dental awareness. Social inequalities was strongly linked with health inequalities with more prevalence of caries, smoking, prolonged TV watching, wrong diet habits and less sealants application among children of low SES. Hence, oral health-related interventions in children sample should be directed at the social structures with more incorporating of oral health promotion programs into other general health promotion programs. This thesis suggests that; the establishment of healthy behaviours such as a regular teeth brushing at school could be one of the most successful ways to involve all children especially children of low SES in dental care. Malocclusion traits were very common in this sample. This underlines the need for more orthodontic preventive programs among children, in order to reducing the risk factors of malocclusion. The association between prevalence of malocclusion and socio economic status could not be established. No generalised conclusion could be drawn about the relationship between caries and malocclusion.
This dissertation evaluates the effects of site conditions and livestock grazing on the vegetation of Azerbaijan’s winter pastures. We improved methods to estimate grazing intensity in vast rangelands and enhanced an approach to detect discontinuities in vegetation changes along environmental gradients. All analyses use field data from the semi-arid rangelands of Gobustan and Jeiranchel, at the foothills of the Greater Caucasus mountains. The data set comprises 313 vegetation relevés, each sized 100 m², based on a pre-stratification using topographical parameters. Additionally, we included data from farm transects and exclosure experiments. For each plot, selected site and soil variables were determined. VEGETATION AND SITE CONDITIONS: By means of cluster analysis, we derived 16 vegetation types with a total of 272 vascular plant species. Our vegetation classification, which is closely linked to site conditions, is an important groundwork for adapted rangeland management and monitoring. The study areas are dominated by semi-deserts with a high coverage of dwarf shrubs, and the mean number of vascular plant species was found to be about 28 per 100 m². According to ordination analysis (NMDS), species composition changes primarily along the altitudinal gradient, gradually proceeding from the Salsola nodulosa semi-deserts of the lowest parts (below 300 m a.s.l.) to the Salsola ericoides and Artemisia lerchiana semi-deserts of the upper regions (300–650 m a.s.l.). Soil salinity and carbonate concentration decrease as altitude increases. A second gradient reflects grazing intensity. One plant community that is typically found on intensively grazed sites in the vicinity of farmyards stands apart from the rest, which are subject to lower grazing and trampling pressures. A third factor that differentiates plant communities is the sand concentration of the soils. Additionally, communities that occur on steep slopes differ from communities that occur on level terrain. EXCLOSURE EXPERIMENTS: Exclosure experiments revealed that short-time abandonment of grazing leads to an increase in the number of annual species, in vegetation coverage, and in the heights of forbs and grasses. Clipping experiments indicated that the herbaceous species show hardly any compensatory growth in response to grazing. ESTIMATING GRAZING INTENSITY: A recurrent theoretical problem in rangeland research is the spatial modelling of grazing intensity around grazing hotspots like farms or watering places, the so called piospheres. In a widely used approach, grazing intensity is assumed to decrease in direct proportion to the distance from a hotspot. The resulting response patterns, which relate characteristics of the vegetation or site conditions to grazing intensity, are often nonlinear, and have been interpreted as indicating threshold changes or diff erent state-and-transitions along grazing gradients. However, we show that these ‘thresholds’ are usually geometrical artefacts. Taking into account the concentric structure of grazing hotspots, we suggest a new approach that approximates grazing intensity as the ratio of the total number of livestock kept at the farm to the distance between a given plot and the hotspot centre. Our approach is a simple yet significant improvement over current approaches because it enables us to merge or compare data from different sampling sites and because the approximation is in direct proportion to other grazing indicators like dung density or soil salinity. SPECIES TURNOVER PATTERNS: Combining our new grazing pressure model with species presence/absence data, we modelled vascular plant species responses, patterns of species richness and species turnover along grazing gradients on farm transects in Gobustan. The derived typical species response pattern along the finite grazing gradient is a sigmoid decrease. Species richness declines monotonically with increasing grazing intensity and thus conforms to generally acknowledged assumptions on the relationship between species richness and grazing pressure in semi-arid rangelands. Species turnover along the gradient was calculated using the slopes of species response curves. At first sight, the resulting pattern gives evidence for a discontinuous change. However, it ranges within the 95 % confidence interval of a null model based on assumptions of the individualistic continuum concept. Thus, species composition seems to change continuously along grazing gradients in Gobustan. This new null model approach can probably be adapted and applied to all ecological gradients and is useful for the validation of individualcontinuum or community concepts.
Impurity ions pose a potentially serious threat to fusion plasma performance by affecting the confinement in various, usually deleterious, ways. Due to the creation of helium ash during fusion reactions and the interaction of the plasma with the wall components, which makes it possible for heavy ions to penetrate into the core plasma, impurities can intrinsically not be avoided. Therefore, it is essential to study their behaviour in the fusion plasma in detail. Within the framework of this thesis, different problems arising in connection with impurities have been investigated. 1. Collisional damping of zonal flows in tokamkas: The effect of impurities on the collisional damping of zonal flows is investigated. Since the Coulomb collision frequency increases with increasing ion charge, heavy, highly charged impurities play an important role in this process. The effect of such impurities on the linear response of the plasma to an external potential perturbation, as caused by zonal flows, is calculated with analytical methods and compared with numerical simulations, resulting in good agreement. 2. Impurity transport driven by microturbulence in tokamaks: Fine scale turbulence driven by microinstabilities is a source of particle and heat transport in a fusion reactor. A semi-analytical model is presented describing the resulting impurity fluxes and the stability boundary of the underlying mode. The results are compared with numerical simulations. Both the impurity flux and the stability boundary are found to depend strongly on the plasma parameters such as the impurity density and the temperature gradient. 3. Pfirsch-Schlüter transport in stellarators: Due to geometry effects, collisional transport plays a much more prominent role in stellarators than in tokamaks. Analytical expressions for the particle and heat fluxes in an impure, collisional plasma are derived from first principles. Contrary to the tokamak case, where collisional transport is exclusively caused directly by friction, in stellarators an additional source of transport exists, namely pressure anisotropy. Since this term is, contrary to the contribution from friction, non-ambipolar, it plays an important role regarding the ambipolar electric field. Furthermore, the behaviour of heavy impurities in the presence of strong radial temperature and density gradients is studied, which lead to a redistribution of the impurities on the flux surfaces. As a consequence, the radial impurity flux is decreased considerably compared with a plasma in which the impurities are evenly distributed on the flux surfaces.
Electromagnetic Drift Waves
(2010)
In the rf-plasma of the linear magnetized VINETA experiment, different types of low-frequency waves are observed. The emphasis in this work is on the interaction mechanism between drift waves on the one and kinetic Alfven waves on the other hand. In the peaked density profile of the plasma column drift waves occur as modulation of the plasma density. As gradient driven instability, they draw their energy from the radial density gradients. Alfven waves as magnetic field fluctuations are stable in the present configuration. They are launched by a magnetic excitation antenna. Parallel conduction currents in the plasma are common to both wave phenoma. A B-dot probe as standard diagnostic tool is used to detect the fluctuating magnetic fields of both wave types. The challenge are the small induced voltages due to the low wave frequency. The probe design with an integrated amplifier close to the probe head takes this into acount. The developed B-dot probe is mounted to different positioning systems to characterize both wave phenomena. For Alfven waves, the dispersion relation is recorded experimentally. It is found to be in good agreement with the prediction of the Hall-MHD theory with included resistive term, accounting for the cold collisional plasma. The fluctuating magnetic field pattern is recorded with azimuthal scans. The current density is obained by Amperes law. It is concentrated in helically twisted current filaments. For the unstable drift waves, similar investigations are done with simultaneously recorded density fluctuations. In the azimuthal plane, the locations of the parallel current filaments and the fluctuating density are found to be in phase, supporting the predicted drive of parallel currents by pressure gradients. A mutual influence of the two wave types is observed in an interaction experiment. Assuming parallel currents as coupling quantity, an interpretation of the experimental findings is given based on the linear theory of drift waves.
“Za Hranetsiu” – “Beyond the Border”: Constructions of Identities in Ukrainian-Canadian Literature
(2010)
Grounded in the literary and cultural studies, the dissertation “Za Hranetsiu” – “Beyond the Border”: Constructions of Identities in Ukrainian-Canadian Literature answers the question how identities of different Ukrainian immigrants and their offspring have been constructed, continuously developed and transformed in contemporary Canadian literature. The study simultaneously presents a discussion of postmodern identities, a concise historical survey of Ukrainian immigration to Canada in the nineteenth and twentieth centuries, and an overall picture of the exceptionally substantial body of Ukrainian-Canadian literature. Detailed literary analyses focus on seven Ukrainian-Canadian works: Sons of the Soil (1939-45/1959) by Illia Kiriak, Yellow Boots (1954) by Vera Lysenko, A Letter to My Son (1981) by George Ryga, The Green Library (1996) by Janice Kulyk Keefer, The Doomed Bridegroom: A Memoir (1998) by Myrna Kostash, Kalyna’s Song (2003) by Lisa Grekul, and The Ladies’ Lending Library (2007) by Janice Kulyk Keefer.
1. "Sole or Whole" – Quilting the Racialized Subject Calgary feminist and scholar Aruna Srivastava tackles the complex question of what it means to be "mixed-race/mixedblood/halfbreed/mestizo/hybrid/hyphenated?" in her editorial to Hyphe Nation (1996). In response to her question, this study of contemporary African-Canadian literature suggests that mixed-race Canadians are often constructed as the Other in Canadian society. In consequence, constructions of "racial hybridity" in African-Canadian literature usually aim at carving out a space that doesnt marginalize "racial" mixing but eviscerates restrictive Manichean constructions of identity in order to promulgate concepts of wholeness and self-definition. In consequence, African-Canadian mixed-race writers create hybrid identities that are infinite, multilayered, fragmented and yet whole. They reflect the processes of shifting, overlapping and re-creation in the process of creating identity and can hence be read as representations of complex, de-central, non-hierarchical identities. They are quilting multidimensional racialized subjects. 2. Signifying the In-Between: "Race", "Racial Hybridity" and Questions of Belonging "Race" is not a biological category. Rather it represents a social construction predicated upon the interpretation of difference. It was designed to establish, justify or perpetuate hegemonic social structures and is adherent to the principle of white supremacy. Contemporary "race" theory often neglects the experiences of racially mixed individuals because it fails to offer flexible models of identity in which bi- and multiracial people find themselves represented. This thesis argues in favor of a poetics of difference that accepts and recognizes the heterogeneity of subjectivities while taking into consideration the various dimensions of class, gender, sexuality and ethnicity. Accordingly, constructions of racial hybridity in contemporary African-Canadian literature demonstrate that racism must be acknowledged as an ideology in which people believe and upon which people act. Thus identities often bear the stamp of various histories of resistance and domination, while tackling the question of belonging and re-defining Canadianness. 3. African-Canadian Borderlands References to interracial contacts and the existence of bi- and multiracial people have been omitted from the official founding narratives of the Canadian nation. African-Canadian writers have therefore been successful chroniclers of the past and filled blanks in Canadian historiography in order to shed light on hitherto repressed or erased knowledge. Part of this process is also the inscription of the mixed-race experience into Canadas past. Along this line, bi- and multiracial Canadians often criticize that the state-proclaimed policy of multiculturalism fails to take their manifold racial and ethnic subjectivities into account. 4. "From Sole to Whole" – African-Canadian Mixed-Race Poetics Mixed-race characters in US-American literature often appear in the literary motif of the "tragic mulatto" and it is often implicitly suggested that the bi- or multiracial protagonist is categorized as black. In contrast, African-Canadian constructions of racial hybridity less frequently adhere to the stereotype of the "tragic mulatto" and leave more space for employing alternative modes of racial and ethnic identification. Canadian writers of mixed descent have created a mixed-race poetics that calls attention to contexts, relationships, intersections and wholes. They encourage sites of inclusiveness, incessant shifting and discontinuity in the process of constructing identities. Nevertheless, modes of identification among racially hybrid writers in Canada vary, ranging from detesting whiteness to claiming a Black Nationalist stance. In general, however, they support the idea of fluid and flexible identities. The answer to the initial question of Srivastava is hence given by a vast variety of African-Canadian subject positions. An essentially "black" or "racially mixed" Canadian subject does not exist. Instead, constructions of racial hybridity in African-Canadian literature offer a holistic view of identity and aim at re-conceptualizing the various senses of self and community in Canada. This strategy provides a significant means of self-empowerment and self-reclamation – making racially mixed African-Canadians "whole" instead of "sole".
A quantum kinetic approach is presented to investigate the energy relaxation of dense strongly coupled two-temperature plasmas. We derive a balance equation for the mean total energy of a plasma species including a quite general expression for the transfer rate. An approximation scheme is used leading to an expression of the transfer rates for systems with coupled modes relevant for the warm dense matter regime. The theory is then applied to dense beryllium plasmas under conditions such as realized in recent experiments. Special attention is paid to the influence of correlation and quantum effects on the relaxation process.
In classical Drude theory the conductivity is determined by the mass of the propagating particles and the mean free path between two scattering events. For a quantum particle this simple picture of diffusive transport loses relevance if strong correlations dominate the particle motion. We study a situation where the propagation of a fermionic particle is possible only through creation and annihilation of local bosonic excitations. This correlated quantum transport process is outside the Drude picture, since one cannot distinguish between free propagation and intermittent scattering. The characterization of transport is possible using the Drude weight obtained from the f-sum rule, although its interpretation in terms of free mass and mean free path breaks down. For the situation studied we calculate the Green's function and Drude weight using a Green's functions expansion technique, and discuss their physical meaning.
In order to clarify the physics of the crossover from a spin-density-wave (SDW) Mott insulator to a charge-density-wave (CDW) Peierls insulator in one-dimensional (1D) systems, we investigate the Hubbard-Holstein Hamiltonian at half filling within a density matrix renormalisation group (DMRG) approach. Determining the spin and charge correlation exponents, the momentum distribution function, and various excitation gaps, we confirm that an intervening metallic phase expands the SDW-CDW transition in the weak-coupling regime.
We present a Green's function based treatment of the effects of electron-phonon coupling on transport through a molecular quantum dot in the quantum limit. Thereby we combine an incomplete variational Lang-Firsov approach with a perturbative calculation of the electron-phonon self energy in the framework of generalised Matsubara Green functions and a Landauer-type transport description. Calculating the ground-state energy, the dot single-particle spectral function and the linear conductance at finite carrier density, we study the low-temperature transport properties of the vibrating quantum dot sandwiched between metallic leads in the whole electron-phonon coupling strength regime. We discuss corrections to the concept of an anti-adiabatic dot polaron and show how a deformable quantum dot can act as a molecular switch.
We discuss a numerical method to study electron transport in mesoscopic devices out of equilibrium. The method is based on the solution of operator equations of motion, using efficient Chebyshev time propagation techniques. Its peculiar feature is the propagation of operators backwards in time. In this way the resource consumption scales linearly with the number of states used to represent the system. This allows us to calculate the current for non-interacting electrons in large one-, two- and three-dimensional lead-device configurations with time-dependent voltages or potentials. We discuss the technical aspects of the method and present results for an electron pump device and a disordered system, where we find transient behaviour that exists for a very long time and may be accessible to experiments.
Pyrrolobenzodiazepines (PBDs) are a group of antitumor antibiotics that exert their biological activity by alkylation of guanine bases within the minor groove of double-stranded DNA through nucleophilic attack of the guanine amino group on the PBD imine functionality. In trying to increase both the binding strength and sequence selectivity for further enhancing their biological activity, PBDs were linked to additional DNA binding moieties. Preliminary DNA melting experiments partly also performed in our lab with a series of closely related PBD-naphthalimide and benzimidazole conjugates revealed extraordinary DNA-binding capability of hybrids PBD-NIM and PBD-BIMZ. These studies also indicated the favorable contribution of the piperazine structure on drug binding to the DNA duplex. Previously, in vitro cytotoxicity studies also showed promising antitumor activity of both compounds with PBD-BIMZ having the largest cytotoxic potential among various examined conjugates. In the present work, the kinetics, thermodynamics and structural details of the drug-DNA interactions have been determined employing a variety of spectroscopic, calorimetric and computational methods. Thus, a high thermal duplex stabilization upon DNA binding could be ascertained for both drugs and attributed to their covalent attachment to the DNA guanine bases. The 1:1 binding stoichiometry as well as the exclusive minor groove binding for the benzimidazole and the mixed minor grove - intercalative type of binding for the naphthalimide hybrid could be verified by several spectroscopic methods including NMR spectroscopy. Furthermore, by using a combination of solution NMR and some of the most recent molecular modeling techniques, the first high-resolution structures of DNA-drug complexes with PBD hybrid drugs could be obtained giving detailed insight into the specific drug-DNA interactions. Thus, details on van der Waals and hydrogen bond contacts within the complex and the tight fit of the benzimidazole hybrid into the DNA minor groove could be revealed. By using recent data analysis techniques like clustering algorithms, the high flexibility of the piperazine moiety within the PBD-BIMZ-DNA complex could be nicely captured and visualized. Additionally, a thermodynamic analysis for the non-covalent drug binding by UV and fluorescence spectroscopy as well as by direct calorimetric methods revealed a 1:1 binding mode driven by enthalpy changes and counteracted by unfavorable entropic contributions to result in moderately strong association constants. Analysis of the solvent-accessible surface area confirmed the importance of hydrophobic effects on drug binding and the combination of these data with ITC measurements allowed for an extensive thermodynamic characterization of the drug binding process. With respect to the influence of the individual drug moieties on DNA binding, the importance of the piperazine ring for drug-DNA interactions and the basis for its capability to enhance drug binding were addressed. Furthermore, it could be shown that the naphthalimide and benzimidazole moieties also impart additional sequence selectivity to the alkylating PBD structural unit and these distinct differences in the sequence selectivity could be linked to the three-dimensional structures of the DNA-drug complexes. Clearly, the combination of detailed structural and thermodynamic data of complex formation allows for a better understanding of the binding mechanism and structure-activity relationship when it comes to drug-DNA interactions. Therefore, the information gathered can assist in the design of more efficient derivatives of this type of alkylating DNA binding drugs in particular and of DNA recognition by ligands composed of several motifs in general.
The aim of this thesis was to validate a method called OSCARR for One-pot, Simple Cassette Randomization and Recombination for focused directed evolution, which had been developed by Dr. Hidalgo. It is based upon the megaprimer PCR method using outer primers differing in TM and including asymmetric cycles before the addition of the forward primer to generate more mutated megaprimer. As mutation-carrying primers, spiked oligonucleotides are employed. These spiked oligonucleotides are designed using an algorithm and have strictly defined composition of nucleotides at each position. An OSCARR library of the Pseudomonas fluorescens esterase I (PFE I) of approximately 8000 clones was generated and screened for altered chain-length selectivity. Two mutants with higher activity towards medium chain length p-nitrophenyl esters were identified, both carried the mutation F126I, which causes the substrate entrance tunnel to be widened, thus facilitating access of bulkier substrates to the active site. One mutant carried the additional mutation G120S which completes a catalytic tetrad which is observed mainly in proteases. F126I had a stronger influence on chain-length specificity, so the further amino acids which form the “bottleneck” to the active site were mutated to further widen the entrance, and mutants with improved activity were found. The bottleneck mutants which consist of single, double, triple and quadruple mutants which are mostly combinations of F126L, F144L, F159L and I225L were then assayed for altered enantioselectivity against chiral acids and secondary alcohols. For substrates 1-phenyl-1-propyl acetate (2), 1-phenyl-2-propyl acetate (3) and 1-phenyl ethyl acetate (4), mutants with increased enantioselectivity were found. I225L plays a crucial role, as it is vital for enantioselectivity against 3, but destroys selectivity against 2, both facts obvious from the comparison of the triple mutant without I225L (mutant T3) and the corresponding quadruple mutant including I225L (mutant Q). However, the single mutant I225L alone does not possess high selectivity against 3, so synergistic effects play an important role. The PFE I wild type already possesses a good enantioselectivity in the hydrolysis of 4, but all mutants which were analyzed in detail surpass the wild type. The program YASARA was then used to calculate docking solutions for both enantiomers of 2 and 3 into the wild type and the best mutant. The results revealed that the mutants’ widened bottleneck allows the phenyl moiety of the substrates to point towards the access tunnel, while only (R)-2 does so in the wild type. Residues 126 and 144 do not come very close to the substrate and are more likely to influence substrate diffusion. Another goal was to find a way to confer promiscuous amidase activity upon the PFE I. In the search for structural homologues, a close structural neighbour with amidase activity was found. The --lactamase from Aureobacterium sp. was named after its activity toward the Vince lactam 2-azabicyclo[2.2.1]hept-5-en-3-one. Biocatalysis experiments with the PFE I and its mutants revealed an excellent enantioselectivity against the ( )-lactam. Specific activities were determined for purified proteins, and the activity of some mutants was within the same order of magnitude as lactamase’s activity.
The focus of this thesis is the engineering and analysis of the enantioselectivity of esterases using 3-phenylbutyric acid (3-PBA) as model substrate. An ultra high throughput assay for identification of enantioselective esterases has been developed, based on the combination of in vivo selection and flow cytometry. The in vivo selection medium consists of a couple of pseudo-enantiomers of 3-PBA; one enantiomer is coupled to glycerol (GE), and hydrolysis of this substrate will enable cell survival. The other enantiomer is coupled to the toxin 2,3-dibromopropanol (BE), the hydrolysis of this substrate will cause cell death. Thus, cell survival is a function of the enantioselectivity of the enzyme expressed. The pseudo-enantiomeric substrates are structurally similar to allow selection for enantioselectivity instead of selection for enzyme substrate affinity. Next, esterase BS2 was chosen as negative control to establish the selection system since it hydrolyses both pseudo-enantiomers with low enantioselectivity (E~3 and 1, respectively). High enantioselective esterases towards 3-PBA: esterases PestE and CL1 (E > 100, both (R)-selective) were identified in a screening and used as positive controls. Further, the hyperthermophilic esterase PestE was crystallized. After elucidation of the enzyme structure, the high enantioselectivity of the enzyme towards 3-PBA could be explained by molecular modelling. The optimal concentration of the pseudo-enantiomeric substrates was set to be 5 mM for GE (higher concentrations were toxic) and 20 mM for BE (lower concentrations did not completely inhibit bacterial growth). The in vivo selection system was established together with the identification of a flow cytometric method to differentiate bacterial physiological status. The combination of Syto9 and PI was chosen as staining technique, because it allowed differentiation of the viable and the dead cell populations, and of these from the background. After viability detection by flow cytometry was established, esterases PestE and BS2 were cultivated in selection ((R)-GE and (S)-BE) and anti-selection medium ((S)-GE and (R)-BE). Clear differences in the culture viability depending on the enantioselectivity of the enzyme expressed appeared: cells expressing the (R)-enantioselective PestE could proliferate in selection medium, but could not proliferate in anti-selection medium. Cells expressing the non-selective BS2 did not grow in any media. Further, cultures containing mixtures of BS2/PestE or BS2/CL1 expressing cells were incubated in selection and anti-selection medium, and the viable clones were detected by flow cytometry analysis, sorted out and plated on agar. When the mixtures were incubated in selection medium, enrichment of the (R)-selective enzyme (PestE or CL1) over the non-selective enzyme (BS2) was observed. When the enzyme mixtures were incubated in anti-selection medium, very few colonies grew on agar, indicating that cell survival was a function of enzyme enantioselectivity. The successfully developed assay was used to identify variants with increased enantioselectivity in a mutant library of esterase PFEI (E ~ 3, (R)-selective) created by saturation mutagenesis. After library expression, 108 clones were in vivo selected and analyzed by flow cytometry. The viable cells were sorted out and plated on agar. The 28 resulting colonies were transferred to one microtiterplate and their activity and enantioselectivity (Eapp) was investigated using p-nitrophenyl derivatives. Four interesting mutants were identified: Table 1. Enantioselectivity of the in vivo selected mutants. Mutant Eapp[a]Etrue[b]Etrue[c]Etrue[d]Etrue[e] Mutations C4 80 4 4 3 1 V121I, F198G, V225A E7 >100 2 n.d. 3 n.d. V121S E8 2 25 16 50 >100 V121S, F198G, V225A F5 5 13 15 18 80 F121I, F198C [a] with separate (R)- or (S)-enantiomers of p-nitrophenyl-3-phenylbutanoate. [b] towards GE with cell lysate or [c] pure enzyme. [d] towards Et-3-PB with cell lysate or [e] pure enzyme. n.d. not determined. The mutants were purified and activity and enantioselectivity were determined in kinetic resolutions towards Et-3-PB and GE (Table 1). Mutants identified as highly enantioselective in the Eapp-assay (C4 and E7) were low selective in kinetic resolutions. On the contrary, mutants E8 and F5, which showed low enantioselectivity towards p-nitrophenyl-3-phenylbutanoate, hydrolyzed the 3-phenylbutyric esters with good to excellent enantioselectivities. This confirms that Eapp values can differ much from Etrue values as “you get what you screen for”, and supports that the here described method is very suitable for identification of enantioselective esterases. In this PhD thesis a novel strategy for identification of enantioselective esterases has been developed. This method allows a very high throughput (≥ 108 mutants/day) and opens the bottleneck of variant analysis, which exists in protein engineering technology.
Tertiary alcohols have become interesting targets for organic synthesis themselves or as building blocks for valuable pharmaceutical compounds. However, the synthesis of optically pure tertiary alcohols is still a challenge both chemical and enzymatic means. Enzymes containing the GGG(A)X motif in the active site region have been known to show activity towards these sterically demanding substrates. Several tertiary alcohols have been resolved with high enantioselectivity by using this biocatalytic synthetic route. This thesis aims at providing a better understanding of enantiorecognition of GGG(A)X motif hydrolases in the enzymatic synthesis of enantiomerically enriched tertiary alcohols. Kinetic resolution of a wide range of tertiary alcohols using hydrolases provided insights on factors that can influence enantioselectivity of GGG(A)X motif enzymes. Additionally, a newly proposed chemoenzymatic method to synthesize protected alpha,alpha-dialkyl-alpha-hydroxycarboxylic acids has broadened the application of these enzymes to synthesize optically pure tertiary alcohols. Newly found biocatalysts through functional screening, database mining and rational protein design approaches provided a better enzyme platform for optically pure tertiary alcohol resolution.
Macrophages are cells of immune system and distributed throughout the body. They provide the first line of defense against microbial pathogen infections. Using bone marrow macrophages (BMMs) which derived from mice of strain BALB/c and strain C57BL/6, this study aimed to identify the changes in proteome of the macrophages due to IFN gamma stimulation and S. aureus infection. Two quantitative proteomic techniques, two-dimensional difference gel electrophoresis (2D-DIGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were applied in this study. The analysis results indicated that many proteins which play important roles in immunological functions of macrophages were changed due to IFN gamma stimulation and S. aureus infection. This study also identified the differences in proteome of macrophages derived from mice of strain BALB/c in comparing to macrophages of strain C57BL/6.
Diabetes mellitus has been linked with an increased risk for oral diseases, especially periodontitis. However, studies results were not consistent. The present study was conducted to evaluate whether both type 1 (T1DM) and type 2 diabetes mellitus (T2DM) are associated with increased prevalence and extent of periodontal disease and tooth loss compared with non-diabetic subjects within a homogeneous adult study population. T1DM, T2DM and non-diabetic subjects were recruited from the population-based Study of Health in Pomerania (SHIP). Additionally, T1DM subjects were retrieved from a Diabetes Centre in the same region. The total study population comprised 145 T1DM and 2,647 non-diabetic subjects aged 20-59 years, and 182 T2DM and 1,314 non-diabetic subjects aged 50-81 years. Multivariable regression revealed an association between T1DM and mean attachment loss (B=0.40 [95% CI; 0.19, 0.61], adjusted). Also, T1DM was positively associated with increased number of missing teeth after full adjustment (p<0.001). The association between T1DM and tooth loss was enhanced in subjects aged 40-49 and 50-59 years (p for interaction=0.01). In T2DM subjects, mean attachment loss was significantly higher compared with non-diabetic subjects (B=0.47 [95% CI; 0.21, 0.73], adjusted). The effect of T2DM was significantly enhanced in 60-69-years-old subjects (p for interaction=0.04). The association between T2DM and number of missing teeth was not statistically significant after adjustment (p=0.25). Analyses showed that the effect of T2DM on tooth loss was pronounced in females compared with males (p for interaction=0.01). In accordance with previous literature, present results suggested that periodontal diseases and tooth loss can been seen as a complication of both types of diabetes. Generally, periodontal diseases are preventable and treatable. Therefore, appropriate goals and strategies for improving periodontal health in subjects with diabetes need to be developed. Further, early detection and careful managed therapeutics with the physician and dentist working hand-in-hand may prove beneficial to the patient–s general health.
This thesis contains results from transcriptome studies on different aspects of host-pathogen interactions. First, liver gene expression profiles from a murine chronic stress model served to elucidate aspects of the influence of stress on metabolism and immune response state. Chronic stress in female BALB/c mice was shown to lead to a hypermetabolic syndrome including induction of gluconeogenesis, hypercholesteremia, and loss of essential amino acids, to the induction of the acute phase response, but also of immune suppressive pathways and to the repression of hepatic antigen presentation. Increased leukocyte trafficking, increased oxidative stress together with counter-regulatory gene expression changes, and an induction of apoptosis were detected. The influence of intra-venous infection on the host kidney gene expression was analyzed in another murine model using the wild type strain Staphylococcus aureus RN1HG and its isogenic sigB mutant. Gene expression profiling indicated a highly reproducible host kidney response to infection. The comparison of infected with non-infected samples revealed a strong inflammatory reaction of kidney tissue, e. g. Toll-like receptor signaling, complement system, antigen presentation, interferon and IL-6 signaling. However, the results of this study did not provide any hints for differences in the pathomechanism of the S. aureus strains RN1HG and ΔsigB, since the host response did not differ between infections with the two strains analyzed. Effects of SigB might be transient, only apparent at earlier time points, or might also be compensated for in the in vivo infection by the interlaced pattern of other regulators. SigB might possess only to a lesser extent characteristics attributed to virulence factors and might act in vivo more like a virulence modulator and fine tune bacterial reactions. In addition to the analysis of tissue samples, different in vitro models were furthermore studied. The third part of this thesis focuses on bone-marrow derived macrophages (BMM) of the two mouse strains BALB/c and C57BL/6, which are described in literature to exhibit genetically determined differences in their reaction to infection. Expression profiling was performed on control and IFN-γ treated samples from a serum-free cultivation system and revealed mainly induction of gene expression after treatment of BMM with IFN-γ. Gene expression changes confirmed known IFN-γ effects like induction of immunoproteasome, antigen presentation, interferon signaling related genes, GTPase/GBPs, and inducible NO synthase. IFN-γ dependent gene expression changes were highly similar in BALB/c and C57BL/6 BMM. Considering gene expression differences between BMM of both strains, a similar expression trend was visible on the level of untreated controls as well as after IFN-γ treatment. Differentially expressed genes between BMM of both strains included immune-relevant genes as well as genes linked to cell death, but the coverage of functional groups was limited. The bronchial epithelial cell line S9 was used as an in vitro model system for the infection with S. aureus RN1HG. The fourth chapter in this thesis includes S9 cell gene expression signatures 2.5 h and 6.5 h after start of infection. At the early time point, only 40 genes were differentially expressed, which nevertheless indicated a beginning pro-inflammatory response, e. g. induction of cytokines (IL-6, IFN-β, LIF) or prostaglandin-endoperoxide synthase 2 (PTGS2), but also counter-regulatory processes, e. g. induction of CD274. The host cell response was dramatically aggravated at the later 6.5 h time point. Differential expression was detected for 1196 genes. These included induced cytokines, pattern recognition receptor signaling, antigen presentation, and genes involved in immune defense (e. g. GBPs, MX, APOL). Negative effects on growth and proliferation were even more enhanced in comparison to the early time point, and signs for apoptotic processes were revealed. Finally, the last chapter addresses amongst others the pathogen’s expression profile in the S9 cell in vitro infection model at the two time points 2.5 h and 6.5 h after start of infection by tiling array gene expression analysis. The pathogen expression profiling revealed the activity of the SaeRS two-component system in internalized staphylococci. Partly dependent on SaeRS, the induction of adhesins (e. g. fnbAB, clfAB), toxins (hlgBC, lukDE, hla), and immune evasion genes (e. g. chp, eap) was observed. Furthermore, expression changes of metabolic genes were recorded (gene induction of amino acid biosynthesis, TCA cycle, gluconeogenesis; gene repression of glycolysis, purine biosynthesis, tRNA synthetases). Expression analysis recorded a distinct bacterial expression program, which supported literature results of a specific, bacterial strain and host cell line dependent transcriptional adaptation of the pathogen.
In the post genomic era, novel “Omics” technologies like genomics and proteomics can be used in powerful screening approaches to provide unbiased lists of candidate genes and proteins and thus facilitate a comprehensive analysis of complex diseases such as cancer, which would not have been possible applying traditional genetic and biochemical approaches alone. During my PhD tenure I applied functional genomics screening technologies including proteomics in combination with traditional biochemical and cell biology approaches in two disease oriented projects: 1. Characterization of the role of BCL11b in Human T cell lymphomas (and) 2. Elucidation of the mechanism of pathophysiology of Johanson Blizzard Syndrome using UBR1 knockout mice and JBS patients’ lymphoblasts cell lines.
1.Characterization of the role of BCL11b in Human T cell lymphomas
: The Bcl11b protein belongs to the C2H2-family of Krueppel-like zinc finger proteins and thus is a member of the largest family of transcription factors in eukaryotes. It was shown to be important for a variety of functions such as T cell differentiation, normal development of central nervous system and DNA damage response. Malignant T cells undergo apoptotic cell death upon BCL11B down-regulation. However, the detailed mechanism of this cell death is not fully understood. Two dimensional difference in-gel electrophoresis (2D-DIGE), mass spectrometry and cell biological experiments were employed to investigate the functional impact of knock down of BCL11B in malignant T cell lines such as Jurkat and huT78. To further confirm the findings of these experiments, changes in protein patterns were also recorded after down-regulation of BCL11B expression in Jurkat cells over expressing BcL-xL and in Jurkat cells over expressing BCL11B. These experiments provide evidence for the involvement of the mitochondrial apoptotic pathway and increased levels of cleavage fragments of known caspase targets such as myosin, spectrin and vimentin were observed after BCL11B knockdown. The findings suggest an involvement of ERM proteins, which were up-regulated and phosphorylated upon BCL11B down-regulation. Besides ERM proteins, PDCD5, a key regulator of apoptosis, was also found at increased levels upon down regulation of BCL11B. Moreover, the levels of several proteins implicated in cell cycle entry, including DUT-N, UCK2, MAT1, CDK6, MCM4 and MCM6 were elevated, which might lead to uncontrolled cell cycle progression, uracil misincorporation and cell death. Interestingly, an inverse regulation pattern, i.e. decreased levels of ERM proteins, DUT-N, UCK2 and PDCD5 was seen upon over expression of BCL11B in Jurkat cells. In summary, proteome analyses revealed several previously unidentified mechanisms which could significantly contribute to the cell death following BCL11B knockdown.
2.Elucidation of the mechanism of pathophysiology of Johanson Blizzard Syndrome using UBR1 knockout mice and JBS patients’ lymphoblasts cell lines
: Johanson-Blizzard syndrome (JBS; OMIM 243,800), which was first described in 1971, is a rare autosomal recessively inherited genetic disorder with a unique combination of congenital abnormalities. The most constant clinical feature of JBS is the loss of exocrine pancreatic function due to progressive destruction of pancreatic acini. Genome wide linkage analysis identified the disease associated locus in the 15q14-q21 chromosome region and high-throughput sequencing of this region revealed several truncated and some missense mutations in the UBR1 gene. UBR1 gene contains 47 exons and spans over 161 kilobases. The UBR1 protein belongs to the E3 ubiquitin ligase family and is an important component of the N-end rule pathway of ubiquitous protein degradation. It was hypothesized that stabilization of direct and unique substrates of UBR1 could be the main cause of the JBS pathophysiology. So far sequencing of the UBR1 gene is the only available diagnostic procedure. However, sequencing might not always allow precise prediction of residual UBR1 activity. Hence, this study was started to develop a protein based diagnostic assay for the detection of subclinical cases of JBS and to identify signalling pathways contributing to the pathophysiology of this complex disorder using a murine UBR1 knockout model. 2D-DIGE proteome analysis was carried out for a comparative evaluation of lymphoblast samples of 14 patients and 11 controls. Principal component Analysis (PCA) clearly discriminated JBS patients from controls. However, 4 JBS patients differed from the rest and resembled controls more closely. Western-blot analysis revealed residual UBR1 levels in these patients, which were linked to a milder phenotype. Hierarchical clustering of the three groups (controls, patients with residual UBR1 levels and patients without UBR1) showed group-specific characteristic differences in the abundance of differentially regulated proteins. Quantification of a panel of five selected protein spots encompassing Interferon-induced GTP binding protein, HLA class II histocompatibility antigen, Annexin A6, FK506-binding protein 4 and GRP78 permitted discrimination of controls and JBS patients with mild phenotypes. Of note, the molecular chaperones GRP78 (BiP) and FK506BP were consistently altered in level in JBS patients and probably constitute UBR1 dependent substrates. This suggested JBS as an ER-stress related disease also indicating a possible way of therapeutic intervention. Comparative proteome analysis of UBR1 knockout and wild type animals after caerulein treatment revealed a significant accumulation of pancreatic proteases such as chymotrypsin B, anionic trypsin and pancreatic elastase in animals lacking UBR1. Furthermore, an up-regulation of ER-stress proteins and inflammation related proteins was observed. Phenotypic characterisation revealed in UBR1 knockout animals significantly increased lipase levels, a significantly increased histological score and significantly increased elastase activity 8h after the onset of pancreatitis. In isolated pancreatic acini of UBR1 knockout animals we found a significant increase in intracellular elastase activation upon supramaximal CCK stimulation, which was associated with a significant rise in the rate of necrosis explaining the more severe phenotype in the UBR1 knock-out animals. A TUNEL assay showed that there was more apoptosis in wild type compared to UBR1 knockout mice. Another set of experiments was designed to identify physiologically important substrates of UBR1. Inhibition of such substrates might then in turn allow reversion or prevention of the severe form of pancreatitis in UBR1 knockout mice. However, using the trypsin specific and reversible inhibitor S-124 it was shown that impaired trypsin degradation and thereby prolonged activation of this protease did not critically influence the phenotype. Calcium analysis after physiological stimulation revealed an increase of pathological Ca2+ signalling events, i.e. significant decrease of spike number and significant increase of spike duration. Of the candidates potentially influencing Ca2+ signalling RGS4 turned out to be of particular importance. Pre-incubation of pancreatic acini of UBR1 knockout animals with a specific RGS4 inhibitor (CCG-4986, 10 µM) normalized Ca2+ patterns, did not affect trypsin activity itself but prevented Ca2+-triggered premature trypsin activation and thus acinar disintegration. In summary, using lymphoblasts samples of JBS patients we were able to deduce a protein panel which could be developed as a possible diagnostic tool for confirmation of JBS syndrome. Furthermore, using UBR1 knockout mice in an experimental model we were able to elucidate the vital function of UBR1 and its direct substrate RGS4 in the defense against pathologic pancreatic damage thereby manifesting JBS as an inflammatory disorder due to an inadequate UBR1 mediated defense.
Staphylococcus (S.) aureus is the most common cause of nosocomial infections and the species is becoming increasingly resistant to antibiotics. In contrast, about 35% of the healthy population are colonized with S. aureus in the anterior nares. The genetic make-up of this species is highly diverse. Mobile genetic elements comprise about 15% of the S. aureus genome. They encode many virulence factors like the 21 different known staphylococcal superantigens (SAgs), highly potent activators of T lymphocytes. Besides their well known causative role in food poisoning and toxic shock syndrome, information about SAg involvement in pathogenesis is limited. On the other hand, the human host and its immune response are also highly diverse. This study focuses on SAgs, because they are potent virulence factors that are highly diverse and therefore mirror of the variability of the species S. aureus. The goals of this work were (i) to identify virulence determinants by comparing the prevalence of SAg genes and phages among colonizing and invasive S. aureus isolates and to correlate it with the clonal background, (ii) to determine the prevalence and the development of anti-SAg antibodies in healthy S. aureus carriers and noncarriers as well as in bacteremia patients, and (iii) to elucidate the reasons for the selective lack of neutralizing serum antibodies specific for a subgroup of SAgs, the egc SAgs. In search for a molecular-epidemiological associations between SAgs and different diseases caused by S. aureus we investigated the distribution of SAg genes and/ or bacteriophages and correlated this with the clonal background, determined by spa genotyping. The analysis of more than 700 S. aureus isolates from nasal colonization, bacteremia or furunculosis revealed that SAg-encoding mobile genetic elements and bacteriophages were strongly associated with the clonal background. As a consequence, each clonal lineage was characterized by a typical SAg gene and phage repertoire. Therefore, we suggest that the simultaneous assessment of virulence gene profiles and the genetic background strongly increases the discriminatory power of genetic investigations into the mechanisms of S. aureus pathogenesis. However, we found no association of SAg genes with bacteremia or furunculosis. While functional neutralization assays closely mimic the protective action of anti-SAg antibodies in vivo, they are labor-intensive and time-consuming. A fast and easy method for the simultaneous quantification of antibody binding to multiple staphylococcal antigens is the Luminex® technology. Using serum samples from persistent carriers and noncarriers we showed a strong correlation between antibody binding and neutralizing capacity against the SAg TSST-1. This assay confirmed the astonishing lack of antibodies against egc SAgs in healthy carriers and noncarriers, which was previously described by Holtfreter and coworkers. Since colonization is probably not sufficient to induce a robust antibody response as revealed by experimental colonization with S. aureus, we propose that (minor) infections are required to induce the high titers of non-egc SAg-neutralizing antibodies in healthy adults. To test this, we investigated whether SAgs elicit a neutralizing antibody response during S. aureus bacteremia. At the acute phase of the disease most patients already had neutralizing antibodies against non-egc SAgs, and antibody titers frequently increased during infection. Notably, egc SAgs did not elicit a boost or de novo generation of specific antibodies. The “egc gap” in the antibody response, which has now been shown in healthy adults, as well as following systemic infection with S. aureus, is astonishing. After all, egc SAgs are by far the most prevalent SAgs. In search for an explanation, the intrinsic properties of three recombinant egc (SEI, SElM, SElO) and non-egc SAgs (SEB, SElQ, TSST-1) were compared in depth. Egc and non-egc SAgs were very similar with regard to induced T cell proliferation, cytokine profiles, and gene expression of human immune cells. However, there was a striking difference in the regulation of the two groups of SAgs by S. aureus in bacterial culture. We conclude that the differential regulation of egc and non-egc SAg has an impact on the immune response. But how are SAgs regulated by S. aureus during its interaction with the host? Up until now most research on regulation of virulence factors has been performed in vitro. The immune response can help to shed light on this problem, because it is an exquisitely specific sensor for the exposure to different antigens. The high prevalence of neutralizing serum antibodies against non-egc SAgs indicates that most healthy adults have been exposed to these toxins during their encounters with S. aureus. For egc SAgs this remains an open question. However, initial data indicate that the egc SAg genes are transcribed during nasal colonization.
Peatlands cover only about 3% of the terrestrial surface but are significant players in the global carbon (C) cycle and the climate system, since they store roughly one quarter of the global soil carbon (C) and are among the largest natural sources of methane (CH4). Since the resulting feedbacks on the climate system are uncertain, research efforts aim at identifying key processes and quantifying the C exchange from ecosystem to regional and global scales. To identify peatland ecosystem dynamics requires analysis of yet different scales. The key scale for their C dynamics is the microform scale, which is the smallest entity of the system. To estimate ecosystem dynamics, up-scaling from the microform scale is needed. Up-scaling demands (1) a correct estimation of the spatial heterogeneity and (2) the correct aggregation. In this thesis, the traditional spatial weighting of microform fluxes by the microform distribution is evaluated by (1) analyzing the flux calculation procedure, (2) investigating the effect of the resolution of the landcover maps on the up-scaling and by (3) cross-evaluating the up-scaling result with the directly measured ecosystem flux. Eventually, it is evaluated how these dynamics are considered in a mechanistic ecosystem model (LPJ-WHyMe). CH4 fluxes were measured on the microform scale with the closed chamber technique and on the ecosystem scale with the eddy covariance (EC) technique. The quantification of microform fluxes relies on the correct flux calculation. Since only few gas samples are taken during the closure period, traditionally the linear regression is applied when calculating CH4 fluxes from chamber measurements. Still, the chamber itself affects the diffusion gradient between peat and chamber atmosphere resulting in a theoretically non-linear concentration increase in the chamber. Using data with six data points per measurement from different microform types it is tested whether the linear or exponential regression fits the data better. In the majority of cases, the linear regression fits best. However, the exponential concentration change might still not be detectable resulting in an underestimation of the ’real‘ flux and the test of different techniqes to estimate the slope of a non-linear function with small sample amounts is recommended. To define the spatial heterogeneity of the peatland surface, the application of remote sensing techniques offer the advantage of supplying area-wide information with less uncertainty when compared to vegetation mapping along transects. However, the required resolution to resolve the microform distribution is <1m which in this study was derived from near-aerial photography. Besides for up-scaling, the resulting high-resolution landcover map was used in combination with a footprint model to analyze (1) the effect of landcover on the directly measured ecosystem flux and (2) its spatial representativeness. It was shown that fluctuations of the measured ecosystem flux over periods of several days could be explained by changes of the landcover composition in the source area of the EC measurements. The estimated budget was slightly biased towards the higher emissions from lawns which could be corrected. Still, the seasonal ecosystem CH4 budget was higher than the estimate derived from the up-scaling of microform fluxes. This is most likely due to an underestimation of microform fluxes by the chamber technique. Generally, the budget estimate derived from EC measurements was more accurate, i.e., characterized by less uncertainty than the up-scaled estimate. The developed approach depends on (1) identification and accurate measurements of all relevant microform types and (2) on spatial information which should be smaller than the footprint size of the EC measurements and available on the scale relevant for the studied process, i.e., the microform scale. The demonstrated effect of microform dynamics on the ecosystem flux highlights the importance of dealing with spatial heterogeneity of ecosystems in mechanistic modelling. For example, in LPJ-WHyMe, the ecosystem flux is simulated with mean input variables as water table level. To investigate its model performance, flux data from the rather homogeneous peatland margin and the more heterogeneous peatland centre were compared with the model output. At the homogeneous peatland margin, the ecosystem flux was clearly dominated (with a contribution of 91%) by one microform flux. In this case, one water table level as input variable could be used to estimate the ecosystem flux. However, for a heterogeneous site such as the peatland centre in this study, only one mean water table would simulate a mean microform flux but not the ecosystem flux. Consequently, it is recommended to incorporate at least one high-emitting and one low-emitting microform type in the model to increase the model performance.
Carbon dioxide (CO2) is one of the most important factors of the Earth’s carbon cycle. Peatlands are well-known to be a long term sink for atmospheric carbon dioxide. Under changing environmental conditions, the carbon balance and hence the CO2 fluxes can be significantly changed, and peatlands may even become a significant atmospheric carbon source. To be able to predict the changes in climatic conditions and their effects on ecosystems, it is important to understand the contemporary CO2 exchange of the ecosystems. Many studies on peatland CO2 fluxes have been conducted in the boreal zone of North America and Scandinavia. Still little scientific evidence is available from peatland ecosystems of boreal Russia. This dissertation presents the detailed investigation of CO2 dynamics and the relevant processes and environmental factors from the boreal peatland site Ust-Pojeg (61°56'N, 50°13'E) in Komi Republic, northwest Russia. On the small spatial scale (microform), the investigated peatland was characterised by high variability in vegetation composition and coverage as well as in water table level which resulted in large variability in CO2 fluxes not only between the microform types but also within one microform type. The cumulative flux over the investigation period for the different microforms ranged from strong CO2 sources to CO2 sinks. An area-weighted estimate for the entire peatland showed that it was a CO2 source for the investigation period, which was characterised by average conditions in terms of precipitation and temperature. The CO2 fluxes were measured at different scales: by the closed chamber method at the microform scale and by the eddy covariance technique at the ecosystem scale. Three different upscaling methods were used to compare the fluxes. Irrespective of the upscaling methods, the discrepancies between the estimates based on the upscaled chamber measurements and estimates based on measurements by the eddy covariance technique were high. The high spatial heterogeneity of the vegetation and the water table level and thus of the CO2 fluxes were recognised as reasons for high potential errors when upscaling CO2 fluxes from the microform to the ecosystem level. Large discrepancies were also observed in comparison between measured CO2 fluxes and CO2 estimates based on the mechanistic ecosystem model LPJ-GUESS. Insufficient model forcing may have led to errors in the timing of the onset and the end of the growing season, and the modelled vegetation did not always reproduce the observed vegetation. These two factors may have led to the discrepancies in the model-measurement comparison. Although the closed chamber technique is widely used for measurements of CO2 fluxes between ecosystems and the atmosphere, the errors which might occur during the measurement itself or which are associated with the used measurement devices as well as the flux calculation from chamber-based CO2 concentration data are still under discussion. The study showed that the CO2 fluxes measured by the closed chamber method can be overestimated during low-turbulence nighttime conditions and can be seriously biased by inappropriate application of linear regression for the flux calculation. The methodological studies were conducted at the boreal peatland Salmisuo in eastern Finland (62°46'N, 30°58'E). The methods developed in this dissertation could contribute significantly to improved CO2 flux estimates. VI
With the development of new functional genomics methods that can access the whole genome, transcriptome, proteome and metabolome more comprehensive insights in cellular processes are possible. Largely based on these advances, our knowledge about molecular constituents for many organisms is increasing at a tremendous rate. Until today, the genomes of several organisms including pathogenic bacteria are already sequenced and pave the way for metabolic network constructions. Interest in metabolomics, the global profiling of metabolites in a cell, tissue or organism, has been rapidly increased. A range of analytical techniques, including nuclear magnetic resonance (NMR) spectroscopy, gas chromatography–mass spectrometry (GC–MS), liquid chromatography–mass spectrometry (LC–MS), Fourier Transform mass spectrometry (FT–MS), high performance liquid chromatography (HPLC) are required in order to maximize the number of metabolites that can be identified in a matrix. With the help of microbial metabolomics (qualification and quantification of a huge variety of metabolites from a bacterium) deciphering of the bacterial metabolism is feasible. The metabolome pipeline or workflow encompasses the processes of (i) sample generation and preparation, (ii) establishment of analytical techniques (iii) collection of analytical data, raw data pre-processing, (iv) data analysis and (v) data integration into biological questions. The present work contributes to the above mentioned steps in a metabolomics workflow. A specific focus was set to the exo- and endometabolome analysis of Gram-positive bacteria
Staphylococcus aureus is a commensal colonizing 20-30% of the population as well as a pathogen causing diverse diseases ranging from skin infections via toxin mediated diseases to life threatening conditions. In its interplay with the human host, this microorganism resorts to an extensive repertoire of both membrane-bound and secreted virulence factors facilitating adhesion to, invasion of, and spreading into various host tissues. Among the numerous virulence factors produced by S. aureus are the staphylococcal superantigens (SAgs). They directly cross-link conserved regions of the T cell-receptor with MHC class II molecules (outside the peptide-binding cleft) on antigen presenting cells. This results in a strong stimulation of up to 20% of all T cells which respond with proliferation and massive cytokine release. Recently, the enterotoxin gene cluster (egc) located on a pathogenicity island was described. The egc-genes are the most prevalent SAg genes in commensal and invasive S. aureus isolates. However, they appear to cause toxic shock only very rarely and their presence is negatively correlated with severity of S. aureus sepsis. Therefore it was suggested that SAgs might differ in their pro-inflammatory potential. In addition to their superantigenicity, SAgs also act as conventional antigens and induce a specific antibody response. In contrast to non-egc SAgs, despite the high prevalence of egc SAgs, neutralizing antibodies against egc SAgs are very rare, even among carriers of egc-positive S. aureus strains. In order to find an explanation for this “egc-gap”, we have tested two non-exclusive hypotheses: (i) egc and non-egc SAgs have unique intrinsic properties and drive the immune response into different directions and (ii) egc and non-egc SAgs are released by S. aureus under different conditions, which shape the immune response to them. To test these hypotheses, we compared the effects of egc and non-egc SAgs on human blood cells. Their T cell-mitogenic potencies, the elicited cytokine profiles as well as their impact on gene expression were highly similar. Both egc and non-egc SAgs induced a very strong pro-inflammatory response. In contrast, the regulation of SAg release by S. aureus differed markedly between egc and non-egc SAgs. Egc-encoded proteins were secreted by S. aureus during exponential growth, while non-egc SAgs were released in the stationary phase. We conclude that the distinct biological behavior of egc and non-egc SAgs is not due to their intrinsic properties, which are very similar, but is caused by their differential release by S. aureus. Traditionally, S. aureus has not been considered as an intracellular pathogen but strong evidence emerged indicating that staphylococci can invade and persist in various cell types. Internalization might constitute a bacterial strategy to evade the host’s defense reactions and the action of antibiotics. The intracellular niche might thus constitute a reservoir for chronic or relapsing infections. Contrary to their potential importance, genome-wide functional genomics analyses of the adaptation reactions of S. aureus to the host cell environment are rare and so far confined to gene expression profiling. Investigations addressing the proteome of internalized S. aureus are still lacking due to the challenge of obtaining a sufficient number of infecting bacteria. The proteome of other pathogens such as Francisella tularensis has been characterized by classical 2-DE approaches. However, the number of bacteria required for such a 2-DE based approach is often exceeding the numbers available from in vivo infection models. Furthermore, this approach does not allow monitoring of time-dependent quantitative changes in protein levels. Here, a workflow allowing time-resolved analysis of internalized S. aureus by combining pulse-chase stable isotope labeling by amino acids in cell culture with high capacity cell sorting, on-membrane digestion, and high-sensitivity mass spectrometry is presented. This workflow permits detection and quantitative monitoring of several hundred staphylococcal proteins from as little as a few million internalized S. aureus cells. This approach has been used to reveal time-resolved changes in levels of proteins in S. aureus RN1HG upon internalization by human bronchial epithelial cells. Proteins involved in stress adaptation as well as protein folding and some components of the phosphotransferase system were upregulated in internalized staphylococci, whereas proteins of the purine biosynthesis pathway and tRNA aminoacylation were downregulated. Furthermore, regulatory adaptive responses of internalized S. aureus to the intracellular milieu were shown as global regulators displayed increased protein abundance levels compared to non-internalized bacteria. Taken together, we observed changes in levels of proteins with functions in protection against oxidative damage and adaptation of cell wall synthesis in internalized S. aureus.
Genome-wide association studies (GWAS) are used to identify genetic markers linked with at least partially heritable diseases or phenotypes without prior knowledge of any disease-associated genetic loci. In summer 2008, all individuals of the population based cohort Study of Health in Pomerania (SHIP) were individually genotyped using the Affymetrix Genome-Wide Human SNP Array 6.0 microarray. The aim of this work was to establish an efficient workflow for GWAS using the more than 4000 individually genotyped samples of the SHIP cohort as well as pooled samples, focusing exclusively on analyzing genetic variations based on single nucleotide polymorphisms (SNPs). Firstly, an optimal array platform for the genotyping analysis had to be chosen that detected most of the available genetic variants at a high level of accuracy. Secondly, extensive quality controls had to be performed starting from DNA extraction and including tests of the generated array data by the analysis software to obtain the most reliable data for the subsequent association studies. For the identification of loci with smaller genetic influences, individual cohorts were meta-analyzed in large nationally and internationally organized consortia (e.g. CHARGE, BPGen, HaemGen, GIANT, CKD Gen). To participate in those meta-analyses, a comparable common set of genetic data had to be generated. This was done by imputation of the data generated by individual array-based genotyping on the basis of a reference panel using chromosomal linkage information. Due to the extensive phenotype information in the SHIP study, it was possible to perform many genome-wide discovery analyses and replication studies of possible susceptibility loci in a short time once the genetic data was available and processed. This resulted in the necessity to set up an efficient workflow for storing the huge amount of genetic data, converting it into different formats readable for specific analysis software, performing the association analyses and processing the results into a human-readable and clear format. This included replications, GWAS and meta-analyses of several cohorts. Many susceptibility loci were newly identified in different association studies with the SHIP data included and were subsequently published. In this work, genetic association studies with the SHIP data included were performed and published on blood pressure, uric acid concentrations, cardiac structure and function, lipid metabolism, hematological parameters, kidney functions, smoking quantity, circulating IGF-I and IGFBP-3 concentrations and thyroid volume including the risk of goiter development. Besides the SHIP cohort, there was a need to use other, especially patient cohorts for GWAS. Since no genotype information from these patient cohorts was available and the individual genotyping of many probands is still expensive and therefore often not affordable, we established the cost-effective allelotyping method that relied on pooling of DNA samples prior to the hybridization with microarrays. After estimating the pooling-specific error of a case-control allelotyping study, the allelotyping approach was used for identifying genetic susceptibility loci associated with aggressive periodontitis. If not referring to work of collaborators, all statistical analyses, data handling and in silico work concerning the SHIP data described in this context was performed by the author of this dissertation.
Behavior of a porous particle in a radiofrequency plasma under pulsed argon ion beam bombardment
(2010)
The behavior of a single porous particle with a diameter of 250 μm levitating in a radiofrequency (RF) plasma under pulsed argon ion beam bombardment was investigated. The motion of the particle under the action of the ion beam was observed to be an oscillatory motion. The Fourier-analyzed motion is dominated by the excitation frequency of the pulsed ion beam and odd higher harmonics, which peak near the resonance frequency. The appearance of even harmonics is explained by a variation of the particles's charge depending on its position in the plasma sheath. The Fourier analysis also allows a discussion of neutral and ion forces. The particle's charge was derived and compared with theoretical estimates based on the orbital motion-limited (OML) model using also a numerical simulation of the RF discharge. The derived particle's charge is about 7–15 times larger than predicted by the theoretical models. This difference is attributed to the porous structure of the particle.
Because of some disadvantages of chemical disinfection in dental practice (especially denture cleaning), we investigated the effects of physical methods on Candida albicans biofilms. For this purpose, the antifungal efficacy of three different low-temperature plasma devices (an atmospheric pressure plasma jet and two different dielectric barrier discharges (DBDs)) on Candida albicans biofilms grown on titanium discs in vitro was investigated. As positive treatment controls, we used 0.1% chlorhexidine digluconate (CHX) and 0.6% sodium hypochlorite (NaOCl). The corresponding gas streams without plasma ignition served as negative treatment controls. The efficacy of the plasma treatment was determined evaluating the number of colony-forming units (CFU) recovered from titanium discs. The plasma treatment reduced the CFU significantly compared to chemical disinfectants. While 10 min CHX or NaOCl exposure led to a CFU log10 reduction factor of 1.5, the log10 reduction factor of DBD plasma was up to 5. In conclusion, the use of low-temperature plasma is a promising physical alternative to chemical antiseptics for dental practice.
In vivo Imaging of Bile Accumulation and Biliary Infarction after Common Bile Duct Ligation in Rats
(2011)
Obstructive cholestasis is caused by mechanical constriction or occlusion leading to reduced bile flow. Serious complications such as jaundice and even death may follow. Little is known about the initial phase of cholestasis and its consequences for the hepatic microarchitecture. This in vivo study aimed to characterize the nature and kinetics of developing obstructive cholestasis and focused on areas with biliary stasis and infarction by visualizing the autofluorescence of bile acids using intravital microscopy of the liver over a period of 30 h after bile duct ligation in rats. The innovation resided in performing fluorescence microscopy without applying fluorescent dyes. In animals subjected to obstructive cholestasis, the most significant changes observed in vivo were the concomitant appearance of (1) areas with bile accumulation increasing in size (6 h: 0.163 ± 0.043, 18 h: 0.180 ± 0.086, 30 h: 0.483 ± 0.176 mm<sup>2</sup>/field) and (2) areas with biliary infarction (6 h: 0.011 ± 0.006, 18 h: 0.010 ± 0.004, 30 h: 0.010 ± 0.050 mm<sup>2</sup>/field) as well as (3) a relation between the formation of hepatic lesions and enzyme activity in serum. The sequential in vivo analysis presented herein is a new method for the in vivo visualization of the very early changes in the hepatic parenchyma caused by obstructive cholestasis.
Purpose: To determine the surface characteristics of porcine corneal lenticules after Femtosecond Lenticule Extraction. Methods: The Carl Zeiss Meditec AG VisuMax® femtosecond laser system was used to create refractive corneal lenticules on 10 freshly isolated porcine eyes. The surface regularity on the corneal lenticules recovered was evaluated by assessing scanning electron microscopy images using an established scoring system. Results: All specimens yielded comparable score results of 5–7 points (SD = 0.59) per lenticule (score range minimum 4 to maximum 11 points). Surface irregularities were caused by tissue bridges, cavitation bubbles or scratches. Conclusion: The Femtosecond Lenticule Extraction procedure is capable of creating corneal lenticules of predictable surface quality. However, future studies should focus on the optimization of laser parameters as well as surgical technique to improve the regularity of the corneal stromal bed.
For surgery in congenital hyperinsulinism (CHI), a distinct surgical strategy and technique is required for focal, diffuse and atypical CHI. In focal CHI, a confined, localized and parenchyma-sparing resection which is guided by the PET-CT is always indicated in order to cure the patient. In diffuse CHI, however, the results of surgical therapy are unpredictable and cure is an exception. Therefore, a strong tendency exists nowadays that medical therapy should be preferred in diffuse CHI. In atypical CHI the situation is more complex: if the focal lesion or the segmental mosaic are not too extensive, cure by resection should be possible. But care must be taken in atypical cases not to resect too much of the gland in order not to induce diabetes.
Background: Therapyrelated mucositis is associated with considerable morbidity. This complication following allogeneic stem cell therapy (alloSCT) is less severe after reduced intense conditioning (RIC); however, even here it may be serious. Methods: 52 patients (male: n = 35 (67%), female: n = 17 (33%)) at a median age of 62 years (35–73 years) underwent alloSCT after RIC. Conditioning was either total body irradiation (TBI)<sub>2Gy</sub>/±fludarabine (n = 33, 63.5%) or chemotherapy based. Graftversushost disease (GvHD) prophylaxis was carried out with cyclosporine A ± mycophenolate mofetil (MMF). 45 patients (87%) received shortcourse methotrexate (MTX). Mucositis was graded according to the Bearman and the World Health Organisation (WHO) scale. A variety of parameters were correlated with mucositis. Results: The Bearman and WHO scales showed excellent correlation. Mucositis was significantly more severe after chemotherapybased conditioning compared to conditioning with TBI<sub>2Gy</sub>/±fludarabine (p < 0.002) as well as in cases with an increase in creatinine levels above the upper normal value (UNV) on day +1 after SCT (p < 0.05). Furthermore, the severity correlated with time to engraftment of leucocytes (correlation coefficient (cc) = 0.26, p < 0.02) and thrombocytes (cc = 0.38, p < 0.001). Conclusions: The conditioning regimen and increased creatinine levels at day +1 were identified as factors predicting the severity of mucositis after RICSCT. Creatinine levels on day +1 after SCT may help identify patients at risk for severe mucositis in the further course of transplantation.
Aim: The efficacy of antimicrobial compounds included in wound dressings has been determined using the quantitative suspension test according to EN 13727 before. However, as suspension tests are not an accurate reflection of the conditions under which wound antiseptics are used, it was investigated if a disc carrier test would yield results simulating practical conditions on wound surfaces. A silver-leaching foam wound dressing was used for evaluation of the disc carrier test method. Method: The disc carriers consisted of circular stainless-steel discs measuring 2 cm in diameter and 1.5 mm in thickness, complying with the requirements of EN 10088-2. Carriers were contaminated with Staphylococcus aureus, methicillin-resistant S. aureus or Pseudomonas aeruginosa, respectively, together with an artificial wound secretion and left to dry at room temperature for 30 min. The wound dressings being tested were placed on the discs for the length of the exposure time, and after neutralization by thioglycolate in phosphate-buffered saline the number of surviving test organisms was then counted. The logarithmic reduction factor was calculated from the difference between the initial inoculum and the number of recovered test organisms. Results: The disc carrier test allowed determination of an antimicrobial efficacy in a realistic setting. It also imposed more stringent requirements on efficacy over time than the quantitative suspension test. The silver foam wound dressing showed a time-dependent antimicrobial efficacy. After 24-hour application time, the reduction factors against S. aureus, P. aeruginosa and the methicillin-resistant S. aureus were 1.9 ± 0.15, 2.1 ± 0.14 and 3.1 ± 0.18, respectively. Conclusion: The disc carrier test was a useful method for testing the antimicrobial efficacy of a foam silver dressing. The antimicrobial dressing exhibited an antimicrobial effect after 3 h and achieved a reduction >2 log against the tested bacterial strains in the presence of a simulated wound secretion after 24 h.
The effect of water-filtered infrared-A radiation (wIRA) on normal skin flora was investigated by generating experimental wounds on the forearms of volunteers utilizing the suction blister technique. Over 7 days, recolonization was monitored parallel to wound healing. Four groups of treatment were compared: no therapy (A), dexpanthenol cream once daily (B), 20 min wIRA irradiation at 30 cm distance (C), and wIRA irradiation for 30 min once daily together with dexpanthenol cream once daily (D). All treatments strongly inhibited the recolonization of the wounds. Whereas dexpanthenol completely suppressed recolonization over the test period, recolonization after wIRA without (C) and in combination with dexpanthenol (D) was suppressed, but started on day 5 with considerably higher amounts after the combination treatment (D). Whereas the consequence without treatment (A) was an increasing amount of physiological skin flora including coagulase-negative staphylococci, all treatments (B–D) led to a reduction in physiological skin flora, including coagulase-negative staphylococci. In healthy volunteers, wIRA alone and in combination with dexpanthenol strongly inhibited bacterial recolonization with physiological skin flora after artificial wound setting using a suction-blister wound model. This could support the beneficial effects of wIRA in the promotion of wound healing.
The exact qualitative and quantitative analysis of wound healing processes is a decisive prerequisite for optimizing wound care and for therapy control. Transepidermal water loss (TEWL) measurements are considered to be the standard procedure for assessing the progress of epidermal wound healing. The damage to the stratum corneum correlates with an increased loss of water through the skin barrier. This method is highly susceptible to failure by environmental factors, in particular by temperature and moisture. This study was aimed at comparing TEWL measurements and in vivo laser scanning microscopy (LSM) for the characterization of the epidermal wound healing process. LSM is a high-resolution in vivo method permitting to analyze the kinetics and dynamics of wound healing at a cellular level. While the TEWL values for the individual volunteers showed a wide scattering, LSM permitted the wound healing process to be clearly characterized at the cellular level. However, a comparison between the two methods was very difficult, because the results provided by LSM were images and not numerical. Therefore, a scoring system was set up which evaluates the stages of wound healing. Thus, the healing process could be numerically described. This method is independent of any environmental factors. Providing morphologically qualitative and numerically quantitative analyses of the wound healing process and being far less vulnerable to failure, LSM is advantageous over TEWL.
Wound healing disorders frequently occur due to biofilm formation on wound surfaces requiring conscientious wound hygiene. Often, the application of conventional liquid antiseptics is not sufficient and sustainable as (1) the borders and the surrounding of chronic wounds frequently consist of sclerotic skin, impeding an effectual penetration of these products, and (2) the hair follicles representing the reservoir for bacterial recolonization of skin surfaces are not affected. Recently, it has been reported that tissue-tolerable plasma (TTP), which is used at a temperature range between 35 and 45°C, likewise has disinfecting properties. In the present study, the effectivity of TTP and a standard liquid antiseptic was compared in vitro on porcine skin. The results revealed that TTP was able to reduce the bacterial load by 94%, although the application of the liquid antiseptic remained superior as it reduced the bacteria by almost 99%. For in vivo application, however, TTP offers several advantages. On the one hand, TTP enables the treatment of sclerotic skin as well, and on the other hand, a sustainable disinfection can be realized as, obviously, also the follicular reservoir is affected by TTP.
Postoperative Immune Suppression in Visceral Surgery: Characterisation of an Intestinal Mouse Model
(2011)
Background: Postoperatively acquired immune dysfunction is associated with a higher mortality rate in case of septic complications. As details of this severe clinical problem are still unknown, animal models are essential to characterise the mechanisms involved. Methods: Mice were laparotomised and the small intestine was pressed smoothly in antegrade direction. For extension of trauma, the intestine was manipulated three times consecutively. Following this, the ex vivo cytokine release of splenocytes was determined. The degree of surgical trauma was analysed by detection of HMGB1 and IL-6 in serum and by neutrophil staining in the muscularis mucosae. Results: We adapted the previously described animal model of intestinal manipulation to provide a model of surgically induced immune dysfunction. Following intestinal manipulation, the mice showed elevated serum levels of HMGB1 and IL-6 and increased infiltration of granulocytes into the muscularis mucosae. Ex vivo cytokine release by splenocytes was suppressed in the postoperative period. The degree of suppression correlated with the extent of surgical trauma. Conclusions: In this study, we describe a surgically induced immune dysfunction animal model, in which a significant surgical trauma is followed by an immune dysfunction. This model may be ideal for the characterisation of the postoperative immune dysfunction syndrome.
Currently, there are no generally accepted definitions for wounds at risk of infection. In clinical practice, too many chronic wounds are regarded as being at risk of infection, and therefore many topical antimicrobials – in terms of frequency and duration of use – are applied to wounds. Based on expert discussion and current knowledge, a clinical assessment score was developed. The objective of this wounds at risk (W.A.R.) score is to allow decision-making on the indication for the use of antiseptics on the basis of polihexanide. The proposed clinical classification of W.A.R. shall facilitate the decision for wound antisepsis and allow an appropriate general treatment regimen with the focus on the prevention of wound infection. The W.A.R. score is based on a clinically oriented risk assessment using concrete patient circumstances. The indication for the use of antiseptics results from the addition of differently weighted risk causes, for which points are assigned. Antimicrobial treatment is justified in the case of 3 or more points.
In the search for bioactive compounds, 32 fungal strains were isolated from Indonesian marine habitats. Ethyl acetate extracts of their culture broth were tested for cytotoxic activity against a urinary bladder carcinoma cell line and for antifungal and antibacterial activities against fish and human pathogenic bacteria as well as against plant and human pathogenic fungi. Bioassay-guided fractionation led to the isolation of bioactive compounds. Altogether 14 compounds were isolated and further elucidated. The compounds were obtained from the ethyl acetate and dichloromethane extracts of six fungal strains. They included 9 polyketides, 2 terpenes, 1 alkaloid and 2 till now undefined structures.
District hospitals are the only solution to guarantee basic health care including life-saving surgeries and hospitalisations in rural SSA areas. Neither regional nor national hospitals, financially and geographically out of reach for the majority of the population, nor rural health care centres, mostly staffed with a nurse only, can cover these tasks adequately. However, only little research exists on care giving processes, cost and efficiency of district hospitals in SSA. The general problem in health economics is that limited resources should be used in order to maximise health effects. This dissertation evaluates the actual treatment pathways and their average provider’s cost per patient for four different diagnoses at Nouna district hospital in Burkina Faso. A total of 95 patient records was analysed in detail and discussed with the health personnel in charge. Cost information for the year 2005 was taken from the well-established provider cost information system. Cost were broken down to the different sequences of the treatment pathway and summed up at the end. Average provider’s cost for paediatric Malaria were U$ 6.71 for outpatients, US$ 60.59 for inpatients with anaemia and US$ 75.11 for inpatients with neurological affection. Average provider’s cost for treating hypertension were US$ 67.94 per year. Average cost for hernia cure were US$ 146.85 under local anaesthesia, US$ 153.08 under spinal anaesthesia and US$ 169.78 under general anaesthesia. Average provider’s cost for Caesarean sections were US$ 140.15 under spinal anaesthesia and US$ 180.41 under general anaesthesia. This means that cost per patient are comparable to or even lower than provider’s cost found for other SSA setting in the literature. Cost would decrease between 20% (a hypertensive outpatient) and 46% for Malaria with neurological affection as complication, if utilisation rates rose from actually 20 to 80%. Patients paid between 35 and 94% of total provider’s cost in form of user fees. If fees would not change and the utilisation rate increased to 80%, cost-recovery for the considered diseases would then be between 63 and 117%. Although this would not allow the hospital to break even in its current configuration, the cost-recovery rate would be considerably higher, especially when taking into account that a full cost analysis was done including all investment cost. The introduction of clinical pathways based on the actual treatment pathways is suggested to improve process structure and documentation and to standardise the treatment according to national and international guidelines.
Protein quality control systems are essential for the viability and growth of all living organisms. They protect the cell from irreversible protein aggregation. Because the frequency of protein misfolding, which ultimately results in protein aggregation, varies with the environmental conditions, the amount and activity of protein quality systems have to be accurately adapted to the rate of protein misfolding. The main goal of this thesis was to gain detailed molecular insights into the transcriptional and post-translational regulation of these protein quality control networks in the ecologically, medically and industrially important phylum of low GC, Gram-positive bacteria. In these bacteria the core protein quality control systems are under the transcriptional control of the global repressor CtsR. In a first study it was demonstrated that the arginine kinase McsB is not responsible for the regulation of CtsR activity during heat stress, as was concluded by others on the basis of previous in vitro data. Rather, it was demonstrated that CtsR acts as an intrinsic thermosensor that adapts its activity to the surrounding temperature. CtsR displays a decreased DNA binding at higher temperatures, which leads to induction of transcription of the protein quality control systems under these conditions. This CtsR feature is conserved in all low GC, Gram-positive bacteria. However, the CtsR proteins of various low GC, Gram-positive species do not have the same temperature optima. CtsR responds to heat in a species-specific manner according to their corresponding growth temperature. Detailed analysis revealed that a highly conserved tetra-glycine loop within the winged helix-turn-helix domain of CtsR is responsible for thermosensing. Dual control of CtsR activity during different stresses was demonstrated for the first time in this work. In addition to heat-dependent de-repression, CtsR is inactivated by thiol-specific stress conditions. This latter de-repression depends on a molecular redox-switch that is independent of CtsR auto-regulation. In Bacillus subtilis and its closest relatives the McsA/McsB stress-sensing complex is responsible for CtsR de-repression during redox stress conditions. McsA is able to sense the redox state of the cell via its highly conserved cysteine residues. When these cysteines are reduced, McsA is able to bind and inhibit McsB. But when these cysteine residues are oxidized, McsB is released from McsA. Thereby, McsB is activated and removes CtsR from the DNA. However, the McsA/McsB complex is not present in all low GC, Gram-positive bacteria. In the species lacking this complex, ClpE is able to act as a redox-sensor probably via its highly conserved N-terminal zinc finger domain. When these cysteine residues are oxidized, ClpE is activated which results in CtsR de-repression. In addition to the transcriptional regulation of CtsR low GC, Gram-positive protein quality control systems are regulated post-transcriptionally. The expression of the McsA/McsB adaptor pair is regulated by CtsR. However, McsB activity is also tightly regulated by three different regulatory proteins (McsA/ClpC/YwlE). McsB is needed to target specific substrates to ClpC, either for refolding or degradation by the ClpCP protease. It was demonstrated that only the auto- phosphorylated form of McsB is able to bind to its substrates. This McsB function is inhibited in non-stressed cells by a direct interaction with ClpC. Consequently, McsB is activated by a release from ClpC during protein stress. In addition, McsB activation depends on the presence of its activator McsA. Accordingly, McsB cannot be activated as an adaptor protein during thiol-specific stress because McsA is no longer able to bind to McsB under these conditions. However, also active McsB is subject to post-translational control. Activated McsB is either de-phosphorylated by McaP or degraded by ClpCP ensuring an appropriate shut-down of the McsB adaptor. Both McaP and ClpC inhibit McsB activity with different intensities. ClpC possesses a stronger impact on McsB activity than McaP but both proteins are needed for an adequate silencing of McsB activity. In addition, it was shown for the first time that B. subtilis McsB is a global adaptor that influences the stability of multiple proteins. The B. subtilis ClpC protein is unlike most members of the Hsp100 family because it not only requires several adaptor proteins for substrate recognition but also for its general ATP- dependent activity. Biochemical analysis revealed how ClpC is activated by distinct adaptor proteins. McsB modulates ClpC activity by regulatory phosphorylation of arginine residues. Moreover, McaP (formerly YwlE) was identified as an arginine phosphatase that modulates the McsB mediated ClpC activity. MecA, another known adaptor protein for ClpC, activates ClpC independently of these arginine phosphorylations, which demonstrates the existence of multiple pathways for ClpC activation.
The pUS3, a serine/threonine protein kinase that is conserved in Alphaherpesvirinae may play an important in phosphorylation and regulation of the activities of viral and cellular proteins. It has also been proposed that pUS3 affects virulence. Whereas many studies of the pUS3 functions of HSV-1 and PrV, a closely related homolog of BHV-1 have supported these assumptions, the role of BHV-1pUS3 is not yet fully understood so far. The aims of this study therefore were to investigate the functions of BHV-1pUS3 for virus replication in cultured cells, effect on apoptosis and identification of protein interactions with cellular proteins and addressed the function of the aminoterminal region by generating a short isoform of BHV-1pUS3 which corresponds in size to the natural short isoforms of PrVpUS3 and HSV-1pUS3. Results of the study are briefly summarized here: -BHV-1pUS3 is, although not essential, beneficial for infectious replication of BHV-1 in-vitro. It also supports direct cell-to-cell spread of BHV-1/Aus12 and prevents the formation of electron dense aggregates with embedded capsids in nuclei of BHV-1 infected cells, a phenotype that may affect nuclear egress of BHV-1 nucleocapsids. -The protein, independent from other BHV-1 encoded functions, located mainly to the nuclei of cells. -In contrast to functions of pUS3 in PrV and HSV-1, the protein of BHV-1 has no anti-apoptotic activity. -Biologically active BHV-1pUS3 physically interacts with the cellular SET protein and overexpression of SET, independent from the expression of the protein, inhibits productive BHV-1 replication in a dose dependent manner. -The aminoterminal 101 amino acids of the protein are dispensable for all in-vitro functions tested whereas kinase activity is required.
The key objective of this dissertation is to study the expected impact of the introduction of the Social Health Insurance (SHI) on the public hospital management and to develop recommendations that will improve this management. In addition to the key objective, this study aims to analyze the health sector financing in Syria, to outline problems affecting on management of public hospitals in Syria. Furthermore, it aims to study the various countries' experience with SHI and analyze key components of the Syrian SHI.
Main drivers for biodiversity loss in terrestrial ecosystems are changes in land use, climate change, enhanced nitrogen deposition and biotic exchange (invasive species). These drivers also affect dry, nutrient-poor open anthropo-zoogenic inland and coastal heathlands which often harbor a high biodiversity. To counteract biodiversity loss in coastal ecosystems, a basic step is the assessment of the various threats. Therefore it is important to select suitable model organisms for analyses of biodiversity dynamics. In this thesis the three arthropod groups Orthoptera (Ensifera and Caelifera), carabid beetles (Coleoptera: Carabidae) and spiders (Araneae) were studied, as they are very useful indicators. Besides sampling of the three arthropod groups vegetation and microclimate parameters were recorded. The studies were done between 2008 and 2010 in the coastal heathland on the Baltic island of Hiddensee, Germany. The main aim of the thesis was to analyze the impact of three drivers of heathland biodiversity loss (succession, grass encroachment, moss invasion) on the selected indicator arthropod groups. Based on this multi-level and -species approach, implications for the conservation of coastal heathlands are given. The results show that successional processes and grass encroachment have strong impact on species richness and abundance, species composition and functional groups, as well as life-history traits and functional diversity of the arthropod groups. Main findings were: Orthoptera species richness was highest in the intermediate stages (heath encroached by grasses and heath with shrubs) because of higher habitat heterogeneity and higher food supply (grasses). Opposed to that, species richness of ground-dwelling carabid beetles and spiders did not differ among the five successional stages, which contradicts the ‘habitat heterogeneity hypothesis’. In contrast to species richness, functional diversity differed among successional stages. The concept of functional diversity – which integrates species life-history trait data – therefore might be particularly suitable for biodiversity research, while the explanatory power of species richness alone might not be sufficient. The species compositions of all three taxa changed remarkably along the coastal heathland gradient indicating a high species turnover. In particular, open, dynamic habitats (‘grey dunes’ and ‘dwarf-shrub heath’) could be separated. Here, several specialized, xerothermic and threatened species occurred due to the extreme habitat conditions, but are displaced during grass and shrub encroachment. On a smaller spatial scale, the invasion of Campylopus introflexus alters habitat conditions in grey dunes and therefore affects carabid beetle and spider species and the dominant Orthoptera species Myrmeleotettix maculatus. Species richness of carabid beetles and spiders, and the abundance of adult M. maculatus grasshoppers were reduced. Species compositions of carabids and spiders changed remarkably with a loss of several species. These negative impacts could be explained by the vegetation structure of the moss which is unsuitable for web-building spiders or large carabid beetles, and by reduced germination of higher plants and therefore reduced food supply for M. maculatus and phytophagous carabid species. Within the open coastal heathland, the mosaic of grey dunes and adjacent dwarf-shrubs is important since many species perform a habitat change during their development and, besides the scarcely vegetated, thermally benefited grey dunes, need denser vegetation of adjacent dwarf-shrubs for shelter, as song posts, or for foraging. As grey dunes harbor a high abundance and species richness of threatened and specialized, mainly xerothermic and geobiont species and are important as oviposition and nymphal habitat, they are regarded as a keystone habitat within the coastal heathland. Besides these ecological studies, two studies focused on the method of pitfall trapping. It could have been shown, that pitfall trapping might be a useful sampling method for Orthoptera in open habitats. The other study demonstrated that sampling interval has a strong influence on the capture efficiency of several arthropod groups (‘digging-in effect’). Conservation practices should aim at maintaining a heterogeneous heathland mosaic with open grey dunes and Calluna stands, in addition to scattered grassy and shrub-encroached heath for the survival of species-rich heathland arthropod assemblages with a high proportion of specialized and threatened species.
Background: Computational tools for the investigation of transcriptional regulation, in particular of transcription factor binding sites (TFBS), in evolutionary context are developed. Existing sequence based tools prediction such binding sites do not consider their actual functionality, although it is known that besides the base sequence many other aspects are relevant for binding and for the effects of that binding. In particular in Eukaryotes a perfectly matching sequence motif is neither necessary nor sufficient for a functional transcription factor binding site. Published work in the field of transcriptional regulation frequently focus on the prediction of putative transcription factor binding sites based on sequence similarity to known binding sites. Furthermore, among the related software, only a small number implements visualization of the evolution of transcription factor binding sites or the integration of other regulation related data. The interface of many tools is made for computer scientists, although the actual interpretation of their outcome needs profound biological background knowledge. Results and Discussion: The tool presented in this thesis, "ReXSpecies" is a web application. Therefore, it is ready to use for the end user without installation providing a graphical user interface. Besides extensive automation of analyses of transcriptional regulation (the only necessary input are the genomic coordinates of a regulatory region), new techniques to visualize the evolution of transcription factor binding sites were developed. Furthermore, an interface to genome browsers was implemented to enable scientists to comprehensively analyze their regulatory regions with respect to other regulation relevant data. ReXSpecies contains a novel algorithm that searches for evolutionary conserved patterns of transcription factor binding sites, which could imply functionality. Such patterns were verified using some known transcription factor binding sites of genes involved in pluripotency. In the appendix, efficiency and correctness of the used algorithm are discussed. Furthermore, a novel algorithm to color phylogenetic trees intuitively is presented. In the thesis, new possibilities to render evolutionary conserved sets of transcription factor binding sites are developed. The thesis also discusses the evolutionary conservation of regulation and its context dependency. An important source of errors in the analysis of regulatory regions using comparative genetics is probably to find and to align homologous regulatory regions. Some alternatives to using sequence similarity alone are discussed. Outlook: Other possibilities to find (functional) homologous regulatory regions (besides whole-genome-alignments currently used) are BLAST searches, local alignments, homology databases and alignment-free approaches. Using one ore more of these alternatives could reduce the number of artifacts by reduction of the number of regions that are erroneously declared homologous. To achieve more robust predictions of transcription, the author suggests to use other regulation related data besides sequence data only. Therefore, the use and extension of existing tools, in particular of systems biology, is proposed.
Computational chemical physics can give important input to astrophysical modelling and other fields of physics, where molecular properties are of importance. Understanding of spectroscopic and reactive behaviour is crucial for many systems of astrophysical interests like stars, interstellar medium and comets. Especially stellar atmospheres are of interest, because the complex physics of stars are not yet completely understood. Stars are in an unstable balance of gravitation and radiation pressure and the atmospheric dynamics have been subject of extensive modelling. Complete and accurate spectroscopic information of the atoms and molecules in these atmospheres is necessary for this attempt. In addition, the only information we have about astrophysical systems is light which is emitted or absorbed by particles in these media. This is not only true for astrophysics. In plasma physics sometimes the usage of invasive diagnostics, like Langmuir probes, is not wanted because they disturb the system. In these cases some information of the system can be regained by passively measuring infrared spectra of the plasma or by active induction of electronic transition like the laser-induced fluorescence method. Another remote sensing application is the measurement of the atmospheric composition on earth. Here, larger particles in the atmosphere as well as greenhouse gases are of current interest. Unfortunately, the experimental spectroscopic data, which is needed for the understanding and interpretation of the measured spectra, is often incomplete. This gap can be, to some extend, filled by computational chemical physics. The aim of this work was to investigate the capabilities and limitations of ab initio based potential energy surfaces for spectroscopic and reactive studies and to apply these methods to problems of rovibrational and rovibronic spectroscopy and reaction dynamics. The choice of ab initio methods and the potential fitting methods is critical for the computational chemical physics, as all further quantities directly depend on their quality. In this work modified versions of the Braams polynomial potential energy surface were used. A high level coupled cluster ab initio method was used to build potentials for a series of small hydrocarbons. Hydrocarbons can be found almost everywhere on earth and in the universe. They exist in laboratory plasmas, stellar and planetary atmospheres and interstellar gases. In all these cases, light emitted or absorbed by the molecules is an important diagnostics of the system. The potential constructed in this work partly included a cluster expansion, which adds reactant configuration spaces to the fits. This could not be done for CH_3 and higher hydrocarbons, because of the limitations of the Coupled Cluster ab initio method, which is well suited for the potential wells, but not for the dissociation regions. The examples of methyl and methane show how the potentials can be used for rovibrational spectroscopy. Results of radiation transport simulations illustrate the importance of as complete-as-possible line lists for radiation transport calculations.\\ The rovibronic spectroscopy of diatomic molecules is another important aspect for the stellar atmospheric modelling. Metal hydrides and oxides add opacity to the atmosphere in the visible light and ultraviolet frequency regions, as well as do the hydrocarbons in the infrared one. In addition the spectra of metal hydrides/oxides can be used to gather information about metal and their isotope abundances. They are used as markers for the conditions in the atmospheres of stars. In this work a new code was developed, that efficiently calculates bound-bound transitions between electronic states and bound-continuum cross sections for diatomic molecules. It also offers an adequate treatment of quasi-bound rovibrational states. One important representative of the diatoms is magnesium hydride, MgH. Before this work, line lists and photodissociation cross section were available involving the three lowest doublet states of MgH. In this work new potential energy curves were calculated and adapted to updated experimental data. This causes changes in the relative energies between the electronic states and therefore shifts in the line lists. These are important, because accurate line positions are needed for the identification of spectral lines. In addition two further electronic states were included in the calculations. This expands the spectral range of MgH into the near ultraviolet region. Radiation transport models showed significant absorption by MgH from the newly added electronic states. A second usage of the diatomic potential energy curves are photodissociation cross sections. As interstellar environments are chemically active, such data is necessary for a complete picture of the ongoing processes. The photodissociation cross sections of MgH reveal a stronger dependence of the underlying potential than the bound-bound lines. In the case of MgH the cross sections are rather weak, besides occasional resonance lines which can be several orders of magnitude stronger. As mentioned, not only spectroscopic, but also reactive behaviour of molecules is important in astrophysics. A current problem connected with this is the abundance of CH^+ in interstellar clouds. Its measured abundances do not fit the predictions from theoretical models. In addition Gerlich and co-workers recently measured low temperature H + CH^+ -> C^+ + H_2 reaction rates, which diverge from the theoretical picture and which could not be explained. In this work a reactive potential energy surface was built for the CH_2^+ system, which was then used to perform extensive calculations with quasi-classical trajectory and quantum scattering methods. It was found out, that the potentials used in previous works are not accurate enough to allow low temperature calculations. Results from these potentials must be taken with care. Furthermore, the results from the new potential energy surface indicate significantly reduced reaction rates compared to previous numerical studies. This is in agreement with the new results of Gerlich and co-workers. Nevertheless, the large error bars in the low temperature range for experimental as well as numerical results strongly suggest refined methods to be developed for both, before a final conclusion can be made. This work demonstrated the possibility of modern computational chemical physics to supply consistent data for spectroscopy and reaction dynamics. These are necessary and important inputs for fields like astrophysics, plasma physics and chemistry.
In this thesis wave propagation in the whistler wave frequency range ωci≤ω≤ωce in the linear magnetized plasma experiment VINETA is investigated. The plasma is generated by a helicon antenna and has a diameter of about 10 cm. Whistler waves are launched by a loop antenna with a diameter of 4.5 cm and the fluctuating magnetic field is mapped by Ḃ-probes. Experiments are carried out for plasma parameters γ≤1/ √ 2 under which the only transversal polarized wave according to plane wave dispersion theory is the whistler wave. Due to the small collision frequencies ν≪1 cyclotron damping of whistler waves in this parameter regime is dominant and depends only on the electron plasma-β. The influence of the inhomogeneous plasma profile and excitation by a loop antenna is investigated by measurements of the fluctuating magnetic field perpendicular to the ambient magnetic field in azimuthal and radial axial planes. A mode characterized by the number of wave lengths m in the azimuthal direction is found. The mode structure is modified by the specific shape of the plasma density profile. Profiles with a homogeneous density inside the plasma radius are found to posses a comparably simple mode structure. An agreement in the mode structure of full-wave simulations in three dimensions, including a Gaussian density profile and excitation of the wave by a loop antenna, with the experimental results is found. Conclusions on the spatial structure of the excited mode are drawn using the simulations which predict excitation of an m=2 mode. The wave is found to be ducted within the plasma radius over a wide parameter range. A Helmholtz decomposition of the simulations electric field exhibits the fluctuating space charge as the dominant source for the electric field, while the contribution due to induction is negligible. The magnetic field is given partially by the electron and displacement current. Both contributions to the magnetic field are of the same order of magnitude. The frequency dependency of the excited modes spatial damping increment is investigated using measurements of the magnetic fluctuations along the symmetry axis of the plasma. In order to illustrate the parameter dependency, the electron plasma-β is varied over two orders in magnitude in the range β = 4·10-4 - 2.4·10-2. The experimental result for the spatial damping increment of the mode yields a strong damping for wave frequencies ω/ωce > 0.5 at maximum plasma-β, which shifts to higher frequencies with decreasing β. The parameter dependency of the damping for a fixed frequency is studied in an axial ambient magnetic field gradient. In both cases an excellent agreement between the experimental result and predictions for cyclotron damping from plane wave dispersion theory is found.
In this thesis, a stereoscopic camera system is presented that is designed for the use on parabolic flights for the investigation of dusty plasmas under microgravity conditions. This camera system consists of three synchronously triggered high-speed cameras observing a common volume of approximately (15 × 15 × 15) mm³ size. In this volume, the three-dimensional trajectories of a large number of particles surrounded by a dense dust cloud were reconstructed. For this task an intricate set of reconstruction algorithms has been developed, including a four-frame linking algorithm and a complex combined 2D/3D tracking algorithm for a reliable tracking of 3D particles. Furthermore, these algorithms effectively suppress so-called ghost particles in the evaluation process which are reconstructed from falsely identified 2D particle correspondences. Dusty plasmas under microgravity conditions are of special interest due to their complex structure and the variety of observable dynamic phenomena. Under typical discharge conditions, a central dust-free void is formed, surrounded by a dense particle cloud. Since the void is inherently dust-free, particles shot into the void can be uniquely identified and used to probe plasma properties inside this region. In the dust cloud itself, processes like self-excited dust-density waves can be observed under suitable experimental conditions. Using the presented camera setup and reconstruction algorithms, two parts of a dusty plasma under microgravity on parabolic flights are investigated. Initially, the force field creating and sustaining the central void is deduced and characterized. The combination of ion drag and electric field force is measured and compared to current models of the ion drag, showing a good agreement with these models. While previous investigations on the forces were limited to two-dimensional slices through the void, our measurements represent the first three-dimensional quantitative analysis of a large fraction of the void region. From this analysis the structure of the force field is determined and separated into a radial and a non-radial (or orthogonal) contribution. It is shown that the radial contribution dominates in the central void, while non-radial forces increase in magnitude close to the void edge. The radial domination is also observed in the velocity distribution of the probe particles which is significantly shifted to radially outward directed velocities for particles leaving the void. Assuming a strictly radial force profile in the horizontal mid-plane of the void, the friction coefficient determining the interaction of the probe particles with the neutral gas background is experimentally determined and shown to match the theoretical expectation. Subsequently, particles at the outer surface of the dust cloud are reconstructed. There, the particles are found to oscillate due to dust-density waves propagating through the high-density dust cloud. For the investigation of the correlation between waves and oscillating particles, the instantaneous wave and oscillation properties are determined and the instantaneous phase difference is obtained. Modeling the probe particles as driven, damped harmonic oscillators, these phase differences between waves and particles are interpreted with respect to the resonance frequency of the oscillating particles. Spatial variations of the phase difference are observed that may be attributed to different frequencies of the dust-density waves, or to changes of the resonance frequency induced by changing local plasma parameters. From a few measurements of particles oscillating at their resonance frequency, information about the surrounding plasma or properties of the particles themselves can be deduced. However, a larger number of reconstructed trajectories is necessary in order to interpret the phase differences on a reliable data basis. The presented camera setup in combination with the evaluation algorithms is a flexible system for the investigation of three-dimensional dusty plasmas. Its robust construction allows the operation of the system in challenging environments such as on parabolic flights, where spatial limitations and vibrations produced by the aircraft make special demands on such a diagnostic tool. This versatility makes our stereoscopic camera setup and the reconstruction process a suitable standard diagnostic for the application with dusty plasmas; this system will therefore be used in future research amongst other things for the investigation of boundary layers in extended three-dimensional dust clouds under microgravity.
Tidal flats represent the transition zone between the terrestrial and marine realm. They are subject to pronounced dynamics due to distinct tidal and seasonal variations of physical, chemical, and biological parameters significantly influencing redox-sensitive element cycles. Thus, redox-sensitive trace metals may be suitable indicators for variations in bioproductivity and microbial activity. Therefore, seasonal and tidal dynamics of manganese, iron, molybdenum, uranium, and vanadium were studied in the water column and sediments of tidal systems of the German Wadden Sea (southern North Sea) in the years 2007 to 2009 involving also previously analysed data from year 2002. To demonstrate the response of the trace metal cycles on phytoplankton blooms and enhanced biological activity time series data of nutrients and phytoplankton dynamics were also involved in this study. Pronounced cycling is seen for pelagic manganese revealing distinctly higher values during low tide. Complex seasonal cycling showing maxima of dissolved manganese in spring and late summer and a depletion period in early summer is caused by benthic-pelagic coupling and reflection of exhaustion and replenishing periods in the surface sediments. Vanadium dynamics are coupled to the manganese cycling due to vanadium scavenging and release during manganese oxide formation and reduction, respectively. Molybdenum and uranium behave almost conservatively following changes in salinity and thus, being slightly enhanced during high tide. Deviations from conservative behaviour are found to occur during breakdowns of summer phytoplankton blooms. In the following, significant enrichments of manganese, molybdenum, iron, and uranium are observed in the shallow pore waters. These coherences are assumed to be caused by a tight coupling of geochemical, biological, and sedimentological processes. Intense release of organic matter during the breakdowns of algae blooms leads together with enhanced bacterial activity in summer to the formation of organic- and trace metal-rich aggregates which are deposited and incorporated into the tidal surface sediments. Microbial decomposition of the aggregates and corresponding shifts in redox-conditions effect a release of dissolved trace metals into the pore water. Subsequently, the trace metals are fixed in the sediment as sulphides, adsorbed to organic compounds or released to the overlying bottom water. Furthermore, two tidal systems, one from the East Frisian and one from the North Frisian Wadden Sea are compared. Although, both areas show different hydrodynamical, sedimentological, and ecological conditions similar manganese dynamics are observed implying that this is a common behaviour in the entire Wadden Sea. However, distinct quantitative differences appear showing a 6-fold higher level of dissolved manganese in the water column of the East Frisian area. This is explained by a higher manganese release from tidal flat sediments and a larger sediment area/water volume ratio compared to the North Frisian area. Detailed time-series data of the nutrients phosphate, silica, and nitrite+nitrate are used to verify model simulations and to calculate nutrient export budgets considering tidal and seasonal variations. The model results imply an export of nutrients from the tidal flats into the open waters of the German Bight which is in the same order of magnitude as the combined discharge of the rivers Elbe, Weser, and Ems. To investigate the importance of the Wadden Sea as a potential manganese source for the North Sea, transects were carried out into several tidal flat areas of the North Frisian Wadden Sea. The results suggest that the North Frisian Wadden Sea is a less important source for dissolved manganese compared to the East Frisian area. In contrary, the export of particulate manganese seems to be more important showing distinctly higher concentrations in the North Frisian study areas in summer. The influence of sediment permeability and bioturbation on trace metal budgets of the pore waters are investigated in natural and experimentally manipulated tidal flat sediments. Advective pore water transport in highly permeable sandy sediments and bioturbation promote exchange processes at the sediment/water interface probably leading to reduced nutrient and trace metal enrichments in the shallow pore waters. Furthermore, the penetration of oxygen into deeper sediment layers induces a release of sulphidic bound molybdenum to the pore water. During laboratory experiments with natural anoxic sediments an effective oxidative molybdenum release is determined during resuspension of the sediments in oxic seawater. Thus, pronounced sediment resuspension during storm events is suggested to cause significant release of molybdate from displaced anoxic sediment components thereby enhancing the molybdate level of the open water column. In addition to the examination of recent biogeochemical processes, the paleo-environmental influence on geochemical and microbiological processes in Holocene and Pleistocene sediments of the East Frisian study area were analysed in an interdisciplinary study. It is found that the microbial abundance and activity are higher in the Holocene than in the Pleistocene sediments. However, this is mainly caused by present environmental conditions. The impact of the paleo-environment on the microbiology is less pronounced. The lithological succession affects hydrological processes which enable the transfer of electron donors and acceptors for present early diagenetic processes into deep sediment layers. The paleo-environmental imprint is still detectable but the modern biogeochemical processes dominate in the sediment-pore water system.
Interactive Visualization for the Exploration of Aligned Biological Networks and Their Evolution
(2011)
Network Visualization is a widely used tool in biology. The biological networks, as protein-interaction-networks are important for many aspects in life. Today biologists use the comparison of networks of different species (network alignment) to understand the networks in more detail and to understand the underlying evolution. The goal of this work is to develop a visualization software that is able to visualize network alignments and also their evolution. The presented software is the first software for such visualization tasks. It uses 3D graphics and also animations for the dynamic visualization of evolution. This work consists of a review of the Related Work, a chapter about our Graph-based Approach for Interactive Visualization of Evolving Network Alignments, an explanation of the Graph Layout Algorithm and some hints for the Software System.
The six extraocular muscles (EOMs) are arranged around the eyeball as agonist-antagonist pairs performing the eye movements. The EOMs comprise a distinct muscle group that is fundamentally different from other skeletal muscle, which is reflected on many levels, such as functionality, anatomy as well as in their molecular make-up. Physiologically EOMs are considered superfast, high endurance muscles that are continuously active. In addition, EOMs contain unusual slow-tonic fibers that share features with amphibian and avian slow-tonic fibers. EOMs also express slow/cardiac isoforms of proteins and genes along with the typical isoforms of fast muscle fibers. Another striking hallmark of EOM is their differential involvement in a number of diseases. For instance, EOMs are preferentially spared in Duchenne Muscular Dystrophy (DMD). DMD is the most common fatal, genetic disease in males clinically characterized by progressive muscle wasting. Mutations in the dystrophin gene result in a destabilization of the muscle membrane causing muscle fiber damage. While all other skeletal muscles deteriorate the EOMs remain morphologically and functionally healthy. In the pathogenesis of DMD elevated Ca2+ levels are believed to be an early event and it has been shown that EOMs are protected from pharmacologically induced Ca2+ damage. The goal of this study was to characterize the spared EOMs, in particular their Ca2+ homeostasis, in the context of DMD pathology to reveal new potential therapeutic targets for the disease. A combination of physiological, molecular and biochemical methods was used to investigate the Ca2+ homeostasis of EOMs to demonstrate clear differences compared with the fast limb muscle tibialis anterior (TA). Ca2+ handling of stimulated cultured EOM myotubes suggested more efficient Ca2+ removal from the cytoplasm after induced Ca2+ influx compared with cultured myoblasts from TA. Subsequent mRNA and protein expression analyses of myoblasts and adult muscle tissue revealed high expression levels of many key Ca2+ regulating and buffering proteins in rodent EOMs compared with TA. Among these Ca2+ proteins were slow/cardiac proteins, which normally are not found in fast muscles. For instance, the sarcoplasmic Ca2+ ATPase SERCA2 was elevated along with its regulator phospholamban (PLN). Further, PLN was preferentially endogenously phosphorylated at Thr17 suggesting continuous activation of SERCA2 and possibly the fast isoform SERCA1, the main Ca2+ pumps responsible for removing Ca2+ from the cytoplasm after muscle contraction. Furthermore, Ca2+ buffers, such as calsequestrin (CASQ2) and parvalbumin (PARV) were elevated. These results suggest that EOMs are endowed with a unique and superior Ca2+ homeostasis that facilitates efficient Ca2+ buffering and removal from the cytoplasm. This is in agreement with their continuous and fast activation cycles, as well as with a potential protective mechanism in prevention of Ca2+ overload in DMD. The extreme activity patterns of EOM suggested that a high activity of store-operated Ca2+ entry (SOCE) plays a critical part to replenish Ca2+ for rapid and continuous cycles of contractions. To extend the data on general Ca2+ homeostasis and because of possible implications of store-operated Ca2+ influx and other Ca2+ influx pathways in DMD, the expression patterns of group 1 transient receptor potential (TRP) channels and the proteins Orai1 and STIM1 were studied. The TRP channels, TRPC1, TRPC6 and TRPV4 channel proteins in addition to STIM1 showed higher expression in EOM compared with TA. High TRPC1, TRPV4 and STIM1 levels could play a significant role in the high fatigue resistance, muscle differentiation and SOCE in EOM. In addition, tissue from the mdx mouse model of DMD was investigated. The only channels differentially expressed in mdx EOM compared with normal EOM were TRPM4 and TRPM7 (decreased in mdx EOM) and TRPV4 (increased in mdx EOM). Although, these changes in mdx EOM were of small magnitude, they could point toward subtle compensatory changes related to the disease process. In general, EOMs seem to be unaffected by the disease and inherently protected. In conclusion, the results in this thesis have improved the understanding of the Ca2+ homeostasis in EOMs and suggest that EOM may be better able to prevent prolonged elevation of cytoplasmic Ca2+ levels. These data may help to design new therapeutic approaches targeting Ca2+ handling proteins to ameliorate muscular dystrophy.
This thesis aims at bridging the gap of deficient understanding of effective buffer zone management. The overall research goal of the thesis is to evaluate buffer zone effectiveness and to identify factors influencing effective buffer zone management in forest biosphere reserves. To address the multi-facetted issue of buffer zone effectiveness an integrative research design was applied. To answer the raised research questions a combination of social science (quantitative and qualitative approaches) and natural science (remote sensing) was chosen. To gain global insights into buffer zone management (research question 1) the quantitative approach of social science research was chosen. As part of a global telephone survey of BR management conducted by the research project in which the thesis was embedded, BR managers were asked to evaluate different management aspects. Between July and December 2006, managers from 225 BRs in 79 countries were interviewed, which corresponds to an overall response rate of 42 %. Answers were statistically analyzed using SPSS 17.0. To obtain detailed information of factors influencing buffer zone management (research question 2) the qualitative social science research approach was applied. A case study was conducted in the Lore Lindu Biosphere Reserve, Sulawesi, Indonesia between March and May 2008. Following the snowball sampling approach 47 semi-structured interviews and seven group discussions were carried out representing the local, sub-national, and national level associated with the BR management. These interviews and discussions provide important insights into the institutional dimensions and their interaction within the context of BR management including e.g. implementation of rules and the distribution of responsibilities for buffer zone management. Interviews were conducted in the national language Bahasa Indonesia, fully recorded, and subsequently transcribed and translated into English. Analysis was carried out with ATLAS.ti to specify categories and to formulate theorems. To evaluate buffer zone effectiveness in terms of reducing deforestation in the core area of Lore Lindu Biosphere Reserve (research question 1), satellite image analysis was performed using a GIS. A time series of LANDSAT scenes from 1972, 1983, 1999, 2002, and 2007 was used to classify homogeneous areas of forest cover to ultimately detect deforestation. Deforestation rate was computed for the periods before and after management establishment in 1998. The combination of all three research methods provided important insights into buffer zone management of BRs. Thus, based on these findings, recommendations to improve buffer zone management (research question 3) could be drawn. Overall, the evaluation of buffer zones depicts their importance for BR management effectiveness. Analysis revealed buffer zone effectiveness as important success factor, while it explicitly depends on both the implementation of the BR concept at the national level and coordination of stakeholders on the local level. As more and more PAs create buffer zones to integrate the local people, they may face similar problems. The case study from Lore Lindu exhibited important preconditions for successful buffer zone management. From a methodological perspective the thesis calls for the need of integrated research approaches across disciplines to adequately assess both buffer zone and PA effectiveness. Generally, it is recommended to pay special attention to pre-Seville BRs in the future, since most of these BRs still lack the three zone scheme. Analysis of the case study area revealed particular weaknesses in implementing central elements for effective BR management, such as the four goals of the Seville Strategy, even 15 years after inauguration. Thus, the thesis shows that not only the quantity of PAs but also the quality of its management and thus effectiveness is an important indicator for global conservation targets. Finally, it can be summarized that the idea of establishing buffer zones within BRs and PAs in general, is the right way forward to enhance PA effectiveness and to achieve global reduction of biodiversity loss. Integrating the people living within and adjacent to PAs, must be given more attention in the future. Establishment of buffer zones, where this integration and cooperation is a necessity, should be the central conservation measure, not only within BRs but also within PAs in general.
Self-similar sets are a class of fractals which can be rigorously defined and treated by mathematical methods. Their theory has been developed in n-dimensional space, but we have just a few good examples of self-similar sets in three-dimensional space. This thesis has two different aims. First, to extend fractal constructions from two-dimensional space to three-dimensional space. Second, to study some of the properties of these fractals such as finite type, disk-likeness, ball-likeness, and the Hausdorff dimension of boundaries. We will use the neighbor graph tool for creating new fractals, and studying their properties.
The dissertation aims at developing means to integrate conservation and development in biosphere reserves in Madagascar. Despite a multitude of concepts such as UNESCO biosphere reserves, Integrated Conservation and Development Projects and community-based natural resource management, gaps between conservation and development remain to exist. In a qualitative case study in Mananara-Nord and Sahamalaza Iles-Radama Biosphere Reserves in Madagascar data was collected on biosphere reserve management, local use of natural resources and socio-cultural aspects that influence natural resource use. Furthermore, natural values local people associate with the forest were investigated. Analysis revealed that management capacities constitute a limiting factor in biosphere reserve management. Collaboration between management, local people and international organisations fosters the achievement of both conservation and development. However, collaboration is only possible if (i) clear rules are formulated and (ii) partners have a vision in common. Based on the theory of social capital, newly introduced and locally existent rules/institutions having an influence on the use of natural resource were categorized in bonding, linking and bridging social capital. Furthermore, the perception of natural values was classified in instrumental and non-instrumental values and assigned to ecosystem services identifying the importance of nature for human well-being. With the capabilities approach Amartya Sen defined human well-being as the achievement of those capabilities a person considers valuable. This includes aspects that assure livelihoods on the one hand and aspects that are conducive to well-being on the other, thus both being relevant for development. In the dissertation capabilities are based on both instrumental and non-instrumental natural values and consequently offer an opportunity to demonstrate and characterise the relationship between nature and human well-being. Social and natural values provide orientation for a biosphere reserve management. The category bonding social capital (social values) describes local socio-cultural aspects in communities and their importance for collaborative processes. Natural values provide the management with guiding principles to foster nature conservation and to integrate locally existent capabilities. Supporting and furthering these capabilities enables the development of new capabilities of all concerned persons. The dissertation demonstrates various possibilities to build bridges between (i) nature conservation and development, (ii) natural and social sciences, (iii) formal regulations and local socio-cultural aspects and (iv) diverse actors. Implementation of a social monitoring is recommended together with local stewards and Malagasy students to collect information about the perception of natural and social values and use them as guiding principles for biosphere reserves. Collaboration with national and international scientific institutions can foster this process.
Staphylococcus aureus is a pathogenic bacterium infecting the human host. It’s multifaced adaptation to various environmental conditions is mediated by a tight regulation of the virulence factors influencing the host’s immune system. In this thesis two regulators of gene expression were analysed: (i) the global influence of the two-component system SaePQRS and (ii) the regulation of superantigen gene expression by the alternative sigma factor σB. At the outset of this thesis, single target genes induced by SaeRS were known (hla, hlb, cap5, fnbA, coa). In order to get a general idea of the Sae-regulon, the influence of SaePQRS on gene-expression was analysed in two strain backgrounds by proteomics and transcriptomics aproaches. Recapitulatory, expression of at least 18 secreted and two covalently cell-wall bound proteins was decreased following inactivation of the Sae-system. Sae-dependently expressed were, amongst others, well decribed virulence factors like the y-hemolysins HlgA, HlgB, HlgC, LukM and LukF, the innate immune system modulating proteins Efb, CHIPS and SCIN-B as well as the enterotoxin SEB. SaeR acts as an activator of its target genes. Some proteins were detected in increased amounts in the extracellular proteome of the Sae-deficient strain. However, these changes did not occur at the transcriptional level. The expression of virulence factors is determined by other global regulators. No influence of SaePQRS on the transcription of five substancial regulators, namely the Agr-system and its effector molecule RNAIII, the alternative sigma factor σB, the two-component system ArlRS and the DNA-binding protein SarA, could be shown. In the second part of this thesis the issue was broached to the regulation of gene-expression of a subgroup of virulence factors, the superantigens (SAgs) of S. aureus by SaePQRS and σB. In contrast to their well described molecule structure and function, the regulation of their gene expression was largely unknown. Six different S. aureus strains (two laboratory strains and four clinical isolates) encoding one to seven SAg-genes each, were used for analysis of a total of twelve SAgs regarding their transcription and mitogenic activity. The transcriptional units were characterized using Northern-Blotting. The expression of SAgs could be correlated to the respective growth phase. While egc-SAgs were expressed mainly at low optical densities, seb was induced during late growth phase. In contrast, the transcription of sea, seh, sek, tst and sep remained constant and growth-phase independent. The transcriptional dataset was verified using T-cell proliferation assays. The expression of seh, tst and the egc-operon was dependent on σB. A potential σB-dependent promotor could be identified preceeding seo, the first gene of the egc-operon. In contrast, the expression of seb was increased in sigB-deficient background. This might be due to indirect effects. Expression of seb required SaePQRS. Transcriptional datasets were verified by Immuno-Blotting and T-cell-proliferation assays. In conclusion, the same mutation in sigB but in different strain backgrounds could result in opposite phenotypes with respect to their mitogenic activity. Besides well characterized virulence factors, some secreted proteins with so far unknown function belong to the Sae-regulon. Given that the influence of SaePQRS was restricted to virulence factors and induced especially modulators of the innate immune system, it can be assumed, that these proteins potentially play a role in virulence of S. aureus. In the third part of this thesis, one of these potential new virulence factors, namely SACOL0908, was analysed in detail. In cooperation with the group of Prof. Stehle, Tübingen, the crystal structure was solved. The protein folding of SACOL0908 is new with only minor similarities to described protein structures. Recombinantly expressed SACOL0908 binds to granulocytes. These cells belong to the innate immune system, incorporate bacteria by phagocytosis and kill them. The receptor for SACOL0908 on the surface of granulocytes could not be identified using immunoprecipitation, antibody-blocking assays and functional assays in cooperation with the group of Prof. Peschel, Tübingen. The gene encoding SACOL0908 was deleted in two S. aureus strain backgrounds (COL and Newman). These mutants are currently in use to characterize their phenotype in mouse-infection studies.
Proteolysis represents the final step in the life of a protein. It is one of the most important cellular processes assisted by chaperone systems and ensures an appropriate protein homeostasis. Protein degradation is essential for the removal of cytotoxic protein aggregates and mis-translated/mal-folded proteins, „unemployed“ and regulatory proteins to enable rapid cell adaptation to altering environmental conditions (Gottesman, 2003; Wiegert & Schumann, 2001; Parker, 1981; Stansfield et al., 1998; Drummond & Wilke, 2008; Goldberg, 1972; Gerth et al., 2008). The bacterial Clp (caseinolytic proteins) protease complexes are analogous to the eukaryotic 26S proteasome and consist of Hsp100/Clp proteins of the AAA+ superfamily and an associated barrel-like proteolytic chamber (e.g. ClpP). The Clp proteases seem to be responsible for the major protein turnover in low GC, Gram+ bacteria. The main goal of this thesis was to develop new methods and tools to investigate global proteolysis more precisely and to get a detailed understanding of protein degradation during starvation conditions and it´s regulation in low GC, Gram-positive bacteria. To analyse protein degradation under starvation conditions the well established glucose starvation model was used. In Bacillus subtilis it could be shown that approximately 200 proteins are selectively degraded in a glucose depletion induced stationary phase. Furthermore radioactive pulse-chase labelling experiments coupled with 2D-PAGE analysis revealed that mainly the ClpCP protease complex is involved in the degradation of proteins in the stationary growth phase. To investigate proteolysis in the human pathogen Staphylococcus aureus in the same way, a newly developed chemically defined medium was established suitable for radioactive pulse-chase labelling experiments under stable glucose starvation conditions. The degradation kinetics of individual 2D spots was significantly better resolved using 14C-BSA as an internal marker protein for the sample normalisation. A rather huge overlap was found within the functional protein classes that were degraded in B. subtilis and S. aureus the stationary phase. Among others, especially proteins involved in amino acid, nucleotide and cell wall biosynthesis were rapidly degraded, whereby not always the same and sometimes another enzymes from a biosynthetic chain were targeted for proteolysis. Despite the resolution power of the 2D-PAGE method, there are some drawbacks such as a limited "protein window" with regard to the molecular weight and isoelectric point, loss of low abundance proteins and a rather low reproducibility for time course experiments. Therefore a mass spectrometry based approach for the simultaneous detection of protein synthesis, accumulation and degradation was developed. This pulse-chase SILAC approach provides a very good reliability with a broad spectrum of proteins that can be analysed. Through the combination with ultracentrifugation even non-soluble and aggregated proteins could be analysed. Several hundred proteins were degraded in S. aureus during glucose starvation. Among them was the functional cluster of ribosomal proteins which is degraded in the early stationary phase. Furthermore proteins belonging to complexes were degraded with the same kinetic (e.g. NrdE, NrdF). In addition selective protein degradation took place according to functional categories (e.g., ribosomal proteins, biosynthetic, glycolytic enzymes) and not to regulatory groups (e.g. CcpA, SigB regulon).The investigation of a clpP deletion mutant in S. aureus revealed a greater susceptibility to aggregation, where the cells try to counteract with the expression of chaperones like GroEL/ES, ClpB and DnaK. The renaturation process is very ATP consuming and only takes place in energy rich phases of growth (e.g. from exponential to transient growth phase). Protein aggregation was found enhanced in the stationary phase. Furthermore, a higher GTP level compared to the wild-type probably resulted in a stronger CodY mediated repression with a rather low level of amino acids in clpP mutant cell. In addition substances like glycerol, which thermodynamically stabilise proteins in refolding processes (Maeda et al., 1996; Feng & Yan, 2008), were found in higher levels compared to the wild-type. A strong response to reactive oxygen species was detected in the clpP mutant strain, which is probably due to ROS production during the early stages of protein aggregation. Altogether, different methods were used for investigation protein degradation at a proteome-wide scale. Hundreds of degradation candidates were identified by gel-based and gel-free approaches in S. aureus wild-type cells. “Unemployed” proteins (e.g. ribosomal proteins, biosynthetic enzymes) were degraded and proteins particularly required and synthesized in glucose-starved cells such as TCA cycle enzymes were stable in the stationary phase. Investigation of the clpP mutant strain supports a proposed model for the pleiotropic phenotype and provides a deeper insight in the fine-tuned protein quality control and the important role of ClpP during starving conditions.
Decision making in everyday purchase situations requires mental processing of factors that are related to the items on display. These influencing factors – called persuasive information – can take various forms, like the price level, the design of the package or the display of certain product attributes. Despite the existence of persuasive information trying to influence our buying behavior, almost nothing is known about the underlying neural mechanisms responsible for processing this information. In this thesis functional magnetic resonance imaging was used to investigate neural activity correlated with product related persuasive information. As persuasive information organic, light and regular labeled food was chosen. The 1st experiment investigated the neural correlates of visually inspected organic and regular labeled food and the influence on willingness to pay (WTP) for the displayed items. It was hypothesized that organic compared to regular labeled food will be perceived as more rewarding which should be visible by an increased activity in the ventral striatum as a central area for reward processing and by a heightened WTP. As organic label information the national German eco emblem 'Bio-Siegel' was chosen (for stimuli details see 2.1). As there is no emblem indicating regular food, an artificially created logo was used for indicating a conventional product. 40 well- known food products (e.g. milk, bread, eggs etc.) were presented to the subjects. These products were marked with the organic emblem and the same 40 products with the regular label. We found that visual inspection of organic labeled food indeed led to an increase in neural activity in the ventral striatum and to a heightened WTP, suggesting a higher subjective value for these products. The 2nd experiment investigated the neural correlates of actually administered food stimuli labeled organic, light or regular and the influence on expected and experienced taste. For organic compared to regular labeled food we hypothesized an increase in expected and experienced taste pleasantness. Furthermore, light compared to regular labeled food should lead to a decrease in expected and perceived pleasantness and intensity ratings. During the active tasting process this should be accompanied by an increase in reward-related (e.g. organic vs. regular; regular vs. light) areas like the ventral striatum medial orbitofrontal cortex or aversion-related (e.g. regular vs. organic; light vs. regular) areas as the lateral orbitofrontal cortex (lOFC) and operculum/insula. As organic label information the national German eco emblem 'Bio-Siegel' was chosen. Light label information was issued in form of the internationally used 'Bewusst-Wählen®' ('Healthy Choice') label (for stimuli details see 2.2). However, inside the scanner the written forms 'Bio', 'Light' or 'Normal' (indicating regular food) were chosen. Subjects were randomly assigned in two groups and were either confronted with the organic or the regular label (organic group) or with the light or the regular label (light group) but otherwise identical milk drink. The results show that organic compared to regular labeling of identical food stimuli indeed led to an increase in expected and experienced taste pleasantness for organic labeled food. Light compared to regular labeling of identical food stimuli led to a decrease in expected and experienced taste pleasantness and intensity for light labeled food. Moreover, taste-related activity was found in aversion related areas like the operculum insula and the lOFC for food labeled regular compared to organic and in reward-related areas like the ventral striatum for food labeled regular compared to light. The results show that persuasive food-related information influences human cognition on the behavioral and neural level; the effects were shown during visual and gustatory evaluation of the stimuli. Taken together the results demonstrate that the same stimulus can vary dramatically in personal valuation depending on the applied information.
Chromosomal abnormalities, like deletions, amplifications, inversions or translocations, are recurrent features in haematological malignancies. However, the precise molecular breakpoints are frequently not determined. Here we describe a rapid analysis of genetic imbalances combining fine tiling comparative genomic hybridization (FT-CGH) and ligation-mediated PCR (LM-PCR). We clarified an inv(14)(q11q32) in a case of T cell acute lymphoblastic leukaemia with a breakpoint in the TRA/D in 68% of cells detected by fluorescence in situ hybridization. FT-CGH showed several mono- and biallelic losses within TRA/D. LM-PCR disclosed a TRA/D rearrangement on one allele. The other allele revealed an inv(14)(q11q32), joining TRDD2 at 21,977,000 of 14q11 together with the IGH locus at 105,948,000 and 3′-sequence of TRAC at 22,092,000 joined together with IGHV4–61 at 106,166,000. This sensitive approach can unravel complex chromosomal abnormalities in patient samples with a limited amount of aberrant cells and may lead to better diagnostic and therapeutic options.
Three-dimensional (3D) dynamical properties of fast particles being injected into the void region of a dusty plasma under microgravity conditions have been measured. For that purpose, a stereoscopic camera setup of three cameras has been developed that is able to track and reconstruct the 3D trajectories of individual dust particles. From more than 500 particle trajectories, the force field inside the void region and its influence on particle movement are derived and analyzed in 3D. It is shown that the force field is dominated by forces pointing radially out of the void and that this radial character is reflected in the velocity distributions of particles leaving the void. Furthermore, the structure of the force field is used for measuring the neutral gas friction for the particles inside the void.
Background: Cardiovascular diseases are the leading cause of death worldwide. Subclinical alterations of the cardiovascular system, such as increased exercise blood pressure or an endothelial dysfunction confer a higher risk of manifest cardiovascular diseases and incident events. Detecting associations between circulating markers of the endocrine-metabolic system and the subclinical cardiovascular phenotypes could be useful to better understand cardiovascular disease progression and to improve risk prediction for manifest cardiovascular diseases. Methods: The associations between (a) serum thyroid-stimulating hormone and increased exercise blood pressure, (b) serum hemoglobin A1c and endothelial dysfunction as well as (c) serum insulin-like growth factor I and endothelial dysfunction were studied using cross-sectional data from around 1400 subjects aged 25 to 85 years collected during the 5-year follow-up of the population-based Study of Health in Pomerania (SHIP-1). Increased exercise blood pressure was defined as a value above the sex- and age-specific 80th percentile measured at the 100 W stage of a symptom-limited bicycle ergometry test. Endothelial dysfunction was defined as an impaired flow-mediated dilation measured as a continuous decrease or below the median of sex-specific distribution. Non-fasting blood samples were drawn from the cubital vein in the supine position. Results: The odds for increased systolic exercise blood pressure (odds ratio 1.24, 95% confidence interval 0.88; 1.76) and diastolic exercise blood pressure (odds ratio 0.98, 95% confidence interval 0.70; 1.39) as well as for exercise-induced increase of systolic and diastolic blood pressure were not significantly different between subjects with high and low serum thyroid-stimulating hormone levels within the reference range. In women without current use of antihypertensive medication, increasing serum hemoglobin A1c levels were associated with decreasing flow-mediated dilation levels (ß = -1.17, 95% confidence interval -2.03; -0.30). Such an association was not found in men. In men, logistic regression analysis revealed an odds ratio of 1.27 (95% confidence interval 1.07; 1.51) for decreased flow-mediated dilation for each decrement of serum insulin-like growth factor I standard deviation. In women, no significant association between serum insulin-like growth factor I levels and flow-mediated dilation was observed (odds ratio 0.88, 95% confidence interval 0.74; 1.05). Conclusions: Based on the presented results it is concluded that (a) serum thyroid-stimulating hormone levels are not associated with exercise blood pressure in the general population, (b) higher serum hemoglobin A1c levels in non-diabetic subjects are inversely associated with flow-mediated dilation in women without antihypertensive medication, but not in men, and (c) lower serum insulin-like growth factor I levels are associated with impaired endothelial function in men, but not in women. Therefore the metabolic marker hemoglobin A1c and the endocrine marker insulin-like growth factor I might be markers facilitating the identification of subjects at high risk of subclinical cardiovascular alterations.
Novel heterocyclic alpha-phosphinoamino acids, by structural relationship named 3-phosphaprolines, were obtained by cyclocondensation of 2-phenylphosphinoethylamines with glyoxylic, pyruvic or phenylglyoxylic acid at room temperature in diethylether. The reactions proceed via primary attack of the P-lone electron pair, as shown by the synthesis of phosphonium glycolates from tertiary phosphines and glyoxylic acid, and addition of PH at the carbonyl group. The ring closure proceeds by replacement of the hydroxy by the amino group and is kinetically controlled. NMR monitoring of the phosphaprolines in CD3OD over several days indicates changes of the diastereoisomer ratios leading to higher contents of the more stable trans-diastereoisomers. The zwitterionic compounds are soluble in part in CD3OD, DMF or DMSO, are somewhat sensitive to air in solution and may undergo hydrolysis with larger amounts of water. The structures are proved by multinuclear NMR spectra and two crystal structure analyses. Suitable phosphaprolines as well phosphonium glycolates and Ni(COD)2 allow to generate precatalysts, activated by NaH for the oligomerisation of ethylene to mainly linear products with methyl and vinyl end groups. Some additional investigations with phosphinophenolates, another type of P-C-C-O- ligands, were performed for comparison. Precatalysts prepared from 2-phosphinophenolesters and Ni(COD)2 at room temperature were characterized by multinuclear NMR but decomposed on heating to stable nickel cis-bis(P,O-chelate) complexes. Heating precatalysts generated from a phosphinophenolester or phosphinophenols and Ni(COD)2 in the presence of ethylene under pressure led to linear ethylene oligomers. These reactions are much faster than the above mentioned conversions with NaH activated P,O-Ni-catalysts. In the presence of 9-decenol with unprotected remote hydroxyl group incorporation of a small amount of isolated hydroxyoctyl side groups takes place, detected by 13C NMR spectroscopy. Finally it is stated that the development of a facile synthesis and the characterization of the properties of the phosphaprolines pave the way for derivatisation and further studies with these novel types of amino acids.
Phosphines are highly versatile ligands for transition metal catalysts because of wide tuning abilites of their stereoelectronic properties. Bulky and basic phosphines, to a smaller extend also π-acidic phosphites were intensively studied whereas dicoordinated trivalent phosphorus compounds were comparatively little investigated in this respect. In part this may go back to the limited stability of many P=C compounds, in the case of the stable benzazaphosphole to low stabilityof complexes with non-zero-valent transition metals. With the availability of suitable chelate complexes this problems may be overcome. Because biaryl phosphines proved particularly useful as chelate ligands this work is focused on the development of convenient syntheses of new biaryl-type N-heterocyclic or functionally aryl substituted 1,3-benzazaphosphole P,N- P,P- and P,O-chelate ligands and the characterization of their structures. The pivotal point was to find an applicable synthetic route to the title ligands. Because currently transition metal catalyzed cross-coupling reactions are a hot field in catalytic research, the initial target of my work was the investigation of the applicability of suitable biaryl coupling reactions on 1,3-benzazaphospholes. There are several types of transition metal catalyzed biaryl couplings. One reaction, which is currently in the main focus by use of non-toxic and air stable coupling partners, often allowing water as environmental friendly solvent, is the Pd-catalyzed Suzuki-Miyaura coupling of an aryl halide with an arylboronic acid. To apply the Suzuki coupling to the synthesis of biaryl-type benzazaphospholes, the synthesis of either benzazaphosphol-2-boronic acids or reactive 2-halogen-benzazaphospholes have to be performed. Because of the successful introduction of functional groups in position 2 of benzazaphospholes via lithiation and reaction with electrophiles, the 2-lithiation of suitably available N-substituted benzazaphospholes and introduction of boryl groups or halogen by reaction with boronic acid esters or with a halogenating reagent like dibromoethane appeared as a realistic route and was chosen for closer study. N-Neopentyl-benzazaphosphole was selected by its relatively easy access and N-mesityl-benzazaphosphole as a N-aryl representative. From the two principal methods developed to synthesize 1,3-benzazaphospholes, only the synthesis and reduction of o-aniline phosphonic acid esters to o-phosphinoanilines and subsequent [4+1] cyclocondensation is promising to access N-substituted 2-CH benzazaphospholes. My first investigations targeted to improve the synthesis of the benzazaphosphole precursors. The invention of a Cu- instead of the earlier used Pd-catalyzed P-C coupling allows a more economical access to anilinophosphonates which were then transformed to 2H-1,3-benzazaphospholes by the established orthoformamide cyclocondensation. Several attempts of the coupling with careful control of dryness of all reagents and solvents were made in order to obtain pure 1,3-benzazaphosphole-2-boronic acid ester and, after mild hydrolysis, to isolate 1,3-benzazaphosphole-2-boronic acid. The coupling worked with N-mesityl-1,3-benzazaphosphole 13e, but the benzazaphosphol-2-boronic acid could not be obtained in pure form because of easy B-C bond cleavage during crystallization, certainly by the two ‘OH groups. For attempts with a reverted methodology, the synthesis of a 2-bromo-substituted benzazaphosphole was studied, which should be coupled with (hetero)arylboronic acids via Suzuki-Mijaura reaction. However, the 2-bromo-benzazaphosphole also could not be obtained in pure form, and a coupling experiment with phenyl boronic acid and catalysis with ligand free Pd/C failed. Therefore, other routes to biaryl-type benzazaphospholes were envisaged. Direct C-H functionalization has emerged over the past few years as an attractive strategy to enhance molecular complexity. This holds also for π-excess-type heterocycles like indoles, benzoxazoles or purines which allow direct CH-arylation in 2-position. These reactions generally involve palladium based catalysts and in some cases rhodium catalysts. In a series of experiments the catalytic arylation, heteroarylation and later also alkylation were studied with 1,3-benzazaphospholes 13a-e as precursors. The initial studies were carried out with iodobenzene, keeping similar reaction conditions as for 2-CH arylation of indoles. Then transition metal catalysts, bases and conditions were varied. The necessity and influence of a catalyst was established by blind experiments without transition metal catalyst which led to strong decrease of the reactivity. However, the transitional metal catalyzed reactions of N-substituted-1,3-benzazaphosphole with aryl- and heteroaryl halides did not give the desired 2-aryl-substituted 1,3-benzazaphosphole biaryl ligands but revealed a novel oxidative addition at the P=C double bond. In the presence of moisture benzazaphospholine-P-oxides are formed. Further exploration of the scope of this reaction showed that it is applicable to several functionally substituted aryl halides and heteroaryl halides. As besides PdX2 (X = Cl, OAc) also Pd(0)(PPh3)4 was found active as catalyst, it can be assumed, that the reaction occurs via a Pd(0) species and oxidative addition of the aryl halide at Pd(0). Because Pd(0) will coordinate stronger to the π-acidic benzazaphosphole than Pd(II) it is assumed that in the first step small equilibrium amounts of a Pd(0)benzazaphosphole complex will be formed which undergo the oxidative addition and then react to benzazaphospholium salt and furnish back a Pd(0) complex with 1,3-benzazaphosphole ligand. The benzazaphospholium salts are highly sensitive to moisture and react with traces of water to form benzazaphospholine-P-oxides 20 and acid, neutralized by the base. A cyclic species RR’P(OH)=CHR”, where the halogen is replaced by OH, may be assumed as intermediate which undergoes a rearrangement to the more stable RR’P(=O)-CH2R” tautomer, driven by the high P=O bond energy. After various investigations of the optimum conditions for the reaction, a number of new functionally substituted P-aryl or P-heteroaryl benzazaphospholine P-oxides and 1,3-dineopentyl-benzazaphospholine-3-oxide were isolated and characterized by 1H, 31P, 13C and HRMS data and two by crystallography. The biaryl-type 2-phenyl-1,3-benzazaphosphole is known since the earliest reports of these heterocycles, synthesized by cyclocondensation of 2-phosphinoaniline with benziminoester hydrochloride or in low yield with benzaldehyde. The latter method was further developed because of the compatibility of the aldehyde group with various donor functions. 2-Phosphinoaniline (12a) and 2-phosphino-4-methylaniline (12b) were heated with pyridine-2-carboxaldehyde under varied conditions, and a crucial role of acid catalyst was observed in the investigation. The results showed that the dehydrogenating cyclocondensation, if catalyzed by a suitable type and amount of acid catalyst, works well for primary phosphinoanilines 12a,b and a variety of reactive aldehydes, including N-heterocyclic and o- or m-functionally substituted arylaldehydes. In an equimolar ratio, on heating usually hydrogen is eliminated, at least formally, to furnish the aromatically stabilized 1H-1,3-benzazaphosphole ring systems of 35 whereas in other cases reductive side reactions occur, e.g. the N-CH2R substitution to 36 in reactions with two equivalents of aldehyde. Thus the synthesis of 1,5-dimethyl-1,3-benzazaphosphole (36a) was achieved by double cyclocondensation of 12b and formaldehyde in a 1:2 molar ratio. This provides the so far shortest way to synthesize N-substituted 1,3-benzazaphospholes and suggests, that the reaction is generally applicable in reactions with two equivalents of monoaldehyde. This puts the question if N-secondary o-phosphinoanilines such as N-neopentyl-2-phosphinoaniline (12d) can be cyclocondensed with aldehydes to benzazaphospholes or if a primary amino group is required. The successful experiment shows that cyclocondensation of N-secondary o-phosphinoanilines with suitable aldehydes is possible. N-Neopentyl-2-pyrido-1,3-benzazaphosphole was obtained in high yield. An interesting extension of the above reaction are cyclocondensations with compounds bearing two aldehyde groups. Double condensation of 12b with o-phthaldialdehyde was performed. It proceeded fast and gave tetracyclic-1,3-benzazaphosphole in high yield. Based on the NMR monitored primary formation of organoammonium phosphino glycolates from amines, phosphines and glyoxylic acid, followed by conversion to phosphinoglycines, it is assumed that the reaction proceeds by initial attack of the primary phosphino group of 12b at the carbonyl carbon atom of R-CHO, polarized with the help of the acid catalyst. The resulting P-C bonded secondary phosphine, containing an α-hydroxy group, may release water after transfer of a proton to oxygen in equilibrium, followed by attack of amine. This leads to formation of the dihydro-intermediate 34, observed by NMR reaction monitoring in several cases. Possible ways are releasing of H2 during reflux, directly giving 2-substituted NH-1,3-benzazaphospholes 35, or hydrogen transfer, connected e.g. with N-substitution leading to 1,2-disubstituted 1,3-benzazaphospholes 36. The second path is observed mainly when excess or double molar quantities of aldehydes are used at the start of the reaction. The two hydrogen atoms at P and C2 are consumed during the second condensation and formation of the NCH2R group and generate the P=C double bond. Finally, cyclocondensation of o-phosphinoanilines with aldehydes has proven as a useful method for the synthesis of biaryl type benzazaphosphole ligands. After thorough investigations, N-primary and secondary phosphino anilines were found cyclisable with various heteroaryl aldehydes upon refluxing in toluene in the presence of a suitable acid catalyst, and 11 new compounds were synthesized following this procedure and characterized by 1H, 31P, 13C NMR and HRMS data. For two compounds crystal structures were also obtained. First attempts to synthesize chelate complexes with the 2-(hetero)aryl-1,3-benzazaphospholes were started. A soluble 2-(o-diphenylphosphinophenyl)-1,3-benzazaphoasphole-Cr(CO)4 chelate complex was detected by NMR spectroscopy, whereas most products of the new ligands with Rh(COD) or NiCp complexes were insoluble in usual NMR solvents and require further efforts for synthesis and full analytical and structural characterization.
Triple helix-forming oligonucleotides (TFOs) are one of the most specific DNA duplex binding agents and offer new perspectives towards oligonucleotide-mediated gene regulation and manipulation. However, the poor thermodynamic stability of DNA triplexes under physiological conditions limits a successful application in the antigene strategy. Thus, the conjugation of TFOs with small triplex-specific binding ligands is a promising approach to stabilize the formed complexes and to enhance their overall binding affinity. The present study focused on the synthesis of novel TFO conjugates with triplex-binding indolo[3,2-b]quinoline derivatives (PIQ) and on their ability to form and stabilize intermolecular triplexes through their recognition of a duplex target. During the course of the work the thermodynamics of conjugate binding and structural aspects of drug-DNA interactions have been characterized by a variety of spectroscopic and calorimetric techniques.
The present work provides new insight concerning histidine phosphorylation in proteins, which is an essential regulatory posttranslational modification. To study histidine phosphorylation, a newly developed NMR approach, the HNP experiment, is presented in this thesis. The HNP experiment provides specific experimental evidence of phosphorylated histidines in proteins. It allows for the determination of the regiochemistry of phosphohistidines on the basis of three individual peak patterns for distinguishing all three phosphohistidines i.e. 1- and 3-phosphohistidine and 1,3-diphosphohistidine. This novel NMR approach allows the investigation of histidine phosphorylation in proteins under physiological conditions without resorting to chemical shift comparisons, reference compounds, or radioactively labelled phosphate. In this thesis, histidine phosphorylation in the regulatory domains PRDI and PRDII of the Bacillus subtilis antiterminator protein GlcT was intensely studied. GlcT is a transcription factor, which regulates the phosphotransferase system (PTS) by modulating the expression level of PTS-enzymes (Enzyme I, HPr, Enzyme II) on a transcriptional level. Upon the phosphorylation of conserved histidines in PRDI and PRDII, the function of GlcT is regulated through its aggregation state. In this thesis, it is shown that histidines in both PRDs are primarily phosphorylated at their N(Epsilon-2), forming 3-phosphohistidine. In addition, we found, by newly optimized mass spectrometry conditions, that both PRDs are dominantly onefold phosphorylated. By using tandem mass spectrometry to study PRDI, we identified histidine 170, which is the second of two conserved histidines (His 111 and His 170), as the phosphorylation site. In this thesis, it is also shown through comprehensive mutational studies that both conserved histidines (His 218 and His 279) in PRDII can be individually phosphorylated. This is in good agreement with mass spectrometry results that indicated an additional twofold phosphorylation in PRDII. This can be explained as follows: an intra-domain phosphate transfer between both conserved histidines in PRDII might be involved in the phosphorylation reaction, finally leading to a mainly onefold phosphorylated PRDII at one of the two conserved histidines. This minor twofold phosphorylation has also been found in PRDI. However, the specific peak pattern in the HNP-spectra of PRDI strongly suggest that this additional phosphorylation originates from a 1,3-diphosphohistidine, most likely at histidine 170. Furthermore, for the first time the existence of 1,3-diphosphohistidine in a protein was found. We also show that the phosphorylation of PRDI can be achieved in the absence of Enzyme II which is in contrast to the literature. Shown by analytical gel filtration, the monomeric aggregation state of PRDI obtained upon Enzyme II-free phosphorylation is identical to the monomeric aggregation state which was proposed for the Enzyme II-dependent phosphorylation of GlcT. As shown in this thesis, the combined results of HNP-NMR, mass spectrometry and analytical gel filtration deepen our understanding of regulatory histidine phosphorylation in the individual PRDI and PRDII domains of the Bacillus sub- tilis GlcT. I anticipate that this approach will be applicable to study histidine phosphorylations in other phosphoproteins.
Background: Alcohol consumption accounts for a high burden of disease. The general population of West Pomerania has been characterized as a population at risk with a high prevalence of behavioural risk factors such as alcohol risk drinking. This is reflected by the high proportion of patients being admitted to general hospitals due to alcohol-attributable diseases. The aims of the present dissertation were (a) to analyze dose-response relations between volume of alcohol drinking and the risk of diseases with different alcohol-attributable fractions (AAF) in general hospital inpatients (study 1); (b) to assess motivation to change drinking behaviour and motivation to seek help for alcohol problems during their hospital stay as well as changes in motivation to change drinking behaviour, motivation to seek help and changes in daily alcohol consumption across time according to diseases with different AAFs (study 2); and (c) to investigate the association of fatty liver disease (FLD) with blood pressure and hypertension in a general population sample and to test for the specific contribution of alcohol consumption to this association (study 3). Methods: For studies 1 and 2, data from 'Early Intervention at General Hospitals', a randomized controlled trial to test the effectiveness of brief intervention for alcohol problem drinking in general hospitals, were used. Study 1 comprised data from 846 inpatients, study 2 comprised data from 294 inpatients aged 18 to 64 years with alcohol problem drinking and alcohol-attributable diseases from four general hospitals in West Pomerania. Hospital diagnoses were classified according to their AAF: (1) diseases wholly attributable to alcohol consumption by definition (AAF=1), (2) diseases partially attributable to alcohol consumption (AAF<1), and (3) diseases with no relation to alcohol consumption or where alcohol consumption has been found to be a protective factor (AAF=0). Study 3 encompassed data from the 'Study of Health in Pomerania', a general population sample of 3191 adults aged 20-79 years. FLD was defined using ultrasound in combination with increased serum alanine aminotransferase levels. Results: Analyses showed that 46.8% of the general hospital inpatients had a disease attributable to alcohol consumption. There was a dose-response relationship between volume of alcohol drinking and the risk of diseases with different AAFs. Inpatients consuming >120 g and inpatients consuming 61-120 g of pure alcohol per day revealed significantly higher odds for diseases with AAF=1 compared to inpatients consuming 31-60 g of pure alcohol per day with odds ratios (OR) of 6.3 (95% CI 3.6-11.3) and 2.9 (95% CI 1.6-5.1), respectively. Regarding diseases with AAF<1, inpatients consuming >120 g of pure alcohol per day had significantly higher odds compared to inpatients consuming 31-60 g of pure alcohol per day (OR 2.0, CI 1.2-3.4). Analyses on motivation to change drinking behaviour and on motivation to seek help at hospitalization revealed that motivation to change drinking behaviour was higher among inpatients with alcohol-attributable diseases than among inpatients without alcohol-attributable diseases (p<.001). Among inpatients with AAF=1, motivation to seek help was higher than among inpatients with AAF<1 and AAF=0 (p<.001). While motivation to change drinking behaviour remained stable within one year after hospitalization in all three AAF groups, motivation to seek help decreased in this time period. The volume of alcohol consumed decreased in all three AAF groups within one year after hospitalization. Data from the general population study revealed that FLD was associated with blood pressure and hypertension at baseline and at five-year examination follow-up. For example, the chance of hypertension at both time points was threefold higher in individuals with FLD (OR 2.8, CI 1.3-6.2; OR 3.1, CI 1.7-5.8, respectively) compared to individuals without FLD. Analyses further revealed that the association of FLD with blood pressure and hypertension was independent of alcohol consumption. Conclusion: The results of the present dissertation provide relevant implications for public health. In view of the high proportion of general hospital inpatients with alcohol-attributable diseases, a screening procedure for problem drinking is needed. Furthermore, appropriate interventions considering the inpatient’s motivational level have to be implemented. The concept of AAFs to classify disease conditions according to their causal relationship with alcohol consumption might be a tool to detect inpatients with problem drinking. The results regarding FLD and its association with blood pressure and hypertension demonstrate that it is important to pay attention to alcohol-attributable diseases in the general population and that alcohol-attributable diseases are associated with subsequent serious sequelae. The results of the present work further indicate that the concept to distinguish between alcoholic and non-alcoholic origin of FLD might be obsolete and should be replaced by a concept that regards FLD as a multifactorial disease condition.
In this thesis, two novel assay systems had been developed, which allow a fast and easy screening for amine transaminase activity as well as the characterization of the amino donor and acceptor specificity of a given amine transaminase. The assays overcome some limitations of previously described assays but of course have some limitations themselves. The relatively low wavelength of 245 nm, at which the production of acetophenone is detected with the spectrophotometric assay, limits the amount of protein/crude extract that can be applied, which eventually results in a decreased sensitivity at higher enzyme loads due to an increased initial absorbance. Otherwise, this assay can be used very easily for the investigation of the amino acceptor specificity and both pH and temperature dependencies of amine transaminases. The conductometric assay is – by its very nature – limited to low-conducting buffers, a neutral pH and constant temperatures. In summary, the assays complement one another very well and the complete characterization of the most important enzyme properties can be accomplished quickly. Furthermore, we developed and applied a novel in silico search strategy for the identification of (R)-selective amine transaminases in sequence databases. Structural information of probably related proteins was used for rational protein design to predict key amino acid substitutions that indicate the desired activity. We subsequently searched protein databases for proteins already carrying these mutations instead of constructing the corresponding mutants in the laboratory. This methodology exploits the fact that naturally evolved proteins have undergone selection over millions of years, which has resulted in highly optimized catalysts. Using this in silico approach, we have discovered 17 (R)-selective amine transaminases. In theory, this strategy can be applied to other enzyme classes and fold types as well and for this reason constitutes a new concept for the identification of desired enzymes. Finally, we applied the seven most promising candidates of the identified proteins to asymmetric synthesis of various optical pure amines with (R)-configuration starting from the corresponding ketones. We used a lactate dehydrogenase/glucose dehydrogenase system for the necessary shift of the thermodynamic equilibrium. For all ketones at least one enzyme was found that allowed complete conversion to the corresponding chiral amine with excellent optical purities >99% ee. Bearing in mind that until last year there was only one (R)-selective amine transaminase commercially available and two microorganisms with the corresponding activity described, the identification of numerous enzymes is a breakthrough in asymmetric synthesis of chiral amines.
Multiple sclerosis (MS) and stroke share a number of mechanisms of neuronal damage. In both cases the balance between neurodestruction and neuroprotection appears modulated by the function of the adaptive immune system. MS is a chronic inflammatory disease of the central nervous system (CNS), leading to permanent disability. It seems certain that an autoimmune response directed against the CNS is central to the pathogenesis of the disease. While these CNS-specific T cells are activated in MS patients, they are inactive and naive in healthy. Therefore it is believed that an activation of autoreactive T cells by cross-reactivity with pathogens occurs outside of the CNS. In consequence T cells express adhesion molecules and proteinases which enable them to cross the blood-brain barrier. In stroke, however, the blood-brain barrier is disturbed in its integrity caused by the decreased blood flow. Cells can freely migrate from the periphery into the brain. CNS autoreactive cells from the periphery can be activated within the CNS and thus contribute to further tissue damage. While the local autoimmune response remains temporary in stroked brains, it is chronically destroyed in MS. The differences between the underlying mechanisms are not understood. This thesis investigated T cell responses in Multiple Sclerosis in response to the therapeutics Mitoxantrone and IFN-b. The induction of a TH1 to TH2 cytokine response appears to be a shared mechanism of action between both therapeutic agents. Primarily the post stroke immune response was investigated. Patients developed a stroke induced immune suppression characterized by monocytic dysfunction and lymphocytopenia explaining the high frequency of post stroke infections. Moreover early post stroke predictors of subsequent infections, like the CD4+ T cell count, were identified. The T cell response of stroke patients appeared primed to proinflammation and unsuppressed after mitogen stimulation. A detailed understanding of post stroke immune alterations may offer new avenues of intervention to improve the clinical fate of stroke victims. In addition, such knowledge could also further our understanding of Multiple Sclerosis, because, while increasing the infection risk, the dampening of the immune system could have an important protective function, if it limits autoimmune brain damage triggered by the massive release of brain antigens during stroke. If these two pathways could be modulated separately it would create the opportunity to develop distinct therapeutic approaches that inhibit autoimmunity and strengthen antibacterial defenses. To further delineate these mechanisms it is crucial to investigate the role of the innate immune system as compared to the adaptive immune system in stroke induced immune suppression.
Histopathologic and Clinical Subtypes of Autoimmune Pancreatitis: The Honolulu Consensus Document
(2011)
Autoimmune pancreatitis (AIP) has been extensively reported from Japan, Europe and the USA. While the descriptions of AIP from Japan have predominantly been based on the presence of a distinct clinical phenotype, reports from Europe and the USA describe at least 2 histopathologic patterns in patients diagnosed with AIP, namely lymphoplasmacytic sclerosing pancreatitis (LPSP) and idiopathic duct-centric pancreatitis (IDCP) or granulocytic epithelial lesion- positive pancreatitis. While the 2 entities share common histopathologic features (periductal lymphoplasmacytic infiltration and peculiar periductal fibrosis), expert pathologists can accurately distinguish them on the basis of other unique histopathologic features. Clinically, the 2 entities have a similar presentation (obstructive jaundice/pancreatic mass and a dramatic response to steroids), but they differ significantly in their demography, serology, involvement of other organs and disease relapse rate. While LPSP is associated with elevation of titers of nonspecific autoantibodies and serum IgG4 levels, IDCP does not have definitive serologic autoimmune markers. All experts agreed that the clinical phenotypes associated with LPSP and IDCP should be nosologically distinguished; however, their terminology was controversial. While most experts agreed that the entities should be referred to as type 1 and type 2 AIP, respectively, others had concerns regarding use of the term ‘autoimmune’ to describe IDCP.
Background: Chronic kidney disease (CKD) and low serum total testosterone (TT) concentrations are independent predictors of mortality risk in the general population, but their combined potential for improved mortality risk stratification is unknown. Methods: We used data of 1,822 men from the population-based Study of Health in Pomerania followed- up for 9.9 years (median). The direct effects of kidney dysfunction (estimated glomerular filtration rate <60 ml/min/ 1.73 m<sup>2</sup>), albuminuria (urinary albumin-creatinine ratio ≧2.5 mg/mmol) and their combination (CKD) on all-cause and cardiovascular mortality were analyzed using multivariable Cox regression models. Serum TT concentrations below the age-specific 10th percentile (by decades) were considered low and were used for further risk stratification. Results: Kidney dysfunction (hazard ratio, HR, 1.40; 95% confidence interval, CI, 1.02–1.92), albuminuria (HR, 1.38; 95% CI, 1.06–1.79), and CKD (HR, 1.42; 95% CI, 1.09–1.84) were associated with increased all-cause mortality risk, while only kidney dysfunction (HR, 2.01; 95% CI, 1.21–3.34) was associated with increased cardiovascular mortality risk after multivariable adjustment. Men with kidney dysfunction and low TT concentrations were identified as high-risk individuals showing a more than 2-fold increased all-cause mortality risk (HR, 2.52; 95% CI, 1.08–5.85). Added to multivariable models, nonsignificant interaction terms suggest that kidney dysfunction and low TT are primarily additive rather than synergistic mortality risk factors. Conclusion: In the case of early loss of kidney function, measured TT concentrations might help to detect high-risk individuals for potential therapeutic interventions and to improve mortality risk assessment and outcome.
Northern peatlands are ecosystems with unique hydrological properties, storing about 400-500 Gt of carbon. As the production rate of organic material is higher than its decomposition, which is slowed down in the wet and cold environment, peatlands store a great amount of carbon. Carbon assimilated from the atmosphere during photosynthesis by plants is partly lost due to autotrophic and heterotrophic respiration as carbon dioxide (CO2), as methane (CH4) or/and as dissolved organic carbon. The proportion of each carbon component is strongly controlled by environmental conditions as temperature, radiation, precipitation and subsequent water table changes and active role of vegetation. With predicted changes in the global climate, changes in the influence of environmental parameters on peatland ecology are expected. Thus thorough research is essential for a better understanding of mechanisms which influence carbon cycling in peatlands. In this thesis, various components of the carbon cycle were studied at two boreal peatland sites (Ust Pojeg in Komi Republic in Russian Federation and Salmisuo in Eastern Finland) using the micrometeorological eddy covariance method. The focus was placed on the temporal changes of the controlling parameters, ranging from a few days during short snow thawing through the rest of the year. At the Salmisuo site, two measurement seasons allowed to address possible inter-annual variation. We observed that diurnal variations in methane emissions which are typically controlled by vegetation during the growing season, might appear during snow melt as a result of the influence of physical factors rather than biological factors. The diurnal pattern in methane emissions was caused by the interaction of the freeze-thaw cycle and near urface turbulence. During the night time, when surface temperatures fell below zero and caused formation of the ice layer, methane emissions were only around 0.8 mg m-2 h-1, however after the increase in temperature and melting of the ice layer they reached peak values of around 3 mg m-2 h-1. The near surface turbulence had a significant influence on methane emissions, however only after the thawing of the ice layer. The effect of changing environmental parameters over the year was further elaborated on a carbon dioxide time series from the Ust Pojeg site. The generally accepted effects of temperature on ecosystem respiration during the night are not stable throughout the year and can change rapidly during the growing season. Using moving window regression analysis I could show that the strength of the exponential relationship between ecosystem respiration and temperature is changing during the year. This was in correspondence with recent publications elaborating on sub-seasonal changes of the controlling parameters. In general, measurements from the Ust Pojeg site represent estimates of annual CO2 and CH4 fluxes with an annual carbon balance of -94.5 g C m-2 and a new contribution to the quantification of trace gases emissions from a Russian boreal peatland. The inter-annual comparison of net ecosystem exchange (NEE) measurements with previously published data on CH4 and DOC flux from the Salmisuo site showed that the NEE of CO2 is the most important component of the carbon balance at this site. However, primary production was not responsible for the inter-annual changes in NEE. Rather, the effects of water table position during the year had a strong influence on ecosystem respiration, which was probably due to the influence on soil respiration, and higher NEE was observed during the year with smaller primary production, but higher water table levels. The effects of higher precipitation and higher water table during the wet year were shown to increase CH4 flux and the export of DOC, but their effects could not compensate for changes in ecosystem respiration. In the presented thesis intra- and inter- annual changes in carbon flux components and their controls, in our case attributed mostly to hydrological conditions in combination with other environmental parameters as temperature and the role of peatland vegetation, are discussed.
IL-10 drives the re-establishment of peritoneal macrophage populations in bacterial peritonitis
(2011)
The aim of this thesis work was to explore the physiological and functional properties of peritoneal macrophage populations in both the steady state and in inflammatory conditions. In the steady state there are two populations of macrophages in the peritoneum which I refer to as the R1 and R2 populations. The R1 cells are a rapidly turning over population which constitute around 20% of the peritoneal macrophages. I show that these cells have the capacity to efficiently present peptides on MHC-II to CD4+ T cells but that they are poor at phagocytosis. Monocytes transferred into the un-infected peritoneum give rise almost exclusively to this R1 population, suggesting that the R1 fate is the default pathway of monocyte development under steady state conditions. In contrast, the R2 population in the peritoneum turns over very slowly in the steady state and is composed of cells which are poor at the presentation of peptide to T cells but which are efficient at phagocytosis. Both of these populations are lost from the peritoneum within an hour of the induction of a poly-microbial peritonitis. A large fraction of the R2 population relocates from the peritoneal wash fraction to the omentum, the fate of the R1 population is less clear. Over the course of the next three days, the macrophage populations in the peritoneum are re-established. Transfer experiments using genetically marked cell populations demonstrated that neither the R1 nor the R2 populations which “disappeared” one hour after infection contributes to the re-established peritoneal wash fraction macrophage pool at day 3. While the re-established R1 population retains the functional properties and the FACS phenotype of the steady state R1 cells, the re-established R2-like population is clearly not identical to the R2 cells present in the pre-infection environment. In particular, this R2-like population can be split into two sub-populations which have non-identical functional properties. In this inflammatory situation monocytes transferred into the peritoneum now acquire the capacity to differentiate not only into R1-like cells but also into R2-like macrophages. I looked for the molecular basis driving this change of monocyte differentiation in the infected peritoneum by using a solid phase cytometry based ELISA procedure to examine the spectrum of cytokines produced in the peritoneum in response to poly-microbial infection. One of the most prominent cytokines produced early in infection is IL-10. To determine whether IL-10 is directly involved in assigning monocyte fate in the peritoneum I looked at the ability of mice carrying a targeted deficiency of either the IL-10 gene or of the IL-10 receptor gene to form the R2-like cells after infection. Neither mouse strain efficiently generates the R2-like population after infection. Adoptive transfer of genetically marked wild type or mutant monocytes into appropriate hosts demonstrated that the effect of IL-10 is not direct. Rather, the IL-10 responding cell produces a mediator which then directs monocyte fate. Thus, the bystander IL-10R deficient monocytes are driven by the mediator produced by wild type monocytes to generate R2 cells with high efficiency. The crucial role of this IL-10 dependent pathway was underscored by supplementation experiments. Mice carrying a targeted deficiency of the IL-10 gene fail to generate the R2 population during peritonitis. However, injection of IL-10 into these animals rescues the capacity to form the R2 population. In addition the normal default pathway of monocyte development in un-infected animals which leads to the R1 population is modulated by injection of IL-10 so that the monocytes can now differentiate into the R2 population. The work presented in this thesis describes the steady state populations of phagocytes in the un-infected peritoneum and the dynamics of these populations during the induction of peritonitis. It also uncovers an IL-10 dependent pathway which regulates the choice of monocyte developmental fate within the peritoneum.
Transition metal complexes play a crucial role in antitumor therapy. Complexes of platinum, ruthenium as well as lanthanum and gallium have been investigated in preclinical as well as in clinical studies. The best known platinum(II) agents approved worldwide, cisplatin or carboplatin, are used in nearly 50% of all cancer therapies. This work focused on the development of new metal-based drugs that could act against human cancer cells. It was motivated in part by previous work with Cu(II) complexes, reporting new coordination compounds of SOD mimicking and cytotoxic activities. On the basis of this work we chose several commercially available heterocyclic ligands to synthesize new metal ion complexes in search of their interesting biological activity. New as well as previously reported Cu(II), Co(II), Pt(II) and Zn(II) complexes were synthesized using various ligands (1-6). Almost all chelating 2:1 ligand-metal complexes were obtained generally in water at room temperature in the reaction of metal(II) chloride with corresponding aromatic nitrogen ligands bearing an O-carboxylate group ligand. The synthesized chelating complexes were characterized by the use of spectroscopic methods, elemental analyses and HPLC chromatography and some by X-ray crystallography. Such coordination compounds are easily formed by transition metals with free orbitals d that can accept the donor electron pairs. The coordination is through the heterocyclic nitrogen and carboxylate oxygen donor atoms, which was shown by analysis of the characteristic functional groups in the IR spectra. The d-d transitions and absorption of visible light in Cu(II) and Co(II) complexes make them highly colored, blue, green or green-blue, respectively. The configuration of the coordination center was established in some cases by X-ray crystallography. Most of the already published structures possess the trans configuration. This led to the assumption that other uncrystallized complexes were also trans configured. However, X-ray data of the Cu(II) complex of 5 showed quite unexpectedly the cis configuration. On the other hand, the LC/MS experiments with the Pt(II) complex of 5 indicated that this complex exists in two isomeric forms, i.e., cis and trans at the Pt(II) center. Through the use of density functional calculations we optimized the structures and calculated the energies and dipole moments. The differences in energy for all complexes were about 6 to 15-fold lower when compared to cis and transplatin. The DFT calculations confirmed that the trans-isomers are more stable than their cis-isomers. UV-Vis stability studies with most of the synthesized complexes as well as some other Cu(II) complexes were performed to study the spectral changes over 24 h in addition of glutathione, a tripeptide present in the cancer cells and ascorbate that were added to the incubations. The results indicated time-dependent changes and instability of the complexes in the cells and their possible decomposition to lose the ligand and release the metal ion. In the case of Cu(II) complexes, reduction of Cu(II) to Cu(I) may take place. New species such as GSSG could arise and the complexes may decarboxylate, but these structures were not elucidated. The synthesized coordination metal(II) complexes were tested for their potential antiproliferative activities by using the crystal violet staining method in a panel of human cancer cell lines. Out of all complexes, three Pt(II) complexes of 2, 5 and 6 showed satisfactory activity and for these complexes the IC50 values were additionally determined in new RT-4, DAN-G and MCF-7 cancer cell lines. Interestingly, the active complexes were the chelating trans complexes which is quite unexpected, based on the difference in activities between cis and transplatin. All of the complexes were tested for their potential antimicrobial activities in comparison to the standard antibiotics on such bacterial strains as Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and yeast Candida maltosa. Co(II) complexes have been especially known to act against bacterial strains. The activity of the Co(II) complexes was indeed the highest of all metal(II) complexes. The ligand 2 (a nicotinic acid isomer) was also found active. This fact could explain why some antibacterial activity was found in the MIC assay. In addition to the complexes synthesized in this work, several novel heterocyclic metal(II) complexes of copper, ruthenium, platinum, gallium, osmium and lanthanum from other research groups were screened for their antiproliferative activity, some of which exhibited very potent activity in the cancer cell lines. In conclusion, Pt(II) complexes with bis-chelating heterocyclic carboxylate ligands represent a particularly interesting new class of compounds from the view point of their structural and biological properties.
Recent climate change has affected the forest system comprehensively. Northern hemisphere elevational treelines are considered as a key environment for monitoring the effects of current anthropogenic climate change. Moreover, trees from these areas are also widely employed in paleo-climate reconstructions. The stability of the tree growth climate relationship under current scenario is crucial for all tree ring based climate researches. It is important to investigate how trees respond to this rapid environmental change at altitudinal treelines. Tree cores from 21 treeline sites of three species (Pinus tabulaeformis, Picea crassifolia, and Sabina przewalskii) from Northeastern Tibetan have been conducted in this thesis. The instable correlations between tree growth and climate are the general response pattern of trees from all study sites in NE Tibetan Plateau. Picea crassifolia shows the most instable response to climate factors (mean monthly temperature and total monthly precipitation). Pinus tabulaeformis and Sabina przewalskii just showed instable and divergent responses to their main limiting climate factors but no clear trend was found which is limited by the few sample sites. Corresponding to divergent responses of Picea crassifolia to mean monthly temperature, most radial growth of Picea crassifolia were inhibited by this climate change type drought, only few trees within same sites grew faster due to temperature increasing during recent decades. The divergence response mainly started in last 30 years in six of eleven sample sites over the Northeastern Tibetan Plateau. North-westerly drier sites showed a large percentage of trees per site with a negative correlation to temperature and mostly southerly moister sites showed more mixed responses with both negatively and positively responding trees within site. Concurrent with the regional pattern, low elevation sites show mostly negative correlations with temperature and high elevation sites show more mixed responses. As the hydrothermal conditions of the investigation area changed to a drier and warmer combination, drought stress on tree growth have been intensifying over time and expanding spatially from the middle to most of our study area during the last half century. The Picea crassifolia tree growth climate relationship conducted on an elevational gradient with four different levels from upper treeline to lower treeline at the NE Tibetan Plateau. Results show that upper treeline trees show divergent growth trends and divergent responses in recent decades. Trees from lower treeline show a strengthening drought stress signal over time and no divergent growth trends within sites. This potential ecological reaction of tree populations to changing environmental conditions shows an implications for using trees to reconstruct climate, since the indiscriminate use of tree ring data from sites showing opposite responses to increasing warming could cause mis-calibration of tree ring based climate reconstructions, and over- or underestimation of carbon sequestration potential in biogeochemical models. The physiological response of Sabina przewalskii tree growth to major limiting climate factors based on the Vaganov-Shashkin (VS) model indicated that precipitation during the early growing season, especially in May and June, has significant effect on tree growth, while temperature mainly affects tree growth by warming-induced drought and by extending the growing season in the NE Tibetan Plateau. Under current and projected climate scenarios, modeling results predict an increase in radial growth of Sabina przewalskii around the Qaidam Basin, with the potential outcome that regional forests will increase their capacity to sequester carbon. However, most Picea crassifolia trees growing at lower elevations than Sabina przewalskii might be continue stressed by the warming induced drought and might decrease radial growth in future.
In this thesis, it was the subject to build a setup to study the interaction of clusters with intense laser light. A magnetron sputter cluster ion source was built to create metal clusters for the planned investigations. Furthermore, a linear Paul trap setup was built in order to allow the investigation of the mentioned interaction at one specific cluster size. The whole apparatus was characterized and first experiments were performed.
Background/Aims: Acute pancreatitis (AP) is characterized by premature zymogen activation, systemic inflammatory response resulting in inflammatory infiltrates, sustained intracellular calcium, neurogenic inflammation and pain. The inhibitory neurotransmitter and cytoprotective amino acid glycine exerts a direct inhibitory effect on inflammatory cells, inhibits calcium influx and neuronal activation and therefore represents a putative therapeutic agent in AP. Methods: To explore the impact of glycine, mild AP was induced in rats by supramaximal cerulein stimulation (10 µg/kg BW/h) and severe AP by retrograde injection of sodium taurocholate solution (3%) into the common biliopancreatic duct. 100/300 mmol glycine was administered intravenously before induction of AP. To elucidate the effect of glycine on AP, we determined pathomorphology, pancreatic cytokines as well as proteases, serum lipase and amylase, pancreatic and lung MPO activity and pain sensation. Results: Glycine administration resulted in a noticeable improvement of pathomorphological alterations in AP, such as a reduction of necrosis, inflammatory infiltrates and cytoplasmic vacuoles in cerulein pancreatitis. In taurocholate pancreatitis, glycine additionally diminished pancreatic cytokines and MPO activity, as well as serum lipase and amylase levels. Conclusions: Glycine reduced the severity of mild and much more of severe AP by attenuating the intrapancreatic and systemic inflammatory response. Therefore, glycine seems to be a promising tool for prophylactic treatment of AP.
Chronic pancreatitis has long been thought to be mainly associated with immoderate alcohol consumption. The observation that only ∼10% of heavy drinkers develop chronic pancreatitis not only suggests that other environmental factors, such as tobacco smoke, are potent additional risk factors, but also that the genetic component of pancreatitis is more common than previously presumed. Either disease-causing or protective traits have been indentified for mutations in different trypsinogen genes, the gene for the trypsin inhibitor SPINK1, chymotrypsinogen C, and the cystic fibrosis transmembane conductance regulator (CFTR). Other factors that have been proposed to contribute to pancreatitis are obesity, diets high in animal protein and fat, as well as antioxidant deficiencies. For the development of pancreatic cancer, preexisting chronic pancreatitis, more prominently hereditary pancreatitis, is a risk factor. The data on environmental risk factors for pancreatic cancer are, with the notable exception of tobacco smoke, either sparse, unconfirmed or controversial. Obesity appears to increase the risk of pancreatic cancer in the West but not in Japan. Diets high in processed or red meat, diets low in fruits and vegetables, phytochemicals such as lycopene and flavonols, have been proposed and refuted as risk or protective factors in different trials. The best established and single most important risk factor for cancer as well as pancreatitis and the one to clearly avoid is tobacco smoke.
Background/Aims: To develop a clinically relevant immunocompetent murine model to study pancreatic cancer using two different syngeneic pancreatic cancer cell lines and to assess MRI for its applicability in this model. Methods: Two cell lines, 6606PDA and Panc02, were employed for the experiments. Cell proliferation and migration were monitored in vitro. Matrigel™ was tested for its role in tumor induction. Tumor cell growth was assessed after orthotopic injection of tumor cells into the pancreatic head of C57/BL6 mice by MRI and histology. Results: Proliferation and migration of Panc02 were significantly faster than those of 6606PDA. Matrigel did not affect tumor growth/migration but prevented tumor cell spread after injection thus avoiding undesired peritoneal tumor growth. MRI could reliably monitor longitudinal tumor growth in both cell lines: Panc02 had a more irregular finger-like growth, and 6606PDA grew more spherically. Both tumors showed local invasiveness. Histologically, Panc02 showed a sarcoma-like undifferentiated growth pattern, whereas 6606PDA displayed a moderately differentiated glandular tumor growth. Panc02 mice had a significantly shorter (28 days) survival than 6606PDA mice (50 days). Conclusion: This model closely mimics human pancreatic cancer. MRI was invaluable for longitudinal monitoring of tumor growth thus reducing the number of mice required. Employing two different cell lines, this model can be used for various treatment and imaging studies.
Background: It has not been investigated whether there are associations between urinary iodine (UI) excretion measurements some years apart, nor whether such an association remains after adjustment for nutritional habits. The aim of the present study was to investigate the relation between iodine-creatinine ratio (ICR) at two measuring points 5 years apart. Methods: Data from 2,659 individuals from the Study of Health in Pomerania were analyzed. Analysis of covariance and Poisson regressions were used to associate baseline with follow-up ICR. Results: Baseline ICR was associated with follow-up ICR. Particularly, baseline ICR >300 µg/g was related to an ICR >300 µg/g at follow-up (relative risk, RR: 2.20; p < 0.001). The association was stronger in males (RR: 2.64; p < 0.001) than in females (RR: 1.64; p = 0.007). In contrast, baseline ICR <100 µg/g was only associated with an ICR <100 µg/g at follow-up in males when considering unadjusted ICR. Conclusions: We detected only a weak correlation with respect to low ICR. Studies assessing iodine status in a population should take into account that an individual with a low UI excretion in one measurement is not necessarily permanently iodine deficient. On the other hand, current high ICR could have been predicted by high ICR 5 years ago.
Heart Rate Reduction by Ivabradine Improves Aortic Compliance in Apolipoprotein E-Deficient Mice
(2012)
Background: Impaired vascular compliance is associated with cardiovascular mortality. The effects of heart rate on vascular compliance are unclear. Therefore, we characterized effects of heart rate reduction (HRR) by I(f) current inhibition on aortic compliance and underlying molecular mechanisms in apolipoprotein E-deficient (ApoE<sup>–</sup>/<sup>–</sup>) mice. Methods: ApoE<sup>–</sup>/<sup>–</sup> mice fed a high-cholesterol diet and wild-type (WT) mice were treated with ivabradine (20 mg/kg/d) or vehicle for 6 weeks. Compliance of the ascending aorta was evaluated by MRI. Results: Ivabradine reduced heart rate by 113 ± 31 bpm (∼19%) in WT mice and by 133 ± 6 bpm (∼23%) in ApoE<sup>–</sup>/<sup>–</sup> mice. Compared to WT controls, ApoE<sup>–</sup>/<sup>–</sup> mice exhibited reduced distensibility and circumferential strain. HRR by ivabradine increased distensibility and circumferential strain in ApoE<sup>–</sup>/<sup>–</sup> mice but did not affect both parameters in WT mice. Ivabradine reduced aortic protein and mRNA expression of the angiotensin II type 1 (AT1) receptor and reduced rac1-GTPase activity in ApoE<sup>–</sup>/<sup>–</sup> mice. Moreover, membrane translocation of p47<sup>phox</sup> was inhibited. In ApoE<sup>–</sup>/<sup>–</sup> mice, HRR induced anti-inflammatory effects by reduction of aortic mRNA expression of IL-6, TNF-alpha and TGF-beta. Conclusion: HRR by ivabradine improves vascular compliance in ApoE<sup>–</sup>/<sup>–</sup> mice. Contributing mechanisms include downregulation of the AT1 receptor, attenuation of oxidative stress and modulation of inflammatory cytokine expression.
Background: To analyze the relation and distribution of mean, systolic and diastolic ocular perfusion pressure (OPP) in telemedical homemonitoring of patients with primary open-angle glaucoma (POAG). Methods: 70 patients with POAG measured intraocular pressure (IOP) and blood pressure at home for a period of 6 months with the Goldmann applanation self-tonometer Ocuton S and the blood pressure device boso medicus PC. Twenty-four-hour profiles were taken every 4 weeks in addition to single measurements in the morning and evening once a week. All measured values were transmitted to an electronic patient record, which calculated OPP by taking systolic, diastolic and mean arterial blood pressure and subtracting IOP. Results: We analyzed 3,282 values of mean, systolic and diastolic OPP. The quantity of values below the risk levels of the Barbados Eye Studies was calculated. We found values lower than the risk levels for LE: 49 (1.5%)/RE: 60 (1.8%) systolic OPP, LE: 1,623 (49.5%)/RE: 1,761 (53.7%) diastolic OPP and LE: 687 (20.9%)/RE: 794 (24.2%) mean OPP. The individual average OPP levels of all 70 patients below the risk levels showed the following distribution: LE: 4 (5.7%)/RE: 6 (8.6%) systolic OPP, LE: 19 (27.1%)/RE: 20 (28.6%) diastolic OPP and LE: 10 (14.3%)/RE: 10 (14.3%) mean OPP. Conclusion: The individual distribution of different OPP values in POAG patients is not easy to interpret for clinical ophthalmologists. Precise practicable guidelines for clinical use still have to be determined.
Background: Alveolar soft-part sarcoma (ASPS) is a rare sarcoma often occurring in young patients that is characterized by the unbalanced translocation der(17)t(X;17) (p11;q25). Although itusuallyshowsan indolent clinical course, the prognosis is usually poor in advanced disease. Since standard chemotherapy regimens used in soft-tissue sarcomas lack efficacy in ASPS, new therapeutic options are needed. We investigated the efficacy of trabectedin, which has demonstrated activity in a variety of cancer types including some of the most prevalent translocation-related sarcomas. Patients and Methods: 7 patients with metastatic or advanced ASPS treated with trabectedin in the Sarcoma Center Berlin-Brandenburg and the University Hospital of Greifswald were analyzed for median progression-free survival (mPFS), overall survival (OS), and therapy-related toxicity. Results: In 6 patients with documented disease progression, disease stabilization was reached with trabectedin; only 1 patient experienced progressive disease. The mPFS and OS were 7 months and 21 months, respectively, since the start of trabectedin treatment. Overall, no severe Common Toxicity Criteria (CTC) grade 3 or 4 toxicity was observed. Conclusions: The poor prognosis of patients with ASPS has so far been due to the unavailability of effective systemic treatments. Trabectedin can be considered the only currently registered drug with clinical activity in this disease.
Background: Among the five somatostatin receptors (sst<sub>1</sub>-sst<sub>5</sub>), the sst<sub>3</sub> receptor displays a distinct pharmacological profile. Like sst<sub>2</sub>, the sst<sub>3</sub> receptor efficiently internalizes radiolabeled somatostatin analogs. Unlike sst<sub>2</sub>, however, internalized sst<sub>3</sub> receptors are rapidly transferred to lysosomes for degradation. Apart from this, very little is known about the clinical relevance of the sst<sub>3</sub> receptor, which may in part be due to the lack of specific monoclonal sst<sub>3</sub> antibodies. Methods: Here, we have extensively characterized the novel rabbit monoclonal anti-human sst<sub>3</sub> antibody UMB-5 using transfected cells and receptor-expressing tissues. UMB-5 was then subjected to immunohistochemical staining of a series of 190 formalin-fixed, paraffin-embedded normal and neoplastic human tissues. Results: Specificity of UMB-5 was demonstrated by detection of a broad band migrating at a molecular weight of 70,000–85,000 in immunoblots from human pituitary. After enzymatic deglycosylation, the size of this band decreased to a molecular weight of 45,000. Tissue immunostaining was completely abolished by pre-adsorption of UMB-5 with its immunizing peptide. In addition, UMB-5 detected distinct cell populations in human tissues like pancreatic islands, anterior pituitary, adrenal cortex, adrenal medulla, and enteric ganglia, similar to that seen with a rabbit polyclonal antibody generated against a different carboxyl-terminal epitope of the sst<sub>3</sub> receptor. In a comparative immunohistochemical study, UMB-5 yielded predominant plasma membrane staining in the majority of pituitary adenomas, pheochromocytomas, and a subset of neuroendocrine tumors. The sst<sub>3</sub> receptor was also present in many glioblastomas, pancreatic, breast, cervix, and ovarian carcinomas. Conclusion: The rabbit monoclonal antibody UMB-5 may prove of great value in the identification of sst<sub>3</sub>-expressing tumors during routine histopathological examinations. Given its unique trafficking properties, these tumors may be potential candidates for sst<sub>3</sub>-directed receptor radiotherapy.
Background: In clinical practice, treatment of genital tract infections is based on administration of either antibiotics or antiseptics. While antibiotics may be applied systemically or topically, antiseptics may be applied only topically. In case of bacterial vaginosis (BV), antibiotic therapy may often be limited and side effects due to systemic administration may develop. Polihexanide (PHMB) is a promising option for the topical treatment of genital tract infections, in particular BV and vaginitis. Method: A systematic search for publications on the use of PHMB for the treatment of genital infections in two electronic databases was performed. Titles, abstracts and citations were imported into a reference database. Duplicates were removed and two reviewers assessed each identified publication separately. Results: Among a total of 204 references, 3 prospective randomized trials were identified. Two trials treated BV infections with PHMB in comparison to clindamycin as antibiotic standard therapy with no significant differences either in safety or in efficacy. The third controlled trial investigated the clinical efficacy of PHMB compared to placebo in the treatment of human papilloma virus. Patients treated with PHMB daily for up to 16-weeks showed significantly higher (52%) clearance of genital warts as compared to patients treated with placebo (4%). Conclusion: PHMB may be a clinically effective alternative for the treatment of BV and human papilloma virus. Although PHMB-based antiseptics are available since the late 90s, controlled trials to investigate its clinical potential for antiseptic treatment are scant. Clinical use of antiseptics for the treatment of infectious diseases should be explored and supported further.
Colonization and infection of wounds represent a major reason for the impairment of tissue repair. Recently, it has been reported that tissue-tolerable plasma (TTP) is highly efficient in the reduction of the bacterial load of the skin. In the present study, the antiseptic efficacy of TTP was compared to that of octenidine hydrochloride with 2-phenoxyethanol. Both antiseptic methods proved to be highly efficient. Cutaneous treatment of the skin with octenidine hydrochloride and 2-phenoxyethanol leads to a 99% elimination of the bacteria, and 74% elimination is achieved by TTP treatment. Technical challenges with an early prototype TTP device could be held responsible for the slightly reduced antiseptic properties of TTP, compared to a standard antiseptic solution, since the manual treatment of the skin surface with a small beam of the TTP device might have led to an incomplete coverage of the treated area.
Effectiveness of Varenicline as an Aid to Smoking Cessation in Primary Care: An Observational Study
(2012)
Aims: Although varenicline is commonly prescribed in primary care, information on smoking-related comorbidities and the effectiveness of varenicline in this context in Germany is scarce. This study assessed the efficacy and safety of varenicline in a large sample of patients seeking smoking cessation treatment through their general practitioners. The frequency of comorbidities was also evaluated. Methods: This was a 12-week, prospective, observational, non-comparative phase IV trial conducted in Germany. Abstinence rates at week 12 were evaluated by verbal reporting using the nicotine use inventory. Results: Overall, 1,391 subjects were enrolled; 1,177 received study medication and were evaluated for effectiveness and safety. At the end of the study, 71.1% (95% confidence interval 68.5–73.7) of subjects were abstinent. There were a total of 205 all-causality adverse events; 2.2% were classified as serious or severe. There were no fatal adverse events. At inclusion, 66.7% of participants had at least 1 concurrent comorbidity, with chronic obstructive pulmonary disease (35.5%), hypertension (29.6%) and depression (10.4%) being the most commonly reported. Conclusion: These real-world data indicate that varenicline is an effective and well-tolerated smoking cessation treatment when used in the primary care setting including patients with smoking-related comorbidities.